Development of a Kit of Single Nucleotide Polymorphism Markers

A set of 180 SNPs were selected in rice chromosomes 1, 2, 3, 5, 6 and 7 and six PCR-multiplex experiments were designed. PCR-multiplex reactions were carried out in two sets of 30 markers, one for each chromosome. Single base extension reactions were carried out in multiplex of 13 to 19 SBE primers. No fluorescent signal was observed for 47 SNPs, eleven were monomorphic and seven were classified as no-specific since one allele could not be recognized alone and was observed always together with the alternate allele as a heterozygote. The remaining 41 SNPs (68%) were scored with high fluorescent signals and were also polymorphic, although two of them did not reproduced the polymorphism between Nipponbare and 93-11. The remaining 114 SNPs (64%) were scored with high fluorescent signals and were also polymorphic, although seven of them did not reproduced the polymorphism between Nipponbare and 93-11. For four markers, alleles could be scored only in O. sativa and O. rufipogon but not in the other species. Several SNPs were found to be polymorphic between the parents of wild x cultivated CSSLs populations (Table 1) and the definition of sets of markers to screen for each population has initiated. We plan to start SNP genotyping of the 312 BC3DH lines from the Caiapo x O. glaberrima population soon.