- Faculty of Biosciences and Medical Engineering

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PHYLOGENETIC ANALYSIS OF MALAYSIAN PINEAPPLE CULTIVARS
USING DNA SEQUENCES OF INTERNAL TRANSCRIBED SPACER(ITS)
REGION OF NUCLEAR RIBOSOMAL DNA (nRDNA)
CHANDRIKA KUPPUSAMY
A dissertation is submitted in partial fulfillment of the requirements for the award of
the degree of Masters of Science (Biotechnology)
Faculty of Bioscience and Bioengineering
University Teknologi Malaysia
JULY 2012
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I would like to dedicate my research founding to God almighty who continues to
make the impossible possible. Thank you Lord.
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ACKNOWLEDGMENT
Foremost, I would like to express my sincere gratitude to my supervisor Dr. Topik
Hidayat for the continuous support of my Masters Study and research, for his
patience, motivation, enthusiasm, and immense knowledge. His guidance helped me
all the time of research and writing of this dissertation.
My gratitude also goes to the ‘Lembaga Perindustrian Nanas Malaysia’ for their
kindness to provide us with pineapple samples that were used in this study. Not to be
forgotten, thanks to University Teknologi Malaysia for making this study possible by
providing its funding to us.
Besides that, I would like to thank and very grateful to my friends who helped and
encouraged me a lot throughout this research especially to Farah Izana Abdullah and
the rest of the laboratory mates. Not also forgetting the laboratory personnel who
were helpful and do not hesitate to give advice throughout the research. I also would
like to express my appreciation to all individuals who had helped me directly or
indirectly in completing this research.
Last but not least, I would like to express my deepest gratitude to my family for their
constant support, emotional understandings, encouragement and financial aids in
successfully completing this research.
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ABSTRACT
The phylogenetic study was conducted to determine the phylogenetic status
and evolutionary relationships among the nine commercial pineapple cultivars using
sequences of the internal transcribed spacer (ITS) region. Genomic DNA was
extracted, and the ITS region was amplified and sequenced. Parsimony analysis
revealed that Malaysian cultivars could be classified into two major groups based on
the ITS region. The first group comprised the cultivars Sarawak Green Local,
Gandul, and N36 whereas the second group consisted of the cultivars Josapine,
Yankee, Morris Bentanggur, Morris Gajah, MD2 and MD2/T. Several combinations
of synapomorphic characters (leaf and fruit) support this classification system,
suggesting the ITS region has the ability to determine the phylogenetic status and
relationships of pineapple cultivars. Since each group has its own similar genetic
pattern and presumably certain specific biochemical properties, the relationships
revealed here can be used as the basis for successful hybridizations to generate new
pineapple cultivars.
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ABSTRAK
Di dalam kajian ini, analisis filogenetik telah dijalankan untuk mengetahui
status dan hubungan evolusi antara sembilan kultivar nanas Malaysia komersial
menggunakan urutan basa DNA ‘internal transcribed spacer’ (ITS). Genomik DNA
diekstrak dan urutan basa ITS diamplikasi dan di hantar untuk penjujukan. Analisis
filogenetik menggunakan kaedah Parsimoni menunjukkan kultivar nanas Malaysia
boleh dikategorikan pada dua kelompok berdasarkan urutan basa ITS. Kelompok
pertama terdiri daripada Sarawak Green Local, Gandul dan N36. Kelompok kedua
pula terdiri daripada kultivar Josapine, Yankee, Morris Bentanggur, Morris Gajah,
MD2 dan MD2/T. Beberapa gabungan karakter sinapomorphik (daun dan buah)
menyokong sistem klasifikasi ini, mencadangkan bahawa urutan basa ITS
mempunyai keupayaan untuk menentukan status filogenetik dan hubungan
kekerabatan antara kultivar nanas. Oleh kerana setiap kelompok mempunyai
keseragaman dalam corak genetik, maka hubungan kekerabatan ini boleh digunakan
sebagai asas untuk hibridisasi dalam usaha untuk menghasilkan kultivar nanas yang
baru.
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TABLE OF CONTENTS
CHAPTER
TITLE
PAGE
TITLE OF RESEARCH
i
DECLARATION
ii
DEDICATION
iii
AKNOWLEDGMENT
iv
ABSTRACT
v
ABSTRAK
vi
TABLE OF CONTENTS
vii
LIST OF TABLES
x
LIST OF FIGURES
xi
LIST OF ABBREVATIONS/ SYMBOLS
xiii
CHAPTER 1 INTRODUCTION
1.1 Study Background
1
1.2 Problem Statement
2
1.3 Objectives of Study
3
1.4 Scope of Study
4
1.5 Significance of Study
4
viii
CHAPTER 2 LITERATURE REVIEW
2.1 Taxonomy Ananas comosus
5
2.1.1 History and Distribution
5
2.1.2 Morphology and Sexual Reproduction
7
2.2 Pineapple Cultivars
9
2.3 Pineapple Breeding
13
2.4 Importances of Pineapple
15
2.4.1 Nutritional Value
15
2.4.2 Food for Livestock
16
2.5 Ananas Genomic Study
17
2.6 Molecular Phylogenetic Analysis
18
2.6.1 Molecular Phylogenetic Analysis in Plants
19
Using ITS Region
CHAPTER 3 METHODOLOGY
3.1 Experimental Design
23
3.2 Sources and Storage of Pineapple Leaves
24
3.3 Genomic DNA Extraction from Pineapple Leaves
24
3.4 Polymerase Chain Reaction (PCR) Amplification
25
3.5 Vector Insertion and Cloning
26
3.5.1 Ligation
26
3.5.2 Transformation into NEB 5 Alpha Strain of
27
Escherichia coli
3.5.3 Blue White Screening
3.6 Plasmid Isolation
28
29
3.6.1 Preparation of E. coli
29
3.6.2 Plasmid Extraction
29
3.6.3 Reconfirmation by Restriction Enzyme (RE) digestion
29
3.7 Bioinformatics Analysis
30
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CHAPTER 4 RESULTS AND DISCUSSION
4.1 Genomic DNA Extraction
31
4.2 Amplifying ITS Region using PCR
33
4.3 Blue White Screening
35
4.4 Plasmid Isolation
37
4.5 Reconfirmation by Restriction Enzyme
40
4.6 Phylogenetic Tree Construction and Analysis
41
CHAPTER 5 CONCLUSIONS AND FUTURE WORK
5.1 Conclusions
49
5.2 Future Work
50
REFERENCES
51
APPENDICES
59
x
LIST OF TABLES
TABLE
TITLE
PAGE
2.1
Nutritional information of pineapple
17
3.1
Primer sequence used in this research
25
3.2
Reagent for ITS region amplification
25
3.3
PCR amplification profile
26
3.4
Preparation of ligation mixture
27
3.5
Preparation of ligation mixture for positive control
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3.6
Digestion mixture preparation for insertion reconfirmation
30
xi
LIST OF FIGURES
FIGURES
TITLE
PAGE
2.1
(a) Whole inflorescence of A. comosus into huge compound
fruit, (b) Structure of a pineapple plant (A. comosus var.
comosus) showing the different types of planting materials
for further vegetative propagation (stem sucker, slip, and
crown).
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2.2
Pictures shows fruits of various pineapple cultivars in
Malaysia (a) Morris Gajah, (b) Josapine, (c) MD2,
(d) N36, (e) MD2/T, (f) Sarawak Green Local, (g) Morris
Bentanggur, (h) Yankee and (i) Gandul
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2.3
Organization of Internal Transcribed Spacer (ITS) region
of nuclear ribosomal DNA (nrDNA).
22
3.1
Flowchart shows summary of experimental design.
23
4.1
The result for genomic DNA(gDNA) extraction L1:1kb DNA
Ladder, L2: Josapine, L3: Morris Gajah, L4: Morris
Bentanggur, L5: MD2, L6: Sarawak Green Local.
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4.2
The results for genomic DNA (gDNA) extraction L1:1kb DNA
Ladder, L2: Yankee, L3: Gandul, L4: MD2/T, L5: N36
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4.3
The result for ITS region amplification using PCR L1:1kb DNA
Ladder, L2: Josapine, L3: Morris Gajah, L4: Morris Bentanggur,
L5: MD2, L6: Sarawak Green Local, L7: Yankee.
33
xii
4.4
The result for ITS region amplification using PCR L1:1kb
DNA Ladder, L2: Gandul, L3: MD2/T, L4: N36.
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4.5
The results for blue white screening where blue colony
indicates control without insert and white colony indicates
insert with gene of interest (a) Josapine, (b) N36, (c) MD2,
(d) MD2/T, (e) Gandul, (f) Morris Bentanggur, (g) Sarawak,
(h) Yankee (i) Morris Gajah
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4.6
The result for plasmid extraction. L1:1kb ladder, L2: Control
from blue colony, L3: Josapine (a), L4: Josapine (b),
L5: Yankee (a), L6: Yankee (b), L7: Mr.Bentanggur (a), L8:
Mr.Bentanggur (b).
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4.7
The result for plasmid extraction. L1:1kb ladder,
L2: Mr. Gajah (a), L3: Mr. Gajah (b), L4: MD2/T (a),
L5: MD2/T (b), L6: Sarawak (a) L7: Control from blue
colony.
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4.8
The result for plasmid extraction. L1:1kb ladder,
L2: Sarawak (b), L3: MD2 (a), L4: MD2 (b), L5: Gandul (a),
L6: Gandul (b) L7: Control from blue colony
L2: Control from blue colony, L3: N36 (a), L4: N36 (b),
L5: N36 (c)
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4.9
The result for plasmid extraction. L1:1kb ladder,
L2: Control from blue colony, L3: N36 (a),
L4: N36 (b), L5: N36 (c)
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4.10
The result of reconfirmation using EcoRI.
L1:1kb ladder, L2: Josapine (a), L3: Josapine (b),
L4: Yankee (a), L5: Morris Bentanggur (a), L6: MD2/T (a),
L7: MD2 (a), L8: Gandul (a)
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4.11
The results of reconfirmation using EcoRI. L1:1kb
DNA ladder, L2: N36 (a), L3: N36 (b), L4: N36 (c),
L5: Sarawak (a), L6: Sarawak (b), L7: Mr. Gajah (a)
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4.12
Strict consensus tree from the parsimony analysis of
the ITS region (3 Most Parsimonious Trees, Length
= 831 steps, CI=0.888, RI= 0.842). The value in the branch
showed the bootstrap support (BS). The abbreviations
of the cultivar were shown in the key
41
xiii
LIST OF ABBREVIATIONS / SYMBOLS
%
percentage
˚C
Degree Celcius
µL
microliter
bp
basepair
cm
centimeter
g
gram
kb
kilobase
kg
kilogram
m
meter
min
minute
mL
milliliter
nm
nanometer
sec
second
x
times
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CHAPTER 1
INTRODUCTION
1.1 Study Background
Pineapple (Ananas comosus (L.) Merr) is the most economically important
plant in the family Bromeliaceae (Sripaoraya et al., 2001). Pineapple industry in
Malaysia was first established in the late 1880s by a European in Singapore. Ananas
comosus has yielded high quantity of cultivars throughout its period of cultivation.
