Single nucleotide polymorphisms in GBBSI and SSa genes in relation to starch physicochemical properties in selected rice (Oryza sativa L.) varieties

Starch quality is one of the most important agronomic traits in rice (Oryza sativa L). In this study, we identified single nucleotide polymorphisms (SNPs) in the Waxy and Alk genes of eight rice varieties and their associations with starch physicochemical properties.vi.e.vamylose content (AC) and gelatinization temperature (GT). Seven Sri Lankan rice varieties, Pachchaperumal, Herathbanda, At 354, Bg 352, Balasuriya, H 6 and Bw 295-5 were detected as high amylose varieties while Nipponbare exhibited low amylose content. In silico analysis of the Waxy gene revealed that all tested Sri Lankan varieties possessed ‘G’ (Wxa allele) instead of ‘T’ in the first intron which could explain varieties with high and intermediate amylose content. All Sri Lankan varieties had ‘A’ instead of ‘C’ in exon 6 of the Waxy gene and this fact was tally with the varieties showing high amylose content. Therefore, possessing the Wxa allele in the first intron and ‘A’ in exon 6 could be used as a molecular marker for the selection of high amylose varieties as validated using several Sri Lankan varieties. All Sri Lankan varieties except, Bw 295-5 exhibited the intermediate type of GT which could not be explained using the so far reported allelic differences in the Alk gene. However, Bw 295-5 which is a low GT variety had two nucleotide polymorphisms in the last exon of the Alk gene, i.e. ‘G’ and ‘TT’ that represent low GT class. Therefore, it can be concluded that sequence variations of Waxy and Alk genes reported in this study are useful in breeding local rice varieties with preferential amylose content and GT class. Key word: Alk gene, amylose content, single nucleotide polymorphism, Waxy gene NTRODUCTION: Rice (Oryza sativa L.) is one of the leading food crops of the world. More than half of the world’s population relies on rice as the major daily source of calories and protein (Sartaj and Suraweera, 2005). After grain yield, quality is the most important aspect of rice breeding. Grain size and shape largely determine the market acceptability of rice, while cooking quality is influenced by the properties of starch. In rice grains starch is the major component that primarily controls rice quality. Starch consists of two forms of glucose polymers, relatively unbranched amylose and a highly branched amylopectin. Starch-synthesizing genes may contribute to variation in starch physicochemical properties because they affect the amount and structure of amylose and amylopectin in rice grain (Kharabian-Masouleh et al., 2012). Amylose content (AC), gelatinization temperature (GT) and gel consistency (GC) is the three most important determinants of eating and cooking quality. Amylose content is the ratio of amylose amount present in endosperm to total starch content. Rice varieties are grouped based on their amylose content into waxy (0-2%), very low (3-9%), low (10-19%), intermediate (20-25%), and high (> 25%) (Kongseree and Juliano, 1972). The most widely used method for amylose determination is a colorimetric assay where iodine binds with amylose to produce a blue-purple color, which is measured spectrophotometrically at a single wavelength (620nm). Low amylose content is usually associated with tender, cohesive and glossy cooked rice; while, high amylose content is associated with firm, fluffy and separate grains of cooked rice. The Waxy (Wx) gene, which encodes granule-bound starch synthase I (GBSSI), is the major gene controlling AC in rice (Nakamura, 2002). The Waxy gene is located on chromosome six and various single nucleotide polymorphisms (SNPs) of Wx were found, including a ‘G’ to ‘T’ SNP of the first intron, ‘A’ to ‘C’ SNP of the sixth exon and ‘C’ to ‘T’ SNP of the tenth exon (Larkin and Park, 2003). The ‘AGGTATA’ sequence at the 5’splicejunction coincides with the presence of the Wxa allele, while the ‘AGTTATA’ sequence coincides with the presence of the Wxb allele. Therefore, all intermediate and high amylose cultivars had ‘G’ nucleotide while low amylose cultivars had ‘T’ nucleotide at the putative leader intron 5′ splice site. The cytosine and thymidine (CT) dinucleotide repeats in the 5’untranslated region (UTR) of the Waxy gene were reported to be a factor associated with AC. However, the relationship between these polymorphisms and amylose contents is not clear. Amylopectin chain length distribution plays a very important role to determine GT in cooked rice. The time required for cooking is determined by the gelatinization temperature of starch. It is important because it affects the texture of cooked rice and it is related to the cooking time of rice. The gelatinization temperature is estimated by the alkali digestibility test. It is measured by the alkali spreading value (ASV). The degree of spreading value of individual milled rice kernels in a weak alkali solution (1.7% KOH) is very closely correlated with gelatinized temperature. According to the ASV, rice varieties may be classified as low (55 to 69°C), intermediate (70 to 74°C) and high (> 74°C) GT classes. In a breeding program ASV is extensively used to estimate the gelatinization I ISSN (Online) = 2522-6754 ISSN (Print) = 2522-6746 www.sciplatform. com Research Manuscript