Single-Strand Conformational Polymorphism of EST-SSRs: A Potential Tool for Diversity Analysis and Varietal Identification in Sugarcane

Abstract

Sugarcane (Saccharum spp.) has a large complex polyploid genome. Assay of molecular variation in the expressed component of its genome has relevance to the analysis of genetic diversity, variety identification and introgression of agronomically useful genes present in different members of the Saccharum complex. The present study was designed to evaluate single-strand conformational polymorphism (SSCP) as a potential tool to detect genetic variation in the expressed sequence tag (EST) derived microsatellites. Twenty primer pairs obtained from EST libraries and one designed from soluble acid invertase gene sequence were used to characterise 21 clones belonging to four different Saccharum species and 22 sugarcane varieties/genotypes. All the markers, including the two, which were reported monomorphic even at the interspecific level in an automated fragment analysis system in a previous study, could be successfully converted into polymorphic ones using SSCP analysis. A broad range of variation could be revealed by this technique. The Saccharum spp. clones could be grouped into distinct clusters, confirming the species relationships postulated earlier using morphological, biochemical and molecular methods. The polymorphic markers could also differentiate all the 22 sugarcane varieties from each other. This is a first report that demonstrates the usefulness of SSCP technique, in obtaining polymorphic microsatellite markers developed from EST sequences for various genetic and breeding applications, in this polyploid species.

Single strand conformation polymorphism of genomic and EST-SSRs marker and its utility in genetic evaluation of sugarcane

“…The higher rate of polymorphism may due to the presence of saccahrum hybrid cultivars developed as inter specific and inter generic hybrids. The Indian sugarcane clone and the genotypes with the 'Co' origin have shown the significant distribution at 50-80 % GS through EST-SSR SSCP technique (Swapna et al 2011) which shows more similar results with the obtained 55-81 % GS in present study. This could reinforce the utility of SSR-SSCP approach for genetic evaluation of more number of tropical and subtropical sugarcane cultivars of Indian origin.…”

“…Optimized 28 sets of microsatellite primers amplified 22 sugarcane genotypes that provided significant information about the elite sugarcane cultivars in terms of the number of alleles, PIC and Rp values. The allelic range obtained in several studies of sugarcane had a considerable similarity with the present alleles found (Swapna et al 2011;Devarumath et al 2012). The Pearson correlation coefficient between the PIC and Rp values considerably showed the reproducibility of selected marker in SSCP procedure.…”

“…By this procedure, sequence characterized amplified region (SCAR) marker can be develop for the specific trait of sugarcane. In account of the sugarcane, there has been less report on the SSCP procedures used for the genetic evaluation of sugarcane DNA by Srivastava et al (2005Srivastava et al ( , 2011 and Swapna et al (2011). It also overcomes the problem of agarose based genetic analysis which shows limited polymorphism.…”

“…SSCP analysis SSCP protocol used as described by Swapna et al (2011) was performed in either 6 % or 8 % denaturating polyacrylamide gel (29:1) depending on the molecular weight of the amplified fragments. SSCP technique was used to separate the different conformers of amplified product of primer pairs.…”

“…This technique was originally developed for rapid analysis of mutations (Gasser et al 2006;Orita et al 1989;Spinardi et al 1991) and has been used extensively in human genetics to detect singlepoint mutations (Hayashi 1992). The application of SSCP to SSR amplified products helps to detect conformational changes in single stranded DNA molecules and hence enhances polymorphism of microsatellite repeats to facilitate molecular genetic diversity analysis (Srivastava et al 2005(Srivastava et al , 2011Swapna et al 2011;Swapna and Srivastava 2012).…”