BackgroundSalinity is a major environmental factor limiting productivity of crop plants including rice in which wide range of natural variability exists. Although recent evidences implicate epigenetic mechanisms for modulating the gene expression in plants under environmental stresses, epigenetic changes and their functional consequences under salinity stress in rice are underexplored. DNA methylation is one of the epigenetic mechanisms regulating gene expression in plant’s responses to environmental stresses. Better understanding of epigenetic regulation of plant growth and response to environmental stresses may create novel heritable variation for crop improvement.Methodology/Principal FindingsMethylation sensitive amplification polymorphism (MSAP) technique was used to assess the effect of salt stress on extent and patterns of DNA methylation in four genotypes of rice differing in the degree of salinity tolerance. Overall, the amount of DNA methylation was more in shoot compared to root and the contribution of fully methylated loci was always more than hemi-methylated loci. Sequencing of ten randomly selected MSAP fragments indicated gene-body specific DNA methylation of retrotransposons, stress responsive genes, and chromatin modification genes, distributed on different rice chromosomes. Bisulphite sequencing and quantitative RT-PCR analysis of selected MSAP loci showed that cytosine methylation changes under salinity as well as gene expression varied with genotypes and tissue types irrespective of the level of salinity tolerance of rice genotypes.Conclusions/SignificanceThe gene body methylation may have an important role in regulating gene expression in organ and genotype specific manner under salinity stress. Association between salt tolerance and methylation changes observed in some cases suggested that many methylation changes are not “directed”. The natural genetic variation for salt tolerance observed in rice germplasm may be independent of the extent and pattern of DNA methylation which may have been induced by abiotic stress followed by accumulation through the natural selection process.

Drought resistance is of enormous importance in crop production. The identification of genetic factors involved in plant response to drought stress provides a strong foundation for improving drought tolerance. Stay-green is a drought resistance trait in sorghum (Sorghum bicolor L. Moench) that gives plants resistance to premature senescence under severe soil moisture stress during the post-flowering stage. The objective of this study was to map quantitative trait loci (QTLs) that control the stay-green and chlorophyll content in sorghum. By using a restriction fragment length polymorphism (RFLP) map, developed from a recombinant inbred line (RIL) population, we identified four stay-green QTLs, located on three linkage groups. The QTLs (Stg1 and Stg2) are on linkage group A, with the other two, Stg3 and Stg4, on linkage groups D and J, respectively. Two stay-green QTLs, Stg1 and Stg2, explaining 13-20% and 20-30% of the phenotypic variability, respectively, were consistently identified in all trials at different locations in two years. Three QTLs for chlorophyll content (Chl1, Chl2, and Chl3), explaining 25-30% of the phenotypic variability were also identified under post-flowering drought stress. All coincided with the three stay-green QTL regions (Stg1, Stg2, and Stg3) accounting for 46% of the phenotypic variation. The Stg1 and Stg2 regions also contain the genes for key photosynthetic enzymes, heat shock proteins, and an abscisic acid (ABA) responsive gene. Such spatial arrangement shows that linkage group A is important for drought- and heat-stress tolerance and yield production in sorghum. High-resolution mapping and cloning of the consistent stay-green QTLs may help to develop drought-resistant hybrids and to understand the mechanism of drought-induced senescence in plants.

