RESEARCH ARTICLE

Transfer of leaf rust and stripe rust resistance from Aegilopsumbellulata Zhuk. to bread wheat (Triticum aestivum L.)

Parveen Chhuneja Æ Satinder Kaur Æ R. K. Goel Æ M. Aghaee-Sarbarzeh ÆM. Prashar Æ H. S. Dhaliwal

Received: 29 June 2007 / Accepted: 8 October 2007 / Published online: 31 October 2007

� Springer Science+Business Media B.V. 2007

Abstract Aegilops umbellulata acc. 3732, an excel-

lent source of resistance to major wheat diseases, was

used for transferring leaf rust and stripe rust resistance

to cultivated wheat. An amphiploid between Ae.

umbellulata acc. 3732 and Triticum durum cv.

WH890 was crossed with cv. Chinese Spring PhI to

induce homoeologous pairing between Ae. umbellulata

and wheat chromosomes. The F1 was crossed to the

susceptible Triticum aestivum cv. ‘WL711’ and leaf

rust and stripe rust resistant plants were selected among

the backcross progenies. Homozygous lines were

selected and screened against six Puccinia triticina

and four Puccinia striiformis f. sp. tritici pathotypes at

the seedling stage and a mixture of prevalent

pathotypes of both rust pathogens at the adult plant

stage. Genomic in situ hybridization in some of the

selected introgression lines detected two lines with

complete Ae. umbellulata chromosomes. Depending

on the rust reactions and allelism tests, the introgres-

sion lines could be classified into two groups,

comprising of lines with seedling leaf rust resistance

gene Lr9 and with new seedling leaf rust and stripe rust

resistance genes. Inheritance studies detected an

additional adult plant leaf rust resistance gene in one

of the introgression lines. A minimum of three

putatively new genes—two for leaf rust resistance

(LrU1 and LrU2) and one for stripe rust resistance

(YrU1) have been introgressed into wheat from Ae.

umbellulata. Two lines with no apparent linkage drag

have been identified. These lines could serve as sources

of resistance to leaf rust and stripe rust in breeding

programs.

Keywords Aegilops umbellulata �Homoeologous pairing � Leaf rust �Puccinia striiformis � Puccinia triticina �Stripe rust � Triticum aestivum � Wheat �Wide hybridization

Introduction

Leaf rust caused by Puccinia triticina Eriks. (Pt) and

stripe rust caused by Puccinia striiformis Westend f.

sp. tritici (Pst) are the most important foliar diseases

P. Chhuneja (&) � S. Kaur � R. K. Goel �M. Aghaee-Sarbarzeh � H. S. Dhaliwal

Department of Plant Breeding, Genetics and

Biotechnology, Punjab Agricultural University,

Ludhiana 141 004, India

e-mail: pchhuneja@rediffmail.com

M. Prashar

Directorate of Wheat Research, Regional Research

Station, Shimla, India

Present Address:M. Aghaee-Sarbarzeh

Agricultural Organization of Kermanshah, Kermanshah,

Iran

Present Address:H. S. Dhaliwal

Indian Institute of Technology, Roorkee, India

123

Genet Resour Crop Evol (2008) 55:849–859

DOI 10.1007/s10722-007-9289-3

of wheat the worldwide. Genetic resistance is the

most effective, economical and environmentally

friendly means to reduce losses due to the rust

diseases. More than 58 leaf rust and 40 stripe rust

resistance genes have been designated so far

(McIntosh et al. 2005; Kuraparthy et al. 2007a, b),

the most of which condition hypersensitive reactions

and interact with respective pathogens in a gene-

for-gene fashion (Flor 1942). As the resistance

conferred by single major resistance genes is not

durable, hence a constant search for new sources of

rust resistance is necessary to combat the highly

variable rust pathogens. In addition, the replacement

of highly variable land races by high yielding pure-

line varieties in many parts of the world has narrowed

down the genetic base of modern wheats. Wild

progenitor and non-progenitor species of wheat are

highly valuable source of additional resistance genes

(Dvorak 1977; Sharma and Gill 1983; Hammer 1985;

Gale and Miller 1987; Jiang et al. 1994; Singh et al.

1998, 2007). Many genes conferring resistance to rust

diseases, powdery mildew, cereal cyst nematode and

insect-pests were transferred from Aegilops species

into wheat (Jiang et al. 1994; Friebe et al. 1996;

Romero et al. 1998; Dhaliwal et al. 2003; Marais

et al. 2005a). Some of these genes transferred from

distantly related species have been exploited com-

mercially but others seem to be associated with yield

penalty due to the linkage drag (Young and Tanksley

1989).

Several strategies have been used for transferring

the alien segments that are smaller than complete

chromosome arms from non-progenitor wild species

into wheat. Sears (1956) transferred a leaf rust

resistance gene (Lr9) from Ae. umbellulata Zhuk. to

wheat through irradiation. Masoudi-Nejad et al.

(2002) used gametocidal genes for the transfer of

alien chromosome segments to wheat. Alien genes

from non-progenitor species have also been trans-

ferred to the wheat through induced homoeologous

chromosome pairing between the wheat and the

alien chromosomes (Jiang et al. 1994; Friebe et al.

