DEPARTMENT OF HORTICULTURE UNIVERSITY OF AGRICULTURAL SCIENCES GKVK, BENGALURU 2009
ESTIMATION OF GENETIC DIVERSITY IN JAMUN (
Syzygium cumini L.) USING MOLECULAR MARKERS
H.S. SIDDARTHA PHK - 714
Thesis submitted to the University of Agricultural Sciences, Bangalore in partial fulfillment of the requirements MASTER OF SCIENCE (HORTICULTURE)
BENGALURU JUNE, 2009
DEPARTMENT OF HORTICULTURE UNIVERSITY OF AGRICULTURE SCIENCES BANGALORE CERTIFICATE This is to certify that the thesis entitled “ESTIMATION OF GENETIC DIVERSITY IN JAMUN ( Syzygium cumini. L.) USING MOLECULAR MARKERS” submitted by H.S. SIDDARTHA PHK-714 for the degree of
MASTER OF SCIENCE (Horticulture) in FRUIT SCIENCE to the University
of Agricultural Sciences, G.K.V.K., Bangalore, is a record of research work done
by him during the period of his study in the University under my guidance and
supervision and the thesis has not previously formed the basis for the award of any
degree, diploma, associateship, fellowship or other similar titles.
Bengaluru (Dr. P. NARAYANASWAMY)
JUNE, 2009 Professor of Horticulture
Chairman of Advisory Committee
Approved by: Chairperson : __________________________ (P. NARAYANASWAMY)
Members : 1. __________________________
(V. NACHEGOWDA) 2.
(B.N. SATHYANARAYANA) 3.
I take this opportunity with pleasure to my deep sense of gratitude and
indebtedness to Dr. P. Narayanaswamy, Professor of Horticulture, UAS, GKVK,
Bangalore and Chairman of my Advisory Committee for his meticulous guidance,
transcendent suggestions, constructive criticism, sustained interest and constant
encouragement right from the conceptualization of the research work to the preparation
of this documentary proof.
I would like to express my sincere thanks to the members of my Advisory
Committee Dr. V. Nachegowda Professor, Department of Horticulture,
Dr. B.N. Sathyanarayana Professor, Department of Horticulture and
Dr. S. Shyamalamma, Associate Professor, Department of Biotechnology, UAS, GKVK
Bangalore, for their valuable help and advice during the course of my study.
I greatly acknowledge the technical help received from Mr. Bharath Kag,
Mrs. Moumita Ghosal and Mrs. Sumana Saha, Mrs. Rhiya, Mrs. Bhavna, Technical
Assistants, Plant Molecular Biology Lab., Division of Horticulture, UAS, GKVK and
Mrs. Sadana Assistant Professor, Presidency College for molecular biology works.
I owe my sincere and heartfelt thanks to my close friends Mrs. Krishnaveni
M.Sc. (Microbiology), TNAU Coimbatore and Mrs. Sathya, B.Sc., (Biotechnology).
I owe my sincere and heartfelt thanks to my beloved hostel mates of both VSFH
and KSV hostels and special thanks to R. Manjunath, he hosted hostel facility in
VSFH four years freely.
I am thankful to the professor G.S.K. Swamy of Horticulture College KRCH
Arabhavi. He helped me in collection of leaf samples.
My special thanks to Mr. Bharath Kag, for mutual help and sharing many
views aspect of molecular biology works.
I express my sincere thanks and affection to my father Mr. K. Shivalingappa
mother Mrs. K. Jayalakshmi grand mother Mrs. Laxhmamma and my uncle
Padmanabha, aunt Sharada brothers S. Hoysala, Sharath, Darshan and family for
their sensible co-operation and encouragement to pursue my studies and moral support.
I thank brothers Mr. Ali Quid Ahmed and Mr. Pampanna Ph.D.(students),
Division of Horticulture, UAS, GKVK, Bangalore for mutual help and sharing many
views on various aspects. I thankful to my beloved junior Mr. Rane Division of
Horticulture (Fruit Science),
I wish to kindly acknowledge the UAS Bangalore for granting me URMS
(university merit scholarship) during the course of this investigation.
I would also like to pay humble regards from my heart for all who have
contributed directly and indirectly to successfully carryout the present study.