The large number of cultivars represents broad extent of variation in an amount of
characters. A small number of these cultivars have been broadly cultivated such as
Cayenne, Spanish and Queen (D' eeckenbrugge et al., 1997). Each of the cultivar has
their own uniqueness and economic importance. Pineapple has a lot of nutritional
value because it is rich with vitamin A, B1, B2 and C. In addition, it also has protein,
carbohydrate and an enzyme called bromelain (www.mpib.gov.my).
Due to a lot benefits from this fruit, study related to the molecular of
pineapple is very crucial. The available data on the molecular phylogenetic diversity
of pineapple is limited and is usually characterized using morphological characters
(Duval et al., 2001). Some studies has been conducted to separate the pineapple
according to main groups seem to appear less successful.
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Phylogenetic tree seem to be a reliable base of the research in many areas
biology especially in plants. A meaningful knowledge into biology can be gained
from comparisons of plant species or gene sequences in a phylogenetic (Hall et al.,
2002). This significance of realization is now readily obvious to researchers in
diverse fields including ecology, molecular biology and physiology. One of the
importances of a phylogenetic is to increase the areas of plant research boundary
such as the value of placing model organisms in the suitable phylogenetic frame to
obtain a better understanding of both patterns and processes of evolution (Soltis and
Soltis, 2003).
Internal Transcribed Spacer (ITS) region of nuclear ribosomal DNA has been
broadly applied in phylogenetic analysis of plants for family and higher levels. It is
also been used for closely linked genera or species. ITS region, which divides the
18S and 26S and the coding sequence region of 5.8S, has become extensively
describe between interspecific and intergeneric level differences (Baldwin et al.,
1995).
1.2 Problem Statement
In Malaysia, many cultivars of pineapple are grown, such as Maspine,
Sarawak, Morris, Josapine, MD2, Yankee, Gandul, and N36 (www.mpib.gov.my).
Each cultivar has distinct economic importance. Currently there are no established
identification tools at molecular level for Malaysian pineapple. Furthermore, few
studies have focused on the molecular phylogenetic relationships of Malaysian
pineapple cultivars. Indeed, most of the work has focused on breeding, agronomy,
physiology, pathology, entomology, and post-harvest management (www.mardi.my).
The popular approach to distinguish cultivars is based on morphological
characteristics, which is not very suitable because of disagreements among
morphologists who use different methods for phylogenetic analysis or for the
3
interpretation of characteristics. This method is therefore time consuming and
inconsistent.
Comparisons of the DNA sequences of various genes between different
organisms can yield much information about the relationships between organisms
that cannot otherwise be inferred from morphology. This is because, in contrast to
morphological characteristics, DNA sequences are relatively consistent (Baldwin et
al., 1995).
Therefore, in the present study, phylogenetic analysis of nine commercial
Malaysian pineapple cultivars was conducted using sequences of the ITS region of
the nrDNA to determine the phylogenetic status and evolutionary relationships
among these cultivars. The ITS has been widely used by plant systematists to
investigate the relationship between closely related taxa, mainly because of its rapid
evolutionary rate, small size, and highly conserved flanking regions (Kress et al.,
2005).
1.3 Objectives of Study
The objectives of this study are:
i) To test utility of ITS region in construction of phylogenetic tree.
ii) To determine the phylogenetic status of nine commercial Malaysian pineapple
cultivars.
iii) To understand the evolutionary relationship of the nine commercial Malaysian
pineapple cultivars.
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1.4 Scope of Study
The scope of this study includes three main parts. The first is sampling of
nine commercial Malaysian pineapple cultivars at ‘Lembaga Perindustrian Nanas
Malaysia’ (LPNM). Leaves for each cultivar were taken during sampling. The study
was carried out in laboratory using validated standard protocol which includes the in
vitro part that comprise of genomic DNA isolation, polymerase chain reaction (PCR)
and cloning. The third part is the in silico section which includes the bioinformatics
analysis.
1.5 Significance of Study
This study is important as it provides information on the evolutionary
relationships of Malaysian pineapple cultivars. Furthermore, molecular phylogenetic
analysis plays a crucial role in revealing basic knowledge on relationship patterns,
based on which genetic sources can be improved by generating new cultivars.
5
CHAPTER 2
LITERATURE REVIEW
2.1 Taxonomy of Ananas comosus
Pineapple is included in the Bromeliaceae family. The Bromeliaceae family is
divided into three subfamilies that are Pitcairnioideae, Tillandsiodeae and
Bromelioideae. Ananas is included in the Bromeliaceae family as one of the most
crucial genus where the species Ananas comosus or more commonly known as
pineapple is embrace in the genus. The edible pineapple fruit is recognized as an
important tropical fruit crop in the world production besides maize, banana and
citrus. The importances of this fruit which include the economic value, benefit and
the nutrient content become the uniqueness of the fruit itself (Claudete et al, 2001).
2.1.1 History and Distribution
The term ‘pine’ in the pineapple was earliest been used to portray the
reproductive organs of the pine cones. Generally, the Spanish people called
6
pineapple by using the word ‘Pina’, the Portugese called it ‘abacaxi’ and ‘nanas’ in
Southern Asia (Morton, 1987).
A. comosus is believed to be native from South America and Paraguay where
at that moment it seems that it was cultivated by the Indians. Before the advent of the
Europeans, the pineapples were slowly diffused by the Indians to South and Central
America to Mexico and the West Indies. Later on in 1493, a European named
Christopher Columbus saw the pineapple for the first time in his second journey on
the colonized island called Guadeloupe (Leal and D' eeckenbrugge, 1996).
In the beginning of 16th century, the Spanish people bring in A. comosus into
Philippines, Hawaii and Guam (Morton, 1987). Subsequent finding of this fruit, it
was then shortly discovered in numerous countries either by chance or by plan so
that the new fruit can be bring for the attraction to the new world. The distinctive and
remarkable characteristics, in addition to the drought resistance of the pineapple
propagules make certain its express flow all the way through the tropics (D’
eeckenbrugge et al., 2011).
Hawaii, Brazil, Malaysia, Taiwan, Mexico, Philippines and South Africa are
the main producing areas of pineapple. Pineapple currently becomes the third crucial
tropical fruit where it is cultivated in all tropical and subtropical countries. From
1980 to 1995 the production of pineapple was boost up to 20% (exceeded 12 million)
where 70% of the pineapple production at that time was consumed as fresh fruit
(Davey et al., 2007).
In Malaysia, pineapple industries were initially started in 1888 by a European
in Singapore. With rubber crop development, in year 1921 pineapple began to be
planted in Singapore, Johor and Selangor as cash crop. Pineapple industry continued
to increase in peat soil area particularly in Johor. Afterward, Malaysian Pineapple
7
Industry Board (MPIB) or generally known as ‘Lembaga Perindustrian Nanas
Malaysia’ (LPNM) was established in 1959. From then on, there has been steady
progress (www.mpib.gov.my).
The main country for fresh pineapple export in the world are Costa Rica,
Ivory and Philippines while canned pineapple export are dominated by Thailand,
Philippines and Indonesia. For pineapple juice industry, Philippines and Thailand
become the largest export. In terms of the production of pineapple, Malaysia is at the
9th position among the other pineapple industry countries. Meanwhile, Malaysia is in
the 10th position among the countries for the production of fresh and canned
pineapple (D’ eeckenbrugge et al., 1997).
In Malaysia, pineapples are broadly
grown in Selangor, Johor, Sarawak and Penang. Malaysian pineapples are exported
to several countries such as Singapore, United Arab Emirates, Saudi Arabia and
Brunei (Wee, 1972).
2.1.2 Morphology and Sexual Reproduction
The genus Ananas has distinctive characteristics in merging the whole
inflorescence into huge compound fruit (Figure 2.1a). A. comosus, the monocot from
Bromeliaceae family is perennial herbaceous plant that has spirally arranged leaves
in a compact rosette covering a short stem. The leaves are commonly spiny but
sometime several cultivars show absolute absence or partial spines (D’ eeckenbrugge
et al., 2011). The spines might be suppressed by dominant mutations, in the same
way as the one that have merely little spines adjacent to the leaf tip. This can also
happen in the one leading the folding of the lower leaf epidermis over the leaf margin
where all the spines suppressed except the terminal one (Duval et al., 2003). The
leaves color varies which is determined based on the cultivar but typically the
dominant color is green, red and purple. The mature pineapple plant has
8
approximately 1.0 to 1.5 meters tall frequently depending on the leaf length (De La
Cruz Medina and Garcia, 2005).
The pineapple plant has a short stocky stem with strong, waxy leaves. It
reproduces frequently from vegetative propagules that extend from the stem (stem
shoots and ground suckers), the peduncle (slips) and fruit crown (Figure 2.1b). The
pineapple plant usually generate up to 200 flowers when making its fruit
(Bartholomew et al., 2003). At the time it flowers, the individual fruits of the
flowers unite collectively to produce the pineapple with many fruitlets. Following the
fruit production, side shoots called suckers are generated in the leaf axils of the main
stem. It is an optional the suckers to be removed for propagation or left to generate
extra fruits on the original plants. The identical plant might therefore give a series of
numerous production cycles. In the planting industry, as a result of reduction in fruit
size and consistency, the pineapple plants are not permitted to make more than two
or three crops. Thus, a new plantation should be frequently set up using the identical
lateral shoots of the previous crop or by the means of other vegetative propagules
(fruit and crown) (Williams and Fleisch, 1993).
A.comosus has a gametophytic self incompatibility system where it is unable
to produce functional gametes thus required out crossing (Brewbaker and Gorrez,
1967). The whole A.comosus possesses diploid amount of 50 small chromosomes.
However, there is still A.comosus that present with triploid, tetraploid and
heteroploid. Pineapple flower are hermaphrodite. Pineapple is also greatly
heterozygous thus enhancement of several different characters is probable (D’
eeckenbrugge et al., 2011).
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(a)
(b)
Figure 2.1 (a) Whole inflorescence of A. comosus into huge compound fruit (Py et
al., 1987), (b) Structure of a pineapple plant (A. comosus var. comosus) showing the
different types of planting materials for further vegetative propagation (stem sucker,
slip, and crown) (D’ eeckenbrugge et al., 2011).
2.2 Pineapple Cultivars
Criteria to distinguishing between pineapple cultivars include habit of plant,
shape of fruit and fruitlets (‘eye’), amount and size of bracts, characteristics of the
flesh and morphological leaf characteristics (existence or nonexistence of spines,
shape whether piping or non-piping) (Py, 1987). Various cultivars such as
‘Cayenne’, ‘Queen’ and ‘Black Antigua’ possess the initial depiction and
categorization. Nearly all of the cultivars have gone and just ‘Cayenne’ and ‘Queen’
stay due to the commercial importance today (D’ eeckenbrugge et al., 1997; Munro,
1835).
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Pineapple cultivars are usually known in diverse names in different countries
depending where they are grown or from where they are created. It seems that they
are insufficient taxonomic or horticultural classification of the pineapple cultivars.
