THE RESEARCH OF WILD CRANBERRY (Vaccinium oxycoccos L

VYTAUTAS MAGNUS UNIVERSITY
Judita Žukauskien÷
THE RESEARCH OF WILD CRANBERRY
(Vaccinium oxycoccos L.) DNA VARIABILITY AND
PHENOLIC COMPOUNDS IN BERRIES
Summary of Doctoral Disertation
Physical Science, Biochemisry (04 P)
Kaunas, 2009
The doctoral disertation was caried out at Vytautas Magnus University in 2005-2009.
Scientific Supervisor:
Assoc. Prof. dr. Algimantas Paulauskas (Vytautas Magnus University, Physical
Science, Biochemistry – 04 P)
Consultant:
Dr. (HP) Remigijus Daubaras (Vytautas Magnus University, Biomedical science,
Biology – 01 B)
The Committee of the doctoral dissertation:
Chairman:
Dr. Rolandas Meškys (Institute of Biochemistry, Physical Science, Biochemistry –
04 P)
Members:
Dr. Prof. dr. Laima Ivanovien÷ (Kaunas medicine university, Physical Science,
Biochemistry – 04 P)
Prof. hab dr. Eugenija Kupčinskien÷ (Vytautas Magnus University, Biomedical
science, Biology – 01 B)
Dr. Juozas Labokas (Institute of Botany of Vilnius University, Biomedical science,
Biology – 01 B)
Dr. Zita Naučien÷ (Vytautas Magnus University, Physical Science, Biochemistry –
04 P)
Opponents:
Dr. Jolita Radušien÷ (Institute of Botany of Vilnius University, Physical Science,
Biochemistry – 04 P)
Dr. Rytis Rugienius (Lithuania Institute of Horticulture, Biomedical science, Biology –
01 B)
The Doctoral Disertation will be beffended on the 9th of December 2009 at 3p.m. in a
public meeting of the Doctoral committee at Vytautas Magnus University, Faculty of
Nature Science, no 101
Adress: Vileikos st. 8, LT-44404, Kaunas, Lithuania
The summery of Doctoral Dissertation was distributed on the 9 of November 2009.
The Disertation can be rewiewed in M. Mažvydas National Librery of Lithuania and at
the Libraries of Vytautas Magnus University and institute of Biochemistry.
VYTAUTO DIDŽIOJO UNIVERSITETAS
Judita Žukauskien÷
PAPRASTOSIOS SPANGUOLöS (Vaccinium oxycoccos L.)
DNR ĮVAIROVöS IR FENOLINIŲ
JUNGINIŲ UOGOSE TYRIMAI
Daktaro Disertacijos Santrauka
Fiziniai mokslai, biochemija (04 P)
Kaunas, 2009
Disertacija rengta 2005 – 2009 m. Vytauto Didžiojo universitete
Mokslinis vadovas:
prof. hab. Dr.Algimantas Paulauskas (Vytauto Didžiojo universitetas, fiziniai mokslai,
biochemija – 04 P)
Konsultantas:
Hab. Dr. R. Daubaras (Vytauto Didžiojo universitetas, biomedicinos mokslai, biologija
– 01B)
Disertacija ginama Vytauto Didžiojo universitete, biochemijos mokslo kryptie
taryboje:
Primininkas:
Dr. Rolandas Meškys (Biotechnologijos institutas, Fiziniai mokslia, Biochemija –
04 P)
Nariai:
Dr. Prof. dr. Laima Ivanovien÷ (Kauno medicinos unoversitetas, Fiziniai mokslia,
Biochemija – 04P)
Prof. hab dr. Eugenija Kupčinskien÷ (Vytauto Didžiojo universitetas, Biomedicinos
mokslai, Biologija – 01 B)
Dr. Juozas Labokas (Vilniaus universiteto botanikos institutas, Biomedicinos mokslai,
Biologija – 01B)
Dr. Zita Naučien÷ (Vytauto Didžioj universitetas, Fiziniai mokslia, Biochemija – 04P)
Oponentai:
Dr. Jolita Radušien÷ (Vilniaus universiteto botanikos institutas, Fiziniai mokslia,
Biochemija – 04P)
Dr. Rytis Rugienius (Lietuvos sodininkyst÷s ir daržininkyst÷s institutas, Biomedicinos
mokslai, Biologija – 01B)
Disertacija bus ginama viešame Biochemijos mokslo krypties tarybos pos÷dyje 2009 m.
gruodžio 10 d. 15 val. Vytauto Didžiojo universiteto II rūmuose, 101 auditorijoje.
Adresas: Vileikos g. 8, LT-44404, Kaunas, Lietuva.
Disertacijos santrauka išsiuntin÷ta 2009 m. lapkričio 9d.
Disertacija galima peržiūr÷ti Lietuvos nacionalin÷je M. Mažvydo bibliotekoje,
Biochemijos instituto ir Vytauto Didžiojo universiteto bibliotekose.
INTRODUCTION
Genetic variability of Lithuanian wild berry plants is determined by weather
conditions. In Lithuanian forests among the most common edible berry plant is
mentioned blueberry (Vaccinium mytrillus L.), cowberry (Vaccinium vitis–idaea L.),
raspberry (Rubus idaeus L.) and strawberry (Fragaria vesca L.). In wetlands quite
abundant wild cranberry (Vaccinium oxycoccos L.) much less – small cranberry
(Vaccinium microcarpum L.).
Wild cranberry is appreciable because of medical characteristic of berries that exhibit a
high antioxidant activity (Wang et al., 1996). The largest cranberry biological effect is
associated with high concentrations of phenolic compounds (Mann, 2003; Huang et al.,
2003). In 1998 in Lithuania began to take an interest about cranberry biochemical
consumption (Povilaityt÷ ir kt., 1998). In comparison of some chemical compounds and
consumption it was found that wild and American cranberries is quite similar. However,
biochemical works with a wild cranberry done slightly. It is established that the
antioxidant activity of the fruit and their products is linked to in qualitative and
quantitative composition of polyphenols which is different in terms of geographical
regions and climatic features (Kahkonen ey al., 2001). Therefore Lithuania cranberry
must be carried out biochemical tests and compare with other cranberries growing in
other climates as thoroughly as possible.
As flavonoids are important chemical compounds in biological systems, there are
performed genetic flavonoids studies in the world. Biosynthesis of flavonoids is linked
to genes that have been isolated from many plant species and has been widely studied
on the molecular level (Hahlbrock and Scheel, 1989; Martin et al., 1991; Holton and
Comish, 1995; Boss et al., 1996; Winkel-Shirley, 2001). But such kind of studies is not
carry out in Lithuania yet.
Vytautas Magnus university Kaunas botanical garden takes part in the National
Biodiversity Conservation Strategy and Action Program which have been prepared and
adopted in 1996. This program provides to preserve the country's main ecosystems,
species and populations and lay the foundations for their sustainable use and
management for future generations. In this convention is mentioned and Čepkeliai,
Kamanos, Viešvil÷, Žuvintas and Girutiškio reserves, where is solving one major
problem - conservation of biological diversity. It was observed that a wild cranberry has
a lot of morphological differences, that’s why has been carried out cranberry
morphological characteristics evaluation (Daubaras and Česonien÷, 2004). However, it
is not clear whether these differences are genetic, or collected forms significantly differ
from forms in natural populations. Therefore it is important and necessary not only
morphological, but also the genetic researches of wild cranberry in these reserves.
Because of economic value of cranberry genetic diversity has long been interested
in breeders. It is important to select natural forms with big productivity, resistance to
adverse environmental factors (diseases), good fruit taste and size (Vorsa, 1994). In the
first stage of cranberry natural forms evaluation focuses on the morphological
characteristics (berry size and shape, color, presence of cuticle wax, leaf shape) (Novy
and Vorsa, 1995). Because of complicated morphological descriptors separating
cranberry clones, upspring of classification errors. Molecular markers allow direct
assessment of genetic diversity in order objectively determine the genetic material
differences (Patamsyt÷ et al., 2008).
Another one aspect is a concern about invasive species of American cranberry
(Vaccinium macrocarpon Aiton.). Because this invasive species increasingly spreading
5
throughout the berry growers in Lithuania increase probability that a wild cranberry
growing in Lithuania ecological niches will be replaced as species by American
cranberry. According to this, we have to investigate naturally growing wild cranberry
genetic resources in Lithuania and neighboring countries.
Following the above-mentioned purposes is necessary not only morphological and
genetic studies of wild cranberry, but also and biochemical. Summarizing the results of
Lithuanian researchers it can be said that in Lithuania, compared to other countries,
cranberry berry biochemical studies are slightly carried out. There were performed the
morphological and biochemical comparisons, but genetic and biochemical data
comparisons were not done. Such kind of comparisons in other countries was not
detected also. Chemical composition of the wild and American cranberry is different as
already mentioned above. However, it is unclear whether these differences are
determined by the growth conditions of phenotypic or genotype. It arose and purpose of
this work.
The aim of study – to investigate wild cranberry V. Oxycoccos DNA variability and
flavonol compounds in berries. Also to establish internecine relations between
morphologic, genetic and biochemic composition.