Finally, I am thankful to University of Agricultural Sciences, Bangalore, for
giving me opportunity to pursue me my post graduation successfully.
June, 2009 (H.S. Siddartha)
THESIS ABSTRACT Jamun is an important minor fruit of tropical and subtropical countries and
widely distributed throughout India. It is a versatile tree for both food and
medicinal values. Totally, sixty jamun individuals were analyzed in the present
investigations. The leaf samples of genotypes were collected from GKVK i.e. in
the Sericulture Department, Farm section and in and around Bangalore. About 32
accessions were collected from KRC college of Horticulture Arabhavi Belgaum.
Randomly Amplified Polymorphic DNA (RAPD) markers have been used to
estimate the genetic diversity among 60 genotypes of jamun. PCR amplifiable
DNA was isolated using the CTAB method and 62 bands were amplified from 9
random 10-mer primers. The genetic dissimilarity matrix calculated based on
Euclidean distance revealed a maximum genetic distance of 78 per cent between
the genotype SERI 5 and SERI 2, while it was minimum (3 %) between Bangalore
Local 1 and Bangalore Local 3.
The present studies showed a moderate (3 %) to high (78 %) genetic
diversity and in the dendrogram almost all the Arabhavi genotypes and FSO
genotypes clustered together compared to others. This technique is useful in
estimation of genetic diversity and also could be a first step towards efficient
germplasm management of jamun in India.
Signature of the Student Signature of the Major Advisor
CONTENTS CHAPTER TITLE PAGE No. I INTRODUCTION
LIST OF TABLES Table No. Title Page No. 1
List of jamun genotypes collected from different location
Preparation of stock solution (extraction)
Preparation of CTAB buffer 25 ml
Preparation of loading dye
Preparation of running buffer (5x)
Preparation of TE buffer
Preparation of gel for DNA electrophoresis (120 ml)
Reagents used for PCR amplification
PCR Amplification procedure
List of nine primers and their sequences showing good
purity of DNA in jamun
Primers and their amplification
Dissimilarity matrix of 60 genotypes of jamun
LIST OF FIGURES Fig. No. Title Between Pages 3.1 Karnataka map showing plant sampling and experimental sites
1 Gel profile showing high molecular weight DNA of jamun
before RNAse treatment
Gel profile showing high molecular DNA of jamun after RNAse
3a RAPD profile of the 60 jamun genotypes according to OPA-13 90-91
3b RAPD profile of the 60 jamun genotypes according to OPA-18 90-91
3c RAPD profile of the 60 jamun genotypes according to OPC-2 90-91
3d RAPD profile of the 60 jamun genotypes according to OPC-7 90-91
3e RAPD profile of the 60 jamun genotypes according to OPD-11 90-91
3f RAPD profile of the 60 jamun genotypes according to OPF-10 90-91
3g RAPD profile of the 60 jamun genotypes according to OPF-16 90-91
3h RAPD profile of the 60 jamun genotypes according to OPH-15 90-91
3i RAPD profile of the 60 jamun genotypes according to OPJ-16 90-91
4 Dendrogram showing genetic relationship among 60 genotypes
based on RAPD markers according to Ward’s method
of 60 jamun genotypes based on
I. INTRODUCTION Jamun (Syzygium cumini L) belongs to the family Myrtaceae, possesses
commercial importance but considered as a minor fruit in tropical and subtropical
countries. It is a versatile tree of both food and medicinal values. Fruits have high
demand for table purpose and for the preparation of wide variety of products.
Jamun is known by different names such as Java plum, Black plum, Kalajam,
Phalinda and Rajamun.
Jamun is native to India (Singh, 1969) and trees are grown almost
throughout the country. Apart from India, it is also grown in Thailand, Philippines,
Madagascar, West Indies, East Indies, West Africa and Israel.
Tree grows tall and is evergreen. It also has ornamental value,
Inflorescences are borne in leaf axils of branchlets. Flowers are bisexual and light
yellow in colour. Jamun is cross pollinated tree. Droping of flowers and fruits is a
serious problem in this crop. Fruits are oblong and round in shape, deep purple or
bluish with juicy sweet pulp with single seeds. Distinct varieties are not available;
the most common type cultivated in India is Rajamun, which produces large sized,
oblong shaped fruits of deep purple colour with juicy sweet pulp and small stones.
