IDENTIFICATION OF CANDIDATE GENE(S) UNDERLYING QTL CLUSTER FOR GRAIN SIZE TRAITS IN BASMATI RICE

Of the traits that determine the quality of Basmati rice, grain size is one of the important character not only from consumer's angle but more so from traders’ and millers’ angle. Though many genes governing grain size traits have been identified in indica and japonica, little work has been done in basmati rice. Earlier, a QTL cluster controlling grain size was identified on chromosome 5 using a population derived from Basmati370 and Jaya. In the present investigation, it was aimed for narrowing down the identified QTL cluster governing grain size traits in basmati rice employing association mapping and QTL mapping approaches besides identification of candidate gene(s) underlying it. The results obtained are presented below: In the association mapping study, a 96 diverse rice germplasm was used (aromatic (27), indica (45), japonica and javanica (19) and aus (5) groups) which differed significantly for grain size traits. The germplasm was screened with a total of 55 markers (21 SSR markers in the QTL cluster (10 Mb), 18 SSR markers covering other chromosomes to avoid spurious associations with an average number of markers per chromosome as 3.25 and 16 gene specific markers tightly linked to 9 genes reported earlier to govern grain size). Diversity analysis showed a total of 224 alleles with average number of alleles per locus as 4.2 and an average PIC value, 0.53. Phylogenetic tree constructed using DARWIN5.0 revealed, Cluster 1 consisting mainly of aromatic group, Cluster 2, indica group and Cluster 3, aus group in one subcluster and having japonica and javanica accessions in the separate subclusters with an admixture of indica varieties. Association mapping was done using TASSEL v 2.1. Out of six SSRs associated with grain size traits, three SSRs, RM 6024 (grain breadth), RM1237 and RM18582 (grain length breadth ratio) were ‘constitutive QTL’ markers as these were associated with same traits in RILs and association mapping panel across two years which covered a physical distance of 889kb. Thus, the QTL cluster was narrowed from 10Mb to 889kb. Of the nine earlier reported genes governing grain size, GS3, GW2, GS5, GW5, GS7, qSS7, QSS7, QGW8 and SRS5, five genes GW2,GS3, GW5, QSS7, QGW8 showed association with grain size traits in accordance with the earlier reports. To further narrow down the fine mapped QTL cluster, QTL mapping was employed in 410 F2 progeny of a cross, Jaya and Basmati370. To map QTLs for grain size in F2, 39 SSR markers were used for parental polymorphism study in the marker interval RM6024-RM18582. Of which, 7 markers showed polymorphism between Basmati370 and Jaya accounting for 18% of polymorphism. The QTLs for grain size, thousand grain weight and panicle number were clustered in the region RM6024RM18550 with a physical distance of 268 kb. However, there were no QTLs found for single plant yield in this region. This region within the QTL cluster is novel as it was not reported earlier to govern grain size in basmati rice. With the help of RICE TOGO browser, 24 genes were found in this narrowed down QTL region of RM6024-RM18550. The candidate genes were predicted using three approaches viz., semiquantitative pcr, qTELLER and nonsynonymous SNPs. Employing semiquantitative PCR technique to find out DEGs (Differentially Expressed Genes) in the QTL cluster between parents, Basmati370 and Jaya, Zinc finger transcription factors (Os05g0389600), Cytochrome p450 (brassinosteroid signalling) (Os05g0372300) and tetratricopeptide like helical domain containing proteins (Os05g0374500) were involved in regulating grain length whereas, ubiquitin mediated protein degradation proteins (Os05g037060, Os05g0371200 and Os05g0372800) and Cytokinin Oxidase1 (Os05t0374200) were predicted to regulate grain breadth in Basmati rice. Besides candidate genes predicted in the fine mapped QTL cluster, earlier reported grain size regulating genes viz., AP2 (Os05g0389000) and Zinc finger, RING type domain (Os05389600) showed high expression in Basmati370 similar to expression pattern reported earlier. These genes were located nearly 1.7 Mb away from the present QTL cluster. Two genes, CaLB domain containing protein and protein kinase domain containing protein were found to be highly expressed at early inflorescence stage utilising qTELLER information. Unfortunately, there were no non-synonymous SNPs found in the genes underlying the fine mapped QTL cluster. However, a non-synonymous SNP was found in VQ domain (Os05g32460) which was 1 Mb far from the fine mapped QTL cluster. From the above investigation, association mapping along with QTL mapping is found to be an effective tool in narrowing down the QTL and the germplasm used for association mapping is an ideal population for diversity and association mapping studies. The associated markers in the association mapping study can be used for MAS. Marker-assisted introgression of this QTL region or candidate genes underlying it after further confirmation into modern cultivars would help us tailor varieties according to consumer preferences as the genes underlying this region are homologous to earlier reported grain size regulating genes.