c© Indian Academy of Sciences

ONLINE RESOURCES

Diversity in Indian barley (Hordeum vulgare) cultivars and identificationof genotype-specific fingerprints using microsatellite markers

S. K. JAISWAL1, SHREE P. PANDEY1,3, S. SHARMA1, R. PRASAD1, L. C. PRASAD1,R. P .S. VERMA2 and ARUN K. JOSHI1,4∗

1Department of Genetics and Plant Breeding, Institute of Agricultural Sciences, Banaras Hindu University,Varanasi 221 005, India

2Directorate of Wheat Research (DWR), Karnal 132 001, India3Present address: Department of Biology, Massachussetts Institute of Technology, 77 Massachusetts Avenue 68–659,

Cambridge 02139, USA4Present address: CIMMYT-South Asia, Post Box 5186, Singha Durbar Marg, Kathmandu, Nepal

Introduction

Barley, Hordeum vulgare L., is one of the principal cerealcrop of the world. There is a recognized need to reliably dis-tinguish varieties of crop plants and establish their purity as aprerequisite for any breeding programme (Russel et al. 1997;Matus and Hayes 2002). Assessment of the extent of geneticvariability within cultivated crop has important consequencesin plant breeding and conservation of genetic resources (Pe-tersen et al. 1994). The systematic evaluation of the molec-ular diversity encompassed in barley genetic resources is aprerequisite for its efficient exploitation in breeding as wellas for development of the strategies for optimal conservationof genetic diversity (Malysheva-Otto et al. 2006). It is partic-ularly useful in the characterization of individual accessionsand cultivars, in detecting duplication of genetic material ingermplasm collection and as a general guide for the choiceof parents for breeding hybrids. In the context of plant im-provement, this information provides basis for making deci-sions regarding selection of parental combinations that willmaximize gain for selection and maintain genetic diversity(Matus and Hayes 2002).

Traditionally, morphological characters have been usedto evaluate distinctness, uniformity and stability, and toestablish the description of a genotype. This method isthought to be often influenced by environmental conditions

*For correspondence. E-mail: a.k.joshi@cgiar.org.[Jaiswal S. K., Pandey S. P., Sharma S., Prasad R., Prasad L. C., Verma R. P.S. and Joshi A. K. 2010 Diversity in Indian barley (Hordeum vulgare) culti-vars and identification of genotype-specific fingerprints using microsatellitemarkers. J. Genet. 89, e46–e54. Online only: http://www.ias.ac.in/jgenet/OnlineResources/89/e46.pdf]

(Russel et al. 1997) as well as being labour intensive. Rapidmolecular tests, eliminating crop-stage dependency havebeen developed. The techniques of DNA fingerprinting havebeen established and various DNA profiling methods are cur-rently available. Multilocus fingerprinting methods based onthe polymerase chain reaction (PCR) have been extensivelyused to study the relationships among varieties and cultivarsof many different plants. The most informative polymorphicmarker system currently available is microsatellite or simplesequence repeats (SSRs) (Tautz and Renz 1984). Microsatel-lites are particularly attractive for distinguishing cultivars,since the level of variation detected at microsatellite loci ishigher than that detected with most other molecular assays(Saghai-Maroof et al. 1994; Becker and Heun 1995; Bow-ers et al. 1996; Powell et al. 1996; Struss and Plieske 1998;Davila et al. 1999; Pillen et al. 2000; Ivandic et al. 2002).

Although there are many studies on variability and di-versity in barley using molecular markers, there is limitedevidence about diversity in Indian barley accessions. Studiescombining the traditional approaches and molecular mark-ers have not been extensively applied in crop improvementprogrammes. To make optimal use of Indian germplasm foreffective breeding, evaluation of genetic diversity appears es-sential. In the current investigation, we compiled data andanalysed the genetic diversity of 69 Indian barley lines byusing a combination of traditional approaches of morpho–physiological traits as well as using a set of 16 SSR markersto generate DNA fingerprints. Our long term goal is to de-termine the utility of molecular marker diversity as a tool forgene discovery and biologically meaningful classification ofgermplasm.

