«Pak. J. Bot., 41(4): 1627-1635, 2009. DEVELOPMENT OF GENETIC LINKAGE MAP OF LEAF HAIRINESS IN GOSSYPIUM HIRSUTUM (COTTON) USING MOLECULAR MARKERS ...»
RAPDs are usually dominant markers and are sensitive to minor changes in reaction conditions. In the present studies, the DNA markers (OPD-19640, OPF-11630, OPG-06980, OPG-17500, OPH-131100, OPN-14890, OPO-11920, OPO-141200[cis] and OPO-141220&1180[trans] were amplified with the random primer OPD-19, OPF-11, OPG-06, OPG-17, OPH-13, OPO-11, OPO-14 and OPN-14. The nucleotide sequences of the primers have a high proportion (60 % to 70 %) of C+G bases and no palindromic sequences. This might be an advantage for its practical use. Such a sequence of primers is robust and reproducible in PCR analysis (Eujayl et al., 1999).
In the present studies, polymorphic markers viz., OPD-19640, OPG-06980, OPG-17500, and OPO-11920 were at a distance of 1.8 cM, while OPN-14 was at a distance of 6.2 cM from the locus. Although the RAPD markers do not normally allow homozygous and heterozygous individuals to be discriminated, there are several strategies to improve the selection efficiency. Thus the use of markers linked in repulsion phase to the gene would allow the discrimination of homozygous and heterozygous individuals. Consequently, the individuals lacking the marker have high probability of being homozygous for the trait of hairiness, although recombination between the marker and the gene cannot be ruled out (Johson et al., 1995). The strategy pursued in our work was to use both coupling and repulsion linked markers. This allows their use as a codominant marker (Johnson et al., 1995; Avila et al., 2003). RFLP map affecting density of leaf and stem trichomes, showed a QTL on chromosome 6 imparts dense leaf pubescence is inferred to the t1 locus. A second QTL on chromosome 25, which is homoeologous to chromosome 6, fits the description of the t2 locus (Wright et al., 1999).
SSRs have been widely employed in genetic diversity analyses of cotton (Lacape et al., 2007; Liu et al., 2006; Rungis et al., 2005; Zhang et al., 2005) and several genetic linkage maps based mostly on SSRs have now been developed (Han et al., 2004,2006;
Song et al., 2005; Guo et al., 2007). Moreover, SSR markers are preferred over RAPDs because of high reproducibility (Jones et al., 1997). In our study a total of 54 SSR primer pairs were used, out of which one SSR marker JESPR-153 was found to be polymorphic and linked with the trait. The heterozygosity values for microsatellite markers are higher than isozymes (Trujillo et al., 1995; Belaj, 1998), RFLP (Wu & Tanksley, 1993) and RAPD markers (Dayanandan et al., 1998).
In the present studies, the allelic variants amplified at SSR locus JESPR-153 were detected on agarose gel electrophoresis. It becomes more convenient when polymorphisms can be detected on agarose gels since radioactive labeling (Wu & Tanksley, 1993; Rehman et al., 2002) or silver staining is not required. In addition, analysis of microsatellite polymorphism is based on PCR reaction (Szewc-McFadden et al., 1996; Davierwala et al., 2000), which requires only tens of nanograms of DNA or even crude DNA extracts.
GENETIC LINKAGE MAP OF LEAF HAIRINESS IN COTTON 1633The usefulness of RAPD marker linked to the gene depends upon the distance and orientation of the markers with respect to the gene and the type of population analysis. In our work no recombinant was detected with RAPD markers (OPC-08700, OPF-11630, OPH-131100, and OPO-141200), which shows high selection efficiency of these markers for hairiness genotypes. However, in most cases some degree of recombination is observed.
The recombinants were detected in RAPD markers (OPD-19640, OPG-06980, OPG-17500, and OPO-11920), which were located 1.8 cM from the locus while the recombination frequency of the OPN-14890 place this marker at the distance of 6.2 cM. SSR marker JESPR-154 that showed linkage at a distance of 15.7 cM from the H2 gene.
The markers for H2 gene may be useful not only for selection for the hairiness trait itself but also for selection regarding the H2 gene. These markers covers17.5 cM of the specific region of the cotton genome and are linked in coupling phase. The markers linked in the coupling phase markers (OPF-11630, OPH-131100, OPD-19640, OPG-06980, OPG-17500, and OPO-11920) are located within 1.8 cM on the either side of the locus and are closely linked from the H2 locus. Since seven of these markers are linked in a distance less than 5 cM to the target gene, all these markers can be used for effective selection.
Thus the DNA marker was not detected within the genetic window of 10 cM or above.
However, theoretically, the BSA could identify the DNA marker within 30 cM genetic window (Michelmore et al., 1991).
The efficiency of MAS can be increased by employing markers flanking the H2 locus e.g., OPD-19 and OPN-14 even though is linked at 4.4 cM to the H2 gene. Markers linked to hairiness allele increase the selection efficiency, even at greater recombination frequencies between the marker and hairiness locus. The increased efficiency results from the ability to select against heterozygous genotypes, leading to the increased portion of selected homozygous genotypes. In addition to close linkage, the predictability of the molecular markers for MAS is determined by its applications in genetic background.
Therefore, the most closely linked RAPD markers increase the selection efficiency, even at greater recombination frequency between marker and the gene. There is therefore need to develop additional markers and integrate linkage maps using RAPD, RFLP and SSR analysis for highly effective comparative genomic studies for the traits of interest.
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