Identification of Molecular Markers Linked to Leaf Rust Resistance Genes in Wheat and Their Detection in the Local Near-Isogenic Line

doi:10.4236/ajps.2011.23049

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American Journal of Plant Sciences, 2011, 2, 433-437

doi:10.4236/ajps.2011.23049 Published Online September 2011 (http://www.SciRP.org/journal/ajps)

433

Identification of Molecular Markers Linked to

Leaf Rust Resistance Genes in Wheat and Their

Detection in the Local Near-Isogenic Line

Navjot Kaur Dhillon1*, Harcharan Singh Dhaliwal2

1Department of Plant Breeding and Genetics, Punjab Agricultural University, Ludhiana, India; 2Akal School of Biotechnology, Baru

Sahib, Himachal Pradesh, India.

Email: *[email protected]

Received May 31st, 2011; revised July 20th, 2011; accepted August 7th, 2011.

ABSTRACT

Sixty-five random amplified polymorphic DNA (RAPD) primers were used for the detection of polymorphism among

recipient and donor parents and their isogenic lines linked to leaf rust resistance genes, Lr9 and the resistant gene in

kharchia local mutant KLM4-3B. Three primers showed polymorphism among recurrent parent, donor parent and

isogenic lines.

Keywords: RAPD, Lr9, Is o genic Lines

1. Introduction

With the introduction of semi-dwarf, photoinsensitive,

fertilizer responsive and the high yielding varieties of

wheat, the wheat production in India has increased from

12 million tonnes in 1966 to 70 million tonnes in the re-

cent years. Wheat exceeds every other grain crop in

acreage and production and is, therefore, the most im-

portant cereal of the world. It is imperative to stabilize

the wheat production by reducing the losses due to vari-

ous diseases including leaf rust, stem rust, yellow rust,

Karnal bunt etc. Among the diseases, leaf rust caused by

Puccinia recondita Roberage ex. Desmaz f.sp. tritici is

one of the most important and devastating foliar diseases

of wheat which cause significant yield losses all over the

world [1-5]. Breeding for resistance against leaf rust is an

economical, efficient and environmentally safe control

measure to reduce these losses. Development of disease

resistant varieties is one of the most economical methods

of control of diseases like leaf rust. However, growing of

rust resistant varieties having single gene for resistance

results in rapid evolution of virulent biotypes of the

pathogen, thereby making the resistance gene ineffective

and the variety susceptible to rust. It is difficult to pyra-

mid two or more disease resistance genes through con-

ventional means, particularly where the resistance genes

in question are effective against all the prevalent patho-

types. However, recent advances in molecular biology

has made it possible to pyramid several genes in single

line using marker assisted selection (MAS) and tagging

of genes with molecular markers is pre-requisite for

MAS.

A number of rust resistance genes, including leaf rust,

have been transferred from wild relatives of wheat into

cultivated wheats [6]. Most of which could not be ex-

ploited because of extensive linkage drag. One of the leaf

rust resistance genes, Lr9 transferred from Aegilops um-

bellulata [7] and located on chromosome 6BL, has no

undesirable effect associated with it [8]. This gene is

effective against all the races of leaf rust currently

prevalent in northern India. Similarly, another leaf rust

resistance gene identified in (Kharchia local mutant―

KLM4-3B) is also effective against all the prevalent leaf

rust pathotypes in northern India.

Keeping this in view the present study was undertaken

to identify molecular markers linked with Lr9 and

KLM4-3B and to pyramid Lr9 with rust resistant gene in

KLM4-3B, as both the genes provide resistance against

most of the leaf rust pathotypes of the Indian subconti-

nent.

2. Materials and Methods

2.1. Plant Material

Near-isogenic lines carrying the leaf rust resistance genes

Lr9 and the Lr gene of KLM4-3B in the background of

Identification of Molecular Markers Linked to Leaf Rust Resistance Genes in Wheat and

434

Their Detection in the Local Near-Isogenic Line

WL711 developed at the School of Biotechnology were

used along with the donor and the recurrent parents for

identifying RAPD markers linked to the two genes.