Keywords. barley; SSR; mircrosatellite markers; genetic diversity; Hordeum vulgare.

Journal of Genetics Vol. 89, Online Resources e46

S. K. Jaiswal et al.

Materials and methodsSeed material

Seeds of 69 barley varieties (table 1) were procured from Ba-naras Hindu University, Varanasi and Directorate of WheatResearch (Indian Council of Agricultural Research), Karnal,India. Single plant selections of each genotype were multi-plied and used.

Evaluation of morpho–physiological traits of 69 barley varieties

Sixty-nine barley cultivars were grown under field condi-tions in three replications and data for different morpho–physiological traits were recorded.

DNA isolation and SSR primers

Leaves of 69 selected Indian varieties were harvested atseedling stage from fields and DNA was extracted by CTABmethod (Saghai-Maroof et al. 1984). Sixteen SSR markers ofwhich 12 (HVM 5, BMS 64, HVWAXY, HVCMA, BLYR-CAB, BMS 30, BMS 02, BMS 32, BMS 18, BMS 40, HVA-CLI and HVLEU) and 4 (HVM 4, HVM 9, HVM 3 and HVM7) were used, derived from published research of Russel etal. (1997) and Saghai-Maroof et al. (1994), respectively, andwere synthesized at Operon Technologies, California, USA.

PCR amplification and fragment analysis

DNA amplifications were carried out in 25 μL reaction mix-tures, each containing a final concentration of 50 ng templateDNA, 1× PCR buffer, 1.5 mM MgCl2, 1 mM dNTP mix, 20μM of each primer and 1 U taq polymerase (Banglore Ge-nie, Bangalore, India) using the following PCR profile in aPTC-200, Thermal Cycler Techne, UK. Thermal cycling pa-rameters consisted of initial denaturation at 95◦C for 5 min,annealing at 55◦C–60◦C for 30 s, repeated cycles 40 followedby final extension at 72◦C for 10 min. The amplified productswere separated on 3.5% agarose gel electrophoresis (Senioret al. 1998).

Statistical analysis

Cluster analysis was done using UPGMA (unweightedpair-group method with arithmetic average) algorithm onNTSYS-PC, version 1.70 (Rohlf 1992). The polymorphicinformation content (PIC) of each microsatellite locus wasevaluated through allelic frequency (Varshney et al. 2007).

PIC = 1 −k∑

i=1

pi2

Where k is the total number of alleles detected for a mi-crosatellite and pi the frequency of the ith allele ingermplasm. Hierarchical clustering of genotypes was un-dertaken using SPSS version 6.0 based on morpho–physiological traits (SPSS, Chicago, USA).

ResultsData for 69 Indian barley varieties on morpho–physiologicaltraits such as plant height, ear attitude, waxiness, purpose ofcultivation, 1000 grain-weight and disease resistance to yel-low rust, spot botch, and aphid were compiled, along withtheir region of cultivation (table 1). Hierarchical clusteringof these genotypes showed the extent of variability. Ten clus-ters were obtained at an ecludian distance of 3 (data notshown). We further accessed the genetic variability by us-ing microsatellite markers, and mapped the results from theanalysis of morpho–physiological variability to the resultsobtained from microsatellite analysis.

In allele observation by 16 microsatellites, a total 52 al-leles were detected. The number of alleles per locus rangedfrom 1 (marker HVACLI) to 8 (marker BMS 40) with an av-erage of 3.25 alleles. The microsatellite primer pair BMS40 showed the maximum polymorphism with PIC value of0.947234, and was able to differentiate eight varieties. It alsoprovides a unique marker for varieties K 24 and Clipper(bands 280 bp and 310 bp, respectively) which were discrim-inated from other 67 accessions (figure 1). The marker BMS40 revealed null alleles (no amplified products) in three vari-eties: BH 393, DL 3 and Karan 92. The other highly polymor-phic primers were BLYRCAB (allele 6) and HVM 3 (allele6) with PIC value 0.898292.

