ANALYSIS OF GENETIC DIVERSITY AMONG RICE

(Oryza sativa) LANDRACES VARIETIES USING SSR

MARKERS

A
DISSERTATION
SUBMITTED TO
CH. CHARAN SINGH UNIVERSITY, MEERUT
FOR
THE AWARD OF THE DEGREE OF

MASTER OF SCIENCE

IN

BIOTEHNOLOGY

Supervised by: DR. RAKESH SINGH Submitted by: SONALIKA

Principal Scientist M.Sc. Biotechnology

Division of Genomic Resources Institute of Management Studies

NBPGR, New Delhi GHAZIABAD,Utter Pradesh

Roll No. 9352310

ANALYSIS OF GENETIC DIVERSITY AMONG RICE
(Oryza sativa) LANDRACES VARIETIES USING SSR
MARKERS

A
DISSERTATION
SUBMITTED TO
CH. CHARAN SINGH UNIVERSITY, MEERUT
FOR
THE AWARD OF THE DEGREE OF

MASTER OF SCIENCE
IN

BIOTEHNOLOGY

Supervised by: DR. RAKESH SINGH Submitted by: Sonalika

Principal Scientist M.Sc. Biotechnology

Division of Genomic Resources I.M.S. COLLEGE

NBPGR, New Delhi GHAZIABAD,(U.P)

Roll No. 9352310

 ACKNOWLEDGEMENT

With my immense and heartfelt gratitude I place on record the efforts, inspiring
guidance and constant supervision of Dr. Rakesh Singh (Principal Scientist)
DIVISION OF GENOMIC RESOURCES, NBPGR in bringing up my project work.

I also extend my sincere thanks to Dr. K.C. Bansal , Director, NBPGR for
providing me this opportunity to carry out my project work at such a reputated
institute.

I place on record my deep sense of gratitude to B.P. Pethiya( Director), &

Sanjeev Sharma (HOD), I.M.S. College Ghaziabad, for suggesting and permitting
me to work at NBPGR, New Delhi.

I am elated to acknowledge the priceless help, encouragement and kind

cooperation rendered by Dr. Debjani Roy Bhattacharya, and Dr.Gunjan Tiwari
Sukla, for the successful completion of the work.

It is a pleasure to thanks Ashwani Kumar, Narendar Kumar for their timely

help and presence during the entire lab work.

I am highly indebted to the motivation and strong support of my respected and

beloved parents in all my endeavors.

SONALIKA
 Dedicated to

My Parents
 ABBREVIATIONS

& And
bp Base pair
conc. Concentration
cM Centimorgan
CTAB Cetyl trimethyl ammonium bromide
DNA Deoxyribonucleic acid
D/W Distilled Water
EDTA Ethylene diamine tetra acetic acid
EtBr Ethidium Bromide
g Gram
HCl Hydrochloric acid
HvSSR Hypervariable simple sequence repeats
L Litre
MAS Marker aided selection
ml Millilitre
M Molar
mg Milligram
mM Milli molar
Mins Minutes
NaCl Sodium chloride

Nm Nanometer

O.D Optical density

PAGE Polyacrylamide gel electrophoresis

PCR Polymerase Chain Reaction

QTL Quantitative trait loci

RAPD Random Amplified Ploymorphic DNA

RFLP Restriction Fragment Length Polymorphism

RNA Ribonucleic acid
RNase Ribonuclease
rpm Revolutions per minute
TAE Tris-acetate-EDTA
TE Tris EDTA
UV Ultraviolet
V Volts

Symbols:

°C Degree Celsius
µl Micro litre
% Percentage
 CONTENT

Chapter 1 Introduction
Chapter 2 Review of Literature
Chapter 3 Material and Methods
Chapter 4 Result
Chapter 5 Discussion
Chapter 6 References
 LIST OF TABLES

Table No. Title

3.1 List of RICE accessions used in the study.

4.1 Selective 38 highly variable SSR (HvSSR) marker loci with repeatlengths

of 55-70bp of rice genome

4.2 List of SSR primers used for genotyping of 37 rice varieties along with

gene diversity, heterozygosity and PIC

LIST OF FIGURES

Figure no. Title

3.1 Nanodrop

3.2 G-Storm thermal cycler

4.1 Genomic DNA

4.2a Amplification profile of 50 rice varieties with HvSSR 04-27

4.2b Amplification profile of 50 rice varieties with HvSSR 08-19

4.3 Dendogram generated for 50 varieties of rice based on HvSSR markers

Chapter 1
INTRODUCTION
 Chapter-1
INTRODUCTION
Rice (Oryza sativa L.) is the staple food for more than half of the world’s population. It
serves as model plant for study of genomics and breeding. It is highly rich in genetic diversity
at both inter-specific and intra-specific levels. It is very difficult to distinguish germplasm on
the basis of morphological and biochemical parameters (Isozyme electrophoretic patterns).