A large fruited type is known as Paras, which is a seedling selection from Gujarat.
Small fruited types are also seen and these fruits are preferred for processing than
for the table purpose. There exist a large number of local seedling strains of this
crop which provide great scope for the selection of better types.
Jamun fruits have considerable nutritive value, they are rich in iron content.
Fruits are relished for their taste and pleasant flavour, and are used for table
purpose. High tannins content is mainly responsible for astringency and purplish
colour which is due to anthocyanin pigments. Three esters viz., dihydrocarvyl
acetate, geanyl butyrate and terpinyl valerate are responsible for the flavor of the
fruits (Vijayanand et al., 2001).
Fruits are used for treatment of diabetics, dysentery and digestive problems.
The fruit syrup is useful for the curing diarrhea. It markedly lowers the blood
pressure. Seed powder is used in treatment of diabetics, which helps to reduce
urine sugar very quickly and permanently. Seeds contain an alkaloid ‘jambosin’
and glycoside ‘jamolin’ which reduce diastatic conversion of starch into sugars.
Jamun leaves have medicinal value and are being used for correcting stomach
Domestication and crop improvement programmes are enhanced by
knowledge of genetic structure of the existing species and collection, which also
helps to capture high level of diversity in breeding programmes. In a perennial
crop like jamun an important way to develop a new high yielding variety with
specific desirable characters is initially by selecting superior genotypes based on
the phenotypic features from among the existing variants and use them directly in
the future breeding programmes. For this, it is essential to identify and effectively
conserve the existing genetic resources with an utmost certainty.
Traditionally, jamun is propagated by seeds, hence there is considerable
variation among the trees, owing to its cross pollination in nature (Purseglove
1981). Thus, a great deal of variation is oberserved in trees and fruits within jamun
populations. Crop improvement work in jamun is through selection of seedlings
trees based on promising horticultural characteristics. Varieties are being selected
and naming is based on origin, fruit shape, colour etc. The actual identity of cultivars is still in question because similar genotypes
grown in different regions often have various names or synonyms.
Usually the maintenance and evaluation of germplasm is based on
phenotypic features which have limited value, as the plants are grown in different
locations, and only specific developmental stages are suitable for screening and
detection of hybridization and pedigree determination. This is because, the genetic
control of any such characters is complex. Sometimes exhibiting delayed gene
expression and demands higher cost and enormous efforts in perennial crops like
jamun. One approach to reduce the cost is to develop a core collection (Frankel
and Brown, 1984), which represents the genetic diversity of a crop species and its
relatives with minimum repetitions. The main purpose of core collection is to
characterize the germplasm that is preserved and eliminate duplicates and it should
represent the diversity at hand. In any case, the immediate priority is to catalogue
the available germplasm and identify duplicates in the germplasm collection
maintained at various centers in India. However, the other problems like
environment, human visual judgments and less genome coverage limit the use of
phenotypic markers. Such limitations can be largely avoided at molecular levels
by using DNA based markers that are seldom influenced by environmental factors.
The studies on the genetic polymorphisms can provide a scientific basis for
utilization of these phenotypes for the efficient crop improvement and also could
provide sufficient information on origin and evaluation of available jamun
germplasm. Large seededness, fruit and flower drop, and astringency in fruits are
the major problems in jamun. There are no improved varieties in jamun so there is
ample scope for developing small seeded varieties or hybrids with more pulp
recovery, less astringent and having better shelf life.
There is an increasing need for molecular methods to characterize, to define
genotypes, to control quality and to find out genuinity for the prevention of
fraudulent commerce. Identification of genotypes through utmost certainty is a
prerequisite to patent and claiming Plant Varietal Rights.
Among different types of molecular markers available RAPDs (Randomly
Amplified Polymorphic DNA) are attractive because of their simplicity,
versatility, modest cost and ability to detect even relatively small amount of
variations (Rago and Hoisington, 1993).
