American Journal of Plant Sciences, 2011, 2, 683-687
doi:10.4236/ajps.2011.25082 Published Online November 2011 (http://www.SciRP.org/journal/ajps)
683
Identification of AFLP Markers Linked to Leaf
Rust Resistance Genes Using Near Isogenic Lines
of Wheat
Navjot Kaur Dhillon1*, Harcharan Singh Dhaliwal2
1Department of Plant Breeding and Genetics, Punjab Agricultural University, Ludhiana, India; 2 Akal School of Biotechnology, Baru
Sahib, Himachal Pradesh, India.
Received August 20th, 2011; revised October 12th, 2011; accepted October 30th, 2011.
ABSTRACT
The present investigation was undertaken to find mo lecular markers linked to leaf ru st resistance genes , Lr9 and Khar-
chia local mu tant KLM4-3B. Preliminary AFLP analysis was carried out with different stocks, a survey of primer com-
binations with different selective nucleotide indicated that for each primer combination, the number of scorable loci
ranged from 34 to 123. Only a limited primer combination used in the set of parental and near isogen ic lines showed a
high level of polymorphism for AFLP marker. Putative AFLP marker were found to be linked to Lr9, Lr19 and KLM4-
3B. The alien genes were readily identified.
Keywords: AFLP, Leaf Rust, Wheat, Lr9, Isogenic Lines
1. Introduction
Wheat exceeds every other grain crop in acreage and
production and is, therefore, the most important cereal of
the world. With the introduction of semi-dwarf, photoin-
sensitive, fertilizer responsive and the high yielding va-
rieties of wheat, the wheat production in India has in-
creased from 12 million tonnes in 1966 to 85 million
tonnes in the recent years. It is imperative to stabilize the
wheat production by reducing the losses due to various
diseases including leaf rust, stem rust, yellow rust, Kar-
nal 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 [1-8]. Breeding for resistance against leaf rust is
an economical, efficient and environmentally safe con-
trol measure to reduce these losses [9]. 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, and thereby makes the resistance gene
ineffective and the variety susceptible to rust. One of the
ways to develop varieties with durable rust resistance is
to pyramid the genes for resistance in a single variety
[10]. It is difficult to pyramid two or more disease resis-
tance genes through conventional means, particularly where
the resistance genes in question are effective against all
the prevalent pathotypes. However, recent advances in
molecular biology has made it possible to pyramid seve-
ral genes in single line using marker assisted selection
(MAS) and tagging of genes with molecular markers is
pre-requisite for MAS [11].
A number of rust resistance genes, including leaf rust,
have been transferred from wild relatives of wheat into
cultivated wheats [12,13]. In India, from the analyses of
2630 samples collected from 17 states, one union terri-
tory and Nepal from 2005 to 2008, 31 races were identi-
fied among which eight were new [14]. Most of which
could not be exploited because of extensive linkage drag.
One of the leaf rust resistance genes, Lr9 transferred
from Aegilops umbellulata [15] located on chromosome
6BL, has no undesirable effect associated with it [16].
This gene is effective against all the races of leaf rust cu-
rrently prevalent in northern India. Similarly, another
leaf rust resistance gene identified in (Kharchia local mu-
tant 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, Lr19 and

Identification of AFLP Markers Linked to Leaf Rust Resistance Genes Using Near Isogenic Lines of Wheat 684
KLM4-3B as these genes provide resistance against most
of the leaf rust pathotypes of the Indian subcontinent.
2. Materials and Methods
2.1. Plant Material
Near-isogenic lines carrying the leaf rust resistance genes
Lr9, Lr19 and the leaf rust resistant gene of KLM4-3B in
the background of WL711 developed at the School of
Biotechnology were used along with the donor and the
recurrent parents for identifying AFLP 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 [17].