Rice is one of the most important food crops of Asia, including India and is feeding
more than 3 billion people. The cultivated rice, Oryza sativa had originated in humid tropical
climate of South East Asia and under the influence of local environment and farmer’s need
has evolved into 88,681 different varieties, out of that 55,615 are land races, 1,171 are wild
races and 32,895 are other varieties. Green revolution is considerably held responsible for
increasing production of food grains in our country and its role in achieving status of self
sufficiency in food grain is beyond any doubt. But high yielding varieties, which are the back
bone of green revolution have indirectly stimulated erosion of land races and wild varieties of
rice. Presently more than 70% of rice cultivation is being done using high yielding varieties
only. Obviously land races are disappearing fast. Importance of landraces is larger than life in
agriculture system, because improvement in existing variety depends upon desirable genes
which are possibly present in land races and wild varieties only. Besides food, rice plays a
key role in religion, culture and rituals in South Asia. Rice provides 23% of global human per
capita energy and 16% per capita protein. Rice protein ranks high in nutritional quality
among cereals, though protein content is modest. Unmilled rice (brown rice) provides 4.3 to
18.2% protein, averaging 9.5% based on 17,587 cultivars in the IRRI germplasm (Rice
Almanac, 1997). Rice also provides minerals, vitamins, and fibre. Milling removes roughly
80% of the thiamine from brown rice. For the majority of Asians who eat rice, the total intake
is 2,531 calories per person per day, with 35% coming from rice, which is considerably high.
However, breeding efforts to increase protein have so far been largely unsuccessful because
of the considerable effects of environment and lack of inheritance properties in the triploid
rice endosperm tissue (Agnihotri, 2002).
 Indian rice possesses wide diversity in its morphological and agronomic characters. Rice
grows in extremely diverse ecologies adapted to different seasons of the year. It has been
estimated that about 40,000 landraces existed in India. Species diversity of rice in India
include: O.sativa, O.nivara, O.rufipogan, O.officinalis, O.granulate, O.malampuzhasnsis and
a wild relative Porteresia coarctata (O.coarctata ).

In recent years, due to advent of high yielding varieties, local landraces have been drastically
replaced. Further, the natural habitats are also distributed a great deal due to various
developmental activities to meet the present and future needs. Thus, it is important to collect
the germplasm and characterize and evaluate the already collected accessions to identify
those carrying genes for desirable traits for use in crop improvement programmes.

Characterisation of germplasm involves recording the data on highly heritable oligogenic

characters, whereas evaluation involves recording the quantitative traits. Conventionally
morphological and agronomic traits have been used for germplasm characterisation and
evaluation. Though simple, their expression particularly those for agronomic and
physiological characters are influenced by the environment. The difficulties and time required
in data collection and lack of knowledge on genetic control of phenotypic traits are other
limitations. Therefore, molecular techniques are being increasingly adopted for germplasm
characterisation in recent years. These techniques directly utilize DNA and potentially
address the limitations associated with morphological and agronomic markers. These are not
affected by the environment and are simply inherited.

Restriction Length Polymorphic Markers (RFLP) are the first to be developed by

Botstein et al, 1980 Later Polymerase chain reaction (PCR) based techniques like Simple
Sequence repeats (SSRs) (Weber et al., 1989), Random amplified polymorphic DNA (RAPD)
(William et al., 1990) and Amplified fragment length polymorphism (AFLP) (Vos et al.,
1995) were developed. Among these RAPD offers a simple, efficient and economic technique
for diversity analysis (Williams et al., 1990) and is most widely used for characterization of
plant genetic resources and analysis (Patterson., 1996). However, it has low reproducibility
and cannot distinguish the closely related accessions. The drawbacks of RAPD have been
overcome by using SSRs (Temnykh et al., 2000). SSRs are rapidly emerging as a marker
system of choice in many crop sciences (Yang et al., 1994). Due to their variability, relative
ease of scoring, potential for automated analysis and co-dominant nature, SSRs are generally
considered as the most powerful among different molecular markers (Harsh et al., 2002).
 The basic technique (RFLP) of classical DNA fingerprinting, involves digesting DNA
with restriction enzymes, separating the resultant DNA fragments by gel electrophoresis,
blotting the fragments to a filter and hybridizing labelled probes to the separated fragment
(Neuhaus et al., 1993). Specific probe enzyme combinations give highly reproducible
patterns and since RFLPs are co-dominant markers, genetic analysis of band profiles is quite
straightforward. Up to 105 are scattered throughout the genome, including the transcription
units.

Simple Sequence Repeat (SSRs), called microsatellites, are randomly interspersed in

eukaryotic genomes. They are highly variable in the number of repeats they contain and are
co-dominantly inherited (Johansson et al., 1992). The primers flanking the microsatellite
locus in the conserved DNA sequences can detect the variation. The microsatellites are often
multialellic and the number of alleles representing a microsatellite locus is highly variable.

