IDENTIFICATION OF MOLECULAR MARKERS LINKED TO YELLOW MOSAIC VIRUS

IDENTIFICATION OF MOLECULAR

MARKERS LINKED TO YELLOW

MOSAIC VIRUS RESISTANCE IN

BLACKGRAM

(Vigna mungo (L) Hepper)

E RAMBABU BSc (Ag)

MASTER OF SCIENCE IN AGRICULTURE ( MOLECULAR BIOLOGY AND BIOTECHNOLOGY)

2016

IDENTIFICATION OF MOLECULAR MARKERS

LINKED TO YELLOW MOSAIC VIRUS

RESISTANCE IN BLACKGRAM


By

E RAMBABU BSc (Ag)

THESIS SUBMITTED TO

PROFESSOR JAYASHANKAR TELANGANA STATE

AGRICULTURAL UNIVERSITY

IN PARTIAL FULFILMENT OF THE REQUIREMENTS

FOR THE AWARD OF THE DEGREE OF


CHAIRPERSON Dr CH ANURADHA

INSTITUTE OF BIOTECHNOLOGY COLLEGE OF AGRICULTURE

RAJENDRANAGAR HYDERABAD-500 030


AGRICULTURAL UNIVERSITY ndash 2016

DECLARATION

I E RAMBABU hereby declare that the thesis entitled ldquoIDENTIFICATION OF

MOLECULAR MARKERS LINKED TO YELLOW MOSAIC VIRUS RESISTANCE

IN BLACKGRAM (Vigna mungo (L) Hepper)rdquo submitted to Professor Jayashankar

Telangana State Agricultural University for the degree of MASTER OF SCIENCE IN

AGRICULTURE in the major field of Plant Molecular Biology and Biotechnology is the

result of original research work done by me I also declare that no material contained in the

thesis has been published earlier in any manner

Date (E RAMBABU)

Place Hyderabad I D No RAM14-95

CERTIFICATE

Mr E RAMBABU has satisfactorily prosecuted the course of research and that thesis

entitled ldquoIDENTIFICATION OF MOLECULAR MARKERS LINKED TO YELLOW

MOSAIC VIRUS RESISTANCE IN BLACK GRAM (Vigna mungo (L) Hepper)rdquo

submitted is the result of original research work and is of sufficiently high standard to

warrant its presentation to the examination I also certify that neither the thesis nor its part

thereof has been previously submitted by her for a degree of any university

Date ( CH ANURADHA)

Place Hyderabad ChairPerson

CERTIFICATE

This is to certify that the thesis entitled ldquoIDENTIFICATION OF MOLECULAR

MARKERS LINKED TO YELLOW MOSAIC VIRUS RESISTANCE IN

BLACKGRAM (Vigna mungo(L) Hepper)rdquo submitted in partial fulfillment of the

requirements for the degree of bdquoMaster of Science in Agriculture‟ of the Professor

Jayashankar Telangana State Agricultural University Hyderabad is a record of the bonafide

original research work carried out by Mr E RAMBABU under our guidance and

supervision

No part of the thesis has been submitted by the student for any other degree or diploma

The published part and all assistance received during the course of the investigations have

been duly acknowledged by the author of the thesis

(CH ANURADHA)

CHAIRPERSON OF ADVISORY COMMITTEE

Thesis approved by the Student Advisory Committee

Chairperson Dr CH ANURADHA

Associate Professor _____________________

Institute of Biotechnology

College of Agriculture

Rajendranagar Hyderabad

Member Dr V SRIDHAR

Scientist ____________________

ARS

Madhira

Khammam

Member Dr S SOKKA REDDY

Professor and University Head ___________________




Date of final viva-voce

ACKNOWLEDGEMENTS

With a deep sense of gratitude I express my heartfelt thanks to my chairman Dr Ch

Anuradha Associate Professor Department of Plant Molecular Biology and

Biotechnology Institute of Biotechnology College of Agriculture Rajendranagar

Hyderabad for her valuable guidance incessant inspiration and wholehearted help and

personal care throughout the course of this study and in bringing out this thesis I am

indeed greatly indebted for the affectionate encouragement and cooperation received from

her

I record my sincere gratitude to members of the advisory committee Dr S Sokka

Reddy Professor Department of Plant Molecular Biology and Biotechnology Institute of

Biotechnology College of Agriculture Rajendranagar Hyderabad for his benign help and

transcendent suggestions during the course of investigation

I wish to express my esteem towards Dr V sridhar Scientist Agriculture Research

Station madhira khammam for his great advice sustained interest and co-operation

I deem it previllege in expressing my fidelity to Dr Kuldeep Singh Dangi Director of

Biotechnology DrChVDurgaRani Professor DrKYNYamini Assistant professor Dr

balram Assistant professor Dr Vanisri professor Dr Prasad ashraf and ankhita

Research Associate for their sustained interest fruitful advice and co-operation

I express my heart full thanks to my classmates Gusha Bkalpana sk maliha d

aleena v mounica gmahesh jraju ajay who have rendered their help during my course

works and I express my thanks to Juniors durga sairavi mouli rama in whose cheerful

company I have never felt my work as burden

I also express my thanks to my loved seniors dravi eramprasad b jeevula naik for

generously helping me in every possible ways to complete my research successfully and also I

express my thanks with pleasure to all my senior friends for their kind guidance and help

rendered during course of studies

I am greatly indebted to my wellwihsers pgopi Krishna yadav ynagaraju prasanna

kumar joseph raju arjunsyam kumarsaidaPraveenraghavasivasiva

naiksantoshrohitRamesh naik hari nayak vijay reddy satyanvesh for their help and

guidance in my life

I also express my thanks to SRFs mahender sir Krishna kanth sir ranjit sir arun sir

jamal sir rajini madam for their help throughout my research work

Endless is my gratitude and love towards my Father Mr ELingaiah Mother

vijayamma and anavamma Sisters krishanaveni and praveena Brother ramakotaiahand

and cousins srilakshmisrilathasobhameriraju for their veracious love showered upon me

and to whom I devote this thesis I am debted all my life to them for their care non-

compromising love steadfast inspiration blessings sacrifices guidance and prayers which

helped me endure periods of difficulties with cheer They have been a great source of

encouragement throughout my life and without their blessings I canrsquot do anything

I am thankful to department staff Prabaker raju and other non teaching staff of the

Institute of Biotechnology for their timely assistance and cooperation

I express my immense and whole hearted thanks to all my near for their cooperation

help during the course of study and research

I am thankful to the Government of telangana and professor jayashankar telangana

state agricultural university Hyderabad for their financial aid for my research work that

supported me a lot

(rambabu)

LIST OF CONTENTS

Chapter Title Page No

I INTRODUCTION

II REVIEW OF LITERATURE

III MATERIALS AND METHODS

IV RESULTS AND DISCUSSION

V SUMMARY AND CONCLUSION

LITERATURE CITED

APPENDICES APPENDICES

LIST OF TABLES

Sl No

Table

No

Title

Page No

1 31 SSR primers used for molecular analysis of MYMV disease

resistance in blackgram

2 32 Scale used for YMV reaction (Bashir et al 2005)

3 33 Components of PCR reaction

4 34 PCR temperature regime

5 41 Mean disease score of parental lines of the cross LBG 759 X

T9 for MYMV in blackgram

6 42

Frequency of F2 segregants of the cross of LBG 759 X T9 of

blackgram showing different grades of

resistancesusceptibility to MYMV

7 43

Chi-Square test for segregation of resistance and

susceptibility in F2 populations during late rabi season 2016

revealing the nature of inheritance to YMV

8 44 List of polymorphic primers of the cross LBG 759 X T9

9 45 Mean range and variance values for eight traits in

segregating F2 population of LBG 759 X T9 in blackgram

10 46

Estimates of components of variability heritability (broad

sense) expected genetic advance and genetic advance over

mean for eight traits in segregating F2 population of LBG

759 X T9 in blackgram

LIST OF FIGURES

Sl No Figure

No

Title of the Figures Page No

1 41

parental polymorphism survey of uradbean lines LBG 759 (1)

times T9 (2) with monomorphic SSR primers The ladder used

was 50bp

2 42 Parental survey of uradbean lines LBG 759 (1) times T9 (2) with

monomorphic SSR primers The ladder used was 50bp


Polymorphic SSR primers The ladder used was 50bp

4 44 Confirmation of F1s (LBG 759 times T9) using SSR marker

CEDG 185

5 45 Bulk segregant analysis with SSR primer CEDG 185

6 46 Confirmation of bulk segregant analysis with SSR primer

CEDG 185


CEDG 185

LIST OF PLATES

Sl No

Plate No

Title

Page No

1

Plate-41

Field view of F2 population

2

Plate-42

YMV disease scoring pattern

3

Plate-43

Screening of segregation material for YMV

disease reaction

LIST OF APPENDICES

Appendix

No

Title Page

No

I List of Equipments

II List of chemicals used

III Buffers and stock solutions

LIST OF ABBREVIATIONS AND SYMBOLS

MYMV

YMV

MYMIV

YMD

CYMV

LLS

SBR

AVRDC

IARI

ANGRAU

VR

BSA

MAS

DNA

QTL

RILS

RFLP

RAPD

SSR

SCAR

CAP

RGA

SNP

ISSR

Mungbean Yellow Mosaic Virus

Yellow Mosaic Virus

Mungbean Yellow Mosaic India Virus

Yellow Mosaic Disease

Cowpea Yellow Mosaic Virus

Late Leaf Spot

Soyabean Rust

Asian Vegetable Research and Development Council

Indian Agricultural Research Institute

Acharya NG Ranga Agricultural University

Vigna radiata

Bulk Segregant Analysis

Marker Assisted Selection

Deoxy ribonucleic Acid Quantitative Trait Loci Recombinant Inbreed Lines Restriction Fragment Length Polymorphism Randomly Amplified Polymorphic DNA Simple Sequence Repeats

Sequence Characterized Amplified Region Cleaved Amplified Polymorphism

Resistant Gene Analogues

Single Nucleotide Polymorphisms

Inter Simple Sequence Repeats

AFLP

AFLP-RGA

STS

PCR

AS-PCR

AP-PCR

SDS- PAGE

CTAB

EDTA

TRIS

PVP

TAE

dNTP

Taq

Mb

bp

Mha

Mt

L ha

Sl no

et al

viz

microl

ml

cm

microM

Amplified Fragment Length Polymorphism

Amplified Fragment Length Polymorphism- Resistant gene analogues

Sequence tagged sites

Polymerase Chain Reaction

Allele Specific PCR

Arbitrarily Primed PCR

Sodium Dodecyl Sulphide-Polyacyramicine Agarose Gel Electrophoresis

Cetyl Trimethyl Ammonium Bromide Ethylene Diamine Tetra Acetic Acid

Tris (hydroxyl methyl) amino methane

Polyvinylpyrrolidone Tris Acetate EDTA

Deoxynucleotide Triphosphate

Thermus aquaticus Mega bases

Base pairs

Million hectares

Million tonnes

Lakh hectares

Serial number

and others

Namely Micro litres Milli litres Centimeter Micro molar Percent

amp

UV

H2O

mM

ng

cm

g

mg

h2

χ2

cM

nm

C

And Per

Ultra violet

Water

Micromolar Nanogram Centimeter Gram Milligram Heritability

Chi-square

Centimorgan

Nanometer

Degree centigrade

Name of the Author E RAMBABU

Title of the thesis ldquoIDENTIFICATION OF MOLECULAR

MARKERS LINKED TO YELLOW MOSAIC

VIRUS RESISTANCE IN BLACKGRAM (Vigna

mungo (L) Hepper)rdquo

Degree MASTER OF SCIENCE IN AGRICULTURE

Faculty AGRICULTURE

Discipline MOLECULAR BIOLOGY AND

BIOTECHNOLOGY


University PROFESSOR JAYASHANKAR TELANGANA

STATE AGRICULTURAL UNIVERSITY

Year of submission 2016

ABSTRACT

Blackgram (Vigna mungo (L) Hepper) (2n=22) is one of the most highly valuable pulse

crop cultivated in almost all parts of india It is a good source of easily digestible proteins

carbohydrates and other nutritional factors Beside different biotic and abiotic constraints

viral diseases mostly yellow mosaic disease is the prime threat for massive economic loss in

areas of production The Yellow Mosaic disease (YMD) caused by Mungbean Yellow

Mosaic Virus (MYMV) a Gemini virus transmitted by whitefly ( Bemesia tabaciGenn) is

one of the most downfall disease that has the ability to cause yield loss upto 85 The

advancements in the field of biotechnology and molecular biology such as marker assisted

selection and genetic transformation can be utilized in developing MYMV resistance

uradbeans

The investigation was carried out to find out the markers linked to yellow mosaic virus

resistance gene MYMV resistant parent T9 and MYMV susceptible parent LBG 759 were

crossed to produce mapping population Parents F1 and 125 F2 individuals of a mapping

population were subjected to natural screening to assess their reaction to against MYMV

This investigation revealed that single recessive gene is governing the inheritance of

resistance to MYMV F2 mapping population revealed segregation of the gene in 95

susceptible 30 resistant ie 13 ratio showing that resistance to yellow mosaic virus is

governed by a monogenic recessive gene

A total of 50 SSR primers were used to study parental polymorphism Of these 14 SSR

markers were found polymorphic showing 28 of polymorphism between the parents These

fourteen markers were used to screen the F2 populations to find the markers linked to the

resistance gene by bulk segregant analysis The marker CEDG185 present on linkage group

8 clearly distinguished resistant and susceptible parents bulks and ten F2 resistant and

susceptible plants indicating that this marker is tightly linked to yellow mosaic virus

resistance gene

F2 population was evaluated for productivity for nine different morphological traits

namely height of the plant number of branches number of clusters days to 50 flowering

number of pods per plant pod length number of seeds per pod single plant yield and

MYMV score The presence of additive gene action was observed in the number of pods per

plant single plant yield plant height number of branches per plant pod length whereas non-

additive genetic variance was observed in number of seeds per pod which indicate the

epistatic and dominant environmental factors controlling the inheritance of these traits

The presence of additive gene indicates the availability of sufficient heritable variation

that could be used in the selection programme and can be easily transferred to succeeding

generations The difference between GCV and PCV for pods per plant and seed yield per

plant were high indicating the greater influence of environment on the expression of these

characters whereas the remaining other traits were least influenced by environment The

increase in mean values in the segregating population indicates scope for further

improvement in traits like number of pods per plant number of seeds per pod and pod length

and other characters in subsequent generations (F3 and F4) there by facilitating selection of

transgressive segregates in later generations

This marker CEDG185 is used to screen the large germplasm for YMV resistance The

material produced can be forwarded by single seed-descent method to develop RILS and can

be used for mapping YMV resistance gene and validation of identified markers High

heritability variability genetic advance as percent mean in the segregating population can be

handled under different selection schemes for improving productivity

Chapter I

Introduction

Chapter I

INTRODUCTION

Pulses are main source of protein to vegetarian diet It is second important constituent of

Indian diet after cereals Total pulse production in india is 1738 million tonnes (FAOSTAT

2015-16) They can be grown on all types of soil and climatic conditions Pulses being

legumes fix atmospheric nitrogen into the soil They play important role in crop rotation

mixed and intercropping as they help maintaining the soil fertility They add organic matter

into the soil in the form of leaf mould They are helpful for checking the soil erosion as they

have more leafy growth and close spacing Some pulses are turned into soil as green manure

crops Majority pulses crops are short durational so that second crop may be taken on same

land in a year Pulses are low fat high fibre no cholesterol low glycemic index high protein

high nutrient foods They are excellent foods for people managing their diabetes heart

disease or coeliac disease India is the world largest pulses producer accounting for 27-28 per

cent of global pulses production Pulses are largely cultivated in dry-lands during the winter

seasons Among the Indian states Madhya Pradesh is the leading pulses producer Other

states which cultivate pulses in larger extent include Udttar Pradesh Maharashtra Rajasthan

Karnataka Andhra Pradesh and Bihar In India black gram occupies 127 per cent of total

area under pulses and contribute 84 per cent of total pulses production (Swathi et al 2013)

Black gram or Urad bean (Vigna mungo (L) Hepper) originated in india where it has

been in cultivation from ancient times and is one of the most highly prized pulses of India

and Pakistan Total production in India is 1610 thousand tonnes in 2014-15 Cultivated in

almost all parts of India (Delic et al 2009) this leguminous pulse has inevitably marked

itself as the most popular pulse and can be most appropriately referred to as the king of the

pulses India is the largest producer and consumer of black gram cultivated in an area about

326 million hectares (AICRP Report 2015) The coastal Andhra region in Andhra Pradesh is

famous for black gram after paddy (INDIASTAT 2015)

The Guntur District ranks first in Andhra Pradesh for the production of black gram

Black gram is very nutritious as it contains high levels of protein (25g100g)

potassium(983 mg100g)calcium(138 mg100g)iron(757 mg100g)niacin(1447 mg100g)

Thiamine(0273 mg100g and riboflavin (0254 mg100g) (karamany 2006) Black gram

complements the essential amino acids provided in most cereals and plays an important role

in the diets of the people of Nepal and India Black gram has been shown to be useful in

mitigating elevated cholesterol levels (Fary2002) Being a proper leguminous crop black

gram has all the essential nutrients which it makes to turn into a fertilizer with its ability to fix

nitrogen it restores soil fertility as well It proves to be a great rotation crop enhancing the

yield of the main crop as well It is nutritious and is recommended for diabetics as are other

pulses It is very popular in the Punjabi cuisine as an ingredient of dal makhani

There are many factors responsible for low productivity ranging from plant ideotype

to biotic and abiotic stresses (AVRDC 1998) Most emerging infectious diseases of plants are

caused by viruses (Anderson et al 1954) Plant viral diseases cause serious economic losses

in many pulse crops by reducing seed yield and quality (Kang et al 2005) Among the

various diseases the Mungbean Yellow Mosaic Disease (MYMD) disease was given special

attention because of its severity and ability to cause yield loss up to 85 per cent (Nene 1972

Verma and Malathi 2003)The yellow mosaic disease (YMD) was first observed in India in

1955 at the experimental farm of the Indian Agricultural Research Institute New Delhi

(Nariani 1960)

Symptoms include initially small yellow patches or spots appear on green lamina of

young leaves Soon it develops into a characteristics bright yellow mosaic or golden yellow

mosaic symptom Yellow discoloration slowly increases and leaves turn completely yellow

Infected plants mature later and bear few flowers and pods The pods are small and distorted

Early infection causes death of the plant before seed set It causes severe yield reduction in all

urdbean growing countries in Asia including India (Biswass et al 2008)

It is caused by Mungbean yellow mosaic India virus (MYMIV) in Northen and

Central Region (Mandal et al 1997) and Mungbean yellow mosaic virus (MYMV) in

western and southern regions (Moringa et al 1990) MYMV have been placed in two virus

species Mungbean yellow mosaic India virus (MYMIV) and Mungbean yellow mosaic virus

(MYMV) on the basis of nucleotide sequence identity (Fauquet et al 2003) It is a

Begomovirus belonging to the family geminiviridae Transmitted by whitefly Bemisia tabaci

under favourable conditions Disease spreads by feeding of plants by viruliferous whiteflies

Summer sown crops are highly susceptible Yellow mosaic disease in northern and central

India is caused by MYMIV whereas the disease in southern and western India is caused by

MYMV (Usharani et al 2004) Weed hosts viz Croton sparsiflorus Acalypha indica

Eclipta alba and other legume hosts serve as reservoir for inoculum

Mungbean yellow mosaic virus (MYMV) belong to the genus begomovirus and

occurs in a number of leguminous plants such as urdbean mungbean cowpea (Nariani1960)

soybean (Suteri1974) horsegram lab-lab bean (Capoor and Varma 1948) and French bean

In blackgram YMV causes irregular yellow green patches on older leaves and complete

yellowing of young leaves of susceptible varieties (Singh and De 2006)

Management practices include rogue out the diseased plants up to 40 days after

sowing Remove the weed hosts periodically Increase the seed rate (25 kgha) Grow

resistant black gram variety like VBN-1 PDU 10 IC122 and PLU 322 Cultivate the crop

during rabi season Follow mixed cropping by growing two rows of maize (60 x 30 cm) or

sorghum (45 x 15cm) or cumbu (45 x 15 cm) for every 15 rows of black gram or green gram

Treat the seeds with Thiomethoxam-70WS or Imidacloprid-70WS 4gkg Spray

Thiamethoxam-25WG 100g or Imidacloprid 178 SL 100 ml in 500 lit of water

An approach with more perspective is marker assisted selection (MAS) which

emerged in recent years due to developments in molecular marker technology especially

those based on the Polymerase chain reaction (PCR ) (Basak et al 2004) Therefore to

facilitate research programme on breeding for disease resistance it was considered important

to screen and identify the sources of resistance against YMV in blackgram Screening for

new resistance sources by one of the genetically linked molecular markers could facilitate

marker assisted selection for rapid evaluation This method of genotyping would save time

and labour Development of PCR based SCAR developed from RAPD markers is a method

of choice to test YMV resistance in blackgram because it is simple and rapid (B V Bhaskara

Reddy 2013) The marker was consistently associated with the genotypes resistant to YMV

but susceptible genotypes without the resistance gene lacked the marker These results are to

be expected because of the linkage of the marker to the resistance gene With the closely

linked marker quick assessment of susceptibility or resistance at early crop stage it will

eliminate the need for maintaining disease for artificial screening techniques

The advancements in the field of biotechnology and molecular biology such as

genetic transformation and marker assisted selection could be utilized in developing MYMV

resistance mungbean (Xu et al 2000) Inheritance of MYMV resistance studies revealed that

the resistance is controlled by a single recessive gene (Singh 1977 Thakur 1977 Saleem

1998 Malik 1986 Reddy 1995 and Reeddy 2012) dominant gene (Sandhu 1985 and

Gupta et al 2005) two recessive genes (Verma 1988 Ammavasai 2004 and Singh et al

2006) and complementary recessive genes (Shukla 1985)

Despite blackgram being an important crop of Asia use of molecular markers in this

crop is still limited due to slow development of genomic resources such as availability of

polymorphic trait-specific markers Among the different types of markers simple sequence

repeats (SSR) are easy to use highly reproducible and locus specific These have been widely

used for genetic mapping marker assisted selection and genetic diversity analysis and also in

population genetics study in different crops In the past SSR markers derived from related

Vigna species were used to identify their transferability in black gram with the use of such

SSR markers two linkage maps were also developed in this crop (Chaitieng et al 2006 and

Gupta et al 2008) However use of transferable SSR markers in these linkage maps was

limited and only 47 SSR loci were assigned to the 11 linkage groups (Chaitieng et al 2006

and Gupta et al 2008) Therefore efforts are urgently required to increase the availability of

new polymorphic SSR markers in blackgram

These are landmarks located near genetic locus controlling a trait of interest and are

usually co-inherited with the genetic locus in segregating populations across generations

They are used to flag the position of a particular gene or the inheritance of a particular

characteristic Rapid identification of genotypes carrying MYMV resistant genes will be

helpful through molecular marker technology without subjecting them to MYMV screening

Different viral resistance genes have been tagged with markers in several crops like soybean

Phaseolus (Urrea et al 1996) and pea (Gao et al 2004) Inter simple sequence repeat (ISSR)

and SCAR markers linked to the resistance in blackgram (Souframanien and Gopalakrishna

2006) has exerted a potential for locating the gene in urdbean Now-a-days this is possible

due to the availability of many kinds of markers viz Amplified Fragment Length

Polymorphism (AFLP) Random Amplified Polymorphic DNA (RAPD) and Simple

Sequence Repeats (SSR) which can be used for the effective tagging of the MYMV

resistance gene Different molecular markers have been used for the molecular analysis of

grain legumes (Gupta and Gopalakrishna 2008)

Among different DNA markers microsatellites (or) Simple Sequence Repeats

(SSRs)Simple Sequence Repeats (SSRs) Microsatellites Short Tandem Repeats (STR)

have occupied a pivotal place because of Simple Sequence Repeat (SSR) markers are locus

specific short DNA sequences that are tandemly repeated as mono di tri tetra or penta

nucleotides in the genome (Toth et al 2000) They are also called as Simple Sequence

Repeats (SSR) or Short Tandem Repeats (STR) The SSR markers are developed from

genomic sequences or Expressed Sequence Tag (EST) information The DNA sequences are

searched for SSR motif and the primer pairs are developed from the flanking sequences of the

repeat region The SSR marker assay can be automated for efficiency and high throughput