The dendrogram prepared through cluster analysis (fig-ure 2) suggested a high level of diversity among 69 varieties.The varieties could be grouped into four major groups, whichwere further characterized into 10 small clusters. We alsoobtained 10 clusters on the basis of their agronomic char-acteristics. Three clusters (I, VI and IX) comprised of onlyone variety i.e., BH 393, Jyoti and K 24 and had geneticsimilarity 0.44, 0.48 and 0.36, respectively. These three va-rieties are recommended for cultivation in eastern Gangeticplains of India but differ in resistance to spot blotch, yel-low rust and aphid. BH 393 is resistant to spot blotch andaphid, Jyoti is highly susceptible to spot blotch and aphid butresistant to yellow rust, whereas K 24 is susceptible to allthe three. Therefore, BH 393 can be considered as a betterdonor parent. Cluster II comprised of 17 varieties, of which,14 were grown in the western Ganetic plains of India. Twovarieties (Sonu and BHS 169) are from hilly zone (HZ) andone (Azad) is from eastern Gangetic plains. All the mem-bers of cluster II displayed the genetic similarity 0.46. Incluster III, five varieties were present with genetic similar-ity of 0.57. Highest genetic similarity was obtained in clusterIV (0.65). This cluster included NDB 1173 and Narendra 3and C 84 and RDB 1 that could not be distinguished by mi-crosatellite primer pairs used. The genetic similarity in thesevarieties can also be substantiated by their similar morpho-logical characters. Two varieties C 84 and RDB 1 showedsusceptibility to yellow rust and spot blotch, while NDB1173 and Narendra 3 were resistant for both diseases. TheNDB 1173 and Narendra 3 also showed similarity for otheragronomical characters such as plant height, ear attitude,

Journal of Genetics Vol. 89, Online Resources e47

Genetic diversity in Indian barley

Tabl

e1.

Mor

pho–

phys

iolo

gica

ltra

itsof

69ba

rley

vari

etie

s.

Dis

ease

resi

stan

ce

Reg

ion

and

Maj

orH

eigh

tE

arFe

ed/m

alt

1000

Yel

low

Spot

No.

Gen

otyp

eso

win

gco

nditi

onPe

digr

eech

arac

teri

stic

s(c

m)

attit

ude

Wax

ines

spu

rpos

egr

ain

wt

rust

blot

chA

phid

1B

H39

3H

arya

na(I

R)

Cal

ifor

nia

Mar

iout

/

RA

TN

A

Hul

led,

6ro

w10

5–11

0Se

mi

erec

tN

WFB

35–4

2S

SS

2A

mbe

rU

P(R

F)K

12/

C29

4H

ulle

d,6

row

100–

110

Non

-dr

oop

WFB

45.7

SS

MR

3N

aren

dra

4U

P(I

R)

RD

2035

/

DL

470

Hul

led,

6ro

w60

–65

Sem

ier

ect

WFB

49–5

4M

RM

RS

4R

atna

NW

PZ(R

F)Se

lect

ion

from

loca

lmat

eria

lH

ulle

d,6

row

100–

105

Non

-dr

oop

NW

FB48

.7S

SM

R

5K

edar

NE

PZ,(

IR)

BG

1×

K17

Hul

led,

6ro

w10

0–10

5Se

mi

erec

tW

FB43

.2M

RS

MS

6N

DB

1173

NE

PZ,N

WPZ

(IR

)Sa

line

soils

BY

T-L

RA

-3(1

994-

95)/

ND

B21

7

Hul

led,

6ro

w65

–75

Sem

ier

ect

NW

FB44

.25

MR

MR

MS

7B

HS

169

NH

Z(R

F)K

aila

sh×

Bri

ggs

Hul

led,

6ro

w92

–100

Dro

opW

FB48

.00

RS

S

8So

nuH

P(R

F)E

B23

3×

Giz

a11

7H

ulle

d,6

row

100–

105

Non

-dr

oop

WFB

48.7

RS

S

9V

LB

1U

P(R

F)N

P10

9×

HB

L62

Hul

led,

6ro

w11

0–11

2N

on-

droo

pW

FB49

.7T

MS

S

10R

D57

Raj

asth

an(I

R)