1996) by using mutant or null alleles of Ph1 gene

(Sears 1972, 1981) or by using PhI, an epistatic

inhibitor of Ph1 gene from Aegilops speltoides

Tausch (Riley et al. 1968; Chen et al. 1994; Aghaee

et al. 2000). The transfers made by homoeologous

recombination are genetically compensating and the

size of the transferred alien segment is often

proportional to the linkage drag (Jiang et al. 1994;

Friebe et al. 1996). The identification and charac-

terization of cytologically undetectable primary

recombinants in wheat-Aegilops geniculata Roth

(syn. Aegilops ovata auct. non L.) introgression

carrying Lr57 and Yr40 (Kuraparthy et al. 2007a)

and wheat-Aegilops triuncialis L. introgression car-

rying Lr58 (Kuraparthy et al. 2007b), demonstrated

the feasibility of transfer of small alien desirable

segments without linkage drag. Resistance for biotic

and abiotic stresses and some quality traits have

been introgressed from progenitor and non-progen-

itor species into T. durum Desf. also using various

chromosome engineering and molecular techniques

(review by Ceoloni and Jauhar 2006). T. durum has

been reported to be equally amenable to alien

introgressions but relatively less buffered for toler-

ating gene transfers as compared to T. aestivum.

Ae. umbellulata, a non-progenitor diploid species

with the UU genome, was earlier found to be an

excellent source of resistance for various diseases

(Valkoun et al. 1985; Dhaliwal et al. 1991; Singh and

Dhaliwal 2000). This paper reports the introgression

of resistance to leaf rust and stripe rust from Ae.

umbellulata acc. 3732 to the hexaploid wheat.

Materials and methods

Plant material

A leaf rust and stripe rust resistant Ae. umbellulata

acc. 3732 from USSR, supplied by Dr J. P. Gustafson,

University of Missouri, Columbia, USA, was used for

developing a synthetic amphiploid with a susceptible

T. durum cv. WH890. The amphiploid T. durum

WH890-Ae. umbellulata acc. 3732 was crossed with

cv. Chinese Spring {CS (PhI)} (Chen et al. 1994) to

induce homoeologous pairing (Fig. 1). The resultant

F1 plants were crossed with a leaf rust and stripe rust

susceptible genotype, WL711(NN), which has kr

(crossability) alleles and is a near-isogenic line of

T. aestivum L. cv. WL711. The resistant F1 plants

from the cross T. durum WH890-Ae. umbellulata//

CSPh1/3/WL711(NN) were selfed and backcrossed to

WL711(NN) to recover the recurrent genotype. Some

of the selected BC1F1 plants were selfed to generate

BC1F6–7 and others were backcrossed to generate

BC2F5. In the segregating generations, selection for

850 Genet Resour Crop Evol (2008) 55:849–859

123

leaf rust resistance was carried out by screening the

genetically enhanced material at the seedling stage

against two most virulent and prevalent Pt pathotypes

(121R63-1 and 21R5). The same plants were

screened for terminal disease severity for stripe rust

and leaf rust at adult plant stage in the field at the

Punjab Agricultural University (PAU), Ludhiana,

India.

Rust screening

Rust tests on homozygous F8, BC1F6–7 and BC2F5

wheat-Ae. umbellulata introgression lines (ILs) along

with the parental lines were performed using five Pt

pathotypes viz. IR5, 109R31-1, 121R63-1, 21R5 and

253R31 (Lr19 virulent) and four Pst pathotypes viz.

46S102, 47S103, 46S119 (Yr9 virulent) and 78S84

(Yr27 virulent) at the two-leaf seedling stage

(Table 1). A leaf rust resistance gene Lr9 was

transferred from Ae. umbellulata by Sears (1956).

To determine whether the leaf rust resistance gene(s)

in the wheat-Ae. umbellulata ILs were different from

Lr9, these ILs were also tested with a Lr9 virulent Pt

pathotype, 121R127 at the Regional Research

Station, Shimla, India. The seedlings were inoculated

Fig. 1 Schematic representation of the crossing strategy

adopted for transferring leaf and stripe rust resistance genes

from Ae. umbellulata to hexaploid wheat T. aestivum cv.

WL711 through induced homoeologous pairing using Chinese

Spring stock with homoeologous pairing inducer gene, PhIgene (Ph inhibitor). T. aestivum cv. WL711 (NN) with kr(crossability) alleles was used as recipient parent

Ta

ble

1A

vir

ule

nce

/vir

ule

nce

afo

rmu

lae

of

leaf

rust

(P.

trit

icin

a)

and

stri

pe

rust

(P.

stri

ifo

rmis

)p

ath

oty

pes

use

dfo

rth

esc

reen

ing

of

wh

eat-

Ae.

um

bel

lula

tain

tro

gre

ssio

nli

nes

Pat

ho

typ

eA

vir

ule

nce

Vir

ule

nce

P.

trit

icin

a

IR5

Lr1

,L

r9,

Lr1

9,

Lr2

0,

Lr2

4,

Lr2

5L

r2,

Lr3

,L

r10,

Lr1

1,

Lr1

4,

Lr1

5,

Lr1

6,

Lr1

7,

Lr1

8,

Lr2

1,

Lr2

3,

Lr2

6

10

9R

31

-1L

r9,

Lr1

9,

Lr2

4,

Lr2

5,

Lr2

6L

r1,

Lr2

,L

r3,

Lr1

0,

Lr1

1,

Lr1

4,

Lr1

5,

Lr1

6,

Lr1

7,

Lr1

8,

Lr2

0,

Lr2

1,

Lr2

3

12

1R

63

-1L

r9,

Lr1

9,

Lr2

4,

Lr2

5L

r1,

Lr2

,L

r3,

Lr1

0,

Lr1

1,

Lr1

4,

Lr1

5,

Lr1

6,

Lr1

7,

Lr1

8,

Lr2

0,

Lr2

1,

Lr2

3,

Lr2

6

12

1R

12

7L

r19,

Lr2

4,

Lr2

5L

r1,

Lr2

,L

r3,

Lr9

,L

r10,

Lr1

1,

Lr1

4,

Lr1

5,

Lr1

6,

Lr1

7,

Lr1

8,

Lr2

0,

Lr2

1,

Lr2

3,

Lr2

6

21

R5

Lr9

,L

r15,

Lr1

9,

Lr2

4,

Lr2

5L

r1,

Lr2

,L

r3,

Lr1

0,

Lr1

1,

Lr1

4,

Lr1

6,

Lr1

7,

Lr1

8,

Lr2

0,

Lr2

1,

Lr2

3,

Lr2

6

25

3R

31

Lr9

,L

r24,

Lr2

5L

r1,

Lr2

,L

r3,

Lr1

0,

Lr1

1,

Lr1

6,

Lr1

7,

Lr1

9,

Lr2

0,

Lr2

1,

Lr2

3,

Lr2

6

P.