Among various DNA markers systems SSR markers are considered the most ideal marker

for genetic studies because they are multi-allelic abundant randomly and widely distributed

throughout the genome co-dominant that could differentiate plants with homozygous or

heterozygous alleles simple to assay highly reliable reproducible and could be applied

across laboratories and amenable for automation

In method of BSA two pools (or) bulks from a segregating population originating

from a single cross contrasting for a trait (eg resistant and susceptible to a particular

disease) are analysed to identify markers that distinguish them BSA in a population is

screened for a character of interest and the genotypes at the two extreme ends form two

bulks Two bulks were tested for the presence or absence of molecular markers Since the

bulks are supposed to contrast for alleles contributing positive and negative effects any

marker polymorphism between the two bulks indicates the linkage between the marker and

character of interest BSA provides a method to focus on regions of interest or areas sparsely

populated with markers Also it is a method of rapidly locating genes that do not segregate in

populations initially used to generate the genetic map (Michelmore et al 1991)

Nowadays there are research reports using SSR markers for mapping the urdbean

genome and locating QTLs Genetic linkage maps have been constructed in many Vigna

species including urdbean (Lambrides et al 2000) cowpea (Menendez et al 1997) and

adzuki bean (Kaga et al 1996) (Ghafoor et al 2005) determining the QTL of urdbean by

the use of SDS-PAGE Markers (Chaitieng et al 2006) development of linkage map and its

comparison with azuki bean (wild) (Ohwi and Ohashi) in urdbean Gupta et al (2008)

construction of linkage map of black gram based on molecular markers and its comparative

studies Recently Kajonphol et al (2012) constructed a linkage map for agronomic traits in

mungbean

Despite the severity of the damage caused by YMV development of sustainable

resistant cultivars against YMV through conventional breeding has not yet been successful in

this part of the globe It is therefore an ideal strategy to search for molecular markers linked

with YMV resistance

Keeping the above in view the present study was undertaken to identify the molecular

markers linked to YMV resistance with the following objectives

1 To study the parental polymorphism

2 Phenotyping and Genotyping of F2 mapping population

3 Identification of SSR markers linked to Yellow Mosaic Virus resistance by Bulk

Segregation Analysis

Chapter II

Review of Literature

Chapter II

REVIEW OF LITERATURE

Blackgram is belongs to the family Fabaceae and the genus Vigna Only seven species of the

genus Vigna are cultivated as pulse crops Blackgram (Vigna mungo L Hepper) is a member

of the Asian Vigna crop group It is a staple crop in the central and South East Asia

Blackgram is native to India (Vavilov 1926) The progenitor of blackgram is believed to be

Vigna mungo var silvestris which grows wild in India (Lukoki et al 1980) Blackgram is

one of the most highly prized pulse crop cultivated in almost all parts of India and can be

most appropriately referred to as the ldquoKing of the pulsesrdquo due to its mouth watering taste and

numerous other nutritional qualities Being a proper leguminous crop it is itself a mini-

fertilizer factory as it has unique characteristics of maintaining and restoring soil fertility

through fixing atmospheric nitrogen in symbiotic association with Rhizobium bacteria

present in the root nodules (Ahmad et al 2001)

Although better agricultural and breeding practices have significantly improved the

yield of blackgram over the last decade yet productivity is limited and could not ful fill

domestic consumption demand of the country (Muruganantham et al 2005) The major yield

limiting factors are its susceptibility to various biotic (viral fungal bacterial pathogens and

insects) (Sahoo et al 2002) and abiotic [salinity (Bhomkar et al 2008) and drought (Jaiwal

and Gulati 1995)] stresses Among different constraints viral diseases mainly yellow mosaic

disease is the major threat for huge economical losses in the Indian subcontinent (Nene

1973) It can cause 100 per cent yield loss if infection occurs at seedling stage (Varma et al

1992 and Ghafoor et al 2000) The disease is caused by the geminivirus - MYMV

(mungbean yellow mosaic virus) The virus is transmitted by white flies (Bemisia tabaci)

Chemical control may have undesirable effect on health safety and cause environmental risks

(Manczinger et al 2002) To overcome the limitations of narrow genetic base the

conventional and traditional breeding methods are to be supplemented with biotechnological

techniques Therefore molecular markers will be reliable source for screening large number

of resistant germplasm lines and hence can be used in breeding YMV resistant lines and

complementary recessive genes (Shukla 1985)s

21 Viruses as a major constrain in pulse production

Blackgram (Vigna mungo (L) Hepper) is one of the major pulse crops of the tropics and sub

tropics It is the third major pulse crop cultivated in the Indian sub-continent Yellow mosaic

disease (YMD) is the major constraint to the productivity of grain legumes across the Indian

subcontinent (Varma et al 1992 and Varma amp Malathi 2003) YMV affects the majority of

legumes crops including mungbean (Vigna radiata) blackgram (Vigna mungo) pigeon pea

(Cajanus cajan) soybean (Glycine max) mothbean (Vigna aconitifolia) and common bean

(Phaseolus vulgaris) causing loss of about $300 millions MYMIV is more predominant in

northern central and eastern regions of India (Usharani et al 2004) and MYMV in southern

region (Karthikeyan et al 2004 Girish amp Usha 2005 and Haq et al 2011) to which Andhra

Pradesh state belongs The YMVs are included in the genus Begomovirus being transmitted

by the whitefly (Bemisia tabaci) and having bipartite genomes These crops are adversely

affected by a number of biotic and abiotic stresses which are responsible for a large extent of

the instability and low yields

In India YMD was first reported in Lima bean (Phaseolus lunatus) in western India

in 1940s Later in 1950 YMD was seen in dolichos (Lablab purpureus) in Pune Nariani

(1960) observed YMD in mungbean (Vigna radiata) in the experimental fields at Indian

Agricultural Research Institute and was subsequently observed throughout India in almost all

the legume crops The loss in yield is more than 60 per cent when infection occurs within

twenty days after sowing

22 Genetic inheritance of mungbean yellow mosaic virus

Black gram is a self-pollinating diploid (2n=2x=22) annual crop with a small genome size

estimated to be 056pg1C (574Mbp) (Gupta et al 2008) The major biotic stress is

Mungbean Yellow Mosaic India Virus (MYMIV) (Mayo 2005) accounts for the low harvest

index of the present day urdbean cultivers YMD is caused by geminivirus (genus

Begomovirus family Geminiviridae) which has bipartite genomes (DNA A and DNA B)

Begmovirus transmitted through the white fly Bemisia tabaci Genn (Honda et al 1983) It

causes significant yield loss for many legume seeds not only Vigna mungo but also in V

radiata and Glycine max throughout the South-Asian countries Depending on the severity of

the disease the yield penalty may reach up to cent percent (Basak et al 2004) Genetic

control of resistance to MYMIV in urdbean has been investigated using different methods

There are conflicting reports about the genetics of resistance to MYMIV claiming both

resistance and susceptibility to be dominant In blackgram resistance was found to be

monogenic dominant (Kaushal and Singh 1988) The digenic recessive nature of resistance

was reported by (Singh et al 1998) Monogenic recessive control of MYMIV resistance has

also been reported (Reddy and Singh 1995) It has been reported to be governed by a single

dominant gene in DPU 88-31 along with few other MYMIV resistant cultivars of urdbean

(Gupta et al 2005) Inheritance of the resistance has been reported as conferred by a single

recessive gene (Basak et al 2004 and Reddy 2009) a dominant gene (Sandhu et al 1985)

two recessive genes (Pal et al 1991 and Ammavasai et al 2004)

Thamodhran et al (2016) studied the nature of inheritance of YMV through goodness

of fit test and noted it as the duplicate dominant duplicate recessive in segregating

populations of various crosses

Durgaprasad et al (2015) revealed that the resistance to YMV was governed by

digenically and involves various interactions includes duplicate dominant and inhibitory

interactions They performed selective cross combinations and tested the nature of

inheritance

Vinoth et al (2014) performed crosses between resistant cultivar bdquoVBN (Bg) 4‟

(Vigna mungo) and susceptible accession of Vigna mungo var silvestris 222 a wild

progenitor of blackgram and observed nature of inheritance for YMV in F1 F2 RIL

populations and noted it as the single dominant gene controls it

Reddy et al (2014) studied the variability and identified the species of Begomovirus

associated with yellow mosaic disease of black gram in Andhra Pradesh India the total DNA

was isolated by modified CTAB method and amplified with coat protein gene-specific

primers (RHA-F and AC abut) resulting in 900thinspbp gene product

Gupta et al (2013) studied the inheritance of MYMIV resistance gene in blackgram

using F1 F2 and F23 derived from cross DPU 88-31(resistant) times AKU 9904 (susceptible) The

results of genetic analysis showed that a single dominant gene controls the MYMIV

resistance in blackgram genotype DPU 88-31

Sudha et al (2013) observed the inheritance of resistance to mungbean yellow mosaic

virus (MYMV) in inter TNAU RED times VRM (Gg) 1 and intra KMG 189 times VBN (Gg) 2

specific crosses of mungbean 3 (Susceptible) 1 (Resistance) was observed in both the two

crosses of all F2 population and it showed that the dominance of susceptibility over the

resistance and the results of the F3 segregation (121) confirm the segregation pattern of the

F2 segregation

Basamma et al (2011) studied the inheritance of resistance to MYMV by crossing TAU-1

(susceptible to MYMV disease) with BDU-4 a resistant genotype The evaluation of F1 F2

and F3 and parental lines indicated the role of a dominant gene in governing the inheritance of

resistance to MYMV

T K Anjum et al (2010) studied the mapping of Mungbean Yellow Mosaic India

Virus (MYMIV) and powdery mildew resistant gene in black gram [Vigna mungo (L)

Hepper] The parents selected for MYMIV mapping population were DPU 88-31 as resistant

source and AKU 9904 as susceptible one For establishment of powdery mildew mapping

population RBU 38 was used as resistant and DPU 88-31 as the susceptible one Parental

polymorphism was assessed using 363 SSR and 24 RGH markers

Kundagrami et al (2009) reported that Genetic control of MYMV- resistance was

evaluated and confirmed to be of monogenic recessive nature

Singh and Singh (2006) reported the inheritance of resistance to MYMV in cross

involving three resistant and four susceptible genotypes of mungbean Susceptible to MYMV

was dominant over resistance in F1 generation of all the crosses Observation on disease

incidence of F2 and F3 generation indicated that two recessive gene imparted resistance

against MYMV in each cross

Gupta et al (2005) examined the inheritance of resistance to Mungbean Yellow

Mosaic Virus (MYMV) in F1 F2 and F3 populations of intervarietal crosses of blackgram

disease severity on F2 plants segregated 31 (resistant susceptible RS) as expected for a

single dominant resistant gene in all resistant x susceptible crosses The results of F3 analysis

confirmed the presence of a dominant gene for resistance to MYMV

Basak et al (2004) conducted experiment on YMV tolerance and they identified a

monogenic recessive control of was revealed from the F2 segregation ratio of 31 susceptible

tolerant which was confirmed by the segregation ratio of the F3 families To know the

inheritance pattern of MYMV in blackgram F1 F2 and F3 generations were phenotyped for

MYMV reaction by forced inoculation using viruliferous white flies

Verma and Singh (2000) studied the allelic relationship of resistance genes for

MYMV in blackgram (V mungo (L) Hepper) The resistant donors to MYMV- Pant U84

and UPU 2 and their F1 F2 and F3 generations were inoculated artificially using an insect

vector whitefly (Bemisia tabaci Germ) They concluded that two recessive genes previously

reported for resistance were found to be the same in both donors

Verma and Singh (1989) reported that susceptibility was dominant over resistance

with two recessive genes required for resistance and similar reports were also observed in

green gram cowpea soybean and pea

Solanki (1981) studied that recessive gene for resistance to MYMV in blackgram The

recessive and two complimentary genes controlling resistance of YMV was reported by

Shukla and Pandya (1985)

221 Symptomology

This disease is caused by the Mungbean Yellow Mosaic Virus (MYMV) belonging to Gemini

group of viruses which is transmitted by the whitefly (Bemisia tabaci) This viral disease is

found on several alternate and collateral host which act as primary sources of inoculums The

tender leaves show yellow mosaic spots which increase with time leading to complete

yellowing Yellowing leads to less flowering and pod development Early infection often

leads to death of plants Initially irregular yellow and green patches alternating with each

other The yellow discoloration slowly increases and newly formed leaves may completely

turn yellow Infected leaves also show necrotic symptoms and infected plants normally

mature late and bear a very few flowers and pods The pods are small and distorted

The diseased plants usually mature late and bear very few flowers and pods The size

of yellow areas on leaves goes on increasing in the new growth and ultimately some of the

apical leaves turn completely yellow The symptoms appear in the form of small irregular

yellow specs and spots along the veins which enlarge until leaves were completely yellowed

the size of the pod is reduced and more frequently immature small sized seeds are obtained

from the pods of diseased plants It can cause up to 100 per cent yield loss if infection occurs

three weeks after planting loss will be small if infection occurs after eight weeks from the

day of planting (Karthikeyan 2010)

222 Epidemology

The variation in disease incidence over locations might be due to the variation in temperature

and relative humidity that may have direct influence on vector population and its migration It

was noticed that the crop infected at early stages suffered more with severe symptoms with

almost all the leaves exhibiting yellow mosaic and complete yellowing and puckering

Invariably whiteflies were found feeding in most of the fields surveyed along with jassids

thrips pod borers and pulse beetles in some of the fields The white fly population increased

with increase in temperature increase in relative humidity or heavy showers and strong winds

in rainy season found detrimental to whiteflies The temperature of insects is approximately

the same as that of the environment hence temperature has a profound effect on distribution

and prevalence of white fly (James et al 2002 and Hoffmann et al 2003)

The weather parameters play a vital role in survival and multiplication of white fly (B

tabaci Genn) and influence MYMV outbreak in Black gram during monsoon season Singh

et al (1982) reported that high disease attack at pod bearing stage is a major setback for black

gram yield and it also delayed the pod maturity There was a significantly positive correlation

between temperature variations and whitefly population whereas humidity was negatively

correlated with the whitefly population (AK Srivastava)

In northern India with the onset of monsoon rain (June to July) population of vector

increased and the rate of spread of virus were also increased whereas before the monsoon rain

the population of B tabaci was non-viruliferous

23 Genetic variability heritability and genetic advance

The main objective for any crop improvement programme is to increase the seed yield The

amount of variability present in a population where selection has to be is responsible for the

extent of improvement of a character Therefore it is necessary to know the proportion of

observed variability that is heritable

Meshram et al (2013) studied pure line seeds of black gram variety viz T-9 TPU-4

and one promising genotype AKU-18 treated with gamma irradiation (15kR 25kR and 35kR)

with the objective to assess the variability in M3 generation Highest GCV and PCV and high

estimates of heritability were recorded for the characters sprouting percentage number of

pods plant-1 and grain yield plant-1(g) High heritability accompanied with high genetic

advance was recorded for number of pods plant-1 governed by additive gene effects and

therefore selection based on phenotypic performance will be useful to improve character in

future

Suresh et al (2013) studied yield and its contributing characters in M4 populations of

mungbean genotypes and evaluated the genotypic and phenotypic coefficient of variations

heritability genetic advance and concluded that high heritability (broad) along with high

genetic advance as per cent of mean was observed for the trait plant height number of pods

per plant number of seeds per pod 100 seed weight and single plant yield indicating that

these characters would be amenable for phenotypic selection

Srivastava and Singh (2012) reported that in mungbean the estimates of genotypic

coefficient of variability heritability and genetic advance were high for seed yield per plant

100-seed weight number of seeds per pod number of pods per plant and number of nodes on

main stem

Neelavathi and Govindarasu (2010) studied seventy four diverse genotypes of

blackgram under rice fallow condition for yield and its component traits High genotypic

variability was observed for branches per plant clusters per plant pods per plant biological

yield and seed yield along with high heritability and genetic advance suggesting effective

improvement of these characters through a simple selection programme

Rahim et al (2010) studied genotypic and phenotypic variance coefficient of

variance heritability genetic advance was evaluated for yield and its contributing characters

in 26 mung bean genotypes High heritability (broad) along with high genetic advance in

percent of mean was observed for plant height number of pods per plant number of seeds

per pod 1000-grain weight and grain yield per plant

Arulbalachandran et al (2010) observed high Genetic variability heritability and

genetic advance for all quantitative traits in black gram mutants

Pervin et al (2007) observed a wide range of variability in black gram for five

quantitative traits They reported that heritability in the broad sense with genetic advance

expressed as percentage of mean was comparatively low

Byregouda et al (1997) evaluated eighteen black gram genotypes of diverse origin for

PCV GCV heritability and genetic advance Sufficient variability was recorded in the

material for grain yield per plant pods per plant branches per plant and plant height High

heritability values associated with high genetic advance were obtained for grain yield per

plant and pods per plant High heritability in conjugation with medium genetic advance was

obtained for 100-seed weight and branches per plant

Sirohi et al (1994) carried out studies on genetic variability heritability and genetic

advance in 56 black gram genotypes The estimates of heritability and genetic advance were

high for 100-seed weight seed yield per plant and plant height

Ramprasad et al (1989) reported high heritability genotypic variance and genetic

advance as per cent mean for seed yield per plant pods per plant and clusters per plant from

the data on seven yield components in F2 crosses of 14 lines

Sharma and Rao (1988) reported variation for yield and yield components by analysis

of data from F1s and F2s and parents of six inter varietal crosses High heritability was

obtained with pod length and 100-seed weight High heritability coupled with high genetic

advance was noticed with pod length and seed yield per plant

Singh et al (1987) in a study of 48 crosses of F1 and F2 reported high heritability for

plant height in F1 and F2 and number of seeds per pod in F2 Estimates were higher in F2 for

all traits than F1 Estimates of genetic advance were similar to heritability in both the

generations

Kumar and Reddy (1986) revealed variability for plant height primary branches

clusters per plant and pods per plant from a study on 28 F3 progenies indicating additive

gene action Pods per plant pod length seeds per pod 100-seed weight and seed yield per

plant recorded low to moderate heritability

Mishra (1983) while working on variability heritability and genetic advance in 18

varieties of black gram having diverse origin observed that heritability estimates were high

for 100 seed weight and plant height and moderate for pods per plant Plant height pods per

plant and clusters per plant had high predicted genetic advance accompanied by high

variability and moderate heritability

Patel and Shah (1982) noticed high GCV heritability coupled with high genetic

advance for plant height Whereas high heritability estimates with low genetic advance was

observed for number of pods per cluster seeds per pod and 100-seed weight

Shah and Patel (1981) noticed higher GCV heritability and genetic advance for plant

height moderate heritability and genetic advance for numbers of clusters per plant and pods

per plant while low heritability was reported for seed yield in black gram genotypes

Johnson et al (1955) estimates heritability along with genetic gain is more helpful

than the heritability value alone in predicting the result for selection of the best individuals

However GCV was found to be high for the traits single plant yield number of clusters per

plant and number of pods per plant High heritability per cent was observed with days to

maturity number of seeds per pod and hundred seed weight High genetic advance as per

cent of mean was observed for plant height number of clusters per plant number of pods per

plant single plant yield and hundred seed weight High heritability coupled with high genetic

advance as per cent of mean was observed for hundred seed weight Transgressive segregants

were observed for all the traits and finally these could be used further for yield testing apart

from utilizing it as pre breeding material

24 Molecular markers for blackgram

Molecular marker technology has greatly accelerated breeding programs for improvement of

various traits including disease resistance and pest resistance in various crops by providing an

indirect method of selection Molecular markers are indispensable for genomic study The

markers are typically small regions of DNA often showing sequence polymorphism in

different individuals within a species and transmitted by the simple Mendelian laws of

inheritance from one generation to the next These include Allele Specific PCR (AS-PCR)

(Sarkar et al 1990) DNA Amplification Fingerprinting (DAF) (Caetano et al 1991) Single

Sequence Repeats (Hearne et al 1992) Arbitrarily Primed PCR (AP-PCR) (Welsh and Mc

Clelland 1992) Single Nucleotide Polymorphisms (SNP) (Jordan and Humphries 1994)

Sequence Tagged Sites (STS) (Fukuoka et al 1994) Amplified Fragment Length

Polymorphism (AFLP) (Vos et al 1995) Simple sequence repeats (SSR) (Anitha 2008)

Resistant gene analogues (RGA) (Chithra 2008) Random amplified polymorphic DNA-

Sequence characterized amplified regions (RAPD-SCAR) (Sudha 2009) Random Amplified

Polymorphic DNA (RAPD) Amplified Fragment Length Polymorphism- Resistant gene

analogues (AFLP-RGA) (Nawkar 2009)

Molecular markers are used to construct linkage map for identification of genes

conferring resistance to target traits in the crop Efforts are being made to identify the

markers tightly linked to the genes responsible for resistance which will be useful for marker

assisted breeding for developing MYMIV and powdery mildew resistant cultivars in black

gram (Tuba K Anjum et al 2010) Molecular markers reported to be linked to YMV

resistance in black gram and mungbean were validated on 19 diverse black gram genotypes

for their utility in marker assisted selection (SK Gupta et al 2015) Only recently

microsatellite or simple sequence repeat (SSR) markers a marker system of choice have

been developed from mungbean (Kumar et al 2002 and Miyagi et al 2004) Simple

Sequence Repeat (SSR) markers because of their ubiquitous presence in the genome highly

polymorphic nature and co-dominant inheritance are another marker of choice for

constructing genetic linkage maps in plants (Flandez et al 2003 Han et al 2005 and

Chaitieng et al 2006)

2411 Randomly amplified polymorphic DNA (RAPD)

RAPDs are DNA fragments amplified by PCR using short synthetic primers (generally 10

bp) of random sequence These oligonucleotides serve as both forward and reverse primer

and are usually able to amplify fragments from 1-10 genomic sites simultaneously The main

advantage of RAPDs is that they are quick and easy to assay Moreover RAPDs have a very

high genomic abundance and are randomly distributed throughout the genome Variants of

the RAPD technique include Arbitrarily Primed Polymerase Chain Reaction (AP-PCR) which

uses longer arbitrary primers than RAPDs and DNA Amplification Fingerprinting (DAF)

that uses shorter 5-8 bp primers to generate a larger number of fragments The homozygous

presence of fragment is not distinguishable from its heterozygote and such RAPDs are

dominant markers The RAPD technique has been used for identification purposes in many

crops like mungbean (Lakhanpaul et al 2000) and cowpea (Mignouna et al 1998)

S K Gupta et al (2015) in this study 10 molecular markers reported to be linked to

YMV resistance in black gram and mungbean were validated on 19 diverse black gram

genotypes for their utility in marker assisted selection Three molecular markers

(ISSR8111357 YMV1-FR and CEDG180) differentiated the YMV resistant and susceptible

black gram genotypes

RK Kalaria et al (2014) out of 200 RAPD markers OPG-5 OPJ- 18 and OPM-20

were proved to be the best markers for the study of polymorphism as it produced 28 35 28

amplicons respectively with overall polymorphism was found to be 7017 Out of 17 ISSR

markers used DE- 16 proved to be the best marker as it produced 61 amplicons and 15

scorable bands and showed highest polymorphism among all Once these markers are

identified they can be used to detect the QTLs linked to MYMV resistance in mungbean

breeding programs as a selection tool in early generations and further use in developing

segregating material

BVBhaskara Reddy et al (2013) studied PCR reactions using SCAR marker for

screening the disease reaction with genomic DNA of these lines resulted in identification of

19 resistant sources with specific amplification for resistance to YMV at 532bp with SCAR

20F20R developed from OPQ1 RARD primer linked to YMV disease

Savithramma et al (2013) studied to identify random amplified polymorphic DNA

(RAPD) marker associated with Mungbean Yellow Mosaic Virus (MYMV) resistance in

mungbean (Vigna radiata (L) Wilczek) by employing bulk segregant analysis in

Recombinant Inbred Lines (RILs) only one primer ie UBC 499 amplified a single 700 bp

band in the genotype BL 849 (resistant parent) and MYMV resistant bulk which was absent

in Chinamung (susceptible parent) and MYMV susceptible bulk indicating that the primer

was linked to MYMV resistance

A Karthikeyan et al (2010) Bulk segregant analysis (BSA) and random amplified

polymorphic DNA (RAPD) techniques were used to analyse the F2 individuals of susceptible

VBN (Gg)2 times resistant KMG 189 to screen and identify the molecular marker linked to

Mungbean Yellow Mosaic Virus (MYMV) resistant gene in mungbean Co segregation

analysis was performed in resistant and susceptible F2 individuals it confirmed that OPBB

05 260 marker was tightly linked to Mungbean Yellow Mosaic Virus resistant gene in

mungbean

TS Raveendran et al (2006) bulked segregation analysis was employed to identity

RAPD markers linked to MYMV resistant gene of ML 267 in a cross with CO 4 OPS 7 900

only revealed polymorphism in resistant and susceptible parents indicating the association

with MYMV resistance

2412 Amplified Fragment Length Polymorphism (AFLP)

A novel DNA fingerprinting technique called AFLP is described The AFLP technique is

based on the selective PCR amplification of restriction fragments from a total digest of

genomic DNA Amplified Fragment Length Polymorphisms (AFLPs) are polymerase chain

reaction (PCR)-based markers for the rapid screening of genetic diversity AFLP methods

rapidly generate hundreds of highly replicable markers from DNA of any organism thus

they allow high-resolution genotyping of fingerprinting quality The time and cost efficiency

replicability and resolution of AFLPs are superior or equal to those of other markers Because

of their high replicability and ease of use AFLP markers have emerged as a major new type

of genetic marker with broad application in systematics path typing population genetics