RS1

7/

PRIO

RH

ulle

d,6

row

90–9

5N

on-

droo

pW

FB42

.00

SS

T

11R

D10

3R

ajas

than

(IR

)R

DB

1/

K18

Hul

led,

6ro

w82

–90

Non

-dr

oop

WFB

35.0

SS

S

12H

BL

113

NH

Z(R

F)Se

lect

ion

from

Zyp

hzee

Hul

led,

6ro

w85

–95

Non

-dr

oop

WFB

42R

SM

S

13B

H75

Har

yana

(IR

)R

D15

0×

AH

OR

31-6

8H

ulle

d,6

row

80–8

5N

on-

droo

pW

FB40

MR

SM

S

14N

aren

dra

3U

P(I

R)

K42

5/

Jyot

iH

ulle

d,6

row

65–7

5Se

mi

erec

tN

WFB

40–4

5M

RR

MS

15D

olm

aN

HZ

(RF)

Sele

ctio

nfr

omU

SA11

5H

ulll

ess,

6ro

w98

–105

Non

-dr

oop

NW

FB42

.2M

RS

S

16C

164

Punj

ab/

Har

yana

(IR

)C

155

/C

141

Hul

led,

6ro

w95

–100

Non

-dr

oop

WFB

50.9

SM

SR

17D

WR

28*

NW

PZ(I

R)

BC

U73

/PL

172

Hul

led,

2ro

w80

–100

Dro

opN

WM

B54

–58

RM

RM

R

Journal of Genetics Vol. 89, Online Resources e48

S. K. Jaiswal et al.

Tabl

e1

(con

td.)

Dis

ease

resi

stan

ce

Reg

ion

and

Maj

orH

eigh

tE

arFe

ed/m

alt

1000

Yel

low

Spot

No.

Gen

otyp

eso

win

gco

nditi

onPe

digr

eech

arac

teri

stic

s(c

m)

attit

ude

Wax

ines

spu

rpos

egr

ain

wt

rust

blot

chA

phid

18D

L47

2N

otre

leas

edN

WPZ

(IR

)D

L26

0/

DL

20H

ulle

d,6

row

88.2

Ere

ctW

FB30

.7R

RS

19PL

172

PUN

JAB

(IR

)R

D17

8×

DW

472

Hul

led,

6ro

w86

–98

Non

-dr

oop

WFB

44R

MR

MR

20B

G25

Har

yana

(IR

)C

138×

CN

170

Hul

led,

6ro

w94

–96

Non

-dr

oop

NW

FB48

.6R

SM

R

21B

allia

Bar

ley

UP

(RF)

Pure

Stra

inlo

cal

Hul

led,

6ro

w96

–105

Non

-dr

oop

WFB

48.3

HS

SS

22K

24U

P(R

F)C

N29

4/

K12

Hul

led,

6ro

w10

0–11

0N

on-

droo

pW

FB50

.4S

SS

23V

LB

56U

ttrak

hand

(R)

MO

RO

CC

O/

VL

B12

2H

ulle

d,6

row

60–6

4Se

mi

droo

pN

WFB

35–4

2T

--

24C

50U

P(R

F)Pu

reSt

rain

loca

lH

ulle

d,6

row

100–

105

Non

-dr

oop

WFB

41.2

SS

S

25C

84U

P(R

F)Pu

reSt

rain

loca

lH

ulle

d,6

row

100–

105

Non

-dr

oop

WFB

41S

SM

S

26K

12U

P(R

F)Pu

reSt

rain

loca

lH

ulle

d,6

row

100–

105

Non

-dr

oop

WFB

46.7

SS

MS

27D

L3

NW

PZ(R

)Not

rele

ased

C16

4/

C50

Hul

led,

6ro

w70

–85

Ere

ctW

FB26

.2S

RS

28H

BL

276

NH

Z(R

F)H

BL

243

/

HB

L23

8H

ulll

ess,

6ro

w11

0–11

5E

rect

WFB

25–3

0R

SS

29R

D20

52R

ajas

than

(IR

)C

CN

infe

sted

soil

{CA

PIC

M67

×SO

727}×

PL10

1}

Hul

led,

6ro

w97

–105

Non

-dr

oop

NW

FB49

.4M

RR

MS

30R

D31

Raj

asth

an(I

R)

