Rice Genetics IV - IRRI books - International Rice Research Institute

Relationship between SSRs and genesTo further characterize the spatial relationship between class I microsatellites andgenes, the distribution of SSRs was investigated in relation to open reading frames(ORFs), untranslated 3’and 5’ regions (UTRs), introns, and intergenic regions. Fromthis analysis, we observed that about 80% of GC-rich tri-nucleotide repeats occurredin predicted exons, whereas AT-rich tri-nucleotide SSRs were distributed in all fourgenomic domains in this dataset. As reported by Temnykh et al (2001), poly di-nucleotideand poly tetra-nucleotide SSRs were predominantly situated in noncoding regions—preferentiallyin intergenic regions, and only more rarely in introns.At a higher level of resolution, 27 SSRs found in expressed sequences were examinedto determine their precise position in cloned and completely sequenced genes.Cho et al (2000) reported that 13 of the SSRs were positioned in UTRs, 6 were foundin introns, and 8 were found in exons or ORFs. The eight markers in ORFs were allGC-rich tri-nucleotide repeats and showed very low levels of polymorphism. On theother hand, microsatellite sequences located in introns or in 5’ and 3’ UTRs werevariable in motif (mostly poly(GA) or AT-rich di- and tri-nucleotide motifs) and tendedto be much more polymorphic. These observations supported the hypothesis that SSRsfound in ORFs experience functional constraints to their variability and therefore themost polymorphic microsatellite sequences are unlikely to be found in the codingportions of genes.Strategies for successful marker designGiven the interest of our laboratory in developing a high-resolution set of microsatellitemarkers for use in genetics and breeding, we aimed to develop an efficient strategyfor selecting useful SSR markers for this purpose. As outlined in Temnykh et al (2001),such a strategy was based on observations about the frequency, size variation potential,and PCR-amplification properties of different types of rice SSRs. Based on ourexperience, the highest rate of successful amplification was achieved for poly(GA)n,poly(GAA)n, and poly(CAT)n microsatellites in rice, and the first two classes werealso highly polymorphic. Though poly(AT)n blocks were the most abundant and variablemicrosatellite sequences in the rice genome, primers designed from regions flankingthis SSR motif frequently failed to amplify. Yet, primers for the commonly encounteredcompound motif, poly(TA)n(CA)n, performed better. In fact, the complexpoly(TA)n(CA)n microsatellites had the longest runs of uninterrupted repeats anddemonstrated the highest level of allelic diversity in our panel. Other types of motifs,such as poly(CA)n, poly-tetra-nucleotides, and GC-rich poly-tri-nucleotides, amplifiedmoderately well but tended to have fewer alleles and lower polymorphism informationcontent (PIC) values than the other classes.Microsatellite markers in rice: . . . 123