DNA fingerprinting and quantitative trait loci (QTL) mapping The reproducibility of AFLP

is ensured by using restriction site-specific adapters and adapter specific primers with a

variable number of selective nucleotide under stringent amplification conditions Since

polymorphism is detected as the presence or absence of amplified restriction fragments

AFLP‟s are usually considered dominant markers

2413 SSR Markers in Black gram

Microsatellites or Simple Sequence Repeats (SSRs) are co-dominant markers that are

routinely used to study genetic diversity in different crop species These markers occur at

high frequency and appear to be distributed throughout the genome of higher plants

Microsatellites have become the molecular markers of choice for a wide range of applications

in genetic mapping and genome analysis (Li et al 2000) genotype identification and variety

protection (Senior et al 1998) seed purity evaluation and germplasm conservation (Brown

et al 1996) diversity studies (Xiao et al 1996)

Nirmala sehrawat et al (2016) designed to transfer mungbean yellow mosaic virus

(MYMV) resistance in urdbean from ricebean The highest number of crossed pods was

obtained from the interspecific cross PS1 times RBL35 The azukibean-specific SSR markers

were highly useful for the identification of true hybrids during this study Molecular and

morphological characterization verified the genetic purity of the developed hybrids

Kumari Basamma et al (2015) genetics of the resistance to MYMV disease in

blackgram using a F2 and F3 populations The population size in F2 was three hundred The

results suggested that the MYMV resistance in blackgram is governed by a single dominant

gene Out of 610 SSR and RGA markers screened 24 were found to be polymorphic between

two parents Based on phenotyping in F2 and F3 generations nine high yielding disease

resistant lines have been identified

Bhupender Kumar et al (2014) Genetic diversity panel of the 96 soybean genotypes

was analyzed with 121 simple sequence repeat (SSR) markers of which 97 were

polymorphic (8016 polymorphism) Total of 286 normal and 90 rare alleles were detected

with a mean of 236 and 074 alleles per locus respectively

Gupta et al (2013) studied molecular tagging of MYMIV resistance gene in

blackgram by using 61 SSR markers 31 were found polymorphic between the parents

Marker CEDG 180 was found to be linked with resistance gene following the bulked

segregant analysis This marker was mapped in the F2 mapping population of 168 individuals

at a map distance of 129 cM

Sudha et al (2013) identified the molecular markers (SSR RAPD and SCAR)

associated with Mungbean yellow mosaic virus resistance in an interspecific cross between a

mungbean variety VRM (Gg) 1 X a ricebean variety TNAU RED Among the 42 azuki bean

SSR markers surveyed only 10 markers produced heterozygotic pattern in six F2 lines viz 3

121 122 123 185 and 186 These markers were surveyed in the corresponding F3

individuals which too skewed towards the mungbean allele

Tuba K Anjum (2013) Inheritance of MYMIV resistance gene was studied in

blackgram using F1 F2 and F23 derived from cross DPU 88-31(resistant) 9 AKU 9904

(susceptible) The results of genetic analysis showed that a single dominant gene controls the

MYMIV resistance in blackgram genotype DPU 88-31

Dikshit et al (2012) In the present study 78 mapped simple sequence repeat (SSR)

markers representing 11 linkage groups of adzuki bean were evaluated for transferability to

mungbean and related Vigna spp 41 markers amplified characteristic bands in at least one

Vigna species Successfully utilized adzuki bean SSRs in amplifying microsatellite sequences

in Vigna species and inferring phylogenetic relationships by correlating the rate of transfer

among them

Gioi et al (2012) Microsatellite markers were used to investigate the genetic basis of

cowpea yellow mosaic virus (CYMV) resistance in 40 cowpea lines A total of 60 simple

sequence repeat (SSR) primers were used to screen polymorphism between stable resistance

(GC-3) and susceptible (Chrodi) genotypes of cowpea Among these only 4 primers were

polymorphic and these 4 SSR primer pairs were used to detect CYMV resistant genes among

40 cowpea genotypes

Jayamani Palaniappan et al (2012) Genetic diversity in 20 elite greengram [Vigna

radiata (L) R Wilczek] genotypes were studied using morphological and microsatellite

markers 16 microsatellite markers from greengram adzuki bean common bean and cowpea

were successfully amplified across 20 greengram genotypes of which 14 showed

polymorphism Combination of morphological and molecular markers increases the

efficiency of diversity measured and the adzuki bean microsatellite markers are highly

polymorphic and can be successfully used for genome analysis in greengram

Kajonpho et al (2012) used the SSR markers to construct a linkage map and identify

chromosome regions controlling some agronomic traits in mungbean Twenty QTLs

controlling major agronomic characters including days to first flower (FLD) days to first pod

maturity (PDDM) days to harvest (PDDH) 100 seed weight (SD100WT) number of seeds

per pod (SDNPPD) and pod length (PDL) were located on to the linkage map Most of the

QTLs were located on linkage groups 7 and 5

Kasettranan et al (2010) located QTLs conferring resistance to powdery mildew

disease on a SSR partial linkage map of mungbean Chankaew et al (2011) reported a QTL

mapping for Cercospora leaf spot (CLS) resistance in mungbean

Tran Dinh (2010) Microsatellite markers were used to investigate the genetic basis of

Cowpea Yellow Mosaic Virus (CYMV) resistance in 40 cowpea lines A total of 60 SSR

primers were used to screen polymorphism between stable resistance (GC-3) and susceptible

(Chrodi) genotypes of cowpea Among these only 4 primers were polymorphic and these 4

SSR primer pairs were used to detect CYMV resistance genes among 40 cowpea genotypes

Wang et al (2004) used an SSR enrichment method based on oligo-primed second-

strand synthesis to develop SSR markers in azuki bean (V angularis) Using this

methodology 49 primer pairs were made to detect dinucleotide (AG) SSR loci The average

number of alleles in complex wild and town populations of azuki bean was 30 to 34 11 to

14 and 40 respectively The genome size of azuki bean is 539 Mb therefore the number of

(AG) n and (AC) n motif loci per haploid genome were estimated to be 3500 and 2100

respectively

2414 SCAR markers

The sequence information of the genome to be study is not required for the number of PCR-

based methods including randomly amplified polymorphic DNA and amplified fragment

length polymorphism A short usually ten nucleotides long arbitrary primer is used in in a

RAPD assay which generally anneals with multiple sites in different regions of the genome

and amplifies several genetic loci simultaneously RAPD markers have been converted into

Sequence-Characterized Amplified Regions (SCAR) to overcome the reproducibility

problem

SCAR markers have been developed for several crops including lettuce (Paran and

Michelmore 1993) common bean (Adam-Blondon et al 1994) raspberry (Parent and Page

1995) grape (Reisch et al 1996) rice (Naqvi and Chattoo 1996) Brassica (Barret et al

1998) and wheat (Hernandez et al 1999) Transformation of RAPD markers into SCAR

markers is usually considered desirable before application in marker assisted breeding due to

their relative increased specificity and reproducibility

Prasanthi et al (2011) identified random amplified polymorphic DNA (RAPD)

marker OPQ-1 linked to YMV resistant among 130 oligonucleotide primers RAPD marker

OPQ-1 linked to YMV resistant was cloned and sequenced Their end sequences were used

to design an allele-specific sequence characterized amplicon region primer SCAR (20fr)

The marker designed was amplified at a specific site of 532bp only in resistant genotypes

Sudha (2009) developed one species-specific SCAR marker for Vumbellata by

designing primers from sequenced putatively species-specific RAPD bands

Souframanien and Gopalakrishna (2006) developed ISSR and SCAR markers linked

to the mungbean yellow mosaic virus (MYMV) in blackgram

Milla et al (2005) converted two RAPD markers flanking an introgressed QTL

influencing blue mold resistance to SCAR markers on the basis of specific forward and

reverse primers of 21 base pairs in length

Park et al (2004) identified RAPD and SCAR markers linked to the Ur-6 Andean

gene controlling specific rust resistance in common bean

2415 Inter simple sequence repeats (ISSRs)

This technique is a PCR based method which involves amplification of DNA segment

present at an amplifiable distance in between two identical microsatellite repeat regions

oriented in opposite direction The technique uses microsatellites usually 16-25 bp long as

primers in a single primer PCR reaction targeting multiple genomic loci to amplify mainly

the inter-SSR sequences of different sizes The microsatellite repeats used as primer can be

di-nucleotides or tri-nucleotides ISSR markers are highly polymorphic and are used in

studies on genetic diversity phylogeny gene tagging genome mapping and evolutionary

biology (Reddy et al 2002)

ISSR PCR is a technique which overcomes the problems like low reproducibility of

RAPD high cost of AFLP the need to know the flanking sequences to develop species

specific primers for SSR polymorphism ISSR segregate mostly as dominant markers

following simple Mendelian inheritance However they have also been shown to segregate as

co dominant markers in some cases thus enabling distinction between homozygote and

heterozygote (Sankar and Moore 2001)

Swati Das et al (2014) Using ISSR analysis of genetic diversity in some black gram

cultivars to assess the extent of genetic diversity and the relationships among the 4 black

gram varieties based on DNA data A total number of 10 ISSR primers that produced

polymorphic and reproducible fragments were selected to amplify genomic DNA of the urad

bean genotypes

Sunita singh et al (2012) studied genetic diversity analysis in mungbean among 87

genotypes from india and neighboring countries by designing 3 anchored ISSR primers

Piyada Tantasawatet et al (2010) for variety identification and estimation of genetic

relationships among 22 mungbean and blackgram (Vigna mungo) genotypes in Thailand

ISSR markers were more efficient than morphological markers

T Gopalakrishna et al (2006) generated recombinant inbreed population and

screened for YMV resistance with ISSR and SCAR markers and identified one marker ISSR

11 1357 was tightly linked to MYMV resistance gene at 63 cM

2416 SNP (Single Nucleotide Polymorphism)

Single base pair differences between individuals of a population are referred to as SNPs SNP

markers are ubiquitous and span the entire genome In human populations it has been

estimated that any two individuals have one SNP every 1000 to 2000 bps Generally there

are an enormous number of potential SNP markers for any given genome SNPs are highly

desirable in genomes that have low levels of polymorphism using conventional marker

systems eg wheat and sorghum SNP markers are biallelic (AT or GC) and therefore are

highly amenable to automation and high-throughput genotyping There have been no

published reports of the development of SNP markers in mungbean but they should be

considered by research groups who envisage long-term plant improvement programs

(Karthikeyan 2010)

25 Marker trait association

Efficient screening of resistant types even in the absence of disease is possible through

molecular marker technology Conventional approaches hindered genetic improvements by

involving complexity in screening procedure to select resistant genotypes A DNA specific

probe has been produced against the geminivirus which has caused yellow mosaic of

mungbean in Thailand (Chiemsombat 1992)

Christian et al (1992) Based on restriction fragment length polymorphism (RFLP)

markers developed genomic maps for cowpea (Vigna unguiculata 2N=22) and mungbean

(Vigna radiata 2N=22) In mungbean there were four unlinked genomic regions accounting

for 497 of the variation for seed weight Using these maps located major quantitative trait

loci (QTLs) for seed weight in both species Two unlinked genomic regions in cowpea

containing QTLs accounting for 527 of the variation for seed weight were identified

RFLP mapping of a major bruchid resistance gene in mungbean (Vigna radiata L Wilczek)

was conducted by Young et al (1993) mapped the TC1966 bruchid resistance gene using

restriction fragment length polymorphism (RFLP) markers Fifty-eight F 2 progeny from a

cross between TC1966 and a susceptible mungbean cultivar were analyzed with 153 RFLP

markers Resistance mapped to a single locus on linkage group VIII approximately 36 cM

from the nearest RFLP marker

Mapping oligogenic resistance to powdery mildew in mungbean with RFLPs was done by

Young et al (1993) A total of three genomic regions were found to have an effect on

powdery mildew response together explaining 58 per cent of the total variation

Lambrides (1996) One QTL for texture layer on linkage group 8 was identified in

mungbean (Vigna radiata L Wilczek) of the cross Berken x ACC41 using RFLP and RAPD

marker

Lambrides et al (2000)In mungbean (Vigna radiata L Wilczek) Pigmentation of the

texture layer and green testa color have been identified on linkage group 2 from the cross

Berken x ACC41 using RFLP and RAPD marker

Chaitieng et al (2002) mappped a new source of resistance to powdery mildew in

mungbean by using both restriction fragment length polymorphism (RFLP) and amplified

fragment length polymorphism (AFLP) The RFLP loci detected by two of the cloned AFLP

bands were associated with resistance and constituted a new linkage group A major

resistance quantitative trait locus was found on this linkage group that accounted for 649

of the variation in resistance to powdery mildew

Humphry et al (2003) with a population of 147 recombinant inbred individuals a

major locus conferring resistance to the causal organism of powdery mildew Erysiphe

polygoni DC in mungbean (Vigna radiata L Wilczek) was identified by using QTL

analysis A single locus was identified that explained up to a maximum of 86 of the total

variation in the resistance response to the pathogen

Basak et al (2004) YMV-tolerant lines generated from a single YMV-tolerant plant

identified in the field within a large population of the susceptible cultivar T-9 were crossed

with T-9 and F1 F2 and F3 progenies are raised Of 24 pairs of resistance gene analog (RGA)

primers screened only one pair RGA 1F-CGRGA 1R was found to be polymorphic among

the parents was found to be linked with YMV-reaction

Miyagi et al (2004) reported the construction of the first mungbean (Vigna radiata L

Wilczek) BAC libraries using two PCR-based markers linked closely with a major locus

conditioning bruchid (Callosobruchus chinesis) resistance

Humphry et al (2005) Relationships between hard-seededness and seed weight in

mungbean (Vigna radiata) was assessed by QTL analysis revealed four loci for hard-

seediness and 11 loci for seed weight

Selvi et al (2006) Bulked segregant analysis was employed to identify RAPD marker

linked to MYMV resistance gene of ML 267 in mungbean Out of 41 primers 3 primers

produced specific fragments in resistant parent and resistant bulk which were absent in the

susceptible parent and bulk Amplification of individual DNA samples out of the bulk with

putative marker OPS 7900 only revealed polymorphism in all 8 resistant and 6 susceptible

plants indicating this marker was associated with MYMV resistance in Ml 267

Chen et al (2007) developed molecular mapping for bruchid resistance (Br) gene in

TC1966 through bulked segregant analysis (BSA) ten randomly amplified polymorphic

DNA (RAPD) markers associated with the bruchid resistance gene were successfully

identified A total of four closely linked RAPDs were cloned and transformed into sequence

characterized amplified region (SCAR) and cleaved amplified polymorphism (CAP) markers

Isemura et al (2007) Using SSR marker detected the QTLs for seed pod stem and

leaf-related trait Several traits such as pod dehiscence were controlled by single genes but

most traits were controlled by between two and nine QTLs

Prakit Somta et al ( 2008) Conducted Quantitative trait loci (QTLs) analysis for

resistance to C chinensis (L) and C maculatus (F) was conducted using F2 (V nepalensis

amp V angularis) and BC1F1 [(V nepalensis amp V angularis) amp V angularis] populations

derived from crosses between the bruchid resistant species V nepalensis and bruchid

susceptible species V angularis In this study they reported that seven QTLs were detected

for bruchid resistance five QTLs for resistance to C chinensis and two QTLs for resistance

to C maculatus

Saxena et al (2009) identified the ISSR marker for resistance to Yellow Mosaic Virus

in Soybean (Glycine max L Merrill) with the cross JS-335 times UPSM-534 The primer 50 SS

was useful to find out the gene resistant to YMV in soybean

Isemura et al (2012) constructed the first genetic linkage map using 430 SSR and

EST-SSR markers from mungbean and its related species and all these markers were mapped

onto 11 linkage groups spanning a total of 7276 cM

Kajonphol et al (2012) used the SSR markers to construct a linkage map and identify

chromosome regions controlling some agronomic traits in mungbean with a mapping

population comprising 186 F2 plants A total of 150 SSR primers were composed into 11

linkage groups each containing at least 5 markers Comparing the mungbean map with azuki

bean (Vigna angularis) and blackgram (Vigna mungo) linkage maps revealed extensive

genome conservation between the three species

26 Bulk segregant analysis (BSA)

Usual method to locate and compare loci regulating a major QTL requires a segregating

population of plants each one genotyped with a molecular marker However plants from such

population can also be grouped according to the phenotypic expression and tested for the

allelic frequency differences in the population bulks (Quarrie et al 1999)

The method of bulk segregant analysis (BSA) was initially proposed by Michelmore et al

1991 in their studies on downy mildew resistance in lettuce It involves comparing two

pooled DNA samples of individuals from a segregating population originating from a single

cross Within each pool or bulk the individuals are identical for the trait or gene of interest

but vary for all other genes Two pools contrasting for a trait (eg resistant and susceptible to

a particular disease) are analyzed to identify markers that distinguish them Markers that are

polymorphic between the pools will be genetically linked to loci determining the trait used to

construct the pools BSA has two immediate applications in developing genetic maps

Detailed genetic maps for many species are being developed by analyzing the segregation of

randomly selected molecular markers in single populations As a genetic map approaches

saturation the continued mapping of polymorphisms detected by arbitrarily selected markers

becomes progressively less efficient Bulked segregate analysis provides a method to focus

on regions of interest or areas sparsely populated with markers Also bulked segregant

analysis is a method of rapidly locating genes that do not segregate in populations initially

used to generate the genetic map (Michelmore et al 1991)

The bulk segregate analysis results in considerable saving of time particularly when used

with PCR based techniques such as RAPD SSR The bulk segregate analysis can be used to

detect the markers linked to many disease resistant genes including Uromyces appendiculatis

resistance in common bean (Haley et al1993) leaf rust resistance in barley (Poulsen et

al1995) and angular leaf spot in common bean (Nietsche et al 2000)

261 Molecular markers associated MYMV resistance using bulk segregant

analysis

Gupta et al (2013) evaluated that marker CEDG 180 was found to be linked with

resistance gene against MYMIV following the bulked segregant analysis This marker was

mapped in the F2 mapping population of 168 individuals at a map distance of 129 cM The

validation of this marker in nine resistant and seven susceptible genotypes has suggested its

use in marker assisted breeding for developing MYMIV resistant genotypes in blackgram

Karthikeyan et al (2012) A total of 72 random sequence decamer oligonucleotide

primers were used for RAPD analysis and they confirmed that OPBB 05 260 marker was

tightly linked to MYMV resistant gene in mungbean by using bulk segregating analysis

(BSA)

Basamma (2011) used 469 primers to identify the molecular markers linked to YMV

in blackgram using Bulk Segregant Analysis (BSA) Only 24 primers were found to be

polymorphic between the parental lines BDU-4 and TAU -1 The BSA using 24 polymorphic

primers on F2 population failed to show any association of a primer with MYMV disease

resistance

Sudha (2009) In this study an F23 population from a cross between ricebean TNAU

RED and mungbean VRM (Gg)1 was used to identify molecular markers linked with the

resistant gene As a result the bulk segregate analysis identified RAPD markers which were

linked with the MYMV resistant gene

Selvi et al (2006) in these studies a F2 population from cross between resistant

mungbean ML267 and susceptible mungbean CO4 is used The bulk segregant analysis was

identified that RAPD markers linked to MYMV resistant gene in mungbean

262 Molecular markers associated with various disease resistances in

other crops using bulk segregant analysis

Che et al (2003) identified five molecular markers link with the sheath blight

resistant gene in rice including three RFLP markers converted from RAPD and AFLP

markers and two SSR markers

Mittal et al (2005) identified one SSR primer Xtxp 309 for leaf blight disease

resistance through bulk segregant analysis and linkage map showed a distance of 312 cM

away from the locus governing resistance to leaf blight which was considered to be closely

linked and 795 cM away from the locus governing susceptibility to leaf blight

Sandhu et al (2005) Bulk segregate analysis was conducted for the identification of

SSR markers that are tightly linked to Rps8 phytophthora resistance gene in soybean

Subsequently bulk segregate analysis of the whole soybean genome and mapping

experiments revealed that the Rps8 gene maps closely to the disease resistance gene-rich

Rps3 region

Malik et al (2007) used PCR technique and bulk segregate analysis to identify DNA

marker linked to leaf rust resistant gene in F2 segregating population in wheat The primer 60-

5 amplified polymorphic molecules of 1100 base pairs from the genomic DNA of resistant

plant

Lei et al (2008) by using 63 randomly amplified polymorphic DNA markers and 113

sets of SSRSTS primers reported molecular markers associated with resistance to bruchids in

mungbean in bulk segregate analysis Two of the markers OPC-06 and STSbr2 were found

to be linked with the locus (named as Br2)

Silva et al (2008) the mapping populations were screened with SSR markers using

the bulk segregate analysis (BSA) to reported four distinct genes (Rpp1 Rpp2 Rpp3 and

Rpp4) that conferred resistance to Asian rust in soybean and expedite the identification of

linked markers

Zhang et al (2008) used Bulk Segregate Analysis (BSA) and Randomly Amplified

Polymorphic DNA (RAPD) methods to analyze the F2 individuals of 82-3041 times Yunyan 84 to

screen and characterize the molecular marker linked to brown-spot resistant gene in tobacco

Primer S361 producing one RAPD marker S361650 tightly linked to the brown-spot

resistant gene

Hyten et al (2009) by using 1536 SNP Golden Gate assay through bulk segregate

analysis (BSA) demonstrated that the high throughput single nucleotide polymorphism (SNP)

genotyping method efficient mapping of a dominant resistant locus to soybean rust (SBR)

designated Rpp3 in soybean A 13-cM region on linkage group C2 was the only candidate

region identified with BSA

Anuradha et al (2011) first report on mapping of QTL for BGM resistance in

chickpea consisting of 144 markers assigned on 11 linkage groups was constructed from

RILs of a cross ICCV 2 X JG 62 map obtained was 4428 cM Three quantitative trait loci

(QTL) which together accounted for 436 of the variation for BGM resistance were

identified and mapped on two linkage groups

Shoba et al (2012) through bulk segregant analysis identified the SSR primer PM

384100 allele for late leaf spot disease resistance in groundnut PM 384100 was able to

distinguish the resistant and susceptible bulks and individuals for Late Leaf Spot (LLS)

Priya et al (2013) Linkage analysis was carried out in mungbean using RAPD marker

OPA-13420 on 120 individuals of F2 progenies from the crossing between BL-20 times Vs The

results demonstrated that the genetic distance between OPA-13420 and powdery mildew

resistant gene was 583 cM

Vikram et al (2013) The BSA approach successfully detected consistent effect

drought grain-yield QTLs qDTY11 and qDTY81 detected by Whole Population Genotyping

(WPG) and Selective Genotyping (SG)

27 Marker assisted selection (MAS)

The major yield constraint in pulses is high genotype times environment (G times E) interactions on

the expression of important quantitative traits leading to slow gain in genetic improvement

and yield stability of pulses (Kumar and Ali 2006) besides severe losses caused by

susceptibility of pulses to biotic and abiotic stresses These issues require an immediate

attention and overall a paradigm shift is needed in the breeding strategies to strengthen our

traditional crop improvement programmes One way is to utilize genomics tools in

conventional breeding programmes involving molecular marker technology in selection of

desirable genotypes

The efficiency and effectiveness of conventional breeding can be significantly improved by

using molecular markers Nowadays deployment of molecular markers is not a dream to a

conventional plant breeder as it is routinely used worldwide in all major cereal crops as a

component of breeding because of the availability of a large amount of basic genetic and

genomic resources (Gupta et al 2010)In the past few years major emphasis has also been

given to develop similar kind of genomic resources for improving productivity of pulse crops

(Varshney et al 2009 2010a Sato et al 2010) Use of molecular marker technology can

give real output in terms of high-yielding genotypes in pulses because high phenotypic

instability for important traits makes them difficult for improvement through conventional

breeding methods The progress made in using marker-assisted selection (MAS) in pulses has

been highlighted in a few recent reviews emphasizing on mapping genes controlling

agronomically important traits and molecular breeding of pulses in general (Liu et al 2007

and Varshney et al 2010) and faba bean in particular (Torres et al 2010)

Molecular markers especially DNA based markers have been extensively used in many areas

such as gene mapping and tagging (Kliebenstein et al 2002) Genetic distance between

parents is an important issue in mapping studies as it can determine the levels of segregation

distortion (Lambrides and Godwin 2007) characterization of sex and analysis of genetic

diversity (Erschadi et al 2000)

Marker-assisted selection (MAS) offers us an appropriate relevant and a non-transgenic

strategy which enables us to introgress resistance from wild species (Ali et al 1997