Main task of the study
• Using morphophysiological, RAPD and SSR methods evaluate genetic
variability of Lithuanian, Estonian, Swedish and Norwegian wild cranberry
populations.
• To evaluate connections of morphologic and genetic features of wild cranberry.
• To evaluate biochemical consumption and genotype connections of wild
cranberry
The scientific novelty of the work. Although, morphologic investigation methods and
some biochemic features of wild cranberry (Vaccinium oxycoccos) were done in
Lithuania. The genetic variability of cranberries weren’t examined in Lithuania. Genetic
analysis of wild cranberry clones from Vytautas Magnus University Kaunas botanical
garden collection were done. Using random amplified DNA and microsatelite methods
were evaluated genetic variability of wild cranberry. Were compared genetic variability
of collection clones and natural populations.
For the first time in Lithuania and in the world were done comparison of
morphologic and biochemical, morphologic and genetic, and genetic and biochemical
internecine relations using statistical methods. There were obtained 3 RAPD and 7 SSR
fragments specific to clones genotype.
Approval of the research work. The main research findings were published in three
international periodical reviewed journal included in the Master Journal List of Institute
of Scientific Information and three publications - in peer reviewed journals. Study
results were presented at nine international and Lithuanian conferences.
Volume of the work. The dissertation is written in Lithuanian and consists of the
Introduction, Material and Methods, Results, Discussions, List of author’s publications
and List of references. The dissertation is comprised of 111 pages. Data of the research
study are presented in the 35 figures and 22 tables.
6
MATERIAL AND METHODS
Sampling of wild cranberries. Fifty-six clones with clearly differing morphologic
features (color, size, shape of berry and productivity) (Daubaras and Česonien÷, 2004)
were collected from three populations in Lithuania bogs (Čepkeliai, Žuvintas, Kamanos
and Aukštaitija) during 1995–1999. The cuttings of collected clones were planted in the
Kaunas Botanical Garden collection into special peat (pH 4.0-5.0) beds. These clones
were selected for further evaluation ex situ The definite geographical positions of their
habitats were established by the GPS Magelan 315 receiver (GARMIN Corporation,
USA). Morphologic wild cranberry clones characterization and evaluation were done
according to descriptor for cranberries (Plekhanova ir Zamorskaja 1993; Budriūnien÷,
1997, 1997a).
For genomic DNA comparison of collection population there were done analysis
and with groups of natural populations. Samples were collected during 2006 – 2008m.
(samples in Sweden were sampled by - R. Daubaras, L. Česonien÷, L. Kardell; in
Norway – A. Paulauskas, O. Rosef; Estijoje – A. Paulauskas ir J. Radzijevskaja, in
Lithuania - R. Daubaras, L. Česonien÷, L, M. Jodinskien÷ ir J. Žukauskien÷). Avoiding
of cluster immixture in natural populations, samples were picked not less than 5 m. from
each other.
For DNA extraction were used 151 shoots of wild cranberry from different
places. In Lithuania cranberries (109 samples) were collected from 4 Lithuania reserves
(Čepkeliai, Žuvintas, Viešvil÷ and Kamanos) and Aukštaitija national park (Table 1).
For Lithuanian populations comparison there were analyzed and 3 wild cranberry
populations (42 samples) from other countries: Estonia, Sweden and Norway (Table
2.1).
Table 1. Locations of wild cranberry samples, quantity of samples, coordinates of
locations. In brackets first number – quantity of clones from collection samples, second
number – quantity of samples from natural population
Eil.
Locality, state
Coordinate
Nr.
North
East
Quantity of
latitude longitude samples per
population
1.
Čepkeliai, (Lithuania)
54o00’
24o25’
34 (13+21)
o
o
2.
Žuvintas, (Lithuania)
54 23’
23 25’
40 (18+22)
o
o
3.
Kamanos, (Lithuania)
56 15’
21 35’
17
4.
Viešvil÷, (Lithuania)
22o55’
10
55o00’
5.
6.
7.
8.
9.
Aukštaitija nationals
park, (Lithuania)
Sooma nationals park,
(Estonia)
Hinibu, (Norway)
Northmayer, (Sweden)
Taglamyren (Sweden)
Total:
55o40’
25o80’
8
58˚26’
25˚06’
10
58˚34’
57˚01’
56º43’
08˚27’
014˚22’
014º25’
9
11
13
151
7
Morphologic research. Eighteen morphologic properties of shoots, leaves and berries
for the morphologic characterization of the clones were used along the descriptor list for
cranberries (Budriūnien÷, 1997). An average weight of a berry was calculated by
assessing the average weight of 50 berries with three replications. Berries were
characterized depending on the size: very small (<0.3g); small (0.3-0.5g); medium (0.61.0g); large (1.1-1.5g); very large (>1.5g). Uprights (flowering shoots) length was
measured at the end of vegetation. The main phenological phases of the investigated
clones were recorded also. The least significant difference values (LSD) corroborated
differences between the investigated clones. During four years (2001, 2002, 2003, 2004)
clones were observed and evaluated using such morphologic parameters as: vegetation
parameters, beginning of blossoming, beginning of berry ripening, end of berry
ripening. The length of generative uprights was also measured, capacity of the berries,
leaf length, leaf width, berry weight. These morphological data were used for statistical
analysis.
Table 2. Primers and their sequences used for RAPD analysis
Primer
Sequence of
oligonucleotide primers
(5‘→3’)
Opa-01
5’-CAGGCCCTTC-3’
Opa-04
5’-AATCGGGCTG-3’
Opa-05
5’-AGGGGTCTTG-3’
Opb-11
5’-GTAGACCCGT-3’
Opa-10
5’-GTGATCGCAG-3’
Opa-09
5’-GGGTAACGCC-3’
Roth-06
5’-GCACGCCGGA-3’
Roth-08
5’-CGCCCTCAGC-3’
ROT-09
5’-GCACGGTGGG-3’
DNA analysis using RAPD method. DNA extraction and RAPD analysis were done
exactly as previously described (Areškevičiūt÷, 2006). Nine 10 nt long primers of
random sequence (Fermentas, Lietuva; Roth, Germany) were used (Table 2). DNA
amplification was performed in a thermocycler (Mastercycler, Eppendorf, Germany)
under the following conditions: initial denaturation for 4 min at 94 °C, 44 cycles of
denaturation for 1 min at 94 °C, primers annealing for 1 min at 35 °C, extension for 2
min at 72 °C followed by a final extension for 5 min at 72 °C.
DNA analysis using SSR method. DNA extraction were done as previously described
(Areškevičiūt÷, 2006). Nine primers (Table 3) 20 – 23 nt long primers (Biomers,
Germany) were used. DNA amplification reaction was performed by modifications of S
Boches et all., (2005) recommendations. Reaction mix made of: 12,5 µl 2xPCR Master
Mix (0,05 U/µl Taq DNA polymerase; 4mM MgCl2; 0,4mM each dNTP), 0,5µl each
primer (concentration pmol/µl ), 7,5µl twice distilled water and 4µl (5ng) DNA. PCR
reaction was performed in a thermocycler (Mastercycler, Eppendorf, Germany)
according to Boches et all., (2005). PCR product were obtained by ABI 3130 xl Genetic
analyzer (Applied Biosystems), length of fragments were detected using standard of
ROX – 500. Fragments were set using program GeneMapper v.3.5. (Applied
Biosystems).
8
Table 3. Primers used for microsatelites analysis
Primer
Sequence of oligonucleotide primers
(5‘→3’)
F:TCCACCCACTTCACAGTTCA
Ca112f
R:GTTTATTGGGAGGGAATTGGAAAC
F:TAGTGGAGGGTTTTGCTTGG
Ca169f
R:GTTTATCGAAGCGAAGGTCAAAGA
F:TCAAATTCAAAGCTCAAAATCAA
Ca421f
R:GTTTAAGGATGATCCCGAAGCTCT
F:GTCTTCCTCAGGTTCGGTTG
Ca483f
R:GAACGGCTCCGAAGACAG
F:CGGTTGTCCCACTTCATCTT
CA794F
R:GTTTGAATTTGGCTTCGGATTC
F:CAATCCATTCCAAGCATGTG
Na800
R:GTTTCCCTAGACCAGTGCCACTTA
F:GCAACTCCCAGACTTTCTCC
Na1040
R:GTTTAGTCAGCAGGGTGCACAA
F:GCGAAGAACTTCCGTCAAAA
Vcc_j9
R:GTGAGGGCACAAAGCTCTC
F:ATTTGGTGTGAAACCCCTGA
Vcc_s10
R:GTTTGCGGCTATATCCGTGTTTGT
Chemical composition. The amount of total phenolics in the cranberry extracts was
determined with the Folin-Ciocalteu reagent according to the method of Slinkard and
Singleton (Slinkard and Singleton, 1977) using gallic acid as a standard. Samples (1.0
ml, two replicates) were introduced into test cuvettes, and then 5.0 ml of FolinCiocalteu’s reagent and 4.0 ml of Na2CO3 (7.5%) were added. The absorbtion of all
samples was measured at 765 nm using the Genesys-10 UV-VIS spectrophotometer
(Thermo Spectronic, Rochester, USA) after incubating at 20°C for 1.0 h. Results were
expressed as milligrams of gallic acid equivalent (GAE) per 100 gram of fresh weight.