Still, the cultivar group ‘Cayenne’, ‘Queen’, ‘Spanish’, ‘Perolera’ and ‘Perola’ and
other group are able to provide a fine basic to evaluate the current status of pineapple
cultivars (Samuels, 1970).
There are several commercial cultivars such as Josapine, N36, MD2, MD2/T
(MD2/Tissue Culture), Gandul, Morris, Sarawak and Yankee grown in Malaysia
(Figure 2.2). In Malaysia, the groups of pineapple that are famously cultivated are
‘Cayenne’, ‘Queen’ and ‘Spanish’. The ‘Cayenne’ group consists of cultivars such as
‘Smooth Cayenne’ and ‘Hilo. In Malaysia the ‘Smooth Cayenne’ is also known as
‘Sarawak’ cultivar. This group of pineapple is grown mainly for both canning and for
fresh fruit consumption (D’ eeckenbrugge et al., 1997).
‘Sarawak’ is a cultivar that has big, egg shaped fruit seized on a small and
tough peduncle. The fruitlets (eyes) are wide and plane (flat). Meanwhile, the
‘Sarawak’ cultivar has smooth leaves with the infrequent spines at the tips.
Furthermore, the flesh of the Sarawak cultivar is very juicy with pale yellow color
and the acidity and sugar content is approximately 0.5-0.9% and 12-16 ˚ Brix. This
cultivar
has
a
long
produce
cycle
compared
to
the
other
cultivars
(www.mpib.gov.my).
On the other hand, ‘ Queen’ group is also one of the crucial group where
these cultivars are very spread in Asia with different recognition such as ‘Mauritius’,
‘Malacca’ and ‘Red Caylon’ (Mendiola et al., 1951). These strains also have been
cultured and cultivated in South Africa and Australia for fresh fruit industry and the
plant of this group is usually small that is approximately 60 to 80cm. It is
characterized by several characteristics such as it has very short and spiny leaves.
Meanwhile, the fruit is preferred to be consumed as fresh fruit rather than canned
11
industry due to it high sugar content (14- 17˚ Brix) (D’ eeckenbrugge et al., 1997). In
Peninsular Malaysia, ‘Queen’ group is generally known as ‘Nanas Moris’ and in
Sarawak it is called ‘Sarikei’.
‘Spanish’ is a group that is not broadly cultivated in the world but it is a
crucial group that is famously cultivated in the South Asia countries particularly in
Malaysia. This is the main canning cultivar in Malaysia due to the excellent adaption
on the peat soil. In Malaysia ‘Spanish’ group cultivar is called in different names
such as ‘Singapore Canning’, ‘Ruby’, ‘Nanas Merah’, ‘Gandul’, ‘Betek’ and
‘Masmerah’ (Wee, 1972).
The ‘Spanish’ is characterized by having dark green leaves with 35 to 70 and
just about 80 to 100 cm length sized leaves. The presence of spine is inconsistent
where some clones might have full spininess to a small number of spines in certain
clone (adjacent to the leaf tip). The ‘Spanish’ group fruit are small around 1kg to 2kg
with cylindrical shape. The fruit is dark purple color and will turn to reddish orange
with ripening (Morton, 1987). In Malaysia, the cultivars that are crucial for canning
is ‘Gandul’ and ‘Masmerah’ while for fresh fruit industry ‘N36’ and ‘Josapine’ are
much more suitable.
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(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
(i)
Figure 2.2 Pictures shows fruits of various pineapple cultivars in Malaysia (a)
Morris Gajah, (b)Josapine, (c) MD2, (d) N36, (e) MD2/T, (f) Sarawak Green Local,
(g) Morris Bentanggur, (h) Yankee and (i) Gandul (Hidayat, unpublished data).
13
2.3 Pineapple Breeding
From the year 1962 to 1970s breeding of pineapple in Malaysia concentrate
on clonal selection where from the year 1965 the breeding of ‘Sarawak’ (Smooth
Cayenne) and ‘Singapore Spanish’ was focused by Malaysian Pineapple Industry
Board (MPIB). These two cultivars were choose because both of it have its own
unique characteristics that is suitable for breeding. In 1974, the study of pineapple
from MPIB was taken over by Malaysian Agricultural Research Division Institute
(MARDI) (Leal and D’ Eecknbruggge, 1996).
‘Josapine’ is a cultivar that was hybrid by Malaysian Agricultural research
and Development Institute (MARDI) at the Integrated Peat Research Station in
Pontian, Johor. This cultivar was introduced in the means of improving the pineapple
quality. At first, four cultivars ‘Sarawak’ (‘Cayenne’), ‘Morris’ (‘Queen’), ‘Johor’
(‘Spanish’) and ‘Masmerah’ (‘Spanish’) were cross over in all pattern to produce 50
000 F1 progenies. After assessment, 300 selections were prepared and additional
assessment was done twice and subsequently six clones were selected in 1991.
These clones were subjected for several field trial tests in various environments to
ascertain the hybrids’ steadiness (Chan, 1997).
As a result from the field trial test, it was observed one hybrid that is A25-34
has excellent fruit characteristics such as good smell, elevated and stable sugar
content, resistant to black heart disease and can be a high quality hybrid for export
industry. Soon after that, this hybrid was named ‘Josapine’ since it was resulted from
the cross between ‘Johor’ and ‘Sarawak’ and officially released by MARDI on 5 th
August 1996 (Bartholomew et al., 2010).
Another cultivar that was released is ‘N36’. This is a hybrid that was chosen
from the crossover of ‘Gandul’ (‘Spanish’) and ‘Sarawak’ (Smooth Cayenne) by
14
MARDI. Additionally, this cultivar has average sized fruit (1.5 – 2 kg) as well as
large crown. ‘N36’ cultivar have an average of 12°–14° brix and a pale yellow color
which able this cultivar suitable for canning (Chan et al., 2003).
As the demand for the pineapple increased, the finding of a new cultivar that
has the good qualities which fulfill the fresh fruit market is really needed. Until the
mid 1900s, the world’s fresh fruit export was comparatively minor and was
depending on cultivar ‘Smooth Cayenne’ (Bartholomew, 2009).
Later on, the
world’s pineapple fresh fruit export industry undergoes an extraordinary revolution
after Del Monte Corporation bring ‘MD2’ pineapple for the first time to customers in
United States and Europe authoritatively in 1996 (Frank, 2003).
In 1970, there were many crosses was made and one of the cross intended to
become successful which was the cross between Pineapple Research Institute of
Hawaii (PRI) hybrids 58-1184 and 59-443. These crossed were planted in 1971 and
in 1973, two clones were chosen because it is believed to have good characteristics
(Bartholomew, 2009). Both of the clones (73-114-now known as MD2, and 73-50),
have parentage that is somewhat a complicated mixture of ‘Smooth Cayenne’,
‘Smooth Guatamelan’, ‘Ruby’ (a ‘Spanish’ group), Queen and ‘Pernambuco’. These
clones were free at 1980 by PRI to the members company Del Monte Corporation for
additional assessment (Bartholomew, 2009; Williams and Fleisch, 1993). Both of
these clones are more than 50% of ‘Smooth Cayenne’. Between both of the clones,
73-114 (MD2) have vast recognition and popularity among the consumers.
‘MD2’ is a dynamic plant with normal disease and pest resistant, great fruit
yield and excellent fruit quality. The excellent fruit quality consist of golden external
shell color, golden flesh color, somewhat soluble solid content than ‘Smooth
Cayenne’, significantly low acidity and higher vitamin C content. It is believed that,
in the international market, there is no other pineapple cultivar that support suitably
under refrigeration as ‘MD2’ (Bartholomew et al., 2010).
15
2.4 Importance of Pineapples
Pineapple is an amazing fruit and people discover it pleasant due to its sugary
and exotic taste. However, people might also enjoy the fruit because it is one of the
beneficial foods that are easily obtainable nowadays. In recent days, pineapple fruits
are famously consumed fresh or canned. The expanding trade of pineapple products
is an excellent approach to improve consumption in the major world markets. Some
of newer advance in pineapple product are dried chips, cocktail drink, dried powder
and canned forms as fruit bars and cubes. For fresh fruit intake, new cultivars have
been brought in to the market such as ‘Josapine’, ‘MD2’ and ‘Gandul’ (De La Cruz
and Garcia, 2005).
2.4.1 Nutritional Value
As pineapples are being consumed mostly as fresh fruit, its composition has
been examined. This fruit hold 81% to 86% moisture. The total solid (sucrose,
glucose and fructose) of pineapple is approximately 13-19%. In addition,
carbohydrates components are up to 85% while fiber makes up for 3%. Pineapple is
a tremendous source of vitamin C, manganese, vitamin B1, B6, copper and
magnesium (USDA, 2008). High content of Vitamin C is crucial for health because it
helps to cure injuries by assimilation of iron minerals into blood vessels. Vitamin
B’s particularly vitamin B1 helps in digestion process whereas vitamin B3 along
with carbohydrate substance is capable to manage a well digestive system. Water
steadiness in an individual can be control by the potassium and sodium in the fruit
(www.mpib.gov.my). A detailed nutrient composition of this fresh fruits is showed in
Table 2.1.
16
Another important property of pineapple is its proteolytic enzyme, bromelain.
Its enzyme is rich with bromelain which assist break down proteins meant for
digestion. In addition, it also has anti-inflammatory characteristics and used to lessen
swellings in situation of physical wound.
This enzyme is also used for meat
tenderizing (Morton, 1987).
With the intention to promote pineapple consumption, several nations
organize campaigns to make the people realize of the medicinal character of the fruit.
Several medicinal properties of this fruit are such as anti parasitic, help stomach
disorder and vermifuge. Another best properties of this fruit is it act as a diuretic
which assist to get ride toxins through urine and also help to lessen constipation
because of the fiber content of the pulp (De La Cruz and Garcia, 2005).
2.4.2 Food for Livestock
The byproducts of pineapple that derived from canning and juice extraction
where utilize as food for livestock. Pineapple crowns generally used to feed horses if
it does not use for replanting. The end product that is the waste from the factory
might be dried out as bran and used to feed cow, chicken and pig (FAO, 2004).
Pineapple is the one of crucial tropical fruit following banana and citrus. Due
to the importance of pineapple, it is relatively important to understand the molecular
genetics of this fruit. However, only little is known about genetics of this fruit and
the used of molecular markers is really needed in breeding programmes. The uses of
these markers could be remarkable if it can be related to agronomic qualities or to
pest resistance.
