Brassica$napus$L. - 植物科学技术学院

Transcription

湖北 ! 武汉
二O一二年十一月
湖北 ! 武汉
二O一二年十一月
究
2012
$ i
!
DOF!
AtDof1.7!RNA!
A9
!...........!2!
!...........................................................................................!3!
Transcriptome!analysis!reveals!significant!differences!in!gene!expression!
between!nearC!isogenic!lines!of!Brassica!napus!L.!with!Sclerotinia!
sclerotiorum!challenge!....................................................................................................!4!
QTL
!.................................................................!5!
7365A
BnaC.Tic40
!................................................................................!6!
Genomic!change,!retrotransposon!mobilization!and!extensive!cytosine!
methylation!alteration!in!Brassica!napus!introgressions!from!two!
intertribal!hybridizations!...............................................................................................!7!
GbTCP,!a!cotton!TCP!transcription!factor,!confers!fibre!elongation!and!root!
hair!development!by!a!complex!regulating!system!...............................................!8!
Comparative!transcript!profiling!analyses!of!gynoecium!development!in!a!
female!sterility!Brassica!napusCOrychophragmus!violaceus!disomic!
addition!line!........................................................................................................................!9!
Towards!understanding!the!molecular!mechanism!of!a!multiallelic!
maleCsterile!locus!in!Brassica!napus!L!....................................................................!10!
Genetic!structure!and!linkage!disequilibrium!pattern!of!a!rapeseed!
(Brassica!napus!L.)!association!mapping!panel!revealed!by!microsatellites
!...............................................................................................................................................!11!
Genetics!of!seeds!per!silique!and!mapping!of!major!QTL!in!Brassica'napus'L.
!...............................................................................................................................................!12!
GenomeCwide!analysis!of!plantCspecific!Dof!transcription!factor!family!in!
tomato!................................................................................................................................!13!
Genetic!Analysis!and!Improvement!of!a!Recessive!Genic!Male!Sterility!Line!
7365A!in!Brassica!napus!..............................................................................................!14!
Fine!mapping!and!candidate!gene!analysis!of!the!nuclear!restorer!gene!Rfp!
for!pol!CMS!in!rapeseed!(Brassica!napus!L.)!..........................................................!15!
qSS7!.....................................................................................!16!
Detection!and!genotyping!of!rest!riction!fragment!associated!
polymorphisms!in!polyploidy!crops!with!a!pseudo!reference!sequence:!A!
case!study!in!tetraploid!Brassica!napus!..................................................................!17!
!
ii$
WRKY,!A!New!Player!in!the!Biosynthesis!of!Terpenoid!Indole!Alkaloids!in!
Catharanthus!roseu!........................................................................................................!18!
7DL
BAC
!...............................................!19!
Cloning!and!analysis!of!MAPK1!and!it’s!promoter!from!Brassica!napus!.....!20!
Computational!Studies!of!PostCtranslational!Modifications!............................!21!
Abundant!microsatellite!diversity!and!oil!content!in!wild!Arachis!species!22!
0431
21RIL
!............................!23!
MYB!
MYB
!.............................................................!24!
!
Identification!and!analysis!of!microRNAs!associated!with!drought!in!
Solanum'tuberosum!........................................................................................................!26!
Cotton!GhAF18!regulates!anther!abortion!by!delaying!tapetal!programmed!
cell!death!via!the!GlcCABACROS!signaling!pathway!..............................................!27!
Mapping!of!Boron!Efficient!QTL!in!Brassica!napus!under!different!boron!
environments!...................................................................................................................!28!
Introgression!of!bacterial!blight!resistance!genes!Xa7,!Xa21!and!Xa23!into!
thermosensitive!genetic!male!sterile!rice!(Oryza!sativa!L.)!by!phenotypic!
and!molecular!markerCassisted!selection!..............................................................!29!
Proteomes!alteration!of!Brassica!napus!in!response!to!phosphorus!
deficiency!..........................................................................................................................!30!
Dissecting!Quantitative!Trait!Loci!for!Boron!Efficiency!across!Multiple!
Environments!in!Brassica!napus!...............................................................................!31!
!.............................................................................!32!
!...................................!33!
early!senescence!leaf!4 esl4
!.................................................................................................................................!34!
Salicylic!acid!analogues!with!biological!activity!may!induce!chilling!
tolerance!of!maize!(Zea!mays)!seeds!.......................................................................!35!
Genetic!and!QTL!analysis!for!seedling!cold!tolerance!in!rice!..........................!36!
Sequence!and!Functional!Divergence!of!FRUITFULL!Homeologs!in!Brassica!
napus!...................................................................................................................................!37!
PnLOX2
!........................................!38!
Blast!resistance!of!Hybrid!rice!Oryza'sativa!L.!.....................................................!39!
2012
$ iii
Tomato!SlDREB!gene!restricts!leaf!expansion!and!internode!elongation!by!
downregulating!key!genes!for!gibberellin!biosynthesis!...................................!40!
Mapping!and!identification!of!QTLs!for!resistance!to!Sclerotinia!
sclerotiorum!and!its!interaction!with!other!traitCrelated!QTLs!or!genes!in!
Brassica!napus!L.!.............................................................................................................!41!
Characterization!of!a!putative!E3!ubiquitin!ligase!gene!OsETOL1!conferring!
drought!and!submergence!stress!in!rice!................................................................!42!
R421
!..............................................!43!
Screening!fluorescent!dyes!for!antiCcounterfeiting!labelling!in!rape!seed!
(Brassica'napus)!..............................................................................................................!44!
!
!.................................................!46!
Characterize!the!genetic!basis!of!selfCincompatible!line!‘SC1300’!in!Brassica!
napus!and!set!up!a!selfCincompatibility!molecular!marker!system!for!
hybrid!breeding!..............................................................................................................!47!
MarkerCassisted!Breeding!of!Recessive!Genic!Male!Sterility!Lines!and!their!
Temporary!Maintainers!in!Rapeseed!(Brassica'napus)!....................................!48!
MCID
DNA
!..........................................................................................................................................!49!
Enhanced!Oil!Accumulation!in!Vegetative!Organs!by!Metabolic!Pathway!
Assembly!in!Tobacco!(N.!tabacum!L.)!for!HighCquality!Renewable!Biofuel!
Oil!.........................................................................................................................................!50!
!......................................................!51!
A!pleiotropic!drug!resistance!gene!involved!in!the!transport!of!plant!
secondary!metabolites!in!Panax!ginseng!...............................................................!52!
!
A!Metabolic!and!Genetic!Analysis!of!Cotton!Fiber!Cell!Development!in!
Response!to!Inhibition!by!the!Flavonoid!Naringenin!.........................................!54!
Characterization!of!six!NAC!transcription!factor!genes!in!Chickpea!(Cicer!
arietinum),!which!are!involved!in!abiotic!stress!responses!and!various!
developmental!processes!............................................................................................!55!
Improvement!of!Minhui3229!for!blast,!bacterial!blight!and!brown!
planthopper!resistance!and!plant!stature!through!morphological!and!
markerCassisted!selection!...........................................................................................!56!
Genetic!analysis!of!grain!filling!rate!using!conditional!QTL!mapping!in!
!
iv$
maize!...................................................................................................................................!57!
!.............................................................................!58!
FAD2
FAD3
FATB
!.!59!
ANS
DFR
!....................!60!
Isolation!and!functional!characterization!of!a!new!bZIP!transcription!factor!
in!waterlogged!seedlings!of!maize!............................................................................!61!
ZmGA3ox2,!a!candidate!gene!for!a!major!QTL,!qPH3.1,!for!plant!height!in!
maize!...................................................................................................................................!62!
Identification!of!a!major!QTL!for!silique!length!and!seed!weight!in!oilseed!
rape!(Brassica!napus!L.)!...............................................................................................!63!
A!StayCGreen!Gene!SGR1!regulates!lycopene!accumulations!by!directly!inter
acting!with!the!key!carotenoid!biosynthetic!enzyme!PSY1!during!fruit!ripen
ing!in!tomato!....................................................................................................................!64!
A!Large!Insertion!in!bHLH!Transcription!Factor!BrTT8!Resulting!in!Yellow!
Seed!coat!in!Brassica!rapa!...........................................................................................!65!
MapCbased!cloning!and!functional!analysis!of!qbpc!which!controls!protein!
content!in!brown!rice!....................................................................................................!66!
A!serine!threonine!protein!kinase!receptor!gene!is!the!potential!candidate!
of!a!quantitative!trait!locus!for!grain!yieldCrelated!trait!in!maize!.................!67!
RIL
QTL
!................!68!
Function!analysis!of!flowering!locus!T!in!a!model!for!the!interaction!of!flow
eringCtime!genes!in!wheat!...........................................................................................!69!
!...........................................................!70!
“
”
QTL
!.....................!71!
Systematic!Metabolic!Engineering!of!ωC7!Fatty!Acids!in!Soybean!Seeds!.....!72!
Opaque2
!..............................................................!73!
The!cloning!and!characterization!of!Shrunkrn4!gene!in!maize!......................!74!
Characterization!and!Identification!of!ZmaNAC36,!a!Novel!NAC!Domain!
Transcription!Factor,!Imply!Its!Potential!Role!in!the!CoCexpression!of!
Starch!Synthesis!Genes!in!Maize!Endosperm!........................................................!75!
Identification!and!characterization!of!microRNAs!in!the!developing!maize!e
ndosperm!..........................................................................................................................!76!
QTL!analyses!for!resistance!to!Sclerotinia!sclerotiorum!in!Brassica!napus!
revealing!its!association!with!flowering!time!.......................................................!77!
Differential!accumulation!of!phenolic!compounds!and!expression!of!related!
genes!between!the!blackC!and!yellowCseeded!Brassica!napus!L!.....................!78!
2012
$ v
Genetic!dissection!of!trichome!locus!(T1)!and!a!major!QTL!controlling!
multiple!fibre!quality!traits!in!upland!cotton!.......................................................!79!
PSKCαsignalling!involve!in!cotton!fiber!elongation!.............................................!80!
QTL
!.......................................!81!
Genetic!Control!of!Seed!Shattering!in!Rice!by!the!APETALA2!Transcription!
Factor!SHATTERING!ABORTION1!..............................................................................!82!
Y
!..........................................................!83!
QTL
!....................!84!
Genetic!analysis!and!fine!mapping!of!the!Ga1CS!gene!region!conferring!cros
sCincompatibility!in!maize!...........................................................................................!85!
Identification!of!a!major!QTL!for!silique!length!and!seed!weight!in!oilseed!
rape!.....................................................................................................................................!86!
!...............................................................................................................................................!87!
C
SSH
!.........................!88!
Screening!Identification!Indexes!of!Drought!Resistance!at!Whole!Growth!
Period!in!Upland!Rice!Varieties!from!Yunnan!Province!...................................!89!
Phenotype!Analysis!and!Gene!Mapping!of!a!Novel!Narrow!Leaf!Mutant!
(nal9(t))!in!Rice!(Oryza!sativa!L.)!..............................................................................!90!
Development!of!chromosome!segment!substitution!lines!in!Brassica!napus!
L.!using!maker!assisted!selection!..............................................................................!91!
A!Cloning!and!Functional!Analysis!of!Dynamic!Narrow!Leaf! DNL1 Gene!in!
Rice!......................................................................................................................................!92!
A!Genome!Wide!Association!Study!Revealed!the!Genetic!Architecture!and!
Important!Genes!for!Regulating!Kernel!Row!Number!in!Maize!Female!
Inflorescence!....................................................................................................................!93!
!
C
!.........................................................!95!
GenomeCwide!identification!and!analysis!of!microRNA!responding!to!
longCterm!waterlogging!in!crown!roots!of!maize!seedlings!.............................!96!
A!Ubiquitin!Ligase!of!Symbiosis!Receptor!Kinase!Involved!in!Nodule!
Organogenesis!.................................................................................................................!97!
208
!................................................................................................!98!
ChinaExpansin,!especially!the!domain1!plays!a!major!role!in!the!cotton!
!
vi$
fiber!development!..........................................................................................................!99!
High!Resolution!Melting!Curve!Analysis:!An!Efficient!Method!for!
Fingerprinting!of!Hybrid!Rice!Cultivars!and!Their!Parental!Lines!..............!100!
!
!
!..........................................................................................................................!101!
2012
$ 1
2$
DOF$
!
AtDof1.7$RNA$
$ $
2012
$ 3
A9
$
4$
!
Transcriptome$analysis$reveals$significant$differences$in$gene$
expression$between$nearE$isogenic$lines$of$Brassica$napus$L.$
with$Sclerotinia$sclerotiorum$challenge$
Sclerotinia sclerotiorum (Lib.) de Bary is a destructive necrotrophic fungal pathogen
causing disease in more than 400 plant species, primarily dicotyledonous, including
grain legumes(soybean, pea and bean) and oilseeds (oilseed rape and sun-flower).It
causes rotting of leaves, stems, and pods in oilseed rape, resulting in a considerable
seed yield loss in China and other regions around the world. The biochemical and
molecular events occurring in host plant tissues during disease progression are not
clear and the molecular basis of plant defense to this pathogen is poorly understood.
To know more about the mechanism and regulatory network of the host reaction to
fungus infection, and detect susceptibility and resistance related genes, transcriptome
analysis based on RNA-Seq was used to study the changes of gene expression after
infected by S. sclerotiorum in the susceptible and the disease-resistant near-isogenic
lines of Brassica napus L. Plants were selected for inoculation and sampling using a
randomized design with three biological replicates for each cultivar. Each replicate
consisted of 60 plants for three time points (0, 12 and 72 hpi). The lesion-surrounding
areas of the leaf were harvested from one biological replicate at each time point were
pooled as one sample for RNA-Seq.At 12hpi, a total of 33108 genes in which 25625
up-regulated and 5415 down-regulated, and 32104 genes with 18927 up-regulatedand
and 13117 down-regulated, were found to be differentially expressed respectively in
the susceptible and the disease-resistant near-isogenic line respectively. At 24hpi, the
number of differentially expressed genes were 21342 and 19024. Genes detected at
the same time point (12-24h)were compared between the near-isogenic lines .7403
genes were up-regulated and 5152 genes were down-regulated at 12hpi,and 5845
up-regulated, 5917 down-regulated at 24hpi in the disease-resistant near-isogenic line
compared to the susceptible parent. The differentially expressed genes were
categorized into groups according to the putative function of each gene, the categories
included proteins involved in defense related pathways, Plant-pathogen interaction,
antioxidation, Plant hormone signal transduction, Natural killer cell mediated
cytotoxicity transport, cell maintenance, calcium signaling pathway, cxidative
phosphorylation, material and energy metabolism, biological regulation and
transcription etc. Further analysis and studies are in progress and it will help us to
know more about the mechanism of the interaction between Sclerotinia sclerotiorum
and its plant host and the genes related to the resistance or susceptibility to Sclerotinia
sclerotiorum will be detected and be used in the molecular breeding of resistant
varieties.
2012
$ 5
QTL
$
$
97B (ZS97)
93-11
(Backcross recombinant inbred lines, BCRIL)
8
5
QTL
QTL
QTL
1
8
!
6$
7365A
BnaC.Tic40
$$
2
S45AB
9012AB
7365A
2
(Bnrfrf)
Bnms3ms3rfrf
BnMs3/Bnms3
BnRf/Bnrf
9012A
BnRf
Bnms3ms3
7365A
7365A
PCD
Real-time
BnQRT3
BnA6
BnMSR66 BnQRT1
7365A
BnMs3
BnaC.Tic40
7365A
BnaC.Tic40
BnaC.Tic40
Tic40
7365A
7365A
A
C
BnMs3
7-749
BnRf
A
C
7365A
2012
$ 7
Genomic$change,$retrotransposon$mobilization$and$extensive$
cytosine$methylation$alteration$in$Brassica$napus$
introgressions$from$two$intertribal$hybridizations$ $
Xueli Zhang1, Xianhong Ge1*, Yujiao Shao2, Genlou Sun3, Zaiyun Li
National Key Laboratory of Crop Genetic Improvement, National Center of Crop Molecular
Breeding Technology
Hybridization and introgression represent an important means for the transfer
and/or de novo origination of traits and play an important role in facilitating
speciation and plant breeding. Two sets of introgression lines in Brassica napus L.
were previously established by its intertribal hybridizations with two wild species and
long-term selection. In this study, amplified fragment length polymorphisms (AFLP),
sequence-specific amplification polymorphism (SSAP) and methylation-sensitive
amplified polymorphism (MSAP) were used to determine their genomic change,
retrotransposon mobilization and cytosine methylation alteration in these lines. The
genomic change revealed by the loss or gain of AFLP bands occurred for ~10% of the
total bands amplified in the two sets of introgressions, while no bands specific for
wild species were detected. The new and absent SSAP bands appeared for 9 out of 11
retrotransposons analyzed, with low frequency of new bands and their total
percentage of about 5% in both sets. MSAP analysis indicated that methylation
changes were common in these lines (33.4-39.8%) and the hypermethylation was
more frequent than hypomethylation. Our results suggested that certain extents of
genetic and epigenetic alterations were induced by hybridization and alien DNA
introgression. The cryptic mechanism of these changes and potential application of
these lines in breeding were also discussed.
8$
!
GbTCP,$a$cotton$TCP$transcription$factor,$confers$fibre$
elongation$and$root$hair$development$by$a$complex$
regulating$system$ $
Juan Hao, Lili Tu, Haiyan Hu, Jiafu Tan, Fenglin Deng, Wenxin Tang, Yichun Nie and Xianlong
Zhang*
National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University,
Wuhan, Hubei 430070, PR China
As the most important natural raw material for textile industry, cotton fibres are
an excellent model for studying single-cell development. Although expression
profiling and functional genomics have provided some data, the mechanism of fibre
development is still not well known. A class I TCP transcription factor (designated
GbTCP), encoding 344 amino acids, was isolated from the normalized cDNA library
of sea-island cotton fibre (from –2 to 25 days post anthesis). GbTCP was
preferentially expressed in the elongating cotton fibre from 5 to 15 days post anthesis.