stri

ifo

rmis

46

S1

19

Yr1

,Y

r5,

Yr1

0,

Yr1

5,

Yr2

4,

Yr2

5,

Yr2

6,

Yr2

7Y

r2,

Yr3

,Y

r4,

Yr6

,Y

r7,

Yr8

,Y

r9,

Yr1

7,

Yr1

8

46

S1

02

Yr1

,Y

r5,

Yr9

,Y

r10,

Yr1

5,

Yr1

7,

Yr2

4,

Yr2

5,

Yr2

6,

Yr2

7Y

r2,

Yr3

,Y

r4,

Yr6

,Y

r7,

Yr8

,Y

r18

47

S1

03

Yr5

,Y

r9,

Yr1

0,

Yr1

5,

Yr1

7,

Yr2

4,

Yr2

5,

Yr2

6,

Yr2

7Y

r1,

Yr2

,Y

r3,

Yr4

,Y

r6,

Yr7

,Y

r8

78

S8

4Y

r1,

Yr5

,Y

r10,

Yr1

5,

Yr1

7,

Yr2

4,

Yr2

5,

Yr2

6Y

r2,

Yr3

,Y

r4,

Yr6

,Y

r7,

Yr8

,Y

r9,

Yr2

7

aA

tth

ese

edli

ng

stag

ein

the

gla

ssh

ou

seu

nd

erst

and

ard

con

dit

ion

s(N

ayar

etal

.1

99

7)

Genet Resour Crop Evol (2008) 55:849–859 851

123

with a mixture of rust urediospores and talc. The

inoculated seedlings were incubated in a dark cham-

ber at 20 ± 1�C at 100% RH for 16 h (Nayar et al.

1997). After the incubation, the trays were shifted to

a green house maintained at 20 ± 2�C. Infection

types (ITs) were recorded 14 days after the inocula-

tion using a 0–4 scale (Stakman et al. 1962) where IT

0; = no uredinia or other macroscopic sign of

infection, ; = no uredinia but small hypersensitive

necrotic or chlorotic flecks present, 1 = small uredi-

nia surrounded by necrosis, 3 = medium uredinia

with or without chlorosis, 3+ = large uredinia with

or without chlorosis and 33+ = large uredinia with-

out chlorosis. For adult plant screening, the

introgression lines were planted in 1.5 m rows with

row-to-row distance of 20 cm and plant-to-plant

distance of 10 cm. Infector rows of the susceptible

cultivar Agra Local were planted all around the

experimental plot and sprayed with a mixture of

urediniospores of Pt pathotypes (109R31-1, 121R63-

1 and 21R5) and Pst pathotypes (46S102, 47S103 and

46S119). At the adult plant stage, data were recorded

according to the modified Cobb’s scale (Peterson

et al. 1948) which included disease severity (percent

leaf area affected) and infection type viz. 0 =

immune; R = resistant, MR = moderately resistant;

MS = moderately susceptible and S = susceptible.

Cytological studies

Spikes were fixed at the pre-booting stage in Cornoy’s

solution II (6 ethanol:3 chloroform:1 glacial acetic

acid) and transferred to ethanol (70%) after 48 h.

Squash preparations of pollen mother cells (PMCs) at

the metaphase I were made in acetocarmine (2%) to

study the chromosome number and pairing behavior

in the selected wheat-Ae. umbellulata introgression

lines. Genomic in situ hybridization (GISH) was used

to detect alien introgression in some of the selected

rust resistant wheat-Ae. umbellulata ILs. GISH was

done as described in Zhang et al. (2001) using Ae.

umbellulata (2n = 14, UU) genomic DNA as the

probe and Chinese Spring (CS) genomic DNA as

blocker. Total genomic DNA was extracted from CS

wheat and Ae. umbellulata using the CTAB method as

modified by Saghai-Maroof et al. (1984). The DNA

from Ae. umbellulata was sheared using a needle to an

average size of 200 bp and CS DNA was autoclaved

for 12 min. The genomic Ae. umbellulata DNA was

labeled with fluorescein (FITC) dUTP using nick

translation. A ratio of 35–40:1 of wheat competitor

DNA to the labeled probe (Ae. umbellulata DNA) was

used in the hybridization mixture. Chromosomes were

counterstained with Propidium Iodide (PI) and

mounted in Vectashield (Vector Laboratories). The

slides were analyzed with an epifluorescence Zeiss

Axioplan 2 microscope and images were captured

using a SPOT CCD (charge-coupled-device) camera

operated with SPOT 2.1 software (Diagnostic Instru-

ments) and processed with Photoshop 5.5 (Adobe

Systems).

Results

Triticum durum cv. WH890, CS (PhI) and the

recurrent parent WL711(NN) were susceptible against

all the P. triticina and P. striiformis pathotypes used at

the seedling stage whereas at the adult plant stage,

both ‘WH890’ and CS (PhI) exhibited low severities

with susceptible responses to leaf rust as well as stripe

rust in different years (Table 2). The recurrent parent

WL711(NN) showed 60S–80S leaf rust and 40S–80S

stripe rust reactions at the adult plant stage. Ae.

umbellulata accession 3732 was resistant to all the

tested Pt and Pst pathotypes at the seedling stage and

showed hypersensitive reactions to both the rusts at

the adult plant stage. The amphiploid was susceptible

to Pt and Pst pathotypes at the seedling stage and

showed a terminal disease severity of 40S for leaf rust

as well as stripe rust at the adult plant stage (Table 2).