The SSR loci have been divided into two classes based on their repeat length and
potential as informative genetic markers: Class I SSRs with repeat lengths of 20 bp or higher,
and Class II SSRs with repeat lengths of 12–19 bp (Temnykh et al., 2001). The rationale for
making the two classes was that the SSRs with larger number of repeats were more
polymorphic than those with less number of repeats as reported in human (Weber et al., 1990;
Xu et al., 2000). The class I SSRs were found more polymorphic than the class II SSR and
denoted as hypervariable marker (Temnykh et al., 2001)

Microsatellites have been increasingly used as molecular marker. Their

polymorphisms have shown high efficiency for many studies. SSRs have been used
successfully for genomic mapping, population and evolutionary studies, as well as for
fingerprinting and pedigree analysis (Hazan et al., 1992; Plaschke et al., 1995; Rongwen et
al., 1995; Guilford et al., 1997). Micro-satellites were found more polymorphic than RFLP
markers in rice (Akkaya et al., 1992; Wu et al., 1993; Bell et al., 1994). There are reports of
positive correlation between SSR length and polymorphism in rice but there is no systematic
genome wide study to validate these assertions (Cho et al., 2000; Temnykh et al., 2001).

Scoring micro-satellite gels or autoradiogram is usually a relatively simple process because

the used electrophoresis systems have a high resolution (to single base pair) and because the
alleles differ in a very predictable way (multiples of microsatellite repeat unit, e.g. two base
pairs). These analysis programmes provide algorithms that separate native allele
automatically from slippage product.
In the present investigation, 37 varieties of rice landraces have been fingerprinted using
HvSSR makers. The diversity present between the varieties was also studied by constructing
the dendogram using UPGMA method.
Chapter 2
REVIEW OF
LITERATURE
 Chapter 2

REVIEW OF LITERATURE

Indian centre of diversity possesses rich diversity in several crop plants and their wild
relatives. Several crops have their centre of diversity in the Indian sub-continent including
rice. It is grown in wide range of environments from the equatorial tropics to subtropical mid
latitudes from low altitude paddy fields to high altitude, terraces and from swamps to upland
rice fields .

As the progenitor of the Asian cultivated rice (O. sativa, O. rufipogan) has been proven to be
valuable gene pool for rice genetic improvement and thus plays a critical role in rice breeding
in the future (Chang., 1984; Khush., 1997), It is well established that wild rice populations
harbor significantly higher genetic diversity than does the cultivated rice (Oka 1988; Provan
et al., 1997; Sun et al., 2001).

Molecular Characterization

Several molecular marker techniques have been used to characterize and study genetic
diversity. An ideal marker should be easy to measure and evaluate, able to discriminate
between individuals, highly heritable, provide comparable results and remain neutral to the
environment. DNA based molecular techniques are powerful and accurate tools for genotype
identification and analysis of genetic diversity (Karp et al., 1997).

Restriction Fragment Length Polymorphism

RFLP is one of the earliest techniques used to study variety polymorphism at DNA level. The
potential use of RFLP for varietal characterization as such is not well exploited but its use in
species identification, classification and establishing phylogenetic relationship is well
established. Zhang et al., (1992) studied the nuclear RFLP variation to identify indica and
japonica rice. Wang et al., (1992) reported the phylogenetic relationship among different
species of the genus Oryza. Ishii et al., (1993) used chloroplast, mitochondrion and nuclear
RFLPs to characterize O. sativa and O. glaberrima

Random Amplified Polymorphic DNA

The power of RAPD technique for detecting genetic variation among genotype and
identifying germplasm is well established in rice (Virk et al., 1995b; Xie et al., 2000). RAPD
has been used for estimation of genetic diversity among landraces (Yu et al., 1994; Cao et al.,
1997).

Simple Sequence Repeats (SSRs)

SSRs are also known as Microsatellites. Microsatellites are simple sequence repeats (SSRs)
of 1-6 nucleotides. They are abundant, dispersed throughout the genome and show higher
levels of polymorphism than other genetic markers. These features coupled with their ease of
detection, have made them useful markers. Their potential for automation and their
inheritance in a co dominant manner are additional advantages when compared with another
type of molecular markers (McCouch et al., 1997). SSRs have recently become important
genetic markers in cereals including wheat and barley (Wu et al., 1993; Panaud et al., 1996).

Variations in the length of tandem repeats can be identified by amplification of the

region containing the repeat via PCR using primers designed to the regions flanking
individual SSRs. Polymorphism are detected based on size differences which result from
differences in the number of repeats. SSR loci are believed to evolve in the step-wise manner
by the addition or subtraction of a single repeat.

PCR primers sequences flanking microsatellite repeats are generally designed to

produce primers 17-22 nucleotides long , with GC content of approximately 50% and a Tm
about 60° C. The ideal size for analysis of SSRs amplification product is approximately 100-
250bp.

Microsatellite markers have been developed and utilized in the study of cultivated
rice, including genetic diversity (Yang et al., 1994; Dairerwala et al., 2000). More than 500
microsatellite markers are available in rice, which cover the genome with an average density
of one SSR per 6 cM (Temnykh et al., 2001). However, the marker density of approximately
one marker for every 15-20 cM had been shown to be sufficient for DNA fingerprinting and
varietal identification (Rongwen et al., 1995). SSRs consisting of AT repeat regions are
highly polymorphic in rice genome (Akagi et al., 1997).
 Genetic diversity and population genetic structure of natural Oryza rufipogan
populations in China were studied by Hai - fei et al., (2003) based on ten microsatellites loci.
For a total of 237 individuals of 12 populations collected from four regions, a moderate to
high level of genetic diversity was observed.