Some expression was also observed in stems, apical buds, and petals. RNAi silencing
of GbTCP produced shorter fibre, a reduced lint percentage, and a lower fibre quality
than the wild-type plants. Overexpression of GbTCP enhanced root hair initiation and
elongation in Arabidopsis and regulated branching. Solexa sequencing and
Affymetrix GeneChip analysis indicated that GbTCP positively regulates the level of
jasmonic acid (JA) and, as a result, activates downstream genes (reactive oxygen
species, calcium signalling, ethylene biosynthesis and response, and several NAC and
WRKY transcription factors) necessary for elongation of fibres and root hairs. JA
content analysis in cotton also confirmed that GbTCP has a profound effect on JA
biosynthesis. In vitro ovule culture showed that an appropriate concentration of JA
promoted fibre elongation. The results suggest that GbTCP is an important
transcription factor for fibre and root hair development by regulating JA biosynthesis
and response and other pathways, including reactive oxygen species, calcium channel
and ethylene signalling.
2012
$ 9
Comparative$transcript$profiling$analyses$of$gynoecium$
development$in$a$female$sterility$Brassica$
napusEOrychophragmus$violaceus$disomic$addition$line$ $
Wenqin Fu, Xianhong Ge, Zaiyun Li
National Key Laboratory of Crop Genetic Improvement, College of Plant Science and
Technology, Huazhong Agricultural University, Wuhan 430070, P. R. China
The gynoecium is one of the most complex organs of angiosperm specialized for
seed production and dispersal. Brassica gynoecium consists of an apical stigma, a
short style and a large central ovary where ovules grow in. Here, we produced a
female sterility Brassica napus-Orychophragmus violaceus disomic addition line (S1)
with an entirely abortive gynoecium at early stage of development. Tissue clearing
observation showed that ovules initiated only one short integument primordia which
undergoes no further development and the female gametophyte development was
blocked at mononuclear embryo sac stage. Meanwhile, the stigmatic papillae and
whole pistil of abortive gynoecium in addition line are shorter than those of B. napus
(H3). To further clarify the genes regulation related with gynoecium development,
comparative transcripts profiling was performed using Illumina sequencing
technology. Using Brassica_95k_unigene as the reference genome, a total of 28,065
and 27,653 unigenes were identified and transcribed in S1 and H3 respectively,
suggesting that newly initiated transcription occurs in S1. Further comparison of the
transcripts abundance between S1 and H3 revealed that 4540 unigenes show more
than two fold expression difference. Moreover, 467 unigenes are up-regulated and
172 are down-regulated at RPKM≥50 level by more than fourfold difference. Pathway
enrichment analysis indicated that 20 metabolic pathways or signal transduction
pathways are significantly rich in the differentially expressed genes. A number of
genes were identified related with important development process of gynoecium,
embryo sac, ovule, integument and pollen tube as well as the interactions between
pollen and pistil. A set of related genes as candidate genes for evalution of roles in
gynoecium development were choose for further transgenic analysis which may
provide new information for the molecular foundation of female sterility in B. napus.
10$
!
Towards$understanding$the$molecular$mechanism$of$a$
multiallelic$maleEsterile$locus$in$Brassica$napus$L$ $
Qiang Xin, Wei Lu, Dengfeng Hong, Guangsheng Yang*
WuHan
HuBei
Huazhong Agricultural University
Hybrid cultivars are popular in crop production due to their increased yield. The
generation of hybrids is greatly facilitated by male-sterile strains, making male
sterility a much sought-after trait for plant breeders. A triallelic male sterility locus,
BnMs5, from a Chinese B. napus two-type line Rs1046AB, was used for the
commercial reproduction of three-line hybrids, though the function of this locus in
microsporogenesis remains unknown so far. In this study, we corresponded BnMs5 to
six candidate genes in a 21-kb interval in A subgenome of B. napus by integration of
various existing genetic maps, identification of the BAC clones and comparative
genome analysis between B. napus, B. rapa, B. oleracea and Arabidopsis. The
genomic sequences of these candidates were successfully isolated from the restorers,
and subsequent complementation experiments showed that one of the six candidates
can restore the male sterility caused by the allele of BnMs5b. This finding laid a solid
foundation towards understanding the interesting biological phenomenon of how the
variations of a multiallelic locus can result in the maintenance and restoration of male
sterility. Key words triallelic male sterility; complementation experiments; candidate
genes validation
2012
$ 11
Genetic$structure$and$linkage$disequilibrium$pattern$of$a$
rapeseed$(Brassica$napus$L.)$association$mapping$panel$
revealed$by$microsatellites$ $
Yingjie Xiao, Dongfang Cai, Wei Yang, Wei Ye, Muhammad Younas, Jiangsheng Wu, Kede Liu
National Key Laboratory of Crop Genetic Improvement, National Center of Plant Gene Research
(Wuhan), Huazhong Agricultural University, Wuhan 430070, China
Understanding the population structure and linkage disequilibrium (LD) is a
prerequisite for association mapping of complex traits in a target population. In this
study, we assessed the genetic diversity, population structure and the extent of LD in a
panel of 192 inbred lines of Brassica napus from all over the world using 451
single-locus microsatellite markers. The inbred lines could be divided into P1 and P2
groups by a model-based population structure analysis. Out of the 142 inbred lines in
the P1 group, 126 lines were from China and Japan, and the remaining 16 lines were
from Europe, Canada and Australia. In the P2 group, 33 out of the 50 lines were from
Europe, Canada and Australia, and the remaining 17 lines were from China. Structure
analysis further divided each group into two subgroups. AMOVA, pairwise FST and
neutrality analyses confirmed the differentiation between groups and subgroups. More
than 80% of the pairwise kinship estimates between inbred lines were less than 0.05,
indicating that relative kinship is weak in our panel. Only 6% linked marker pairs
showed LD, suggesting the low level of LD in this association panel. The LD decayed
within 0.5-1 cM at the genome level, and varied considerably across each group and
subgroup, due to the population size, genetic background and genetic drift. The
characterization of the population structure and LD patterns would be useful for
performing association studies for complex agronomic traits in rapeseed.
12$
!
Genetics$of$seeds$per$silique$and$mapping$of$major$QTL$in$
Brassica'napus'L.'
Shipeng Li, Liwu Zhang, Dengfeng Hong, Guangsheng Yang*
Huazhong Agricultural University, Wuhan,430070,China
Seeds per silique (SS) is one of the three important components of yield in
oilseed rape (Brassica napus L.). In the study, We chose parental varieties that
showed highly significant differences in Seeds per silique. We crossed , Y106,one
stable high-SS and high combining ability line , HZ396, one stable low-SS and high
combining ability line to identify the QTL. An experiment with the orthogonal and
reciprocal F1generations was conducted for investigating the cytoplasmic effects of
this trait. Results revealed that SS more or less unaffected by environments, that SS
was controlled by nuclear genes instead of cytoplasmic genes, and the higher-SS
genotype was almost completely dominant over the lower-SS genotype. The
frequency distribution of the F2 generation deviated from a normal distribution and
appeared to have a multi-modal pattern, indicating the influence of major genes mixed
with polygenes.
To dissect the genetic basis of SS, we used one F1 plant of the cross of HZ396
×Y106 to develop double haploid (DH) population. The DH population, consisted of
140 lines, was used for map construction and QTL analysis. A linkage map
comprising 19 linkage groups and covering 1833.9 cM was constructed. In field
experiments across three seasons and two locations in China 140 doubled haploid
lines and their corresponding parents were evaluated for silique-traits. Quantitative
Trait Loci (QTL) meta-analysis revealed that 3 consensus QTL for SS. For the unique
QTL in the linkage group C9, the additive effects of cqSS.C9, which explained
57.77% of the phenotypic variance of SS and favorable alleles originated from Y106.
Near-isogenic line(BC3F2) for cqSS.C9 was used to consecutively back cross
with the recurrent parent HZ396.A total of 152 BC3F2 plants and their parents were
evaluated for silique-traits. A Chi-square test showed that the ratio of the frequency
distribution fit the Mendelian segregation ratio (3:1)of a single gene (χ2= 0.355,
P>0.05), suggesting that one locus existed in the BC3F2 population. QTL analysis
based on the BC3F2 population showed that this locus was located between the marker
SRC9-022 and SCC9-005. The QTL peak was near SRC9-022 marker at a distance of
0.60 cM. Furthermore this locus explained 85.8% of phenotypic variance with
additive and dominant effects of 6.1 and 5.7 SS, respectively. Two SSR markers
(SRC9-27 and SRC9-298) closely linked to cqSS.C9 were identified by using high-SS
bulk and low-SS bulk. To precisely map the cqSS.C9, we assayed 2,058 plants
showing the recessive phenotype for low-SS from a BC4F2 population with two SSR
markers, SRC9-27 and SRC9-298, narrowing down to a 267-kb region.
2012
$ 13
GenomeEwide$analysis$of$plantEspecific$Dof$transcription$
factor$family$in$tomato$ $
Xiaofeng Cai, Chanjuan Zhang, Tingyan Zhang, Yuyang Zhang, Tixu Hu, Jie Ye, Junhong
Zhang,Taotao Wang, Hanxia Li, Zhibiao Ye
The Key Laboratory of Horticultural Plant Biology, Ministry of Education, Huazhong
Agricultural University, Wuhan 430070, China
The Dof (DNA binding with One Finger) family encodes single zinc finger
proteins has been known to be a family of plant-specific transcription factors. They
are involved in a variety of functions of importance for different biological processes
in the plants. In current study, we identified 34 Dof family genes in tomato,
distributed on 11 chromosomes. A complete overview of SlDof genes in tomato was
presented, including the gene structures, chromosome locations, phylogeny, protein
motifs and evolution pattern. In addition, a comparative analysis between these genes
in tomato, Arabidopsis and rice was also performed. The gene duplication pattern
indicated that segmental duplication is predominant for the SlDof genes and tandem
duplication is also involved in tomato genome; the Ks, Ka and Ks/Ka ratio analysis
suggested that the tomato Dof family expansion was date to recent duplication events
and these Dof genes retained their function after duplication. It is the first step
towards genome wide analyses of the Dof genes in tomato; our study provides a very
useful reference for cloning and functional analysis the members of this gene family
in tomato and other species.
14$
!
Genetic$Analysis$and$Improvement$of$a$Recessive$Genic$Male$
Sterility$Line$7365A$in$Brassica$napus$
XIA Shengqian, ZU Feng, ZHU Yun, DUN Xiaoling, ZHOU Zhengfu, YI Bin, WEN Jing, SHEN
Jinxiong, MA Chaozhi, TU Jinxing(*), FU Tingdong
National Key Laboratory of Crop Genetic Improvement, National Center of Rapeseed
Improvement, Huazhong Agricultural University, Wuhan 430070, China
To utilize heterosis in Brassica napus, both the pollination control system and
genetic distance are major factors. Recessive epistatic genic male sterile (REGMS)
three-line system 7365ABC were widely applied to hybrid seed production because it
can produce 100% sterile population. Previous study has illustrated the system related
to three genes model: Bnms3, Bnms4 and BnRf. Near-isogenic lines 736512AB and
7365AC correspond to the BnMs4 and BnRf were respectively constructed for
mapping. Comparison of mapping results, six markers were linked to both BnRf and
BnMs4. Both genes were located in the top of the N7 linkage group, and might be
multiple alleles. We propose a new method of reproducing the male sterile line using
self-pollinated seeds generated by heat shock. The seeds were crossed with the
maintainer line to drive a 100% sterile population for hybrid seed production. We
further study the relation between genetic difference of two parents and F1 yield. We
transfered the target genes into two subgenomic (ArArCcCc) materials, and bred new
A and B lines in REGMS system. The combining ability analysis of yield and
yield-related characters were done in the crosses from the new A lines and 10
restorers. The results demonstrated that the improved sterile line showed positive
effects of value in the general combining ability (GCA).
2012
$ 15
Fine$mapping$and$candidate$gene$analysis$of$the$nuclear$
restorer$gene$Rfp$for$pol$CMS$in$rapeseed$(Brassica$napus$L.)$
Zhi Liu, Pingwu Liu, Guangsheng Yang
National Key Lab of Crop Genetic Improvement, National Center of Rapeseed Improvement in
Wuhan, Huazhong Agricultural University, Wuhan 430070, P.R. China
The polima (pol) system of cytoplasmic male sterility (CMS) in rapeseed is widely
used in China for commercial hybrid seed production. During the utilization of pol
CMS three-line or two-line system, breeding of elite restorer lines is a very important
aspect. Therefore, the researchers at home and abroad have been paying much
attention to the studies on the fertility restorer Rfp gene. In order to fine map the Rfp
for gene cloning, a near isogenic line (NIL) comprising 3,662 individuals of BC14F1
generation segregating for the Rfp gene was created. Based on the previous study in
our laboratory and the synteny region of Brassica napus and other Brassica species,
nineteen markers strongly linked with the Rfp gene were identified. Afterwards, a
genetic linkage map around the Rfp gene was constructed by using 13 of them.
Linkage analysis showed that all these 13 markers spanned a genetic region of 1.04
cM, and the Rfp gene was mapped between two nearest markers with the distance of
0.1 cM and 0.02 cM, respectively. By integrating three of these markers to the
published linkage map, the Rfp gene was mapped on linkage group N9 of B. napus.
Using these markers, the Rfp locus was narrowed down to a 29.2-kb DNA fragment
after sequence alignment. Seven ORFs were predicted in this target region, out of
which, ORF2, encoding a PPR protein, was the most likely candidate of the Rfp gene.
DNA sequencing analysis revealed that there were some nucleotide polymorphisms
between two parents in the coding region of ORF2. Subsequently, a pair of primers
was designed based on the nucleotide polymorphisms. Genotype analysis among the
NIL population revealed that the genotypes exhibited co-segregation with phenotypes.
The genomic sequence of the candidate was successfully isolated from the restorer
line. Complementation experiment was undergoing, and some positive transformants
have been obtained. These results lay a solid foundation for map-based cloning of the
Rfp gene and will be helpful for marker-assisted selection (MAS) of elite CMS
restorer lines.Key words Brassica napus, cytoplasmic male sterility, fertility
restoration, collinearity, PPR
16$
!
qSS7$
2012
$ 17
Detection$and$genotyping$of$restriction$fragment$associated$p
olymorphisms$in$polyploidy$crops$with$a$pseudo$reference$seq
uence:$A$case$study$in$tetraploid$Brassica$napus
Xun Chen1, Xuemin Li1, Muhammad Younas1, Bing Zhang2, Jinsong Xu1, Zhikun Wu1, H
aitao Li1, Lei Huang1, Yingfeng Luo2, Jiangsheng Wu1, Songnian Hu2, Kede Liu1*
Wuhan 430070, China; and Key Laboratory of Rapeseed Genetic Improvement, the Ministry of
Agriculture
The presence of homoeologous sequences and absence of a reference genome seq
uence make discovery and genotyping of single nucleotide polymorphism (SNP) more
challenging in all kinds of polyploidy crops compared to their diploid progenitor spec
ies. To address this challenge, we developed a technique called restriction fragment as
sociated polymorphism (RFAP), and designed a bioinformatics pipeline that consisted
of three modules i.e., assembly of a pseudo-reference sequence, SNP identification an
d discrimination of allelic SNPs from homoeologous sequence variations. To illustrate
our modeling approach, a common set of restriction fragments across a double haploi
d (DH) population (BnaNZDH) of highly established allotetraploid Brassica napus an
d its two parents were sequenced. Allelic SNPs and the presence/absence variations (P
AVs) were identified using this pipeline. Two parallel linkage maps, one SNP bin ma
p containing 8780 SNP loci and one PAV linkage map containing 12,423 dominant lo
ci, were constructed. By aligning these linkage maps to the B. rapa reference genome
sequence, we assigned 44 unassembled sequence scaffolds comprising 8.15 Mb onto t
he B. rapa chromosomes, and also identified 14 instances of misassembly and eight in
stances of mis-ordered sequence scaffolds. These results suggested that RFAP is a cos
t-effective, efficient and simple methodology to genotype tens of thousands SNPs and
PAV markers in a large population, and the related bioinformatics pipeline is a power
ful tool to mine allelic SNPs from homoeolgous sequences, therefore they are generall
y applicable in either diploid or polyploidy species with or without a reference genom
e sequence.
18$
!
WRKY,$A$New$Player$in$the$Biosynthesis$of$Terpenoid$Indole$
Alkaloids$in$Catharanthus$roseu$
$
Institute of Molecular Agriculture and Bioenergy, Shanxi Agricultural University, Taigu 030801
China
A medical plant Catharanthus roseus produces a lot of terpenoid indole alkaloids
(TIAs) that are an important source of natural or semisynthetic anticancer drugs such
as vinblastine, vincristine,
vindesine, and vinorelbine.