However, plants with rust resistance were recovered

from the selfed and the backcross progenies.

Homozygous wheat-Ae. umbellulata ILs viz.

PAU#315-5, 329-1, 333-4, 339-4, 357-1, 367-4,

380-3, 388-5, 393-4 and 403-1 with leaf and/or stripe

rust resistance were selected and analyzed for the

chromosome number, chromosome pairing and the

effectiveness of resistance against the most virulent

and prevalent Pt and Pst pathotypes. The cytogenetic

characterization of the introgression lines using

meiotic analysis detected normal chromosome num-

ber and pairing in most of the ILs. The ILs 329-1,

357-1, 367-4, 380-3, 388-5, 393-4 and 403-1 had

2n = 42 and showed normal chromosome pairing

with 17–19 ring and 2–4 rod bivalents (Fig. 2a–c) at

the metaphase I. IL315-5 had 2n = 41 and IL333-4

852 Genet Resour Crop Evol (2008) 55:849–859

123

Ta

ble

2L

eaf

rust

(P.

trit

icin

a)

and

stri

pe

rust

(P.

stri

ifo

rmis

)re

acti

on

(see

dli

ng

and

adu

ltp

lan

tst

age)

of

the

sele

cted

intr

og

ress

ion

lin

es(I

Ls)

fro

mth

ecr

oss

T.

du

rum

cv.

WH

89

0/A

e.u

mb

ellu

lata

acc.

37

32

//C

SP

hI /3

/n*

WL

71

1(N

N)

Cu

ltiv

ar/I

Ls

Ch

rN

o.

Gen

erat

ion

See

dli

ng

reac

tio

na

agai

nst

leaf

rust

pat

ho

typ

es

See

dli

ng

reac

tio

na

agai

nst

stri

pe

rust

pat

ho

typ

es

Ru

stre

acti

on

atad

ult

pla

nt

stag

eb

Lea

fru

stS

trip

eru

st

IR5

10

9R

31

-11

21

R6

3-1

12

1R

12

7c

21

R5

25

3R

31

c4

6S

11

9d

46

S1

02

46

S1

03

78

S8

4c

20

04

20

05

20

06

20

04

20

05

20

06

T.

aes

tivu

mcv

.

WL

71

1

42

33

33

33

33

33

80

S6

0S

80

S4

0S

80

S6

0S

Ae.

um

bel

lula

taac

c.3

73

2

14

;;

;;

;n

t0

;;

;n

t0

0n

t0

0n

t

CS

(Ph

I )4

23

3+

33

33

33

33

32

0S

20

S2

0S

10

S1

0S

10

S

T.

du

rum

cv.

WH

89

0

28

33

+3

3n

t3

nt

33

3n

t1

0S

10

S1

0S

10

MS

20

S1

0S

Am

ph

iplo

id4

2n

t3

nt

nt

3+

nt

nt

33

nt

40

S4

0S

40

S4

0S

60

S4

0S

32

9-1

42

BC

2F

4;

;0

;3

+1

;3

33

30

00

40

S4

0S

40

S

33

3-4

44

F8

;;

;–

;;

3n

tn

t3

00

04

0S

60

S4

0S

36

7-4

42

BC

2F

5n

tn

t0

;3

3+

;;

33

33

00

04

0S

40

S4

0S

38

0-3

42

BC

3F

4;

0;

0;

3;

;3

33

30

00

40

S4

0S

40

S

31

5-5

41

F8

;;

;;

;;

nt

nt

nt

nt

00

00

00

33

9-4

42

BC

1F

7n

tn

t;1

11

nt

0;

;0

;n

t0

00

00

0

35

7-1

42

BC

1F

6;1

;;1

;;1

;S

eg.

;;

;0

00

00

0

38

8-5

42

BC

1F

6;1

;1;1

;1;1

;1

nt

nt

30

00

00

0

39

3-4

42

BC

2F

5;1

0;

;1;

;1–

;;1

;n

t;

00

00

00

40

3-1

42

BC

2F

5n

tn

t;

1;1

;0

;;

;3

00

00

00

nt—

no

tte

sted

;S

eg.—

seg

reg

atin

ga

ITs

of

seed

lin

gs

wer

esc

ore

dac

cord

ing

toth

em

od

ified

scal

eo

fS

tak

man

etal

.(1

96

2)

bA

tth

ead

ult

pla

nt

stag

e,ra

tin

gs

wer

eb

ased

on

the

mo

difi

edC

ob

bsc

ale

(Pet

erso

net

al.

19

48

).T

he

adu

ltp

lan

tsc

reen

ing

was

do

ne

agai

nst

am

ixtu

reo

fle

afru

st(1

09

R3

1-1

,

12

1R

63

-1an

d2

1R

5)

and

stri

pe

rust

pat

ho

typ

es(4

6S

11

9,

47

S1

03

and

46

S1

02

)c

Tes

ted

inis

ola

tio

nat

DW

R,

Reg

ion

alR

esea

rch

Sta

tio

n,

Flo

wer

dal

e,S

him

lad

Tes

ted

atD

WR

,R

egio

nal

Res

earc

hS

tati

on

,F

low

erd

ale,

Sh

imla

asw

ell

asat

PA

U,

Lu

dh

ian

a

Genet Resour Crop Evol (2008) 55:849–859 853

123

was observed to be an addition line with 2n = 44.

