Summary

Breeding for salt tolerant rice varieties accumulated with salt tolerant genes is the most promising approach to utilize saline soil, especially coastal areas of Sri Lanka. The promising strategy to develop salt tolerant rice is to map genes responsible for salt tolerance followed by breeding with elite rice varieties. So far only the Saltol region of chromosome 1 has been identified as the major QTL or gene cluster associated with salt tolerance and it is the only locus used in breeding varieties for salt tolerance. However, there are many other QTLs mapped to the other locations of the rice genome giving rise salt tolerance. The difficulty of finding out genes responsible for these QTLs is that those QTL maps, developed from SSR markers, were not saturated enough to demarcate the closest region to proceed map based sequencing.

We were able to develop a mapping population for salt tolerance with At354 x Bg352 with the research grant offered by National Science Foundation (NSF), Sri Lanka and also we were able to map many QTLs responsible for salt tolerance in At354 x Bg352 cross employing high-throughput SNP marker technology using Illumina Infinium rice 6K SNP chip sponsored by International Rice Research Institute (IRRI). Unlike SSR markers, 1135 SNP markers were found polymorphic between At354 and Bg352 thereby, highly saturated genetic map, with an average interval of 1.29 cM between marker loci was developed indicating possible applications in gene discovery. Therefore, we propose this project as an extension of our previous studies conducted with the assistance of NSF and IRRI, to proceed towards discovery of salt tolerant candidate genes underlined by the mapped QTLs.

In this project we plan to validate previously identified QTLs under Sri Lankan environment. Ninety four recombinant inbred lines of At354 x Bg352 that were previously identified as highly salt tolerant and salt susceptible lines of F5 generation, will be grown in the hydroponic system. High-throughput whole-genome sequencing will be performed on At354 and Bg352 parents. QTLs that are validated and contained high LOD score will be compared with the genomic region associated with each QTL using the reference sequence (Nipponbare) corresponding to the QTL. Coding sequence regions derived from At354 and Bg352 will be interrogated to detect mutated sequences. Therefore, two parental sequences corresponding to the QTL should reveal the allelic variation that cause salt tolerance or susceptibility. Among the genes harbouring mutated sequences, only the genes for which the mutation caused amino acid changes will be selected as candidate genes. Candidate genes that show regulatory region based difunctionality will be selected and assessed by RT PCR based differential expression. With these experiments, we expect to reveal minimum of 2 or 3 novel genes with their functional mutations that cause either salinity tolerance or susceptibility, to the world.

This project improve the human capacity by offering facilities for a PhD and undergraduate researches in the area by plant molecular breeding which is a lacking area in Sri Lanka. Also this project improves the science and technology capacity of Sri Lanka, as it deals with latest technologies such as high through put next generation sequencing. Also this project reveal novel information on the candidate genes of salt tolerance which would be useful to rice breeders in the Department of Agriculture, Sri Lanka and the foreign world to develop resilient salt-tolerant rice cultivars.

Objectives

1. To validate previously identified salinity tolerant QTL hotspots in At354 x Bg352 rice cross under Sri Lankan environment

2. To find candidate genes for the major and validated salinity tolerant QTLs in At354 x Bg352

3. To find particular alleles leading to salinity susceptibility and salinity tolerance

4. To predict the protein structures for mutated alleles to confirm their functionality on salt tolerance

5. To assess the differential expression of the selected candidate genes under different salt levels

Major Equipment Facilitated by Grant