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Drought resistance is of enormous importance in crop production. The identification of genetic factors involved in plant response to drought stress provides a strong foundation for improving drought tolerance. Stay-green is a drought resistance trait in sorghum (Sorghum bicolor L. Moench) that gives plants resistance to premature senescence under severe soil moisture stress during the post-flowering stage. The objective of this study was to map quantitative trait loci (QTLs) that control the stay-green and chlorophyll content in sorghum. By using a restriction fragment length polymorphism (RFLP) map, developed from a recombinant inbred line (RIL) population, we identified four stay-green QTLs, located on three linkage groups. The QTLs (Stg1 and Stg2) are on linkage group A, with the other two, Stg3 and Stg4, on linkage groups D and J, respectively. Two stay-green QTLs, Stg1 and Stg2, explaining 13-20% and 20-30% of the phenotypic variability, respectively, were consistently identified in all trials at different locations in two years. Three QTLs for chlorophyll content (Chl1, Chl2, and Chl3), explaining 25-30% of the phenotypic variability were also identified under post-flowering drought stress. All coincided with the three stay-green QTL regions (Stg1, Stg2, and Stg3) accounting for 46% of the phenotypic variation. The Stg1 and Stg2 regions also contain the genes for key photosynthetic enzymes, heat shock proteins, and an abscisic acid (ABA) responsive gene. Such spatial arrangement shows that linkage group A is important for drought- and heat-stress tolerance and yield production in sorghum. High-resolution mapping and cloning of the consistent stay-green QTLs may help to develop drought-resistant hybrids and to understand the mechanism of drought-induced senescence in plants.

SummaryThe physiological role of a vacuolar ATPase subunit c1 (SaVHAc1) from a halophyte grass Spartina alterniflora was studied through its expression in rice. The SaVHAc1-expressing plants showed enhanced tolerance to salt stress than the wild-type plants, mainly through adjustments in early stage and preparatory physiological responses. In addition to the increased accumulation of its own transcript, SaVHAc1 expression led to increased accumulation of messages of other native genes in rice, especially those involved in cation transport and ABA signalling. The SaVHAc1-expressing plants maintained higher relative water content under salt stress through early stage closure of the leaf stoma and reduced stomata density. The increased K + ⁄ Na + ratio and other cations established an ion homoeostasis in SaVHAc1-expressing plants to protect the cytosol from toxic Na + and thereby maintained higher chlorophyll retention than the WT plants under salt stress. Besides, the role of SaVHAc1 in cell wall expansion and maintenance of net photosynthesis was implicated by comparatively higher root and leaf growth and yield of rice expressing SaVHAc1 over WT under salt stress. The study indicated that the genes contributing toward natural variation in grass halophytes could be effectively manipulated for improving salt tolerance of field crops within related taxa.

By combining the amplified fragment length polymorphism (AFLP) technique with selective genotyping, we constructed a linkage map for rice and assigned each linkage group to a corresponding chromosome. The AFLP map, consisting of 202 AFLP markers, was generated from 74 recombinant inbred lines (RIL) which were selected from both extremes of the population (250 lines) with respect to the response to complete submergence. Map length was 1756 cM, with an average interval size of 8.5 cM. To assign linkage groups to chromosomes, we used 50 previously mapped AFLP markers as anchor markers distributed over the 12 chromosomes. Other AFLP markers were then assigned to specific chromosomes based on their linkage to anchor markers. This AFLP map is equivalent to the RFLP/AFLP map constructed previously as the anchors were in the same order in both maps. Furthermore, tests with two restriction fragment length polymorphism (RFLP) markers and two sequence-tagged site (STS) markers showed that they mapped in the expected positions. Using this AFLP map, a major gene for submergence tolerance was localized on chromosome 9. Quantitative trait loci (QTL) associated with submergence tolerance were detected on chromosomes 6, 7, 11, and 12. We conclude that the combination of AFLP mapping and selective genotyping provides a much faster and easier approach to QTL identification than the use of RFLP markers.

We exploited the newly developed amplified fragment length polymorphism (AFLP) technique to study the polymorphism, distribution and inheritance of AFLP markers with a doubled haploid rice population derived from 'IR64'/'Azucena'. Using only 20 pairs of primer combinations, we detected 945 AFLP bands of which 208 were polymorphic. All 208 AFLP markers were mapped and distributed over all 12 chromosomes. When these were compared with RFLP markers already mapped in the population, we found the AFLP markers to be highly polymorphic in rice and to follow Mendelian segregation. As linkage map of rice can be generated rapidly with AFLP markers they will be very useful for marker-assisted backcrossing.