R51

7/

PRIO

RH

ulle

d,6

row

85–9

0Se

mi

erec

tW

FB37

–40

TM

SM

R

31V

ijaya

Wes

tern

UP

(IR

)K

12/

C25

1H

ulle

d,6

row

100–

105

Non

-dr

oop

WFB

42.9

SS

MS

32PL

426

Punj

ab(I

R)

KA

RA

N92

/

PL10

1H

ulle

d,6

row

95–1

02N

on-

droo

pN

WFB

42.6

SS

S

33K

409

UP

(IR

-LS)

Jyot

ixD

L65

Hul

led,

6ro

w94

–100

Non

-dr

oop

WFB

44–4

8S

SS

34N

aren

dra

2U

P(I

R)

DL

470

/

RD

2035

Hul

led,

6ro

w75

–93

Sem

ier

ect

WFB

36–3

9M

RM

SM

R

35R

DB

1R

ajas

than

(IR

)G

amm

ara

ym

utan

tofR

S17

Hul

led,

6ro

w90

–95

Non

-dr

oop

WFB

46.4

MS

SM

S

Journal of Genetics Vol. 89, Online Resources e49

Genetic diversity in Indian barley

Tabl

e1

(con

td.)

Dis

ease

resi

stan

ce

Reg

ion

and

Maj

orH

eigh

tE

arFe

ed/m

alt

1000

Yel

low

Spot

No.

Gen

otyp

eso

win

gco

nditi

onPe

digr

eech

arac

teri

stic

s(c

m)

attit

ude

Wax

ines

spu

rpos

egr

ain

wt

rust

blot

chA

phid

36B

G10

5H

arya

na(I

R)

C14×

MO

NT

LO

CI

Hul

led,

6ro

w90

–98

Non

-dr

oop

WFB

54.9

SS

S

37R

anje

etPu

njab

(IR

)B

G1×

ME

X5-

13H

ulle

d,6

row

95–1

05N

on-

droo

pW

FB40

.5S

MS

MS

38B

ilara

2R

ajas

than

(IR

)sa

line

soil

RS

17×

C25

1H

ulle

d,6

row

100–

105

Non

-dr

oop

NW

FB36

.5H

SS

S

39PL

419

Punj

ab(R

F)PL

101

/PH

182

Hul

led,

6ro

w9–

95N

on-

droo

pN

WFB

43.6

SS

S

40B

H87

NW

PZ(I

R)N

otre

leas

edC

D40×

CN

51lo

calc

ontr

olH

ulle

d,6

row

90–9

5Se

mi

droo

pN

WFB

48–5

2M

RS

MS

41K

14U

P(R

)Pu

reSt

rain

loca

lH

ulle

d,6

row

96–1

05N

on-

droo

pW

FB43

.1S

SS

42H

iman

iN

HZ

(RF)

Atla

s54

/B

HS

15-8

0/

Kai

lash

Hul

led,

6ro

w65

–72

Dro

opW

FB21

SS

S

43K

141

UP

(RF)

K18

xIB

254

Hul

led,

6ro

w10

5–11

0N

on-

droo

pW

FB49

.2H

SH

SS

44N

aren

dra

1U

P(I

R)

Kar

an15

/P4

08H

ulle

d,6

row

84–1

02Se

mi

erec

tW

FB35

–37

RM

RM

R

45N

DB

1180

Not

rele

ased

BH

S265

/

Kar

an18

Hul

led,

6ro

w45

–50

Sem

idr

oop

NW

FB39

-42

TM

RS

46K

aran

92N

otre

leas

edA

zam

(dw

arf)

1/

K12

5/

DL

85

Hul

led,

6ro

w85

–95

Ere

ctW

FB30

–40.