The pattern of diversity within the two rice subspecies Indica and japonica using SSR
markers showed that japonica group had significantly higher genetic diversity on
chromosome 6 and 7, and considerably lower diversity on chromosome 2 in comparison to
Indica group (Ni et al., 2002). Indica and japonica, both form exactly separate groups, with
japonica further divided into temperate and tropical types.

Australian breeding lines of rice (Oryza sativa L.) were identified by the use of
microsatellite polymorphism. Garland et al., (1999) analyzed 10 microsateliite loci for 43
cultivars or breeding lines of rice. These microsatellite markers were useful for cultivar
identification and assessment of genetic relationships. Rice microsatellites have also
demonstrated utility for gene - tagging and marker - assisted selection (Chen et al., 1997;
McCouch et al.., 1997) and are polymorphic between (Akagi et al., 1996; Akagi et al., 1997;
Chen et al., 1997; Olufowote et al., 1997; Panaud et al., 1996; Wu et al., 1993; Yang et al.,
1994 ) and within rice varieties (Olufowote et al., 1997 ).

Mohapatra and Srinivasan analyzed 79 non-aromatic rice cultivars by using 48 SSR

markers (NRC DNAF, 2000).The results revealed high degree of similarity between
varieties having related ancestors.

Song et al., (2003) estimated genetic diversity of the residual northern populations of
Oryza rufipogan by SSR markers. A total of 232 individuals from six populations were
analysed including three from Dongxiang ( Jiangxi Province ) and three from Chaling
(Human Province) in China. In this study, the 23 rice SSR primers pairs selected from the
Rice Genes Database detected a total of 115 alleles, indicating that all the SSR loci were
polymorphic and the Donxiang populations showed higher diversity than the Chaling
populations.

Rabiei et al., (2004) performed the SSR mapping for the identification of
quantitative trait loci (QTLs), controlling grain size (grain length and breadth ) and shape
(length/breadth ratio) using an F2 populations of a cross between two Iranian cultivars,
Domsephid and Gerden, comprising of 192 individuals. A total of 11 intervals carrying 18
QTLs for three traits were identified, that included five QTLs for grain length, seven QTLs
for grain breadth, and six QTLs for grain shape.

Thirty rice SSR primer pairs well distributed on all the 12 chromosomes were
utilized on the basis of the published rice microsatellites framework (Temnykh et al., 2000)
for evaluating the genetic diversity and patterns of relationship among the 18 rice genotypes
representative of the traditional Basmati, Cross - bred basmati and Non Basmati (indica and
japonica) rice varieties.

The genetic diversity and DNA fingerprinting of 15 elite rice genotypes using 30
SSR primers on chromosome number 7-12 was investigated by Chakravarthi and
Naravaneni, (2006). The results revealed that all the primers showed distinct polymorphism
among the cultivars studied indicating the robust nature of micro-satellite in revealing
polymorphism.

In Maritime Guinea the genetic diversity of two cultivated rice species O.sativa and
O.glaberrima showed interspecific recombination. Barry et al., (2007) collected one hundred
seventy accession and genotype with 11 SSR markers and phenotype with 26 morph-
physiologic descriptors. The study detected an original genetic compartment, highlighting the
occurrence of O.glaberrima and O. sativa hybridization.

Kibria et al., (2008) assessed the genetic diversity among aromatic rice genotypes
using simple sequence repeat (SSR) and randomly amplified polymorphic DNA (RAPD)
markers through marker aided selection (MAS). Considering the genetic distance values the
genotypes was genetically different from each other which could be used in breeding
programme to have potential genetic gains.
Singh et al., (2010) described a genome wide set of 436 validated highly variable SSR
(HvSSR) markers with repeat lengths of 51–70 bp for their consistent amplification and high
polymorphism which are suitable for QTL mapping and fingerprinting studies in rice
employing agarose gels. Kaushik et al., (2011) developed a DNA fingerprint database for 50
rice genotypes using 50 SSR and 30 TE based markers and the study demonstrated that SSR
are best markers for differentiating between closely related basmati, indica and japonica
varieties. A rice core collection consisting of 150 varieties which was established by
genotyping from 2260 varieties of rice germplasm with 274 SSR markers. (Zhang et al.,
2011). Singh et al in 2013 conducted a study between SSR and SNP markers. Later A core
collection was developed by Choudhury et al in 2014 using 36 SNP markers from 6984 rice
accessions originating from of the Northeastern region of India.

Chapter 3

MATERIAL AND
METHODS
 Chapter 3

MATERIAL AND METHODS

The present investigation was carried out on rice genotypes (Oryza sativa) at National Bureau
of Plant Genetic Resources (NBPGR) Pusa, New Delhi. The objective of the study was
characterization and analysis of genetic diversity among 37 rice varieties using HvSSR
markers.