Total anthocyanin assay. The pigments were extracted from 5g of fresh cranberry with
95% (v/v) food grade ethanol acidified with 0.1 M HCl with a purpose to assay the total
amount of anthocyanins (Rubinskiene et al., 2005). The berries were ground with
quartz sand and the extraction was continued with 20 ml portions of solvent until the
sample became colorless. The extract was diluted with acidified ethanol until 100 mL
pH of the extracts was 1.2. The absorption was measured on a spectrophotometer
Genesys-10 UV-VIS (Thermo Spectronic, Rochester, USA) at 535 nm. The amount of
anthocyanins was expressed as prevailing cyaniding-3-galactoside and calculated in
mg/100g.
HPLC analysis of anthocianins. The extracts were analyzed by HPLC using a reversed
phase C18 LiChrospher R_100 RP 18e column (125 × 4 mm, 5 µm) (Merck, Darmstadt,
Germany). The eluents were (A) 4% H3PO4 in water, and (B) 100% HPLC-grade
acetonitrile (Merck) (Rubinskiene and others 2005). Gradien: 0 min - 10% B, 4 min –
14% B, 10 min - 16% B, 25 min - 30% B, 26 min - 10% B. Flow rate was 0.8 mL/min,
20 µL was injected. The samples were filtered through a 0.45-µm cellulose syringe filter
before analysis. Detection was performed using a UV detection system Agilent 1200
(Agilent Technologies, JAV), at 520 nm.
9
Radical scaverin test. Radical scavenging activity (RSA) against stable 2,2-diphenyl-1picrylhydrazyl hydrate (DPPH∗) was determined spectrophotometrically (Blois 1958).
When DPPH∗ (2 mL) reacts with an antioxidant compound (50 µL), which can donate
hydrogen, it is reduced. The changes in color (from deep-violet to light-yellow) were
measured at 515 nm after 20 min. The RSA was calculated by the following formula:
RSA = [( Ab − Aa ) / Ab ]× 100%
where AB is the absorption of blank sample (t =0min) and AS is the absorption of tested
sample.
Statistical methods. Relationships among V.oxycoccos individuals were evaluated
using a dendrogram based on Nei and Li’s (1979) genetic distances. It was generated by
the UPGMA (unweighted pair group method) cluster analysis method. Calculation of
genetic distances and UPGMA cluster analyses were performed with the TREECON
program for Windows V 1.3b (Nei and Li, 1979). PC analysis was performed with the
GenAlEx 6 program (Peakall and Smouse, 2006). Calculation of the observed number
of alleles, Nei’s (Peer et all., 1994; Nei and Nalt, 1973) gene diversity (H), Shannon’s
Information Index (I), total gene diversity (Ht), gene diversity within populations (Hs),
gene diversity among populations (Gst = (Ht–Hs) / Ht), gene flow (Nm = 0.5 (1–Gst) /
Gst) and generation of a Nei’s genetic distance based dendrogram were carried out with
POPGENE V 1.31 software (Yeh and Yang 1999).Stepwise multiple correlations (twotailed significance level) were used to determine which combinations of genetic
variables were associated with morphologic traits (SPSS 13.0 for Windows). The test
for the significance of each RAPD product band was done using the Pearson Chi-Square
(Kish, 1987). The results were statistically analyzed and the significance of differences
was calculated using ANOVA for Excel vers.3.1. The correlation (r) and variation (V)
coefficients were calculated using STAT for Excel vers.1.5 (Tarakanovas and
Raudonius, 2003).
RESULTS AND DISCUSSION
Investigation of wild cranberries. For Lithuanian wild cranberries analysis was chosen
3 different populations groups: Vytautas Magnus University Kaunas Botanical garden
(VMU KBG) clones populations (56 clones from Čepkeliai, Žuvintas, Kamanos
reserves and Aukštatija national park), Lithuanian natural populations (53 samples from
Žuvintas, Čepkeliai and Viešvil÷ rezerves) and not Lithuanian natural populations (42
samples from Estonia, Sweden and Norvay).
Firs of all using different measurements and carry out observations were
examined morphologic variability of VMU KBG collection clones.
The second stage of work was evaluation of morphologic variability. Genetic
variability were evaluated for all three wild cranberry groups. Genetic variability were
checked using two method: random amplified polymorphic DNA (RAPD) and simple
sequence repeats (SSR)
Third stage of work was to carry out flavonoids consumption and RSA analysis
with VMU KBG clones. This analysis was done using chromatographic and
spectrophotometric methods.
10
Last stage of the work was correlations search of all three work stages. Looking
for correlations between biochemical and genetic, biochemical and morphologic and
genetic and morphologic features.
Morpfhologic research of wild cranberries (V. oxycoccos). The determination of
berry shape of European cranberry revealed high variability. The most common were
clones with round or oblate berries (Table 4). The average width and height of a berry
varied from 1.00 to 1.35 cm and from 0.99 to 1.41 cm, respectively. The color of berries
was red or dark red at full maturation. Only two clones (98-Č-19 and 98-Č-20) were
distinguished for pink while the berries of clone 99-Ž-10 for purple color. The berries of
clones 98-Č-06 and 99-Ž-09 were covered with a waxy coat.
Table 4. Morphological characteristics of berries of V. oxycoccoss
Clone
Length x width of a
Prevailing Color of a berry
berry, cm
shape of a
berry
99-Ž-03
1.11 x 1.12
round
dark red
99-Ž-04
1.19 x 1.21
round
dark red
99-Ž-07
1.38 x 1.29
cylindrical
red
99-Ž-09
1.02 x 1.04
round
dark red
99-Ž-10
1.00 x 1.28
oblate
purple
99-Ž-11
1.21 x 1.29
oblate
red
99-Ž-12
1.35 x 1.25
ovale
dark red
99-Ž-13
1.29 x 1.21
ovale
dark red
99-Ž-16
1.16 x 1.32
oblate
dark red
99-Ž-18
0.99 x 1.28
oblate
dark red
98-Č-01
1.17 x 1.22
oblate
red
98-Č-04
1.21 x 1.17
round
dark red
98-Č-06
1.17 x 1.24
round
dark red
1.16 x 1.17
cylindrical
dark red
98-Č-15
98-Č-15A
1.11 x 1.35
oblate
red
98-Č-17
1.15 x 1.00
ovale
dark red
98-Č-19
1.31 x 1.17
ovale
pink
98-Č-20
1.41 x 1.34
cylindrical
pink
Reliable differences in the average weight of a berry were ascertained since
Fisher’s criterion was F05(fact)=42.04>F05(theor)=1.7 (Figure 1). An average berry weight
of clones investigated was 0.94g (LSD05=0.052). It has been found that the berries of
twelve clones fall into the group of medium-sized (0.6-1.0g), the berries of five clones
were large (1.1-1.5g). Only the clone from Čepkeliai (98-Č-17) was distinguished by
small berries. The average berry weight of clones 99-Ž-03, 99-Ž-11, 99-Ž-13, 98-Č-01,
98-Č-06 and 98-Č-19 varied slightly, variation coefficient V<10%. The largest variation
of berry weight was detected in the clone 99-Ž-03 (V=19.3%).
The collection of cranberry clones during expeditions in situ was based upon the
selection of samples conspicuous in berry shape, coloration and size. The latest
evaluation of clones of European cranberry revealed high variability in berry shape and
weight ex situ as well. The berries of four clones 99-Ž-07, 99-Ž-11, 99-Ž-12, 99-Ž-16
from Žuvintas and one clone 98-Č-20 from Čepkeliai were distinguished for the largest
11
average weight of a berry (1.16g, 1,11g, 1.09g, 1.08 and 1.15g, respectively). The
berries of these clones could be equal to the some cultivars of American cranberry
(Budriūnien÷, 1998).
Figure 1. The average weight of a berry of O. palustris clones in 2003-2005 in grams,
LSD05=0.052
During four years (2001, 2002, 2003, 2004) clones were observed and evaluated
using such morphologic parameters as: beginning of vegetation and blossoming,
beginning of berry ripening, end of berry ripening, the length of generative uprights also
was measured, capacity of the berries, leaf length, leaf width, berry weight. These
morphological data were used for statistical analysis.
Biochemical research of wild cranberry (V. oxycoccos). The consumption of wild
cranberry anthocyanins was calibrated during earlier analysis. Using method of mass
spectrometry was confirmed pigments elution secession (Jasutien÷ et al., 2006). The
cxromatogtram of cranberries antocianyns extract is shown in Figure 2 and quantative
and qualitative consumption of anthocyanins cranberry in Table 5.