Understanding the
mechanism underlying the biosynthesis and regulation network of TIAs in C. roseus
is crucial for developing an effective metabolic engineering strategy to increase levels
of TIAs, particularly vinblastine and vincristine. Here, we describe the isolation and
functional characterization of a WRKY transcriptional factor (TF), CrWRKY3, from
C. roseus. This TF is preferentially expressed in roots and induced by the
phytohormones jasmonate, gibberellic acid, and ethylene. The overexpression of
CrWRKY3 in C. roseus hairy roots up-regulated several key TIA pathway genes,
especially Tryptophan Decarboxylase (TDC), as well as the transcriptional repressors
ZCT1 (for zinc-finger C. roseus transcription factor 1), ZCT2, and ZCT3. However,
CrWRKY3 overexpression repressed the transcriptional activators ORCA2, ORCA3,
and CrMYC2. Overexpression of a dominant-repressive form of CrWRKY3, created
by fusing the SRDX repressor domain to CrWRKY3, resulted in the down-regulation
of TDC and ZCTs but the up-regulation of ORCA3 and CrMYC2. CrWRKY3 was
detected to bind to the Wbox elements of the TDC promoter in electrophoretic
mobility shift, yeast one-hybrid, and C. roseus protoplast assays. Up-regulation of
TDC increased TDC activity, tryptamine concentration, and resistance to 4-methyl
tryptophan inhibition of CrWRKY3 transgenic hairy roots. Compared with control
roots, CrWRKY3 hairy roots accumulated up to 4-fold higher levels of vinblastine
and vincristine. The current results indicate that CrWRKY3 is a positive regulator in
determining the root-specific accumulation of vinblastine and vincristine in C. roseus
plants. Furthermore, bioinformatics tools were used to analyses the database of C.
roseus genomic DNA and expression sequences available so far. Total of 17 candidate
CrWRKY TFs were identified. Those TF exhibited different expression patterns in
various tissues/organs and developmental stages. Combining our evidences including
microarray and other known data, we propose a working model to illustrates the
interaction of CrWRKYs with indole pathway genes, AS (Anthranilate synthase) and
TDC, terpenoid pathway genes, DXS (1-Deoxy-D-xylulose 5-phosphate Synthase)
abd SLS (Secologanin synthase), and downstream genes, STR (Strictosidine synthase)
and SGD (Strictosidine β-D-glucosidase), as well as the ORCA, CrMYC2, and ZCT
regulators.
2012
7DL
$ 19
BAC
$
20$
!
Cloning$and$analysis$of$MAPK1$and$it’s$promoter$from$
Brassica$napus$ $
Jun-Xing Lu, Kai Zhang, Kun Lu, Ying Liang, You-Rong Chai*
Chongqing Key Laboratory of Crop Quality Improvement; College of Agronomy and
Biotechnology, Southwest University, Chongqing 400716
Mitogen-activated protein kinase (MAPK) is a large family of serine/threonine
protein kinase, contains 11 conserved subdomains. Mitogen-activated protein kinase
pathways play a central role in transfer information from diverse receptors/sensors to
a wide range of cellular responses in plants. Signaling through MAP kinase cascade
could lead to cellular responses including cell division, development, hormone,
physiology, as well as response to a broad variety of biotic and abiotic stresses. In this
study, the full-length cDNA and promoter of BnMAPK1 were isolated from Brassica
napus. BnMAPK1 is 1632 bp encoding a 369-aa protein. RT-PCR detection revealed
that BnMAPK1 transcribed in the 10 organs tested, while more abundant in root and
leaf. BnMAPK1 showed inhibited expression when treated with ABA, 6-BA and heat,
while up-regulated under SA, GA3, NaCl and PEG8000 induction. Several
cis-elements associated with stress and hormone-induction were predicted in
BnMAPK1 promoter, which indicated that BnMAPK1 most likely played important
roles in response to abiotic stress through JA, heat and drought signal pathways. This
study would contribute to further discuss the molecular mechanism of stress
resistance of Brassica and lay the foundation for Brassica quality improvement in the
future
2012
$ 21
Computational$Studies$of$PostEtranslational$Modifications$
Through temporally and spatially modified proteins, post-translational
modifications (PTMs) greatly expand the proteome diversity and play critical roles in
regulating the biological processes. Identification of site-specific substrates is
fundamental for understanding the molecular mechanisms and functions of PTMs,
which is still a great challenge under current technique limitations. Development of
computational approaches has promoted studies of PTMs, since in silico predictions
and analysis could rapidly generate useful information for later experimental
investigations. Recently, our group contributed a number of studies to this area, which
are focused on three aspects as follows: 1) Improving the GPS algorithm and
employed it to implement a series of softwares including GPS-CCD, GPS-PUP,
GPS-YNO2 and GPS-Polo to predict PTMs sites including calpain cleavage,
pupylation, tyrosine nitration and Plk-mediated phosphorylation and phospho-binding,
respectively; Extending the GPS algorithm to develop predictors of GPS-MBA and
GPS-ARM for prediction of MHC Class II Epitopes and APC/C recognition motif,
respectively. Through comprehensive comparison, the predictors were demonstrated
to be better than other methods. 2) Systematic studies of PTMs to dissect their
biological roles including comparison of cysteine S-nitrosylation and tyrosine
nitration (PTN), comprehensive analysis of potential D-boxes and KEN-boxes and
systematic analysis of the Plk-mediated phosphoregulation in eukaryotes. The
large-scale analyses together with statistical analysis provide systematic insights into
the biological roles of PTMs, especially a global view. Furthermore, the following
experimental assay verified our predictions and analyses. 3) Integrating the
experimental protein lysine acetylation information into a comprehensive database of
CPLA, followed by systematical construction and analysis of human lysine
acetylation regulatory network. We proposed the potential triplet mechanism of
acetylation-mediated regulation through network analysis. Token together, we believe
that computational analysis backed up with subsequent experimental identification
can propel systematic studies of PTMs into a new and highly productive phase
22$
!
Abundant$microsatellite$diversity$and$oil$content$in$wild$
Arachis$species$$
Li Huang, Huifang Jiang, Xiaoping Ren, Yuning Chen, Yingjie Xiao, Xinyan Zhao, Mei Tang,
Jiaquan Huang, Boshou Liao
Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences, Wuhan, 430062,
China; Huazhong Agricultural University, Wuhan, 430070, China
The peanut (Arachis hypogaea) is an important oil crop. Breeding for high oil
content is becoming increasingly important. Wild Arachis species have been reported
to harbor genes for many valuable traits that may enable the improvement of
cultivated Arachis hypogaea, such as resistance to pests and disease. However, only
limited information is available on variation in oil content. In the present study, a
collection of 72 wild Arachis accessions representing 19 species and 3 cultivated
peanut accessions were genotyped using 136 genome-wide SSR markers and
phenotyped for oil content over three growing seasons. The wild Arachis accessions
showed abundant diversity across the 19 species. A. duranensis exhibited the highest
diversity, with a Shannon-Weaver diversity index of 0.35. A total of 129 unique
alleles were detected in the species studied. A. rigonii exhibited the largest number of
unique alleles (75), indicating that this species is highly differentiated. AMOVA and
genetic distance analyses confirmed the genetic differentiation between the wild
Arachis species. The majority of SSR alleles were detected exclusively in the wild
species and not in A. hypogaea, indicating that directional selection or the hitchhiking
effect has played an important role in the domestication of the cultivated peanut. The
75 accessions were grouped into three clusters based on population structure and
phylogenic analysis, consistent with their taxonomic sections, species and genome
types. A. villosa and A. batizocoi were grouped with A. hypogaea, suggesting the
close relationship between these two diploid wild species and the cultivated peanut.
Considerable phenotypic variation in oil content was observed among different
sections and species. Nine alleles were identified as associated with oil content based
on association analysis, of these, three alleles were associated with higher oil content
but were absent in the cultivated peanut. The results demonstrated that there is great
potential to increase the oil content in A. hypogaea by using the wild Arachis
germplasm.
2012
0431
$ 23
21RIL
$
24$
!
MYB$
MYB
$$
2012
$ 25
26$
!
Identification$and$analysis$of$microRNAs$associated$with$
drought$in$Solanum'tuberosum$
Yang Jiangwei1
1
Ma Congyu1,2
Zhang Ning2 Si Huaijun1,2
∗
Wangdi1
Gansu Provincial Key Laboratory of Aridland Crop Science, Gansu Key Laboratory of Crop
Genetic and Germplasm Enhancement, Gansu Agricultural University, Lanzhou 730070; 2College
of Life Science and Technology, Gansu Agricultural University, Lanzhou 730070
Drought is one of major abiotic stresses which can greatly give an impact to
crops growth, development, geographical distribution and adversely affect reduction
or total loss of crop production. MicroRNAs (miRNAs) are a class of non-coding
small RNAs which regulate gene expression of target mRNAs at the
post-transcriptional level. In recent years, researches showed that miRNAs play
critical roles in plant stress response, and hundreds of stress-associated miRNAs and
their target sequences have been identified in plants by cloning and bioinformatics
methods. The potato (Solanum tuberosum L.) is the fourth largest agricultural crop in
the world. Up to now, little is known about miRNAs related to drought that have been
identified in potato. In order to make better understand how miRNAs contribute to
drought tolerance in potato. Two small RNA libraries were constructed from both
control and drought treatment potato samples, and generated two deep sequencing
small RNA reads. Based on sequence similarity and hairpin structure prediction, we
found 471 known miRNAs in drought treatment and 458 known miRNAs in control,
and 674 novel miRNAs in drought treatment and 566 novel miRNAs in control. The
differential expression analysis showed that 45 miRNAs were up-regulated and 14
miRNAs were down-regulated among 89 drought-associated known miRNAs under
drought condition. Meanwhile, 239 novel miRNAs of differential expression was
found, which contained 127 up-regulated miRNAs and 112 down-regulated miRNAs
under drought condition. The putative function for novel miRNAs was assessed and
defined in potato. 10584 miRNA targeted records of 566 novel miRNA in control and
15115 miRNA targeted records of 674 novel miRNA in drought treatment were
produced. Those predicted targets genes encoded transcription factors, enzymes or
proteins, which involved in plant growth and development, metabolism, signal
transduction, transcriptional regulation and stress responses. miRNA targets genes
also encoded proteins whose function are largely unclear. miRNAs of regulation of
proline accumulation under drought stress were predicted and analyzed by
bioinformatics and RNA sequencing. The result provides molecular evidence for the
possible involvement of miRNAs in the process of drought response and/or tolerance
in potato.
2012
$ 27
Cotton$GhAF18$regulates$anther$abortion$by$delaying$tapetal$
programmed$cell$death$via$the$GlcEABAEROS$signaling$
pathway$ $ $
Ling Min, Longfu Zhu, Lili Tu, Fenglin Deng, Yichun Nie, Daojun Yuan, Xiaoping Guo, and
Xianlong Zhang*
Huazhong Agricultural University, Wuhan, 430070, China
Infertility of anthers under high temperature (HT) has become a critical factor in
yield loss in cotton. Previous studies showed altered carbohydrate metabolism or
disrupted tapetal cell programmed cell death (PCD) underlie anther sterility. Through
large-scale expression profile sequencing we studied the effect of HT on cotton anther
development and found that the GhAF18 gene, was induced by HT in a HT-sensitive
cotton line. Our studies showed Gh AF18 causes glucose (GLU) accumulation in
early-stage anthers by inactivating starch synthase via phosphorylation. Subsequent
feedback inhibition of glucose assimilation and induction of starch synthesis cause a
deficiency in glucose in late-stage anthers. The early accumulation of glucose also
promotes excessive amounts of abscisic acid (ABA) in early anthers and alters the
balance of reactive oxygen species (ROS) scavenging and accumulation in late
anthers, causing delayed PCD in the tapetum leading to anther indehiscence. The
timings of tapetal PCD and anther dehiscence are critical for fertilization, and
GhAF18-GLU-ABA-ROS pathway may regulates these timings.
28$
!
Mapping$of$Boron$Efficient$QTL$in$Brassica$napus$under$
different$boron$environments$ $
Didi Zhang, Yingying Li ,Zunkang Zhao,Lei Shi, Fangsen Xu
Boron (B) is an essential mineral nutrient for the growth and development of
higher plants, which involves in a series of important physiological functions.
Brassica napus is a major oil crop in China, and is sensitive to boron deficiency. The
typic B deficiency symptom is “flowering without seed setting” in B. napus. Wide
genotype differences exist in response to B deficiency in Brassica napus. In this study,
A B. napus DH population developed from a cross between B-efficient ‘Qingyou
10’and B- inefficient‘Westar’was used for phenotypic investigation and QTL analysis.
Biomass shoot fresh weight root fresh weight shoot dry weight shoot dry weight
and root elongation length were investigated in three hydroponic experiments under
low B and normal B conditions, and QTLs were indentified by composite interval
mapping. There was significant genetic variation in all of the detected traits and
correlation between most of traits were observed. A total of 128 putative QTLs were
detected, 43 under the normal boron condition and 85 under the low boron condition,
respectively. Among these QTLs there was a QTLs clustered on chromosome A3
detected specifically under low boron condition. These results showed different
genetic determinants controlled B efficiency-related traits in B. napus. The QTLs
detected under low boron condition may provide useful information for improving
boron tolerance of B. napus.
2012
$ 29
Introgression$of$bacterial$blight$resistance$genes$Xa7,$Xa21$
and$Xa23$into$thermosensitive$genetic$male$sterile$rice$(Oryza$
sativa$L.)$by$phenotypic$and$molecular$markerEassisted$
selection$$
Jiefeng Jiang, Tongmin Mu
National Key Lab of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan,
430070, China
Bacterial blight (BB) of rice caused by X. oryzae pv. oryzae (Xoo) is the most
destructive diseases in rice production. C815S is an elite thermo-sensitive genic male
sterile (TGMS) line which is used widely in China. Xa7, Xa21 and Xa23, the
broad-spectrum bacterial blight resistance genes, were introgressed into C815S which
was susceptible to BB by molecular marker-assisted selection and backcrosses. Five
breeding lines with different BB resistance genes and backcross generations,
Hua1006S (Xa23), Hua1009S (Xa23), Hua1002S (Xa7, Xa21), Hua1005S (Xa7,
Xa21) and Hua1001S (Xa7, Xa23) were developed. The results of resistance
identification showed that they were resistant against all of inoculated seven Xoo
strains, ZHE173, GD1358, FuJ1, YuN24, HeN11 from China and PXO61, PXO99
from Philippines. All of 15 hybrids from five TGMS lines crossed with three restorer
lines were also against above seven Xoo strains. Hua1006S, Hua1009S, Hua1002S
and Hua1005S had a stable complete sterility period of longer than 30 days under
natural field conditions at Wuhan. The results of fertility identification in growth
chamber showed that the critical sterile point (CSP) of fertility alteration were at 23°C
of daily mean temperature in which the maximum and minimum temperature were
26°C in day and 19°C in night, respectively. The recorded data of agronomic traits
showed that these four TGMS lines were similar to that of C815S (recurrent parent).
The improved BB resistance TGMS lines could have significant role in promoting the
two-line hybrid rice breeding
30$
!
Proteomes$alteration$of$Brassica$napus$in$response$to$
phosphorus$deficiency$ $
Chen Shuisen, Wang Zhenhua, Xu Fangsen
National Key Laboratory of Crop Genetic Improvement, and Microelement Research Centre,
Huazhong Agricultural University, Wuhan, China
430070
Phosphorus (P) is the most widely studied macronutrient due to its low available in
many soil of the word. In this study, comparative proteomics were performed to study
the roots and leaves proteomes changes of two genotype Brassica napus, P-efficiency
(97081) and P-inefficiency (97009), in long-term low phosphorus (LP) and short-term
phosphorus starvation conditions (NP), and classified the identified proteins into
seven categories as follows: defense and stress, carbohydrate and energy metabolism,
signaling and regulation, amino acid and fatty acid metabolism, protein process,
biogenesis and cellular component, and function unknown. Regardless of the roots
and leaves proteome alternation in response to phosphorus deficiency are different in
two genotype Brassica napus. Due to the differential expressed proteins identified in
LP or NP in 97009 were inconsistent with 97081, regardless of roots or leaves
proteins, we suppose that there have different adaptive mechanism in response to
phosphorus deficiency in two Brassica napus. To further understand the adaptive
mechanism can help us to breed the phosphorus efficiency Brassica napus cultivars.
2012
$ 31
Dissecting$Quantitative$Trait$Loci$for$Boron$Efficiency$across$
Multiple$Environments$in$Brassica$napus$$
Zunkang Zhao, Likun Wu, Fuzhao Nian, Guangda Ding, Taoxiong Shi, Didi Zhang, Lei Shi,
Fangsen Xu, Jinling Meng
High yield is the most important goal in crop breeding, and boron (B) is an essential
micronutrient for plants. However, B deficiency, leading to yield decreases is an
agricultural problem worldwide. Brassica napus is one of the most sensitive crops to
B deficiency, and considerable genotypic variation exists among different cultivars in
response to B deficiency. To dissect the genetic basis of tolerance to B deficiency in B.
napus, we carried out QTL analysis for seed yield and yield-related traits under low
and normal B conditions using the double haploid population (TNDH) by two-year
and the BQDH population by three-year field trials. In total, 80 putative QTLs and 42
epistatic interactions for seed yield, plant height, branch number, pod number, seed
number, seed weight and B efficiency coefficient (BEC) were identified under low
and normal B conditions, singly explaining 4.15-23.16% and 0.53-14.38% of the
phenotypic variation. An additive effect of putative QTLs was a more important
controlling factor than the additive-additive effect of epistatic interactions. Four
QTL-by-environment interactions and 7 interactions between epistatic interactions
and the environment contributed to 1.27-4.95% and 1.17-3.68% of the phenotypic
variation, respectively. The chromosome region on A2 of SYLB-A2 for seed yield
under low B condition and BEC-A2 for BEC in the two populations was equivalent to
the region of a reported major QTL, BE1. The B. napus homologous genes of
Bra020592 and Bra020595 mapped to the A2 region and were speculated to be
candidate genes for B efficiency. These findings reveal the complex genetic basis of B
efficiency in B. napus. They provide a basis for the fine mapping and cloning of the B
efficiency genes and for breeding B-efficient cultivars by marker-assisted selection
(MAS).