The meiotic analysis of the F1 plants from the cross

of IL393-4 with cv. PBW343, a widely adapted wheat

cultivar, detected 21II consisting of 19 ring and 2 rod

bivalents (Fig. 2d) suggesting the presence of a small

alien introgression. The GISH analysis of IL 315-5

detected four Ae. umbellulata chromosomes, one

submetacentric and three with subterminal centro-

meres (Fig. 3). GISH analysis of IL333-4 detected a

pair of Ae. umbellulata chromosomes with subtermi-

nal centromeres (Fig. 3). GISH analysis was also

conducted for IL339-4 with 2n = 42, however, no

alien chromatin was detected (results not shown).

Rust screening of wheat-Ae. umbellulata

introgression lines

To test the effectiveness of putatively new rust

resistance genes, 10 selected homozygous wheat-Ae.

umbellulata ILs were screened as seedlings and adult

plants against the most virulent pathotypes of Pt and

Pst prevalent in the Indian subcontinent. All the ILs

were resistant to Pt pathotypes IR5, 109R31-1,

121R63-1, 21R5 and 253R31 at the seedling stage

(Table 2). Three ILs 329-1, 367-4 and 380-3 showed

susceptible seedling responses against pathotype

121R127, indicating the presence of Lr9. All the

other ILs (IL333-4 could not be tested for 121R127)

were resistant to 121R127, suggesting the introgres-

sion of leaf rust resistance genes, other than Lr9, from

Ae. umbellulata. All the selected ILs showed com-

plete leaf rust resistance at the adult plant stage

(Table 2) for 3 years (Fig. 4).

These ILs were also tested at the seedling stage

against Pst pathotypes 46S102, 47S103, 46S119 and

78S84. Genotypes including ILs 339-4, 357-1, 388-5,

393-4 and 403-1 were resistant (Table 2) at the

seedling stage indicating the presence of a seedling

resistance gene for stripe rust in these ILs. In contrast,

Fig. 2 Pollen mother cells

in wheat-Ae. umbellulataintrogression lines (a) 380-3

(2n = 42) with 19 ring and

2 rod bivalents, (b) 393-4

with 19 ring and 2 rod

bivalents, (c) 403-1

(2n = 42) with 17 ring and

4 rod bivalents and (d) F1

plant from the cross of

IL393-4 with bread wheat

cv. PBW343 (2n = 42)

with 19 ring and 2 rod

bivalents

Fig. 3 Genomic in situhybridization (GISH) of the

mitotic metaphase

chromosomes of wheat-Ae.umbellulata interspecific

lines T315-5 (a) and T333-4

(b) using total genomic

DNA of Ae. umbellulata as

a probe, visualized by

yellow-green FITC

fluorescence; chromosomes

were counterstained with

propidium iodide and

fluoresce red

854 Genet Resour Crop Evol (2008) 55:849–859

123

ILs 388-5 and 403-1 showed susceptible infection

types against the Pst pathotype 78S84 and need to be

retested. Remaining ILs viz. 329-1, 333-4, 367-4 and

380-3 were susceptible at the seedling stage. IL 315-5

was not tested at the seedling stage. Introgression

lines 329-1, 333-4, 367-4 and 380-3 showed moder-

ate to high adult plant stripe rust responses (40S–

60S). On the contrary, ILs 315-5, 339-4, 357-1,

388-5, 393-4 and 403-1 exhibited complete stripe rust

resistance at the adult plant stage (Fig. 4).

Inheritance studies for leaf rust resistance

Introgression lines 367-4 (leaf rust resistant and stripe

rust susceptible, proposed to carry Lr9) and 403-1

(leaf rust and stripe rust resistant, proposed to carry

putatively new genes) were crossed with the recurrent

parent WL711 and segregation for resistance was

studied in the F2 population. F1 derived from the

IL367-4/WL711 cross was resistant to leaf rust at the

seedling and adult plant stage. The F2 population

showed monogenic segregation at both the seedling

and the adult plant stage (Table 3). In F3, out of 79

progenies screened at the seedling and adult plant

stage, 17 progenies were homozygous resistant, 43

segregating and 19 homozygous susceptible for leaf

rust (v2(1:2:1) = 0.66) confirming the segregation of a

single leaf rust resistance gene. The F1 derived from

IL 403-1/WL711 cross was resistant to both leaf rust

and stripe rust indicating that the resistance was

dominant. When tested for leaf rust seedling resis-

tance, F2 population segregated into 104 resistant (R)

and 48 susceptible (S) plants with v2(3:1) = 3.5

indicating segregation of a single dominant gene.

All the 152 plants (both resistant and susceptible)

were transplanted in the field and screened for

terminal leaf rust severity at the adult plant stage.

Out of the 48 plants that were susceptible at the

seedling stage, 28 were resistant at the adult plant

stage, indicating the presence of an adult plant

resistance (APR) gene for leaf rust resistance. At

the adult plant stage, this F2 population segregated

into 132R: 20S plants with v2(15:1) = 12.4 (Table 3).

Only selected F3 progenies with seedling and/or APR

genes were evaluated for leaf rust resistance. The

Table 3 Segregation of leaf rust (P. triticina) resistance in the F2 from the crosses of the introgression lines derived from the cross T.durum cv. WH890/Ae. umbellulata acc. 3732//CS PhI/3/3*WL711(NN) with the recurrent parent WL711 (NN)

IL Stage No. of plants in F2 Expected ratio Calculated v2 valuec

Resistant Susceptible Total

367-4 Seedling a 68 27 95 3:1 0.59

Adult plantb 67 24 91 3:1 0.09

403-1 Seedlinga 104 48 152 3:1 3.5

Adult plantb 132 20 152 15:1 12.4

a Seedling screening with leaf rust pathotype 121R63-1b Adult plant screening in the field using a mixture of leaf rust and stripe rust pathotypesc v2 value for significance at P = 0.05 is 3.84 (df = 1)

Fig. 4 Leaf and stripe

reaction of the parents and

introgression lines

developed from the cross: T.durum cv. WH890/Ae.umbellulata acc. 3732//CS

PhI/3/*nWL711 (NN) at the

adult plant stage against a

mixture of leaf rust and

stripe rust pathotypes

Genet Resour Crop Evol (2008) 55:849–859 855

123

progeny testing of the 30 selected leaf rust resistant

F2 plants confirmed the transfer of a seedling

resistance gene and an APR gene for leaf rust in

IL403-1 from Ae. umbellulata.