12
Absorbtion
n
ele
av
W
t,
gh
,
Time
min.
nm
Figure 2. Ethanols extract chromatograme of Kaunas botanical garden of Vytautas
Magnus university wild cranberry clones, registered in diapazone of visible wavelength
Table 5 Wild cranberry V. oxycoccos extract quantitative consumption of anthocyanins
in VMU Kaunas botanical garden collection Žuvintas and Aukštaitija populations
clones
Peonidin-3glucoside
Peonidin-3arabinoside
1,65±0,32
Not found
7,18±1,29
1,18±0,31
5,88±1,46
2,37±2,45
0,28±0,48
2,22±0,01
3,39±0,72
0,13±0,12
1,13±0,48
1,01±0,03
1,13±0,44
4,03±0,12
2,98±0,18
Peonidin-3galactoside
14,88±0,45
17,95±1,54
15,58±1,16
22,00±1,56
17,44±2,00
25,22±2,14
17,61±1,21
23,28±1,42
14,23±0,88
20,84±0,58
24,70±0,02
26,77±0,13
18,97±0,18
15,12±0,22
20,65±0,48
Cyanidin-3arabinoside
99-ž-04
99-ž-05
99-ž-08
99-ž-10
99-ž-11
99-ž-13
99-ž-15
99-ž-16
99-ž-17
99-ž-18
95-a-03
95-a-04
95-a-05
95-a-07
95-a-09
Cyanidin-3glucoside
Sample
Cyanidin-3galactoside
Quantity of anthocyan in percents (%.)
17,49±1,78
21,17±0,17
17,39±0,77
21,12±0,15
17,76±2,56
22,82±0,25
22,70±0,93
25,11±0,04
40,49±0,52
21,65±0,60
22,00±0,30
22,81±0,65
23,19±0,74
17,13±0,08
20,07±0,53
31,63±2,18
36,48±1,35
21,33±0,64
34,79±2,54
27,76±2,89
29,25±1,21
34,30±2,59
26,08±0,15
18,17±1,19
33,02±0,61
32,73±0,12
31,08±3,44
32,24±0,17
35,34±4,97
32,21±0,48
12,09±0,17
2,98±1,03
23,33±0,87
3,26±3,21
17,15±2,32
5,07±0,27
1,92±1,06
5,78±0,92
8,69±0,47
2,25±0,81
2,80±1,15
3,42±2,45
1,85±0,19
4,84±2,64
6,92±1,21
22,27±1,63
21,41±0,88
15,19±0,79
17,65±1,09
14,01±5,90
15,27±0,63
23,19±0,69
17,53±0,32
15,03±0,47
22,11±0,85
16,64±0,16
14,91±0,17
22,62±0,58
23,15±0,09
17,17±0,31
13
Cranberry and specific components within their berries are being associated with
human health attributes, such as maintenance of urinary tract health and antioxidant
status (Vorsa et al., 2002). The berries of European cranberry are one of the best sources
of phenolic compounds if compare with the other berry plants, such as strawberry, black
currant, raspberry etc. (Moyer et al., 2002). The results of this study corroborated
significant variability for total phenolics as well as for anthocyanins amounts. The
Kaunas botanical garden clones of European cranberry were compared according to the
total amount of phenolic compounds. The clones accumulated from 224 (clone 99-ž-04)
to 482 mg/100g (clone 95-a-07) of phenolic compounds The total amount of phenolics
in the clones from Aukštaitija on average attained 432 mg/100g, the same in clones
from Žuvintas 299 mg/100g.
In accordance with total amount of anthocyanins the clone 99-ž-16 from Žuvintas
was distinguished (103mg/100g). The amount of anthocyanins attained from 41 to 103
mg/100g in Žuvintas clones and the same from 79 to 99 mg/100g in Aukštaitija clones.
The clones of European cranberry accumulated on average 78 mg/100g of total amounts
of anthocyanins.
Radical scavenging activity (RSA) against stable 2.2-diphenyl-1-picrylhydrazyl
hydrate (DPPH∗) were evaluated ethanol extracts from berries. All analyzed samples
were characterized of antioksidant activity, radical scavenging activity after one hour
were from 85% (99-ž-04) to 77% (95-a-5). Average RSA of analyzed 15 samples was
82%.
In wild and American cranberries dominate the same 6 anthocyanins (cianidin-3galactoside, cianidin-3-glucoside, cianidin-3-arabinoside, peonidin-3-galactoside,
peonidin-3-glucoside ir peonidin-3-arabinoside) (Fuleki and Francis, 1968; Jacquemart,
1997; Vorsa et all., 2005). Quantitative wild cranberry analysis of anthocyanins has
shown different consumption in separate cranberries clones. Clone 99-ž-05 haven had
cianidin-3-gliucoside. In another sample from Žuvintas reserve revealed that the same
athocianin constitute 7.18%. On average biggest part of anthocyanins constitute
peonidin-3-galactoside (30.43±2.9%), the smallest part – cianidin-3-glucoside
(2.47±0.6%). Comparing dominant anthocyanins of wild and American cranberries
anthocyanins (Vorsa et all., 2005) with biggest and smallest amounts were the same.
But comparing them with main anthocianns of others wild cranberries detected others
authors (Jacquemart, 1997) cianidin-3-glucoside were attributed to biggest amounts
anthocyanins. But in our researched wild cranberries clones this not proved out.
Genetic research of V. oxycoccos using RAPD and SSR. In our study, RAPD markers
proved to be a powerful method for the detection of spatial genetic variation based on
the literature (Nei and Li, 1979). To analyse all three wild cranberry groups we chose
nine Opa and Roth primers.
Evaluating RAPD results of fifty-six V. oxycoccos clones from Vytautas Magnus
University Kaunas botanical garden collection, we obtained 213 fragments (Figure 3),
reflecting a rich allelic diversity among the populations. The size of the amplified
fragments ranged from 100 to 2750 bp, 99.53 % of loci were polymorphic. The number
of bands per primer ranged from 15 (OPB-11) to 31 (Roth-09) (Table 6).
In Lithuanian natural population from Viešvil÷, Čepkeliai and Žuvintas (53
samples) were obtained 209 fragments. The size of the amplified fragments ranged from
14
125 to 2750 bp, 98.56 % of loci were polymorphic. The number of bands per primer
ranged from 15 (OPB-11) to 31 (Roth-08).
In not Lithuanian natural populations from Estonia, Sweden and Norway (42
samples) were obtained 240 fragments. The size of the amplified fragments ranged from
125 to 3000 bp, 96.39 % of loci were polymorphic. The number of bands per primer
ranged from 15 (OPB-11) to 46 (Roth-08).
For SSR analysis all analysed groups (VMU Kaunas botanical garden clones)
were chosen nine primers (Ca112f, Ca169f, Ca421f, Ca483f, CA794f, Na800, Na1040,
Vcc_j9, Vcc_s10).
Evaluating SSR results of all analysed groups were obtained 62 fragments. The
size of the amplified fragments ranged from 46 to 328 bp. Most different fragments (18)
were obtained in VMU KBG clones with CA794f primer. Least fragments (3) were
found with Ca112f, Ca169f and Vcc_s10 primers.
Table 6. Genetic characteristic of wild cranberry population groups. In sharpen fount
marked data of each investigated group
Population
groups.
Population Shanon’s
Information
Index (I)
KBG
collection
populations
Čepkeliai
Žuvintas
Kamanos
Aukštatija
Total
Čepkeliai
Žuvintas
Viešvil÷
Total
Norway
Sweden
Estonia
Total
Natural
Lithuanian
populations
Natural not
Lithuanian
populations
0,22±0,24
0,12±0,19
0,09±0,13
0,19±0,24
0,20±0,13
0,16 ±0,15
0,14±0,13
0,12±0,17
0,16±0,10
0,22±0,23
0,17±0,15
1,52±0,50
0,22±0,14
Nei’s gene
Observet
diversity (H) mean
number of
alleles (Na)
0,14±0,17
0,07±0,12
0,05±0,08
0,12±0,16
0,10±0,09
0,08±0,09
0,08±0,09
0,07±0,10
0,08±0,07
0,14±0,15
0,09±0,09
0,13±0,15
0,12±0,09
0,15±0,50
1,37±0,48
1,37±0,48
1,43±0,50
2,00±0,07
1,69±0,46
1,63±0,49
1,39±0,49
1,99±0,12
1,56±0,50
1,75±0,44
0,20±0,23
1,97±0,19
The number The
of
percentage
polymorphic of
loci
polymorphic
loci %(P)
111
79
78
92
212
145
131
81
206
140
186
129
240
52,11
37,09
36,62
43,19
99,53
69,38
62,68
38,76
98,56
56,22
74,70
51,81
96,39
15
16
Figure 3. RAPD agarose gel electrophoresis profiles of different Vaccinium oxycoccos population’s individuals. Lines marked with letters and numbers (k1,
k2…) represents the individual that belong to different population. M – DNA size markers is given in base pairs (bp). a) Estonia and Žuvintas natural
populations individuals profiles with Opa-10 primer; b) Norway natural population individuals profiles with Opa-01 primer; c) Sweden natural population
individuals profiles with Opa-05 primer; d) VMU Kaunas botanical garden Kamanos collection clones profiles with Opa-09 primer; e) VMU Kaunas botanical
garden Aukštaitija collection clones profiles with Opa-09 primer.