32$
!
$$
Auxin has pervasive roles in many aspects of plant growth and development, and
its regulatory mechanisms have been well studied. However, auxin’s role in leaf
senescence remains uncertain. Some of the external application experiments showed
that auxin was able to inhibit leaf senescence while some other experiments indicated
auxin could promote leaf senescence. Here we report the identification and
characterization of an Arabidopsis leaf senescence-associated gene named SAG201.
SAG201 is highly up-regulated during leaf senescence and can be induced by
1-naphthaleneacetic acid (NAA, a synthetic auxin species). It encodes an
uncharacterized small auxin-up RNA (SAUR) that has been annotated as SAUR36.
Leaf senescence in the saur36 knockout lines caused by T-DNA insertion was
significantly delayed as revealed by analyses of chlorophyll contents, Fv/Fm ratio (a
parameter for photosystem II activities), ion leakage, and the expression of leaf
senescence marker genes. In contrast, transgenic Arabidopsis plants overexpressing
SAUR36 (without its 3’-UTR) displayed an early leaf senescence phenotype.
However, plants overexpressing SAUR36 with its 3’-UTR were normal and didn’t
exhibit the early senescence phenotype. These data suggest that SAUR36 mediates
auxin-induced leaf senescence and that the 3’-UTR containing a highly conserved
downstream (DST) renders the instability of the SAUR36 transcripts in young leaves.
2012
$ 33
$
5-
34$
!
early$senescence$leaf$4
esl4
$ $
Abstract In this study, a rice early senescence mutant, early senescence leaf 4
esl4 , which was derived from ethylmethane sulfonate (EMS)-treated Jinhui10
(Oryza sativa L.ssp. indica). The mutants’ leaf opex and leaf edge were yellowing
from booting stage. During booting stage, chla, chlb, total chl and cart of 1-3 leaf in
the mutants were significantly lower than those of the wild type. Consistently,
compared with the wild type, net photosynthetic rate, stomatal conductance and
transpiration rate were significantly decreased in mutant. The genetic analysis
revealed that the mutational trait was controlled by a single recessive gene. By bulked
segregation analysis (BSA) and 900 F2 mutants, the ESL4 gene was finally located
between the IN/DEL marker S4-25 and S4-53 on chromosome 4, with 199 kb
physical distances. These results provided a foundation of map-based cloning and
function analysis of ESL4 gene.
2012
$ 35
Salicylic$acid$analogues$with$biological$activity$may$induce$
chilling$tolerance$of$maize$(Zea$mays)$seeds$$
Yang Wang, Jin Hu, Guochen Qin, Huawei Cui, and Qitian Wang
One kind of biologically active salicylic acid (SA) analogue (acetylsalicylic acid,
ASA) and two inactive compounds (4-aminosalicylic acid and 4-aminobenzoic acid),
along with SA were chosen to evaluate their role in inducing chilling tolerance of two
different chilling-tolerant maize (Zea mays L.) inbred lines. These compounds were
applied as seed treatments or as a hydroponic application. The results showed that
four compounds had no significant effect on germination of maize seeds; however,
SA or ASA soaking treatments significantly increased the root length, shoot height,
and shoot and root dry weights of seedlings grown under chilling stress. Hydroponic
applications of SA or ASA significantly alleviated the accumulation of
malondialdehyde, hydrogen peroxide, and superoxide radicals in roots and leaves of
both lines under chilling stress, and the applications also increased the photosynthetic
pigments, including chlorophyll a, chlorophyll b, and carotenoids. However,
4-aminosalicylic acid and 4-aminobenzoic acid applications had no significant effect
in ameliorating the growth inhibition of seedlings under chilling stress. This study
showed that SA and ASA significantly induced the chilling tolerance of maize;
however, 4-aminosalicylic acid and 4-aminobenzoic acid were not effective in
inducing tolerance to chilling stress. The results suggest that only SA analogues with
biological activity may have the ability to induce chilling tolerance of maize.
36$
!
Genetic$and$QTL$analysis$for$seedling$cold$tolerance$in$rice$ $
Canyang Li,
Guo Zong,
Ahong Wang,
Lu Wang,
and Bin Han
National Center for Gene Research, Institute of Plant Physiology and Ecology, CAS, Shanghai
200233, China
Seedling cold tolerance is of particular importance to direct seeding culture
systems as planting typically coincides with the lowest temperatures of the season. Of
the two major subspecies of O. sativa, indica and japonica, general phenomenon is
that japonicas possess better cold tolerance than indicas. To determine the basis for
the difference in low temperature tolerance between japonica and indica, we
developed a recombinant inbred line (RIL) mapping population using the cold tolerant
temperate japonica cultivar Nipponbare and the cold sensitive indica cultivar 93-11.In
this study, a high-throughput re-sequencing method was used to genotype the 150
RILs. For phenotyping, 14-day old seedlings were treated with 8 °C for 7 days and
then recovered under 24 °C for 14 days. We used the lethal percentage to describe the
cold tolerance. A seedling was considered to be dead if its newly appeared leaf is
wilting. Analysis of this population led to the identification of three number of
Nipponbare derived QTL that confer tolerance to cold-induced death observed in the
cold-sensitive indica, 93-11. They were named as qCtss2, qCtss4 and qCtss12
according to their position on chromosomes, and accounted for 6.63%, 6.17% and
9.55% phenotypic variation respectively. We then used chromosome segment
substitution lines (CSSL) to confirm the effect of the three QTLs, and found qCtss12
from Nipponbare had significant effect on cold tolerance while the other two QTLs
had minor effect. In the next, we will use the F2 population derived from the CSSL
and Nipponbare to fine mapping the qCtss12 locus.
2012
$ 37
Sequence$and$Functional$Divergence$of$FRUITFULL$Homeologs$
in$Brassica$napus$ $
Pengfei Peng1 • Yunchang Li1 • Desheng Mei1 • Joseph Colasanti2 • Li Fu1 • Jia Liu1 • Yufeng
Chen1 • Qiong Hu1
"1Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture, Oil
Crops Research Institute of the Chinese Academy of Agricultural Sciences, No. 2 Xudong 2nd RD,
Wuhan, 430062, Hubei, People’s Republic of China
2Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, N1G
2W1, Canada
Resistance to pod shattering is a key seed crop trait that can substantially improve
yield by preventing seeds from dropping to the ground. To improve pod shatter
resistance in the important oilseed crop Brassica napus, the phenotypic diversity of B.
napus was exploited using eighty B. napus varieties was evaluated for pod shatter
resistance by a random impact test. Among these, R1-1 was identified as resistant and
R2, 8908B as susceptible. To understand the molecular basis for this phenotype
difference, base on the candidate gene approach, B. napus FRUITFULL (FUL)
homologs were identified and characterized. Two FUL loci, BnaA.FUL.a and
BnaC.FUL.a, in the A and C genomes of B. napus were found, respectively. In the
susceptible variety, both BnaA.FUL.a and BnaC.FUL.a were expressed in the same
tissues. However, the expression level of BnaC.FUL.a was different in varieties with
different pod shatter resistance. In the most resistant variety R1-1, only BnaA.FUL.a
was expressed, and BnaC.FUL.a was silenced. Ectopic over-expression of
BnaA.FUL.a in A. thaliana resulted in elevated resistance to pod shatter, as well as
earlier flowering. Ectopic overexpression of BnaC.FUL.a in Arabidopsis also caused
shatter resistance, but no pleiotropic phenotypes. Therefore, the functional divergence
and differing expression of BnaX.FUL.a homeologs may significantly affect
phenotypic variation, which is an important consequence of allopolyploid evolution.
Moreover, BnaA.FUL.a could prove useful to improve pod shatter resistance in B.
napus breeding programs, thereby improving yield for this important oilseed crop.
38$
!
PnLOX2
$
Aspergillus flavus
is one of the major diseases of peanut. The objective of the study was to clone
PnLOX2 gene from peanut seed coat and analyze its expression. Real-time RT-PCR
analysis indicated that the gene was overexpressed in the challenge of Aspergillus
flavus and the greatest expression occurred 10 days after inoculation. The full length
of PnLOX2 gene was 2592bp and encoded a 97.5kDa protein containing 863 amino
acids. The fusion protein (apparent 121.5kDa) only existed in the precipitation and the
maximum was obtained by inducing at 37ºC for 3.5h. As a result of response to the
challenge of Aspergillus flavus, PnLOX2 gene was more greatly expressed in the
resistant genotype than in the susceptible genotype. It indicated that PnLOX2 gene
was closely related to Aspergillus flavus-resistance of peanut. Further investigations
were needed to bacteriostatically identify the gene in vitro.
2012
$ 39
Blast$resistance$of$Hybrid$rice$Oryza'sativa$L.$
Haichao Jiang, Yutao Feng, Liang Bao, Guanjun Gao, Qinglu Zhang, Jinghua Xiao, Caiguo Xu,
Yuqing He
National Key Laboratory of Crop Genetic Improvement, National Center of Plant Gene Research
(Wuhan) and National Center of Crop Molecular Breeding, Huazhong Agricultural University,
430070, Wuhan, China
Rice blast is one of the most serious diseases in rice (Oryza sativa L.) worldwide.
Jin 23B is the maintainer line, a parent for a number of hybrid rice varieties used
widely in China. However, Jin 23B is highly susceptible to rice blast. In this study,
Pi1, Pi2 and D12 were introgressed to improve the blast resistance of Jin 23B and its
derived hybrids, Jinyou 402 and Jinyou 207, by marker-assisted selection (MAS). The
improved Jin 23B, which carried single-, two- and three-genes, were evaluated for
their resistance to rice blast using natural inoculation methods in disease nursery of
Xianfeng, Hubei, China. The results showed that the more genes contained in the
improved Jin 23B and hybrids, the higher resistance to rice blast they showed. Pi1,
Pi2 and D12 showed a strong dosage effect on the resistance to blast in the hybrid
background during the entire growth duration in the field condition, being very useful
for breeding blast-resistant hybrids. The result of examining agronomic traits showed
that the improved Jin 23B and its derived hybrid rice were taller than or similar to the
controls, when there was no disease stress.
40$
!
Tomato$SlDREB$gene$restricts$leaf$expansion$and$internode$
elongation$by$downregulating$key$genes$for$gibberellin$
biosynthesis$$
Jinhua Li, Wei Sima, Bo Ouyang, Taotao Wang, Khurram Ziaf, Zhidan Luo, Lifeng Liu,
Hanxia Li, Mingluan Chen, Yunqing Huang, Yuqi Feng, Yanhong Hao and Zhibiao Ye*
Plants have evolved and adapted to different environments. Dwarfism is an
adaptive trait of plants that helps them avoid high energy costs under unfavorable
conditions. The role of gibberellin (GA) in plant development has been well
established. Several plant dehydration-responsive element-binding proteins (DREBs)
have been identified and reported to be induced under abiotic and biotic stress
conditions. A tomato DREB gene named SlDREB, which is a transcription factor and
was cloned from cultivated tomato M82, plays a negative role in tomato plant
architecture and enhances drought tolerance. Tissue expression profiles indicated that
SlDREB is expressed mainly in the stem and leaf, and could be induced by abscisic
acid (ABA), but suppressed by GA and ethylene. SlDREB altered plant morphology
by restricting leaf expansion and internode elongation when over-expressed, and the
resulting dwarfism of tomato plants could be recovered by application of exogenous
GA3. Transcriptional analysis of transgenic plants revealed that over-expression of
SlDREB caused dwarf phenotype by down-regulating key genes involved in GA
biosynthesis such as ent-copalyl diphosphate synthase (SlCPS) and GA 20-oxidases
(SlGA20ox1, 2, and 4), thereby decreasing endogenous GA levels in transgenic plants.
A yeast activity assay demonstrated that SlDREB specifically binds to
dehydration-responsive element/C-repeat (DRE/CRT) elements of the SlCPS
promoter region. Taken together, these data demonstrate that SlDREB can
down-regulate the expression of key genes required for GA biosynthesis and acts as a
positive regulator in drought stress responses by restricting leaf expansion and
internode elongation.
2012
$ 41
Mapping$and$identification$of$QTLs$for$resistance$to$
Sclerotinia$sclerotiorum$and$its$interaction$with$other$
traitErelated$QTLs$or$genes$in$Brassica$napus$L.$
Fengqi Zhang, Junyan Huang, Xiaohui Cheng, Yueying Liu, Caihua Dong, Lingyan Liu,
Shengyi Liu*
Institute of Oil Crops Research, Chinese Academy of Agricultural Sciences, Wuhan, 430062
Sclerotinia stem rot, caused by Sclerotinia sclerotiorum, is an important limiting
factor in canola breeding, which annually causes yield loss ranging from 5 to 30%
with incidence of 5%-80%. The history of breeding canola cultivars with high levels
of durable resistance was more than 60 years at home and abroad, cultivars with
completely resistance to S. sclerotiorum were no reported
only some with partial
resistance have been identified in recent years. The incidence of Sclerotinia stem rot
under nature enviroment was difference between years, and associated with other
plant traits, such as plant height, flowering time etc. Exploiting resistant-related
molecular markers, cloning resistant-related genes and finding its interaction with
other traits is urgent need at present. In this study, we identified the resistant level of
more than 200 RILs, which derived from a cross between M083 (resistant) and 888-5
(sensitive), using artificial inoculation(stem inoculation with mycelium plugs and
parafilm, detached leaf inoculation), artificial disease nursery and sclerotia-seeded
semi-natural field evaluations under 3 different environments in 2010-2011 and
2011-2012. Through comprehensive assessment of each line resistant to Sclerotinia
sclerotiorum and its interaction with other traits or environment, combined with using
molecular markers gained by Illumina infinium assay based on Brassica napus
genome sequence, major QTLs for resistance to Sclerotinia sclerotiorum or the other
plant traits will be identified. The disease identification results indicated that
significant differences were observed (P<0.01) among the RILs responsive to
Sclerotinia sclerotiorum. Flowering time, plant height, the first branching height also
showed obvious difference (P<0.01) in the population. We also found that correlation
between disease index and flowering time or plant height was showed significantly
(P<0.01) negative relationship. Based on the Brassica napus genome sequence and the
population parents resequencing information, infinium SNP assay contained 60K
locus has been customized. At prsent, the work is to perform the SNP genotyping for
the RILs, constructing high density linkage mapping, finally identifying the QTLs or
gene related to the resistance to Sclerotinia sclerotiorum and other traits QTLs or
genes in Brassica napus.
42$
!
Characterization$of$a$putative$E3$ubiquitin$ligase$gene$
OsETOL1$conferring$drought$and$submergence$stress$in$rice$$
Hao Du, Wu Nai, Fei Cui, Lei You, Li Xianghua and Lizhong Xiong*
"National Key Laboratory of Crop Genetic Improvement
National Center of Plant Gene Research (Wuhan)
Huazhong Agricultural University
Wuhan, 430070, China"
Submergence and drought are major limiting factors for rice (Oryza sativa),
production. To identify stress related candidate genes, we performed drought
screening of mutant lines in our T-DNA insertion mutant library. Here we present the
identification and functional analysis of a drought-resistant mutant Osetol1. The
mutant phenotype was caused by impairing of a gene encoding a putative E3 ubiquitin
ligase, and two allelic mutants (Zhonghua 11 background) of this gene, Osetol1-1 and
Osetol1-2, showed increased resistance to drought stress at panicle development stage
as shown higher seed setting rate. Interestingly, Osetol1-1 mutant showed
significantly faster growth rate under submergence stress at both the seedling and
panicle development stages. Overexpression of OsETOL1 in rice resulted in reverse
phenotype as mutants shown. The analysis of tempo-spatial expression pattern imply
higher transcript of OsETOL1 in matured tissue at panicle development stages, and
OsETOL1 gene was strongly induced by abiotic stress, and the responses of
OsETOL1 to environmental stimuli are distinctive from other OsETOL1 homologues
in rice. The determination of transcription and physiology level display that
carbohydrate catabolism and fermentation related genes and pruduction were changed
by OsETOL1, imply that OsETOL1 function as a regulator of energy metabolism.
Yeast two-hybrid analysis found OsETOL1 can interact with OsACS2 proteins, and
the interaction was further confirmed by BiFC experiments, the fluorescence was
strongly detected in Arabidopsis protoplasts and it was clearly localized in the cytosol
with the localization of OsETOL1. Furthermore, in the mutants Osacs2 and OsETOL1
overexpression line the ACC and ethylene content was significant decreased,
implying that the nagtive regulation of ethylene synthesis mechanism is conserved in
ETOL families, however, genetic study display the functional redundancy and
specificity of OsETOL family in Arabidopsis and rice. So far, very few genes have
been reported for functions in both drought and submergence stress. The OsETOL1
may be very useful for studying crosstalk between drought and submergence
responses.
2012
$ 43
R421
$
44$
!
Screening$fluorescent$dyes$for$antiEcounterfeiting$labelling$in$
rape$seed$(Brassica'napus)$
Yixin Tian
Zhejiang University
Five fluorescent dyes – rhodamine B (RB), rhodamine 6G (R6G), lissamine
rhodamine B (LRB), 3, 6-diamincacridine hulmisulfate (DH) and safranine T (ST) –
were used to label seeds as an anti-counterfeiting technology. The effects of the dyes
on physiological parameters of rape and the patterns of fluorescence of rape seedlings
were investigated. The results showed that soaking seeds for 24 hours in 0.1 mg RB
ml-1, 0.1 mg LRB ml-1 or 0.1 mg ST ml-1 had no significant effects on seed
germination, seedling growth, seedling protective enzyme activities or chlorophyll
content. RB and LRB emitted bright red and bright orange fluorescence under a
fluorescence microscope, when excited by green light (546 nm) and blue light (495
nm), respectively, whereas ST showed little difference compared with the control.
Moreover, the vascular bundles of stem, seedling roots and aerial parts of seedlings
treated with RB all emitted brilliant fluorescence for a long time, which could be used
as a marker in seedlings. Our results suggest that labelling rape seeds with RB dye at
a suitable concentration can be used as an anti-counterfeiting technique.