Allelic tests

Evaluation of wheat-Ae. umbellulata ILs at the

seedling stage with different Pt isolates led to the

identification of two groups of lines, one group with

Lr9 and other with a putatively new leaf rust

resistance gene effective at the seedling stage. To

further test that the leaf rust seedling resistance genes

in the two groups were different, crosses were made

between IL 393-4, resistant to all leaf rust and stripe

rust pathotypes and IL 380-3, susceptible to leaf rust

pathotype 121R127 and all stripe rust pathotypes. All

the F1 plants were resistant to both the rusts. In the F2,

however, out of a total of 85 plants, 82 were resistant

to leaf rust at the seedling as well as at the adult plant

stage (Table 4) and three were susceptible, indicating

segregation of two dominant genes (v2(15:1) = 1.07).

The F2 population was also scored for stripe rust

reaction at the adult plant stage in the field, which

segregated into 69R and 16S plants (v2(3:1) = 1.73)

indicating a single gene for stripe rust resistance in

the IL393-4. F2 population from the cross of two ILs

(T393-4 and T357-1) with putatively new leaf rust

and stripe rust resistance genes did not show any

segregation for leaf rust and stripe rust indicating that

the same genes were present in both the ILs.

Discussion

In the present study, we are reporting the transfer of

putatively new leaf rust and stripe rust resistance

genes from a non-progenitor species Ae. umbellulata

to bread wheat through induction of homoelogous

pairing. An accession of Ae. umbellulata with

multiple disease resistance was hybridized with a

susceptible durum cultivar to produce a synthetic

amphiploid. The amphiploid was, however, suscep-

tible, indicating the presence of suppressors in the A

and/or B genomes of durum wheat (Aghaee et al.

2001). Ae. umbellulata leaf rust and stripe rust

resistance genes were recovered following the crosses

of the amphiploid with the bread wheat cv. WL711

due to the segregation of the suppressors of the durum

genomes. Based on the leaf and stripe rust reaction of

the seedling and adult plant stages and the tests of

allelism, the introgression lines could be grouped into

two broad categories. Introgression lines 329-1, 333-

4, 367-4 and 380-3 carried seedling effective leaf rust

resistance gene only, similar to Lr9, transferred

previously from Ae. umbellulata into the wheat as a

homoeologous chromosome transfer 6BS.6BL-6UL

(Sears 1961; Friebe et al. 1995). The inheritance

studies in the IL 367-4 also confirmed the transfer of

a single leaf rust resistance gene only. All the other

six ILs viz. 315-5, 339-4, 357-1, 388-5, 393-4 and

403-1 carried putatively new seedling leaf rust and

stripe rust resistance gene(s). Inheritance studies in

the IL 403-1 showing monogenic segregation at the

seedling stage and digenic segregation at the adult

Table 4 Allelic tests of wheat-Ae. umbellulata introgression lines T393-4 with T380-3 (leaf rust resistant and stripe rust susceptible)

and T357-1 (leaf and stripe rust resistant)

No. of plants in F2 Expected ratio Calculated v2 value

Resistant Susceptible Total

F2: T393-4/T380-3

Seedlinga 82 3 85 15:1 1.07

Adult plant leaf rustb 82 3 85 15:1 1.07

Adult plant stripe rustb 69 16 85 3:1 1.73

F2: T393-4/T 357-1

Seedling leaf rust 111 0 111 – –

Adult plant leaf rust and stripe rust 111 0 111 – –

a Seedling screening with leaf rust pathotype 121R63-1b Adult plant screening in the field using a mixture of leaf and stripe rust pathotypesc v2 value for significance at P = 0.05 is 3.84 (df = 1)

856 Genet Resour Crop Evol (2008) 55:849–859

123

plant stage indicated that this introgression line

carried a gene each for seedling and adult plant leaf

rust resistance. The segregation for the seedling leaf

rust resistance gene fitted well to the monogenic ratio.

However, the two gene segregation observed at the

adult plant stage was distorted suggesting that the

APR gene for leaf rust might be carried on a larger

alien segment. Such distortions in the transmission of

alien genes have often been observed (Endo 1990;

Prins and Marais 1999; Marais et al. 2005b). APR

gene for leaf rust in IL403-1 appeared to have been

masked due to the presence of the seedling resistance

gene indicating that some other ILs with seedling

resistance gene for leaf rust might also carry the APR

gene. The cv. Chinese Spring parent used for the

induction of homoeologous pairing carried an APR

gene for leaf rust, Lr34 (McIntosh 1992; Singh and

Gupta 1992). Chinese Spring showed terminal leaf

rust severity of 20S in the field while the F2 plants

and one of the ILs homozygous for APR gene (data

not given) developed 5MR leaf rust indicating that

the APR gene identified in the IL403-1 was different

from Lr34. The standard Ae. umbellulata genome has

three chromosomes with submedian and four chro-

mosomes with subterminal centromeres (Friebe et al.

1995). The Ae. umbellulata chromosome, with sub-

terminal centromere, present in IL333-4 might be 6U

which is known to carry Lr9 (Sears 1956). Hence the

putatively new rust resistance genes might be located

on other subterminal/submetacentic chromosome(s)

observed in 315-5. The other introgression lines,

derived from the same cross amphiploid/CS Ph1//

WL711(NN), might be carrying different sizes of the

introgressed alien segments. Linkage drag was

apparent from the variation in the plant type of these

leaf and stripe rust resistant interspecific lines. The

plant type of the IL315-5 was the most affected, with

very poor, grass-like phenotype with narrow leaves,

reduced height, small spikes and delayed flowering.