In natural Lithuanian and not Lithuanian populations most different fragments
(14 and 9) were obtained with CA794f. Least fragments (3) were found with primers
Ca112f (in both groups of natural populations) and Ca169f, Na800 and Na1040 (in
natural not Lithuanian populations group). With 3 SSR primers (Vcc_s10, Na800 ir
Na1040) in natural Lithuanian populations were obtained no bands. In Lithuanian
natural populations primers Vccj_9 and ca169 were monomorphic. Primer Ca169f were
monomorphic in two VMU KBG clones collections –and primer ca169 – Čepkeliai and
Aukštaitija were monomorphic in Aukštaitija and Žuvintas clones populations. Other
authors (Boches et al., 2005) obtained most fragments with Na800 primer and least with
Ca112f ir Ca169f
To estimate genetic variation between populations, the values of Shannon’s
Information Index (I), Nei’s gene diversity (H) and the observed number of alleles per
locus (Na), number and percentage of polymorphic loci were calculated (Table 6). For
all populations Nei’s genetic diversity was 0.08. And for each population group ranged
from 0.08 (Lithuanian natural populations) to 0.10 (KBG collection clones) to 0.12
(natural not Lithuanian populations). These data shows that proliferation of all these
groups are vegetative. This kind of reproduction is mentioned and other authors (Bartish
et al., 2000a). Meaning of Shannon’s Information Index for all groups for KBG clones –
0.2, natural Lithuanian populations – 0.16 and natural not Lithuanian populations – 0.22
also point up the way of proliferation.
A low genetic diversity within but a high diversity among populations is
expected (Kreher, 2000). Plants with highest genetic diversity within and among
populations can better adapt to different environmental conditions. Analyzing molecular
variance of Vaccinium and other plant species were observed that biggest part of
molecular variance were within populations (Stewart and Excoffier, 1996; Jordano and
Godoy 2000; Jürgens et all., 2007). Average molecular variance within Vaccinium
species populations were 87.7% and within populations were 27.7%. Highest molecular
variance was detected within American cranberry (V. macrocarpon) populations (more
than 91%) (Stewart and Excoffier, 1996). In V. ulingosum – 90.3% (Kreher et all., 2000)
in V. myrtillus – 86.19% (Albert et all., 2004, Garkava-Gustavson et all., 2005).
Comparing RAPDs results Lithuanian KBG collection V. oxycoccos samples appears to
maintain a quite low level of the genotypic variance within (75%) (Figure 4). But
genotypic variation of natural populations was close to other Vaccinium species.
In this work using AMOVA analysis genetic variability among KBG clones
populations were 25% (RAPD) and 12% (SSR). Genetic variability among V. oxycoccos
clones was lower than in others Vaccinium species (75% by RAPD data and 88% by
SSR). According to literature in other Vaccinium species genetic variability ranged from
86 to 96% (according to AMOVA-RAPD) (Stewart and Exocoffier 1996; Person and
Gustavsson 2001; Albert et al., 2005a; Albert et all. 2005b). And according to SSR data
in others clonal species genetic variability ranged from 71 to 86% (Godt and Hamrick,
1999; Reusch et al., 2000; Bockelmann et al., 2003; Tsyusko 2005). Determined genetic
variability values in VMU KBG colonal populations were similar to others clonal
species. Comparing results of both methods (RAPD and SSR) biggest clonal genetic
variability were determined using RAPD than SSR method.
Comparing analysed natural populations, biggest part of molecular variance
were within than among analyzed natural wild cranberry populations and ranged from
93% (in natural lithuanian) to 87% (natural not Lithuanian populations) as and in others
Vaccinium species (Stewart and Excoffier, 1996; Jordano and Godoy, 2000; Kreher et
17
al., 2000; Persson and Gustavsson, 2001; Albert et al., 2004, Garkava-Gustavsson et al.,
2005; Jürgens et al., 2007). Genetic variability distribution among regions were also not
big – 7% (Figure 5). Although regions are geographically far each other, but genetic
variability among them are marginally small. So small regional partitioning can reflect
wild cranberry outspread. There is prediction, that distribution of molecular variance
among and within populations in total searched samples is influenced by genetic
variability of total analysed samples. Within Hippophae rhamnoides ssp. Rhamnoides
populations genetic variability were 15% (Bartish et al., 1999a) but when research wee
done with all group of genes variability increased till 59.6% (Bartish et al., 2000b). But
in our research this prediction do not proof out. Although number of samples in
Lithuanian natural populations were bigger than in natural not Lithuanian populations
but genetic variability were lower. This could bee explained by intensive cranberry
picking in Lithuania. In this way was decreased genetic variability of Lithuanian
cranberry. While in other counties (especially in Norway and Sweden) cranberries
picking are not so intensiv.
Fiure 4. Molecular variance in percents distribution among and within investigated wild
cranberries populations by RAPD
Figure 5. Molecular variance in percents distribution: among and within natural wild
cranberries populations, among and within individuals and among regions by SSR
18
To estimate the relationship between V. oxycoccos populations, Nei’s genetic
distance between pairs of populations was calculated. To generate graphs principal
coordinate analysis (PCA) was used. Graphs show genetic distances among samples and
populations. The PCA and UPGMA analyses by individuals have revealed that
Čepkeliai populations of V.oxycoccos had homogenous genotype and all samples are in
one lineage. Kamanos, Žuvintas and Aukštaitija populations go to one site and have
mixed genotypes (Figure 6 and 8). The observed different and mixed lineages confirm
the prediction that these two Lithuanian V. oxycoccos populations are ancestral to one
population before glaciation and that the Čepkeliai population differed from them. In
the Lithuanian glaciation stage of pomeranija only asmall part of Lithuania was not
covered by ice, which includes Čepkeliai Reserve. Thus, the Čepkeliai represents the
first bog uncovered by ice. In the next stage of ice retreat the Žuvintas Reserve Bog was
uncovered (South Lithuanian ice retreat stage) later Aukštatija National Park (Midle
Lithuania ice retreat stage) and followed by the Kamanos Reserve Bog. According to
Lithuania deglaciation periods, these four isolated populations genetical separated into
different lineages (Figure 7). The resulting different and mixed lineages can confirm
prediction that these three Lithuanian V. oxycoccos populations were derived from one
population before glaciation`s and the Čepkeliai population differed from them. This
prediction also confirms and PCA analysis (Figure 6). Postglacial decolonization one
more factor that could have influenced these differences is because the populations
Čepkeliai, Žuvintas, Kamanos and Aukštatija (Webb and Bartelein, 1992).
Figure 6. UPGMA dendrogram based on Nei’s genetic distances between individuals of
V. oxycoccos from VMU Kaunas botanical garden collection clones
19
Figure 7. Locations of sampling of VMU Kaunas botanical garden populations of V.
oxycoccos from Lithuania (1 – Čepkleliai , 2 – Žuvintas, 3 – Kamanos rezerves, 4 –
Aukštatija national park). Different lines on Lituania map show different Lithuania
glaciation stages. Line with dots – North Lithuania, line with square – middle Lithuania,
line with dashes – Pomeranija glaciation stage.
According to RAPD results samples distribution in PCA plane by different
examined groups showed distinction of three populations from others. VMU KBG
Čepkeliai collection samples and from not Lithuanian natural population group Norway
and Estonian populations. All others samples from different populations made mixed
groups (Figure 8).
According to SSR results samples distribution in PCA plane by different
examined groups showed one population VMU KBG Kamanos clones distinction from
others populations. Lithuanian natural populations were in one mixed group and
distribute in all two-dimensional plane. There do not made groups by populations. All
natural populations samples also do not made groups by populations, but Norway
populations were in one two-dimensional plane side with Viešvil÷ population. That kind
of group formation show biggest genetic similarity of these two populations (Figure 9).
This similarity confirms and distribution by populations (Figure 11).
20
Figure 8. Principal coordinate analysis of all three wild cranberries researched groups
(VMU Kaunas botanical garden collection, natural Lithuanian, natural not Lithuanian
populations) genetic similarity by individuals using RAPD
Figure 9. Principal coordinate analysis of all three wild cranberries researched groups
(VMU Kaunas botanical garden collection, natural Lithuanian, all natural populations)
genetic similarity by individuals using SSR
Figure 10. Principal coordinate analysis of all three wild cranberries researched groups
(VMU Kaunas botanical garden collection, natural Lithuanian, all natural populations)
genetic similarity by populations using RAPD
21
According to RAPD results distribution by populations in PCA plane by
different examined groups most genetical similar in VMU KBG group were Kamanos
and Aukštaitija clones populations. Farthest in this group were Čepkeliai population as
like in distribution in PCA plane by individuals (Figure 10). According to Nei genetic
distance genetical farthest in natural Lithuanian populations group were Čepkeliai and
Viešvil÷ populations. Genetical nearest in all natural populations group were Norway
population with all natural Lithuanian populations. According to genetic distances most
similar populations distinction from each other were 0.001.
PCA analysis by SSR results revealed different results from RAPD in VMU
KBG group. Most similar populations were Žuvintas and Kamanos (0.096), and most
distinct Aukštaitija and Žuvintas (0.243) clones populations (Figure 11). In natural
Lithuanian populations biggest genetic distance was among Žuvintas and Viešvil÷
(0.86) and smallest genetic distance were among Čepkeliai and Žuvintas (0.57).
According to Nei genetic distance biggest distinct among all natural populations were
among Čepkeliai and Norway (2.28) and smallest among Norway and Viešvil÷ (0.637)
populations.
Figure 11. Principal coordinate analysis of all three wild cranberries researched groups
(VMU Kaunas botanical garden collection, natural Lithuanian, all natural populations)
genetic similarity by populations using SSR
22
Figure 12. UPGMA dendrogram based on Nei’s genetic similarities between V.
oxycoccos all natural populations.
