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.
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).
REFERENCES
[1] M. G. Eversmeyer and L. E. Browder, “Effect of Leaf and
Stem Rust on 1973 Kansaswheat Yields,” Plant Disease
Reporter, Vol. 58, No. 5, 1974, pp. 469-471.
[2] R. G. Saini and A K Gupta, “Genes for Resistance to
Brown Rust Puccinia recondita) in Wheat. II. Lr Genes in
Frontana, WG138 and E6360,” Cereal Research Commu-
nications, Vol. 7, 1979, pp. 289-291.
[3] D. Anand, R. G. Saini and A. K. Gupta, “Slow Leaf Rust
Development Due to Combination of Some Genes in
Wheat,” Plant Disease Reporter, Vol. 3, 1988, p. 97. Figure 1. Genomic DNA amplification with primer OPA-19.
Lane1: WL711; lane2: Lr9; lane3: KLM4-3B; lane4: WL711
+ KLM4-3B; lane5: WL711 + Lr9. [4] M. Seck, A. P. Roelfs and P. S. Teng, “Effect of Leaf
Rust Puccinia recondite triticii on Yield of Four Isogenic
Wheat Lines,” Crop Production, Vol. 7, No. 1, 1988, pp.
39-43. doi:10.1016/0261-2194(88)90036-1
[5] K. V. Subha Rao, J. P. Snow and G. T. Berggren, “Effect
or Growth Stage and Initial Inoculum Level on Leaf Rust
Development and Yield Loss Caused by Puccinia recon-
dita f. sp. tritici,” Journal of Phytopathology, Vol. 127,
No. 3, 1989, pp. 200-210.
doi:10.1111/j.1439-0434.1989.tb01130.x
[6] M. Baum, E. S. Laguadah and R. Appels, “Wide Crosses
in Cereals,” Annual Review of Plant Physiology and Plant
Molecular Bi ology, Vol. 43, 1992, pp. 117-143.
doi:10.1146/annurev.pp.43.060192.001001
[7] E. R. Sears, “The Transfer of Leaf Rust Resistance from
Aegilops umbellulata into Wheat,” Brookhaven Symposia
in Biology, Vol. 9, 1956, pp. 1-21.
[8] A. S. Soliman, E. Y. Heyne and C. O. Johnston, “Resis-
tance to Leaf Rust in Wheat Derived from Chinese Ae-
gilops umbellulata Translocation Lines,” Crop Science,
Vol. 3, No. 3, 1963, pp. 254-256.
doi:10.2135/cropsci1963.0011183X000300030025x
Figure 2. Genomic DNA amplification with primer OPD-12.
Lane1: WL711; lane2: Lr9; lane3: KLM4-3B; lane4: Wl711
+ KLM4-3B; lane5: WL711 + Lr9. [9] S. L. Dellaporta, J. Wood and J. B. Hicks, “A Plant DNA

Identification of Molecular Markers Linked to Leaf Rust Resistance Genes in Wheat and 437
Their Detection in the Local Near-Isogenic Line
Mini Preparation. Version 11,” Plant Molecular Biology
Reporter, Vol. 1, No. 4, 1983, pp. 19-21.
doi:10.1007/BF02712670
[10] J. G. K. Williams, A. R. Kubelik, K. J. Livak, J. A. Ra-
fulski and S. V. Tingey,“ DNA Polymorphisms Ampli-
fied by Arbitrary Primers Are Useful Genetic Marker,”
Nucleic Acids Research, Vol. 18, No. 22, 1990, pp. 6531-
6535. doi:10.1093/nar/18.22.6531
[11] G. B. Martin, J. G. K. Williams and S. D. Tanksley,
“Rapid Identification of Markers Linked to a Pseudomo-
nas Resistance Gene in Tomato by Using Random Prim-
ers and Near Isogenic Lines,” Proceedings of the Na-
tional Academy of Sciences of the USA, Vol. 88, No. 6,
1991, pp. 2236-2340. doi:10.1073/pnas.88.6.2336
[12] L. R. M. Klein, A. Vermunt, R. Weide, T. Liharska and R.
Zabel, “Isolation of Molecular Markers for Tomato (L.
esculentum) Using Random Amplified Polymorphic DNA
(RAPD),” Theoretische und Angewandte Genetik, Vol. 83,
No. 1, 1991, pp. 108-114.
[13] P. C. Ronald, B. Albano, R. Tabien, L. Abenes, K. S. Wu,
S. McCouch and S. D. Tanskley, “Genetic and Physical
Analysis of the Rice Bacterial Blight Disease Resistance
Locus, Xa21,” Molecular & General Genetics, Vol. 236,
No. 1, 1992, pp. 113-120.
[14] S. Yoshimura, A. Yoshimura, N. Iwata, S. R. McCouch,
M. L. Abenes, M. R. Baraoiden, T. W. Mew and K. J.
Nelson, “Tagging and Combining Bacterial Blight Resis-
tance Genes in Rice Using RAPD and RFLP Markers,”
Molecular Breeding, Vol. 1, No. 4, 1995, pp. 375-387.