17
Table 2.1: Nutritional information of pineapple (USDA, 2008)
Composition
Amount milligrams (mg)
Manganese
2.56
Vitamin C
23.87
Vitamin B1(thiamine)
0.14
Copper
0.17
Dietary Fiber
Vitamin B6 (pyridoxine)
1860.0
0.13
Calcium
6.2-37.2
Nitrogen
38.0-98.0
Phosphorous
6.6-11.9
Iron
0.27-1.05
Ascorbic Acid
27.0-165.2
2.5 Ananas Genomic Study
As mention before, Ananas is one of the crucial genus in Bromeliaceae
family. Until now, there is only modest advancement in the pineapple genomics. The
existing data on Ananas is inadequate and majority diversity research generally
related on phenetic approaches based on morphology, isozyme and DNA markers
(Duval et al., 2003). The markers that had been used for the pineapple genetic
correlation are such as isozyme, RAPD, RFLP and chloroplast DNA in which the
majority works done are related to the genetic correlation of seven Ananas species
and the adjacent genus Pseudoananas with the purpose to find out their position
within Bromeliaceae family. This was done to elucidate the taxonomy and
phylogenetic analysis (Botella and Smith, 2008). Elucidation of genetic diversity in
pineapple is important to assist the improvement of breeding strategies as well as to
increase the results of hybridization programmes (Davey et al., 2007).
18
In addition to the study of relation between Ananas and Pseudoananas, there
were also small numbers of studies conducted to differentiate among different A.
comosus varieties. Ananas comosus cultivar that cultivated for fruits are categorized
on few groups based on the resemblance of morphological characters (Botella and
Smith, 2008). A study using RAPD conducted to analyze three commercial cultivars
groups of Cayenne, Queen and Spanish. This study showed that the first two groups
mentioned seem to in separate clusters in the dendogram while unsuccessful to place
the latter group in separate clusters (Sripaorya et al., 2001). On the contrary,
amplified fragment length polymorphism (AFLP) markers appear to be further
successful than RLFP for the evaluation of genetic diversity. A study by Kato et al.
(2004) using AFLP showed that this marker revealed a high percentage of genetic
variation among this species, however still ineffective to differentiate the most
important cultivar group.
2.6 Molecular Phylogenetic Analysis
Molecular phylogenetics is derived from the conventional method for
categorization of organisms corresponding to their resemblance and dissimilarity as
primarily practiced by Linnaeus in the 18th century. Then, the classification system
set up by Linnaeus turn out to be re-explain as phylogeny demonstrating not only the
resemblance among the species, however also their evolutionary relationship (Nei
and Kumar, 2000).
Whether the purpose is to build a categorization organisms or to conclude a
phylogeny, the significant information are gained by investigating variable characters
in the organisms being compared. Initially, the characters that have been compared
were morphological characters. However, later on, molecular data were used
(Brown, 2002).
19
Molecular phylogenetic is the study of the hereditary molecular diversity
primarily in DNA sequences. This is crucial because through this molecular
phylogenetic study the evolutionary relationship of species can be gathered. The
outcome of the molecular phylogenetic study is conveyed in phylogenetic tree (Fitch,
1971).
2.6.1 Molecular Phylogenetic Analysis in Plants using ITS Region
It seem most of the work done on Ananas previously were done to analyze
the genetic correlation of Ananas species and the adjacent genus Pseudoananas.
Some studies also have been conducted to separate the pineapple according to main
groups seem to appear less successful. Thus, a new approach to study the
phylogenetic relationship of pineapple is crucially needed.
Phylogenetic study is the most favored method to construct evolutionary
relationship of organisms with the purpose to understand the biodiversity. Cladistics
is one of the most general approaches used to understand evolutionary relationship.
In this approach, a group of organisms that have most of the characters shares
together is believed to be intimately connected and understood to be derived from
same ancestor. The ancestors and all its descendents will form monophyletic group
(Hennig, 1965). Previously, morphological characters have been used as for most of
the phylogenetic study but with current development of molecular techniques, the
DNA sequences have rather been used in phylogenetic study (Hambry and Zimmer,
1992).
When genetic materials such as DNA are studied, the external factor
influenced such as environment can be avoided. Genetic material has been passed by
ancestral to their offspring. Certainly, the DNA inherited by the offspring will reflect
20
their ancestor. Cultivar which is highly similar in their morphology and difficult to
be identified also can be distinguished by molecular method due to presence of
variation in DNA sequence among species (Vijayan and Tsou, 2010).
The use of outgroup in phylogenetic analysis are necessary which cause
polarization condition of apomorphic and plesiomorphic. Apomorphic condition is
derivative characters that appear in an ingroup while plesiomorphic condition is
ancient (primitive) character that appears in outgroup. Synapomorphic condition is
characters that are shared together between the ingroups (Fitch, 1971).
Phylogenetic analysis at DNA level is carried out using two fundamental
approaches where the first is by restriction analysis and the second one is using DNA
sequences. The latter is done by using PCR and DNA sequencing. There are three
genomes in plants accessible for research which is chloroplast, mitochondria and
nucleus. Therefore, several question arise on which is the proper genome must be
selected to explain the specific phylogenetic? This is due to that each gene evolved
not in the same rates and thus offers various level of genetic resolution among plants
groups (Hidayat and Pancoro, 2006).
Regulatory and coding sequences have tendency to be highly conserved and
normally show variability when comparing plants belonging to different genera,
families order, classes and divisions because more sequence variation seem to be
observed at higher levels. Examples of these loci comprise of rbcL chloroplast gene
and the nuclear ribosomal DNA gene. Nevertheless, most important controversies
regarding plant classification above family level are uncommon where adequate
morphological differences exists to position plants in the correct categories
The major controversies in plant systematic occur at the lower taxonomic
levels such as genus and species where morphological similarities can be so great as
21
to confuse proper classification. Therefore, when comparing plants at these levels it
is crucial to study the loci that can gather mutations very rapidly without having
deleterious effect on the organisms (Brinegar, 2009).
ITS region is one of beneficial DNA regions that have turn out to be familiar
in phylogeny analysis of many group of organisms. The ITS region of nrDNA has
been broadly apply in phylogenetic analysis of plants for family and higher levels. It
is also been used for closely linked genera or species. ITS region, which divides the
18S and 26S and the coding sequence region of 5.8S (Figure 2.3), has become
extensively describe between interspecific and intergeneric level differences
(Baldwin et al., 1995).
The ITS region are generally conserved within a species but show enough
variation between species and genera to be useful in the construction of phylogenetic
trees. The ITS 1 and ITS 2 are generally amplified and sequenced together with the
small 5.8S rDNA region, have been tremendously helpful in delimiting plant taxa
ITS region becomes the most important sequenced locus for plant molecular
systematic research (Blattner, 1999). Studies using ITS region have shown
confirmation on species level discrimination in the majority phylogenetic study.
There are huge amount of sequence data that by now presence for this ITS region
(Kress et al., 2005).
There are numerous common characteristics of ITS region that enable it to be
used in phylogeny study for angiosperms. Primarily, the ITS region is greatly
repeated in the plant nuclear genome where it exist in thousands copies which is
organized in a tandem repeat form. The great copy number of the ITS region make it
possible for detection, amplify, cloning and sequencing of nrDNA. Next, the ITS
region is believed to experiences fast concerted evolution which will be very
beneficial for phylogenetic study (Hamby and Zimmer, 1992). In addition, the small
size of the ITS region approximately 500-700bp in angiosperm and existence of two
22
conserved sequences flanking spacer enable this region to be easily amplified
(Baldwin et al., 1995).
Figure 2.3 Organization of Internal Transcribed Spacer (ITS) region of nuclear
ribosomal DNA (nrDNA
23
CHAPTER 3
METHODOLOGY
3.1 Experimental Design
The summary of experimental design is shown in the flowchart.
Sampling of pineapple leaves
In vitro
In silico




Genomic DNA extraction and purification
ITS region amplification by PCR
Ligation, transformation and blue white screening
Plasmid isolation





BLAST analysis
Edit and contig sequence by CODONCODE
Complete alignment by CLUSTAL-X
Phylogenetic tree construction by MEGA-4
Tree analysis
Figure 3.1 Flowchart shows summary of experimental design.
24
3.2 Sources and Storage of Pineapple Leaves
A total nine pineapple cultivars that were used in this study are Morris
Bentanggur (Mr. Bentanggur), Morris Gajah (M. Gajah), Sarawak Green Local
(Sarawak), MD2, MD2/Tissue Culture (MD2/T), Josapine, N36, Yankee and Gandul.
The sampling of the pineapple leaves was done at ‘Jana Plasma, Pusat Pembangunan
Taman Teknologi Nanas’(PPTTN), Pekan Nanas, Pontian, Lembaga Perindustrian
Nanas Malaysia’ (LPNM). During sampling, young and fresh leaves were selected,
labeled and kept in an appropriate storage bag. In the laboratory, these leaves were
stored in -20˚C freezer to maintain its freshness. The cultivars that have been
selected are the commercial pineapple cultivars in Malaysia.
3.3 Genomic DNA Extraction from Pineapple Leaves
Young leaves were grind in the presence of liquid nitrogen using mortar and
pestle. Even though leaves should be completely crushed before adding extraction
buffer, it is vital not to grind the leaves into a very fine powder as it results in
shearing of DNA (Lodhi et al., 1994). Total DNA were extracted from fresh material
plant tissue with a QIAGEN DNeasy Mini Plant Kit following the manufacturer’s
instructions with slight modifications. Genomic DNA isolation was qualitatively
analyzed by performing agarose gel electrophoresis. Purity of DNA was analyzed
using Nanodrop.
25
3.4 Polymerase Chain Reaction (PCR) Amplification
The ITS region was amplified using specific primer AB101 (forward) and
AB102 (reverse). Sequences of the respective primers were given in Table 3.1. The
PCR tubes that contain the reaction as in Table 3.2 were gently mixed and PCR
reaction was carried out by using MyCycler Thermal Cycler System from Bio-Rad.
The PCR cycle profile used is depicted in Table 3.3.
After PCR, 5µL of PCR products were analyzed on 1% Agarose gel by Gel
electrophoresis which was conducted at 80 volts, 240 watts, 240 amp for 50 minutes
Subsequently, the DNA band were visualized under UV Gene Flash to verify the
presence of the PCR products. PCR products were purified using HiYield Gel/PCR
DNA Mini Kit according to the manufacture’s instruction.
Table 3.1: Primer sequence used in this research
Primer
Sequence
AB101
5- ACG AAT TCA TGG TCC GGT GAA GTG TTC G-3
AB102
5- GAA TTC CCC GGT TCG CTC GCC GTT AC- 3
Table 3.2: Reagent for ITS region amplification
Solution
Volume (µL)
10x Standard Taq reaction buffer
5.00
10µM forward primer
1.00
10µM reverse primer
1.00
10µM dNTPs
1.00
Sterile distilled water
39.75
Taq DNA polymerase
0.25
Template DNA
2.00
TOTAL
50.00
26
Table 3.3: PCR amplification profile
Steps
No of Cycles
Initial denaturation
1
Denaturation
Annealing
30
Extension
Temperature
Duration
94˚ C
2 min
94˚ C
50 sec
55˚ C
1 min
72˚ C
30 sec
Final extension
1
72˚ C
7 min
Hold
1
4˚ C
∞ (forever)
3.5 Vector Insertion and Cloning
3.5.1 Ligation
One of the most important steps in the cloning process is the ligation of linear
DNA into a cloning vector. The standard reaction for ligation mixture were prepared
by mixing 5.0 µL of purified PCR with 5.0 µL of 2x rapid ligation buffer, 1.0 µL
pGEM-t easy cloning vector, 1.0 µL T4-DNA ligase. The reaction mixture then
mixed by pipeting and incubated in ice water for overnight (16 hours) in 4 ˚C
refrigerator. The summary of the reagents is shown in Table 3.4.