5S

TT

47K

aran

280

Not

rele

ased

Aza

m(d

war

f)1

/K

125

/D

L85

Hul

led,

6ro

w80

–95

Ere

ctSW

FB36

.5S

TT

48R

S6

Raj

asth

an(I

R)

RS

17/

NP

21H

ulle

d,6

row

65–7

2E

rect

WFB

20.5

SM

RS

49B

CU

73N

WPZ

,NE

PZ(I

R)

WU

M14

3H

ulle

d,2

row

95–9

8N

on-

droo

pW

MB

55M

SM

RS

50R

D20

35N

WPZ

(IR

)R

D13

7/

PL10

1H

ulle

d,6

row

95–1

00N

on-

droo

pN

WFB

44.1

SS

S

51A

zad

UP

(RF)

K12

/K

19H

ulle

d,6

row

110–

115

Non

-dr

oop

WFB

49S

SM

R

52K

603

NE

PZ(R

F)K

257

/C

138

Hul

led,

6ro

w10

0–10

5N

on-

droo

pN

WFB

45.5

RS

MR

53K

aran

16N

WPZ

(IR

)A

zam

(dw

arf)

1×

EB

7576

Hul

lles

s,6

row

75–8

0N

on-

droo

pW

FB36

.6S

SM

R

Journal of Genetics Vol. 89, Online Resources e50

S. K. Jaiswal et al.

Tabl

e1

(con

td.)

Dis

ease

resi

stan

ce

Reg

ion

and

Maj

orH

eigh

tE

arFe

ed/m

alt

1000

Yel

low

Spot

No.

Gen

otyp

eso

win

gco

nditi

onPe

digr

eech

arac

teri

stic

s(c

m)

attit

ude

Wax

ines

spu

rpos

egr

ain

wt

rust

blot

chA

phid

54C

lippe

rN

WPZ

(IR

)In

trod

uctio

nfr

omA

ustr

alia

Hul

led,

2ro

w90

–95

Non

-dr

oop

WM

B47

.8R

MR

S

55K

560

NE

PZ(R

F)K

404

/D

L47

9H

ulle

d,6

row

100–

110

Non

-dr

oop

NW

FB46

.4R

MR

MR

56R

D25

52N

WPZ

,NE

PZ(I

R)

PD20

35/

DL

472

Hul

led,

6ro

w95

–100

Non

-dr

oop

WFB

45.3

RT

MR

57K

aran

15N

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Journal of Genetics Vol. 89, Online Resources e51

Genetic diversity in Indian barley

Figure 1. Microsatellite pattern differentiating among 69 varieties of barley asamplified with primer pair BMS 40; M is the 100-bp DNA marker (MBI Fer-mentas) 1, BH393; 2, Amber; 3, Narendra4; 4, Ratna; 5, K-24; 6, NDB1173;7, BHS169; 8, Sonu; 9, VLB1; 10, RD57; 11, RD103; 12, HBL1; 13, BH 75;14, Narendra 3; 15, Dolma; 16, C164; 17, DWR28; 18, DL472; 19, PL172;20, BG25; 21, Ballia Barley; 22, Kedar; 23, VLB56; 24, C50; 25, C84; 26,K12; 27, DL3; 28, HBL276; 29, RD2052; 30, RD31; 31, Vijya; 32, PL426;33, K409; 34, Narendra 2; 35, RDB1; 36, BG105; 37, Ranjeet; 38, Bilara 2;39, PL419; 40, BH87; 41, K14; 42, Himani; 43, K 141; 44, Narendra1; 45,NDB1180; 46, Karan92; 47, Karan 280; 48, RS6; 49, BCU73; 50, RD2035; 51,Azad; 52, K603; 53, Karan16; 54, Clipper; 55, K560; 56, RD2552; 57, Karan15;58, Karan4; 59, DL88; 60, Manjula; 61, K-551; 62, Karan 741; 63, Lakhan; 64,Karan 521; 65, C-138; 66, Karan19; 67, RD2503; 68, Jyoti; 69, RD2624.

leaf sheath, waxiness and 1000 grain weight. Cluster V, VII,VIII and X showed the genetic similarity values 0.53, 0.46,0.44 and 0.34, respectively. The value of genetic similaritybetween varieties varied from 13% (BG 105 and BH 393) to100% (Narendra 3 and NDB 1173, C 84 and RDB 1).