Thirty-seven varieties of Oryza sativa (rice) belonging to family Poaceae considered for this
study (Table 3.1)

Table 3.1: List of genotypes used for molecular characterization using HvSSR markers
3.1 Genomic DNA Isolation

Rice landraces were collected from remote areas through extensive survey and farmers were
consulted about the local varieties they have and they plant in their fields. Information
gathered was cross verified by other means as well. Seeds of local landraces were collected
and were planted on 15th march 2016 by drawing two to three deep lines on small study plots
with suitable and uniform spacing in between two plants (20cm in a row and 25cm in a
column) in control condition without giving any synthetic agrochemical manure. On 1 st April
2016 Plantlets are removed from the soil and washed it by Tap running water to clean the soil
from plantlet.

For genomic DNA isolation, CTAB method was used. CTAB ( Cetyl trimethyl ammonium
bromide) is a cationic detergent, which solubilizes cell membranes and forms a complex with
DNA. Plantlets are crushed in mortar and pestle using liquid nitrogen and transferred to the
2.0ml of eppendorf tube with pre warmed (65°C) CTAB DNA extraction buffer. Samples
were mixed well at 10 minutes interval five to six times. Then samples were incubated for 1
hour in a water bath at 65°C. An equal volume of Chloroform: Isoamyl alcohol (24:1) was
added to the tubes and mixed gently for 5-10 minutes by gently inverting the tubes. The
eppendorf were centrifuged at 15,000 rpm for 15 minutes. Supernatant was transferred to
fresh eppendorf and Isopropanol 0.6 volumes (30 minutes, -80 ˚C chilled) was added and
mixed for 10 minutes. The eppendorf tubes were centrifuged at 10,000 rpm for 10 minutes.
After centrifugation, supernatant was discarded and pellet was washed with 70% ethanol.
Finally the air-dried pellet was dissolved in 100µl of TE buffer.
3.2 DNA Purification
To 100µl of DNA solution, 15.0µl of RNase (10 mg/ml) was added and incubated at 37°C
for 1 hour. Equal volume of C: I (24:1) was added to the samples and mixed gently by
inverting the tubes. Tubes were centrifuged at 15,000 rpm at 20°C for 15 minutes.
Supernatant was transferred to fresh tube carefully without disturbing whitish layer of
impurities. Equal volume of isopropanol (30 minutes, -80 chilled) was added and centrifuged
at 10,000 rpm for 10 minutes. Supernatant was discarded by saving fine pellets. Pellet was
dried at room temperature for 20 minutes. Dried pellet was dissolved in 100µl of TE buffer
and stored at 4°C for further experiments.

3.3 Quantification of Genomic DNA

3.3.1 Quantification by Nanodrop 1000 spectrophotometer

The genomic DNA dissolved in TE buffer was taken for quantification by UV absorbance at
260 nm. To measure the concentration, Thermo Scientific Nanodrop 1000 spectrophotometer
was used. Reference was set against TE and then the sample was measured at 260 nm and
280 nm. The ratio of OD260/ OD 280 provides an estimate of purity of nucleic acid. DNA
has the ratio between 1.8 and 2.0 use in experiment.

Fig.3.1 NanoDrop
.
3.3.2 Quantification by Gel Electrophoresis
Agarose gel electrophoresis of the isolated genomic DNA also performed to know about the
quality of DNA. Larger molecules migrate slower because of greater frictional drag and
because they form their way through the pores of gel less efficiently than smaller molecules.
0.8 % gel was used to visualize the genomic DNA.

3.4 Dilution of DNA sample

A part of the DNA sample was diluted with appropriate amount of sterilized water to yield a
working concentration of 10ng/μl stored at -20°C until use for PCR amplification.

3.5 PCR Reaction for SSR Markers

DNA amplification was carried out in a 96 well thermo cycler (Applied Biosystem - G-
Storm Thermal Cycler) in a volume of 10μl each containing 5µl PCR grade Millipore water,
2µl of 10-20ng template DNA, 0.2ul of 5µm/µl of each primer, 0.2 µl of 10mM of dNTPs
mix, 1.2μl of 25mM MgCl2, 1µl of 1X Taq buffer and 0.2μl of 5units/μl Fermentas Taq
DNA Polymerase.

Fig. 3.2 G- Storm Thermal Cycler (PCR)

The PCR amplification profile for SSR marker analysis followed is as follows:

1. Initial extended step of denaturation at 94°C for 5 min, followed by 36 cycles of

denaturation at 94°C for 30 sec.
2. Primer annealing at 50°C -61°C for 1 min..
3. Elongation at 72°C for 2 min. with a final extension step of 10 min at 72°C.
4. Final hold at 4°C till electrophoresis.
The choice of 50°C -61°C for annealing was according to the information provided for the
primers.

