Department of horticulture university of agricultural sciences gkvk, bengaluru



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ESTIMATION OF GENETIC DIVERSITY 
IN JAMUN (
Syzygium cumini L.) 
USING 
MOLECULAR MARKERS 
 
 
 
 
 
H.S. SIDDARTHA 
PHK - 714 
 
 
 
 
 
 
 
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) 
in 
FRUIT SCIENCE 
 
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. 
__________________________ 
  
(S. 
SHYAMALAMMA)                                 

ACKNOWLEDGEMENT 
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. 
 
 
Bangalore 
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 

II REVIEW 
OF 
LITERATURE 

III MATERIALS 
AND 
METHODS 
65 
IV EXPERIMENTAL 
RESULTS 
83 
V DISCUSSION 
96 
VI SUMMARY 
106 
VII REFERENCES 
108 
 APPENDIX 
 
 
 
 
 
 

 
 
 
LIST OF TABLES 
Table 
No. 
Title Page 
No. 

List of jamun genotypes  collected from different location 
66 

Preparation of stock solution (extraction) 
68 

Preparation of CTAB buffer 25 ml 
68 

Preparation of loading dye 
69 

Preparation of running buffer (5x) 
69 

Preparation of TE buffer 
69 

Preparation of gel for DNA electrophoresis (120 ml) 
76 

Reagents used for PCR amplification 
79 

PCR Amplification procedure  
79 
10 
List of nine primers and their sequences showing good 
amplification 
81 
11 Concentration 
and 
purity of DNA in jamun 
84 
12 
Primers and their amplification  
89 
13 
Dissimilarity matrix of 60 genotypes of jamun 
94 
 
 
 
 
 
 

 
 
 
 
LIST OF FIGURES 
Fig. 
No. 
Title 
Between 
Pages 
3.1  Karnataka map showing plant sampling and experimental sites 
67-68 
1   Gel profile showing  high molecular  weight DNA of  jamun  
before RNAse treatment  
83-84 

Gel profile showing high molecular DNA of jamun after RNAse 
treatment 
83-84 
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  
92-93 
5 Principal 
Component 
Analysis 
of 60 jamun genotypes based on 
RAPD markers 
95-96 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
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 
disorders. 
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   
specific objectives. 
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 
distributed 
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 



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