Associating diversity in resistant starch content of selected indica rice (Oryza sativa L.) accessions with SNP variations of genes related to starch metabolic pathway

Resistant starch (RS) as a grain component characteristic, is gaining recognition because food containing higher RS are found to have lower glycemic index, which in turn provide health benefits to consumers apart from energy sources for the body. In this study, 310 indica rice accessions were selected from the 3000 rice genome sequencing project (3K RGP) based on diversity in grain quality phenotypes, similarity in flowering time and availability of high-quality reads genome sequence. This diversity panel was assayed for RS, digestible carbohydrates (DC) contents and other grain characteristics, which resulted to five (5) population clustering of the sampled rice accessions. RS grain content of the samples ranged from 0.169%-3.366%, while DC grain content ranged from 69.94%-82.59%. RS and amylose content (AC) were positively correlated at 0.44, while DC and protein content (PC) were negatively correlated at -0.51. For targeted genome association (targeted GWAS), 33 lines with contrasting grain characteristics were selected. This sub-set diversity panel represents each of the five population clusters classified based in all phenotyped grain characteristics. Genes involved in starch biosynthesis and degradation, and in synthesis and regulation of non-starch polysaccharides (NSPs) as previously reported were used in the targeted associations. Using Efficient Mixed-Model Association eXpedited (EMMAX) to do the targeted genome associations, 66 SNPs associated to rice grain RS content (p0.01) were identified. The Waxy locus at chromosome 6, which encodes for granule bound starch synthase, had 28 accumulated SNPs. Non-synonymous SNVs associated to grain RS were detected each in exon 2 of LOC_Os08g36910, a gene locus responsible for encoding alpha-amylase isozyme, and exon 2 of LOC_Os10g20260, a gene locus encoding a cellulose synthase family protein. At p0.01 significance level, 75 SNPs could be associated to rice grain DC content. Most SNPs (52) associated to grain DC are in LOC_Os05g33570, the gene locus that encodes pyruvate phosphate dikinase. The identification of SNPs at these loci and their effects on rice grain RS content has to be further validated in gene expression assays. For rice accessions with very contrasting grain quality phenotypes but no significant SNPs detected, further genome-wide association (GWAS) should be conducted with other genes outside the starch biosynthetic pathway. Markers for marker--assisted breeding shall be developed for validated SNPs especially those in gene clusters and with cumulative positive effect rice grain RS.