3.6 Preparation of Metaphor Gel

4% Metaphor gel was prepared by suspending 20g Metaphor in 500ml chilled 0.5X TAE
buffer and mix with magnetic stirrer. Then boil in microwave oven untill it get completely
dissolved. Cooled and 16l ethidium bromide was added and then gel was cast in a gel
loading tray kept on horizontal surface. Combs were placed in such a way that 2 mm gap was
maintained between the bottom of the gel and the comb tip. The gel was allowed to solidify
and then combs were removed. Gel was placed in the electrophoresis chamber and 0.5x TAE
buffer was added to fill the chamber and flood the surface of the gel.

3.6.1 Metaphor Gel Electrophoresis

The amplified DNA samples were mixed with 6X DNA loading dye in 5:1 proportion and
electrophoresis was carried out on 4% Metaphor Gel in 0.5X TAE buffer at 120 volt for 4
hours.

3.7 Data Analysis

Bands appeared in the gel were examined critically under Biochem Life Sciences gel
documentation system and their size were noted down. The amplified products were scored
across the lanes comparing their respective molecular weights with 100 bp DNA ladder in the
marker lane. Each band was treated as one SSR allele. Homology of bands was based on
distance of migration in the gel. Presence of band was scored as "1", absence of band as "0". .
The major allele frequency, gene diversity, heterozygosity and PIC for each locus were
calculated for SSR markers using Power Marker 3.5 (Liu and Muse 2005). In addition,
genetic distances (Nei et al. 1983) across the genotypes and neighbor-joining (NJ) tree were
calculated using Power Marker 3.5 (Liu and Muse 2005). The dissimilarity matrix generated
by Power marker was used to make un-weighted neighbour joining tree using DARwin
software 5.0.158 (Perrier and Jacquemoud-Collet, 2006).
Chapter 4
RESULTS
 Chapter 4

RESULTS

4.1 DNA extraction, purification and estimation

Using CTAB as polysaccharide chelating agent good quality of plant genomic DNA was
obtained that amenable to optimum PCR amplification. The concentration of DNA obtained
was up to 200 ng/µl in the samples.

149 250 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

Fig 4.1 Genomic DNA isolated from 37 Rice varieties

4.2 PCR amplification with Hv SSR markers

Fifty genotypes were studied with ten HvSSR markers (Table 4.1). All primers showed
good amplification and were easily scorable. For all the ten primers number of bands
produced varied from 2 to 4 (Fig 4.2a, 4.2b). Temperature of Amplification (Ta) was
standardized for all HvSSR primers by gradient PCR and Ta varied from 55˚C to 61˚C (Table
4.1)

Primer Product Locus SSR SSR Forward primer Reverse primer No of Annealing
Size(bp) ID motif length alleles temp (°C)
(bp) amplified
55.6
AAACTGGAGAT GTAACGAACTAGAGCA
HvSSR01-32 250 RM580 CTT 57 GAACTCGAA TGGG 2
59
RM108 TGAGTGAGACTT AGTTAACACCAATGCTG
HvSSR01-41 348 64 GT 55 GACAGTGC ACC 3

RM129 TAATGCACGCAC TATAGAATGCTGACTGG

HvSSR02-33 355 75 TA 65 AACTTTAC GCT 2 60.1
55
RM144 GTACACAACGTC ACTGTGGCATATGTTCG
HvSSR03-10 280 73 GA 56 ACAACAGC ATT 2
58
RM169 ATGGATTTAGGC ATACTGCGAAGGTGAA
HvSSR04-27 318 43 AT 58 TTGTTTGA GAGA 3

RM174 GGCGCGCTTATA CGATTGCGTGGTGTAAC

HvSSR04-46 179 67 AT 62 TATGTACT TAT 4 61.2
57.8
RM192 CTAGGGAATCAG GCTCTCTTGTCCTTCTTC
HvSSR06-03 212 55 GAA 67 CGGTTAG TTC 2

RM200 CTCTTCCGTGGT CACTGGTATGATCTCCG

HvSSR06-40 385 66 ATA 65 TAAAGAAA ACT 3 61
55.6
RM827 CATCTCTTGAGA TGTGCATTTCGTCTTTC
HvSSR08-19 221 1 AG 64 AATCTGCC ATA 2

RM270 TTACTCCGCATA ATTTGACACCAAGTTGA

HvSSR09-55 382 5 AT 66 TATCCATGT TCC 4 61

Table 4.1 Selective 10 highly variable SSR (HvSSR) marker loci with repeat lengths of
55-70bp of rice genome
Fig4.2a Amplification profile of 37 rice accessions with HvSSR04-27 , 100 bp DNA
Ladder, Expected size : 318 bp