Estonia
Sweden
Norway
Viešvil÷
Žuvintas
Čepkeliai
Table 7. Genetic distances by Nei (1978) among wild cranberry all natural populations.
In sharpen fount marked data genetical most similar and most different populations
Čepkeliai 0.0000
Žuvintas
0.0036 0.0000
0.0053 0.0042
0.0000
Viešvil÷
0.0064 0.0063
0.0065
0.0000
Norway
0.0141 0.0131
0.0150
Sweden
0.0165 0.0000
0.0087 0.0087
0.0087
0.0093 0.0141 0.0000
Estonia
Estimating relationships among natural populations of V. oxycoccos to generate
dendrogram UPGMA analysis were used (Figure 12). Lithuanian natural populations
made one cluster from Norway and Sweden populations. Estonian population made
separate cluster from all others populations from this group. Least genetic distance
among natural populations was detected among Žuvintas and Čepkeliai populations
(0.0036) and biggest genetic distance were among Norway and Sweden (0.0165). Not
far from biggest genetic distance meaning were and Sweden and Estonian also Estonian
and Čepkeliai natural populations (0.0141) (Table 7).
Comparing RAPD and SSR results, SSR showed more differences among
individual than RAPD. That kind of results could be because of mutations occurrence
during time (chromosome aberrations, aneuploidy, polyploidy etc.), witch were chosen
because of vegetative propagation in plants. Simple sequence repeats is more sensitive
method that could reveal mutation by 1 nucleotide. That’s why we can see biggest
distinction in collection clones.
Different scientists analyzing different plant collections by using RAPS and SSR
methods remark that in such research is important to find individual DNR fragments.
Such fragments can be used in further selection works and searching for genes coding
some features (Garkava-Gustavsson et al., 2005). In this work after VMU Kaunas
botanical garden clones collection analysis with 9 RAPD primers were identified 3
specific fragments. These fragments can be used for identification of different clones:
clone Žuvintas 5 (99-Ž-05) – Roth 61200, clone Kamanos 4 (96-K-04) – Opa 5100, and
clone Kamanos 9 (96-K-09) – Opa 1125.
In previous Kaunas botanical garden wild cranberry collection researches
(Budriūnien÷ 1998, Daubaras et al., 2004) were marked late (95-A-3, 96-K-07) and
early (96-K-2, 96-K-3, 96-K-4 96-K-15 and 96-K-17) clones also clones with big
productivity (95-A-5, 95-A-9, 96-K-8, 96-K-10, 96-K-13). There were especially
important specific fragments in these separated clones. As it is mentioned above
fragment Opa 5100,is specific for clone Kamanos 4 (96-K-04) and this clone is attached
to early clones.
With SSR primers for late, early and with big productivity clones were
obtained 7 specific fragments: Aukštaitija 3 (95-A-03) - ca 421150, ca 483304, Kamanos 2
23
and 15 (96-K-02 and 96-K-15) - ca 483316, Kamanos 3 (96-K-03) - ca794233, Kamanos 7
(96-K-07) - Na800227, Kamanos 8 (96-K-08) - Vccj 9139, Kamanos 10 (96-K-10) Ca794251.
According to clones separation in to these three groups, we decided to check do
they genetical separate into different groups. For that reason according to genetic
distances and using UPGMA method were draw dendrogrames (Figure 12 and 13). As
we can see from dendrogrames cluster according to early, late phases and big
productivity did not separated.
Figure 13.UPGMA dendrogram based on Nei’s genetic distances between Kaunas
botanical garden collection 7 V. oxycoccos early (96-K-k-2, 96-K-3, 96-K-4 96-K-15
and 96-K-17) and late (95-A-3 and 96-K-07) clones using RAPD. Bootstraps are shown
in percents.
Figure 14. UPGMA dendrogram based on Nei’s genetic distances between Kaunas
botanical garden collection 5 V. oxycoccos clones with biggest productivity (96-K-13
(k13), 96-K-8 (k8), 96-K-10 (k10), 95-A-09 (a9) and 95-A-05 (a5)) and 6 clones with
smallest productivity (99-Ž-03 (z3), 99-Ž-09 (z9), 99-Ž-13 (z13) 98-Ž-05 (c5), 98-Ž-06
(c6) ir 98-Ž-17 (c17)). Bootstraps are shown in percents.
Correlation analysis of biochemical, molecular and morphologic features in
Vytautas Magnus University Kaunas botanical garden collection clones. After
conducting correlative analysis it has been established that there exists the negative
relationship between average berry weight and total amount of anthocyanins. The
coefficient of correlation was r= -0.657 (at the level of probability α=0.01). The clones
with small and dark red berries (98-Č-17, 98-Č- 06, 99-Ž-09, 99-Ž-13) accumulated the
largest amounts of anthocyanins (Table 4, Figure 1). This data motivate the selection of
clones with putative large amount of anthocyanins in situ and the preservation in the
collection ex situ with the purpose to use of them in the further breeding works.
Anthocyanins form a part of phenolics. So it is expected to find correlations
between phenols and anthocyanins quantities (Table 8). That kind of correlations was
24
detected in American cranberry (Kalt et al., 1999; Sun et al., 2002; Vinson et al., 2001),
plums and peaches (Bolívar et al., 2006). We also established linear dependence
between total amounts of anthocyanins and phenolics .
Table 8. Phenolics and anthocyanins quantity correlation analysis of wild cranberry
clones from VMU Kaunas botanical garden Aukštaitija and Žuvintas populations (p –
reliability, r – correlation coefficient, dash “-“ not significant correlation coefficient)
r=0.60
r=0.68
r=0.54
populations
p=0.03
p=0.01
p=0.05
r=0.61
mg/100g
berries
p=0.03
Anthocyanins
r=0.70
r=0.57
mg/ml
p=0.01
p=0.04
mg/ml
mg/100g
mg/ml mg/100g
berries
berries
Phenols
Anthocyanins
Roth-08
Opb-11
Roth-06
Opa-10
Opa-09
Opa-05
Opa-01
Opa-04
Roth-09
Table 9. Morphologic and RAPD fragments correlation analysis of wild cranberry
clones from VMU Kaunas botanical garden populations. Numbers show how many
correlations were obtained (dash “-“ not significant correlation meanings when p>0.05).
Total number
of correlations
with
morphologic
Morphologic
features
characteristics
Start
of
4
5 1 4
3
3
6 3 1
30
vegetation
Beginning
of
3
4 - 1
2
3
5 2 1
21
blossom
Beginning
of
1
1 - 1
2
- 5
berries ripening
End of berries
1
- 1 1
1
1 - 1
6
ripening
Length
of
generative
2
2 - 1
1 6
uprights
- 1
1 5
Volume of berry 3
2
1 1 2
1
1
1 1 1
11
Leaf length
4
3 1 3
3
3 17
Leaf width
5
1
2
1
4
1
14
Berry mass
Total number of
25 16 5 14 10 16 18 7 4
115
correlations
Using chi-square test, 119 out of 213 RAPD band products, were identified as
significantly contributing (p<0.05). We made correlations search between RAPD
fragments and biochemical features in VMU KBG collection Žuvintas and Aukštaitija
25
clones. There were detected 19 correlations. Biggest part of correlations were with
peonidin-3-galactoside. Analyzing total amount of phenol and anthocyanins were find 5
correlations. That kind of correlations could be related with biosynthesis ways of
flavonoids. This process in biggest part is depended of phenological stages and variety
of flavonoids (Vvedenskava and Vorsa, 2004).
There were found some correlations between RAPD fragments and 9 different
morphologic features. Biggest part of them was beginning of vegetation. Biggest part of
genetic correlations was found with Roth-09 praimer (Table 9). After some more
investigations these correlations could be used in the further breeding works. It could
release detection of early and late clones.
CONCLUSIONS
1. Because of carried out samples with best features selection during collection
formation in Vytautas Magnus university Kaunas botanical garden wild cranberry (V.
oxycccsos) collection clones genetic variability within populations is lower than in
natural Lithuanian populations (in collection populations – 75%, in Lithuanian natural
populations – 93%).
2. Defined lower genetic diversity in Lithuanian natural populations than in not
Lithuanian natural populations reveal, about intensive cranberries picking in Lithuania
(different than in Norway, Sweden and Estonia analyzed places), markedly reduce
genetic diversity of Lithuanian cranberries.
3. Established negative relationship between average berry weight and total
amount of anthocyanins, motivate select clones which predicted have biggest
anthocyanins amounts in situ, and carry out to preserve them in ex situ collection for
further selections works.
4. Established amount of phenols in wild cranberry clones of Vytautas Magnus
University Kaunas botanical garden collection, showed, that purposeful wild cranberry
selection let reach results close to American cranberries (Vaccinium macrocarpon
Aiton) rates.
5. Reviewed biochemic and genetic correlations between Vytautas Magnus
University Kaunas botanical garden collection Žuvintas and Aukštaitija collection
populations were revealed 19 correlations with 16 different fragments. Biggest part of
them was with peonidin-3-galaktosides, and biggest part of genetic data correlations
were with Roth-09 primer. These correlations can be used for selection works.