To confirm the performance of the reaction components, a positive control
ligation reaction was prepared to run concurrently with the experimental reaction.
Substitute the PCR product with 2 µL of control insert vector and add 1 µL of sterile
distilled water. The reaction composition for positive control ligation mixture is
depicted in Table 3.5.
27
Table 3.4: Preparation of ligation mixture
Solution
Volume (µL)
2x rapid ligation buffer
5.0
pGEM-t easy cloning vector
1.0
T4- DNA ligase
1.0
PCR product
5.0
TOTAL
12.0
Table 3.5: Preparation of ligation mixture for positive control
Solution
Volume (µL)
2x rapid ligation buffer
5.0
pGEM-t easy cloning vector
1.0
T4- DNA ligase
1.0
Control insert vector
2.0
Sterile distilled water
1.0
TOTAL
10
3.5.2 Transformation into NEB 5 Alpha Strain of Escherichia coli
Prior starting the transformation procedure, the incubator shaker was
preheated at 37 ˚C and water bath was set at 42 ˚C. In addition, the SOC media was
left to be equilibrated in room temperature. The ligation mixture was taken out from
refrigerator and equilibrated at room temperature for 1 minute. In the transformation
step, the 50 µl NEB-5-Alpha competent cell was thawed in ice for 5 minutes and 6 µl
of the ligation mixture was added to the competent cell. The mixture was carefully
flicked 4-5 times without vortexing. The mixture was placed on ice for 30 minutes.
Subsequently, heat shock the cells at 42 ˚C for 30 seconds and placed it again onto
ice for 5 minutes. Express changes in the temperature such as adding the plasmid to
the cells on ice makes the plasmid adhere to the cell wall. While the heat shock does
28
open the pores (made by the preparation of competent cells) and gets the plasmid to
enter the cell. Returning the cells on ice after the shock closes the pores and inhibits
the plasmid to escape (Subrata et al., 2008).
After that, SOC medium was added to the mixture and was followed by
incubation for one hour on the incubator shaker (250 rpm) at 37 ˚C. While waiting
for the incubation of the mixture, the LAIX plate (LB+ Ampicilin+ IPTG+ X-GAL)
was prepared. After one hour, the cell from the incubator shaker was mix thoroughly
by flicking and inverting the tube and 10 fold serial dilutions was prepared in SOC
medium. Then, the dilution was spread onto the LAIX plates using hockey stick.
Later on, the plates then were incubated overnight in incubator at 37 ˚C for 16 hours.
3.5.3 Blue White Screening
After incubation of 16 hours, the plates were screened for the selection of
white colony which is the possible positive colony that had insertion gene of interest.
The white colony that had been picked was subculture into another plate for the
purpose of backup. This was done by picking up the white colony using a sterile
pipette tips and put a drop in a new labeled grid plate. The grid plates then incubated
in 37 ˚C for 16 hours prior to be stored at 4 ˚C. The same white colony then was
dipped into the 5 mL LB broth for the purpose of plasmid extraction.
29
3.6 Plasmid Isolation
3.6.1 Preparation of E. coli
The white colony were picked and inoculated in 5 mL LB broth and
incubated overnight (16 hours) at 37 ˚C in a shaking incubator for 150rpm. After
that, the bacterial cell was harvested by centrifuging it at 4000 x g (4000 rpm) at 4 ˚C
for 15 min. Subsequently, after centrifuged, all the supernatant were removed and
continue with plasmid extraction.
3.6.2 Plasmid Extraction
Plasmids were extracted using QIAmp Spin Miniprep using manufacturer’s
instruction with slight modifications. The plasmid isolation was qualitatively
analyzed by performing Agarose gel electrophoresis.
3.6.3 Reconfirmation by Restriction Enzyme (RE) Digestion
The restriction enzyme digestion using EcoRI was done on the sample to
make sure the plasmid has the insert. The digestion mixture were added in 1.5 mL
micro centrifuge tube and mixed by pipeting prior EcoRI added. After adding the
restriction enzyme, the mixtures were gently mixed by pipeting.
Later on, the
mixture was incubated for 4 hours at 37˚ C water bath. Then, the mixture was
qualitatively analyzed by performing Agarose gel electrophoresis. The detailed of
digestion mixture is shown in Table 3.6.
30
Table 3.6: Digestion mixture preparation for insertion reconfirmation
Solution
Volume (µL)
Sterile distilled water
16.3
RE 10x buffer
2.0
Acetylated BSA
0.2
Plasmid
1.0
Restriction enzyme
0.5
TOTAL
20.0
3. 7 Bioinformatics Analysis
The plasmid samples were sent to 1st Base Laboratories for DNA
sequencing. The sequencing was done in two way using the primer SP6 and T7. The
obtained DNA sequences were edited and assembled using CodonCode Aligner
(http://www.codoncode.com/aligner/).
Then, the sequences were saved in fasta
format and followed by multiple alignments using ClustalX. Subsequently the
sequences were edited using BIOEDIT. The total nine samples sequences with an
outgroup sequences were subjected to phylogenetic analyses. The 10 aligned
sequences were used to construct the phylogenetic tree using MEGA version 4
(www.megasoftware.net). Phylogenetic analysis was done using the Parsimony
method. The principle of this method is that the differences observed among the
cultivar under the study were identified by minimization of character transformations
of a tree (Hidayat and Pancoro, 2008). The outgroup Encholirium scrutor (GenBank
ID: JN016950) which the data is available in the Gene Bank was used because it has
been recognized as sister group to the family Bromeliaceae (Terry et al., 1997).
31
CHAPTER 4
RESULTS AND DISCUSIONS
4.1 Genomic DNA Extraction
The band of genomic DNA extracted from nine pineapples samples are shown in
Figures 4.1 and 4.2.
L1
L2
L3
L4 L5 L6
L7
L8
bp
10000
8000
6000
gDNA
5000
4000
3000
2000
1500
1000
500
Figure 4.1 The result for genomic DNA(gDNA) extraction L1:1kb DNA Ladder,
L2: Josapine, L3: Morris Gajah, L4: Morris Bentanggur, L5: MD2, L6: Sarawak
Green Local
32
L1 L2
L3
L4
L5 L6
L7 L8
bp
10000
8000
gDNA
6000
5000
4000
3000
2000
1500
1000
500
Figure 4.2 The results for genomic DNA (gDNA) extraction L1:1kb DNA Ladder,
L2: Yankee, L3: Gandul, L4: MD2/T, L5: N36
The results were viewed by 1.5% Agarose gel electrophoresis. The size of
bands is approximately more than 10 Kb. Then the bands were cut and purified using
HiYield Gel/PCR DNA Mini Kit prior to PCR.
The ratio absorbance at 260/280 nm results that used to measure the purity of
DNA and RNA depicted that all the nine pineapple cultivars’ absorbance reading
were approximately 1.6-1.8. Generally the estimated ratio of 1.8 is accepted as pure
DNA while ratio about 2.0 accepted as pure RNA. Meanwhile, if the ratio is
noticeably lower in either case, it might be a sign of existence of protein, phenol or
other contaminants that absorb strongly at or near 280 nm (Van der Vlies, 2010).
During the sampling, appropriate selection of leaves is important because leaf
tissue is very crucial for DNA extraction. The use of very mature leaves should be
avoided and thus young leaves are much more preferred. In the latter leaves, the
contents of polysaccharides and polyphenols are in reduced amount. At the stage of
grinding the leaf samples, phenolics turn out to be oxidized and unite with protein
33
and nucleic acid irreversibly which happen to inappropriate for downstream work in
molecular study (Qiang et al., 2004). Polysaccharides make the DNA sticky, glue
like and not amplifiable during PCR reaction where it will prevent Taq enzyme
activity (Shankar et al., 2011).
4.2 Amplifying ITS Region using PCR
The results for the ITS region amplification were shown in the Figures 4.3
and 4.4.
L1
L2
L3 L4
L5
L6
L7 L8
bp
10000
8000
6000
5000
4000
3000
2000
1500
600bp
1000
500
Figure 4.3 The result for ITS region amplification using PCR L1:1kb DNA Ladder,
L2: Josapine, L3: Morris Gajah, L4: Morris Bentanggor, L5: MD2, L6: Sarawak
Green Local, L7: Yankee.
34
L1
L2
L3
L4
L5
L6
L7
L8
bp
10000
8000
6000
5000
4000
3000
2000
600bp
1500
1000
500
Figure 4.4 The result for ITS region amplification using PCR L1:1kb DNA Ladder,
L2: Gandul, L3: MD2/T, L4: N36.
From the Figures, it was shown the band was positioned at the size of ITS
gene which is approximately 600 bp. The sequences were amplified using forward
primer AB101 and reverse primer AB102 (Hidayat and Pancoro, 2010). In most of
the previous study, amplification of ITS region were done using primers ITS 5 and
ITS 4, but when amplification is not sufficient, AB101 and AB102 can be used
(Shimai et al., 2007).
The major challenge in this step was optimization of the annealing
temperature. Previous study of molecular phylogenetic of Orchidaceae using these
primers shows that 60 ˚C might be the good annealing temperature (Hidayat et al.,
2005), but in this research the suitable annealing temperature was 55 ˚C. The PCR
products for each cultivar were subjected to the cloning procedure by ligation of ITS
region sequence into pGEM-t easy cloning vector and subsequently transformed to
competent cell.
35
4.3 Blue White Screening
Figure 4.5 shows the results for blue white screening of the nine Malaysian
pineapple cultivars.
(a)
(d)
(g)
(b)
(c)
(e)
(f)
(h)
(i)
Figure 4.5 The results for blue white screening where blue colony indicates control
without insert and white colony indicates insert with gene of interest (a) Josapine, (b)
N36, (c) MD2, (d) MD2/T, (e) Gandul, (f) Morris Bentanggur, (g) Sarawak, (h)
Yankee (i) Morris Gajah
36
The plates were observed after overnight incubation (16 hours). Two to three
white colonies per sample were picked and prepared for plasmid isolation and subcultured onto another plate for backup purpose.
In the cloning method, blue white screening is one of the crucial procedures
that need to be done to ensure our ligation and transformation step end up successful
or not. Blue white screening frequently applied when attempting to clone a gene of
interest into a vector or plasmid. The vectors or plasmid have a gene identify as βgalactosidase (β-gal) and a gene for antibiotic interest. In the cloning plasmid, gene
of interest will be inserted in the middle of the β-gal thus inhibit the function of the
β-gal (Keese and Graf, 1996).