2012
$ 45
46$
!
$ $
2012
$ 47
Characterize$the$genetic$basis$of$selfEincompatible$line$
‘SE1300’$in$Brassica$napus$and$set$up$a$selfEincompatibility$
molecular$marker$system$for$hybrid$breeding$ $ $
Self-incompatibility (SI) is one of the most important strategies developed by
flowering plants to prevent inbreeding and promote out-crossing. In Brassica, B.
napus (AACC=38) is self-compatible (SC) though its two ancestor species B. oleracea
(CC=18) and B. rapa (AA=20) are SI. ‘S-1300’ is a self-incompatible line in B.napus
generated by introgressing an S haplotype from B. rapa ‘Xishuibai’ into a B. napus
line ‘Huayou No. 1’. Hybrids from ‘S-1300’ as mother line have been delivered in
China, but the knowledge on the SI of ‘S-1300’ is very limited. In this study, genetic
analysis using ‘S-1300’ and a self-compatible line ‘8400’ and sequence alignment of
S locus genes SRK (S-locus receptor kinase gene) and SCR (S-locus cysteine-rich
protein gene) showed that the S haplotype BnS-1300 on A genome controls the SI
phenotype of ‘S-1300’. BnSP11-1300 and BnSRK-1300 were cloned by
homology-based candidate gene method, sharing 100% identity of the coding
sequence(CDS) with those of BrS-60. The expression of BnSP11-1300 was detected
only in ‘S-1300’, while the transcripts of BnSRK-1300 was detected in both ‘S-1300’
and ‘S-1300×8400’, which further confirmed that BnS-1300 controls the SI in
‘S-1300’. Based on the SI of ‘S-1300’ and the sequence of S haplotypes in B. napus, a
SI molecular marker system was set up. It contains three SCAR markers: SpeS1
linked to the restoration of self-incompatibility (a self-compatible B. napus line which
gives rise to self-compatible F1 hybrid), SRKc linked to the self-incompatibility of SI
line ‘S-1300’ and SpeS7 linked to the maintenance of self-incompatibility (a
self-compatible B. napus line which gives rise to self-incompatible F1 hybrid). SI line
‘S-1300’, 84 self-compatible cultivated B. napus lines, F1 hybrids (derived from the
crossing between ‘S-1300’ and the 84 self-compatible cultivated B.napus lines) and a
segregation population of (‘S-1300’×‘8400’)×(‘S-1300’×‘8403’) were used to check
the applicability of the SI molecular marker system. With expectation, the phenotypes
of the plants were all in line with the genotypes identified by the SI molecular marker
system. So, the SI molecular marker system can be used in and accelerate the process
of self-incompatibility hybrid breeding.
48$
!
MarkerEassisted$Breeding$of$Recessive$Genic$Male$Sterility$
Lines$and$their$Temporary$Maintainers$in$Rapeseed$(Brassica'
napus)$ $
FM Dong, DF Hong, L Kang, SH Huang, Y Yang, GS YANG
Improvement (Wuhan Branch), Huazhong Agricultural University, Wuhan 430070
Corresponding author, E-mail: [email protected]
The spontaneous recessive genic male sterility (RGMS) line 9012AB was firstly
reported in China and is regarded to have great potential in rapeseed heterosis
utilization because of their stable and complete sterility, extensive distribution of
restorers, and diverse cytoplasmic sources. In our breeding programs, 9012AB and
its temporary maintainer line T45 were individually utilized as the donor parents for
introgression of the male sterility genes ms3 or Rf into the excellent polima
cytoplasmic male sterility (CMS) lines (DH206A, DH303A, DH987A and
DH195A-14) and open-pollination (OP) cultivars (Zhongshuang9, Zheshuang72,
Huyou15), using a marker assisted selection (MAS) strategy. Massive dominant and
co-dominant molecular markers linked to ms3 and Rf were identified in map-based
cloning of these two genes, and they were screened between the donor and the
recipient parents. Using those markers displaying stable and repeatable polymorphism
for foreground selection, we picked out the individuals heterozygous at developed of
both the ms and Rf loci, and analyzed their genetic backgrounds with a lot of AFLP
and SSR markers. After six generations’ selection in three years, we have obtained
eight new recessive genic male sterility lines T45AB, ZS9AB, 987AB, 195-14AB,
206AB, ZS72AB, HY15AB and 303AB also with their own homologous temporary
maintainer lines. Genotypic analysis showed that the genome of the sterility lines and
the temporary maintainer lines comprises above 96% of the recurrent parents DNA.
The preliminary analysis of agronomy characteristics and quality showed that they
have exhibited high similarities with their corresponding recurrent parents. The
breeding of the other lines (Zheyou50, Zhongshuang11, Youyan9 and so on) were
ongoing as well as expected. These results demonstrated that MAS is an effective and
convenient approach for breeding of new RGMS lines and their homologous
temporary maintainers.
Key words: Brassica napus, recessive genic male sterility (RGMS), temporary
maintainer line, marker assisted selection
2012
MCID
$ 49
DNA
$
50$
!
Enhanced$Oil$Accumulation$in$Vegetative$Organs$by$Metabolic$
Pathway$Assembly$in$Tobacco$(N.$tabacum$L.)$for$HighEquality$
Renewable$Biofuel$Oil$
$
Changyong Gao, Xiaoyun Jia, Xue Mao, Li Zhang, Runzhi Li*
China
With the current concerns on a challenging set of interrelated global issues
including energy crises and depletion of natural resources, scientific community and
plant biomass industry have gained increasing attention for developing alternative
resources for chemicals and biofuels to meet market demands. When grown for
energy production instead for smoking, tobacco can generate a large amount of
inexpensive biomass more efficiently than almost any other agricultural crop. In this
work, we explored metabolic engineering approaches to enhance the oil content in
tobacco green tissues including leaf and stem for potential biofuel production. First, a
Vernonia cDNA clone (VgDGAT1) encoding diacylglycerol acyltransferase, a key
enzyme in triacylglycerol (TAG) biosynthesis, was over-expressed in tobacco
vegetative tissues. This modification resulted in a 23- and 15-fold increase in TAG
accumulation in tobacco leaves and stems, respectively. Abundant oil droplets were
visible in the vegetative tissues. Second, we engineered tobacco to ectopically
overproduce the transcription factor WRINKLED1 (RcWRI1) isolated from castor
developing seed. This WRI1 encoding an AP2/EREBP transcription factor regulates a
subset of genes involved in glycolysis and the incorporation of sucrose into TAG.
This modification alone increased TAG accumulation by 18 and 14 folds in leaves
and stems. Te transgenic tissues also showed an increase in sugar level. Third,
plastid-targeting expression of a fungus (Pleurotus ostreatus) acyl-CoA-Δ9 desaturase
(ScΔ9D), a cytosolic enzyme converting palmitic acid(16:0) into palmitoleic acid
(16:1Δ9), led to the level of ω-7 fatty acids up to 21.6% and 18.7% in the
transgenic leaves and stems from <0.5% in the wild-type tobacco plants. ω-7 fatty
acids including palmitoleic acid and its elongated product cis-vaccenic acid (18:1Δ11)
are the best lipid sources for high-quality biodiesel production. Combination of the
three approaches described above will have the potential to generate the transgenic
tobacco that accumulate both high oil and ω-7 fatty acids in the vegetable
tissues/organs, which benefits the production of high-quality biodiesel. The present
study developed a new strategy for metabolic pathway modification to increase the
energy density of biomass in vegetative organs by diverting carbon from starch to oil
biosynthesis.
2012
$ 51
$$
52$
!
A$pleiotropic$drug$resistance$gene$involved$in$the$transport$of$
plant$secondary$metabolites$in$Panax$ginseng$ $
Ru Zhang, Jie Zhu, Hongzhe Cao, Zhiyong Luo
Molecular Biology Research Center, School of Biological Science and Technology, Central South
University, Changsha 410078, China
A famous Chinese traditional medicine, Panax ginseng can produce a large
number of secondary metabolites ginsenosides which have various functions
including protection against pathogens and UV light, and have been used as natural
medicines for humans utilizing their diverse biological activities. In order to
accomplish these functions, biosyntheses and accumulation of ginsenosides are highly
regulated in a temporal and spatial manner in plant organs where they can
appropriately accumulate. Pleiotropic drug resistance (PDR) proteins, a subfamily of
the ATP-binding cassette (ABC) transporters, have been recently shown to play a role
in plant defense against various stresses and transportation the secondary metabolites.
However, nothing is known about their function in P. ginseng. In this study, we have
firstly cloned a PDR transporter gene from P. ginseng using cDNA-amplified
fragment length polymorphism (cDNA-AFLP and rapid-amplification of cDNA ends
(RACE) technology. The gene designated PgPDR3 has 4515 bp in length containing a
4137 bp open reading frame (ORF). The deduced amino acid sequence of PgPDR3
shares high similarity to other plant pleiotropic drug resistance (PDR) transporter and
have the characteristic domain of plant PDR transporter Walker A, Walker B and
ABC signature both at the N- and C-terminal end respectively. PgPDR3 was
expressed at a high level in the roots and adventitious roots than in seeds and buds,
and its expression and the content of the ginsenosides were up-regulated strongly by
100 µM MeJA in a time-dependent manner, as is well know MeJA is a signaling
molecule that mediates the biosynthesis and accumulation of secondary metabolites.
In conclusion, the results demonstrate a potential role of the PgPDR3 gene in
stimulation the transportation of secondary metabolites and might eventually be used
to enhance secondary metabolite production in ginseng. Better understandings of this
transporter as well as the biosynthetic genes of secondary metabolites will be
important for metabolic engineering aiming to increase the production of
commercially valuable secondary metabolites in plant cells and provide a new method
to produce a high quality ginseng plant.
2012
$ 53
54$
!
A$Metabolic$and$Genetic$Analysis$of$Cotton$Fiber$Cell$
Development$in$Response$to$Inhibition$by$the$Flavonoid$
Naringenin$ $
Jiafu Tan, Lili Tu, Fenglin Deng, Haiyan Hu, Yichun Nie, and Xianlong Zhang*
National Key Laboratory of Crop Genetic Improvement and National Plant Gene Center
(Wuhan), Huazhong Agricultural University, Wuhan, Hubei 430070, China
The cotton fiber is a unique elongated cell that is useful for investigating cell
differentiation. Previous studies have demonstrated the importance of factors such as
sugar metabolism, the cytoskeleton and hormones, which are commonly known to be
involved in plant cell development, while the secondary metabolites have been less
regard. By mining public data and comparing analyses of two cotton species, we
found that the flavonoid pathway is very active in early fiber cell development.
Different flavonoids exhibited distinct effects on fiber development during ovule
culture; among them, naringenin (NAR) was an obvious inhibitor of fiber
development. Naringenin is a substrate of flavanone 3-hydroxylase (F3H), and
silencing the F3H gene significantly increased the NAR content of fiber cells. Fiber
development was suppressed following F3H silencing, but the over-expression of
F3H caused no obvious effects. Exogenous dihydrokaempferol (DHK) and
kaempferol (K) which downstream of F3H, could not restore fiber development in
F3H-RNAi plants, But NAR caused a more extensive inhibition in vitro. Introducing
the F3H-RNAi segment into the high-flavonoid brown fiber cotton T586 line caused a
much greater accumulation of NAR as well as much shorter fibers in vivo and in vitro.
An inhibitor of phenylalanine ammonia-lyase (PAL) which upstream of flavonoids
pathway and NAR metablism, 2- aminoindan-2-phosphonic acid (AIP), could
promote fiber growth, and partially recover the inhibition of fiber growth in
F3H-RNAi plants. These results demonstrated that secondary metabolism represents a
potentially novel pathway with the potential to be engineered to genetically modify
cotton fiber quality.
2012
$ 55
Characterization$of$six$NAC$transcription$factor$genes$in$
Chickpea$(Cicer$arietinum),$which$are$involved$in$abiotic$
stress$responses$and$various$developmental$processes$
Xingwang Yu, Hui Peng, and Hao Ma
Nanjing Agricultural University
1. NAC transcription factors have been found to play important roles in plant
development and responses to environmental stresses. Based on two chickpea
drought-related cDNA libraries, six NAC gene, CarNAC1 6, were isolated and
characterized. Phylogenetic analysis showed that CarNAC1 6 belonged to different
subgroup of the NAC protein family. Multiple sequences alignment revealed that the
N-terminus of CarNAC1 6 proteins had conserved NAC domain. All CarNAC
proteins were localized in the nucleus and possessed the trans-activation activity in
the C-terminus. These data suggested that CarNAC1 6 were typical NAC genes and
their corresponding proteins probably were transcriptional activators in chickpea.
2. The CarNAC genes had different tissue-specific expression profiles and
showed differential expression patterns with cooperativity during developmental
processes. Moreover, Transcription of CarNAC1 6 genes was enhanced by drought,
high salinity, high and low temperature, mechanical wound, respectively.
Additionally, Some CarNAC genes were significantly up-regulated or down-regulated
by chemical treatments. These findings suggested that functions of all six CarNAC
transcription factors were involved in plant growth and development regulation as
well as environmental stress responses.
3. Transgenic Arabidopsis plants expressing CarNAC2 showed some
abnormalities such as germination delay, shorter stem of seedling, longer root of
seedling, dwarfish aerial part, early blossoming and low propagation coefficient. Both
transgenic Arabidopsis plants expressing CarNAC3 and CarNAC6 showed enhanced
tolerance to drought stress and high sensitivity to ABA, indicating that they are
ABA-dependent genes involved in stress responses.
56$
!
Improvement$of$Minhui3229$for$blast,$bacterial$blight$and$
brown$planthopper$resistance$and$plant$stature$through$
morphological$and$markerEassisted$selection$
TIAN Dagang1,2
CHEN Songbiao1
ZHOU Yuanchang2
WANG Feng1
1.Key Laboratory of Ministry of Education for Genetice, Breeding and Multiple Utilization of
Crops, Fujian Agricultural & Forestry University, Fuzhou 350002, China;
Blast, bacterial blight (BB) and brown planthopper (BPH) are three major threat
to rice production. IIyou 3229 and Minyou 3229 have high output and good qualities
when plant in south China rice-producing areas; however, these lines are susceptible
to blast, BB, and BPH. In order to improve the resistance of Minhui 3229 and its
derived hybrids such as IIyou 3229 and Minyou 3229 to the three diseases and insect
pests. We utilized the strategy of foreground marker-assisted backcrossing for
introgression of Pi9, Xa23 and BPH14/15, which express, respectively, highly
resistant to blast, BB and BPH. The whole breeding process coupled with
phenotype-based selection for improvement of their plant type and yield. C682, C751
and C60 are the three donors for Pi9, Xa23 and BPH14/15, which were improved
from wild rice O.Minutar, O.rufipogon and O.officinalis Wall. After 3 crossing with
the three donors, 3 backcrosses and 3 self-crosses, we obtained 3 three-gene pyramid
lines (Pi9+Xa23+BPH14) and 2 four-gene pyramid lines (Pi9+Xa23+BPH14/15) with
stable agronomic traits. By artificial inoculation of 21 blast isolates which came from
different areas of Fujian and an high poisonous BB stain P6 in the greenhouse, the
five pyramid lines had high resistance to blast and BB and exhibited the similar
resistant spectrum as the donor variety C682 and C751. The effect of BPH14/15 were
evaluted using seedbox screening with biotype I, II, and III, the results showed that
the 3 three-gene lines expressed moderately resistance to BPH, while the 2 four-gene
lines were resistance to BPH. Under conditions of blast, BB or BPH infection, these
five pyramid lines exhibited a significant yield advantage over Minhui 3229 and its
derived hybrids. Most importantly, these lines retain the excellent agronomical
propeties without compromising the yield as determined in multi-location trials. This
work demonstrates the successful application of marker-assisted selection in
conjunction with phenotype-based selection for targeted introgression of multiple
resistance genes into Minhui3229 along with improvement of their plant stature and
yield. Key words: Minhui 3229; Blast; BB; BPH; Marker-assisted Selection; Pyramid
lines
2012
$
$ 57
Genetic$analysis$of$grain$filling$rate$using$conditional$QTL$
mapping$in$maize$ $
Zhang Zhanhui, Liu Zonghua, Cui Zitian, Hu Yanmin, Wang Bin, Tang Jihua
The grain filling rate (GFR) is an important dynamic trait that determines the
final grain yield and is controlled by a network of genes. The GFR can be affected by
several environmental factors. To determine the genetic basis of the GFR, a
conditional quantitative trait locus (QTL) analysis method was conducted using
time-related phenotypic values of the GFR collected from a set of 243 immortalized
F2 (IF2) population, which were evaluated at two locations over 2 years. The GFR
gradually rose in the 0-15 days after pollination (DAP) and 16-22 DAP, reaching a
maximum at 23-29 DAP, and then gradually decreasing. The variation of kernel
weight (KW) was mainly decided by the GFR, and not by the grain filling duration
(GFD). Thirty-three different unconditional QTLs were identified for the GFR at the
six sampling stages over 2 years. Among them, QTLs qGFR7b, qGFR9 and qGFR6d
were identified at the same stages at two locations over 2 years. In addition, 14
conditional QTLs for GFR were detected at five stages. The conditional QTL qGFR7c
was identified at stage V|IV (37-43 DAP) at two locations over 2 years, and qGFR7b
was detected at the sixth stage (44-50 DAP) in all four environments, except at
Anyang location in 2009. QTLs qQTL7b and qQTL6f were identified by
unconditional and conditional QTL mapping at the same stages, and might represent
major QTLs for regulating the GFR in maize in the IF2 population. Moreover, most of
the QTLs identified were co-located with QTLs from previous studies that were
associated with GFR, enzyme activities of starch synthesis, soluble carbohydrates,
and grain filling related genes. These results indicated that the GFR is regulated by
many genes, which are specifically expressed at different grain filling stages, and the
specific expression of the genes between16-35 DAP might be very important for
deciding the final kernel weight.