6. Reviewed correlations among 9 morphologic features and RAPD fragments
detected with 9 primers, biggest part of morphologic correlations were revealed with
beginning of vegetation and blossoming, and biggest part of genetic correlations were
with Roth-09 primer, these correlations can be used for selection works.
Suggestion: on purpose that VMU KBG collection populations reflected
biodiversity if wild cranberry, collection populations should be added of accidentally
picked samples.
26
SANTRAUKA
IVADAS. Paprastoji (Vaccinium oxycoccos L.) spanguol÷ yra vertinama d÷l
gydomosiomis savyb÷mis pasižyminčių uogų, kurios išsiskiria dideliu antioksidaciniu
aktyvumu (Wang et al., 1996). Didžiausias spanguolių biologinis efektas yra susijęs su
didel÷mis koncentracijomis fenolinių junginių (Mann, 2003; Huang et al., 2003).
Lietuvoje spanguolių biochemine sud÷timi prad÷ta dom÷tis dar 1998 m (Povilaityt÷ ir
kt., 1998).. Tačiau biocheminių darbų su paprastąja spanguole atlikta nedaug. Yra
nustatyta, kad vaisių, uogų bei jų produktų antioksidacinis aktyvumas susijęs su
polifenolių kokybine ir kiekybine sud÷timi, kuri yra skirtinga d÷l geografinių regionų ir
klimato sąlygų ypatumų (Kahkonen ey al., 2001). Tod÷l reikia kuo nuodugniau atlikti
Lietuvoje augančių paprastųjų spanguolių biocheminius tyrimus, bei palyginti su
spanguol÷mis augančiomis kitomis klimato sąlygomis.Kadangi flavonoidai yra svarbūs
cheminiai junginiai biologin÷se sistemose, pasaulyje atliekami ir genetiniai flavonoidų
tyrimai. Flavanoidų biosintez÷ yra susieta su genais, kurie buvo išskirti iš daugelio
augalų rūšių ir buvo plačiai tyrin÷jami molekuliniame lygmenyje (Hahlbrock and
Scheel, 1989; Martin et al., 1991; Holton and Comish, 1995; Boss et al., 1996; WinkelShirley, 2001). Tačiau toki tyrimai Lietuvoje dar n÷ra atlikti. Spanguolių genetin÷
įvairov÷ jau senokai domina selekcininkus, kadangi jos turi ūkinę reikšmę. Svarbu
atrinkti tokias gamtines formas, kurios pasižym÷tų produktyvumu, atsparumu
nepalankiems aplinkos veiksniams (ligoms), geru uogų skoniu ir dydžiu (Vorsa, 1994).
Pirminiame spanguolių gamtinių formų vertinime d÷mesys kreipiamas į morfologinius
požymius (Novy and Vorsa, 1995). Molekuliniai žymenys sudaro galimybę tiesioginiam
genetin÷s įvairov÷s vertinimui, siekiant objektyviai nustatyti genetin÷s medžiagos
skirtumus (Patamsyt÷ et al., 2008).
Buvo pasteb÷ta, jog paprastoji spanguol÷ turi labai daug morfologinių skirtumų, d÷l to
buvo atliktas spanguolių morfologinių požymių įvertinimas (Daubaras and Česonien÷,
2004). Tačiau ar tie skirtumai yra genetiniai, ar surinktos formos daug skiriasi nuo
gamtinių populiacijų – neaišku. D÷l to yra svarbūs ir reikalingi ne tik morfologiniai, bet
ir genetiniai šių rezervatų paprastosios spanguol÷s tyrimai. Dar vienas keliantis
susirūpinimą aspektas yra invazin÷ stambiauog÷s spanguol÷s (Vaccinium macrocarpon
Aiton.) rūšis. Kadangi invazin÷ stambiauog÷s spanguol÷s rūšis Lietuvoje vis daugiau
plinta tarp uogų augintojų, did÷ja tikimyb÷, kad Lietuvoje augančių paprastosios
spanguol÷s ekologines nišas užims stambiauog÷ spanguol÷ ir išstums ją kaip rūšį. Tuo
tikslu turime ištirti natūraliai Lietuvoje ir kaimynin÷se šalyse augančių spanguolių
genetinius išteklius. Sekant aukščiau pamin÷tais tikslais, yra reikalingi ne tik
morfologiniai ir genetiniai paprastosios spanguol÷s tyrimai, bet ir biocheminiai. Kaip
jau yra min÷ta aukščiau aprašytuose darbuose, chemin÷ paprastosios ir stambiauog÷s
spanguolių sud÷tis yra skirtinga. Tačiau neaišku, ar šie skirtumai yra nulemti fenotipo
augimo sąlygų, ar genotipo. Iš to ir kilo šio darbo tikslas
Darbo tikslas: ištirti paprastosios spanguol÷s V. oxycoccos DNR kintamumą ir fenolių
sud÷tį uogose bei nustatyti morfologinių, genetinių požymių ir fenolinių junginių
tarpusavio ryšius.
Darbo uždaviniai:
1.
Įvertinti skirtingų (lietuviškų, estiškų, švediškų ir norvegiškų) spanguolių
populiacijų genetinę įvairovę panaudojus morfofiziologinius, APPD ir SSR žymenis.
2.
Ištirti fenolinių junginių kiekius spanguolių uogose
Įvertinti spanguolių biochemin÷s sud÷ties ir genotipo sąsajas.
3.
27
4.
Įvertinti morfologinių ir genetinių požymių sąsajas.
Mokslinis naujumas ir praktin÷ reikšm÷. Lietuvoje iki šiol buvo tirtos paprastųjų
spanguolių morfologin÷s savyb÷s bei keletas darbų atlikta tiriant paprastosios
spanguol÷s biochemines savybes.
Šiame darbe pirmą kartą Lietuvoje įvertinta paprastosios spanguol÷s (Vaccinium
oxycoccos L.) genetin÷ įvairov÷.
Atlikta VDU Kauno botanikos sodo paprastosios spanguol÷s kolekcijos genetin÷
analiz÷. Naudojant atsitiktinai pagausintos polimorfin÷s DNR ir mikrosatelitų metodus,
įvertinus paprastosios spanguol÷s genetinį kintamumą, kuris buvo mažesnis Lietuviškų
gamtinių populiacijų nei užsienietiškų. Palyginus Vytauto Didžiojo universiteto Kauno
botanikos sodo kolekcijos klonų genetinę įvairovę su gamtin÷mis populiacijomis,
kolekcijos vidupopuliacin÷ imčių genetin÷ įvairov÷ buvo ženkliai mažesn÷ nei
lietuviškose gamtin÷se populiacijose.
Pirmą kartą Lietuvoje bei pasaulyje buvo atlikta paprastosios spanguol÷s, morfologin÷s
ir genetin÷s sąsajos (daugiausiai jų aptikta su Roth–09 pradmeniu ir tarp vegetacijos ir
žyd÷jimo pradžios) bei genetinių ir biocheminių požymių ryšių analiz÷ (daugiausiai
koreliacijų aptikta su peonidin–3 galaktozidais, o daugiausiai genetinių duomenų sąsajų
aptikta su Roth–09 pradmeniu).
Paprastoji spanguol÷ (V. oxycoccos) yra vienas vertingiausių laukinių uoginių augalų,
aptinkamų Lietuvoje. Ji yra vertinama visame pasaulyje d÷l gydomųjų savybių. Atlikta
genotipų sąsajų paieška su biocheminiais ir morfologiniai duomenimis, gali būti
panaudota kryptingam kolekcijos pavyzdžių tvarkymui bei naujų paprastosios
spanguol÷s veislių selekcijai. Patikrinus biochemines ir genetines sąsajas, tarp Kauno
botanikos sodo kolekcijos Žuvinto ir Aukštaitijos populiacijų imčių, buvo aptikta 19
sąsajų su 16 skirtingų fragmentų iš kurių daugiausiai fragmentų koreliacijų aptiktų su
peonidin–3 galaktozidais, o daugiausiai genetinių duomenų sąsajų aptikta su Roth–09
pradmeniu, šias sąsajas galima naudoti selekciniams tyrimams. Norint, kad VDU Kauno
botanikos sodo kolekcijos populiacijų imtys atspind÷tų paprastosios spanguol÷s gamtinę
bioįvairovę, kolekcijos populiacijų imtis reik÷tų papildyti atsitiktinai rinktais
pavyzdžiais.
Darbo aprobavimas. Darbo rezultatai paskelbti 6 publikacijose, iš jų trys paskelbtos
žurnaluose įrašytuose į Mokslin÷s informacijos instituto sąrašą (ISI), trys – kituose
Lietuvos ir užsienio moksliniuose žurnaluose. Disertacijos tema pristatyta 9 pranešimai
vietin÷se ir tarptautin÷se konferencijose.
Išvados
1.
Vytauto Didžiojo universiteto Kauno botanikos sodo paprastosios spanguol÷s
(V. oxycccsos) kolekcijos vidupopuliacin÷ imčių genetin÷ įvairov÷ yra ženkliai mažesn÷
nei lietuviškose gamtin÷se populiacijose (kolekcijos populiacijų imtyse – 75%,
lietuviškose gamtin÷se populiacijose – 93%), d÷l kolekcijos formavimo metu vykdytos
kryptingos spanguolių uogų su geresn÷mis savyb÷mis atrankos.