The GISH analysis confirmed that this IL had very

high amount of the alien chromatin. IL393-4

appeared to carry the smallest alien segment with

leaf rust and stripe rust resistance genes since it did not

have any apparent linked, deleterious phenotypic

effects. Presence of only bivalents in the F1 derived

from 393-4/PBW343 cross also indicated that 393-4 did

not carry complete Ae. umbellulata chromosome.

Another IL339-4 carried cytologically undetectable

introgression. The IL393-4 has been selected for further

studies on the molecular mapping of putative novel leaf

rust and stripe rust resistance genes and for transferring

these genes to other elite wheat backgrounds.

Except for 1BS-1RL wheat-rye translocation in

Veery series of CIMMYT wheat lines, most of the alien

introgressions had limited use in practical breeding

because of cytological instability of alien chromosome

segments incorporated in non-homoeologous regions or

because of the linkage of the undesirable genes on the

large alien segments (Friebe et al. 1996). However, by

screening a large number of recombinants, we were

successful in recovering the IL 393-4 with apparently

least linkage drag and IL 339-4 with cytologically

undetectable alien segment. Identification of the wheat-

Ae. umbellulata introgression lines with no apparent

linkage drag clearly suggests that it is possible to

transfer novel and useful genetic variability from wild

species with little undesirable genetic information.

During the present investigation, three putatively

new rust resistance genes, one seedling and one APR

gene for leaf rust temporarily designated as LrU1 and

LrU2 and one seedling gene for stripe rust, temporarily

designated as YrU1 have been transferred from a single

accession of Ae. umbellulata to T. aestivum cv. WL711.

In addition to these new genes, a leaf rust resistance

gene that may be similar to Lr9 has been transferred.

Transfer of three novel rust resistance genes including

one APR gene from single Ae. umbellulata accession

clearly demonstrates the presence of a high level of

useful variability among related species and the

importance of these sources for germplasm enhance-

ment. Mapping of these resistance genes using

molecular markers is in progress and will facilitate

their cataloguing and utilization for wheat germplasm

enhancement, especially in the Indian subcontinent.

Acknowledgements Financial assistance provided by the

USDA-ARS under the Project IN-ARS-842 and Grant Number

FG-In-792 to carry out this research work is gratefully

acknowledged. We thank Dr. B S Gill, Distinguished

Professor and Dr. Bernd Friebe, WGGRC, KSU, USA, for

providing laboratory facilities for GISH analysis. We are also

grateful to Dr. H S Bariana, Australian Cereal Rust Control

Program, University of Sydney, Australia for a critical review

of the manuscript and valuable suggestions.

References

Aghaee-Sarbarzeh M, Singh H, Dhaliwal HS (2000) Ph1 gene

derived from Ae. speltoides induces homoeologous

Genet Resour Crop Evol (2008) 55:849–859 857

123

pairing in wide crosses of Triticum aestivum. J Heredity

91:417–421

Aghaee-Sarbarzeh M, Dhaliwal HS, Chhuneja P, Singh H

(2001) Suppression of rust resistance genes from distantly

related species in Triticum durum-Aegilops amphiploids.

Wheat Inf Ser 92:12–16

Ceoloni C, Jauhar PP (2006) Chromosome engineering of the

durum wheat genome: strategies and applications of

potential breeding value. In: Singh RJ, Jauhar PP (eds)

Genetic resources, chromosome engineering and crop

improvement: Cereals. CRC Press, Taylor & Francis

Group, London, pp 27–59

Chen PD, Tsujimoto H, Gill BS (1994) Transfer of PhI genes

promoting homoeologous pairing from Triticum spelto-ides to common wheat. Theor Appl Genet 88:97–101

Dhaliwal HS, Singh H, Gupta S, Bagga PS, Gill KS (1991)

Evaluation of Aegilops and wild Triticum species for

resistance to leaf rust (Puccinia recondita f.sp. tritici) of

wheat. Intern J Trop Agric 9:118–121

Dhaliwal HS, Chhuneja P, Gill RK, Goel RK, Singh H (2003)

Introgression of disease resistance genes from related

species into cultivated wheats through interspecific

hybridization. Crop Improv 29:1–18

Dvorak J (1977) Transfer of leaf rust resistance from Aegilopsspeltoides to Triticum aestivum. Can J Genet Cytol

19:133–141

Endo TR (1990) Gametocidal chromosomes and their induc-

tion of chromosome mutations in wheat. Jpn J Genet

65:135–152

Flor HH (1942) Inheritance of pathogenicity in Melampsoralini. Phytopathology 32:653–669

Friebe B, Jiang J, Tuleen N, Gill BS (1995) Standard karyotype

of Aegilops umbellulatum and the characterization of the

derived chromosome addition and translocation lines in

common wheat. Theor Appl Genet 90:150–156

Friebe B, Jiang J, Raupp WJ, McIntosh RA, Gill BS (1996)

Characterization of wheat-alien translocations conferring

resistance to diseases and pests: current status. Euphytica

91:59–87

Gale MD, Miller TE (1987) The introduction of alien genetic

variation into wheat. In: Lupton FGH (ed) Wheat

breeding: its scientific basis. Chapman and Hall, UK,

pp 173–210

Hammer K (1985) Studies towards a monographic treatment of

wild plant collections: Aegilops L. – resistance tests.