Lambrides et al 1999 and Humphry et al 2002) Indirect selection using molecular markers

linked to resistance genes could be one of the alternate approaches as they enable MAS to

overcome the inaccuracies in the field evaluation (Selvi et al 2006) The use of molecular

markers for resistance genes is particularly powerful as it removes the delay in breeding

programmes associated with the phenotypic analysis (Karthikeyan et al 2012)

Chapter III

Materials and Methods

Chapter

MATERIAL AND METHODS

The present study entitled ldquoIdentification of molecular markers linked to

yellow mosaic virus resistance in blackgram (Vigna mungo (L) Hepper)rdquo was conducted

during the year of 2015-2016 The plant material and methods followed to conduct the present

study are described in this chapter

31 EXPERIMENTAL MATERIAL

311 Plant Material

The identified resistant and susceptible parents of blackgram for yellow mosaic virus

ie T-9 and LBG-759 respectively were procured from Agriculture Research Station

PJTSAU Madhira A cross was made between T9 and LBG 759 F2 mapping population was

developed from this cross was used for screening against YMV disease incidence

312 Markers used for polymorphism study

A total of 50 SSR (simple sequence repeats) markers were used for blackgram for

polymorphic studies and the identified polymorphic primers were used for genotyping

studies List of primers used are given in table 31

313 List of equipments used

Equipments and chemicals used for the study are mentioned in the appendix I and

appendix II

32 DEVELOPMENT OF MAPPING POPULATION

Mapping population for studying resistance to YMV disease was developed from the

crosses between the susceptible parent of LGG-759 used as female parent and the resistant

variety T9 used as a pollen parent The crosses were affected during kharif 2015-16 at the

College farm PJTSAU Rajendranagar The F1s were selfed to produce F2 during rabi 2015-

16 Thus the mapping population comprising of F2 generation was developed The mapping

populations F2 along with the parents and F1 were screened for yellow mosaic virus resistance

at ARS Madhira Khammam during late rabi (summer) 2015-16 One twenty five (125)

individual plants of the F2 population involving the above parents namely susceptible (LGG-

759 and the resistant T9 were developed in ARS Madhira Khammam) were screened for

YMV incidence

33 PHENOTYPING OF F2 MAPPING POPULATION

Using the disease screening methodology the F2 population along with the parents

and F1 were evaluated for yellow mosaic virus resistance under field conditions

331 Disease Screening Methodology

F2 population parents and F1 were screened for mungbean yellow mosaic virus

resistance under field conditions using infector rows of the susceptible parent viz LBG-759

during late rabi 2015-16 at ARS Madhira Khammam As this Madhira region is hotspot for

YMV incidence The mapping population (F2) was sown in pots filled with soil Two rows of

the susceptible check were raised all around the experimental pots in order to attract white fly

and enhance infection of MYMV under field conditions All the recommended cultural

practices were followed to maintain the experiment except that insecticide sprays were not

given to encourage the white fly population for the spread of the disease

Thirty days after sowing whitefly started landing on the plants the crop was regularly

monitored for the presence of whitefly and development of YMV Data on number of dead

and surviving plants were recorded Infection and disease severity of MYMV progressed in

the next 6 weeks and each plant was rated on 0-5 scale as suggested by Bashir et al (2005)

which is described in Table 32 The disease scoring was recorded from initial flowering to

harvesting by weekly intervals

Table 32 Scale used for YMV reaction (Bashir et al 2005)

SEVERITY INFECTION INFECTION

CATEGORY

REACTION

GROUP

0 All plants free of virus

symptoms

Highly Resistant HR

1 1-10 infection Resistant RR

2 11-20 infection Moderately resistant MR

3 21-30 infection Moderately Suseptible MS

4 30-50 infection Susceptible S

5 More than 50 Highly susceptible HS

332 Quantitative Traits

1 Height of the plant (cm) Height measured from the base of the plant to the tip of

the main shoot at harvesting stage

2 Number of branches per

plant

The total number of primary branches on each plant at the

time of harvest was recorded

3 Number of clusters (cm) The total number of clusters per branch was counted in

each of the branches and recorded during the harvest

4 Pod Length (cm) The average length of five pods selected at random from

each of the plant was measured in centimeters

5 Number of pods per plant The total number of fully matured pods at the time of

harvest was recorded

6 Number of seeds per pod This was arrived at counting the seeds from five randomly

selected pods in each of five plants and then by calculating

the mean

7 Days to 50 flowering Number of days for the fifty percent flowering

8 Single plant yield (g) Weight of all well dried seeds from individual plant

35 STATISTICAL ANALYSIS

The data recorded on various characters were subjected to the following

statistical analysis

1 Chi-Square Analysis

2 Analysis of variance

3 Estimation of Genetic Parameters


Test of significance among F2 generation was done by chi-square method2 Test was

applied for testing the deviation of the observed segregation from theoretical segregation

Chi-square was calculated using the formula

E

EO 22 )(

Where

O = Observed frequency

E = Expected frequency

= Summation of the data

If the calculated values of 2 is significant at 5 per cent level of significance is said

to be poor and one or more observed frequencies are not in accordance with the hypotheses

assumed and vice versa So it is also known as goodness of fit The degree of freedom (df) in

2 test is (n-1) Where n = number of classes

352 Analysis of Variance

The mean and variances were analyzed based on the formula given by Singh and

Chaudhary (1977)

3521 Mean

n

1 ( sum yi )

Y = n i=1

3522 Variance

n

1 sum(Yi-Y)2

Variance = n-1 i=1

Where Yi = Individual value

Y = Population mean

sum d2

Standard deviation (SD) = Variance = N

Where

d = Deviation of individual value from mean and

N = Number of observations

353 Estimation of genetic parameters

Genotypic and phenotypic variances and coefficients of variance were computed

based on mean and variance calculated by using the data of unreplicated treatments

3531 Phenotypic variance

The individual observations made for each trait on F2 population is used for calculating the

phenotypic variance

Phenotypic variance (2p) = Var F2

Where Var F2 = variance of F2 population

3532 Environmental variance

The average variance of parents and their corresponding F1 is used as environmental

variance for single crosses

Var P1 + Var P2 + Var F1

Environmental Variance (2e) = 3

Where

Var P1 = Variance of P1 parent

Var P2 = Variance of P2 parent and

Var F1 = variance of corresponding F1 cross

3533 Genotypic and phenotypic coefficient of variation

The genotypic and phenotypic coefficient of variation was computed according to

Burton and Devane (1953)

2g

Genotypic coefficient of variation (GCV) = --------------------------------------- times100

Mean

2p

Phenotypic coefficient of variation (PCV) = ------------------------------------ times100

Mean

Where

2g = Genotypic variance

2p = Phenotypic variance and X = General mean of the character

3534 Heritability

Heritability in broad sense was estimated as the ratio of genotypic to phenotypic

variance and expressed in percentage (Hanson et al 1956)

σsup2g

hsup2 (bs) = ------------

σsup2p

Where

hsup2(bs) = heritability in broad sense


2p = Phenotypic variance

As suggested by Johnson et al (1955) (hsup2) estimates were categorized as

Low 0-30

Medium 30-60

High above 60

3535 Genetic advance (GA)

This was worked out as per the formula proposed by Johnson et al (1955)

GA = k 2p H

Where

k = Intensity of selection

2p = Phenotypic standard deviation

H = Heritability in broad sense

The value of bdquok‟ was taken as 206 assuming 5 per cent selection intensity

3536 Genetic advance expressed as percentage over mean (GAM)

In order to visualize the relative utility of genetic advance among the characters

genetic advance as percent for mean was computed

GA

Genetic advance as percent of mean = ---------------- times 100

Grand mean

The range of genetic advance as percent of mean was classified as suggested by

Johnson et al (1955)

Low Less than 10

Moderate 10-20

High More than 20

34 STUDY OF PARENTAL POLYMORPHISM

341 Preparation of Stocks and Buffer solutions

Preparation of stocks and buffer solutions used for the present study are given in the

appendix III

342 DNA extraction by CTAB method (Doyle and Doyle 1987)

The genomic DNA was isolated from leaf tissue of 20 days old F2 population

MYMV susceptible LBG-759 and the MYMV resistant T9 parents and following the protocol

of Doyle and Doyle (1987)

Method

The leaf samples were ground with 500 μl of CTAB buffer transferred into an

eppendorf tubes and were kept in water bath at 65degC with occasional mixing of tubes The

tubes were removed from the water bath and allowed to cool at room temperature Equal

volume of chloroform isoamyl alcohol mixture (24 1) was added into the tubes and mixed

thoroughly by gentle inversion for 15 minutes by keeping in rotator 12000 rpm (eppendorf

centrifuge) until clear separation of three layers was attained The clear aqueous phase

(supernatant) was carefully pipette out into new tubes The chloroform isoamyl alcohol (241

vv) step was repeated twice to remove the organic contaminants in the supernatant To the

supernatant cold isopropanol of about 05 to 06 volumes (23rd

of pipette volume) was

added The contents were mixed gently by inversion and keep at 4degC for overnight

Subsequently the tubes were centrifuged at 12000 rpm for 12 min at 24degC temperature to

pellet out DNA The supernatant was discarded gently and the DNA pellet was washed with

70 ethanol and centrifuged at 13000 rpm for 4-5 min This step was repeated twice The

supernatant was removed the tubes were allowed to air dry completely and the pellet was

dissolved in 50 μl T10E1 buffer DNA was stored at 4degC for further use

343 Quantification of DNA

DNA was checked for its purity and intactness and then quantified The crude

genomic DNA was run on 08 agarose gel stained with ethidium bromide following a

standard method (Sambrook et al 1989) and was visualized in a gel documentation system

(BIO- RAD)

Quantification by Nanodrop method

The ratio of absorbance at 260 nm and 280 nm was used to assess the purity of DNA

A ratio of ~18 is generally accepted as ldquopurerdquo for DNA a ratio of ~20 is generally

accepted as ldquopurerdquo for RNA If the ratio is appreciably lower in either case it may indicate

the presence of protein phenol or other contaminants that absorb strongly at or near 280

nm The quantity of DNA in different samples varied from 50-1350 ng μl After

quantification all the samples were diluted to 50 ng μl and used for PCR reactions

344 Molecular analysis

Molecular analysis was carried out by parental polymorphism survey and

genotyping of F2 population with PCR analysis

345 PCR Confirmation Studies

DNA templates from resistant and susceptible parent were amplified using a set of 50

SSR primer pairs listed in table 31 Parental polymorphism genotyping studies on F2

population and bulk segregation analysis were conducted by using PCR analysis PCR

amplification was carried out on thermal cycler (AB Veriti USA) with the components and

cycles mentioned below in tables 32 and 33

Table 33 Components of PCR reaction

PCR reaction was performed in a 10 μl volume of mix containing the following

Component Quantity Reaction volume

Taq buffer (10X) with Mg Cl2 1X 10 microl

dNTP mix 25 mM 10 microl

Taq DNA polymerase 3Umicrol 02 microl

Forward primer 02 μM 05 microl

Reverse primer 02 μM 05microl

Genomic DNA 50 ngmicrol 30 microl

Sterile distilled water 38 microl

Table 34 PCR temperature regime

SNO STEP TEMPERATURE TIME Cycles

1 Initial denaturation 95o C 5 minutes 1

2 Denaturation 94o C 45 seconds

35cycles 3 Annealing 57-60 o

C 45 seconds

4 Extension 72o C 1 minute

5 Final extension 72o C 10 minutes 1

6 4˚c infin

The reaction mixture was given a short spin for thorough mixing of the cocktail

components PCR samples were stored at 4˚C for short periods and at -20

˚C for long duration

The amplified products were loaded on ethidium bromide stained agarose gels (3 ) and

polymorphic primers were noted

346 Agarose Gel Electrophoresis

Agarose gel (3) electrophoresis was performed to separate the amplified products

Protocol

Agarose gel (3) electrophoresis was carried out to separate the amplified DNA

products The PCR amplified products were resolved on 3 agarose gel The agarose gel was

prepared by adding 3 gm of agarose to 100ml 10X TAE buffer and boiled carefully till the

agarose completely melted Just before complete cooling 3μ1 ethidium bromide (10 mgml)

was added and the gel was poured in the tray containing the comb carefully avoiding

formation of air bubbles The solidified gel was transferred to horizontal electrophoresis

apparatus and 1X TAE buffer was added to immerse the gel

Loading the PCR products

PCR product was mixed with 3 μl of 6X loading dye and loaded in the agarose gel well

carefully A 50 bp ladder was loaded as a reference marker The gel was run at constant

voltage of 70V for about 4-6 hours until the ladder got properly resolved Gel was

photographed using the Gel Documentation system (BIORAD GEL DOC XR + Imaging

system)

347 PARENTAL POLYMORPHISM AND SCREENING OF MAPPING

POPULATION

A total number of 50 SSR primers (table no 31) were screened among two parents

for a parental polymorphism study 14 primers were identified as polymorphic (Table)

between two parents and they were further used for screening the susceptible and resistant

bulks through bulked segregant analysis Consistency of the bands was checked by repeating

the reaction twice and the reproducible bands were scored in all the samples for each of the

primers separately As the SSR marker is the co dominant marker bands were present in both

resistant and susceptible parents

348 BULK SEGREGANT ANALYSIS (BSA)

Bulk segregant analysis was used to identify the SSR markers that are associated with

MYMV resistance for rapid selection of genotypes in any breeding programme for resistance

Two bulks of extreme phenotypes resistant and susceptible were made for the BSA analysis

The resistant parent (T9) the susceptible parent (LBG 759) ten F2 individuals with MYMV

resistant score ndash 1 of 13 plants and the ten F2 individuals found susceptible with MYMV

susceptible score ndash 5 of 17 plants were separately used for the development of bulks of the

cross Equal quantities of DNA were bulked from susceptible individuals and resistant

individuals to give two DNA bulks namely resistant bulks (RB) and susceptible bulks (SB)

The susceptible and resistant bulks along with parents were screened with polymorphic SSR

which revealed polymorphism in parental survey The polymorphic marker amplified in

parents and bulks were tested with ten resistant and susceptible F2 plants Individually

amplified products were run on an agarose gel (3)

Chapter IV

Results amp Discussion

Chapter IV

RESULTS AND DISCUSSION

The present study was carried in Department of Molecular Biology and Biotechnology to tag

the gene resistance to MYMV (Mungbean yellow mosaic virus) in Blackgram In present

study attempts were made to develop a population involving the cross between LBG-759

(MYMV susceptible parent) and T9 (MYMV resistant parent) MYMV resistant and

susceptible parents were selected and used for identifying molecular markers linked to

MYMV resistance with the following objectives

1) To study the Parental polymorphism

2) Phenotyping and Genotyping of F2 mapping population

3) Identification of SSR markers linked to Yellow mosaic virus resistance by Bulk

Segregant analysis

The results obtained in the present study are presented and discussed here under

41 PHENOTYPING AND STUDY OF INHERITANCE OF MYMV

DISEASE RESISTANCE

411 Development of Segregating Population

Blackgram MYMV resistant parent T9 and blackgram MYMV susceptible parent LBG-759 were

selected as parents and crossing was carried out during kharif 2015 The F1 obtained from that

cross were selfed to raise the F2 population during rabi 2015 F2 populations and parents were also

raised without any replications during late rabi 2015-16 The field outlook of the F2 population

along with parents developed for segregating population is shown in plate 41

412 Phenotyping of F2 Segregating Population

A total of 125 F2 plants along with parents used for the standard disease screening Standard

disease screening methodology was conducted in F1 and F2 population evaluated for MYMV

resistance along with parents under field conditions as mentioned in materials and method

Plate 41 Field view of F2 population

Resistant population Susceptible population

Plate 42 YMV Disease scorring pattern

HIGHLY RESISTANT-0 MODERATELY SUSEPTIBLE-3

RESISTANT-1 SUSEPTIBLE-4

MODERATELY RESISTANT-2 HIGHLY SUSCEPTIBLE-5

Plate 43 Screening of segregating material for YMV disease reaction

times

T9 LBG 759

F1 Plants

Resistant parent T9 selected for crossing showed a disease score of 1 according to the Basak et al

2005 and LBG-759 was taken as susceptible parent showed a disease score of 5 whereas F1 plants

showed the mean score of 2 (table 41)

F1 s seeds were sowned and selfed to produce F2 mapping population F2 seed was sown during

late rabi 2015-16 F2 population was screened for disease resistance under field conditions along

with parents Of a total of 125 F2 plants 30 plants showed the less than 20 infection and

remaining plants showed gt50 infection respectively The frequency of F2 segregants showing

different scores of resistancesusceptibility to MYMV are presented in table 42 The disease

scoring symptoms are represented in plate 42

413 Inheritance of Resistance to Mungbean Yellow Mosaic Virus

Crossings were performed by taking highly resistant T9 as a male parent and susceptible LBG-

759 as female parent with good agronomic background The parents F1 were sown at College of

Agriculture Rajendranagar and F2 population of this cross sown at ARS Madhira Khammam in

late rabi season of 2015-16

The inheritance study of the 30 resistant and 95 susceptible F2 plants showing a goodness

of fit to expected 13 (Resistant Suceptible) ratio These results of the chai square test suggest a

typical monogenic recessive gene governing resistance and susceptibility reaction against MYMV

(Table 43 Plate 43)

Such monogenic recessive inheritance of YMV resistance is compared with the results

reported by Anusha et al(2014) Gupta et al (2013) Jain et al (2013) Reddy (2009)

Kundagrami et al (2009) Basak et al (2005) and Thakur et al (1977) However reports

indicating the involvement of two recessive genes in controlling YMV resistance in urdbean by

Singh (1990) verma and singh (2000) singh and singh (2006) Single dominant gene

controlling resistance to MYMV has been reported by Gupta et al (2005) and complementary

recessive genes are reported by Shukla 1985

These contradictory results can be possible due to difference in the genotype used the

strains of virus and interaction between them Difference in the nature of gene contributing

resistance to YMV might be attributed to differences in the source of resistance used in study

42 STUDY OF PARENTAL POLYMORPHISM AND

IDENTIFICATION OF MOLECULAR MARKERS LINKED TO YELLOW

MOSAIC VIRUS RESISTANCE BY BULK SEGREGANT ANALYSIS

(BSA)

In the present study the major objective was to tag the molecular markers linked to yellow mosaic

virus using SSR marker in the developed F2 population obtained from the cross between LBG 759

times T9 as follows

421 Checking of Parental Polymorphism Using SSR markers

The LBG 759 (MYMV susceptible parent) and T9 (MYMV resistant parent) were initially

screened with 50 SSR markers to find out the markers showing polymorphism between the

parents Out of these 50 markers used for parental survey 14 markers showed polymorphism

between the parents (Fig 43) and the remaining markers were showed monomorphic (Fig 42)

28 of polymorphism was observed in F2 population of urdbean The sequence of polymorphic

primers annealing temperature and amplification are represented in the table 44 Similarly the

confirmation of F1 progeny was carried out using 14 polymorphic markers (Fig 44)

422 Bulk Segregant Analysis (BSA)

The polymorphism study between the parents of LBG-759 and T9 was carried out using 50 SSR

markers Of which 14 markers namely viz CEDG073 CEDG075 CEDG091 CEDG092

CEDG097 CEDG116 CEDG128 CEDG139 CEDG147 CEDG154 CEDG156 CEDG176

CEDG185 CEDG199 showed polymorphism with a different allele size (bp) (Table 44) Bulk

segregant analysis was carried with these polymorphic markers to identify the markers linked to

the gene conferring resistance to MYMV For the preparation of susceptible and resistant bulks

equal amounts of DNA were taken from ten susceptible F2 individuals (MYMV score 5) and ten

resistant F2 individuals (MYMV score 1) respectively These parents and bulks were further

screened with the 14 polymorphic SSR markers which showed polymorphism in parental survey

using same concentration of PCR ingredients under the same temperature profile

Out of these 14 SSR markers one marker CEDG185 showed the polymorphism between the bulks

as well as parents (Fig 44) When tested with ten individual resistant F2 plants CEDG185 marker

amplified an allele of 160 bp in the susceptible parent susceptible bulk (Fig 46) This marker

found to be amplified when tested with ten individual resistant F2 plants (Fig 46) Similarly same

marker amplified an allele of 190 bp in resistant parent resistant bulk

This marker gave amplified 170 bp amplicon when tested with ten individual susceptible F2

plants (Fig 45) The amplification of resistant parental allele in resistant bulk and susceptible

parental allele in susceptible bulk indicated that this marker is associated with the gene controlling

MYMV resistance in blackgram Similar results were found in mungbean using 361 SSR markers

(Gupta et al 2013) Out of 361 markers used 31 were found to be polymorphic between the

parents The marker CED 180 markers were found to be linked with resistance gene by the bulk

segregant analysis (Gupta et al 2013) Shoba et al (2012) identified the SSR marker PM384100

allele for late leaf spot disease resistance by bulked segregant analysis Identified SSR marker PM

384100 was able to distinguish the resistant and susceptible bulks and individuals for late leaf spot

disease in groundnut

In Blackgram several studies were conducted to identify the molecular markers linked to YMV

resistance by using the RAPD marker from azukibean which shows the specific fragment in

resistant parent and resistant bulk which were absent in susceptible parent and susceptible bulk

(Selvi et al 2006) Karthikeyan et al (2012) reported that RAPD marker OPBB05 from

azukibean which shows specific amplified size of 450 bp in susceptible parent bulk and five

individuals of F2 populations and another phenotypic (resistant) specific amplified size of 260 bp

for resistant parent bulk and five individuals of F2 population One species-specific SCAR marker

was developed for ricebean which resolved amplified size of 400bp in resistant parent and absent

in the bulk (Sudha et al 2012) Karthikeyan et al (2012) studied the SSR markers linked to YMV

resistance from azukibean in mungbean BSA Out of 45 markers 6 showed polymorphism

between parents and not able to distinguish the bulks Similar results were found in blackgram

using 468 SSR markers from soybean common bean red gram azuki bean Out of which 24 SSR

markers showed polymorphism between parents and none of the primer showed polymorphism

between bulks (Basamma 2011)

In several studies conducted earlier molecular markers have been used to tag YMV

resistance in many legume crops like soybean common bean pea (Gao et al 2004) and

peanut (Shoba et al 2012) Gioi et al (2012) identified and characterized SSR markers

Figure 41 parental polymorphism survey of uradbean lines LBG 759 (1) times T9 (2) with monomorphic SSR

primers The ladder used was 50bp

50bp 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1

2

CEDG076 CEDG086 CEDG099 CEDG107 CEDG111 CEDG113 CEDG115 CEDG118 CEDG127 CEDG130

200bp

Figure 42 Parental survey of uradbean lines LBG 759 (1) times T9 (2) with monomorphic SSR primers The ladder

used was 50bp

50bp 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2

CEDG132 CEDG0136 CEDG141 CEDG150 CEDG166 CEDG168 CEDG171 CEDG174 CEDG180 CEDG186 CEDG200 CEDG202

CEDG202

200bp

50bp 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2

CEDG073 CEDG185 CEDG075 CEDG091 CEDG092 CEDG097 CEDG116 CEDG128 CEDG139 CEDG147 CEDG154 CEDG156 CEDG199

Figure 43 Parental survey of uradbean lines LBG 759 (1) times T9 (2) with Polymorphic SSR primers The

ladder used was 50bp

200bp

Table 44 List of polymorphic primers of the cross LBG 759 X T9

Sl No Primer

name

Primer sequence Annealing

temperature(degc)

Allele size (bp)

S R

1

CEDG073

F- CCCCGAAATTCCCCTACAC

60

150 250

R- AACACCCGCCTCTTTCTCC

2

CEDG075

F- GCGACCTCGAAAATGGTGGTTT

60

150 200

R- TCACCAACTCACTCGCTCACTG

3

CEDG091

F- CTGGTGGAACAAAGCAAAAGAGT

57

150 170

R- TGGGTCTTGGTGCAAAGAAGAAA

4

CEDG092

F- TCTTTTGGTTGTAGCAGGATGAAC

57

150 210

R- TACAAGTGATATGCAACGGTTAGG

5

CEDG097

F- GTAAGCCGCATCCATAATTCCA

57

150 230

R- TGCGAAAGAGCCGTTAGTAGAA

6

CEDG116

F- TTGTATCGAAACGACGACGCAGAT

57

150 170

R- AACATCAACTCCAGTCTCACCAAA

7 F- CTGCCAAAGATGGACAACTTGGAC 150 180

CEDG128 R- GCCAACCATCATCACAGTGC 60

8

CEDG139

F- CAAACTTCCGATCGAAAGCGCTTG

60

150 190

R- GTTTCTCCTCAATCTCAAGCTCCG

9

CEDG147

F- CTCCGTCGAAGAAGGTTGAC

60

150 160

R- GCAAAAATGTGGCGTTTGGTTGC

10

CEDG154

F- GTCCTTGTTTTCCTCTCCATGG

58

150 180

R- CATCAGCTGTTCAACACCCTGTG

11

CEDG156

F- CGCGTATTGGTGACTAGGTATG

58

150 210

R- CTTAGTGTTGGGTTGGTCGTAAGG

12

CEDG176

F- GGTAACACGGGTTCAGATGCC

60

150 180

R- CAAGGTGGAGGACAAGATCGG

13

CEDG185

F- CACGAACCGGTTACAGAGGG

60

160 190

R- CATCGCATTCCCTTCGCTGC

14 CEDG199 F- CCTTGGTTGGAGCAGCAGC 60 150 180

R- CACAGACACCCTCGCGATG

R=Resistant parent S= Susceptible parent

200bp

50bp P1 P2 1 2 3 4 5 6 7 8 9 10

Figure 44 Conformation of F1 s using SSR marker CEDG185 P1 P2 indicate the parents Lanes 1-