[15] S. Yoshimura, A. Yoshimura, R. J. Nelson, T. W. Mew
and N. Iwata, “Tagging Xa-l the Bacterial Blight Resis-
tant Gene in Rice, by Using RAPD Markers,” Breed Sci-
ence, Vol. 45, 1995, pp. 81-85.
[16] I. Dweikat, H. Ohm, S. MacKenzie, F. Patterson, S. Cam-
bron and R. Rateliffe, “Association of a DNA Marker
with Hessian Fly Resistance Gene H9 in Wheat,” Theo-
retical and Applied Genetics, Vol. 89, No. 7-8, 1994, pp.
964-968. doi:10.1007/BF00224525
[17] J. D. Procunier, T. F. Townley Smith, S. Fox, S. Prashar,
M. Gray, W. K. Kim, Czarnecki and P. L. Dyck, “PCR-
Based RAPD/DGGE Markers Linked to Leaf Rust Resis-
tance Genes Lr29 and Lr25 in Wheat (T. aestivum),”
Journal of Genetics and Breeding, Vol. 49, No. 1, 1995,
pp. 87-91.
[18] G. Schachermayr, M. Messmer, C. Feuillet, H. Winzeler,
M. Winzeler and B. Keller, “Identification of Molecular
Markers Linked to the Agropyron elongatum-Derived
Leaf Rust Resistance Gene Lr24 in Wheat,” Theoretical
and Applied Genetics, Vol. 90, No. 7-8, 1995, pp. 982-
990. doi:10.1007/BF00222911
[19] L. E. Talbert, P. L. Bruckner, L. Y. Smith, R. Sears and T.
J. Martin, “Development of PCR Markers Linked to Re-
sistance to Wheat Streak Mosaic Virus in Wheat,” Theo-
retical and Applied Genetics, Vol. 93, No. 3, 1996, pp.
463-467. doi:10.1007/BF00223191
[20] S. Chao, J. Sharp, A. J. Worland, E. J. Warham, R. Koeb-
ner and M. D. Gale, “RFLP-Based Genetic Maps of Wheat
Homoeologous Group 7 Chromosomes,” Theoretical and
Applied Genetics, Vol. 78, No. 4, 1998, pp. 495-504.
doi:10.1007/BF00290833
[21] L. N. W. Kam-Morgan, B. S. Gill and S. Muthukrishan,
“DNA Restriction Fragment Length Polymorphisms: A
Strategy for Genetic Mapping of D-Genome of Wheat,”
Genome, Vol. 32, No. 4, 1989, pp. 724-732.
[22] N. A. Dadkhodaie, H. Karaoglou, C. R. Wellings and R.
F. Park, “Mapping Genes Lr53 and Yr35 on the Short
Arm of Chromosome 6B of Common Wheat with Mi-
crosatellite Markers and Studies of Their Association
with Lr36,” Theoretical and Applied Genetics, Vol. 122,
No. 3, 2010, pp. 479-487.
doi:10.1007/s00122-010-1462-y
[23] G. F. Marais, P. E. Badenhorst, A. Eksteen and Z. A.
Pretorius, “Reduction of Aegilops sharonensis Chromatin
Associated with Resistance Genes Lr56 and Yr38 in
Wheat,” Euphytica, Vol. 171, No. 1, 2010, pp. 15-22.
[24] V. Kuraparthy, S. Sood, D. R. See and B. S. Gill, “De-
velopment of a PCR Assay and Marker-Assisted Transfer
of Leaf Rust and Stripe Rust Resistance Genes Lr57 and
Yr40 into Hard Red Winter Wheats,” Crop Science, Vol.
49, No. 1, 2009, pp. 120-126.
doi:10.2135/cropsci2008.03.0143
[25] G. F. Marais, B. Mccallum and A. S. Marais, “Wheat
Leaf Rust Resistance Gene Lr59 Derived from Aegilops
peregrin,” Plant Breeding, Vol. 127, No. 4, 2008, pp.
340-345. doi:10.1111/j.1439-0523.2008.01513.x
[26] F. Marais, A. Marais, B. Mccallum and Z. Pretorius,
“Transfer of Leaf Rust and Stripe Rust Resistance Genes
Lr62 and Yr42 from Aegilops neglecta Req. ex Bertol. to
Common Wheat,” Crop Sc ienc e, Vol. 49, No. 3, 2009, pp.
871-879. doi:10.2135/cropsci2008.06.0317
[27] G. F. Marais, T. A. Bekker, A. Eksteen, B. Mccallum, T.
Fetch and A. S. Marais, “Attempts to Remove Gameto-
cidal Genes Co-Transferred to Common Wheat with Rust
Resistance from Aegilops speltoides,” Euphytica, Vol.
171, No. 1, 2010, pp. 71-85.