Development of Novel KASP Markers for Improved Germination in Deep-Sown Direct Seeded Rice.

BACKGROUND: The lack of stable-high yielding and direct-seeded adapted varieties with better germination ability from deeper soil depth and availability of molecular markers are major limitation in achieving the maximum yield potential of rice under water and resource limited conditions. Development of high-throughput and trait-linked markers are of great interest in genomics-assisted breeding. The aim of present study was to develop and validate novel KASP (Kompetitive Allele-Specific PCR) markers associated with traits improving germination and seedling vigor of deep sown direct seeded rice (DSR). RESULTS: Out of 58 designed KASP assays, four KASP assays did not show any polymorphism in any of the eleven genetic backgrounds considered in the present study. The 54 polymorphic KASP assays were then validated for their robustness and reliability on the F1s plants developed from eight different crosses considered in the present study. The third next validation was carried out on 256 F3:F4 and 713 BC3F2:3 progenies. Finally, the reliability of the KASP assays was accessed on a set of random 50 samples from F3:F4 and 80-100 samples from BC3F2:3 progenies using the 10 random markers. From the 54 polymorphic KASP, based on the false positive rate, false negative rate, KASP utility in different genetic backgrounds and significant differences in the phenotypic values of the positive (desirable) and negative (undesirable) traits, a total of 12 KASP assays have been selected. These 12 KASP include 5 KASP on chromosome 3, 1 on chromosome 4, 3 on chromosome 7 and 3 on chromosome 8. The two SNPs lying in the exon regions of LOC_Os04g34290 and LOC_Os08g32100 led to non-synonymous mutations indicating a possible deleterious effect of the SNP variants on the protein structure. CONCLUSION: The present research work will provide trait-linked KASP assays, improved breeding material possessing favourable alleles and breeding material in form of expected pre-direct-seeded adapted rice varieties. The marker can be utilized in introgression program during pyramiding of valuable QTLs/genes providing adaptation to rice under DSR. The functional studies of the genes LOC_Os04g34290 and LOC_Os08g32100 possessing two validated SNPs may provide valuable information about these genes.

Integrating Association Mapping, Linkage Mapping, Fine Mapping with RNA Seq Conferring Seedling Vigor Improvement for Successful Crop Establishment in Deep Sown Direct-Seeded Rice.

BACKGROUND: Ongoing large-scale shift towards direct seeded rice (DSR) necessitates a convergence of breeding and genetic approaches for its sustenance and harnessing natural resources and environmental benefits. Improving seedling vigour remains key objective for breeders working with DSR. The present study aims to understand the genetic control of seedling vigour in deep sown DSR. Combined genome-wide association mapping, linkage mapping, fine mapping, RNA-sequencing to identify candidate genes and validation of putative candidate genes were performed in the present study. RESULTS: Significant phenotypic variations were observed among genotypes in both F3:4:5 and BC2F2:3 populations. The mesocotyl length showed significant positive correlation with %germination, root and shoot length. The 881 kb region on chromosome 7 reported to be associated with mesocotyl elongation. RNA-seq data and RT-PCR results identified and validated seven potential candidate genes. The four promising introgression lines free from linkage drag and with longer mesocotyl length, longer root length, semi-dwarf plant height have been identified. CONCLUSION: The study will provide rice breeders (1) the pre breeding material in the form of anticipated DSR adapted introgression lines possessing useful traits and alleles improving germination under deep sown DSR field conditions (2) the base for the studies involving functional characterization of candidate genes. The development and utilization of improved introgression lines and molecular markers may play an important role in genomics-assisted breeding (GAB) during the pyramiding of valuable genes providing adaptation to rice under DSR. Our results offer a robust and reliable package that can contribute towards enhancing genetic gains in direct seeded rice breeding programs.