Asian Journal of Agricultural Sciences 2(3): 117-119, 2010
ISSN: 2041-3890
© M axwell Scientific Organization, 2010
Submitted Date: December 30, 2009
Accepted Date: January 25, 2010
Published Date: July 05, 2010
Identification of RAPD Primers Specific for Wheat Homoeologous
Group 1 and 3 Chromosomes
1
Ikram Muhamm ad, 1 Habib A hmad, 1 Sajid Ghafoor, 1 Sahib Gul Afridi,
1
Khaist Begum and 2 Imtiaz Ahmad Khan
1
Department of Genetics, Hazara University, Mansehra, Pakistan
2
Department of Genetics, University of Karachi, Pakistan
Abstract: Mapping of homoeologous chromosomes of common wheat (Triticum aestivum L.) has always been
a top priority for w heat breeders. During pre sent w ork, easier, che aper and quicke r RA PD (Ran dom ly
Amplified Polymorphic DNA) primers were mapped on homoeologous group 1 and 3 chromosomes of common
wheat. W heat genetic stocks viz; N ullisom ic-tetrasomic lines were used for mapping of RAPD primers. A total
of 43 RAPD primers were tested, out of which, five primers viz; GLA-10, GLA -11, GLA-14, GLA-20 and
GLB-15 were foun d useful and these p rimers amp lified DNA fragm ents specific for chromosomes 1D, 3D, 1B,
3B and 1D, respectively. These five RAPD primers can reliably be used as markers for the wheat
homoeologous chromosomes 1B, 3B, 1D and 3D.
Key w ords: Homoe olgou s chromosome s, mapping , Nullisomic-tetrasom ic lines, R AP D, Triticun aestivum
INTRODUCTION
Bread/common wheat (Triticum aestivum L.) is one
of the world’s most important cereal crop and is planted
on more than 200 million hectares with an annual
production of approximately 600 million tons
(Anonym ous, 2007). Cy tologically wheat is an
allohe xap loid (2n=6x=42) havin g three rela ted
(Homoe ologous) genom es ‘A’, ‘B’ an d ‘D’ (Sea rs, 1952).
This triplication of genomes enabled late E. R . Sears to
develop gene tically compe nsating nullisomic-tetrsom ic
(NT) lines of wheat cultivars ‘Chinese Spring’
(Sears, 1966). These genetic stocks of wheat have been
widely used for chromosome mapping studies. Most of
the mapping w ork done in past was based on Restriction
Fragment Length Polymorphism (Chao et al., 1989;
Gill et al., 1991). During present study attempts were
made to identify RAPD primers specific for group 1 and
3 homoeologous chromosomes of wheat using nullisomicterrsom ic lines.
MATERIALS AND METHODS
Plant material used during present study comprising
NT-1A1D , -1B1D, -1D1A, -3A3B, -3B3D, -3D3A lines
of wheat cultivar ‘Chinese Spring’, was kindly provided
by Dr John Rau pp, Kansas State University, USA.
Because of unavailability of seeds of euploid Chinese
Spring originally used by Sears, NT-4D4B wa s used as
positive contro l during present study. Plants were grown
in pots at the D epartm ent of G enetics, Hazara U niversity
Mansehra, during rabi (winter) season 2007-2008.
Recommended agricultural practices were used during the
grow th period. Total genomic DNA was isolated from 3-4
weeks old seedlings using small scale DNA isolation
procedu re (Weining and Langridge 1991). PCR reactions
were carried out using standard procedure (Khan
et al., 2000 ). Forty-three RAPD primers obtained from
Gene-Link, Inc, USA were used to amplify genomic DNA
isolated from wheat NT lines. An annealing tem perature
of 34 de gree w as use d for all the reactions. PCR products
were separated on 2.0 % Agrose/TBE gels and visualized
by staining w ith Ethedium bomide using ‘UviTech’ gel
documentation system. All the reactions were repeated
twice using indep endently iso lated D NA samples.
