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Professor Jayashankar Telangana Agricultural University, Hyderabad
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ThesisItem Open Access IDENTIFICATION OF CANDIDATE GENE(S) UNDERLYING QTL CLUSTER FOR GRAIN SIZE TRAITS IN BASMATI RICE(PROFESSOR JAYASHANKAR TELANGANA STATE AGRICULTURAL UNIVERSITY. HYDERABAD, 2015) ANNE KITTY DEBORAH, D; ANURADHA, GOf 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 RM6024-RM18550 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.ThesisItem Open Access CONFIRMATION AND FINE MAPPING OF MAJOR QTL FOR GRAIN SIZE IN BASMATI RICE(ACHARYA N.G. RANGA AGRICULTURAL UNIVERSITY, 2011) ANNE KITTY DEBORAH, D; ANURADHA, GBasmati rice is a unique varietal group that has gained wider acceptance as a speciality rice all over the world by virtue of its unique quality traits. Grain size plays a crucial role in determining the grain quality in Basmati rice. Genetic control of basmati grain quality traits is quite complex, but breeding of new varieties having Basmati grain quality characters can be greatly facilitated by the use of molecular markers tightly linked to these traits. Hence, in the present investigation 155 recombinant inbred lines (RILs) developed from a cross between Basmati370 and Jaya were used for confirmation and fine mapping of a major QTL for grain size in Basmati rice using SSR markers derived from inside and near the genomic region associated with grain size in the marker interval of RM289 and RM18600 on chromosome 5, that was already identified by Vemireddy (2008) using F2 population developed from the same cross. The parents used for mapping, Basmati370 and Jaya differed significantly with respect to all the three traits viz., grain length (GL), grain breadth (GB) and Length- Breadth ratio (LBR). Transgressive segregation of 45.8 % in grain length, 21.93 % in grain breadth and 12.25 % in LB ratio was observed in the RILs. All the three traits exhibited normal distribution in the RIL population indicating quantitative inheritance of these traits. Correlation analysis among the grain characters studied in RILs revealed a highly significant negative association of LB ratio with grain breadth and significant positive correlation with grain length. Out of 52 SSR and 1 INDEL markers used, 23 (43.40 %) were polymorphic, 27 (39.62 %) were monomorphic and 9 (16.98 %) were not amplified between Basmati370 and Jaya. The RILs were genotyped for these 23 polymorphic markers and the linkage map employing phenotypic and genotypic data of the RILs was constructed using MAPMAKER v 3.0. The QTLs were identified using interval mapping (IM) and composite interval mapping (CIM) methods of QTL Cartographer v 2.5 software with 2.5 as LOD threshold for detecting a QTL. A minor QTL for grain length qGL5.1 was identified by IM in the marker interval of RM6024 and RM1237 with Phenotypic Variance (PVE) of 3.7 %. A single QTL designated as qGB5.1 was detected for grain MAPMAKER v 3.0. The QTLs were identified using interval mapping (IM) and composite interval mapping (CIM) methods of QTL Cartographer v 2.5 software with 2.5 as LOD threshold for detecting a QTL. A minor QTL for grain length qGL5.1 was identified by IM in the marker interval of RM6024 and RM1237 with Phenotypic Variance (PVE) of 3.7 %. A single QTL designated as qGB5.1 was detected for grain breadth in the marker interval of RM1237 and RM18582 with PVE of 3.58 % in CIM and 4.51 % in IM and a QTL qGLB5.1 for Length- Breadth Ratio was identified with PVE of 11.8 %. The genetic distance of the flanking markers harbouring QTL cluster in the previous study conducted by Vemireddy (2008) was 26.5 cM whereas in the present study it was narrowed down to 15.7 cM. The physical distance also has come down from 11,128 kb to 685 kb. Comparision of rice genome database revealed that LOC_OS05g31920 (zinc ion binding protein), LOC_OS05g31930 (retrotransposon protein, putative, unclassified), LOC_OS05g31940 (retrotransposon protein, putative, unclassified), LOC_OS05g31950 (retrotransposon protein, putative, Ty3-gypsy subclass), LOC_OS05g31959 (hypothetical protein) and LOC_OS05g31970 (expressed protein) genes are present at this region. Interestingly, two predicted genes closure to this region viz., AP2 transcription factor and RING E3 ligase were reported to be involved in controlling the seed size and weight by earlier studies. In the present study, one microsatellite marker i.e., RM18582 showed close association with the grain size QTLs. This marker has potential to be used in markerassisted improvement of the grain size in Basmati rice. Though the present study was carried out in single environment, together with the results of F2 population of the same cross, it could be concluded that these three QTLs associated with grain size traits of Basmati could be considered as stable QTLs. These QTLs, apart from their suitability in improvement of the traits concerned, can also serve as potential candidates for fine mapping. These stable QTLs also facilitate development of Near‐isogenic lines and advanced breeding lines.