2.2. Genomic DNA Isolation

Approximately 5 g fresh weight of young leaves were

harvested from plants grown in the field and DNA was

extracted as per the method of Dellaporta [9].

2.3. Random Amplified Polymorphic DNA

(RAPD) Analysis

RAPD analysis was carried out as described by Williams

[10] using 10 base pair (bp) primers from Operon Tech-

nologies Inc., Alameda, California. PCR was performed

in a reaction volume of 25 l, containing 2.5 l of 1 mM

dNTPs, 3.2 l of 15 mM MgC12, 2.5 l of 10X buffer

containing 10 mM tris-HC1 pH 8.3, 2.0 mM MgC12, 50

mM KC1, 1.5 l (20 ng) of single 10-base primer, 1 l

(30 ng) of template DNA and 0.5 l (1 unit) of Taq po-

lymerase (Stratagene). The reaction mixture was topped

with 50 l of sterilized mineral oil. Template DNA was

initially denatured at 94˚C for 5 minutes followed by 45

cycles of PCR amplification under following parameters.

One minute denaturation at 94˚C, one minute primer an-

nealing at 36˚C and 2 minutes primer extension at 72˚C

further followed by 5 minutes final extension at 72˚C on

a Perkin Elmer Cetus Thermal Cycler.

2.4. Gel Electrophoresis and Photography

5 l loading buffer consisting of 0.5% bromophenol blue,

0.5% xylene cynole FF and 50% glycerol in 1XTAE

buffer (0.04M Tris-acetate, 0.00lM EDTA) was added to

the PCR amplification products for visualization of gel

run. Aliquots of 25 l of DNA products from the PCR

amplification were loaded in 1.5% agarose gel prepared

in l × TAE buffer at 3 V/cm. Gel was stained with

ethidium bromide and photographed under UV light.

3. Results and Discussion

In the present investigation, to identify molecular mark-

ers linked with Lr9 and KLM4-3B and to pyramid Lr9

with rust resistant gene in KLM4-3B, RAPD markers

were tried on near-isogenic lines carrying the leaf rust

resistance genes Lr9 and the Lr gene of KLM4-3B in the

background of WL711 along the recipient and donor

parents of the isogenic lines for the two genes in WL711.

The results pertaining to these studies are presented here.

3.1. RAPD Analysis

65 random primers were tried to study polymorphism

among recurrent and donor parents and their isogenic

line (Table 1). Out of 65 primers tested, 42 primers gave

amplification (64.62%). Total number of loci amplified

with 42 primers tested in WL711 , KLM4-3B, isogenic

line of Lr KLM4-3B, Thatcher + Lr9 and isogenic line of

Lr9 were 148, 145, 140, 126 and 126, respectively. Lim-

ited polymorphism was detected among WL 711, That-

cher + Lr9, WL 711 + Lr9, Lr KLM4-3B, WL 711 + Lr

KLM4-3B when genomic DNA was amplified with the

RAPD primers.

Table 1. Number of RAPD loci in recurrent parent, donor parent and isogenic lines.

Number of RAPD loci

Primer Primer

Sequence of the

primer

5' to 3' WL 711KLM 4-3BWL711 + KLM4-3BThatcher + Lr9 WL711 + Lr9

OPA-02 TGCCGAGCTC 3 3 3 3 3

OPA-14 TCTGTGCTGG 2 1 1 1 1

OPA-15 TTCCGAACCC 2 2 2 2 2

OPA-16 AGCCAGCGAA 2 2 2 3 3

OPA-18 AGGTGACCGT 3 3 3 2 2

OPA-19 CAAACGTCGG 2 3 2 1 1

OPB-05 TGCGCCCTTC 5 5 5 5 5

OPB-06 TGCTCTGCCC 3 3 3 3 3

OPB-15 GGAGGGTGTT 6 3 3 3 3

OPB-17 AGGGAACGAG 5 3 3 3 3

OPC-02 GTGAGGCGTC 2 2 2 2 2

OPC-04 CCGCATCTAC - 2 - - -

OPC-16 CACACTCCAG 2 3 5 5 3

OPC-20 ACTTCGCCAC 5 5 5 3 5

OPD-l2 CACCGTATCC 3 1 3 3 4

OPD-l4 CTTCCCCAAG 2 2 2 - -

Identification of Molecular Markers Linked to Leaf Rust Resistance Genes in Wheat and 435