2.3. Amplified Fragment Length Polymorphism
(AFLP) Analysis
AFLP analysis was carried out according to procedures
of Vos et al. (1995) [18] with minor modifications. The
genomic DNA was restricted with two enzymes, a 6-base
(rare) cutter Pstl and a 4-base (frequent) cutter Mse1 at
37˚C. The Pst1 and Mse1adapters were ligated to the
fragment ends; amplifying a subset of Msel-Pstl frag-
ments with primers that match the adapter and contain
additional selective nucleotide at the 3' end; and separat-
ing the fragments on denaturing polyacrylamide gel (6%).
Sequence of adapters and the primers used for AFLP
analysis are given in Table 1. To achieve selective am-
plification of a subset of these fragments, 10 cycles of
PCR amplification under following parameters. Thirty
seconds denaturation at 94˚C, thirty seconds primer an
nealing at 65˚C and decreasing one degree temperature
in every subsequent cycles and one minute primer exten-
tion at 72˚C.
2.4. Separation of Amplified Fragments on
Denaturing Polyacrylamide Gel
An equal vo lume of formamide load ing buffer (96% for-
mamide, 10 mM EDTA pH 8.0, % 0.1 fuchsin) was
added to the samples and denatured at 94˚C at 1.5 min. A
25 cm, 8% denaturing polyacrylamide gel (Long Ranger)
was prepared and preheated for 20 min. 1.0 _L of each
samples was loaded on to the gel and electrophoresis was
conducted in 1 x Long Run TBE buffer at 1.500 V, 40 W,
40 mA and 50˚C using a Li-Cor DNA Gene Readir 4200
(MWG Biotech. Ebersberg/Germany).
3. Results and Discussion
AFLP Analysis
Preliminary AFLP analysis was carried out with different
stocks including recurrent parent WL711, Thatcher + Lr9
and LrKLM4-3B and isogenic lines i.e. WL 711 + Lr9
and WL 711 + Lr KLM4-3B. KLM4-3B, along with these
stocks, analysis of HD 2329, Agatha (Lrl9) and isogenic
line WL 711. A survey of primer combinations with dif-
ferent selective nucleotide indicated that for each primer
combination, the number of scorable loci ranged from 34
to 123 (Table 2). Total number of marker loci scored
with differen t primer combination were 682 for WL 711,
629 for HD 2329, 611 fo r Thatcher+Lr9, 515 for Agatha
(Lr19), 582 for Lr KLM4-3B, 629 for WL 711 + Lr9,
516 for WL 711 + Lr19 and 599 for WL 711 + Lr
KLM4-3B (Table 2). So, minimum number of marker
loci (515) amplified were from Agatha (Lr19) stock and
Table 1. Sequence of the adapters and primers used for pre-amplification and selective amplification.
Purpose Oligonucleotide sequences
Adapters
Pstl-Adapter-Sequence 5' -CTCGTAGACTGCGTACATGCA-3'
3' -CATCTGACGCATGT-5'
Msel-Adapter-Sequence 5' -GACGATGAGTCCTGAG- 3'
3' -TACTCAGGACTCAT-5'
Primers for preamplification
Pstl-primer 5' -GACTGCGTACATGCAGA-3'
Msel-primer 5' -GATGAGTCCTGAGTAAC-3'
Primers for selective amplification
Pstl-primer + ACT 5' -GACTGCGTACATGCAGACT- 3'
Pstl-primer + ACC 5' -GACTGCGTACATGCAGACC-3'
Msel-primer + CAA 5' -GATGAGTCCTGAGTAACAA-3'
Msel-primer + CTA 5' -GATGAGTCCTGAGTAACTA-3'
Msel primer + CTG 5' -GATGAGTCCTGAGTAACTG-3'
Msel primer + CTT 5' -GATGAGTCCTGAGTAACTT-3'

Identification of AFLP Markers Linked to Leaf Rust Resistance Genes Using Near Isogenic Lines of Wheat685
Table 2. Number of AFLP loci scored in different stocks using different primer combinations.