1 2 3 4 5 6 7 8 9 10 11 12 M 13 14 15 16 17 18 19 20 21 22 23 24

Fig4.2b Amplification profile of 37 rice accessions with HvSSR08-19, 100 bp DNA

Ladder, Expected size : 221 bp

25 26 27 28 29 30 31 32 33 34 35 36 M 37 38 39 40 41 42 43 44 45 46 47 48

4.3 Genetic diversity analysis

M 49 50
To study the diversity present in the collection of 37 accessions of rice, all the bands
produced were scored across all the varieties based on the presence (1), absence (0) and
missing value (9). The weak bands of negligible intensity and smeared bands were excluded
from the final data analysis. Thirty- eight primers were used for final analysis on the basis of
easily scoreable amplified bands. The number of bands amplified by each of the thirty-eight
primers ranged from 2 to 4. A total of 27 alleles were amplified with an average of 2.7 alleles
per locus in 50 genotypes. Maximum of four alleles were observed for HvSSR02-33, three
alleles were observed for HvSSR01-41, HvSSR03-10, HvSSR04-46, HvSSR06-03 and
HvSSR08-19 and two alleles were observed for HvSSR01-32, HvSSR04-27, HvSSR06-40
and HvSSR09-55.
PIC value for 10 primers ranged from 0.07 (for HvSSR03-10) to 0.50 (HvSSR02-33) with an average of 0.31.Gene diversity ranged from 0.07
(for HvSSR03-10) to 0.53 (HvSSR02-33) with an average of 0.3772. Heterozygosity was also calculated for all 10 primers which ranged from
0.02 (for HvSSR03-10) to 0.84 (for HvSSR06-03) with an average value of 0.26. Major allele frequency was calculated for 10 primers and it
ranged from 0.52 ( for HvSSR04-46) to 0.95 ( for HvSSR03-10) with an average value of 0.70.

Marker Major.Allele.Frquency AlleleNo GeneDiversity Heterozygosity PIC

HvSSR01-32 0.9184 2.0000 0.1499 0.0408 0.1387
HvSSR01-41 0.7600 3.0000 0.3918 0.1400 0.3561
HvSSR02-33 0.6458 4.0000 0.5397 0.3542 0.5025
HvSSR03-10 0.9592 3.0000 0.0789 0.0204 0.0770
HvSSR04-27 0.5400 2.0000 0.4968 0.3200 0.3734
HvSSR04-46 0.5200 3.0000 0.5176 0.2400 0.4028
HvSSR06-03 0.5200 3.0000 0.5496 0.8400 0.4510
HvSSR06-40 0.9490 2.0000 0.0968 0.0612 0.0921
HvSSR08-19 0.5900 3.0000 0.4994 0.5000 0.3931
HvSSR09-55 0.6556 2.0000 0.4516 0.1556 0.3496
Mean 0.7058 2.7000 0.3772 0.2672 0.3136

Table 4.2 List of SSR primers used for genotyping of 50 rice varieties along with gene
diversity, heterozygosity and PIC

4.4 Cluster analysis

All the HvSSR amplicons generated across 50 varieties were taken for calculation of
genetic distance and the dissimilarity matrix was used for the cluster development using
neighbour joining (NJ) method. In the dendogram rice genotypes were grouped into three
major clusters. Further cluster1 contained 2 varieties, cluster2 was sub-grouped into three
sub clusters; cluster 2a, cluster 2b and cluster 2c with 1, 8 and 14 varieties respectively.
Cluster 3 was the largest containing 25 varieties in cluster 3a, 6 varieties and 19 varieties
in cluster 3b. FR- 43- B and GAR-13 showed common parentage in cluster 1 but they are
further apart from the rest of the varieties.
 Fig 4.3 Dendogram generated for 50 varieties of rice based on HvSSR primers

Chapter 5
DISCUSSION
 Chapter 5
DISCUSSION

Molecular mapping and tagging the genes is essential aspect of molecular breeding. The
parents selected for the development of a mapping population should be genetically divergent
enough to exhibit sufficient polymorphisms. The parents are then crossed to produce the
segregating mapping population, which could be an F2, backcross inbred lines (BILs),
recombinant inbred lines (RILs), in double haploid (DH) lines. The primary step to identify
divergent parents starts at morphological as well as molecular level. The DNA based genetic
diversity study is more reliable because the environment does not affect it. The availability of
PCR amplification techniques had made easier to screen large number of markers in more
effective manner.
 In the present study fifty accessions of rice has been taken and screened with 10
HvSSR markers. These are Highly variable Simple Sequence repeat markers. The present
study focused on a comparative evaluation of marker polymorphism emphasizing SSRs
designed from different rice chromosomes. Presence of high level of variability suggested
that these micro-satellite sequences experiences less selective constraint and offer
opportunities to investigate biological significance of micro-satellite expansion and
contraction on functional aspects.

The gene diversity in the present analysis varied from 0.07-0.94 which is comparable
to the work conducted by Singh et al in 2013 having gene diversity of 0.07-0.58 which shows
the present set of rice accession has broad range of genetic diversity. Also the PIC ranged
from 0.07-0.5 as seen in the paper published by Singh et al in 2013 with values of 0.04-0.5.
Similar study by the using morphological isozyme and molecular marker, such as RFLPs,
RAPDs AFLPs, and micro-satellites, have been used to determine genetic diversity and
phylogenetic relationships in Oryza (Tateoka 1962; Morinag 1964; Second 1982; Dally and
Second, 1990; Wang et. al., 1992; Aggarwal et al., 1999). Cluster analysis grouped all the
fifty accessions of rice into three clusters. Two varieties (FR- 43- B and GAR- 13) were
grouped in one cluster away from all other fourty eight varieties .This indicates that these
fourty eight are sharing common parentage or have common origin of cultivation and are
very different from FR- 43- B and GAR- 13 varieties . Dendogram helps in such
interpretations.