At the ligation step, not every plasmid gets a gene incorporate into it. All of
the ligation mixture added into bunch bacteria and spread into plates which contain
X-Gal, IPTG and antibiotic. Three things will happen after this step where if the
bacteria have no plasmid it will not grow while if the bacterial cells have plasmid
with no gene of interest inserted the β-gal on the plasmid will combine with X–Gal
and turn into blue colony. Meanwhile, the bacterial cell with plasmid which has the
inserted gene will produce white colony due to the β-gal gene function that has been
disturbed by the insert gene (Steege, 1977).
37
4.4 Plasmid Isolation
The Figures 4.6 to 4.9 shows the results of plasmid isolation.
L1
L2
L3
L4
L5
L6
L7
L8
bp
10000
8000
6000
Inserted plasmid
5000
4000
3000
2000
1500
1000
500
Figure 4.6 The result for plasmid extraction. L1:1kb ladder, L2: Control from blue
colony, L3: Josapine (a), L4: Josapine (b), L5: Yankee (a), L6: Yankee (b) L7:
Mr.Bentanggur (a), L8: Mr.Bentanggur (b).
L1
L2
L3
L4
L5
L6
L7
bp
10000
8000
6000
5000
4000
Inserted plasmid
3000
2000
1500
1000
500
Figure 4.7
The result for plasmid extraction. L1:1kb ladder, L2: Mr. Gajah (a), L3:
Mr. Gajah (b), L4: MD2/T (a), L5: MD2/T (b), L6: Sarawak (a), L7: Control from
blue colony.
38
L1
L2
L3
L4
L5
L6
L7
bp
10000
8000
6000
Inserted plasmid
5000
4000
3000
2000
1500
1000
500
Figure 4.8 The result for plasmid extraction. L1:1kb ladder, L2: Sarawak (b), L3:
MD2 (a), L4: MD2 (b), L5: Gandul (a), L6: Gandul (b) L7: Control from blue colony
L1
L2
L3
L4
L5
bp
10000
8000
6000
5000
4000
Inserted plasmid
3000
2000
1500
1000
500
Figure 4.9 The result for plasmid extraction. L1:1kb ladder, L2: Control from blue
colony, L3: N36 (a), L4: N36 (b), L5: N36 (c)
39
For each cultivar, two or three white colonies plasmids were isolated for the
purpose of backup. However, only one successful inserted plasmid from each
cultivar was send for sequencing. From the figure, the plasmid bands were compared
with a control which is the blue colony (without insert). The inserted plasmid bands
were higher than the control.
The size of the band shown in the Figures 4.6 to 4.9 is less a bit from the total
size of the inserted plasmid. The total size of the plasmid is 3010 bp whereas the
insert is 600bp. The total combination of the inserted plasmid should be
approximately 3610 bp. However, from the figure, the inserted plasmid size shown
around 2500 bp. This is because the plasmid is in circular and not in the linearised
form.
It is crucial to make sure that the inserts have ligated properly into the vector.
Thus, electrophoresis procedure is required to show combined insert and vector
where before electrophoresis, the vector should be linearised. This is because, at the
time the insert is ligated properly into the vector, it causes the vector change into a
circular plasmid. This structure is important in the transformation step for the
introduction of the DNA into the cells. But, this causes troubles at the time to prove
it in a gel electrophoresis because circular DNA and super coiled DNA run at
dissimilar rates to a linear piece of DNA of the same size. Thus, the inserted plasmid
should be digested using restriction enzyme to ensure the size of the band (Willshaw
et al., 1979).
40
4.5 Reconfirmation by Restriction Enzyme
The Figures 4.10 and 4.11 shows the results for reconfirmation using
digestion enzyme.
bp
10000
L1
L2
L3 L4 L5
L6
L7 L8
8000
6000
5000
4000
3010bp
3000
2000
1500
1000
600bp
500
Figure 4.10 The result shows reconfirmation using EcoRI. L1:1kb ladder, L2:
Josapine (a), L3: Josapine (b), L4: Yankee (a), L5: Morris Bentanggur (a), L6:
MD2/T (a), L7: MD2 (a), L8: Gandul (a)
L1
L2
L3
L4
L5
L6
L7
bp
10000
8000
6000
5000
4000
3010bp
3000
2000
1500
600bp
1000
500
Figure 4.11 The results shows reconfirmation using EcoRI. L1:1kb DNA ladder,
L2: N36 (a), L3: N36 (b), L4: N36 (c), L5: Sarawak (a), L6: Sarawak (b), L7: Mr.
Gajah (a)
41
EcoR1 is a restriction endonuclease found in Escherichia coli which cut DNA
at a specific sequence. pGEM-T Vector Systems used in this experiment have
recognition sites for EcoRI. Results of restriction digestion are shown in the Figures
4.10 and 4.11. From the figure, the presence of two bands which were the plasmid
without insertion (3010bp) and the insert (approximately 600bp) confirmed the
complete digestion by EcoR1. Only one colony per cultivar with a successful insert
was chosen before send for sequencing.
4.6 Phylogenetic Tree Construction and Analysis
The Figure 4.12 shows the consensus phylogenetic tree that was constructed
using MEGA-4 software.
Figure 4.12 Strict consensus tree from the parsimony analysis of the ITS region (3
Most Parsimonious Trees, Length= 831 steps, CI=0.888, RI= 0.842). The value in
the branch shows the bootstrap support (BS). The abbreviations of the cultivar were
shown in the key.
42
Nine clones from libraries of ITS region were sent to sequencing and
analyzed
by
numerous
bioinformatics
software
such
as
CLUSTAL-X,
CODONCODE and Molecular Evolutionary Genetics Analysis (MEGA-4).
The phylogenetic analysis was based on the maximum parsimony (MP) and
bootstrap reliability tests with 1000 replicates. The phylogenetic tree was constructed
using MEGA 4. Insertions and deletions were treated as missing data. The results
showed that the ITS region comprises 561 characters. Of these, 449 were potentially
informative and 112 characters were constant.
From the analysis, three phylogenetic trees were constructed. The consensus
tree based on these three constructed trees is shown in Figure 4.12. The consensus
tree was build with a consistency index (CI) of 0.888 and a retention index (RI) of
0.842 (Figure 4.12). The CI value will equivalents to 1 at time character reveal no
homoplasy. Therefore CI decreases as the quantity of homoplasy increases. Another
index to determine the robustness of the tree is the retention index. The RI of 1
suggest that the character is totally steady with phylogeny (it represents no
homoplasy), while an RI of 0 suggest the maximum quantity of homoplasy that is
probable (Masatoshi and Sudhir, 2000).
Furthermore, from Figure 4.12, it can be observed that the nine cultivars are
clearly separated into two major clades. Sarawak Green Local, Gandul, and N36
form the first clade whereas Josapine, Yankee, Morris Bentanggur, Morris Gajah,
MD2, and MD2 Tissue Culture (MD2/T) are in the second clade; both clades have
100% bootstrap support (BS).
As a general rule, if the BS value for a given interior branch is 90% or higher,
then the topology at that branch is considered statistically significant (Nei and
Kumar, 2000). The CI, RI and bootstrap values showed that the phylogentic tree
43
constructed for the nine commercial pineapple cultivar have a high confidence level
and is also robust . This showed that, ITS region can be a good candidate to study
the phylogenetic status and evolutionary relationship of pineapple cultivars.
The first clade shows that the cultivars Sarawak, Gandul, and N36 have a BS
of 100%. The Gandul cultivar is united with N36 (100 % BS), and the Sarawak
cultivar is grouped together with both of these cultivars (100 % BS) (Figure 4.12).
The second clade is further divided into two subclades, both of which have
100% BS. In the first subclade, the Josapine cultivar is united with the Yankee
cultivar with 100% BS. Morris Bentanggur and Morris Gajah cultivars form a sister
group to these cultivars. Meanwhile, the second subclade consists of MD2 and
MD2T cultivars, with 100% BS (Figure 4.12).
The second clade has Josapine, Yankee, Morris Bentanggur, Morris Gajah,
MD2, and MD2T grouped together to form a monophyletic group. In phylogenetic
analysis, a group of organisms that have a high similarity in their characteristics are
assumed to be closely related and to derive from a single ancestor, forming a
monophyletic group (Hidayat and Pancoro, 2008). In the second clade, all of the
cultivars shared synapomorphic characteristics such as presence of spines along the
leaf margin except for MD2 (spine at base only) and MD2T (spine at tip and base
only). It also have subulate leaf apex (Hidayat, unpublished data). This might be why
these six cultivars are grouped together in a clade.
The leaf margin is important in distinguishing between cultivars. Six leaf
margin types were originally described by Collins (1960): spiny, spiny tip, scallop,
smooth, piping, and sandpaper. The environment is known to influence the
expression of spines in some genotypic backgrounds, and this, if not properly
understood, can result in erroneous varietal descriptions. However, the leaf margin
44
type remains an important descriptor in pineapple, and is one of the distinguishing
characteristics of cultivars and botanical varieties. A spiny leaf margin can be
considered the usual condition in pineapple. It is easily identifiable, as the entire leaf
margin on all leaves is completely spiny. Spine shape, size, and density are highly
variable among cultivars (D’eeckenbrugge and Sanewski, 2011).
The consensus tree in Figure 4.12 shows that the Josapine cultivar is a sister
cultivar to Yankee although Yankee is from Queen group. In addition, there are some
morphological similarities between these two cultivars. For example, these two
cultivars have rough and long spines arranged along the margin. In addition both
cultivars have linear shape and smooth surface of leaves. The leaf color for upper and
beneath surface of both of these cultivars is brownish green and reddish green
(Hidayat, unpublished data).
However, there are still some morphological differences between these two
cultivars. The spines in the Josapine and Yankee cultivars are distantly and closely
arranged, respectively. The fruit shapes are also different: Josapine bears cylindrical
fruit, whereas Yankee bears tapered fruit. The eye shapes are projected in Josapine
but flat in Yankee (Hidayat, unpublished data).
In the consensus tree from Figure 4.12, it is seen that the Morris Bentanggur
(Mr. Bentanggur) and Morris Gajah (Mr. Gajah) cultivars are separated into two
different groups, even though both of them are derived from the same group. The BS
values for these two cultivars were low in the three phylogenetic trees generated (tree
not shown). There are many similarity in their morphologies such as have closely
arranged spine, with acuminate leaf shape and rough leaf surface and tapered shape
fruit with projected eyes (Hidayat, unpublished data). This might be because the
number of cultivars used in this study may have been insufficient. In phylogenetic
analysis, increasing the number of samples can help overcome this difficulty.
Therefore, further phylogenetic analysis is needed after more extensive sampling
(Hidayat et al., 2008).
45
From Figure 4.12, it can be observed that MD2 and MD2/T form the second
subclade in the second clade. The morphology of both of the cultivars is almost
similar such as it has fine and short spines. It also has smooth and shiny leaves with
cylindrical fruit shape and green orange fruit when ripe (Hidayat, unpublished data).