58$
!
$$
2012
$ 59
FAD2
FAD3
FATB
$
60$
!
ANS
DFR
$
2012
$ 61
Isolation$and$functional$characterization$of$a$new$bZIP$
transcription$factor$in$waterlogged$seedlings$of$maize$
Wei Sun, Jun Huang, Feng Yu, Lei Liu, Fazhan Qiu, Yonglian Zheng and Zuxin Zhang
Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan
430070, China.
Waterlogging is one of the major limition factors for maize production in the
south China. Therefore, the effort to reveal the molecular mechanism and to identify
stress-related candidate genes is very important for maize cultivation and genetic
improvement. Here we present the isolation and function analysis of a transcription
factor, ZmbZIP which was found to be highly associated with waterlogging tolerance
of inbred lines by a high-through cDNA microarray in previous studies. Our studies
showed ZmbZIP is located in the nucleus in onion epidermal cells by bombarding
transformation. Yeast one-hybrid revealed that ZmbZIP had transcriptional activation
in vitro and combined with anaerobic response element (ARE, Adh1 promoter
sequence). The candidate gene association study to 21 waterlogging-related
quantitative traits in 205 temperature lines found the polymorphic sites in coding
region were significantly associated with waterlogging tolerance of maize.
62$
!
ZmGA3ox2,$a$candidate$gene$for$a$major$QTL,$qPH3.1,$for$
plant$height$in$maize$ $
Feng Teng1, Lihong Zhai1, Ruixiang Liu2, Wei Bai2, Liqiu Wang1, Dongao Huo1, Yongsheng Tao2,
Yonglian Zheng1, Zuxin Zhang1
1National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University,
Wuhan 430070, China;
2College of Agronomy, Hebei Agricultural University, Baoding 071001,
Chinahin a 12.6 kb interval. ZmGA3ox2, an ortholog of OsGA3ox2, which encodes a
GA3 β–hydroxylase, was positionally cloned. Association mapping identified two
polymorphisms in ZmGA3ox2 that were significantly associated with PHT across two
environments. Quantitative RT-PCR showed that SL15 had a higher ZmGA3ox2
expression relative to Zong3. The resultant higher GA1 accumulation, would lead to
longer internodes in SL15 because of longitudinally increased cell lengths. Moreover,
a large deletion in the coding region of ZmGA3ox2 is responsible for dwarf mutant
d1-6016. The result supports the theory that qualitative loci share the same loci as
quantitative traits. The successfully isolated qPH3.1 provides information on the
genetic basis of plant height in maize, and provides an opportunity for the
improvement of plant architecture in maize breeding.
2012
$ 63
Identification$of$a$major$QTL$for$silique$length$and$seed$
weight$in$oilseed$rape$(Brassica$napus$L.)$
Pu Yang, Zejun Yuan, Liuliu Shi, Chang Shu, Lin Chen, Jinsong Xu, Jiangsheng Wu, Kede Liu*
National Key Laboratory of Crop Genetic Improvement, Wuhan 430070, China
Silique length (SL) and seed weight (SW) are two important yield-related traits
controlled by quantitative trait loci (QTL) in oilseed rape (Brassica napus L.). The
genetic bases underlying these two traits are largely unknown at present. In this study,
we conducted QTL analyses for SL and SW using 186 recombinant inbred lines (RILs)
derived from a cross between S1, an EMS mutant with extremely long siliques and
large seeds, and S2, an inbred line with regular silique length and seed size. RILs
were grown in Wuhan in the 2008/2009 (SS09) and 2009/2010 (SS10) growing
seasons, and mean SL and SW for each line were investigated. Ten non-redundant
QTL were identified for SL. Of these, a major QTL, cqSLA9, consistently explained
as much as 53.4% of SL variation across environments. The others are minor QTL
and individually explained less than 10% of the SL variation. Nine non-redundant
QTL were identified for SW. Of which, one major QTL, cqSWA9, explained as much
as 28.2% of the total SW variation in the SS09 and SS10 environments. In addition,
three additive by additive interactions with small effects were detected for SL, and no
interactions were detected for SW. Interestingly, the two major QTL, cqSLA9 for SL
and cqSWA9 for SW colocalized in the same chromosomal region and were integrated
into a unique QTL, uqA9. The S1 allele at this locus increases both SL and SW,
suggesting that uqA9 has pleiotropic effects on both SL and SW. The existence and
effect of uqA9 was confirmed in genetically different RILs derived from the cross
between S1 and No2127, a resynthesized DH line having regular silique length and
seed size. Individuals in one residual heterozygous line (RHL) for cqSLA9, L133,
showed significant difference in silique length. Molecular and progeny verification
showed the S1 allele homozygous plant and two S2 allele homozygous plants in this
line are near isogenic lines (NIL) for cqSLA9. A F2 population comprising of 5235
individuals derived from the cross between the NILs were produced. The genetic
interval of cqSLA9 was firstly verified between marker Na10-B07 and BoGMS116 by
genotyping each F2 individuals by using seven mostly linked markers. SL for each
plant was measured and 355 recombinant plants were detected based on the genotype
of Na10B07 and BoGMS116. The genetic composition of each individual was scored
using 14 new developed polymorphism SSR markers. Finally, cqSLA9 was delimited
to a 237kb region, between the marker BrGMS4931 and BrGMS4942, on B. rapa A9
chromosome. The results in this study revealed that silique length in the S1 mutant is
mainly controlled by the cqSLA9 locus. Fine mapping of cqSLA9 will greatly
accelerate the process of cqSLA9 cloning and finally assist our understanding of the
genetic basis for silique and seed development.
64$
!
A$StayEGreen$Gene$SGR1$regulates$lycopene$accumulations$by
$directly$interacting$with$the$key$carotenoid$biosynthetic$enzy
me$PSY1$during$fruit$ripening$in$tomato$ $
Lycopene is a powerful antioxidant in plant and animal. As the main sources of ly
copene for human nutrients and health, flesh fruits synthesize such carotenoid by conv
ersion of chlorophyll during the ripening. SlSGR1, a stay-green gene, plays critical rol
es in retardance chlorophyll degradation in leaves and fruits of tomato (Solanum lyco
persicum). Here, we elucidate a new mechanism on SlSGR1 regulating the lycopene a
ccumulation. Through inhibiting the activity of a key carotenoid synthetic enzyme SlP
SY1 by direct protein-protein interaction, SlSGR1 can mediate the accumulations of l
ycopene during fruit development and ripening in tomato. This first confirmed in the e
ngineered bacteria harboring vector pACCRT-EBI the SlSGR1 could dramatically red
uce the lycopene biosynthesis. Knock-down of SlSGR1 in transgenic tomato fruits the
accumulation patterns of phytoene and lycopene were altered along with elevating th
e transcriptional level of SlPSY1 and accelerating plastid conversion at the early stage
s of fruit ripening, and eventually enhanced both lycopene and β-carotene contents up
to 4 and 9-folds in red ripe fruits compared to the wild-type. Furthermore, our results i
ndicated that SlSGR1 mediates ethylene signal transduction with altering their gene e
xpressions, such as ethylene receptor genes ETR1~6 and ethylene-induced genes PG2
a, PE and EXP1. The texture of pericarp was arranged more firmly and the fruit shelflife significantly extended in transgenic tomatoes. These demonstrated that SlSGR1 pl
ays pivotal regulatory roles in color formation and fruit ripening in tomato.
2012
$ 65
A$Large$Insertion$in$bHLH$Transcription$Factor$BrTT8$Resulting$
in$Yellow$Seed$coat$in$Brassica$rapa$ $
Xia Li1, Li Chen2, Meiyan Hong1, Yan Zhang3, Feng Zu4, Jing Wen1, Bin Yi1, Chaozhi Ma1,
Jinxiong Shen1, Jinxing Tu1*, Tingdong Fu1
Improvement in Wuhan, Huazhong Agricultural University, Wuhan, P.R. China
Yellow seed is a desirable quality trait of the Brassica oilseed species. Previously,
several seed coat color genes have been mapped in the Brassica species, but the
molecular mechanism is still unknown. In the present investigation, map-based
cloning method was used to identify a seed coat color gene, located on A9 in B. rapa.
Blast analysis with the Arabidopsis genome showed that there were 22 Arabidopsis
genes in this region including at4g09820 to at4g10620. Functional complementation
test exhibited a phenotype reversion in the Arabidopsis thaliana tt8-1 mutant and
yellow-seeded plant. These results suggested that the candidate gene was a homolog
of TRANSPARENT TESTA8 (TT8) locus. BrTT8 regulated the accumulation of
proanthocyanidins (PAs) in the seed coat. Sequence analysis of two alleles revealed a
large insertion of a new class of transposable elements, Helitron in yellow sarson. In
addition, no mRNA expression of BrTT8 was detected in the yellow-seeded line. It
indicated that the natural transposon might have caused the loss in function of BrTT8.
BrTT8 encodes a basic/helix-loop-helix (bHLH) protein that shares a high degree of
similarity with other bHLH proteins in the Brassica. Further expression analysis also
revealed that BrTT8 was involved in controlling the late biosynthetic genes (LBGs) of
the flavonoid pathway. Our present findings provided with further studies could assist
in understanding the molecular mechanism involved in seed coat color formation in
Brassica species, which is an important oil yielding quality trait.
66$
!
MapEbased$cloning$and$functional$analysis$of$qbpc$which$
controls$protein$content$in$brown$rice$
$
Bo Peng, Huili Kong, Yibo Li, Guanjun Gao, Qinlu Zhang, Xianghua Li, Yuqing He*
National Key Laboratory of Crop Genetic Improvement and National Centre of Plant Gene
Research (wuhan), Wuhan 430070, China
Rice protein is one of the most important nutritional quality, and also is the second
most abundant constituent of milled rice, following starch. As one of the staple food,
rice protein is one of the main sources of protein intake for people. Rice protein
content is typical quantitative traits, with the advent of molecular marker,
introgression of genes controlling good quality by marker-assisted selection has been
proved to be an effective choice to improve the quality of cultivars. However, the
genetic basis of protein is complex and often controlled by quantitative trait loci. Thus,
fine mapping and cloning those QTLs underlying protein is needed for rice genetic
improvement.
2012
$ 67
A$serine$threonine$protein$kinase$receptor$gene$is$the$
potential$candidate$of$a$quantitative$trait$locus$for$grain$
yieldErelated$trait$in$maize$
Zuxin Zhang, Ruixiang Liu, Xiaoliang Cao, Jun Huang, Fazhan Qiu, Yongsheng Tao
Maize grain yield is a highly complex quantitative trait that is controlled by multiple
quantitative trait loci (QTLs) with small effects, and is frequently influenced by
multiple genetic and environmental factors. Thus, it is challenging to finely map and
clone a QTL for grain yield in the large maize genome. Previously, we identified a
major QTL for kernel number per row (KNPR) across multiple environments, defined
as qKNPR6, and developed two nearly isogenic lines, SL57-6 and Ye478, which
differ only in the allelic constitution at the short chromosome segment harboring the
QTL. Recently, qKNPR6 was re-evaluated in new segregating populations derived
from SL57-6/Ye478, and was narrowed down to a 2.8 cM interval, which explained
56.3% of phenotypic variance of KNPR in 201 F2families. The QTL also
simultaneously affected ear length, kernel weight and grain yield. Furthermore, a
large F2 population
!
68$
RIL
QTL
$
2012
$ 69
Function$analysis$of$flowering$locus$T$in$a$model$for$the$intera
ction$of$floweringEtime$genes$in$wheat$ $
Bo Lv, Shuyun wang, Xiuli Han, Kun Li, Jiajie Wu and Daolin Fu
State Key Laboratory of Crop Biology, Shandong Key Laboratory of Crop Biology, Shandong Ag
ricultural University, Tai’an, Shandong, China, 271018
In flowering plants, the phase transition from vegetative to reproductive develop
ment is a critical event in the life cycle. Florescence is complex and influenced by ma
ny factors in plants. FLOWERING LOCUS T (FT) gene plays important roles on the
floral development. FT can promote flowering in the plant photoperiod pathway and
also facilitates vernalization flowering pathways and other way to promote flowering.
The protein encoded by FT gene, which is able to transfer long distance, is a flowerin
g hormone, and it has key functions on the process of flower bud formation. The silen
ce expression of FT in a transgenic spring wheat (Bobwhite) caused extremely late he
ading and some of transgenic plants can’t flowering. From QT-PCR results, VRN1, a
n APETALA1/FRUITFULL homolog in wheat, was downregulated in these transgeni
c plants. While the overexpression of FT in transgenic plant caused early heading with
the upregulation of VRN1. From RT-PCR results show that Bobwhite has null alleles
of VRN2. Base on these results, we present a model for a genetic network of flowerin
g-time gene in wheat leaves, in which FT is upstream of VRN1 and has key functions
on the process of flower bud formation.
!
70$
$
(Fusarisum Head Blight, FHB)
GC/MS
5000
127
5
100
661
2012
$ 71
“
”
QTL
$
250010
72$
!
Systematic$Metabolic$Engineering$of$ωE7$Fatty$Acids$in$
Soybean$Seeds$ $ $
Jinai Xue, Xue Mao, Xiaoyun Jia, Yongmei Wu, Runzhi Li*
China
Plant oils containing large amounts of ω-7 fatty acids such as palmitoleic acid
(16:1Δ9) and cis-vaccenic acid (18:1Δ11) are high-valued for human health, medicine
and industry. For commercial utilization of such important fatty acids (FA), oilseeds
can be used as the target to be genetically modified by metabolic engineering to
produce high level of those unusual monounsaturated lipids. In this study, we
performed a series of systematic metabolic engineering experiments to enhance the
accumulation of ω-7 FA and simultaneously decrease the saturated fatty in soybean
seeds. For this goal, three enzyme genes were selected as targets to be modified. The
enzyme responsible for the conversion of palmitic acid (16:0) to palmitoleic acid
(16:1Δ9) is acyl-Δ9 desaturase, which have two classes, acyl-ACP-Δ9 desaturase
worked in plastid and acyl-CoA-Δ9 desaturase acted in cytosol/ER. We isolated and
functionally characterized a cDNA clone (MuACP-Δ9D) of acyl-ACP-Δ9 desaturase
from developing seeds of cat’s claw (Macfadyena unguis-cati), a natural high
accumulator of 16:1Δ9. A cDNA clone (ScCoA-Δ9D) encoding acyl-CoA-Δ9
desaturase was isolated from yeast (Saccharomyees cerevisiae). Soybean acyl-ACP
thioesterase FatB (GmFatB) can selectively transport acyl-ACP 16:0 from plastid to
cytosol. Down-regulation of this FatB could increase 16:0 content in plastid, and
subsequently provide high level of 16:0 substrate for acyl-ACP- Δ9 desaturase.
Seed-specific expression of either MuACP-Δ9D or ScCoA-Δ9D increased ω-7 FA
accumulation from <1% to 15.7% and 6.1% in soybean seed oil, respectively. Small
RNA silencing technique was employed to down-regulate GmFatB only in seed
development, resulting in ω-7 FA enhancement by 8.4% in mature seeds. Notably,
co-expression of MuACP-Δ9D in plastid and ScCoA-Δ9D in cytosol, together with
down-regulating GmFatB, led to ω-7 FA increase up to 34.7% in soybean seed oil.
Moreover, saturated 16:0 FA dropped from 15% down to 8% in the best transgenic
line seeds. Such engineered soybean seeds with high level of ω-7 FA could be used
for commercial production of those unusual FA for human food, health and industry.
Additional modifications on other genes related to FA biosynthesis are under way.
Overall, the present results demonstrate that the stacking of multiple traits by
systematic metabolic can successfully optimize the accumulation of a variety of
unusual FA in any established oilseed.
2012
$ 73
Opaque2
$
$
Opaque-2 O2
bZIP
22kD α
O2
Yeast
two-hybridization
2
ZmTaxilin
O2
Taxilin
GST Pull-down
O2
O2
O2
MADS
MADS
Mu
opaque
O2
74$
!
The$cloning$and$characterization$of$Shrunkrn4$gene$in$maize$
Zeins and starches are major storage components of maize seeds.The composition
and content of zeins or starches determined the quality or productivity of maize
seeds.Shrunken4 is one of the important mutants.Studies have shown that,the
mutations affecting the activity of starch systhesis pathway Related Enzymes caused
the mutant phenotype. The cloning and functional analysis of the gene for improved
maize grain quality traits.First, map-based cloning of SH4 and the gene sequence
analysis showed that the gene encodes an AVP protein; followed by biochemical and
cytological studies,we fined the gene mutation significantly affect the synthesis of
starch and protein, the germination rate, also the development and aleurone layer;
ultimately, we believe that the gene mainly affect grain development through
regulating the transport of sucrose. We follow-up gradually reveal the role Sh4 play in
the storage material anabolic process ,and its value by genetically modified.