2.
Nustatytas mažesnis lietuviškų gamtinių populiacijų, lyginant su užsienietiškom
populiacijom genetinis kintamumas parodo, kad Lietuvoje vykdomas intensyvus
spanguolių rinkimas (skirtingai nei Norvegijos, Švedijos ir Estijos tirtose vietov÷se),
žymiai sumažino lietuviškų spanguolių genetinę įvairovę.
3.
Aptikta bendro antocianinų kiekio neigiama koreliacija su uogos dydžiu skatina
atrinkti tuos klonus, kurie sp÷jama, turi didelį antocianinų kiekį in situ ir toliau siekti
juos išsaugoti kolekcijoje ex situ tolimesniems selekciniams darbams.
28
4.
Aptikti fenolių kiekiai Vytauto Didžiojo universiteto Kauno botanikos sodo klonų,
parod÷, kad kryptinga paprastosios spanguol÷s atranka leidžia pasiekti rezultatus
artimus stambiauogių spanguolių (Vaccinium macrocarpon Aiton) rodikliams.
5.
Patikrinus biochemines ir genetines sąsajas, tarp Kauno botanikos sodo kolekcijos
Žuvinto ir Aukštaitijos populiacijų imčių, buvo rasta 19 sąsajų su 16 skirtingų
fragmentų, iš kurių daugiausiai fragmentų koreliacijų aptikta su peonidino-3galaktozidais, o daugiausiai genetinių duomenų sąsajų – su Roth-09 pradmeniu. Šias
sąsajas galima naudoti selekciniams tyrimams.
6.
Atlikus su 9 pradmenimis aptiktų fragmentų ir 9 skirtingų morfologinių požymių
duomenų sąsajų paiešką, nustatyta, kad daugiausiai morfologinių duomenų sąsajų rasta
su vegetacijos ir žyd÷jimo pradžia, o daugiausiai genetinių duomenų sąsajų – su Roth09 pradmeniu, šios sąsajos gali būti panaudotos ankstyvųjų ir v÷lyvųjų klonų paieškai.
Pasiūlymai: norint kad VDU KBS kolekcijos populiacijos imtys atspind÷tų
paprastosios spanguol÷s bioįvairovę, kolekcijos populiacijų imtis reik÷tų papildyti
atsitiktinai rinktais pavyzdžiais.
Paskelbti straipsniai disertacijos tema
1. L. Česonien÷, R. Daubaras, J. Areškevičiūt÷, P. Viškelis. 2006. Evaluation of
Morphological Peculiarities, Amount of Total Phenolics and Anthocyanins in
Berries of European Cranberry (Oxycoccos palustris). Baltic Forestry, 12 (1): 5963.
2. J. Areškevičiūt÷, A. Paulauskas, L. Česonien÷, R. Daubaras. 2006. Genetic
characteristic of wild cranberry (Vaccinium oxycoccos) collected from Čepkeliai
reserve using RAPD method. Biologija.1: 5-7
3. J. Žukauskien÷, A. Paulauskas, L. Česonien÷, R. Daubaras. 2009. Genetic
structure of isolated (Vaccinium oxycoccos) populations in Lithuania. Proceedings
of the Latvian academy of science. Section B. 63(1/2): 33-36.
4. J. Areškevičiūt÷, A. Paulauskas, R. Daubaras, L. Česonien÷. 2005. Atsitiktinai
amplifikuotos polimorfin÷s DNR metodo pritaikymas paprastosios spanguol÷s
(Oxycoccos palustris) įvairov÷s tyrimams. Lietuvos biologin÷ įvairov÷: būkl÷,
struktūra, apsauga. Vilnius. 6-13. ISBN 1822-2781
5. Česonien÷ L., Daubaras R., Areškevičiūt÷ J. 2007. Investigation of genetic
resources of European cranberry Oxycoccos palustris (Ericaceae) in Lithuania.
Monographs of Botanical Gardens. 1: 61-64.
6. J.E. Paičiūt÷, I. Dežicait÷, J. Žukauskien÷, A. Paulauskas. 2007. Genetic
characteristic of morphologic different clones of Vaccinium oxycoccos collected
from Žuvintas, Čepkeliai. Kamanos rezerve and Aukštaitija national park. The Vital
Nature Sign. 10-14. ISBN 978-9955-12-213-5
Konferencijų pranešimų tez÷s:
1. Areškevičiūt÷ J., Paulauskas A., Česonien÷ L., Daubaras R. 2005. Genetic
characteristic of wild cranberry (Vaccinium oxycoccos) of morphologically
different forms collected form Žuvintas reserve. International conference:
Biodiversity, molecular ecology and toxicology., Palanga. 7.
2. Areškevičiūt÷ J., Daubaras R., Česonien÷ L. 2005. Phenotypic diversity of wild
cranberry Vaccinium oxycoccos in Žuvintas and Čepkeliai reserves in Lithuania. 3th
29
3.
4.
5.
6.
7.
8.
9.
30
International Conference: Research and conservation of biological diversity in
Baltic Region. Daugavpils. 20.
Areškevičiūt÷ J., Paulauskas A., Česonien÷ L., Daubaras R. 2006. Čepkelių
rezervate surinktų paprastosios spanguol÷s (Vaccinium oxycoccos) genetin÷
charakteristika atliekant AAPD analizę. Kur gamta siejasi su mokslu. Kaunas. 1617.
Česonien÷ L., Daubaras R., Žukauskien÷ J. 2007. Investigation of genetic
resources of European cranberry in Lithuania. The 2nd International Conference of
Eastern and Central European Botanic Gardens. Poland. 47.
Paičiūt÷ E., Dežicait÷ I., Žukauskien÷ J., Paulasukas A.. 2007. Genetic
characteristic of morphologic different clones of Vaccinium oxycoccos collected
from Žuvintas, Čepkeliai. Kamanos rezerve and Aukštaitija national park. The Vital
Nature Sign. Kaunas. 10-14.
Žukauskien÷ J., Paulauskas A., Česonien÷ L., Daubaras R. 2007. Genetic structure
of isolated Vaccinium oxycoccos populations. 4th International Conference:
Research and conservation of biological diversity in Baltic Region. Daugavpils.
137.
Žukauskien÷ J., Paulauskas A. 2007. Genetic variation and phylogeographic
relationship among Lithuanian wild cranberries (Vaccinium oxycoccos). 4th Baltic
genetical congress. Daugavpils. 183.
Žukauskien÷ J., Paulauskas A., Daubaras R.. 2008. Genetic variation and
development between morphologic features in Vaccinium oxycoccos. International
scientific conference actualities in plant physiology. Lithuanian University of
Agriculture. Babtai.
Žukauskien÷ J., Paulauskas A., Česonien÷ L., Daubaras R. 2009. Vaccinium
oxycoccos Morphology and RAPD Marker Characterizations NJF seminar and
international scientific conference „Vaccinium ssp. and less known small fruit:
challenges and risks”. Jelgava
Judita Žukauskien÷
Vytauto Didžiojo universiteto biologijos katedros doktorant÷
Darbo adresas:
Namų adresas:
Molekulin÷s ekologijos ir genetikos laboratorija
S. Žukausko 37-48
Biologijos katedra
LT Kaunas
Gamtos mokslų fakultetas
Tel.: +37068661332
Vytauto Didžiojo universitetas
Vileikos 8
LT 44404
Tel.: 8-37 327902
Faksas: 8-37 327916
El. paštas: [email protected]
Gimimo vieta ir data: 1981 m. kovo 15, Kaunas Lietuva.
Išsilavinimas: 1999-2003 m. Vytauto Didžiojo universitetas gamtos mokslų fakultetas
– biologijos bakalaur÷; 2003-2005 m. Vytauto Didžiojo universitetas gamtos mokslų
fakultetas – molekulin÷s biologijos ir biotechnologijos magistr÷;
2006-2007 m. Vytauto Didžiojo universitetas edukologijos ir pedagogikos fakultetas –
pedagogikos profesinis išsilavinimas.
Darbin÷ veikla: 2004-2008 m. Ekologin÷s genetikos būrelio vadov÷ Kauno moksleivių
aplinkotyros centre ir Vytauto Didžiojo universitete.
2004-2008 m. biologijos katedros laborant÷.
Stažuot÷s: Mokslin÷ stažuot÷ Lenkijoje Varšuvos botanikos centro, bioįvairov÷s
saugojimo ir tyrimų centre 2007 06 04 – 2007 07 04.
Tyrimų sritis: paprastosios spanguol÷s molekuliniai bei biocheminiai tyrimai.
31
Judita Žukauskien÷
THE RESEARCH OF WILD CRANBERRY
(Vaccinium oxycoccos L.) DNA VARIABILITY AND
PHENOLIC COMPOUNDS IN BERRIES
Summary of Doctoral Dissertation
Išleido ir spausdino – Vytauto Didžiojo universiteto leidykla
(S. Daukanto g. 27, LT-44249 Kaunas)
Užsakymo Nr. 132. Tiražas 50 egz. 2009 11 09.
Nemokamai.