Molecular markers are derived from Polymerase Chain Reaction (PCR)
amplification of genomic DNA and are an important part of the tool kit of
evolutionary genetists. RAPDs and Inter Simple Sequence Repeat (ISSR), Simple
Sequence Repeats (SSR) polymorphisms allow analysis of species for which
previous DNA sequence information is lacking (Holsinger et al., 2002). Among
myriad of markers, RAPD is simple, fast and an easy assay, which acts as an
excellent tool to characterize the germplasm, study phylogenetic relationships and
gene tagging. Such precise and refined techniques appear to have not been utilized
so far in the genus Syzygium cumini and hence, the present investigation.
An investigation entitled “Estimation of genetic diversity in jamun
Syzygium cumini L)
using molecular markers” was carried out with the following
1. Standardization of procedures for RAPD in jamun
2. RAPD characterization of jamun cultivars and estimation of genetic diversity
II. REVIEW 0F LITERATURE 2.1. Importance Jamun belongs to Myrtraceae family consists of about 90 genera and 2800
species. Jamun is a large ever-green tree of approximately 3.6 m girth and 30 m
high. Generally two main varieties of jamun are distinguished based on the type of
fruit. The Raa Jamun fruit has sweet flesh with a central cavity containing small
seeds. While desi jamun fruit has relatively large seeds and acidic flesh. Generally
this tree is cultivated on the main boundaries of fruit gardens. The annual
production of this fruit in Pakistan is about 5772 tones. The fruit size is variable in
size up to 2.5 mm (Purohith, 1985).
Jamun seed can be used as a concentrate for animals because it is rich in
protein, carbohydrates and calcium. Its wood is used for railway sleepers,
buildings, agricultural implements and furniture’s making as the wood resists alien
of water (Purohith, 1985). It is widely distributed in tropical and subtropical parts
of India, Sri Lanka, Malaysia, Thailand, Australia and Philippines (Popenoe,
1920). Jamun has been successfully introduced in to many subtropical countries.
In India, maximum number of jamun cultivation is found scattered through out the
tropical and subtropical regions. It is also growing in lower ranges of Himalaya up
to an elevation of 1300 meters and in the Kumayun hills up to 1600 meters.
The jamun fruits are good source of iron, minerals and proteins. The fruits
are tasty and pleasantly flavoured and hence very much liked by masses. They are
mostly used for desert purpose and also in preparations of delicious beverages,
jellies, jam, squash, wine, vinegar and pickles (Oschse et al., 1961). The nutrient
content of 100 g of fresh fruits are 19.7 g carbohydrate, 0.7g protein, 0.1 g fat, 1.0
g iron, 0.02 g calcium, 0.01 g phosphorus and 0.9 g fibre. Besides taken as a
dessert fruit, it is also used for making value added products like beverages,
squash, jam, jelly and wine.
Jamun juice is very refreshing drink in summer season. Little quantity of
fruit syrup is useful for curing diarrhea and diabetes. Vinegar prepared from the
juice extracted from slightly unripe fruits can cure stomach-ache, carmative and
diuretic apart from having cooling and digestive properties (Thaper, 1958).
2.2 Origin and distribution Jamun is native to India (Singh, 1969) and tress are seen almost throughout
the country. According to De Candole, and Vavilo (Decandolle. 1904) and others
it is originated in Indonesia and cultivation extended towards India Apart from
India, this fruit crop is grown in Pakistan, Thailand, Philippines, Madagascar,
West Indies, East Indies and West Africa, Israel, Malaysia, Australia and tropical
America It also grown to some extent in California Florida, Mexico, South Africa
and Mediterranean countries, Egypt and other parts of the North Africa, Arab,
Palestine, Persia. It thrives well in all tropical countries. Syzygium species of horticultural interest found in India (Chaturvedi et al., 2004)
Species Distribution S. cumini Indo-Gangetic plains of north India, Tamil Nadu in south, widely
S.arnottianum Western Ghats, the Nilgiris, Palni and Anamalai Hills
S. bracteatum Western Ghats, Eastern/north-eastern India
S.operculatum Grows wild in Nilgiri Hills of Tamil Nadu, Western Ghats
S. aqueum Mainly in Assam, Sikkim and Meghalaya, Eastern/ north-eastern India
S. fruiticosum Grows as an avenue tree, widely distributed