Kulturpflanze 33:123–131

Jiang J, Friebe B, Gill BS (1994) Recent advances in alien gene

transfer in wheat. Euphytica 73:199–212

Kuraparthy V, Chhuneja P, Dhaliwal HS, Kaur S, Bowden RL,

Gill BS (2007a) Characterization and mapping of cryptic

alien introgression from Aegilops geniculata with novel

leaf rust and stripe rust resistance genes Lr57 and Yr40 in

wheat. Theor Appl Genet 114:1379–1389

Kuraparthy V, Sood S, Chhuneja P, Dhaliwal HS, Kaur S,

Bowden RL, Gill BS (2007b) A cryptic wheat-Aegilopstriuncialis translocation with leaf rust resistance gene

Lr58. Crop Sci 47:1995–2003

Marais GF, McCallum B, Snyman JE, Pretorius ZA, Marais AS

(2005a) Leaf rust and stripe rust resistance genes Lr54 and

Yr37 transferred to wheat from Aegilops kotschyi. Plant

Breed 124:538–541

Marais GF, Pretorius ZA, Wellings CR, McCallum B, Marais

AS (2005b) Leaf rust and stripe rust resistance genes

transferred to common wheat from Triticum dicoccoides.

Euphytica 143:115–123

Masoudi-Nejad A, Nasuda S, McIntosh RA, Endo TR (2002)

Transfer of rye chromosome segments to wheat by a ga-

metocidal system. Chromosome Res 10:349–357

McIntosh RA (1992) Close genetic linkage of genes conferring

adult plant resistance to leaf rust and stripe rust in wheat.

Plant Pathol 41:523–527

McIntosh RA, Devos KM, Dubcovsky J, Rogers WJ, Morris

CF, Appels R, Anderson OD (2005) Catalogue of gene

symbols: 2005 supplement. In: KOMUGI—Integrated

Wheat Science Database. http://www.grs.nig.ac.jp/wheat/

komugi/genes/macgene/supplement2005.pdf

Nayar SK, Prashar M, Bhardwaj SC (1997) Manual of current

techniques in wheat rusts. Research Bull No. 2, 32 pp,

Regional Station. Flowerdale, Shimla 171002, India

Peterson RF, Campbell AB, Hannah AE (1948) A diagram-

matic scale for rust intensity on leaves and stems of

cereals. Can J Res 26:496–500

Prins R, Marais GF (1999) A genetic study of the gametocidal

effect of the Lr19 translocation of common wheat. S Afr J

Plant Soil 16:10–14

Riley R, Chapman V, Johnson R (1968) The incorporation of

alien disease resistance in wheat by genetic interference

with the regulation of meiotic chromosome synapsis.

Genet Res Camb 12:198–219

Romero M, Montes MJ, Sin E, Lopez-Brana I, Duce A, Martin-

Sanchez JA, Andres MF, Delibes A (1998) A cereal cyst

nematode (Heterodera avenae) resistance gene transferred

from Aegilops triuncialis to hexaploid wheat. Theor Appl

Genet 96:1135–1140

Saghai-Maroof MA, Soliman KM, Jorgensen RA, Allard RW

(1984) Ribosomal DNA spacer length polymorphisms in

barley: Mendelian inheritance, chromosomal location, and

population dynamics. Proc Natl Acad Sci USA 81:8014–

8018

Sears ER (1956) The transfer of leaf rust resistance from

Aegilops umbellulata to wheat. Brookhaven Symp in Biol.

No. 9, Genetics in Plant Breeding, pp 1–22

Sears ER (1961) Identification of the wheat chromosome car-

rying leaf rust resistance from Aegilops umbellulata.

Wheat Inf Serv 12:12–13

Sears ER (1972) Chromosome engineering in wheat. In:

Stadler Genetics Symp. 4. Univ. of Missouri, Columbia,

USA, pp 23–38

Sears ER (1981) Transfer of alien genetic material to wheat. In:

Evans LT, Peacock WJ (eds) Wheat science today and

tomorrow, pp 75–89

Sharma HC, Gill BS (1983) Current status of wide hybridiza-

tion in wheat. Euphytica 32:17–31

Singh H, Dhaliwal HS (2000) Intraspecific genetic diversity for

resistance to wheat rusts in wild Triticum and Aegilopsspecies. Wheat Inf Serv 90:21–30

Singh RP, Gupta AK (1992) Expression of wheat leaf rust

resistance gene Lr34 in seedlings and adult plants. Plant

Dis 76:489–491

Singh H, Grewal TS, Dhaliwal HS, Pannu PPS, Bagga PS

(1998) Sources of leaf rust and stripe rust resistance in

wild relatives of wheat. Crop Improv 25:26–33

858 Genet Resour Crop Evol (2008) 55:849–859

123

Singh K, Chhuneja P, Ghai M, Kaur S, Goel RK, Bains NS,

Keller B, Dhaliwal HS (2007) Molecular mapping of leaf

and stripe rust resistance genes in Triticum monococcumand their transfer to hexaploid wheat. In: Buck H, Nisi JE,

Solomon N (eds) Wheat production in stressed environ-

ments. Developments in Plant Breeding, vol 12. Springer,

Netherlands, pp 779–786

Stakman EC, Stewart DH, Loegering WQ (1962) Identification

of physiologic races of Puccinia graminis var. tritici.USDA Agri Res Serv No E617 (Rev.), p 53

Valkoun J, Hammer K, Kucerova D, Bartos P (1985) Disease

resistance in the genus Aegilops L. – stem rust, leaf rust,

stripe rust and powdery mildew. Kulturpflanze 33:

133–153

Young ND, Tanksley SD (1989) RFLP analysis of the size of

chromosomal segment retained around TM-2 locus of

tomato during backcross breeding. Theor Appl Genet

77:353–359

Zhang P, Friebe B, Lukaszewski AJ, Gill BS (2001) The

centromere structure in robertsonian wheat-rye translo-

cation chromosomes indicates that centric breakage-

fusion can occur at different positions within the primary

constriction. Chromosoma 110:335–344

Genet Resour Crop Evol (2008) 55:849–859 859

123