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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 (NGS) 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 aim of the project was to identify, characterize and validate the salt tolerant determinants in the rice landrace Horkuch. This rice landrace is popular among farmers in the southwestern areas of coastal Bangladesh, known as Satkhira. This area is north of the Sundarbans and is known to be affected by salinity stress. The study used a reciprocal population derived from the tolerant Horkuch and sensitive IR29 to locate genetic loci segregating with tolerance and sensitivity. Application of NGS tools was considered the best and most sensitive approach to identify loci, their linked markers, and genes associated with tolerance to salinity. The following focus areas had been addressed by the time the project was completed at the end of May 2016:
  1. Discovery of QTLs linked to tolerance by combining the phenotype and genotype data (RADgenotyping) in both seedling and reproductive stages: For this a linkage map was produced successfully and QTLs identified under stress at both seedling and reproductive stages. The PI and her team have identified several progenies in the F5 stage that have multiple QTLs, including a combination of seedling and reproductive. These plants will be further validated and incorporated into breeding programs in Bangladesh.
  2. Observation of differential expression of genes in Horkuch and IR29 and their derived reciprocal populations (RNA-Seq) under salt stress in different tissues, time points, and developmental stages: Among other findings, from this work the researchers have identified interesting differences in response to stress by tolerant and sensitive progenies.
  3. Discovery of eQTLs (expression QTLs) by combining above expression and genotyping data: Preliminary analysis showed that when the effect of salt was considered, most of the trans-eQTL hotspots (indicative or regulatory proteins) belonged to genes involved in photosynthesis. Further analysis of the data and fine-tuning is still under way.
  4. Whole genome resequencing and transcriptome analysis of the Parents, Horkuch and IR29: More than 70% of both genomes could be annotated and are being aligned with their respective transcriptomes with and without stress. These data along with the QTL and eQTL are expected to give insights into the mechanism of salt tolerance in Horkuch.
Dr. Seraj and her team have completed at least seven papers on their work that they expect to submit for publication during the summer of 2016. The PI has also received two grants this year to help continue the work pursued with PEER funds. The first, for a project on “Validating role of the Drought and Salinity Tolerant (DST) Transcription Factor for a better understanding of high yields and abiotic stress tolerance mechanisms in two Bangladeshi rice varieties,” was awarded by the Third World Academy of Sciences. The second, from the Bangladesh Academy of Sciences and U.S. Department of Agriculture, Bangladesh Chapter (BAS-USDA), will support three years of work on “Development of high yielding abiotic stress tolerant rice by introducing Ideal Plant Architecture (IPA) into salt tolerant landrace and functional validation of introduced transgenes."

In summing up the overall results and impacts of her PEER funded work over the past 2.5 years, Dr. Seraj notes that “the mentorship of the U.S. partner has been excellent and has led to a standard of work for my lab which we were previously unable to reach (prior to PEER). We are hoping to publish seven research articles in prestigious international journals from this PEER grant. This will bring a tremendous boost to the reputation of Dhaka University. By the time that all the papers are published, we will hopefully be able to propose a mechanism for salt tolerance of the traditional landrace Horkuch. This will not only increase knowledge but also enhance our salinity tolerance breeding programs. Moreover it is envisaged that the output of this work will lead to salt tolerance breeding programs which will incorporate multiple salt tolerance QTLs in single progenies and hopefully produce greater tolerance levels.”

On the capacity building side, two of the research associates who worked under PEER are now PhD students under the supervision of the U.S. partner Dr. Juenger at the University of Texas at Austin (UTA). One of them, Samsad Razzaque, began his studies in the fall of 2015, and the other, Taslima Haque, will enroll in the fall of 2016. When the PEER project began, the third research associate, Sabrina Elias, was already in the Monsanto, Beachell, Borlaug International Scholar Program (MBBISP) pursuing her PhD under the University of Dhaka, UTA, and the University of Nebraska-Lincoln. Sabrina’s thesis will officially document the work under PEER as a record under the University of Dhaka. In addition, Sabrina will be visiting the International Rice Research Institute in October 2016, where she will be doing a comparative analysis of Horkuch with other salt tolerance donors like Pokkali and Nona Bokra under the guidance of Dr. Abdelbagi Ismail and Dr. Damien Platten. Dr. Seraj reports that there was a mutual benefit to both MBBISP and PEER under this project, as the PEER award allowed the sequence analysis of a big population, while the automated phenotyping done at UNL will help validate the results obtained from the PEER work.