Their Detection in the Local Near-Isogenic Line

Number of RAPD loci

Primer Primer Sequence of the primer

5' to 3' WL 711KLM 4-3BWL711 + KLM4-3BThatcher + Lr9 WL711 + Lr9

OPE-10 CACCAGGTGA 1 - - - -

OPE-20 AACGGTGACC 4 4 2 4 4

OPG-13 CTCTCCGCCA 3 3 3 3 3

OPG-l4 GGATGAGACC 4 4 4 4 4

OPG-l7 ACGACCBACA 2 2 2 2 2

OPJ-01 CCCGGCATAA 6 6 6 6 6

OPJ-05 CTCCATGGGG 1 1 1 - -

OPJ-10 AAGCCCGAGG 7 6 6 7 7

OPJ-12 GTCCCGTGGT 2 2 2 2 2

OPK-10 GTGCAACGTG 10 10 10 10 10

OPK-13 GGTTGTACCC 1 1 1 1 1

OPK-15 CTCCTGCCAA 3 - - - -

OPK-16 GAGCGTCGAA 3 3 3 3 3

OPO-10 TCAGAGCGCC 4 4 4 3 3

OPO-11 GACAGGAGGT 1 1 1 1 1

OPP-01 GTAGCACTCC 6 6 6 6 6

OPP-05 CCCCGGTAAC 4 4 4 - -

OPP-06 GTGGGCTGAC 4 4 4 2 2

OPP-10 TCCCGCCTAC 5 5 5 3 3

OPP-19 GGCTTGGCCT 6 6 6 6 6

OPZ-12 TCAACGGGAC 3 3 3 3 3

OPZ-15 CAGGGCTTTC 1 1 1 1 1

OPZ-17 CCTTCCCACT 5 5 5 5 5

OPBA-06 ATATTTGGCC 4 4 4 3 3

OPBA-12 GCCCTTAGCA 3 3 3 2 2

OPBA-14 GCAATGGGAT 3 3 3 3 3

Total Number of Loci 148 145 140 126 126

Identification of Molecular Markers Linked to Leaf Rust Resistance Genes in Wheat and

Their Detection in the Local Near-Isogenic Line

436

Some genes of agronomic importance, however, have

been tagged with RAPD markers in tomato [11,12], in

rice [13-15], and in wheat [16-19]. The three primers

(OPA-19, OPD-12, OPJ-10) were polymorphic between

WL711 and other stocks. One RAPD locus amplified

with primer OPA-19 was observed in Thatcher + Lr9 and

its isogenic lines. The amplification product was absent

in WL711 (Figure 1). This indicated association of this

RAPD marker with Lr9.

Amplification of a distinct RAPD loci associated with

Lr9 with RAPD, primer OPA-19 and its absence in donor

and isogenic lines containing LrKLM4-3B vs RAPD

primer OPD-12 which amplified a distinct loci associated

with LrKLM4-3B and absent in lines with Lr9 further

indicate that Lr9 and LrKLM4-3B are non-allelic.

Bread wheat has a narrow genetic base. This was also

shown by limited polymorphism for many molecular

markers [20,21]. This limited polymorphism along with

higher ploidy level and high repetitive DNA content has

impeded genetic linkage mapping in wheat. RAPD mar-

kers behave as dominant markers because polymorphism

is detected as the presence/absence of bands. RAPD

markers provide a quick and cost effective method for

generating genetic maps and analyzing population. MAS

could be useful in the development of highly resistant

germplasm based on new combinations of Lr genes are

Lr53 [22], Lr56 [23], Lr57 [24], Lr59 [25], Lr62 [26],

Lr63 and Lr66 [27].

Similarly, with RAPD primer OPD-12, one locus was

specifically amplified in KLM4-3B and the isogenic line

carrying Lr KLM4-3B. The amplification product was

absent in WL711 (Figure 2).

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