SSCP-SNP in pearl millet—a new marker system for comparative genetics

Abstract

A considerable array of genomic resources are in place in pearl millet, and marker-aided selection is already in use in the public breeding programme at ICRISAT. This paper describes experiments to extend these publicly available resources to a single nucleotide polymorphism (SNP)-based marker system. A new marker system, single-strand conformational polymorphism (SSCP)-SNP, was developed using annotated rice genomic sequences to initially predict the intron-exon borders in millet expressed sequence tags (ESTs) and then to design primers that would amplify across the introns. An adequate supply of millet ESTs was available for us to identify 299 homologues of single-copy rice genes in which the intron positions could be precisely predicted. PCR primers were then designed to amplify approximately 500-bp genomic fragments containing introns. Analysis of these fragments on SSCP gels revealed considerable polymorphism. A detailed DNA sequence analysis of variation at four of the SSCP-SNP loci over a panel of eight inbred genotypes showed complex patterns of variation, with about one SNP or indel (insertion-deletion) every 59 bp in the introns, but considerably fewer in the exons. About two-thirds of the variation was derived from SNPs and one-third from indels. Most haplotypes were detected by SSCP. As a marker system, SSCP-SNP has lower development costs than simple sequence repeats (SSRs), because much of the work is in silico, and similar deployment costs and through-put potential. The rates of polymorphism were lower but useable, with a mean PIC of 0.49 relative to 0.72 for SSRs in our eight inbred genotype panel screen. The major advantage of the system is in comparative applications. Syntenic information can be used to target SSCP-SNP markers to specific chromosomal regions or, conversely, SSCP-SNP markers can be used to unravel detailed syntenic relationships in specific parts of the genome. Finally, a preliminary analysis showed that the millet SSCP-SNP primers amplified in other cereals with a success rate of about 50%. There is also considerable potential to promote SSCP-SNP to a COS (conserved orthologous set) marker system for application across species by more specifically designing primers to precisely match the model genome sequence.