10 indicate F1 plants The ladder used was 50bp

200bp

50bp SP RP SB RB SB RB SB RB

Figure 45 Bulk segregant analysis with SSR primer CEDG 185 SP RP indicates susceptible and

resistant parents SB RB indicates susceptible and resistant bulks The ladder used is 50bp

200bp

50bp SP RP SB RB 1 2 3 4 5 6 7 8 9 10

Figure 46 Conformation of Bulk segregant analysis with SSR primer CEDG 185 SP RP indicates

susceptible and resistant parents SB RB indicates susceptible and resistant bulks The lanes 1-10

indicates F2 resistant plants The ladder used is 50bp

50bp SP RP SB RB 1 2 3 4 5 6 7 8 9 10



indicates F2 suceptible plants The ladder used is 50bp ladder

200bp

linked to YMV resistance gene in cowpea by using 60 SSR markers The interval QTL mapping

showed 984 per cent of the resistance trait mapped in the region of three loci AGB1 VM31 amp

VM1 covered 321 cM in which 95 confidence interval for the CYMV resistance QTL

associated with VM31 locus was mapped within only 19 cM

Linkage of a RGA marker of 445 bp with YMV resistance in blackgram was reported by Basak et

al (2004) The resistance gene for yellow mosaic disease was identified to be linked with a SCAR

marker at a map distance of 68 cm (Souframanien and Gopalakrishna 2006) In another study a

RGA marker namely CYR1 was shown to be completely linked to the MYMIV resistance gene

when validated in susceptible (T9) and resistant (AKU9904) genotypes (Maiti et al 2011)

Prashanthi et al (2011) identified random amplified polymorphic DNA (RAPD) marker OPQ-1

linked to YMV resistant among 130 oligonucleotide primers Dhole et al (2012) studied the

development of a SCAR marker linked with a MYMV resistance gene in Mungbean Three

primers amplified specific polymorphic fragments viz OPB-07600 OPC-061750 and OPB-

12820 The marker OPB-07600 was more closely linked (68 cM) with a MYMV resistance gene

From the present study the marker CEDG185 showed the polymorphism between the parents and

bulks and amplified with an allele size 190 bp and 160 bp in ten individual of both resistant and

susceptible plants respectively which were taken as bulks This marker CEDG185 can be

effectively utilized for developing the YMV resistant genotypes thereby achieving substantial

impact on crop improvement by marker assisted selection resulting in sustainable agriculture

Such cultivars will be of immense use for cultivation in the northern and central part of India

which is the major blackgram growing area of the country

44 EVALUATION OF QUANTITATIVE TRAITS IN F2

SEGREGATING POPULATION

A total of 125 plants in the F2 generation were evaluated for the following morphological traits

viz height of the plant number of branches number of clusters days to 50 per cent flowering

number of pods per plant length of the pod number of seeds per pod single plant yield along with

MYMV score The results are presented as follows

441 Analysis of Mean Range and Variance

In order to assess the worth of the population for isolating high yielding lines besides looking for

resistance to YMV the variability parameters like mean range and variance were computed for

eight quantitative traits viz height of the plant number of branches number of clusters days to

50 per cent flowering number of pods per plant length of the pod number of seeds per pod

single plant yield and the MYMV score (in field) in F2 population of the crosses LBG 759 X T9

The results are presented in Table 45

Mean values were high for days to 50 flowering (4434) and plant height (2330) number of

pods per plant (1491) Less mean was observed in other traits lowest mean was observed in single

plant yield (213)

Height of the plant ranged from20 to 32 with a mean of 2430 Number of branches ranged from 4

to 7 with a mean of 516 Number of clusters ranged from 3 to 9 with a mean of 435 Days to 50

flowering ranged from 38 to 50 with a mean of 4434 Number of pods per plant ranged from 10 to

21 with a mean of 1492 Pod length ranged from 40 to 80 with a mean of 604 Number of seeds

per pod ranged from 3 to 6 with a mean of 532 Seed yield per plant ranged from 08 to 443 with

a mean of 213

The F2 populations of this cross exhibited high variance for single plant yield (3051) number of

clusters (2436) pod length (2185) Less variance was observed for the remaining traits The

lowest variation was observed for the trait pod length (12)

The increase in mean values as a result of hybridization indicates scope for further improvement

in traits like number of pods per plant number of seeds per pod and pod length and other

characters in subsequent generations (F3 and F4) there by facilitating selection of transgressive

segregants in later generations The results are in line with the findings of Basamma et al (2011)

The critical parameters are range and variance which decide the higher extreme value of the cross

The range observed was wider for number of pods per plant number of seeds per plant pod

length number of branches per plant plant height number of clusters days to 50 flowering and

single plant yield in F2 population Similar results were obtained by Salimath et al (2007) in F2

and F3 population of cowpea

442 Variability Parameters

The genetic gain through selection depends on the quantum of variability and extent to which it is

heritable In the present study variability parameter were computed for eight quantitative traits


number of pods per plant length of the pod number of seeds per pod single plant yield and the

MYMV score in F2 population The results are presented in Table 46

4421 Phenotypic and Genotypic Coefficient of Variation

High PCV estimates were observed for single plant yield (2989) number of clusters(2345) pod

length(2072)moderate estimates were observed for number of pods per plant(1823) number of

seeds per pod(1535)lowest estimates for days to flowering(752)

High GCV estimates were observed for single plant yield (2077) number of clusters(1435) pod

length(1663)Moderate estimates were observed for number of pods per plant(1046) number of

seeds per pod(929) lowest estimates for days to flowering(312)

The genotypic coefficients of variation for all characters studied were lesser than phenotypic

coefficient of variation indicating masking effects of environment (Table 46) showing greater

influence of environment on these traits These results are in accordance with the finding of Singh

et al (2009) Konda et al (2009) who also reported similar effects of environment Number of

seed per pod and number of pods per pod had moderate GCV and PCV values in the F2

populations Days to 50 flowering had low PCV and GCV values Low to moderate GCV and

PCV values for above three characters indicate the influence of the environment on these traits and

also limited scope of selection for improvement of these characters

The high medium and low PCV and GCV indicate the potentiality with which the characters

express However GCV is considered to be more useful than PCV for assessing variability since

it depends on the heritable portion of variability The difference between GCV and PCV for pods

per plant and seed yield per plant were high indicating the greater influence of environment on the

expression of these characters whereas for remaining other traits were least influenced by

environment

The results of the above experiments showed that variability can be created by hybridization

(Basamma 2011) However the variability generated to a large extent depends on the parental

genotype and the trait under study

4422 Heritability and Genetic advance

Heritability in broad sense was high for pod lenghth (8026) plant height(750) single plant

yield(6948) number of branches per plant(6433)number of clusters(6208) number of seeds per

pod(6052) Moderate values were observed for number of pods per plant (5573) days to

flowering(4305)

Genetic advance was high for number of pods per plant (555) days to flowering(553) plant

height(404) pod length(256) number of clusters(208) Low values observed for number of

branches per plant(179) number of seeds per pod(161) single plant yiield(130)

Genetic advance as percent of mean was high for number of clusters(4792)pod length(4234)

number of pods per plant(3726) single plant yiield(3508) number of branches per plant(3478)

number of seeds per pod(3137) low values were observed for plant height(16) days to

flowering(147)

In this study heritability in broad sense and genetic advance as percent of mean was high for

number of pods per plant single plant yield number of branches per plant pod length indicating

that these traits were controlled by additive genes indicating the availability of sufficient heritable

variation that could be made use in the selection programme and can easily be transferred to

succeeding generations Similar results were found by Rahim et al (2011) (Arulbalachandran et

al 2010) (Singh et al 2009) and Konda et al (2009)

Moderate genetic advance as percent of mean values and moderate heritability in broad sense was

observed in number of seeds per pod which indicate that the greater role of non-additive genetic

variance and epistatic and dominant environmental factors controlling the inheritance of these

traits Similar results were found by Ghafoor and Ahmad (2005)

High heritability and moderate genetic advance as percent of mean was observed in days to 50

flowering indicating that these traits were controlled by dominant epistasis which was similar to

Muhammad Siddique et al (2006) Genetic advance as percent of mean was high for number of

clusters and shows moderate heritability in broad sense

Future line of work

The results of the present investigation indicated the variability for productivity and disease

related traits can be generated by hybridization involving selected diverse parents

1 In the present study hybridized population involving two genotypes viz LBG 759 and T9

parents resulted in increased variability heritability and genetic advance as percent mean values

These populations need to be handled under different selection schemes for improving

productivity

2 SSR marker tagged to yellow mosaic virus resistant gene can be used for screening large

germplasm for YMV resistance

3 The material generated can be forwarded by single seed descent method to develop RILS

4 It can be used for mapping YMV resistance gene and validation of identified marker

Table 41 Mean disease score of parental lines of the cross LBG 759 X T9 for

MYMV in Black gram

Disease Parents Score

MYMV T9

LBG 759

F1

1

5

2

0-5 Scale

Table 42 Frequency of F2 segregants of the cross LBG 759 times T9 of blackgram showing

different grades of resistancesusceptibility to MYMV

Resistance Susceptibility

Score

Reaction Frequency of F2

segregants

0 Highly Resistant 2

1 Resistant 12

2 Moderately Resistant 16

3 Moderately Suseptible 40

4 Suseptible 32

5 Highly Suseptible 23

Total 125

Table 46 Estimates of components of Variability Heritability(broad sense) expected Genetic advance and Genetic

advance over mean for eight traits in segregating F2 population of LBG 759 times T9

PCV= Phenotypic coefficient of variance GCV= Genotypic coefficient of variance

h 2 = heritability(broad sense) GA= Genetic advance

GAM= Genetic advance as percent mean

character PCV GCV h2 GA GAM

Plant height(cm) 813 610 7503 404 16 Number of branches

per plant 1702 1095 6433 119 3478

Number of clusters

(cm) 2345 1456 6208 208 4792

Pod length (cm) 2072 1663 8026 256 4234 Number of pods per

plant 1823 1016 5573 555 3726

No of seeds per pod 1535 929 6052 161 3137 Days to 50

flowering 720 310 4305 653 147

Single plant yield(G) 2989 2077 6948 130 3508

Table 45 Mean SD Range and variance values for eight taits in segregating F2 population of blackgram

character Mean SD Range Variance Coefficient of

variance

Standard

Error Plant height(cm) 2430 266 8 773 1095 010 Number of

branches per

plant

516 095 3 154 1841 0045

Number of

clusters(cm)

435 106 3 2084 2436 005

Pod length(cm) 604 132 4 314 2185 006 Number of pods

per plant 1491 292 11 1473 1958 014

No of seeds per

pod 513 0873 3 1244 1701 0

04 Days to 50

flowering 4434 456 12 2043 1028 016

Single plant yield

(G) 213 065 195 0812 3051 003

Table 43 chai-square test for segregation of resistance and susceptibility in F2 populations during rabi season 2016

revealing nature of inheritance to YMV

F2 generation Total plants Yellow mosaic virus Ratio

S R ᵡ2 ᵖvalue observed expected

R S R S

LBG 759times T9 125 30 95 32 93 3 1 007 0796

R= number of resistant plants S= number of susceptible plants significant value of p at 005 is 3849

Chapter V

Summary amp Conclusions

Chapter V

SUMMARY AND CONCLUSIONS

In the present study an attempt was made to identify molecular markers linked to Mungbean

Yellow Mosaic Virus (MYMV) disease resistance through bulk segregant analysis (BSA) in

Blackgram (Vigna mungo (L) Hepper) This work was preferred in order to generate required

variability by carefully selecting the parental material aiming for improvement of yield and

disease resistance of adapted cultivar Efforts were also made to predict the variability created

by hybridization using parameters like phenotypic coefficient of variation (PCV) and

genotypic coefficient of variation (GCV) heritability and genetic advance and further to

understand the inter-relationship among the component traits of seed yield through

correlation studies in blackgram in F2 population The field work was carried out at

Agricultural Research Station College of Agriculture PJTSAU Madhira Telangana

Phenotypic data particular to quantitative characters viz pods per plant number of seeds per

pod pod length and seed yield per plant were noted on F2 populations of cross LBG 759 X

T9 The results obtained in the present study are summarized below

1 In the present study we selected LBG 759 (female) as susceptible parent and T9

(resistant ) as resistant parent to MYMV Crossings were performed to produce F1 seed F1s

were selfed to generate the F2 mapping population A total of 125 F2 individual plants along

with parents and F1s were subjected to natural screening against yellow mosaic virus using

standard disease score scale

2 The field screening of 125 F2 individuals helped in identification of 12 MYMV resistant

individuals 16 moderately MYMV resistant individuals 40 MYMV moderately susceptible

individuals 32 susceptible individuals and 23 highly susceptible individuals

3 Goodness of fit test (Chi-square test) for F2 phenotypic data of the cross LBG 759 X T9

indicated that the MYMV resistance in blackgram is governed by a single recessive gene in

the ratio of 31 ie 95 susceptible 30 resistant plants Among 50 primers screened fourteen

primers were found to be polymorphic between the parents amounting to a polymorphic

percentage 28 showed polymorphism between the parents

4 The polymorphic marker CEDG 185 clearly expressed polymorphism between PARENTS

BULKS in bulk segregant analysis with a unique fragment size of 190bp AND 160 bp of

resistant and susceptible bulks respectively and the results confirmed the marker putatively

linked to MYMV resistance gene This marker can be used for mapping resistance gene and

marker validation studies

5 F2 population was evaluated for productivity for nine different morphological traits



MYMV score

6 Heritability in broad sense and Genetic advance as percent of mean was high for number of

pods per plant single plant yield plant height number of branches per plant and pod length

indicating that these traits were controlled by additive genes and can easily be transferred to

succeeding generations

7 Moderate genetic advance as percent of mean values and moderate heritability in broad

sense was observed in number of seeds per pod which indicate that the greater role of non-

additive genetic variance and epistetic and dominant environmental factors controlling the

inheritance of these traits

8 For some traits like number of pods per plant single plant yield the difference between

GCV and PCV were high reveals the greater influence of environment on the expression of

these characters whereas other traits were least affected by environment The increase in

mean values as a result of hybridization indicates an opportunity for further improvement in

traits like number of pods per plant number of seeds per pod and pod length test weight and

other characters in subsequent generations (F3 and F4) there by gives a chance for selection

of transgressive segregants in later generations

9 This SSR marker CEDG 185 can be used to screen the large germplasm for YMV

resistance The material generated can be forwarded by single seed-descent method to

develop RILS and can be used for mapping YMV resistance gene and validation of identified

markers

Literature cited

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Chiemsombat P 1992 Mungbean yellow mosaic disease in Thailand A reviewInSK Green

and D Kim (ed) Mungbean yellow mosaic disease Proceedings of the Internation

Workshop 92-373 pp 54-58

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Nadu Agriculture University Coimbatore India 48pp

Christian AF Menancio-Hautea D Danesh D and Young ND 1992 Evidence for

orthologous seed weight genes in cowpea and mungbean based on RFLP mapping


Cobos MJ Fernandez MJ Rubio J Kharrat M Moreno MT Gil J and Millan T

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and Applied Genetics 110 1347ndash1353

Comstock RE and Robinson HF 1952 Genetic parameter their estimation and significance

Proceedings of Internation Gross Congrs 284-291

Department of Economics and Statistics 2013-14

Delic D Stajkovic O Kuzmanovic D Rasulic N Knezevic S and Milicic B 2009 The

effects of rhizobial inoculation on growth and yield of Vigna mungo L in Serbian soils

Biotechnology in Animal Husbandry 25(5-6) 1197-1202

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Dhole VJ and Kandali SR 2013 Development of a SCAR marker linked with a MYMV

resistance gene in mungbean (Vigna radiata L Wilczek) Plant Breeding 132 127ndash

132

Doyle JJ and Doyle JL 1987 A rapid DNA isolation procedure for small quantities of fresh

leaf tissue Phytochemical Bulletin 1911-15

Durga Prasad AVS and Murugan e and Vanniarajan c Inheritance of resistance of

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8(4)413-417

East FM 1916 Studies on seed inheritance in nicotine Genetics 1 164-176

El-Hady EAAA Haiba AAA El-Hamid NRA and Al-Ansary AEMF 2010

Assessment of genetic variations in some Vigna species by RAPD and ISSR analysis

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Erschadi S Haberer G Schoniger M and Torres-Ruiz RA 2000 Estimating genetic

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Fatokun CA Danesh D Menancio HDI and Young ND 1992a A linkage map of

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(ed) Genome Maps Cold Spring Harbor Laboratory New York pp 6256 - 6258

Fary FL 2002 New opportunities in vigna pp 424- 428

Flandez-Galvez H Ford R Pang ECK and Taylor PWJ 2003 An intraspecific linkage

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microsatellite site and resistance gene analog markers Theoretical and Applied

Genetics 106 1447ndash1456

Food and Agriculture Organisation of the United Nations (FAOSTAT) 2011

httpwwwfaostatfaoorgcom

Fukuoka S Inoue T Miyao A Monna L Zhong HS Sasaki T and Minobe Y 1994

Mapping of sequence-tagged sites in rice by single strand conformation polymorphism

DNA Research 1 271-277

Ghafoor A Ahmad Z and Sharif A 2000 Cluster analysis and correlation in blackgram

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Gioi TD Boora KS and Chaudhary K 2012 Identification and characterization of SSR

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unguiculata) International Journal of Plant Research 2(1) 1-8

Giriraj K 1973 Natural variability in greengram (Phaseolus aureus Roxb) Mys Journal of

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5210

Gupta PK Kumar J Mir RR and Kumar A 2010 Marker assisted selection as a

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Gupta SK and Gopalakrishna T 2008 Molecular markers and their application in grain

legumes breeding Journal of Food Legumes 21 1-14

Gupta SK Singh RA and Chandra S 2005 Identification of a single dominant gene for

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Gupta SK Souframanien J and Gopalakrishna T 2008 Construction of a genetic linkage

map of black gram Vigna mungo (L) Hepper based on molecular markers and

comparative studies Genome 51 628ndash637

Haley SD Miklas PN Stavely JR Byrum J and Kelly JD 1993 Identification of

RAPD markers linked to a major rust resistance gene block in common bean

Theoretical and Applied Genetics 85961-968

Han OK Kaga A Isemura T Wang XW Tomooka N and Vaughan DA 2005 A

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Theoretical and Applied Genetics 111 1278ndash1287

Hanson CH Robinson HG and Comstock RE 1956 Biometrical studies of yield in

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Hearne CM Ghosh S and Todd JA 1992 Microsatellites for linkage analysis of genetic

traits Trends in Genetics 8 288-294

Hernandez P Martin A and Dorado G 1999 Development of SCARs by direct sequencing

of RAPD products A practical tool for the introgression and marker assisted selection

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Holeyachi P and Savithramma DL 2013 Identification of RAPD markers linked to mymv

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1409-1411

Humphry ME Konduri V Lambrides CJ Magner T McIntyre CL Aitken EAB and

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comparison with lablab (Lablab purpureus) reveals a high level of co-linearity between

the two genomes Theoretical and Applied Genetics 105 160 -166

Humphry ME Lambrides CJ Chapman A Imrie BC Lawn RJ Mcintyre CL and

Lili CJ 2005 Relationships between hard-seededness and seed weight in mungbean

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Humphry ME Magner CJ Mcintyr ET Aitken EABCL and Liu CJ 2003

Identification of major locus conferring resistance to powdery mildew in mungbean by

QTL analysis Genome 46 738-744

Hyten DL Smith JR Frederick RD Tucker ML Song Q and Cregan PB 2009

Bulked segregant analysis using the goldengate assay to locate the Rpp3 locus that

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Indiastat 2012 httpwwwindiastatcom

Isemura T Kaga A Konishi S Ando T Tomooka N Han O K and Vaughan D A

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1053ndash1071

Isemura T Kaga A Tabata S Somta P and Srinives P 2012 Construction of a genetic

linkage map and genetic analysis of domestication related traits in mungbean (Vigna

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mosaic virus resistance in mungbean (Vigna radiata (L) Wilczek) Trends in

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Johannsen WL 1909 Elements directions Exblichkeitelahre Jenal Gustar Fisher

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483

Jordan SA and Humphries P 1994 Single nucleotide polymorphism in exon 2 of the BCP

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Kaga A Ohnishi M Ishii T and Kamijima O 1996 A genetic linkage map of azuki bean

constructed with molecular and morphological markers using an interspecific

population (Vigna angularis times V nakashimae) Theoretical and Applied Genetics 93

658ndash663 doi101007BF00224059

Kajonphol T Sangsiri C Somta P Toojinda T and Srinives P 2012 SSR map

construction and quantitative trait loci (QTL) identification of major agronomic traits in

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44 (1) 71-86

Kalo P Endre G Zimanyi L Csanadi G and Kiss GB 2000 Construction of an improved

linkage map of diploid alfalfa (Medicago sativa) Theoretical and Applied Genetics

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Kang BC Yeam I and Jahn MM 2005 Genetics of plant virus resistance Annual Review

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Coimbatore India

Karthikeyan A Sudha M Senthil N Pandiyan M Raveendran M and Nagrajan P 2011

Screening and identification of random amplified polymorphic DNA (RAPD) markers

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DOI101080032354082011592016

Karthikeyan A Sudha M Senthil N Pandiyan M Raveendran M and Nagarajan P 2012

Screening and identification of RAPD markers linked to MYMV resistance in

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Karuppanapandian T Karuppudurai T Sinha TPM Hamarul HA and Manoharan K

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Kasettranan W Somta P and Srinivas P 2010 Mapping of quantitative trait loci controlling

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mungbean Legume Research 26(1) 69-70

Khajudparn P Prajongjai1 T Poolsawat O and Tantasawat PA 2012 Application of

ISSR markers for verification of F1 hybrids in mungbean (Vigna radiata) Genetics and

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Khattak AB Bibi N and Aurangzeb 2007 Quality assessment and consumers acceptibilty

studies of newly evolved Mungbean genotypes (Vigna radiata L) American Journal of

Food Technology 2(6)536-542

Khattak GSS Haq MA Rana SA Srinives P and Ashraf M 1999 Inheritance of

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Lakhanpaul S Chadha S and Bhat KV 2000 Random amplified polymorphic DNA

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Milla SR Levin JS Lewis RS and Rufty RC 2005 RAPD and SCAR Markers linked to

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Construction of bacterial artificial chromosome libraries and their application in

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Parent JG and Page D 1995 Evaluation of SCAR markers to identify raspberry cultivars

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Prakit Somta Kaga A Tomooka N Kashiwaba K Isemura T and Chaitieng B 2008

Development of an interspecific Vigna linkage map between Vigna umbellate (Thunb)

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Prasanthi L Bhaskara BV Rekha RK Mehala RD Geetha B Siva PY and Raja

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Applied Genetics 88 (8) 945-948

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scope for yield attributes in M2 mungbean (Vigna radiata l) genotypes Romanian

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Salimath PM Suma B Linganagowda and Uma MS 2007 Variability parameters in F2

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Sato S Isobe S and Tabata S 2010 Structural analyses of the genomes in legumes Current

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Saxena P Kamendra S Usha B and Khanna VK 2009 Identification of ISSR marker for

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Sharma RN 1999 Heritability and character association in non segregating populations of

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Shoba D Manivannan N Vindhiyavarman P and Nigam SN 2012 SSR markers

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Silva DCG Yamanaka N Brogin RL Arias CAA Nepomuceno AL Mauro AOD

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mapping of two loci that confer resistance to Asian rust in soybean Theoretical and

Applied Genetics 11757-63

Singh DP 1980 Inheritance of resistance to yellow mosaic virus in blackgram (Vigna mungo

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Singh SK and Singh MN 2006 Inheritance of resistance to mungbean yellow mosaic virus

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Singh T Sharma A and Ahmed FA 2009 Impact of environment on heritability and genetic

gain for yield and its component traits in mungbean Legume Research 32(1) 55- 58

Solanki IS 1981 Genetics of resistance to mungbean yellow mosaic virus in blackgram

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Souframanien J and Gopalakrishna T 2004 A comparative analysis of genetic diversity in

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Souframanien J and Gopalakrishna T 2006 ISSR and SCAR markers linked to the mungbean

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mutants in blackgram as revealed by random amplified polymorphic DNA and inter-

