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Asian cultivated rice shows allelic variation in sodium transporter, OsHKT1;5, correlating with shoot sodium exclusion (salinity tolerance). These changes map to intra/extracellularly-oriented loops that occur between four transmembrane-P loop-transmembrane (MPM) motifs in OsHKT1;5. HKT1;5 sequences from more recently evolved Oryza species (O. sativa/O. officinalis complex species) contain two expansions that involve two intracellularly oriented loops/helical regions between MPM domains, potentially governing transport characteristics, while more ancestral HKT1;5 sequences have shorter intracellular loops. We compared homology models for homoeologous OcHKT1;5-K and OcHKT1;5-L from halophytic O. coarctata to identify complementary amino acid residues in OcHKT1;5-L that potentially enhance affinity for Na+. Using haplotyping, we showed that Asian cultivated rice accessions only have a fraction of HKT1;5 diversity available in progenitor wild rice species (O. nivara and O. rufipogon). Progenitor HKT1;5 haplotypes can thus be used as novel potential donors for enhancing cultivated rice salinity tolerance. Within Asian rice accessions, 10 non-synonymous HKT1;5 haplotypic groups occur. More HKT1;5 haplotypic diversities occur in cultivated indica gene pool compared to japonica. Predominant Haplotypes 2 and 10 occur in mutually exclusive japonica and indica groups, corresponding to haplotypes in O. sativa salt-sensitive and salt-tolerant landraces, respectively. This distinct haplotype partitioning may have originated in separate ancestral gene pools of indica and japonica, or from different haplotypes selected during domestication. Predominance of specific HKT1;5 haplotypes within the 3 000 rice dataset may relate to eco-physiological fitness in specific geo-climatic and/or edaphic contexts.

The genome of Asian cultivated rice (Oryza sativa L.) shows the presence of six organelle-specific and one plasma membrane (OsNHX1-7) NHX-type cation proton antiporters. Of these, vacuolar-localized OsNHX1 is extensively characterized. The genus Oryza consists of 27 species and 11 genome-types, with cultivated rice, diploid O. sativa, having an AA-type genome. Oryza NHX1 orthologous regions (gene organization, 5′ upstream cis elements, amino acid residues/motifs) from closely related Oryza AA genomes cluster distinctly from NHX1 regions from more ancestral Oryza BB, FF and KKLL genomes. These sequence-specific distinctions also extend to two separate intron retention (IR) events involving Oryza NHX1 transcripts that occur at the 5′ and 3′ ends of the NHX1 transcripts. We demonstrate that the IR event involving the 5′ UTR is present only in more recently evolved Oryza AA genomes while the IR event governing retention of the 13th intron of Oryza NHX1 (terminal intron) is more ancient in origin, also occurring in halophytic wild rice, Oryza coarctata (KKLL). We also report presence of a retro-copy of the OcNHX1 cDNA in the genome of O. coarctata (rOcNHX1). Preferential species and tissue specific up- or down-regulation of the correctly spliced NHX1 transcript/5′ UTR/13th intron-retaining splice variants under salinity was observed. The implications of IR on NHX1 mRNA stability and ORF diversity in Oryza spp. is discussed.

Naturally evolved saline tolerant rice landraces found along the coastline of India are a valuable genomic resource to explore the complex, polygenic nature of salinity tolerance. In the present study, a set of 28 genome wide SSR markers, 11 salt responsive genic SSR markers and 8 Saltol QTL linked SSR markers were used to estimate genetic relatedness and population structure within a collection of 47 rice landraces (including a tolerant and 2 sensitive checks) originating from geographically divergent coastal regions of India. All three marker types identified substantial genetic variation among the landraces, as evident from their higher PIC values (0.53 for genomic SSRs, 0.43 for Genic SSRs and 0.59 for Saltol SSRs). The markers RM431, RM484 (Genomic SSRs), OsCAX (D), OsCAX (T) (Genic SSRs) and RM562 (Saltol SSR) were identified as good candidates to be used in breeding programs for improving salinity tolerance in rice. STRUCTURE analysis divided the landraces into five distinct populations, with classification correlating with their geographical locations. Principal coordinate and hierarchical cluster analyses (UPGMA and neighbor joining) are in close agreement with STRUCTURE results. AMOVA analysis indicated a higher magnitude of genetic differentiation within individuals of groups (58%), than among groups (42%). We also report the development and validation of a new Cleavage Amplified Polymorphic Sequence (CAPS) marker (OsHKT1;5V395) that targets a codon in the sodium transporter gene OsHKT1;5 (Saltol/SKC1 locus) that is associated with sodium transport rates in the above rice landraces. The CAPS marker was found to be present in all landraces except in IR29, Kamini, Gheus, Matla 1 and Matla 2. Significant molecular genetic diversity established among the analyzed salt tolerant rice landraces will aid in future association mapping; the CAPS marker, OsHKT1;5V395 can be used to map rice landraces for the presence of the SNP (Single Nucleotide Polymorphism) associated with increased sodium transport rates and concomitant salinity tolerance in rice.