MICROSATELLITES AND A SINGLE-NUCLEOTIDE POLYMORPHISM DIFFERENTIATE APPARENT AMYLOSE CLASSES IN AN EXTENDED PEDIGREE OF US RICE GERM PLASM

The Waxy gene (Wx) encodes the granule-bound starch synthase responsib le for the synthesis of amylose in rice (Oryza sativa). Recently, a po lymorphic microsatellite sequence closely linked to the Wx gene was re ported. To determine whether polymorphism in this sequence correlates with variation in apparent amylose content, we tested an extended pedi gree of 92 current and historically important long-, medium- and short -grain US rice cultivars representing the efforts of many breeders ove r more than 80 years. Seven Wx microsatellite alleles were identified which together explained 82.9% of the variation in apparent amylose co ntent of the 89 non-glutinous rice cultivars tested. Similar results w ere also obtained with 101 progeny of a cross between low- and interme diate-amylose breeding lines. An additional, unique microsatellite all ele, (CT)(16), was detected in one glutinous cultivar, CI 5309. Howeve r, the other glutinous cultivars, Calmochi 101 and Tatsumi mochi, were in the (CT)(17) class along with three other cultivars that contained 15-16.5% amylose. We sequenced a 200-bp PCR-amplified fragment contai ning the CT microsatellite and the putative 5' splice site of the Wx l eader intron from a subset of 42 cultivars representing all eight micr osatellite alleles. All of the cultivars with 18% or less amylose had the sequence AG (T) under bar TATA at the putative leader intron 5' sp lice site, while all cultivars with a higher proportion of amylose had AG (G) under bar TATA. This single nucleotide substitution could also be assayed by AccI digestion of the amplified fragment. Overall, this single nucleotide polymorphism could explain 79.7% of the variation i n the apparent amylose content of the 89 non-glutinous cultivars teste d. Interestingly, cultivars in the (CT)(19) microsatellite classes tha t differed substantially in amylose content still showed the correlati on between this G-T polymorphism and apparent amylose content. The G-T polymorphism at this site was not, however, able to explain the very low amylose contents of the three glutinous cultivars tested, all of w hich had the sequence AG (T) under bar TATA.