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Sudha M Anusuyaa P Nawkar GM Karthikeyana A Nagarajana P Raveendrana M

Senthila N Pandiyanb M Angappana K and Balasubramaniana P 2013 Molecular

studies on mungbean (Vigna radiata (L) Wilczek) and ricebean (Vigna umbellata

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Phytopathology and Plant Protection 101080032354082012745055 46(5)503-517

Sudha M Karthikeyan A Anusuya1 P Ganesh NM Pandiyan M Senthil N

Raveendran N Nagarajan P and Angappan K 2013 Inheritance of resistance to

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Sudha 2009 An investigation on mungbean yellow mosaic virus (MYMV) resistance in

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1132-1134

Thamodhran g and Geetha s and Ramalingam a 2016 Genetic study in URD bean (Vigna

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Marker-assisted selection in faba bean (Vicia faba L) Field Crops Research 115 243mdash

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Toth G Gaspari Z and Jurka J 2000 Microsatellites in different eukaryotic genomes survey

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Tuba Anjum K Sanjeev G and Datta S2010 Mapping of Mungbean Yellow Mosaic India


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Varma A and Malathi VG 2003 Emerging geminivirus problems a serious threat to crop

production Annals of Applied Biology 142 pp 145ndash164

Varshney RK Penmetsa RV Dutta S Kulwal PL Saxena RK Datta S Sharma

TR Rosen B Carrasquilla-Garcia N Farmer AD Dubey A Saxena KB Gao

J Fakrudin J Singh MN Singh BP Wanjari KB Yuan M Srivastava RK

Kilian A Upadhyaya HD Mallikarjuna N Town CD Bruening GE He G

May GD McCombie R Jackson SA Singh NK and Cook DR 2010a Pigeon

pea genomics initiative (PGI) an international effort to improve crop productivity of

pigeon pea (Cajanus cajan L) Molecular Breeding 26 393mdash408

Varshney R Mahendar KT May GD and Jackson SA 2010b Legume genomics and

breeding Plant Breeding Review 33 257mdash304

Varshney RK Close TJ Singh NK Hoisington DA and Cook DR 2009 Orphan

legume crops enter the genomics era Current Opinion in Plant Biology 12 1mdash9

Verdcourt B 1970 Studies in the Leguminosae-Papilionoideae for the Flora of Tropical East

Africa IV Kew Bulletin 24 507ndash569

Verma RPS and Singh DP 1988 Inheritance of resistance to mungbean yellow mosaic

virus in Greengram Annals of Agricultural Research Vol 9 No 3 pp 98-100


virus in blackgram Indian Journal of Genetics 49 321-324

Verma RPS and Singh DP 2000 The allelic relationship of genes giving resistance to

mungbean yellow mosaic virus in blackgram Theoretical and Applied Genetics 72

737-738 17 165

Varma A and Malathi VG (2003) Emerging geminivirus problems A serious threat to crop

production Ann Appl Biol 142 145-164

Verma S 1992 Correlation and path analysis in black gram Indian Journal of Pulses

Research 5 71-73

Vikas Paroda VRS and Singh SP 1998 Genetic variability in mungbean (Vigna radiate

(L) Wilczek) over environments in kharif season Annual of Agriculture Bioscience

Research 3 211- 215

Vikram P Mallikarjun BPS Dixit S Ahmed H Cruz MTS Singh KA Ye G and

Arvind K 2012 Bulk segregant analysis An effective approach for mapping

consistent-effect drought grain yield QTLs in rice Field Crops Research 134 185ndash

192

Vinoth r and jayamani p 2014 Genetic inheritance of resistance to yellow mosaic disease in

inter sub-specific cross of blackgram (Vigna mungo (L) Hepper) Journal of Food

Legumes 27(1) 9-12

Vos P Hogers R Bleeker M Reijans M Van De Lee T Hornes M Frijters A Pot

J Peleman J and Kuiper M 1995 AFLP A new technique for DNA fingerprinting

Nucleic Acids Research 23 4407-4414

Urrea C A PN Miklas J S Beaver and R H Riley1996 a co dominant RAPD marker

used for indirect selection of bean golden mosaic virus resistant in common bean

HortSience1211035-1039

Wang XW Kaga A Tomooka N and Vaughan DA 2004 The development of SSR

markers by a new method in plants and their application to gene flow studies in azuki

bean [Vigna angularis (Willd) Ohwi amp Ohashi] Theoretical and Applied Genetics

109 352- 360

Welsh J and Mc Clelland M 1992 Fingerprinting genomes using PCR with arbitrary

primers Nucleic Acids Research 19 303 - 306

Xu RQ Tomooka N Vaughan DA and Doi K 2000 The Vigna angularis complex

genetic variation and relationships revealed by RAPD analysis and their implications

for in-situ conservation and domestication Genetic Resources and Crop Evolution 46

136 -145

Yoon MS Kaga A Tomooka N and Vaughan DA 2000 Analysis of genetic diversity in

the Vigna minima complex and related species in East Asia Journal of Plant Research

113 375ndash386

Young ND Danesh D Menancio-Hautea D and Kumar L 1993 Mapping oligogenic

resistance to powdery mildew in mungbean with RFLPs Theoretical and Applied

Genetics 87(1-2) 243-249

Zhang HY Yang YM Li FS He CS and Liu XZ 2008 Screening and characterization

a RAPD marker of tobacco brown-spot resistant gene African Journal of

Biotechnology 7 2559- 2561

Zhao D Cheng X Wang L Wang S and Ma YL 2010 Constructing of mungbean

genetic linkage map Acta Agronomy Science 36(6) 932-939

Appendices

APPENDIX I

EQUIPMENTS USED

Agarose gel electrophoresis system (Bio-rad)

Autoclave

DNA thermal cycler (Eppendorf master cycler gradient and Peltier thermal cycler)

Freezer of -20ordmC and -80ordmC (Sanyo biomedical freezer)

Gel documentation system (Bio-rad)

Ice maker (Sanyo)

Magnetic stirrer (Genei)

Microwave oven (LG)

Microcentrifuge (Eppendorf)

Pipetteman (Thermo scientific)

pH meter (Thermo orion)

UV absorbance spectrophotometer (Thermo electronic corporation)

Nanodrop (Thermo scientific)

UV Transilluminator (Vilber Lourmat)

Vaccum dryer (Thermo electron corporation)

Vortex mixer (Genei)

Water bath (Cintex)

APPENDIX II

LIST OF CHEMICALS

Agarose (Sigma)

6X loading dye (Genei)

Chloroform (Qualigens)

dNTPs (Deoxy nucleotide triphosphates) (Biogene)

EDTA (Ethylene Diamino Tetra Acetic acid) (Himedia)

Ethidium bromide (Sigma)

Ethyl alcohol (Hayman)

Isoamyl alcohol (Qualigens)

Isopropanol (Qualigens)

NaCl (Sodium chloride) (Qualigens)

NaOH (Sodiun hydroxide) (Qualigens)

Phenol (Bangalore Genei)

Poly vinyl pyrrolidone

Taq polymerase (Invitrogen)

Trizma base (Sigma)

50bp ladder (NEB)

MgCl2 buffer (Jonaki)

Primers (Sigma)

APPENDIX III

BUFFERS AND STOCK SOLUTIONS

DNA Extraction Buffer

2 (wv) CTAB (Nalgene) - 10g

100 Mm Tris HCl pH 80 - 100 ml of 05 M Tris HCl (pH 80)

20 mM EDTA pH 80 - 20 ml of 05 M EDTA (pH 80)

14 M NaCl - 140 ml of 5 M NaCl

PVP (Sigma) - 200 mg

All the above ingredients except CTAB were added in respective quantities and final volume

was made up to 500ml with double distilled water the solution was autoclaved The solution

was allowed to attain room temperature and 10g of CTAB was dissolved by intense stirring

stored at room temperature

EDTA (05M) 200ml

Weigh 3722g of EDTA dissolve in 120ml of distilled water by adding 4g of NaoH pellets

Stirr the solution by adding another 25ml of water and allow EDTA to dissolve completely

Then check the pH and try to adjust to 8 by adding 2N NaoH drop by drop Then make the

volume to 200ml

Phenol Chloroform Isoamyl alcohol (25241)

Equal parts of equilibrated phenol and Chloroform Isoamyl alcohol (241) were mixed and

stored at 4oC

50X TAE Buffer (pH 80)

400 mM Tris base

200 mM Glacial acetic acid

10 mM EDTA

Dissolve in appropriate amount of sterile water

Tris-HCl (1 M)

121g of tris base is dissolved in 50 ml if distilled water then check the pH using litmus

paper If pH is more than 8 then add few drops of HCL and then adjust pH

to 8 then make up

the volume to 100ml

IDENTIFICATION OF MOLECULAR MARKERS

LINKED TO YELLOW MOSAIC VIRUS

RESISTANCE IN BLACKGRAM


By

E RAMBABU BSc (Ag)

THESIS SUBMITTED TO


AGRICULTURAL UNIVERSITY

IN PARTIAL FULFILMENT OF THE REQUIREMENTS

FOR THE AWARD OF THE DEGREE OF


CHAIRPERSON Dr CH ANURADHA

INSTITUTE OF BIOTECHNOLOGY COLLEGE OF AGRICULTURE

RAJENDRANAGAR HYDERABAD-500 030


AGRICULTURAL UNIVERSITY ndash 2016

DECLARATION








Date (E RAMBABU)


CERTIFICATE







Date ( CH ANURADHA)


CERTIFICATE







supervision




(CH ANURADHA)








Member Dr V SRIDHAR

Scientist ____________________

ARS

Madhira

Khammam







ACKNOWLEDGEMENTS







her






















guidance in my life
















supported me a lot

(rambabu)

LIST OF CONTENTS


I INTRODUCTION





LITERATURE CITED


LIST OF TABLES

Sl No

Table

No

Title

Page No








6 42




7 43







10 46





LIST OF FIGURES

Sl No Figure

No


1 41



was 50bp






CEDG 185



CEDG 185


CEDG 185

LIST OF PLATES

Sl No

Plate No

Title

Page No

1

Plate-41


2

Plate-42


3

Plate-43


disease reaction

LIST OF APPENDICES

Appendix

No

Title Page

No





MYMV

YMV

MYMIV

YMD

CYMV

LLS

SBR

AVRDC

IARI

ANGRAU

VR

BSA

MAS

DNA

QTL

RILS

RFLP

RAPD

SSR

SCAR

CAP

RGA

SNP

ISSR


Yellow Mosaic Virus




Late Leaf Spot

Soyabean Rust




Vigna radiata








AFLP

AFLP-RGA

STS

PCR

AS-PCR

AP-PCR

SDS- PAGE

CTAB

EDTA

TRIS

PVP

TAE

dNTP

Taq

Mb

bp

Mha

Mt

L ha

Sl no

et al

viz

microl

ml

cm

microM





Allele Specific PCR








Base pairs

Million hectares

Million tonnes

Lakh hectares

Serial number

and others


amp

UV

H2O

mM

ng

cm

g

mg

h2

χ2

cM

nm

C

And Per

Ultra violet

Water


Chi-square

Centimorgan

Nanometer

Degree centigrade







Faculty AGRICULTURE


BIOTECHNOLOGY





ABSTRACT










uradbeans















resistance gene






















Chapter I

Introduction

Chapter I

INTRODUCTION












































(Nariani 1960)













































































































mungbean




with YMV resistance







Chapter II


Chapter II











































































inheritance


















F2 segregation




resistance to MYMV



































221 Symptomology


















222 Epidemology
































future










main stem



































generations

































































































mungbean































































among them






40 cowpea genotypes




























respectively

2414 SCAR markers







problem








































bean genotypes



















(Karthikeyan 2010)
























marker














































to C maculatus







































analysis









(BSA)





resistance





















Rps3 region




plant








linked markers





resistant gene





























desirable genotypes


























Chapter III


Chapter







311 Plant Material











appendix II











YMV incidence





















CATEGORY

REACTION

GROUP


symptoms

Highly Resistant HR










plant











the mean













E

EO 22 )(

Where










Chaudhary (1977)

3521 Mean

n

1 ( sum yi )

Y = n i=1

3522 Variance

n

1 sum(Yi-Y)2

Variance = n-1 i=1


Y = Population mean

sum d2


Where








phenotypic variance








Where







2g


Mean

2p


Mean

Where



3534 Heritability



σsup2g

hsup2 (bs) = ------------

σsup2p

Where





Low 0-30

Medium 30-60

High above 60



GA = k 2p H

Where








GA


Grand mean



Low Less than 10

Moderate 10-20

High More than 20




appendix III





Method





















(BIO- RAD)
































C 45 seconds



6 4˚c infin








Protocol













system)


POPULATION





















Chapter IV


Chapter IV











Segregant analysis



DISEASE RESISTANCE


















times

T9 LBG 759

F1 Plants


















(Table 43 Plate 43)














(BSA)



times T9 as follows






















































50bp 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1

2


200bp


used was 50bp

50bp 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2


CEDG202

200bp

50bp 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2




200bp


Sl No Primer

name


temperature(degc)

Allele size (bp)

S R

1

CEDG073


60

150 250


2

CEDG075


60

150 200


3

CEDG091


57

150 170


4

CEDG092


57

150 210


5

CEDG097


57

150 230


6

CEDG116


57

150 170




8

CEDG139


60

150 190


9

CEDG147


60

150 160


10

CEDG154


58

150 180


11

CEDG156


58

150 210


12

CEDG176


60

150 180


13

CEDG185


60

160 190





200bp

50bp P1 P2 1 2 3 4 5 6 7 8 9 10



200bp




200bp

50bp SP RP SB RB 1 2 3 4 5 6 7 8 9 10




50bp SP RP SB RB 1 2 3 4 5 6 7 8 9 10




200bp





































plant yield (213)






a mean of 213







































environment








flowering(4305)







flowering(147)















Future line of work






productivity






MYMV in Black gram


MYMV T9

LBG 759

F1

1

5

2

0-5 Scale




Score


segregants


1 Resistant 12



4 Suseptible 32


Total 125








per plant 1702 1095 6433 119 3478

Number of clusters

(cm) 2345 1456 6208 208 4792


plant 1823 1016 5573 555 3726


flowering 720 310 4305 653 147




variance

Standard


branches per

plant

516 095 3 154 1841 0045

Number of

clusters(cm)

435 106 3 2084 2436 005


per plant 1491 292 11 1473 1958 014

No of seeds per

pod 513 0873 3 1244 1701 0

04 Days to 50

flowering 4434 456 12 2043 1028 016

Single plant yield

(G) 213 065 195 0812 3051 003





R S R S

LBG 759times T9 125 30 95 32 93 3 1 007 0796


Chapter V


Chapter V




































MYMV score



















markers

Literature cited

LITERATURE CITED













































174ndash184



















































132





8(4)413-417
































5210




























1409-1411


















1053ndash1071










483



483






658ndash663 doi101007BF00224059




44 (1) 71-86



100 641ndash657








Coimbatore India





DOI101080032354082011592016








51214 -1219






























109 227-234





Qld Australia



Adelaide Australia










58 815-824












145505ndash510




123



18 No 1 pp 189-198







Breeding 8(4) 1357-1361 2013










159











110 151- 156











Genome 39 26 - 30



60p














45 175ndash188



99











Genetics 27 561-571



Bulletin 998 56

































































75299-102















































1132-1134






Euphytica 26765



Science 9 43-44






252






























737-738 17 165




Research 5 71-73



Research 3 211- 215




192



Legumes 27(1) 9-12










109 352- 360






136 -145



113 375ndash386



Genetics 87(1-2) 243-249






Appendices

APPENDIX I

EQUIPMENTS USED


Autoclave




Ice maker (Sanyo)


Microwave oven (LG)









Water bath (Cintex)

APPENDIX II

LIST OF CHEMICALS

Agarose (Sigma)














Trizma base (Sigma)

50bp ladder (NEB)


Primers (Sigma)

APPENDIX III












EDTA (05M) 200ml




volume to 200ml



stored at 4oC


400 mM Tris base


10 mM EDTA


Tris-HCl (1 M)



to 8 then make up

the volume to 100ml

DECLARATION








Date (E RAMBABU)


CERTIFICATE







Date ( CH ANURADHA)


CERTIFICATE







supervision




(CH ANURADHA)








Member Dr V SRIDHAR

Scientist ____________________

ARS

Madhira

Khammam







ACKNOWLEDGEMENTS







her






















guidance in my life
















supported me a lot

(rambabu)

LIST OF CONTENTS


I INTRODUCTION





LITERATURE CITED


LIST OF TABLES

Sl No

Table

No

Title

Page No








6 42




7 43







10 46





LIST OF FIGURES

Sl No Figure

No


1 41



was 50bp






CEDG 185



CEDG 185


CEDG 185

LIST OF PLATES

Sl No

Plate No

Title

Page No

1

Plate-41


2

Plate-42


3

Plate-43


disease reaction

LIST OF APPENDICES

Appendix

No

Title Page

No





MYMV

YMV

MYMIV

YMD

CYMV

LLS

SBR

AVRDC

IARI

ANGRAU

VR

BSA

MAS

DNA

QTL

RILS

RFLP

RAPD

SSR

SCAR

CAP

RGA

SNP

ISSR


Yellow Mosaic Virus




Late Leaf Spot

Soyabean Rust




Vigna radiata








AFLP

AFLP-RGA

STS

PCR

AS-PCR

AP-PCR

SDS- PAGE

CTAB

EDTA

TRIS

PVP

TAE

dNTP

Taq

Mb

bp

Mha

Mt

L ha

Sl no

et al

viz

microl

ml

cm

microM





Allele Specific PCR








Base pairs

Million hectares

Million tonnes

Lakh hectares

Serial number

and others


amp

UV

H2O

mM

ng

cm

g

mg

h2

χ2

cM

nm

C

And Per

Ultra violet

Water


Chi-square

Centimorgan

Nanometer

Degree centigrade







Faculty AGRICULTURE


BIOTECHNOLOGY





ABSTRACT










uradbeans















resistance gene






















Chapter I

Introduction

Chapter I

INTRODUCTION












































(Nariani 1960)













































































































mungbean




with YMV resistance







Chapter II


Chapter II











































































inheritance


















F2 segregation




resistance to MYMV



































221 Symptomology


















222 Epidemology
































future










main stem



































generations

































































































mungbean































































among them






40 cowpea genotypes




























respectively

2414 SCAR markers







problem








































bean genotypes



















(Karthikeyan 2010)
























marker














































to C maculatus







































analysis









(BSA)





resistance





















Rps3 region




plant








linked markers





resistant gene





























desirable genotypes


























Chapter III


Chapter







311 Plant Material











appendix II











YMV incidence





















CATEGORY

REACTION

GROUP


symptoms

Highly Resistant HR










plant











the mean













E

EO 22 )(

Where










Chaudhary (1977)

3521 Mean

n

1 ( sum yi )

Y = n i=1

3522 Variance

n

1 sum(Yi-Y)2

Variance = n-1 i=1


Y = Population mean

sum d2


Where








phenotypic variance








Where







2g


Mean

2p


Mean

Where



3534 Heritability



σsup2g

hsup2 (bs) = ------------

σsup2p

Where





Low 0-30

Medium 30-60

High above 60



GA = k 2p H

Where








GA


Grand mean



Low Less than 10

Moderate 10-20

High More than 20




appendix III





Method





















(BIO- RAD)
































C 45 seconds



6 4˚c infin








Protocol













system)


POPULATION





















Chapter IV


Chapter IV











Segregant analysis



DISEASE RESISTANCE


















times

T9 LBG 759

F1 Plants


















(Table 43 Plate 43)














(BSA)



times T9 as follows






















































50bp 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1

2


200bp


used was 50bp

50bp 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2


CEDG202

200bp

50bp 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2




200bp


Sl No Primer

name


temperature(degc)

Allele size (bp)

S R

1

CEDG073


60

150 250


2

CEDG075


60

150 200


3

CEDG091


57

150 170


4

CEDG092


57

150 210


5

CEDG097


57

150 230


6

CEDG116


57

150 170




8

CEDG139


60

150 190


9

CEDG147


60

150 160


10

CEDG154


58

150 180


11

CEDG156


58

150 210


12

CEDG176


60

150 180


13

CEDG185


60

160 190





200bp

50bp P1 P2 1 2 3 4 5 6 7 8 9 10



200bp




200bp

50bp SP RP SB RB 1 2 3 4 5 6 7 8 9 10




50bp SP RP SB RB 1 2 3 4 5 6 7 8 9 10




200bp





































plant yield (213)






a mean of 213







































environment








flowering(4305)







flowering(147)















Future line of work






productivity






MYMV in Black gram


MYMV T9

LBG 759

F1

1

5

2

0-5 Scale




Score


segregants


1 Resistant 12



4 Suseptible 32


Total 125








per plant 1702 1095 6433 119 3478

Number of clusters

(cm) 2345 1456 6208 208 4792


plant 1823 1016 5573 555 3726


flowering 720 310 4305 653 147




variance

Standard


branches per

plant

516 095 3 154 1841 0045

Number of

clusters(cm)

435 106 3 2084 2436 005


per plant 1491 292 11 1473 1958 014

No of seeds per

pod 513 0873 3 1244 1701 0

04 Days to 50

flowering 4434 456 12 2043 1028 016

Single plant yield

(G) 213 065 195 0812 3051 003





R S R S

LBG 759times T9 125 30 95 32 93 3 1 007 0796


Chapter V


Chapter V




































MYMV score



















markers

Literature cited

LITERATURE CITED













































174ndash184



















































132





8(4)413-417
































5210




























1409-1411


















1053ndash1071










483



483






658ndash663 doi101007BF00224059




44 (1) 71-86



100 641ndash657








Coimbatore India





DOI101080032354082011592016








51214 -1219






























109 227-234





Qld Australia



Adelaide Australia










58 815-824












145505ndash510




123



18 No 1 pp 189-198







Breeding 8(4) 1357-1361 2013










159











110 151- 156











Genome 39 26 - 30



60p














45 175ndash188



99











Genetics 27 561-571



Bulletin 998 56

































































75299-102















































1132-1134






Euphytica 26765



Science 9 43-44






252






























737-738 17 165




Research 5 71-73



Research 3 211- 215




192



Legumes 27(1) 9-12










109 352- 360






136 -145



113 375ndash386



Genetics 87(1-2) 243-249






Appendices

APPENDIX I

EQUIPMENTS USED


Autoclave




Ice maker (Sanyo)


Microwave oven (LG)









Water bath (Cintex)

APPENDIX II

LIST OF CHEMICALS

Agarose (Sigma)














Trizma base (Sigma)

50bp ladder (NEB)


Primers (Sigma)

APPENDIX III












EDTA (05M) 200ml




volume to 200ml



stored at 4oC


400 mM Tris base


10 mM EDTA


Tris-HCl (1 M)



to 8 then make up

the volume to 100ml

CERTIFICATE







Date ( CH ANURADHA)


CERTIFICATE







supervision




(CH ANURADHA)








Member Dr V SRIDHAR

Scientist ____________________

ARS

Madhira

Khammam







ACKNOWLEDGEMENTS







her






















guidance in my life
















supported me a lot

(rambabu)

LIST OF CONTENTS


I INTRODUCTION





LITERATURE CITED


LIST OF TABLES

Sl No

Table

No

Title

Page No








6 42




7 43







10 46





LIST OF FIGURES

Sl No Figure

No


1 41



was 50bp






CEDG 185



CEDG 185


CEDG 185

LIST OF PLATES

Sl No

Plate No

Title

Page No

1

Plate-41


2

Plate-42


3

Plate-43


disease reaction

LIST OF APPENDICES

Appendix

No

Title Page

No





MYMV

YMV

MYMIV

YMD

CYMV

LLS

SBR

AVRDC

IARI

ANGRAU

VR

BSA

MAS

DNA

QTL

RILS

RFLP

RAPD

SSR

SCAR

CAP

RGA

SNP

ISSR


Yellow Mosaic Virus




Late Leaf Spot

Soyabean Rust




Vigna radiata








AFLP

AFLP-RGA

STS

PCR

AS-PCR

AP-PCR

SDS- PAGE

CTAB

EDTA

TRIS

PVP

TAE

dNTP

Taq

Mb

bp

Mha

Mt

L ha

Sl no

et al

viz

microl

ml

cm

microM





Allele Specific PCR








Base pairs

Million hectares

Million tonnes

Lakh hectares

Serial number

and others


amp

UV

H2O

mM

ng

cm

g

mg

h2

χ2

cM

nm

C

And Per

Ultra violet

Water


Chi-square

Centimorgan

Nanometer

Degree centigrade







Faculty AGRICULTURE


BIOTECHNOLOGY





ABSTRACT










uradbeans















resistance gene






















Chapter I

Introduction

Chapter I

INTRODUCTION












































(Nariani 1960)













































































































mungbean




with YMV resistance







Chapter II


Chapter II











































































inheritance


















F2 segregation




resistance to MYMV



































221 Symptomology


















222 Epidemology
































future










main stem



































generations

































































































mungbean































































among them






40 cowpea genotypes




























respectively

2414 SCAR markers







problem








































bean genotypes



















(Karthikeyan 2010)
























marker














































to C maculatus







































analysis









(BSA)





resistance





















Rps3 region




plant








linked markers





resistant gene





























desirable genotypes


























Chapter III


Chapter







311 Plant Material











appendix II











YMV incidence





















CATEGORY

REACTION

GROUP


symptoms

Highly Resistant HR










plant











the mean













E

EO 22 )(

Where










Chaudhary (1977)

