Use of degenerate primers in rapid generation of microsatellite markers in Panicum maximum

摘要

Guineagrass (Panicum maximum Jacq.) is an important forage grass oltropical andsemi-tropical regions, largely apomictic and predominantly exist in tetraploid form. For molecular breeding work, it is prerequisite to develop and design molecular markers for characterization of genotypes, development of linkage map and marker assisted selection. Hence, it is an important researchable issue to develop molecular markers in those crops where such information is scanty. Among many molecular markers, microsatellites or simple sequence repeat (SSR) markers are preferred markers in plant breeding. Degenerate primers bearing simple sequence repeat as anchor motifs can be utilized in rapid development of SSR markers; however selection of suitable degenerate primers is a prerequisite for such procedure so that SSR enriched genomic library can be made rapidly. In the present study seven degenerated primers namely KKVRVRV(AG)_(10) KKVRVRV(GGT)_5 KKVRVRV(CT)_(10), KKVRVRV(AAT)_6, KKVRVRV(GTG)_6, KKVRVRV(GACA)_5 and KKVRVRV(CAA)_6 were used in amplification of Panicum maximum genomicDNA. Primers with repeat motifs (GGT)_5and(AA)_s have not reacted whereas (A G)_(10), (GACA)_5 and (CAA)_5 highly informative as they have generated many DNA fragments ranging from 250 to 1600 bps as revealed from the results obtained with restriction digestion of recombinant plasmids. Primer with (CT)_(10) anchor repeat, amplified fragments of high molecular weight where as (GTG)_6 primer generated only six bands with low concentration indicating less suitability of these primer in SSR markers development in P. maximum.