RESULTS AND DISCUSSION
An exam ple of PCR amplification of genomic DNA
isolated from wheat genetic stocks using RAPD primer
GLB-15 is presented in Fig. 1. Five out of 43 p rimers
(GLA-10, GL A-11, GLA-14, GLA-20 and GLB-15)
amplified spec ific DNA fragments for wheat
homoeologous chromosome used during present study
(Table 1). RAPD primer GLA-10, GLA-11, GLA-14,
GLA -20 and GL B-15 was polymo rphic for chromosome
1D, 3D, 1B, 3B and 1D, respectively. Molecular size of
polym orphic bands (estimated using 100 bp DN A ladder,
Gene Link Inc, USA) ranged from 800-1300 bp.
Chromosome mapp ing (genetic as w ell as ph ysical)
is an important prerequisite for understanding of genome
structure and better utilization of usefu l genes in
Corresponding Author: Imtiaz Ahmad Khan, Department of Genetics, University of Karachi, Pakistan
117
Asian J. Agr. Sci., 2(3): 117-119, 2010
Table 1: Identification of RAPD primers specific for homoeologous group 1 and 3 chromo somes of comm on wheat
S iz e r an g e o f D N A
Nu mb er of p olym orph ic Estim ated size of po lymo rphic
S .N o
O l ig o na m e
Seq uenc e (5-3 ’)
fragments amplified
DN A fra gm ents
DNA fragment
1
GLA -10
G T G A T C G CA G
700-1300
1
1200
2
GLA -11
C A A T C G C CG T
500-1300
1
1000
3
GLA -14
T C T G T GC T G G
1200-1400
1
1300
4
GLA -20
GTTGCGATCC
600-1300
1
800
5
GLB-15
GGAGG GTGTT
900-1300
1
100
Chromo some for which sp ecific
DN A fragment w as amplified
1D
3D
1B
3B
1D
Fig. 1: PCR amplification profile of genetic stocks of wheat cultivar Chinese Spring using RAPD primer GLB-15. 1 = NT-1A1D,
2 = NT-1B1D, 3 = NT-1D1A, 4 = NT-3A3B, 5 = NT-3B3D, 6 = NT-3D3A, 7 = NT-4D4B. M = Molecular size marker. Size
of the fragments (in bp) is presented on right. Arrow indicates polymorphic band for chromosome 1D of common wheat
comm ercial agriculture. Earlier, most of the mapping of
wheat chrom osom es w as done using (tho ugh highly
reliable but) relatively more expensive, time consuming
and potentially hazardous technique of Restriction
Fragment Length Polymorphism (R FLP ). W ith the
development of Polymerase Chain Reaction, various
kinds of comparatively easier, cheape r and mo re user’s
friendly PCR based assays have been used for mapping
purposes. Kojima et al. (1998) using ISSR and RAPD
primers showed that the marker orders between the maps
of Einkorn an d com mon wheat coincided in gene ral. PCR
based assays along with RFLPs have also been used for
comparative mapping studies in triticeaeg (Spielmeyer
and Richards 2004). Expressed Sequenc e Tags (ESTs)
derived Simple Sequence Repeat (eSSR ) markers w ere
used for characterization of rye chromosome add ed to
wheat genome (Li-Fang et al., 2008 ).
Use of RAPD during present study had an added
advantage that RAPD s do not need prior knowledge of
sequence information on the target genome and hence can
be useful for potentially any part of the chromosomes
(Williams et al., 1990 ). Identification of approximately
11.6% (5 out of 43) polymorphic RAPD primers during
present work indicate that RAPDs can be used to tag
individual w heat chrom osom es.
Giving due consideration to easy and cheape r nature
of the assay procedure, it is recommended that rather than
using technically difficult and more expensive procedu re
of RFLPs, RAPDs shou ld be used for breeding programs
especially in under developed countries (like Pakistan)
whe re because of financial constraints and scarcity of
infrastructure, other mapping techniques can not be used
routinely.
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