3521 Mean

n

1 ( sum yi )

Y = n i=1

3522 Variance

n

1 sum(Yi-Y)2

Variance = n-1 i=1


Y = Population mean

sum d2


Where








phenotypic variance








Where







2g


Mean

2p


Mean

Where



3534 Heritability



σsup2g

hsup2 (bs) = ------------

σsup2p

Where





Low 0-30

Medium 30-60

High above 60



GA = k 2p H

Where








GA


Grand mean



Low Less than 10

Moderate 10-20

High More than 20




appendix III





Method





















(BIO- RAD)
































C 45 seconds



6 4˚c infin








Protocol













system)


POPULATION





















Chapter IV


Chapter IV











Segregant analysis



DISEASE RESISTANCE


















times

T9 LBG 759

F1 Plants


















(Table 43 Plate 43)














(BSA)



times T9 as follows






















































50bp 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1

2


200bp


used was 50bp

50bp 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2


CEDG202

200bp

50bp 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2




200bp


Sl No Primer

name


temperature(degc)

Allele size (bp)

S R

1

CEDG073


60

150 250


2

CEDG075


60

150 200


3

CEDG091


57

150 170


4

CEDG092


57

150 210


5

CEDG097


57

150 230


6

CEDG116


57

150 170




8

CEDG139


60

150 190


9

CEDG147


60

150 160


10

CEDG154


58

150 180


11

CEDG156


58

150 210


12

CEDG176


60

150 180


13

CEDG185


60

160 190





200bp

50bp P1 P2 1 2 3 4 5 6 7 8 9 10



200bp




200bp

50bp SP RP SB RB 1 2 3 4 5 6 7 8 9 10




50bp SP RP SB RB 1 2 3 4 5 6 7 8 9 10




200bp





































plant yield (213)






a mean of 213







































environment








flowering(4305)







flowering(147)















Future line of work






productivity






MYMV in Black gram


MYMV T9

LBG 759

F1

1

5

2

0-5 Scale




Score


segregants


1 Resistant 12



4 Suseptible 32


Total 125








per plant 1702 1095 6433 119 3478

Number of clusters

(cm) 2345 1456 6208 208 4792


plant 1823 1016 5573 555 3726


flowering 720 310 4305 653 147




variance

Standard


branches per

plant

516 095 3 154 1841 0045

Number of

clusters(cm)

435 106 3 2084 2436 005


per plant 1491 292 11 1473 1958 014

No of seeds per

pod 513 0873 3 1244 1701 0

04 Days to 50

flowering 4434 456 12 2043 1028 016

Single plant yield

(G) 213 065 195 0812 3051 003





R S R S

LBG 759times T9 125 30 95 32 93 3 1 007 0796


Chapter V


Chapter V




































MYMV score



















markers

Literature cited

LITERATURE CITED













































174ndash184



















































132





8(4)413-417
































5210




























1409-1411


















1053ndash1071










483



483






658ndash663 doi101007BF00224059




44 (1) 71-86



100 641ndash657








Coimbatore India





DOI101080032354082011592016








51214 -1219






























109 227-234





Qld Australia



Adelaide Australia










58 815-824












145505ndash510




123



18 No 1 pp 189-198







Breeding 8(4) 1357-1361 2013










159











110 151- 156











Genome 39 26 - 30



60p














45 175ndash188



99











Genetics 27 561-571



Bulletin 998 56

































































75299-102















































1132-1134






Euphytica 26765



Science 9 43-44






252






























737-738 17 165




Research 5 71-73



Research 3 211- 215




192



Legumes 27(1) 9-12










109 352- 360






136 -145



113 375ndash386



Genetics 87(1-2) 243-249






Appendices

APPENDIX I

EQUIPMENTS USED


Autoclave




Ice maker (Sanyo)


Microwave oven (LG)









Water bath (Cintex)

APPENDIX II

LIST OF CHEMICALS

Agarose (Sigma)














Trizma base (Sigma)

50bp ladder (NEB)


Primers (Sigma)

APPENDIX III












EDTA (05M) 200ml




volume to 200ml



stored at 4oC


400 mM Tris base


10 mM EDTA


Tris-HCl (1 M)



to 8 then make up

the volume to 100ml

CERTIFICATE







supervision




(CH ANURADHA)








Member Dr V SRIDHAR

Scientist ____________________

ARS

Madhira

Khammam







ACKNOWLEDGEMENTS







her






















guidance in my life
















supported me a lot

(rambabu)

LIST OF CONTENTS


I INTRODUCTION





LITERATURE CITED


LIST OF TABLES

Sl No

Table

No

Title

Page No








6 42




7 43







10 46





LIST OF FIGURES

Sl No Figure

No


1 41



was 50bp






CEDG 185



CEDG 185


CEDG 185

LIST OF PLATES

Sl No

Plate No

Title

Page No

1

Plate-41


2

Plate-42


3

Plate-43


disease reaction

LIST OF APPENDICES

Appendix

No

Title Page

No





MYMV

YMV

MYMIV

YMD

CYMV

LLS

SBR

AVRDC

IARI

ANGRAU

VR

BSA

MAS

DNA

QTL

RILS

RFLP

RAPD

SSR

SCAR

CAP

RGA

SNP

ISSR


Yellow Mosaic Virus




Late Leaf Spot

Soyabean Rust




Vigna radiata








AFLP

AFLP-RGA

STS

PCR

AS-PCR

AP-PCR

SDS- PAGE

CTAB

EDTA

TRIS

PVP

TAE

dNTP

Taq

Mb

bp

Mha

Mt

L ha

Sl no

et al

viz

microl

ml

cm

microM





Allele Specific PCR








Base pairs

Million hectares

Million tonnes

Lakh hectares

Serial number

and others


amp

UV

H2O

mM

ng

cm

g

mg

h2

χ2

cM

nm

C

And Per

Ultra violet

Water


Chi-square

Centimorgan

Nanometer

Degree centigrade







Faculty AGRICULTURE


BIOTECHNOLOGY





ABSTRACT










uradbeans















resistance gene






















Chapter I

Introduction

Chapter I

INTRODUCTION












































(Nariani 1960)













































































































mungbean




with YMV resistance







Chapter II


Chapter II











































































inheritance


















F2 segregation




resistance to MYMV



































221 Symptomology


















222 Epidemology
































future










main stem



































generations

































































































mungbean































































among them






40 cowpea genotypes




























respectively

2414 SCAR markers







problem








































bean genotypes



















(Karthikeyan 2010)
























marker














































to C maculatus







































analysis









(BSA)





resistance





















Rps3 region




plant








linked markers





resistant gene





























desirable genotypes


























Chapter III


Chapter







311 Plant Material











appendix II











YMV incidence





















CATEGORY

REACTION

GROUP


symptoms

Highly Resistant HR










plant











the mean













E

EO 22 )(

Where










Chaudhary (1977)

3521 Mean

n

1 ( sum yi )

Y = n i=1

3522 Variance

n

1 sum(Yi-Y)2

Variance = n-1 i=1


Y = Population mean

sum d2


Where








phenotypic variance








Where







2g


Mean

2p


Mean

Where



3534 Heritability



σsup2g

hsup2 (bs) = ------------

σsup2p

Where





Low 0-30

Medium 30-60

High above 60



GA = k 2p H

Where








GA


Grand mean



Low Less than 10

Moderate 10-20

High More than 20




appendix III





Method





















(BIO- RAD)
































C 45 seconds



6 4˚c infin








Protocol













system)


POPULATION





















Chapter IV


Chapter IV











Segregant analysis



DISEASE RESISTANCE


















times

T9 LBG 759

F1 Plants


















(Table 43 Plate 43)














(BSA)



times T9 as follows






















































50bp 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1

2


200bp


used was 50bp

50bp 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2


CEDG202

200bp

50bp 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2




200bp


Sl No Primer

name


temperature(degc)

Allele size (bp)

S R

1

CEDG073


60

150 250


2

CEDG075


60

150 200


3

CEDG091


57

150 170


4

CEDG092


57

150 210


5

CEDG097


57

150 230


6

CEDG116


57

150 170




8

CEDG139


60

150 190


9

CEDG147


60

150 160


10

CEDG154


58

150 180


11

CEDG156


58

150 210


12

CEDG176


60

150 180


13

CEDG185


60

160 190





200bp

50bp P1 P2 1 2 3 4 5 6 7 8 9 10



200bp




200bp

50bp SP RP SB RB 1 2 3 4 5 6 7 8 9 10




50bp SP RP SB RB 1 2 3 4 5 6 7 8 9 10




200bp





































plant yield (213)






a mean of 213







































environment








flowering(4305)







flowering(147)















Future line of work






productivity






MYMV in Black gram


MYMV T9

LBG 759

F1

1

5

2

0-5 Scale




Score


segregants


1 Resistant 12



4 Suseptible 32


Total 125








per plant 1702 1095 6433 119 3478

Number of clusters

(cm) 2345 1456 6208 208 4792


plant 1823 1016 5573 555 3726


flowering 720 310 4305 653 147




variance

Standard


branches per

plant

516 095 3 154 1841 0045

Number of

clusters(cm)

435 106 3 2084 2436 005


per plant 1491 292 11 1473 1958 014

No of seeds per

pod 513 0873 3 1244 1701 0

04 Days to 50

flowering 4434 456 12 2043 1028 016

Single plant yield

(G) 213 065 195 0812 3051 003





R S R S

LBG 759times T9 125 30 95 32 93 3 1 007 0796


Chapter V


Chapter V




































MYMV score



















markers

Literature cited

LITERATURE CITED













































174ndash184



















































132





8(4)413-417
































5210




























1409-1411


















1053ndash1071










483



483






658ndash663 doi101007BF00224059




44 (1) 71-86



100 641ndash657








Coimbatore India





DOI101080032354082011592016








51214 -1219






























109 227-234





Qld Australia



Adelaide Australia










58 815-824












145505ndash510




123



18 No 1 pp 189-198







Breeding 8(4) 1357-1361 2013










159











110 151- 156











Genome 39 26 - 30



60p














45 175ndash188



99











Genetics 27 561-571



Bulletin 998 56

































































75299-102















































1132-1134






Euphytica 26765



Science 9 43-44






252






























737-738 17 165




Research 5 71-73



Research 3 211- 215




192



Legumes 27(1) 9-12










109 352- 360






136 -145



113 375ndash386



Genetics 87(1-2) 243-249






Appendices

APPENDIX I

EQUIPMENTS USED


Autoclave




Ice maker (Sanyo)


Microwave oven (LG)









Water bath (Cintex)

APPENDIX II

LIST OF CHEMICALS

Agarose (Sigma)














Trizma base (Sigma)

50bp ladder (NEB)


Primers (Sigma)

APPENDIX III












EDTA (05M) 200ml




volume to 200ml



stored at 4oC


400 mM Tris base


10 mM EDTA


Tris-HCl (1 M)



to 8 then make up

the volume to 100ml

ACKNOWLEDGEMENTS







her






















guidance in my life
















supported me a lot

(rambabu)

LIST OF CONTENTS


I INTRODUCTION





LITERATURE CITED


LIST OF TABLES

Sl No

Table

No

Title

Page No








6 42




7 43







10 46





LIST OF FIGURES

Sl No Figure

No


1 41



was 50bp






CEDG 185



CEDG 185


CEDG 185

LIST OF PLATES

Sl No

Plate No

Title

Page No

1

Plate-41


2

Plate-42


3

Plate-43


disease reaction

LIST OF APPENDICES

Appendix

No

Title Page

No





MYMV

YMV

MYMIV

YMD

CYMV

LLS

SBR

AVRDC

IARI

ANGRAU

VR

BSA

MAS

DNA

QTL

RILS

RFLP

RAPD

SSR

SCAR

CAP

RGA

SNP

ISSR


Yellow Mosaic Virus




Late Leaf Spot

Soyabean Rust




Vigna radiata








AFLP

AFLP-RGA

STS

PCR

AS-PCR

AP-PCR

SDS- PAGE

CTAB

EDTA

TRIS

PVP

TAE

dNTP

Taq

Mb

bp

Mha

Mt

L ha

Sl no

et al

viz

microl

ml

cm

microM





Allele Specific PCR








Base pairs

Million hectares

Million tonnes

Lakh hectares

Serial number

and others


amp

UV

H2O

mM

ng

cm

g

mg

h2

χ2

cM

nm

C

And Per

Ultra violet

Water


Chi-square

Centimorgan

Nanometer

Degree centigrade







Faculty AGRICULTURE


BIOTECHNOLOGY





ABSTRACT










uradbeans















resistance gene






















Chapter I

Introduction

Chapter I

INTRODUCTION












































(Nariani 1960)













































































































mungbean




with YMV resistance







Chapter II


Chapter II











































































inheritance


















F2 segregation




resistance to MYMV



































221 Symptomology


















222 Epidemology
































future










main stem



































generations

































































































mungbean































































among them






40 cowpea genotypes




























respectively

2414 SCAR markers







problem








































bean genotypes



















(Karthikeyan 2010)
























marker














































to C maculatus







































analysis









(BSA)





resistance





















Rps3 region




plant








linked markers





resistant gene





























desirable genotypes


























Chapter III


Chapter







311 Plant Material











appendix II











YMV incidence





















CATEGORY

REACTION

GROUP


symptoms

Highly Resistant HR










plant











the mean













E

EO 22 )(

Where










Chaudhary (1977)

3521 Mean

n

1 ( sum yi )

Y = n i=1

3522 Variance

n

1 sum(Yi-Y)2

Variance = n-1 i=1


Y = Population mean

sum d2


Where








phenotypic variance








Where







2g


Mean

2p


Mean

Where



3534 Heritability



σsup2g

hsup2 (bs) = ------------

σsup2p

Where





Low 0-30

Medium 30-60

High above 60



GA = k 2p H

Where








GA


Grand mean



Low Less than 10

Moderate 10-20

High More than 20




appendix III





Method





















(BIO- RAD)
































C 45 seconds



6 4˚c infin








Protocol













system)


POPULATION





















Chapter IV


Chapter IV











Segregant analysis



DISEASE RESISTANCE


















times

T9 LBG 759

F1 Plants


















(Table 43 Plate 43)














(BSA)



times T9 as follows






















































50bp 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1

2


200bp


used was 50bp

50bp 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2


CEDG202

200bp

50bp 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2




200bp


Sl No Primer

name


temperature(degc)

Allele size (bp)

S R

1

CEDG073


60

150 250


2

CEDG075


60

150 200


3

CEDG091


57

150 170


4

CEDG092


57

150 210


5

CEDG097


57

150 230


6

CEDG116


57

150 170




8

CEDG139


60

150 190


9

CEDG147


60

150 160


10

CEDG154


58

150 180


11

CEDG156


58

150 210


12

CEDG176


60

150 180


13

CEDG185


60

160 190





200bp

50bp P1 P2 1 2 3 4 5 6 7 8 9 10



200bp




200bp

50bp SP RP SB RB 1 2 3 4 5 6 7 8 9 10




50bp SP RP SB RB 1 2 3 4 5 6 7 8 9 10




200bp





































plant yield (213)






a mean of 213







































environment








flowering(4305)







flowering(147)















Future line of work






productivity






MYMV in Black gram


MYMV T9

LBG 759

F1

1

5

2

0-5 Scale




Score


segregants


1 Resistant 12



4 Suseptible 32


Total 125








per plant 1702 1095 6433 119 3478

Number of clusters

(cm) 2345 1456 6208 208 4792


plant 1823 1016 5573 555 3726


flowering 720 310 4305 653 147




variance

Standard


branches per

plant

516 095 3 154 1841 0045

Number of

clusters(cm)

435 106 3 2084 2436 005


per plant 1491 292 11 1473 1958 014

No of seeds per

pod 513 0873 3 1244 1701 0

04 Days to 50

flowering 4434 456 12 2043 1028 016

Single plant yield

(G) 213 065 195 0812 3051 003





R S R S

LBG 759times T9 125 30 95 32 93 3 1 007 0796


Chapter V


Chapter V




































MYMV score



















markers

Literature cited

LITERATURE CITED













































174ndash184



















































132





8(4)413-417
































5210




























1409-1411


















1053ndash1071










483



483






658ndash663 doi101007BF00224059




44 (1) 71-86



100 641ndash657








Coimbatore India





DOI101080032354082011592016








51214 -1219






























109 227-234





Qld Australia



Adelaide Australia










58 815-824












145505ndash510




123



18 No 1 pp 189-198







Breeding 8(4) 1357-1361 2013










159











110 151- 156











Genome 39 26 - 30



60p














45 175ndash188



99











Genetics 27 561-571



Bulletin 998 56

































































75299-102















































1132-1134






Euphytica 26765



Science 9 43-44






252






























737-738 17 165




Research 5 71-73



Research 3 211- 215




192



Legumes 27(1) 9-12










109 352- 360






136 -145



113 375ndash386



Genetics 87(1-2) 243-249






Appendices

APPENDIX I

EQUIPMENTS USED


Autoclave




Ice maker (Sanyo)


Microwave oven (LG)









Water bath (Cintex)

APPENDIX II

LIST OF CHEMICALS

Agarose (Sigma)














Trizma base (Sigma)

50bp ladder (NEB)


Primers (Sigma)

APPENDIX III












EDTA (05M) 200ml




volume to 200ml



stored at 4oC


400 mM Tris base


10 mM EDTA


Tris-HCl (1 M)



to 8 then make up

the volume to 100ml




guidance in my life
















supported me a lot

(rambabu)

LIST OF CONTENTS


I INTRODUCTION





LITERATURE CITED


LIST OF TABLES

Sl No

Table

No

Title

Page No








6 42




7 43







10 46





LIST OF FIGURES

Sl No Figure

No


1 41



was 50bp






CEDG 185



CEDG 185


CEDG 185

LIST OF PLATES

Sl No

Plate No

Title

Page No

1

Plate-41


2

Plate-42


3

Plate-43


disease reaction

LIST OF APPENDICES

Appendix

No

Title Page

No





MYMV

YMV

MYMIV

YMD

CYMV

LLS

SBR

AVRDC

IARI

ANGRAU

VR

BSA

MAS

DNA

QTL

RILS

RFLP

RAPD

SSR

SCAR

CAP

RGA

SNP

ISSR


Yellow Mosaic Virus




Late Leaf Spot

Soyabean Rust




Vigna radiata








AFLP

AFLP-RGA

STS

PCR

AS-PCR

AP-PCR

SDS- PAGE

CTAB

EDTA

TRIS

PVP

TAE

dNTP

Taq

Mb

bp

Mha

Mt

L ha

Sl no

et al

viz

microl

ml

cm

microM





Allele Specific PCR








Base pairs

Million hectares

Million tonnes

Lakh hectares

Serial number

and others


amp

UV

H2O

mM

ng

cm

g

mg

h2

χ2

cM

nm

C

And Per

Ultra violet

Water


Chi-square

Centimorgan

Nanometer

Degree centigrade







Faculty AGRICULTURE


BIOTECHNOLOGY





ABSTRACT










uradbeans















resistance gene






















Chapter I

Introduction

Chapter I

INTRODUCTION












































(Nariani 1960)













































































































mungbean




with YMV resistance







Chapter II


Chapter II











































































inheritance


















F2 segregation




resistance to MYMV



































221 Symptomology


















222 Epidemology
































future










main stem



































generations

































































































mungbean































































among them






40 cowpea genotypes




























respectively

2414 SCAR markers







problem








































bean genotypes



















(Karthikeyan 2010)
























marker














































to C maculatus







































analysis









(BSA)





resistance





















Rps3 region




plant








linked markers





resistant gene





























desirable genotypes


























Chapter III


Chapter







311 Plant Material











appendix II











YMV incidence





















CATEGORY

REACTION

GROUP


symptoms

Highly Resistant HR










plant











the mean













E

EO 22 )(

Where










Chaudhary (1977)

3521 Mean

n

1 ( sum yi )

Y = n i=1

3522 Variance

n

1 sum(Yi-Y)2

Variance = n-1 i=1


Y = Population mean

sum d2


Where








phenotypic variance








Where







2g


Mean

2p


Mean

Where



3534 Heritability



σsup2g

hsup2 (bs) = ------------

σsup2p

Where





Low 0-30

Medium 30-60

High above 60



GA = k 2p H

Where








GA


Grand mean



Low Less than 10

Moderate 10-20

High More than 20




appendix III





Method





















(BIO- RAD)
































C 45 seconds



6 4˚c infin








Protocol













system)


POPULATION





















Chapter IV


Chapter IV











Segregant analysis



DISEASE RESISTANCE


















times

T9 LBG 759

F1 Plants


















(Table 43 Plate 43)














(BSA)



times T9 as follows






















































50bp 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1

2


200bp


used was 50bp

50bp 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2


CEDG202

200bp

50bp 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2




200bp


Sl No Primer

name


temperature(degc)

Allele size (bp)

S R

1

CEDG073


60

150 250


2

CEDG075


60

150 200


3

CEDG091


57

150 170


4

CEDG092


57

150 210


5

CEDG097


57

150 230


6

CEDG116


57

150 170




8

CEDG139


60

150 190


9

CEDG147


60

150 160


10

CEDG154


58

150 180


11

CEDG156


58

150 210


12

CEDG176


60

150 180


13

CEDG185


60

160 190





200bp

50bp P1 P2 1 2 3 4 5 6 7 8 9 10



200bp




200bp

50bp SP RP SB RB 1 2 3 4 5 6 7 8 9 10




50bp SP RP SB RB 1 2 3 4 5 6 7 8 9 10




200bp





































plant yield (213)






a mean of 213







































environment








flowering(4305)







flowering(147)















Future line of work






productivity






MYMV in Black gram


MYMV T9

LBG 759

F1

1

5

2

0-5 Scale




Score


segregants


1 Resistant 12



4 Suseptible 32


Total 125








per plant 1702 1095 6433 119 3478

Number of clusters

(cm) 2345 1456 6208 208 4792


plant 1823 1016 5573 555 3726


flowering 720 310 4305 653 147




variance

Standard


branches per

plant

516 095 3 154 1841 0045

Number of

clusters(cm)

435 106 3 2084 2436 005


per plant 1491 292 11 1473 1958 014

No of seeds per

pod 513 0873 3 1244 1701 0

04 Days to 50

flowering 4434 456 12 2043 1028 016

Single plant yield

(G) 213 065 195 0812 3051 003





R S R S

LBG 759times T9 125 30 95 32 93 3 1 007 0796


Chapter V


Chapter V




































MYMV score



















markers

Literature cited

LITERATURE CITED













































174ndash184



















































132





8(4)413-417
































5210




























1409-1411


















1053ndash1071










483



483






658ndash663 doi101007BF00224059




44 (1) 71-86



100 641ndash657








Coimbatore India





DOI101080032354082011592016








51214 -1219






























109 227-234





Qld Australia



Adelaide Australia










58 815-824












145505ndash510




123



18 No 1 pp 189-198







Breeding 8(4) 1357-1361 2013










159











110 151- 156











Genome 39 26 - 30



60p














45 175ndash188



99











Genetics 27 561-571



Bulletin 998 56

































































75299-102















































1132-1134






Euphytica 26765



Science 9 43-44






252






























737-738 17 165




Research 5 71-73



Research 3 211- 215




192



Legumes 27(1) 9-12










109 352- 360






136 -145



113 375ndash386



Genetics 87(1-2) 243-249






Appendices

APPENDIX I

EQUIPMENTS USED


Autoclave




Ice maker (Sanyo)


Microwave oven (LG)









Water bath (Cintex)

APPENDIX II

LIST OF CHEMICALS

Agarose (Sigma)














Trizma base (Sigma)

50bp ladder (NEB)


Primers (Sigma)

