MOLECULAR DISSECTION AND FINE MAPPING OF MAJOR QTL GOVERNING DROUGHT TOLERANCE TRAITS IN RICE

Drought is considered as one of the major obstacles to rice for its yields and stability, especially in rainfed conditions. Development of rice cultivars with inherent capacity to withstand drought stress would improve rainfed rice production. The genomic regions harbouring drought tolerant QTLs could be of great targets to identify the candidate genes by finemapping. To this end, a QTL region on Chromosome 8 with marker interval of RM38 and RM331 governing important drought tolerant traits viz., root dry weight, root shoot weight ratio have been identified in a recombinant inbred population (RIL) derived from the cross between a semi-dwarf variety IR64 and landrace INRC10192, was targeted for fine mapping. Saturating this region with more polymorphic markers helped to delimit the QTL cluster to manageable size and identification of candidate genes underlying this QTL region by employing combination of QTL mapping and RNA-Seq analyses were the objectives of present investigation. Parents IR64 and INRC10192 used in the present study exhibited significant differences for traits like shoot length (SL) and root dry weight (RDW). The mapping population consisted of 153 F10 RILs and 435 BC1F2 population developed from a cross between a semi dwarf variety IR64 and a landrace INRC10192 was used as material for finemapping. Transgressive segregants in RI population were ranged from 45.1 % (root length) to 91.5% (root to shoot weight ratio) under control conditions, while under stress, 35.29% (shoot length) to 98.69% (root to shoot weight ratio), whereas, the relative parameters showed the transgressive segregants in the range of 62.75% (shoot length) to 95.42% (root to shoot ratio). Out of 159 rice microsatellite markers, 25 (15.72%) showed polymorphism. RILs were genotyped using these polymorphic markers and linkage map was constructed from the genotypic and phenotypic data using MAPMAKER V 3.0. Total map length of 45.6 cM using the kosambi mapping function, resulted in an average of one marker per every 2.53 cM distance. QTL mapping using RIL population revealed, 3 QTLs namely qrdwrs8.1 for relative root dry weight, qrlrs8.1 for relative root length and qslrs8.1 for shoot length with phenotypic variance of 15.95%, 15.88% and 11.62%, respectively. QTL cluster RM38-RM331 on chromosome 8 was narrowed down from 10.17 Mb to 715.67 Kb with an interval RM6999-RM7080. Backcross segregating population was developed to further delimit the QTL region identified for root dry weight using RIL population, in the present study. To reduce the background noise of the donor parent, QTL introgression was followed, wherein, the complete introgression of QTL region in BC1F1 was assessed through foreground and recombinant selections with internal (RM7080) and flanking markers (RM38 and RM331) of the targeted QTL. The BC1F1 plant having maximum recovery of recurrent parent allele was selfed to generate BC1F2 population and was subjected to stress under field conditions. Transgressive segregants observed in BC1F2 population were in the range of 8.97 % (plant yield) to 84.83 % (root dry weight). All the examined traits for the BC1F2 individuals displayed a continuous variation with the skewness ranging from −0.05 to 1.32, indicating quantitative inheritance. Correlation studies in BC1F2 population for root dry weight showed significant positive correlation with traits, viz., relative water content (0.124**) and leaf rolling score (0.142**). QTL mapping using genotypic and phenotypic data generated using BC1F2 population showed the consensus QTL for root dry weight and region was further finemapped from 715.67 Kb to 366.75 Kb with new marker interval RM22483-RM7080. In silico analysis showed 33 annotated genes within this interval. RNA-Seq for two parental root tissues was done on next generation platform (Ion torrent) to identify differentially expressed genes (DEGs) within the QTL region between parents. Total number of reads obtained through RNA-Seq was 4.7 million in INRC10192 and 4.06 million in IR64. Through RNA seq, it was found that 9,795 genes were expressed exclusively in INRC10192 and 6,901 genes were expressed only in the parent IR64, whereas 2455 genes were expressed commonly in both the parents. Through RNA-Seq approach it was found that 18 genes were differentially expressed within the targeted QTL region. Semi-qPCR was performed for all shortlisted 18 genes, but out of which only five showed differential expression. Semi-qPCR results showed that the genes, LOC_Os08g06110 (MYB related Transcription factor), LOC_Os08g07060 (Choloro respiratory reduction 6 protein) and LOC_Os08g08090 (Wound inducible protein) were up-regulated under PEG induced stress compared to control in IR64, while the genes LOC_Os08g06500 (Pentacoripeptide repeats protein) and LOC_Os08g06430 (NADH ubiquinone oxidoreductase) showed very negligible/no change under both control and PEG induced stress in IR64. On the other side, LOC_Os08g06430 (NADH ubiquinone oxidoreductase) and LOC_Os08g06500 (Pentacoripeptide repeats protein) showed down-regulation in parent INRC10192 under stress conditions when compared to control. LOC_Os08g06110 (MYB related Transcription factor), LOC_Os08g07060 (Choloro respiratory reduction 6 protein), LOC_Os08g08090 (Wound inducible protein) showed up regulation in INRC10192 under PEG induced stress compared to control. Study of functional association of gene/s with target trait was performed for all 5 genes but it was confirmed only for LOC_Os08g06110 (MYB related transcription factor). The results from this study indicated that, an integrated strategy which combines QTL-mapping and RNA-seq analysis of parents was very successful at identifying candidate gene/s for root dry weight. In combination with them, a functional association study was a welcome addition for dissecting likely candidate genes for trait of interest. Marker-assisted introgression of this QTL region or candidate genes underlying in it into modern cultivars would pave the way for increasing the yield ceiling under rainfed conditions.