2012
$ 75
Characterization$and$Identification$of$ZmaNAC36,$a$Novel$
NAC$Domain$Transcription$Factor,$Imply$Its$Potential$Role$in$
the$CoEexpression$of$Starch$Synthesis$Genes$in$Maize$
Endosperm$ $
Jiang Chen, Dan Peng, Yufeng Hu, Yinghong Liu, Yong Gu, Yangping Li, Junjie Zhang, Hanmei
Liu, Yubi Huang
Maize Research Institute of Sichuan Agricultural University, Ministry of Agriculture, Wenjiang,
Chengdu, People’s Republic of China
Starch is an essential commodity that is valued for its uses in food, feed, fuel, and
industry. The regulation of starch synthesis is not completely known to us. It had been
reported that the starch synthesis genes were co-expressed during the maize
endosperm development, but the mechanism controlling the co-expression was rarely
reported. By homologous clone strategy, we cloned a gene, ZmaNAC36, which
potentially took part in the co-expression of starch synthesis. In the present research,
we firstly made characterization of ZmaNAC36. It was nuclear localization, had the
transcriptional activation and could bind to the common cite as most NAC families
did. Then, the expression patterns of ZmaNAC36 were analyzed, and it was strongly
expressed in the maize endosperm and was co-expressed with most of starch synthesis
genes. Lastly, ZmaNAC36 was transient over-expressed in the maize endosperm by
particle bombardment, and some starch synthesis genes were up-regulated. All our
results indicated that ZmaNAC36, a novel NAC domain transcription factor, played a
potential role in the co-expression of starch synthesis genes in maize endosperm.
76$
!
Identification$and$characterization$of$microRNAs$in$the$develo
ping$maize$endosperm$ $
Yong Gu ,Yinghong Liu,Hai Du,Jiang Chen, Yubi Huang*
Maize Research Institute, Sichuan Agricultural University, Chengdu, Sichuan,
611130, China
MicroRNAs (miRNAs) are non-coding RNAs that are approximately 20–22 nucle
otides long. miRNAs have been shown to be important regulators that control a large
variety of biological functions in eukaryotic cells including development, viral
defense, metabolism and apoptosis. In maize, the endosperm development involves a r
ange of biological processes, many of which may be regulated, at least in part, by miR
NAs.To investigate the roles of miRNAs in maize endosperm development, two small
RNA libraries of maize endosperm at two developmental stages were sequenced. A t
otal of 17,773,394 and 18,586,523 small RNA raw reads were obtained, respectively.
Further analysis identified and characterized 95 known miRNAs belonging to 20 miR
NA families. In addition, 232 novel miRNAs were identified and grouped into 139 fa
milies. Twenty-one of the 232 novel miRNAs were detected in two data sets . Potentia
l targets for 105of the novel miRNA families were successfully predicted. we had also
identified 26 corresponding miRNA*s of these novel miRNAs. In summary, we inves
tigated expression patterns of known miRNA in maize endosperm at key development
al stages and identified novel miRNAs.
2012
$ 77
QTL$analyses$for$resistance$to$Sclerotinia$sclerotiorum$in$
Brassica$napus$revealing$its$association$with$flowering$time$ $
Dayong Wei, Wangjie Xu, Hongli Dong, Jiana Li, Wei Qian
Engineering Research Center of South Upland Agriculture of Ministry of Education, College of
Agronomy and Biotechnology, Southwest University, Chongqing 400716, P.R.China
Sclerotinia stem rot of oilseed rape (Brassica napus) has a strong negative impact
on seed yield and quality. In this study, quantitative trait loci (QTL) analyses
concerning the Sclerotinia resistance and flowering time were carried out in 2010 and
2011 in a doubled haploid (DH) population of rapeseed which consisted of 261 lines.
Significant positive correlation was found between stem resistance (SR) tested under
controlled condition and natural resistance (NR) assisted in the field, while notable
negative correlations were detected for flowering time (FT) with stem resistance and
natural resistance to Sclerotinia stem rot. In total, nine, seven and 16 QTL were
identified for SR, NR and FT over two years, jointly explained 30.5% and 20.6%,
22.7% and 40.0%, and 85.1% and 62.0% of the phenotypic variation of the
corresponding trait in 2010 and 2011, respectively. Of these, two QTL for SR, four
for NR and four for FT were found to be clustered in a 9.8-cM region on linkage
group N12. The N12-QTL region was extensively compared with previous studies,
and the association of flowering time with Sclerotinia resistance is discussed.
78$
!
Differential$accumulation$of$phenolic$compounds$and$
expression$of$related$genes$between$the$blackE$and$
yellowEseeded$Brassica$napus$L$
Cunmin Qu, Kun Lu, Fuyou Fu, Kai Zhang, Rui Wang, Min Wang, Jiana Li
Southwest University
Developing yellow-seeded rapeseed is a major breeding goal for better
nutritional and processing quality in oil production in rapeseed. The intermediate
metabolites and final metabolites of the phenylprolmnoid metaholk and flavonoids
pathway not only affects oil quality traits, but are also involved in the seed coat color
in B. napus. In this research, we analyzed the accumulation of phenolic compounds in
the seed coat of the typical black-seeded (DH ZY821) and yellow-seeded (DH GH06)
genetic types of B. napus in China. With toluidine blue O and LC-UV-MS, we
showed that there are histochemical and biochemical differences in the accumulation
of phenolic compounds between DH ZY821 and DH GH06. The results of cytological
observation also showed that polymeric phenols compounds began to accumulate in
the hilum of seed during the early development stages, and mainly distributed in the
layer of palisade and pigment of seed coat during the middle-to-late stages. Moreover,
proanthocyanins and polyphenols content is higher in the black-seeded (DH ZY821)
than in the yellow-seeded (DH GH06) of B. napus. Another, results of LC-UV-MS
showed that flavonoid and derivatives mainly accumulated in black-seeded, including
epicatechin, quercetin, isorhametin, and kaemferol derivates. In addition, thirty-five
phenolic compounds were detected in the flavonoid extracts from both DH ZY821
and DH GH06 seed coat according to the retention time analysis of LC-UV-MS.
Interestingly, 2 and 13 compounds could be detected only in DH ZY821 and DH
GH06, respectively. Moreover, epicatechin derivates were not detected in seed coat
extracts of DH GH06. Using qRT-PCR, we revealed that nine structural genes
(BnPAL, BnC4H, BnTT4, BnTT6, BnTT3, BnTT18, BnTT12, BnTT10 and BnUGT2)
and three transcription factors (BnTTG1 BnTTG2 and BnTT8) function in the
flavonoid biosynthetic pathway display a significant difference between the yellowand black-seeded B. napus. BnPAL, BnC4H and BnTT12 encode enzymes
specifically involved in the steps of the pathway leading to the biosythesis of
precursor of polyphenols and flavonoids compounds and proantnocyanins, and may
be the key genes for formation seed coat colour. The BnTT4 is also the first key gene
in the flavonoid pathway in B. napus, and downstream genes were regulated by the
upstream genes in the flavonoid biosynthetic pathway. In addition, BnTTG1 can also
be directly regulated by BnTTG2, and the co-expression of BnTT2, BnTT8 and
BnTTG1 regulates flavonoid biosynthesis pathway in B. napus. Therefore, the
function of and the expression patterns of transcription factors in B. napus were also
similar with Arabidopsis thaliana. These dataset provide a comprehensive expression
profile for the laws of pigments accumulation and expressions patterns of genes in B.
napus, and also provide a useful resource and reference for formation and regulation
mechanism of seed coat colour in B. napus.
2012
$ 79
Genetic$dissection$of$trichome$locus$(T1)$and$a$major$QTL$
controlling$multiple$fibre$quality$traits$in$upland$cotton$ $
Dexin Liu, Jian Zhang , Shiyi Tang, Zhengsheng Zhang*
Fibers and leaf trichomes are large branched single cells that initiate and develop
on epidermal cells, they are likely to have common regulatory mechanisms. RIL 118
( chosen from a recombinant inbred population crossing a high quality cultivar
Yumian 1 and the multiple dominant marker line T586) backcrossed with yumian1
to develop to populations 1440 F2 and 4313 F3 to fine-map a major effecting multiple
fiber qualities and trichome QTL in cotton .A total of 95 markers were identified in
360 F2 population to construct a genetic map that spanned 127.2cM. Trichomes (T1)
locus was mapped between in two markers and co-segregated with the SWU06-77 .
Designed the primers of the other 380 new microsatellites based D genomes ,8
markers were polymorphic in the region. To further define the QTL position ,19
markers were employed to construct a genetic map that spanned 0.463 cM in 1440 F2
population . The T1 locus explained 54.7% of the fiber length phenotypic Variation
and LOD value was 211.1, additive effect was 2.65. The T1 locus explained 40.5% of
micronaire value phenotypic Variation and LOD value was 144.8, additive effect was
-0.41. The T1 locus explained 30.1 of fiber uniformity phenotypic Variation , LOD
value was 100.1 and additive effect was 1.83. The T1 locus explained 193.16 of fiber
strength phenotypic Variation , LOD value was 193.16 and additive effect was
2.91.The 19 and 12 markers , separately ,were co-segregated with T1 in F27
recombinant inbred population and F2(including F23) populations.
80$
!
PSKEαsignalling$involve$in$cotton$fiber$elongation$ $
Jie Han,Jiafu Tan,Lili Tu
Huazhong Agricultural University, Wuhan 430070, China
Phytosulfokine-α (PSK-α), a 5-amino acid sulfated peptide that has several biological
activities including plant cell development, stress response and reproduction via
binding to the membrane-localized PSK receptor. A PSK Precusor gene has been
indentifed from cotton fiber library, named Gossypium hirsutum Phytosulfokine
Precusor(GhPSKP). Real-time reverse transcription PCR (RT-PCR) revealed that
GhPSKP expressed preferentially in elongating fibers. Over-expression GhPSKP
shows fiber lengths was increased compared with the control in different elongate
stages including 10dpa, 15dpa ,20dpa and matrued, which demonstrates that
up-regulation of the GhPSKP gene expression promoted fiber cell elongation, while
RNAi GhPSKP has no remarkable changed. Correspond to this phenotype, we
mensurate the content of PSK-αin 10dpa fiber by LC-MS and the result demonstrated
that over-expression GhPSKP increases the endogenous PSK-αlevels. Subsequently,
we find that adding PSK-αinto cotton ovlue cultured system promotes fiber cell
elongation in vitro. These results indicate that PSK-αsignalling involve in cotton fiber
elongation.
2012
$ 81
QTL
$ $
82$
!
Genetic$Control$of$Seed$Shattering$in$Rice$by$the$APETALA2$
Transcription$Factor$SHATTERING$ABORTION1$ $
Yan Zhou, Danfeng Lu, Canyang Li, Jianghong Luo, Bofeng Zhu,
Zixuan Wang, and Bin Han
National Center for Gene Research, Institute of Plant Physiology and Ecology, CAS, Shanghai
200233, China
Reduced seed-shattering is an important agricultural trait in rice domestication.
Although two rice shattering genes have been identified, the pathway in the regulation
of seed shattering in rice remains unknown. In order to find other genes involve in the
shattering network, we screened for non-shattering rice mutants through mutagenesis
of a shattering line SL4, which harbors SH4 (grain shattering quantitative trait locus
on chromosome 4) and qSH1 (quantitative trait locus of seed shattering on
chromosome 1) loci simultaneously. We isolated two non-shattering mutants shat1
(shattering abortion 1) and shat2 (shattering abortion 2). Through phenotype
characterizing, we found that disruption of abscission zone (AZ) resulted in
non-shattering phenotype in both shat1 and shat2. Through map-based cloning,
SHAT1 was restricted to a 9.0 kb region which contains only one ORF encoding an
AP2 transcriptional factor. SHAT2 was localized to a 9.7 kb region which contains
only one known shattering related gene SH4. In order to distinguish the mutant allele
of SH4 in cultivated rice, we renamed the shat2 allele as sh4-2 in the following text.
Down regulation the expression of SHAT1 in a reduced-shattering variety Kasalath
through RNAi experiment, we observed the BTS value increasing with the decreasing
of SHAT1 mRNA in the transgenic plants.
2012
$ 83
Y
$$
No.2127-17
821
DH
DH
4260
Y5
F1
DH
Y47
F2
AFLP
63
SSR
SSR
C8
DH
11
821
Y27
7500
BC4F2
3:1
APLF
InDel
120K
SSR
BC4F2
InDel
YS52
BC4F2
84$
!
$
QTL
$
2012
$ 85
Genetic$analysis$and$fine$mapping$of$the$Ga1ES$gene$region$co
nferring$crossEincompatibility$in$maize$ $
Cross-incompatibility genes known as gametophyte factors (ga) are numerous in maiz
e. Many popcorn strains carry these genes and cannot be fertilized by pollen of dent a
nd flint maize strains although the reciprocal crosses are successful. A Chinese popcor
n strain SDGa25 carries the strongest allele of Ga1 (Ga1-S) and the majority of Chine
se dent and flint maize germplasm are incompatible with SDGa25. The incompatibilit
y is due to pollen tube growth obstruction 2 h after pollination. The pollen tube is arre
sted in the silk segment 5.5 cm distal to the pollination area and never reaches the ovu
le. The Ga1-S carried by SDGa25 behaves as a single dominant gene. This gene was
mapped between markers SD3 on BAC AC200747 0.827 cM apart on the telomere si
de and SD12 on BAC AC204382 0.709 cM apart on the centromere side. The genetic
region mapped spanning the Ga1-S locus was estimated to be 1.5 cM in length and the
physical distance is 2,056,343 bp on ctg156 based on the B73 RefGen_v2 sequence.
Gametophyte factors influence gene flow direction and the strongest Ga1-S allele is us
eful for isolating one category of commercial varieties from another. The eight tightly
linked markers to Ga1-S developed in this study would greatly improve marker-assist
ed introgression efficiency and the fine mapping would facilitate the isolation of the G
a1-S.
86$
!
Identification$of$a$major$QTL$for$silique$length$and$seed$
weight$in$oilseed$rape
Pu Yang, Zejun Yuan, Liuliu Shi, Chang Shu, Lin Chen, Jinsong Xu, Jiangsheng Wu, Kede
Liu*
National Key Laboratory of Crop Genetic Improvement,
Huazhong Agricultural University, Wuhan 430070, China
Silique length (SL) and seed weight (SW) are two important yield-related traits c
ontrolled by quantitative trait loci (QTL) in oilseed rape (Brassica napus L.). The gene
tic bases underlying these two traits are largely unknown at present. In this study, we
conducted QTL analyses for SL and SW using 186 recombinant inbred lines (RILs) d
erived from a cross between S1, an EMS mutant with extremely long siliques and larg
e seeds, and S2, an inbred line with regular silique length and seed size. RILs were gr
own in Wuhan in the 2008/2009 (SS09) and 2009/2010 (SS10) growing seasons, and
mean SL and SW for each line were investigated. Ten non-redundant QTL were identi
fied for SL. Of these, a major QTL, cqSLA9, consistently explained as much as 53.4
% of SL variation across environments. The others are minor QTL and individually ex
plained less than 10% of the SL variation. Nine non-redundant QTL were identified fo
r SW. Of which, one major QTL, cqSWA9, explained as much as 28.2% of the total S
W variation in the SS09 and SS10 environments. In addition, three additive by additiv
e interactions with small effects were detected for SL, and no interactions were detect
ed for SW. Interestingly, the two major QTL, cqSLA9 for SL and cqSWA9 for SW co
localized in the same chromosomal region and were integrated into a unique QTL, uq
A9. The S1 allele at this locus increases both SL and SW, suggesting that uqA9 has pl
eiotropic effects on both SL and SW. The existence and effect of uqA9 was confirmed
in genetically different RILs derived from the cross between S1 and No2127, a resynt
hesized DH line having regular silique length and seed size. Individuals in one residua
l heterozygous line (RHL) for cqSLA9, L133, showed significant difference in silique
length. Molecular and progeny verification showed the S1 allele homozygous plant a
nd two S2 allele homozygous plants in this line are near isogenic lines (NIL) for cqSL
A9. A F2 population comprising of 5235 individuals derived from the cross between t
he NILs were produced. The genetic interval of cqSLA9 was firstly verified between
marker Na10-B07 and BoGMS116 by genotyping each F2 individuals by using seven
mostly linked markers. SL for each plant was measured and 355 recombinant plants w
ere detected based on the genotype of Na10B07 and BoGMS116. The genetic compos
ition of each individual was scored using 14 new developed polymorphism SSR mark
ers. Finally, cqSLA9 was delimited to a 237kb region, between the marker BrGMS49
31 and BrGMS4942, on B. rapa A9 chromosome. The results in this study revealed th
at silique length in the S1 mutant is mainly controlled by the cqSLA9 locus. Fine map
ping of cqSLA9 will greatly accelerate the process of cqSLA9 cloning and finally assi
st our understanding of the genetic basis for silique and seed development.
2012
$ 87
$$
9
36
2009-2011
36
SSR
9
SRAP
7.77% - 63.53%,
14.81% - 227.64%
-27.52% - 24.77%
10.62% - 35.01%,
4
28.36% - 88.06%
7.77% - 33.26%,
×5
4
-18.96% - 12.21% 5
22.97% - 75.26%
-15.16% - 22.30%
10.42% - 63.53%,
-27.52% - 24.77%
14.81% - 227.64%
8D129 X 8C343
8D129
8C108
8C108 X 8E001
0.89
368
335
350
SRAP
PIC
0.74 – 0.87
SSR
0.41
SRAP
76
SRAP
SSR
0.43
0.31 – 0.87
106
0.24-0.46
SSR
53
206
10
PIC
0.30-0.53
SSR
,
0.44
!