APPENDIX III












EDTA (05M) 200ml




volume to 200ml



stored at 4oC


400 mM Tris base


10 mM EDTA


Tris-HCl (1 M)



to 8 then make up

the volume to 100ml

LIST OF CONTENTS


I INTRODUCTION





LITERATURE CITED


LIST OF TABLES

Sl No

Table

No

Title

Page No








6 42




7 43







10 46





LIST OF FIGURES

Sl No Figure

No


1 41



was 50bp






CEDG 185



CEDG 185


CEDG 185

LIST OF PLATES

Sl No

Plate No

Title

Page No

1

Plate-41


2

Plate-42


3

Plate-43


disease reaction

LIST OF APPENDICES

Appendix

No

Title Page

No





MYMV

YMV

MYMIV

YMD

CYMV

LLS

SBR

AVRDC

IARI

ANGRAU

VR

BSA

MAS

DNA

QTL

RILS

RFLP

RAPD

SSR

SCAR

CAP

RGA

SNP

ISSR


Yellow Mosaic Virus




Late Leaf Spot

Soyabean Rust




Vigna radiata








AFLP

AFLP-RGA

STS

PCR

AS-PCR

AP-PCR

SDS- PAGE

CTAB

EDTA

TRIS

PVP

TAE

dNTP

Taq

Mb

bp

Mha

Mt

L ha

Sl no

et al

viz

microl

ml

cm

microM





Allele Specific PCR








Base pairs

Million hectares

Million tonnes

Lakh hectares

Serial number

and others


amp

UV

H2O

mM

ng

cm

g

mg

h2

χ2

cM

nm

C

And Per

Ultra violet

Water


Chi-square

Centimorgan

Nanometer

Degree centigrade







Faculty AGRICULTURE


BIOTECHNOLOGY





ABSTRACT










uradbeans















resistance gene






















Chapter I

Introduction

Chapter I

INTRODUCTION












































(Nariani 1960)













































































































mungbean




with YMV resistance







Chapter II


Chapter II











































































inheritance


















F2 segregation




resistance to MYMV



































221 Symptomology


















222 Epidemology
































future










main stem



































generations

































































































mungbean































































among them






40 cowpea genotypes




























respectively

2414 SCAR markers







problem








































bean genotypes



















(Karthikeyan 2010)
























marker














































to C maculatus







































analysis









(BSA)





resistance





















Rps3 region




plant








linked markers





resistant gene





























desirable genotypes


























Chapter III


Chapter







311 Plant Material











appendix II











YMV incidence





















CATEGORY

REACTION

GROUP


symptoms

Highly Resistant HR










plant











the mean













E

EO 22 )(

Where










Chaudhary (1977)

3521 Mean

n

1 ( sum yi )

Y = n i=1

3522 Variance

n

1 sum(Yi-Y)2

Variance = n-1 i=1


Y = Population mean

sum d2


Where








phenotypic variance








Where







2g


Mean

2p


Mean

Where



3534 Heritability



σsup2g

hsup2 (bs) = ------------

σsup2p

Where





Low 0-30

Medium 30-60

High above 60



GA = k 2p H

Where








GA


Grand mean



Low Less than 10

Moderate 10-20

High More than 20




appendix III





Method





















(BIO- RAD)
































C 45 seconds



6 4˚c infin








Protocol













system)


POPULATION





















Chapter IV


Chapter IV











Segregant analysis



DISEASE RESISTANCE


















times

T9 LBG 759

F1 Plants


















(Table 43 Plate 43)














(BSA)



times T9 as follows






















































50bp 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1

2


200bp


used was 50bp

50bp 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2


CEDG202

200bp

50bp 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2




200bp


Sl No Primer

name


temperature(degc)

Allele size (bp)

S R

1

CEDG073


60

150 250


2

CEDG075


60

150 200


3

CEDG091


57

150 170


4

CEDG092


57

150 210


5

CEDG097


57

150 230


6

CEDG116


57

150 170




8

CEDG139


60

150 190


9

CEDG147


60

150 160


10

CEDG154


58

150 180


11

CEDG156


58

150 210


12

CEDG176


60

150 180


13

CEDG185


60

160 190





200bp

50bp P1 P2 1 2 3 4 5 6 7 8 9 10



200bp




200bp

50bp SP RP SB RB 1 2 3 4 5 6 7 8 9 10




50bp SP RP SB RB 1 2 3 4 5 6 7 8 9 10




200bp





































plant yield (213)






a mean of 213







































environment








flowering(4305)







flowering(147)















Future line of work






productivity






MYMV in Black gram


MYMV T9

LBG 759

F1

1

5

2

0-5 Scale




Score


segregants


1 Resistant 12



4 Suseptible 32


Total 125








per plant 1702 1095 6433 119 3478

Number of clusters

(cm) 2345 1456 6208 208 4792


plant 1823 1016 5573 555 3726


flowering 720 310 4305 653 147




variance

Standard


branches per

plant

516 095 3 154 1841 0045

Number of

clusters(cm)

435 106 3 2084 2436 005


per plant 1491 292 11 1473 1958 014

No of seeds per

pod 513 0873 3 1244 1701 0

04 Days to 50

flowering 4434 456 12 2043 1028 016

Single plant yield

(G) 213 065 195 0812 3051 003





R S R S

LBG 759times T9 125 30 95 32 93 3 1 007 0796


Chapter V


Chapter V




































MYMV score



















markers

Literature cited

LITERATURE CITED













































174ndash184



















































132





8(4)413-417
































5210




























1409-1411


















1053ndash1071










483



483






658ndash663 doi101007BF00224059




44 (1) 71-86



100 641ndash657








Coimbatore India





DOI101080032354082011592016








51214 -1219






























109 227-234





Qld Australia



Adelaide Australia










58 815-824












145505ndash510




123



18 No 1 pp 189-198







Breeding 8(4) 1357-1361 2013










159











110 151- 156











Genome 39 26 - 30



60p














45 175ndash188



99











Genetics 27 561-571



Bulletin 998 56

































































75299-102















































1132-1134






Euphytica 26765



Science 9 43-44






252






























737-738 17 165




Research 5 71-73



Research 3 211- 215




192



Legumes 27(1) 9-12










109 352- 360






136 -145



113 375ndash386



Genetics 87(1-2) 243-249






Appendices

APPENDIX I

EQUIPMENTS USED


Autoclave




Ice maker (Sanyo)


Microwave oven (LG)









Water bath (Cintex)

APPENDIX II

LIST OF CHEMICALS

Agarose (Sigma)














Trizma base (Sigma)

50bp ladder (NEB)


Primers (Sigma)

APPENDIX III












EDTA (05M) 200ml




volume to 200ml



stored at 4oC


400 mM Tris base


10 mM EDTA


Tris-HCl (1 M)



to 8 then make up

the volume to 100ml

LIST OF TABLES

Sl No

Table

No

Title

Page No








6 42




7 43







10 46





LIST OF FIGURES

Sl No Figure

No


1 41



was 50bp






CEDG 185



CEDG 185


CEDG 185

LIST OF PLATES

Sl No

Plate No

Title

Page No

1

Plate-41


2

Plate-42


3

Plate-43


disease reaction

LIST OF APPENDICES

Appendix

No

Title Page

No





MYMV

YMV

MYMIV

YMD

CYMV

LLS

SBR

AVRDC

IARI

ANGRAU

VR

BSA

MAS

DNA

QTL

RILS

RFLP

RAPD

SSR

SCAR

CAP

RGA

SNP

ISSR


Yellow Mosaic Virus




Late Leaf Spot

Soyabean Rust




Vigna radiata








AFLP

AFLP-RGA

STS

PCR

AS-PCR

AP-PCR

SDS- PAGE

CTAB

EDTA

TRIS

PVP

TAE

dNTP

Taq

Mb

bp

Mha

Mt

L ha

Sl no

et al

viz

microl

ml

cm

microM





Allele Specific PCR








Base pairs

Million hectares

Million tonnes

Lakh hectares

Serial number

and others


amp

UV

H2O

mM

ng

cm

g

mg

h2

χ2

cM

nm

C

And Per

Ultra violet

Water


Chi-square

Centimorgan

Nanometer

Degree centigrade







Faculty AGRICULTURE


BIOTECHNOLOGY





ABSTRACT










uradbeans















resistance gene






















Chapter I

Introduction

Chapter I

INTRODUCTION












































(Nariani 1960)













































































































mungbean




with YMV resistance







Chapter II


Chapter II











































































inheritance


















F2 segregation




resistance to MYMV



































221 Symptomology


















222 Epidemology
































future










main stem



































generations

































































































mungbean































































among them






40 cowpea genotypes




























respectively

2414 SCAR markers







problem








































bean genotypes



















(Karthikeyan 2010)
























marker














































to C maculatus







































analysis









(BSA)





resistance





















Rps3 region




plant








linked markers





resistant gene





























desirable genotypes


























Chapter III


Chapter







311 Plant Material











appendix II











YMV incidence





















CATEGORY

REACTION

GROUP


symptoms

Highly Resistant HR










plant











the mean













E

EO 22 )(

Where










Chaudhary (1977)

3521 Mean

n

1 ( sum yi )

Y = n i=1

3522 Variance

n

1 sum(Yi-Y)2

Variance = n-1 i=1


Y = Population mean

sum d2


Where








phenotypic variance








Where







2g


Mean

2p


Mean

Where



3534 Heritability



σsup2g

hsup2 (bs) = ------------

σsup2p

Where





Low 0-30

Medium 30-60

High above 60



GA = k 2p H

Where








GA


Grand mean



Low Less than 10

Moderate 10-20

High More than 20




appendix III





Method





















(BIO- RAD)
































C 45 seconds



6 4˚c infin








Protocol













system)


POPULATION





















Chapter IV


Chapter IV











Segregant analysis



DISEASE RESISTANCE


















times

T9 LBG 759

F1 Plants


















(Table 43 Plate 43)














(BSA)



times T9 as follows






















































50bp 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1

2


200bp


used was 50bp

50bp 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2


CEDG202

200bp

50bp 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2




200bp


Sl No Primer

name


temperature(degc)

Allele size (bp)

S R

1

CEDG073


60

150 250


2

CEDG075


60

150 200


3

CEDG091


57

150 170


4

CEDG092


57

150 210


5

CEDG097


57

150 230


6

CEDG116


57

150 170




8

CEDG139


60

150 190


9

CEDG147


60

150 160


10

CEDG154


58

150 180


11

CEDG156


58

150 210


12

CEDG176


60

150 180


13

CEDG185


60

160 190





200bp

50bp P1 P2 1 2 3 4 5 6 7 8 9 10



200bp




200bp

50bp SP RP SB RB 1 2 3 4 5 6 7 8 9 10




50bp SP RP SB RB 1 2 3 4 5 6 7 8 9 10




200bp





































plant yield (213)






a mean of 213







































environment








flowering(4305)







flowering(147)















Future line of work






productivity






MYMV in Black gram


MYMV T9

LBG 759

F1

1

5

2

0-5 Scale




Score


segregants


1 Resistant 12



4 Suseptible 32


Total 125








per plant 1702 1095 6433 119 3478

Number of clusters

(cm) 2345 1456 6208 208 4792


plant 1823 1016 5573 555 3726


flowering 720 310 4305 653 147




variance

Standard


branches per

plant

516 095 3 154 1841 0045

Number of

clusters(cm)

435 106 3 2084 2436 005


per plant 1491 292 11 1473 1958 014

No of seeds per

pod 513 0873 3 1244 1701 0

04 Days to 50

flowering 4434 456 12 2043 1028 016

Single plant yield

(G) 213 065 195 0812 3051 003





R S R S

LBG 759times T9 125 30 95 32 93 3 1 007 0796


Chapter V


Chapter V




































MYMV score



















markers

Literature cited

LITERATURE CITED













































174ndash184



















































132





8(4)413-417
































5210




























1409-1411


















1053ndash1071










483



483






658ndash663 doi101007BF00224059




44 (1) 71-86



100 641ndash657








Coimbatore India





DOI101080032354082011592016








51214 -1219






























109 227-234





Qld Australia



Adelaide Australia










58 815-824












145505ndash510




123



18 No 1 pp 189-198







Breeding 8(4) 1357-1361 2013










159











110 151- 156











Genome 39 26 - 30



60p














45 175ndash188



99











Genetics 27 561-571



Bulletin 998 56

































































75299-102















































1132-1134






Euphytica 26765



Science 9 43-44






252






























737-738 17 165




Research 5 71-73



Research 3 211- 215




192



Legumes 27(1) 9-12










109 352- 360






136 -145



113 375ndash386



Genetics 87(1-2) 243-249






Appendices

APPENDIX I

EQUIPMENTS USED


Autoclave




Ice maker (Sanyo)


Microwave oven (LG)









Water bath (Cintex)

APPENDIX II

LIST OF CHEMICALS

Agarose (Sigma)














Trizma base (Sigma)

50bp ladder (NEB)


Primers (Sigma)

APPENDIX III












EDTA (05M) 200ml




volume to 200ml



stored at 4oC


400 mM Tris base


10 mM EDTA


Tris-HCl (1 M)



to 8 then make up

the volume to 100ml

LIST OF FIGURES

Sl No Figure

No


1 41



was 50bp






CEDG 185



CEDG 185


CEDG 185

LIST OF PLATES

Sl No

Plate No

Title

Page No

1

Plate-41


2

Plate-42


3

Plate-43


disease reaction

LIST OF APPENDICES

Appendix

No

Title Page

No





MYMV

YMV

MYMIV

YMD

CYMV

LLS

SBR

AVRDC

IARI

ANGRAU

VR

BSA

MAS

DNA

QTL

RILS

RFLP

RAPD

SSR

SCAR

CAP

RGA

SNP

ISSR


Yellow Mosaic Virus




Late Leaf Spot

Soyabean Rust




Vigna radiata








AFLP

AFLP-RGA

STS

PCR

AS-PCR

AP-PCR

SDS- PAGE

CTAB

EDTA

TRIS

PVP

TAE

dNTP

Taq

Mb

bp

Mha

Mt

L ha

Sl no

et al

viz

microl

ml

cm

microM





Allele Specific PCR








Base pairs

Million hectares

Million tonnes

Lakh hectares

Serial number

and others


amp

UV

H2O

mM

ng

cm

g

mg

h2

χ2

cM

nm

C

And Per

Ultra violet

Water


Chi-square

Centimorgan

Nanometer

Degree centigrade







Faculty AGRICULTURE


BIOTECHNOLOGY





ABSTRACT










uradbeans















resistance gene






















Chapter I

Introduction

Chapter I

INTRODUCTION












































(Nariani 1960)













































































































mungbean




with YMV resistance







Chapter II


Chapter II











































































inheritance


















F2 segregation




resistance to MYMV



































221 Symptomology


















222 Epidemology
































future










main stem



































generations

































































































mungbean































































among them






40 cowpea genotypes




























respectively

2414 SCAR markers







problem








































bean genotypes



















(Karthikeyan 2010)
























marker














































to C maculatus







































analysis









(BSA)





resistance





















Rps3 region




plant








linked markers





resistant gene





























desirable genotypes


























Chapter III


Chapter







311 Plant Material











appendix II











YMV incidence





















CATEGORY

REACTION

GROUP


symptoms

Highly Resistant HR










plant











the mean













E

EO 22 )(

Where










Chaudhary (1977)

3521 Mean

n

1 ( sum yi )

Y = n i=1

3522 Variance

n

1 sum(Yi-Y)2

Variance = n-1 i=1


Y = Population mean

sum d2


Where








phenotypic variance








Where







2g


Mean

2p


Mean

Where



3534 Heritability



σsup2g

hsup2 (bs) = ------------

σsup2p

Where





Low 0-30

Medium 30-60

High above 60



GA = k 2p H

Where








GA


Grand mean



Low Less than 10

Moderate 10-20

High More than 20




appendix III





Method





















(BIO- RAD)
































C 45 seconds



6 4˚c infin








Protocol













system)


POPULATION





















Chapter IV


Chapter IV











Segregant analysis



DISEASE RESISTANCE


















times

T9 LBG 759

F1 Plants


















(Table 43 Plate 43)














(BSA)



times T9 as follows






















































50bp 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1

2


200bp


used was 50bp

50bp 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2


CEDG202

200bp

50bp 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2




200bp


Sl No Primer

name


temperature(degc)

Allele size (bp)

S R

1

CEDG073


60

150 250


2

CEDG075


60

150 200


3

CEDG091


57

150 170


4

CEDG092


57

150 210


5

CEDG097


57

150 230


6

CEDG116


57

150 170




8

CEDG139


60

150 190


9

CEDG147


60

150 160


10

CEDG154


58

150 180


11

CEDG156


58

150 210


12

CEDG176


60

150 180


13

CEDG185


60

160 190





200bp

50bp P1 P2 1 2 3 4 5 6 7 8 9 10



200bp




200bp

50bp SP RP SB RB 1 2 3 4 5 6 7 8 9 10




50bp SP RP SB RB 1 2 3 4 5 6 7 8 9 10




200bp





































plant yield (213)






a mean of 213







































environment








flowering(4305)







flowering(147)















Future line of work






productivity






MYMV in Black gram


MYMV T9

LBG 759

F1

1

5

2

0-5 Scale




Score


segregants


1 Resistant 12



4 Suseptible 32


Total 125








per plant 1702 1095 6433 119 3478

Number of clusters

(cm) 2345 1456 6208 208 4792


plant 1823 1016 5573 555 3726


flowering 720 310 4305 653 147




variance

Standard


branches per

plant

516 095 3 154 1841 0045

Number of

clusters(cm)

435 106 3 2084 2436 005


per plant 1491 292 11 1473 1958 014

No of seeds per

pod 513 0873 3 1244 1701 0

04 Days to 50

flowering 4434 456 12 2043 1028 016

Single plant yield

(G) 213 065 195 0812 3051 003





R S R S

LBG 759times T9 125 30 95 32 93 3 1 007 0796


Chapter V


Chapter V




































MYMV score



















markers

Literature cited

LITERATURE CITED













































174ndash184



















































132





8(4)413-417
































5210




























1409-1411


















1053ndash1071










483



483






658ndash663 doi101007BF00224059




44 (1) 71-86



100 641ndash657








Coimbatore India





DOI101080032354082011592016








51214 -1219






























109 227-234





Qld Australia



Adelaide Australia










58 815-824












145505ndash510




123



18 No 1 pp 189-198







Breeding 8(4) 1357-1361 2013










159











110 151- 156











Genome 39 26 - 30



60p














45 175ndash188



99











Genetics 27 561-571



Bulletin 998 56

































































75299-102















































1132-1134






Euphytica 26765



Science 9 43-44






252






























737-738 17 165




Research 5 71-73



Research 3 211- 215




192



Legumes 27(1) 9-12










109 352- 360






136 -145



113 375ndash386



Genetics 87(1-2) 243-249






Appendices

APPENDIX I

EQUIPMENTS USED


Autoclave




Ice maker (Sanyo)


Microwave oven (LG)









Water bath (Cintex)

APPENDIX II

LIST OF CHEMICALS

Agarose (Sigma)














Trizma base (Sigma)

50bp ladder (NEB)


Primers (Sigma)

APPENDIX III












EDTA (05M) 200ml




volume to 200ml



stored at 4oC


400 mM Tris base


10 mM EDTA


Tris-HCl (1 M)



to 8 then make up

the volume to 100ml

LIST OF PLATES

Sl No

Plate No

Title

Page No

1

Plate-41


2

Plate-42


3

Plate-43


disease reaction

LIST OF APPENDICES

Appendix

No

Title Page

No





MYMV

YMV

MYMIV

YMD

CYMV

LLS

SBR

AVRDC

IARI

ANGRAU

VR

BSA

MAS

DNA

QTL

RILS

RFLP

RAPD

SSR

SCAR

CAP

RGA

SNP

ISSR


Yellow Mosaic Virus




Late Leaf Spot

Soyabean Rust




Vigna radiata








AFLP

AFLP-RGA

STS

PCR

AS-PCR

AP-PCR

SDS- PAGE

CTAB

EDTA

TRIS

PVP

TAE

dNTP

Taq

Mb

bp

Mha

Mt

L ha

Sl no

et al

viz

microl

ml

cm

microM





Allele Specific PCR








Base pairs

Million hectares

Million tonnes

Lakh hectares

Serial number

and others


amp

UV

H2O

mM

ng

cm

g

mg

h2

χ2

cM

nm

C

And Per

Ultra violet

Water


Chi-square

Centimorgan

Nanometer

Degree centigrade







Faculty AGRICULTURE


BIOTECHNOLOGY





ABSTRACT










uradbeans















resistance gene






















Chapter I

Introduction

Chapter I

INTRODUCTION












































(Nariani 1960)













































































































mungbean




with YMV resistance







Chapter II


Chapter II











































































inheritance


















F2 segregation




resistance to MYMV



































221 Symptomology


















222 Epidemology
































future










main stem



































generations

































































































mungbean































































among them






40 cowpea genotypes




























respectively

2414 SCAR markers







problem








































bean genotypes



















(Karthikeyan 2010)
























marker














































to C maculatus







































analysis









(BSA)





resistance





















Rps3 region




plant








linked markers





resistant gene





























desirable genotypes


























Chapter III


Chapter







311 Plant Material











appendix II











YMV incidence





















CATEGORY

REACTION

GROUP


symptoms

Highly Resistant HR










plant











the mean













E

EO 22 )(

Where










Chaudhary (1977)

3521 Mean

n

1 ( sum yi )

Y = n i=1

3522 Variance

n

1 sum(Yi-Y)2

Variance = n-1 i=1


Y = Population mean

sum d2


Where








phenotypic variance








Where







2g


Mean

2p


Mean

Where



3534 Heritability



σsup2g

hsup2 (bs) = ------------

σsup2p

Where





Low 0-30

Medium 30-60

High above 60



GA = k 2p H

Where








GA


Grand mean



Low Less than 10

Moderate 10-20

High More than 20




appendix III





Method





















(BIO- RAD)
































C 45 seconds



6 4˚c infin








Protocol













system)


POPULATION





















Chapter IV


Chapter IV











Segregant analysis



DISEASE RESISTANCE


















times

T9 LBG 759

F1 Plants


















(Table 43 Plate 43)














(BSA)



times T9 as follows






















































50bp 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1

2


200bp


used was 50bp

50bp 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2


CEDG202

200bp

50bp 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2




200bp


Sl No Primer

name


temperature(degc)

Allele size (bp)

S R

1

CEDG073


60

150 250


2

CEDG075


60

150 200


3

CEDG091


57

150 170


4

CEDG092


57

150 210


5

CEDG097


57

150 230


6

CEDG116


57

150 170




8

CEDG139


60

150 190


9

CEDG147


60

150 160


10

CEDG154


58

150 180


11

CEDG156


58

150 210


12

CEDG176


60

150 180


13

CEDG185


60

160 190





200bp

50bp P1 P2 1 2 3 4 5 6 7 8 9 10



200bp




200bp

50bp SP RP SB RB 1 2 3 4 5 6 7 8 9 10




50bp SP RP SB RB 1 2 3 4 5 6 7 8 9 10




200bp





































plant yield (213)






a mean of 213







































environment








flowering(4305)







flowering(147)















Future line of work






productivity






MYMV in Black gram


MYMV T9

LBG 759

F1

1

5

2

0-5 Scale




Score


segregants


1 Resistant 12



4 Suseptible 32


Total 125








per plant 1702 1095 6433 119 3478

Number of clusters

(cm) 2345 1456 6208 208 4792


plant 1823 1016 5573 555 3726


flowering 720 310 4305 653 147




variance

Standard


branches per

plant

516 095 3 154 1841 0045

Number of

clusters(cm)

435 106 3 2084 2436 005


per plant 1491 292 11 1473 1958 014

No of seeds per

pod 513 0873 3 1244 1701 0

04 Days to 50

flowering 4434 456 12 2043 1028 016

Single plant yield

(G) 213 065 195 0812 3051 003





R S R S

LBG 759times T9 125 30 95 32 93 3 1 007 0796


Chapter V


Chapter V




































MYMV score



















markers

Literature cited

LITERATURE CITED













































174ndash184



















































132





8(4)413-417
































5210




























1409-1411


















1053ndash1071










483



483






658ndash663 doi101007BF00224059




44 (1) 71-86



100 641ndash657








Coimbatore India





DOI101080032354082011592016








51214 -1219






























109 227-234





Qld Australia



Adelaide Australia










58 815-824












145505ndash510




123



18 No 1 pp 189-198







Breeding 8(4) 1357-1361 2013










159











110 151- 156











Genome 39 26 - 30



60p














45 175ndash188



99











Genetics 27 561-571



Bulletin 998 56

































































75299-102















































1132-1134






Euphytica 26765



Science 9 43-44






252






























737-738 17 165




Research 5 71-73



Research 3 211- 215




192



Legumes 27(1) 9-12










109 352- 360






136 -145



113 375ndash386



Genetics 87(1-2) 243-249






Appendices

APPENDIX I

EQUIPMENTS USED


Autoclave




Ice maker (Sanyo)


Microwave oven (LG)









Water bath (Cintex)

APPENDIX II

LIST OF CHEMICALS

Agarose (Sigma)














Trizma base (Sigma)

50bp ladder (NEB)


Primers (Sigma)

APPENDIX III












EDTA (05M) 200ml




volume to 200ml



stored at 4oC


400 mM Tris base


10 mM EDTA


Tris-HCl (1 M)



to 8 then make up

the volume to 100ml

IDENTIFICATION OF MOLECULAR MARKERS LINKED TO YELLOW MOSAIC VIRUS

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Transcript of IDENTIFICATION OF MOLECULAR MARKERS LINKED TO YELLOW MOSAIC VIRUS