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Partnerships for enhanced engagement in research (PEER) SCIENCE
Cycle 2 (2012 Deadline)

Validation of salt tolerance determinants in rice (Oryza sativa L. indica) landrace Horkuch and its segregating population by 2b-RAD sequencing and RNA-seq analysis under stress

PI: Zeba I. Seraj (University of Dhaka), with co-PI Abdelbagi Ismail (International Rice Research Institute)
U.S. Partner: Thomas Juenger (University of Texas at Austin)
Project Dates: August 2013 to May 2016

Bangladesh is the world’s fourth-largest rice-producing country and is an enriched germplasm reservoir with 6,500 varieties of wild accessions, landraces, and modern varieties. Salt-tolerant rice landraces are of particular interest as donors of salt tolerance traits. The Bangladesh Rice Research Institute has released six slightly to moderately tolerant modern rice varieties, but for various reasons most have not be widely adopted by farmers. In view of the predicted increase in salinity levels in Bangladesh, more tolerant varieties are needed. Horkuch is a rice landrace popular with some farmers in the southwestern coastal areas in Satkhira, but it has low yields. Farmers in this area cannot grow modern high-yielding varieties due to salinity in the soil. Horkuch has been identified as salt tolerant at the seedling stage, and subsequently its yield-related traits under stress were also found to be superior. In order to determine exactly which genes from Horkuch could be integrated most productively into existing rice varieties, intensive study of the Horkuch landrace is essential.
 

Bangladesh Partnership Picture 1 The rice plants are set up for F2 phenotyping (Photo courtesy Zeba Seraj).

Bangladesh Partnership Picture 2 During the phenotyping of F3 plants, the research team measures SES values (Photo courtesy Zeba Seraj).

As part of this project, next-generation sequencing methods will be used to map a population of several hundred individual plants in weeks rather than the usual months or years required. The ultimate goal will be to develop a list of candidate genes to be targeted for introgression into popular but sensitive varieties of rice to make them more salt tolerant. If these determinants can be identified and introduced into more sensitive rice varieties, this will result in the production of salt-tolerant rice for the coastal areas of Bangladesh. Even a modest increase in rice production in the moderate saline zones would go a long way toward ensuring food security for the local landless farmers in the saline zone.

Summary of Recent Activities
 
The sequencing of the large inset libraries for the two parents, IR29 and Horkuch was completed at the University of Arizona during the first quarter of 2015. The sequencing reads have been quality filtered and trimmed to obtain high quality raw reads. Along with the previously sequenced insert library data, this data is now being processed in a genome assembly pipeline. In order to obtain complete information about the gene content of the parents, the team decided to sequence the transcriptome of both parents under control and stress conditions. This is expected to validate the gene sequences obtained from re-sequencing of the parents as well as the RNA-Seq data of the F3 population. It will also help locate any splice variants of the genes, particularly under stress conditions.

As of March, roughly 40 DNA samples of the F2 population with less than a million RAD-genotyping reads have been resequenced using new libraries. The team now has 191 samples with at least 1 million reads. Using O. sativa japonica as the reference genome the team is now analyzing these data with the GATK tool for calling SNPs. These high quality SNPs will be used for a fresh linkage construction, one for the seedling stage and another for the reproductive stage and a paper will be prepared based on the results.

Additionally, the team has finished analyzing 28 different contrasting models for the seedling stage samples in both leaf and root tissues. The significance of differential gene counts has already been identified using different models. The tolerant and sensitive categories and their corresponding contrasts with respect to time difference as well as treatment had more information compared to the other models. The team was able to select some genes which are differentially expressed significantly in tolerant and sensitive leaf and root tissues.