DiscussionThe aim of the present investigation was to explore the ge-netic variability in Indian germplasm of cultivated barleyusing morpho–physiological traits and SSR markers. SSRmarkers are more specific because of the long primer se-quences and high annealing temperature during PCR am-plification. The characterization and assessment of geneticdiversity among the barley genotypes would be important

for designing breeding strategies for quantitative and qual-itative traits. During the past five years an extensive amountof data have been produced concerning the evaluation of ge-netic diversity with SSR markers in different crops such aswheat sorghum, tomato, potato, rice, maize and barley. Anoverview of the reported results for barley (Maestri et al.2002; Matus and Hayes 2002; Baek et al. 2003; Koebner etal. 2003; Malysheva-Otto et al. 2006) indicated that diversityparameters varied significantly among studies. We selected69 cultivated barley (six rows / two rows) accessions fromdifferent regions of India, estimated the extent of variabilityin morpho–physiological traits important for adaptation, dis-ease resistance, and yield, and surveyed microsatellite DNApolymorphisms at 16 loci to study the genetic divergence.

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S. K. Jaiswal et al.

Figure 2. Dendrogram derived from banding pattern of microsatellite analysis of 69 Indian varieties of barley.

Genomic diversity was estimated according to allelic rich-ness, i.e. number of detected alleles and occurrence of uniquealleles.

A total of 52 alleles were observed from 16 SSR markersamong all the selected 69 barley varieties used and the num-ber of SSR alleles varied from one (HVACLI), to eight (BMS40). The SSR marker (BMS 40) having di-nucleotide motifrepeats (CA)21, (GA)21 amplified maximum number of alle-les (eight) with highest PIC value of 0.947234 which provedthat loci with larger number of tandem repeats are likely tohave larger number of alleles (Struss and Plieske 1998).

Accessions K 24 and Clipper gave unique fragments ofsize 280 and 310 bp respectively at BMS 40 locus. Uniquealleles are important because they may be diagnostic of aparticular inbred line or for regions of the genome specificto a particular type of genotype (Senior et al. 1998). The oc-currence of the unique allele is an indication of the diver-sity present in a germplasm and its potential as a reservoir ofnovel alleles for crop improvement (Matus and Hayes 2002).Similar to results reported by Matus and Hayes (2002) andPillen et al. (2000), the highest PIC value (0.947234) wasdisplayed by SSR marker (BMS 40) having highest num-ber of alleles. However, same PIC value (0.947) was also

reported by several workers for SSR markers varying in num-ber of alleles (Saghai-Maroof et al. 1994; Struss and Plieske1998; Matus and Hayes 2002). The UPGMA analysis for 16SSR markers used in the study showed a high level of diver-sity among all the barley genotypes. All varieties representedmainly four major (I, II, III and IV) and 10 minor clusters.Similarly 69 varieties assembled in 10 groups after hierar-chical clustering of their agro-morphological traits. This highlevel of genetic diversity among all barley genotypes maybe due to their geographical pattern, agronomical characters,growth behaviour and different pedigree. This diversity leadsto allelic richness that may represent various morphologicaltraits in the population.

Assessing genetic diversity in 69 barley varieties of Indiausing morpho–physiological traits as well as SSR markersgave overlapping results. It is concluded that the five primerpairs (BMS 40, BLYRCAB, BMS 30, HVCMA and HVM 3)could be used as a potent tool to discriminate Indian barleyvarieties investigated. Although further search can be madefor even better SSR markers, the three or five primers ob-tained in this study provide an effective way to increase theefficiency of germplasm evaluation and to identify duplicateaccessions in the barley germplasm collection.

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Genetic diversity in Indian barley

Acknowledgement

This research has been supported by Indian Council of AgriculturalResearch, New Delhi, India.

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Received 11 November 2009, in revised form 3 December 2009; accepted 18 June 2010Published on the Web: 6 December 2010

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