88$
C
SSH
$
,
CMS
C48-2
N48-2
Trizoi
mRNA
RNA
T
A
C48-2
(1)
tester
N48-2
N48-2
PCR
driver
N48-2
C48-2
tester
C48-2
driver
PMD-19
425
SSH
220
PCR
300 bp
SSH
200-750 bp
(3)
PCR
C48-2
N48-2
cDNA
C48-2
11
2l
EST
Blast
EST
11
Ca2+
N48-2
12
EST
(4)
EST
1
CMS-C
(2)
22
14
EST
RNA
10
Genbank
6
EST
2012
$
$ 89
Screening$Identification$Indexes$of$Drought$Resistance$at$
Whole$Growth$Period$in$Upland$Rice$Varieties$from$Yunnan$
Province$ $
Xu JianXin, Wang YunYue* , Hu XiangWei , Su ZiHan
Ministry of Education Key Laboratory for Agrobiodiversity and Plant Disease
Management, Yunnan Agricultural University, 650201, Kunming; *Corresponding
author, E-mail: [email protected]
Upland rice production as one of the
effective ways to alleviate water shortages under the situation of global warming and
surge in the world's population has been increasingly valued by human. In-depth
study on the effects of water stress on morphological index and agronomic characters
of upland rice and selecting identification index of drought resistance during bud
seedling and mature stage were foundation to breed drought tolerant varieties and
promote genetic improvement The results of study on drought resistance of Yunnan
upland rice varieties during germinating stage showed that relative germ roughness
relative radicle length and relative radicle roughness could be used as indexes of
assessing the drought resistance at germinating stage of upland rice. The results of
study on drought resistance of Yunnan upland rice varieties during seedling stage
showed that relative value of the length of first leaf under the central leaf the length
of second leaf under the central leaf the width of second leaf under the central leaf
root dry weight and root-shoot ratio were significantly or very significantly correlated
with the survival percentage under repeated condition, and these five indexes could be
used as the morphological indexes to identify the drought resistance. The drought
resistance of 24 upland rice varieties was evaluated by regression equation and the
results indicated that it was feasible to predict the drought resistance in rice during
seedling stage using integrated identification index. Through correlation analysis, path
analysis and regression analysis between major agronomic traits and drought
resistance of upland rice varieties under water stress, we found that principal factor
which influenced rice yield under the dry cultivation condition was effective fringe
number filled grains number and 103 Grain weight. To achieve the purpose of high
yield under water stress, the high-yield production measures like stabilizing the
number of effective fringe and increasing the number of filled grains per panicle and
the weight of 103 Grain should be applied in the cultivation process of upland rice.
90$
!
Phenotype$Analysis$and$Gene$Mapping$of$a$Novel$Narrow$
Leaf$Mutant$(nal9(t))$in$Rice$(Oryza$sativa$L.)$ $
MaLing, Xianchun Sang, Shuang Guo, Xiaoyan Zhu ,Guanghua He
College of Agronomy and Biotechnology, Southwest University, Chongqing 400715 A
novel narrow leaf mutant nal9(t) was obtained from the rice (Oryza sativa L.) restorer
line Jinhui 10 treated by ethyl methyl sulphonate (EMS). At mature stage, the length
of the first, second and third upper leaf in the mutant respectively were 1.34,1.49,1.25
times of wild type Jinhui 10. And the width were 0.68 0.67 0.58 of those of Jinhui 10,
respectively.The height of the mutant was 1.13 times of the wild type. Furthermore,
some important agronomic traits such as booting, effective panicles per plant were
significantly higher than those of Jinhui 10. While the number of grains per plant was
a little less than that of Jinhui 10. Genetic analysis indicated that the mutant traits was
controlled by a single recessive gene. The gene NAL9(t) was finally mappeded on the
chromosome 3 between the Indel markers N.Swu 3 and N.Swu 4, and the physical
distance was 82 kb. These results will lay a good foundation for molecular cloning
and functional analysis of the NAL9(t).
2012
$
$ 91
Development$of$chromosome$segment$substitution$lines$in$
Brassica$napus$L.$using$maker$assisted$selection$ $
Bao Zhang, Baohua Hong, Gang Liu, Jiangsheng Wu, Kede Liu
Chromosome segment substitution lines (CSSLs), with single or multiple
particular chromosome segments from the donor parent in the genetic background of
recurrent parent, are useful resources for genetic studies, such as detection and fine
mapping of QTLs for traits of agronomic importance in crops. To enhance the
detection of QTLs by genetic studies and application of marker-assisted breeding in
Brassica napus, we developed a set of CSSLs using Zhongyou821 (ZY821), an elite
cultivar, as recurrent parent and No2127, a resynthesised rapeseed line, as donors.
From a population of double haploid lines (DHs) derived from the cross of ZY821
and NO2127, 88 DHs were genotyped with more than 1000 SSR markers. A total 300
SSRs corresponding to 314 loci were used for MAS from BC2F1 to BC5F2. Finally
90 CSSLs with 97 target segments were obtained from BC5F2 individuals. Each
CSSL contains 1-6 substitution chromosome segments from NO2127, with average of
2.34 segments per CSSL. These segments overlapped each other and covered the
whole genome of Brassica napus except the end of A8, C02 and C07 spanning a total
41.0 cM. Significant difference of several important traits such as flowering time,
seed size and seed color were observed in these CSSLs.
92$
!
A$Cloning$and$Functional$Analysis$of$Dynamic$Narrow$Leaf$
DNL1
Gene$in$Rice$ $
Guo Min, Yang Hailing, Xu Rugen, Yan Changjie
gricultural College, Yangzhou University; Key Lab of Crop genetics and
physiology in Jiangsu
Province; Key Lab of Plant Functional Genomics, MOE
Rice leaf morphology is one of important components in determining the plant
architecture, whereby photosynthesis occurs. And rice leaf morphology is considered
to indirectly affect the yield as well as grain quality. However, up to date, the
genetical mechanism underlying the leaf development remains obscure. In present
study, we obtained a dynamic narrow leaf mutant, named dynamic narrow leaf 1
(dnl1), derived from the indica cultivar 93-11, by radiating with 60Co-γ ray.
Compared with wild type (WT) 93-11, the widths of leaves of dnl1 are similar to
those of WT after seeding. However, some significantly narrow leaves can be seen
when the tillers appeared in dnl1, and the last one or two leaves become normal again.
The transverse section of blade analysis shows that, the number of vascular bundles of
narrow leaves in dnl1 significantly decrease when compared with 93-11, on the
contrast, the normal leaves of dnl1 have the same number of bundles as 93-11 in same
stage, which indicates the reduction of the number of vascular bundles may be the
reason causing the narrow leaf. In addition, we find the number of root hair decreases
significantly in dnl1 compared to that of WT, but the lateral root number is similar in
both WT and dnl1. Genetic analysis suggests that the phenotype of dynamic narrow
leaf mutant is controlled by a recessive gene. We cloned the DNL1 gene through
map-based cloning strategy, which codes an Aux/IAA protein. In dnl1, three single
bases and one single base substitution are found in introns and exon, respectively; and
a base substitution and two bases insertion in promoter region are also observed.
qRT-PCR result indicated that DNL1 expressed highly in leaves. In addition, we
found when the average dairy temperature is higher than 25 , the new growth leaves
of dnl1 turns narrow; in contrast, when it is lower than 25 , the leaves will be normal,
suggesting the expression of dnl1 is mediated by temperature. This result provides an
important clue to understand the mechanism of rice leaf development.
2012
$ 93
A$Genome$Wide$Association$Study$Revealed$the$Genetic$
Architecture$and$Important$Genes$for$Regulating$Kernel$Row$
Number$in$Maize$Female$Inflorescence$ $
Lei Liu1#, Xiaohong Yang2#, Xiaoliang Cao3, Feng Li1, Haitao Jia1, Lihong Zhai1, Yongsheng
Tao3, Juan Huang1, Bing Yue1, Yanfang Du1, Yonglian Zheng1, Zuxin Zhang1*, Jianbing Yan1*
"1 National Key Laboratory of Crop Genetic and Improvement, Huazhong Agricultural
University, , Wuhan 430070, China
2 National Maize Improvement Center of China, Beijing Key Laboratory of Crop Genetic
Improvement, China Agricultural University, Beijing 100193, China
3 College of Agronomy, Hebei Agricultural University, Baoding 71001, China" Unveiling
the genetic basis of grain yield and yield-related traits is useful for guiding the
genetic improvement of crop plants. Kernel row number (KRN) per ear is an
important yield component, which directly affects grain yield of maize. Here, we
identified 31 single nucleotide polymorphisms (SNPs) representing 17 quantitative
trait loci (QTLs) that are significantly associated with KRN of maize, using a
genome-wide association study. A linkage disequilibrium (LD) block on chromosome
4 was placed in a previously identified consensus QTL interval, and was also
identified by linkage mapping using an F2:3 population. From the LD region on
chromosome 4, two SBP-box protein genes, zmKRN4-1 and zmKRN4-2, were cloned
and re-sequenced. Candidate gene association analysis revealed four allelic variation
sites in zmKRN4-1 that were significantly associated with KRN, with one haplotype
markedly increasing KRN. Expression analysis revealed that zmKRN4-1 is highly
expressed in reproductive organs, and two alleles from the parents were co-expressed
in heterozygotes, while zmKRN4-2 was constitutively expressed. Nucleotide diversity
revealed that the SBP-box encoding region and microRNA156 binding region of
zmKRN4-1 were strongly selected during domestication of maize. Synteny of genes
within the LD region was also detected in grass. The results suggest that a set of loci
with moderate effect sizes and fewer loci with small effect sizes are responsible for
genetic control of KRN in maize. Natural variation of zmKRN4-1 plays an important
role in regulating maize KRN by modulating the initiation and maintenance of SPMs
and SMs in female inflorescence of maize.
94$
!
2012
$ 95
E
$
$
96$
!
GenomeEwide$identification$and$analysis$of$microRNA$
responding$to$longEterm$waterlogging$in$crown$roots$of$maize$
seedlings$$
Lihong Zhai, Zhijie Liu, Xiling Zou, Yuanyuan Jiang, Fazhan Qiu, Yonglian Zheng, Zuxin Zhang
430070, China
MicroRNAs (miRNAs) are critical post-transcriptional modulators of gene expression
involving in plant responses to abiotic stress. However, the regulation of miRNA in
the morphological response to waterlogging is poorly understood in maize. In this
study, we detected miRNAs and their targets that expressed in waterlogged crown
roots of maize seedlings in two inbred lines (Hz32 and Mo17) by RNA sequencing. A
total of 61 mature miRNAs were found including 36 known maize (zma) miRNAs
and 25 potential novel miRNA candidates. Comparison of miRNA expression in both
waterlogged and control crown roots revealed 32 waterlogging-responsive miRNAs,
most were consistently downregulated under waterlogging in the two inbred lines. We
identified the miRNA targets through degradome sequencing. Many known miRNA
targets involving in transcription regulation and reactive oxygen species elimination
were found in the degradome libraries,
2012
$ 97
A$Ubiquitin$Ligase$of$Symbiosis$Receptor$Kinase$Involved$in$
Nodule$Organogenesis$
Songli Yuana, Hui Zhua, Honglan Goua, Weiwei Fua, Lijing Liub, Tao Chena, Danxia Kea, Qi
Xieb, Zonglie Hongc and Zhongming Zhang*
The symbiosis receptor kinase, SymRK, is required for morphological changes of
legume root hairs triggered by rhizobial infection. How protein turnover of SymRK is
regulated and how the nodulation factor signals are transduced downstream of
SymRK are not known. In this report, a SymRK-interacting E3 ubiquitin ligase, SIE3,
was shown to bind and ubiquitinate SymRK. The SIE3 - SymRK interaction and the
ubiquitination of SymRK were shown to occur in vitro and in planta. SIE3 represents
a new class of plant-specific E3 ligases that contain a unique pattern of the conserved
CTLH, CRA, and RING domains. Expression of SIE3 was detected in all tested
tissues of Lotus japonicus plants, and its transcript level in roots was enhanced by
rhizobial infection. The SIE3 protein was localized to multiple subcellular locations
including the nuclei and plasma membrane, where the SIE3 - SymRK interaction took
place. Overexpression of SIE3 promoted nodulation in transgenic hairy roots, whereas
down-regulation of SIE3 transcripts by RNA interference (RNAi) inhibited infection
thread development and nodule organogenesis. These results suggest that SIE3
represents a new class of E3 ubiquitin ligase, acts as a regulator of SymRK and is
involved in rhizobial infection and nodulation in L. japonicus.
98$
!
$
208
$ $
2012
$ 99
ChinaExpansin,$especially$the$domain1$plays$a$major$role$in$
the$cotton$fiber$development$
Yang Li, Lili Tu, Fenglin Deng, Jafu Tan, Xianlong Zhang*
430070, China
Cell wall plays an important role in plant growth and development, it is not only to
provide strength and protection to plant cells, and also involve in morphogenesis.
Cotton fiber from the ovule outer epidermal cells which undergoes four stages:
initiation, elongation, secondary wall thickening and maturation is a single cell
trichome. Therefore, it is an ideal model to study cell elongation, cell wall synthesis.
GbEXPA1 and GbEXPATR that cloned from a normalized fiber cDNA library of G.
barbadense 3-79 were specific expressed in fiber. Protein structure prediction showed
that GbEXPATR lacked expansin domain2. GbEXPA1, GbEXPATR protein were
detected in cell wall and cytoplasm. The GbEXPA1 and GbEXPATR promoter,
differing in the lengths of the5’ flanking regions (pGbEXPA1, 839-bp; pGbEXPATR,
1405-bp), were predominantly expressed during fiber development.GbEXPA1 RNAi
transgenic plants which had a lower expansin rate in the stage of elongation showed a
short mature fiber (5-10% reduced to control). Over-expression GbEXPA1 plants had
a greater extension in the phase of elongation, but the mature fiber were less affected.
Because some genes related secondary wall synthesis (GhCTL1 GhCELA1)
expressed higher than control in the stage of secondary wall thickening, finally it
accelerated elongation termination. GbEXPATR, specifically in 3-79, from elongation
to maturation over-expression plants had a longer fiber (5-8% increased to
control).Therefore, in cotton fiber development expansion changed extension rate to
affect the length of fiber. Especially, GbEXPATR, only domain1, had a major
influence in elongation.
100$
!
High$Resolution$Melting$Curve$Analysis:$An$Efficient$Method$
for$Fingerprinting$of$Hybrid$Rice$Cultivars$and$Their$Parental$
Lines$ $
Yanfang Zhu, Liwei Zhu, Jin Hu*, Yixin Tian, Tingting Wen, Shuijin Zhu and Yajing Guan
Seed Science Center, College of Agriculture and Biotechnology, Zhejiang University,
Hangzhou,
People’s Republic of China Rice (Oryza sativa L.) is one of the most important
crops in the world. It is essential to develop an efficient method to build the
fingerprinting database of commercial hybrid rice cultivars and their parental lines in
seed market for rapid and unambiguous cultivar identification and seed purity analysis
as new similar cultivars increasing yearly. The objectives of this investigation were to
fingerprint three commercial hybrid rice cultivars and their parental lines using SSR
molecular markers by agarose gel electrophoresis, capillary electrophoresis, and high
resolution melting curve analysis, and to develop an efficient fingerprinting method
for rice cultivar identification and seed purity analysis in agriculture production. Nine
SSR primer pairs which produced stable, distinct and polymorphic amplicons as
visualized on agarose gel were selected for capillary electrophoresis and high
resolution melting curve analysis to compare their fingerprinting efficiency. Six
(RM206, RM209, RM211, RM212, RM219, and RM297), four (RM72, RM209,
RM212, and RM276) and six (RM190, RM206, RM211, RM212, RM219, and
RM297) were successfully used to fingerprint the three hybrid rice cultivars,
YLiangYou689, ZhuLiangYou06, and QianYou1, respectively. The result suggested
that capillary electrophoresis and high resolution melting curve analysis could be used
as potential, efficient and valuable methods for fingerprinting hybrid rice cultivars,
and high resolution melting curve analysis should be given priority compared with
capillary electrophoresis for its high accuracy and high efficiency.
2012
tan jiafu
$ 101
[email protected]
[email protected]
15972986261
[email protected]
13096363851
[email protected]
13554339312
m
[email protected]
15527835128
m
[email protected]
18618252196
m
[email protected]
.edu.cn
[email protected]
15928538737
[email protected].
13277919621
edu.cn
[email protected]
13720378249
zau.edu.cn
[email protected]
18234487354
[email protected]
13963620039
[email protected]
13036153583
[email protected]
13618046477
[email protected]
[email protected]
15387027516
[email protected]
[email protected]
13996380997
m
[email protected]
2787283955
[email protected]
13163277618
om.cn
[email protected]
18639771546
102$
!
[email protected]
13407127017
m
jiangjiefeng@webmai
13545385869
l.hzau.edu.cn
[email protected]
u.edu.cn
lengbofeng@foxmail.
13037122020
com
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miRNA芯片筛选干旱胁迫下的差异表达miRNA。
结果分析：测序发现胡杨中324条保守miRNA和72条特异miRNA，并利用
降解组测序鉴定出47条miRNA靶基因，这些基因与转录调控和胁迫应答等
生理过程相关；通过定制miRNA芯片（含有已报道植物miRNA和测序发现
miRNA）筛选出131条干旱胁迫应答miRNA。
作者评论：将二代测序和微阵列芯片结合有效减少了实验工作和生物信息
学分析工作，加速了其研究项目进程。
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Analysis of conserved microRNAs in floral tissues of sexual and apomictic Boechera
species.
Amiteye S, Corral JM, Vogel H, Sharbel TF. (2011) BMC Genomics 12(1), 500.
研究背景与目的：将单性生殖引入作物育种，有利于固定并传播作物的优
良性状。作者通过研究筷子芥（Boechera）miRNA从有性生殖到单性生殖
转换中的表达谱和调控的靶基因，以期揭示miRNA在此转换过程中的重要
调控功能。
实验方法：预测筷子芥miRNA→定制miRNA芯片鉴定预测的miRNA→靶
基因预测与功能分析。
结果分析：首次全面地分析了筷子芥有性生殖和单性生殖物种中的miRNA
表达谱，鉴定出51个在被子植物中保守的miRNA family和6个miRNA调控
转录因子，其中miR156/157调控的SPL11在单性生殖样本中出现显著上
调。
更多研究案例，请访问www.lc-bio.com或咨询800-857-1452。
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Brassica$napus$L. - 植物科学技术学院

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