Chapter 6

REFERENCES
 Chapter 6

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ANNEXURE-I

A. Solution, Chemical and Reagents Used for DNA Extraction

1. Liquid Nitrogen (-1960°C)
2. Cetyl Trimethyl Ammonium Bromide (CTAB) buffer for DNA extraction.
i. CTAB (10%) - 10g CTAB was dissolved in sterile distilled water volume was
made upto 100 ml. with sterile distilled water. The solution was then autoclaved.

ii. Sodium Chloride (Nacl, 4M) - 292.2g of NaCl was dissolved in sterile
distilled water and volume was made upto 100 ml. The solution was autoclaved
prior to use.
 iii. Tris: Buffer (pH 8.0, 1M) - 12.11 g of Tris salt was dissolved in distilled
water and volume was made upto 100 ml and pH was adjusted to 8.0 using 1 N
HCl. The solution was autoclaved prior to use.

iv. Ethylene Diamine Tetra Acetic Acid (EDTA, 0.5M) - 18.62 g EDTA was
dissolved in sterile distilled water. The pH of the solution was adjusted to 8.0 by
using 1 N NaOH. The solution was stirred vigorously on a magnetic stirrer. The
volume was made upto 100 ml using sterile distilled water and the solution was
autoclaved.

v. β Mercaptoethanol 2%
vi. DNA Extraction Buffer
DNA extraction buffer was prepared using following using following volume of stock solutions
of the individual component:

S/No. Component Stock Working Vol. of stock taken to

solution buffer prepare 500 ml buffer
1. CTAB 10% 2% 100ml
2. NaCl 4M 1.4M 175ml
3. Tris 1M 100mM 50ml
4. EDTA 0.5M 20mM 20ml
5. β 2% 2% 10ml
Mercatoethanol
6. Distilled Water -- -- 145ml

3. Isopropanol (Propane – 2-0l)

4. Chloroform: Isoamyl Alcohol (24:1) mixture - 96ml of cloroform was mixed with
4ml of isoamyl alcohol. It was stored in amber coloured bottle.
5. 70% Ethanol - 70 ml of absolute ethanol was mixed well with 30 ml of sterile water
and stored in a stoppered bottle until use.

B. DNA Purification

1. Phenol : Chloroform: Isoamylalochol (25 : 24 : 1 ) mixture - 100ml of Tris

saturated Phenol was added to a mixture of 96 ml Chloroform and 4ml
 isoamylalcohol. The mixture was mixed well prior to use and stored in amber
coloured bottle.

2. Solvent for DNA- Tris : EDTA (TE) buffer (10mM Tris: 1mM EDTA, pH
8.0) was mixed with sterile distilled water and volume made upto 100 ml. The
solution was autoclaved priors to use.

C. Gel Electrophoresis

Loading dye (6x) solution

1. Bromophenol Blue 0.25%

2. Xylene Cyanol FF 0.25%

3. Glycerol 50%

Tris : Acetate : EDTA (TAE) Buffer – 50 x (stock) solution (ph 8.0)

1. Tris base 242g

2. EDTA 100 ml (0.5M, pH8)

3. Glacial acetic acid 57.1 ml

D. PCR Master Mix

1. Taq DNA polymerase - A stock solution of 5 units/μl was provided by the

manufacture (Fermentas) was stored – 200C.

2. 10X Assay buffer- 10X PCR assay buffer for Taq DNA polymerase provided by
the manufacturer (Fermentas) was used. Stored at – 200C.

3. Deoxyribonucleotide Triose Phosphate (dNTPs)- dATP (10mM), dGTP (10mM),

dCTP (10mM), and dTTP (10mM), were mixed in equal volumes and stored at –
200C till use.
4. Magnesium Chloride (MgCl2) - A solution of 25 mM provided by the
manufacturer, stored at – 200C was used.

5. Primers - The primers was provided by the manufacturer in a lyophilized form.

Based on the molecular weight of a given primer, a solution of 100μl was prepared
by adding the required amount of sterile water. Storage was at – 200C.

ANNEXURE-II

Instruments used in DNA Extraction and Quantification

 Micropipettes : Nichiroyo

 Water bath : Orbitek

 Centrifuge : Thermo Scientific

 Tissue Lyser : Qiagen

 Deep freezer (-200C) : Kelvinater

 Refrigerator : Kelvinator

 Nanodrop1000 Spectrophotometer : Thermo Scientific

 Water purification system : Millipore

Instruments used in SSR analysis

 PCR plates : Axygen

 PCR machine : G-Storm thermal cycler

Instruments used for electrophoresis

 Microwave oven : Samsung

 Gel electrophoresis unit : Scientific system

 Gel Documentation System : Biochem LifeSciences

 Power Pack : Bio Rad