A few differences remain in the morphology of MD2/T and MD2 because MD2/T is
a tissue culture cultivar. Tissue cultures have shown that cells in long-term cultures
are genetically unstable. Genetic variations can also occur in the morphological traits
of regenerated plants. Thus, tissue culture sources are direct sources of genetic
variability (Glenda, 2008). Some variations are a result of specific genetic changes or
mutations and are transmitted to the progeny. Such genetically controlled variability
is known as somaclonal variation. This may hamper clonal propagation but at the
same time generate desirable somaclonal variants that can be selected for the
development of novel cell lines (Virginia and Ronald, 1992).
Furthermore, the source of explants has often been considered a critical
variable for somaclonal variation. Pineapple plants rose from the callus of slip,
crown, and axillary buds showed alterations only in spine characters, while those
rose from the callus of the syncarp showed variations in leaf color, spine wax, and
foliage (Radzan, 2003).
The cultivar MD2 and MD2/T were found to be more closely related to the
Queen and Spanish group than to the Smooth Cayenne. The history of MD2 cultivar
showed that it has parentage that is somewhat a complicated mixture of ‘Smooth
Cayenne’, ‘Smooth Guatamelan’, a ‘Spanish’ group, ‘Queen’ and ‘Pernambuco’.
This cultivar has more than 50% of ‘Smooth Cayenne’ parentage (Bartholomew,
2009; Williams and Fleisch, 1993). The relatedness of MD2 and MD2/T to the
Queen and Spanish group rather than to the Cayenne might be because the
environment adaption of this plant. Such outcome agrees with Aradhya et al. (1994)
who described and reported that the differentiation between species of Ananas might
be because of ecological adaptations.
46
The first clade shows that the cultivars Sarawak, Gandul, and N36 have a
high BS. There no much morphological similarities that are shared by these three
cultivars. But, both Gandul and N36 are united with Sarawak Green Local with
100% BS. The presence of these three cultivars in one clade might also be due to the
close relationship between them, because the N36 cultivar is a hybrid selected from a
cross between Gandul (Spanish) and Sarawak (Smooth Cayenne) (Chan et al., 2003).
The N36 cultivar, which was developed by the Malaysian Agricultural
research and Development Institute (MARDI), has special characteristics that render
it suitable for export by sea. One of the special characteristics of this variety is its
resistance to black heart disease. This is definitely beneficial for the industry,
especially for the fresh fruit market. With an average of 12°–14° Brix and a pale
yellow color, this variety is also suitable for canning (www.mpib.gov.my).
Even though N36 is a hybrid between Gandul and Sarawak, N36 is much
more closely related to Gandul with 100% BS. Figure 4.12 suggests that Gandul
could be a sister group to N36, because both these cultivars have similar
morphological characteristics on several parts of the leaves: both these cultivars have
long and fine spines that are closely arranged, and the leaf apex is subulate in both
cultivars (Hidayat, unpublished data).
Furthermore, both these cultivars show similarities in the cylindrical fruit
shape. The size of the fruit in both cultivars is medium and the weight is
approximately 1.8 kg. Moreover, both of these cultivars have crispy pulp
(http://www.mpib.gov.my/). This might be the reason why the cultivars Gandul and
N36, both from the Spanish group, are more closely related than N36 and the
Sarawak Green Local.
47
However, N36 cultivar also has some similarities with the Sarawak cultivar
in its pale yellow pulp color, acidity content, which is around 0.5–0.9%, and sugar
content (12°–16° Brix) (Chan et al., 2003).
Uses of outgroup in a phylogenetic study is very important because it causes
polarization condition of apomorphic and plesiomorphic. Encholirium scrutor is used
as outgroup in this study. This species is from Bromeliaceae family and subfamily of
Pitcairnioideae. As we know, A. comosus is also from the group Bromeliaceae and
subfamily Bromelioideae. A study shows that this Encholirium from the subfamily
Pitcairnioideae has been resolved as the sister group to the Bromelioideae. Character
state reconstructions for ovary position, fruit type, carbon metabolism and growth
habit indicate that the ancestral state for these characters in the Bromeliaceae is most
probably same as the most Pitcairnioids (Terry et al., 1997).
From the phylogenetic study, we can predict which is the group evolved
earlier compared to others. From the consensus tree (Figure 4.12), we can predict
that the second clade is the clade that evolved earliest. Within the second clade, the
first subclade is believed to have evolved first, followed by the second subclade. This
might be because the Josapine cultivar was developed earlier than the other hybrids
developed at the MARDI Integrated Peat Research Station in Pontian, Johor in 1984.
The name ‘Josapine’ was derived from ‘Johore’ and ‘Sarawak’, the parent cultivars,
and it was officially released by MARDI on 5 August 1996. Josapine was derived
from a deliberate cross and is the first successful commercial pineapple hybrid in the
world. Meanwhile, MD2 is a new cultivar in Malaysia and is believed to have been
developed by Pineapple Research Institute of Hawaii (Chan et al., 2003).
It is hypothesized that hybridization is possible among the different cultivars
from the same clade rather than between the two main clades. This is because
hybridization results in the combination of two or more attributes from different
cultivars. Therefore, the cultivars should have a similar genetic pattern and a
48
tendency to follow the same evolutionary path (Li and Graur, 1991). In other words,
plants that are hybridized should have a close evolutionary relationship.
In the context of phylogenetic analysis, plants should have a monophyletic
relationship, i.e. same ancestry. If the evolutionary relationship of the plants to be
hybridized is not monophyletic, hybridization is less or not feasible as they are not
genetically matched, as is usually the case for most hybridizations (Hidayat and
Pancoro, 2008).
49
CHAPTER 5
CONCLUSIONS AND FUTURE WORK
5.1 Conclusion
From the topology of the phylogenetic tree, the nine pineapple cultivars were
separated clearly into two main clades. From this research, it reveals that as the
cultivars are derived from the same species (A. comosus), the subgroups form a
monophyletic group, and the ITS region is believed to have the ability to determine
the phylogenetic status and relationship among pineapple cultivars.
From the phylogenetic tree constructed, it reveals that in the first clade,
Gandul cultivar is sister group to N36 cultivar and both of this cultivar united with
Sarawak cultivar suggesting that these cultivars might have same evolutionary
patterns. The second clade consists of Josapine, Yankee, Morris Bentanggur, Morris
Gajah, MD2, and MD2/T cultivar grouped together to form a monophyletic group.
The results also showed that the cultivars that are grouped together have a
similar genetic pattern, and therefore have a tendency to follow the same
evolutionary path. This information can be used as basic knowledge for successful
50
hybridization to generate new cultivars. Although the study is considered
preliminary, it provides new molecular data on the phylogenetic analysis of
commercial Malaysian pineapple cultivars by using DNA sequences of the ITS
region of nrDNA, as the existing molecular data on our Malaysian pineapple
cultivars is inadequate.
5.2 Future Work
There were several recommendations that can be used for the improvement of
this study. First of all, the extensive number of samples should be used such as all the
Malaysian pineapple cultivars can be used for the analysis. Next, several genetic
markers can be used and compared. Genetic marker from chloroplast such as matK
and rbcL can be studied and analyzed with the purpose to find the most appropriate
marker in order to study the evolutionary relationship of pineapple cultivars. Both of
increase in the number of samples and uses of different markers can produce a more
robust tree.
51
REFERENCES
Aradhya, M. K., Zee, F. and Manshardt,
R. M. (1994). Isozyme Variation in
Cultivated and Wild Pineapple. Euphytica. 79, 87–99.
Baldwin, B.G., Sanderson, M.J., Porter, J.M., Wojciechowski, M.F., Campbell, C.S.
and Donoghue, M.J. (1995). The ITS Region of Nuclear Ribosomal DNA: A
Valuable Source of Evidence on Angiosperm Phylogeny. Annals of the
Missouri Botanical Garden. 82(2), 247-277.
Bartholomew, D.P. (2009). MD2 Pineapple Transforms the World’s Pineapple Fresh
Fruit Export Industry.
Newsletter of the Pineapple Working Group,
International Society for Horticultural Science. July 2009.16, 2-5.
Bartholomew, D.P., Coppens d’eeckenbrugge, G. and Chen, C.C. (2010). Register of
New Fruit and Nut Cultivars List 4. HortScience, 45(5), 740-742.
Bartholomew, D.P., Paull, R.E. and Rohrbach, K.G. (2003). The Pineapple: Botany,
Production and Uses. Wallingford, UK: CABI Publishing.
Blattner, F.R. (1999). Direct Amplification of the Entire ITS Region from Poorly
Preserved Plant Material using Recombinant PCR. BioTechniques, 27, 11801185.
Botella, J.R. and Smith, M. (2008). Genomics of Pineapple, Crowning the King of
Tropical Fruit. In Moore, P. H. and Ming, R. (Eds.) Genomics of Tropical
Crop Plants (pp. 441-451). New York, USA: Springer.
52
Brewbaker, J.L. and Gorrez, D. D. (1967). Genetics of Self-Incompatibility in the
Monocot Genera, Ananas (Pineapple) and Gasteria. American Journal
Botany. 54, 611–616.
Brinegar, C. (2009). Assessing Evolution and Biodiversity in Plants at the Molecular
Level. Journal of Science. 5(2), 149-159.
Brown, T. A. (2002). Chapter 16: Molecular Phylogenetics. In Genomes. (2nd ed).
NCBI Bookshelf. A service of the National Library of Medicine, National
Institutes of Health 2002 Oxford: Wiley-Liss. Retrieved May 25, 2012 from
http://www.ncbi.nlm.nih.gov/books/NBK21122/.
Chan, Y.K. (1997). Josapine: A New Pineapple Hybrid Developed at MARDI.
Newsletter of the Pineapple Working Group, International Society for
Horticultural Science. March 1997. 3(1), 11-12.
Chan, Y.K., Coppens d’eeckenbrugge, G. and Sanewski, G.M. (2003). Chapter 3:
Breeding and Variety Improvement. In Bartholomew, D. P., Paull, R. E. and
Rohrbach, K.G. (Eds.) The Pineapple: Botany, Production and Uses. (pp.
33-51). Wallingford, UK: CABI Publishing.
Claudete de, F. P., Paulo, M.R. and Jose Renato, S.C. (2001). Assestment of
Genetic Relatedness of the genera Ananas and Pseudoananas Confirmed by
RAPD markers. Euphytica. 119, 245-252.
CodonCode Corporation, Better Software for DNA Sequencing.
Retrived
April
20, 2012 from http://www.codoncode.com/aligner/.
Collins, J. (1960). The Pineapple: Botany, Cultivation and Utilization. (pp. 294).
New York: Interscience Publishers.
53
D’ eeckenbrugge, C.G. and Sanewski, G. M. (2011). Leaf Margin in Pineapple.
Newsletter of the Pineapple Working Group, International Society for
HortScience. July 2011. 18, 32-37.
D’ eeckenbrugge, C.G., Leal, F. and Duval, M.F. (1997). Germplasm Resource of
Pineapple. In Jules, J. (Ed.) Horiculture Review. 21,133-175. Oxford, UK :
John Wiley & Sons, Inc.
D’ eeckenbrugge, C.G., Sanewski, G.M., Smith, M.K., Duval, M.F. and Leal, F.