Primer combination T. aestivum
WL 711 T. aestivum HD 2329 Thatcher + Lr9 Agatha (Lr19) KML 4-3BWL711 + Lr9 WL711 + Lr19 WL711 + LrKLM4-3B
Pst1 + ACC/Mse1 + CTB 110 98 107 70 104 111 78 110
Pst1 + ACC/Mse1 + CTA 119 123 98 98 121 100 103 108
Pst1 + ACC/Mse1 + CAA 98 51 96 34 49 97 40 50
Pst1 + ACC/Mse1 + CTG 67 67 43 59 52 49 49 63
Pst1 + ACC/Mse1 + CTA 79 82 75 94 71 79 89 77
Pst1 + ACC/Mse1 + CTT 108 110 92 84 91 94 82 85
Pst1 + ACC/Mse1 + CTA 101 98 100 76 94 99 75 96
Total number of loci 682 629 611 515 582 629 616 599
maximum (682) in case of WL 711.
Number of conserved sequence markers were scored
separately for different primer combination and it was
found that primer Pstl + ACC/Mse1 +CTG amplified 35
sequences common to all the used, whereas Pstl + ACC/
Mse1 + CTG amplified 56, Pstl + ACT/Mse1 + CAA
amplified 24, Pstl + ACT/Mse1 + CTG amplified 26 an d
Pstl + ACTI Msel + CTA amplified 32 common se-
quences. Out of 110 scorable markers (Figure 1) ampli-
fied by Pstl + ACC/Mse1 + CTG, only one marker (Fig-
ure 1(a)) was found to be specific in LrKLM4-3B and
WL 711 + LrKLM4-3B and were not amplified in any of
the other stocks. Further, it was seen that primer combi-
nation Pstl + ACT/Msel + CTT amplified three markers
(Figures 2(b)-(d)) which were specifically amplified in
Agatha (Lr19) and in isogenic line, WL 711 + Lr19 and
were not amplified in any of the other stocks (Figure
2).Only a limited primer combination used in the set of
parental and near isogenic lines showed a high level of
polymorphism for AFLP marker as compared to RAPD
[19]. Putative AFLP marker linked to Lr9 and Lr19, the
alien genes were readily identified. These primer combi-
nations need to be tried on the relevant F2 population or
RILs for estimation of extent of association before de-
velopment of STS pr i mers for MAS.
This technique was utilized to clone and map variety
specific rice genomic DNA sequence [20]. Many other
workers has used this technique in the past for detecting
polymorphism, DNA fingerprinting, molecular typing
[21,22], genome mapping [19,23], gene tagging [24], ge-
netic diversity analysis [25] and gene expression analysis
[26]. AFLP technique for classification of rice germ-
plasm by fingerprinting cytoplasmic male sterile lines of
rice was performed and found that the banding pattern of
AFLP markers were remarkably consistent [27]. The
duplicated CMS lines shared every AFLP band and were
thus confirmed as identical genotypes. Thus AFLP ana-
lysis conducted in the present study were found to be
useful tools in identificatio n of putatively linked markers
Figure 1. AFLP markers amplified by primer combination
Pst1 + ACC/Mse1 + CTG. Lanes 1-8: WL711, HD2329,
Thatcher +Lr9, LrKLM4-3B, Agatha (Lr19), WL711 + Lr9,
WL711 + LrKLM4-3B, WL711 + Lr19.

Identification of AFLP Markers Linked to Leaf Rust Resistance Genes Using Near Isogenic Lines of Wheat 686
Figure 2. AFLP markers amplified by primer combination
Pst1 + ACT/Mse1 + CTT. Lanes 1-8: WL711, HD2329,
Thatcher +Lr9, Agatha (Lr19), LrKLM4-3B, WL711 + Lr9,
WL711 + Lr19, WL711 + LrKLM4-3B.
to different leaf rust resistant genes. This high reproduce-
bility, rapid generation and high frequ ency of id entifiab le
AFLP polymorphic bands makes AFLP analysis an at-
tractive approach for molecular analysis in different or-
ganisms.
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