(2011). Chapter 2: Ananas. In Kole, C. (Ed.) Wild Crop Relatives: Genomic
and Breeding Resources, Tropical and Subtropical Fruits. (pp. 21- 41).
Berlin Heidelberg: Springer.
Davey, M.R., Sripaoraya, S., Anthony, P., Lowe, K.C. and Power, J.B. (2007).
Pineapple. Biotechnology in Agriculture and Forestry. In Pua, E.C. and
Davey, M.R. (Eds.) Transgenic Crops. (pp. 97-127). Berlin Heidelberg:
Springer-Verlag.
De La Cruz, M. J. and Garcia, H.S. (2005). Pineapple: Post-harvest Operations.
Organization: Instituto Tecnologico de Veracruz (http://www.itver.edu.mx).
Last reviewed: 13/11/2005.
Dull, G. G. The Pineapple: General. (1971). In Hulme, A. C. (Ed.) The Biochemistry
of Fruits and Their Products. (pp. 303-331). London: Academic Press.
Duval, M.F., Buso, G.S.C., Ferreira, F.R., Noyer, J.L. and Coppens d’Eeckenbrugge,
G. (2003). Relationship in Ananas and Other Related Genera Using
Chloroplast DNA Restriction Site Variation. Genome. 46, 990-1004.
Duval, M. F., Noyer, J.L., Perrier, X., D’Eeckenbrugge, G.C. and Hamon, P. (2001).
Molecular Diversity in Pineapple Assessed by RFLP markers. Theorotical
Applied Genetics. 102, 83-90.
54
Fitch, W.M. (1971). Toward Defining The Course Of Evolution: Minimal Change
For A Specific Tree Topology. Systematic Zoolology. 20, 406-416.
Food and Agriculture Organizational (FAO). (2004). Retrieved 10 May 2012, from
http://www.fao.org/livestock/agap/frg/afris/espanol.
Frank, R. (2003, October 2003). Going for 'The Gold' Turns Pineapple World Upside
Down.Retrieved
May
25,
2012,
from
http://www.economics.utoronto.ca/ahussain/Baboserver/Articles/pineapple_p
atent.pdf.
Glenda, E.G. (2008). Chapter 4: Plant development and Physiology. In: Stewart, C.
N. (Ed.) Plant Biotechnology and Genetics: Principles, Techniques, and
Applications. Hoboken, NJ, USA: John Wiley & Sons, Inc.
Hall, A. E., Fiebig, A. and Preuss, D. (2002). Beyond The Arabidopsis Genome:
Opportunities for Comparative Genomics.
Plant Physiology. 129, 1439–
1447.
Hambry , R.K. and Zimmer, E.A. (1992). Ribosomal RNA as a Phylogenetic Tool
in Plant Systematic.
In Soltis, P.S., Soltis, D.E. and Doyle, J.J. (Eds.)
Molecular Systematics of Plants. (pp. 50-91). New York: International
Thompson Publishing.
Hennig, W. (1965). Phylogenetic Systematic. Annual Revision Entomology. 10, 97116.
Hidayat, T., Yukawa, T. and Ito, M. (2005). Molecular Phylogenetics of Subtribe
Aeridinae (Orchidaceae): Insights from Plastid matk and Nuclear Ribosomal
ITS Sequences. Journal of Plant Research. 118,271–284. Tokyo: The
Botanical Society of Japan and Springer-Verlag.
55
Hidayat, T. (2012).
Morphological Characteristic and Phylogeny of Malaysian
Pineapple Cultivar. Unpublished Research Report. University Technologi
Malaysia.
Hidayat, T. and Pancoro, A. (2006). Short Communication: DNA Technology and
Studies in Phylogenetic Relationship of Tropical Plant: Prospect in Indonesia.
Paper presented at International Conference on Mathematics and Natural
Science on 29- 30 November 2006.
Hidayat, T. and Pancoro, A. (2008). Kajian Filogenetika Molekular dan Perananya
Dalam Menyediakan Informasi Dasar Untuk Meningkatkan Kualitas Sumber
Genetik Anggrek. Jurnal Agrobiogen. 4(1), 35-40.
Hidayat, T. and Pancoro, A. (2010). Single Strand Conformation Polymorphism
Method for Initial Detection DNA Sequences Homogeneity. Hayati Journal
of Biosciences. 17(1), 50-52.
Hidayat, T., Kusumawaty, D.K., Yati, D.D., Agusthina, A.M. and
(2008).
Analisis
(Euphorbiaceae)
Filogenetik
Menggunakan
Molekuler
Urutan
Pada
Basa
Mariana, D.
Phyllanthus
DNA
Daerah
niruri
Internal
Transcribed Spacer (ITS). Jurnal Matematika Dan Sains, Maret. 13(1), 1621.
Keese, P. and Graf, L. (1996). A positive screen for cloning PCR products. Nucleic
Acids Research. 24(17), 3474-3475.
Kress, W. J., Wurdack, K. J., Elizabeth, A. Z., Weigt, L. A. and Janzen, D. H.
(2005). Use of DNA Barcodes to Identify Flowering Plants. Proceedings of
the National Academy of Sciences. 102(23), 8369–8374.
Leal, F.J. and D’Eeckenbrugge, C.G. (1996). Chapter 9: Pineapple. In Janick, J. and
Moore, J. N. (Ed.) Fruit Breeding,Vol 1, Tree and Tropical Fruits. (pp. 565606). New York: John Wiley & Sons.
56
Li, W.H. and Graur, D. (1991). Fundamentals of Molecular Biology. (2nd ed.)
Sinauer Sunderland Associates: Massachusetts.
Lodhi, M. A., Ye, G.N., Weeden, N. F. and Reisch, B. I. (1994). A Simple and
Efficient Method for DNA Extraction from Grapevine Cultivars, Vitis
Species and Ampelopsis. Plant Molecular Biology Reporter. 12(1), 6-13.
Malaysian Agricultural Research and Development. Retrieved March 12, 2012 from
www.mardi.my.
Malaysian Pineapple Industrial Board. Kultivar Nanas. Retrieved March 12, 2012
from from http://www.mpib.gov.my/web/guest/asal_usul_nanas.
Mendiola, N.B., Capinpin, J.H. and Mercado, T.M. (1951). Pineapple Breeding in
the Philipines. Philippine Journal Agricultural. 16, 51-84.
Molecular Evolutionary Genetics Analysis. Retrieved from April 20, 2012 from
www.megasoftware.net.
Morton, J. (1987). Pineapple. In Morton, J.F. Fruits of Warm Climates (pp. 18–28).
Miami: Morton Collectanea.
Munro, D. (1835). Classification of Pineapple Varities. Transactions of London
Horiculture Society. 1,1-34.
Nei, M. and Kumar, S. (2000). Chapter 7: Phylogenetic Inference: Maximum
Parsimony Method. In Nei, M. and Kumar, S. Molecular Evolution and
Phylogenetics. (pp. 115-143). Newyork: University Press Incorporation.
Py, C., Lacoeuilhe, J. J. and Teisson, C. (1987).
Part 1: The Plant and the
Environment. In Py, C., Lacoeuilhe, J. J. and Teisson, C. The
Pineapple: Cultivation and Uses. (pp. 29-72). Paris: Maisonneuve et Larose.
57
Qiang, X., Xiaopeng, W. and Deng, X. (2004). A Simple Protocol for Isolating
Genomic DNA From Chestnut Rose (Rosa roxburghii Tratt) for RFLP and
PCR Analyses. Plant Molecular Biology Reporter. 22, 301-301.
Radzan, M.K. (2003). Introduction to Plant Tissue Culture. (2nd ed.) Science
Publishers. Isbn 1-57808-237-4.
Samuels, G. (1970). Pineapple Cultivars. Pro. Tropical Region America Society
Horticultural Science. 14, 13-24.
Shankar, K., Chavan, L., Shinde, S. and Patil, B. (2011). An Improved DNA
Extraction Protocol from Four in vitro Banana Cultivars. Asian Journal of
Biotechnology, 3, 84-90.
Shimai, H., Masuda, Yu., Valdes Panfet, C.M. and Katsuhiko, K. (2007).
Phylogenetic Analysis of Cuban pinguicula
Internal
Transcribed
Spacer
(ITS)
(Lentibulariaceae) based on
Region.
Chromosoime
Botany.
International Society of Chromosome Botany. 2, 151-158.
Soltis, D. E. and Soltis, P. S. (2003). The Role of Phylogenetics in Comparative
Genetics.Plant Physiology. American Society of Plant Biologists. 132, 17901800.
Sripaoraya, S., Blackhall, N.W., Marchant, R., Power, J.B., Lowe, K.C. and Davey,
M.R. (2001). Relationships in Pineapple by Random Amplified Polymorphic
DNA (RAPD) Analysis. Plant Breeding. 120, 265–267.
Steege, D.A. (1977). 5′-Terminal Nucleotide Sequence of Escherichia coli Lactose
Repressor mRNA: Features of Translational Initiation and Reinitiation Sites.
Proceedings of the National Academy of Science of the United States of
America. 74(10), 4163–4167.
58
Subrata, P., Pulakesh, A., Bimal, J. and Tarakdas, B. (2008). How Does Plasmid
DNA Penetrate Cell Membranes in Artificial Transformation Process of
Escherichia coli? Molecular Membrane Biology. 25(5), 411-422.
Terry, R.G., Brown, G.K. and Olmstead, R.G. (1997). Examination of Subfamilial
Phylogeny in Bromeliaceae Using Comparative Sequencing of the Plastid
Locus ndhf. American Journal Botany. 84(5), 664–670.
United States Department of Agriculture (USDA) National Nutrient Database for
Standard
Reference.
(2008).
Retrieved
April
20,
2012
from
http://www.ars.usda.gov/Services/docs.htm?docid=8964.
Van der Vlies, P. (2010). Guidelines DNA Quantity and Quality for Infinium and
GoldenGate projects. Version 1.
UMCG Genetica – Genome Analysis
Facility.
Vijayan, K. and Tsou, C.H. (2010). DNA barcoding in Plants: Taxonomy in a New
Perspective. Current Science. 99, 1530–1541.
Virginia M.P. and Ronald, L.P. (1992). Genetic Implications of Somaclonal
Variation in Plants. In John G. S, Theodore R. W. (Eds.) Advances in
Genetic. 30, 157-168.
Wee, Y. C. (1972). Some Common Pineapple Cultivars in West Malaysia. Malaysia
Pineapple. 2, 7-13.
Williams, D.D.F. and Fleisch, H. (1993). Historical Review of Pineapple Breeding in
Hawaii. Acta Horticulturae. 334, 67-76.
Willshaw A.G., Smith, H. R. and Anderson, E. S. (1979). Application of Agarose
Gel Electrophoresis to the Characterization of Plasmid DNA in Drug-resistant
Enterobacteria. Journal of General Microbiology. 114, 15-25.
59
APPENDICE
Appendix A: Complete alignment of the nine sequences of Malaysian pineapple
cultivar using ClustalX software. The black color region in the alignment shows the
consensus sequence of the nine pineapple cultivars.