Identification of genes for quantitative traits is difficult using any single approach due to complex inheritance of the traits and limited resolving power of the individual techniques. Here a combination of genetic mapping and bulked transcriptome profiling was used to narrow down the number of differentially expressed salt-responsive genes in rice in order to identify functional polymorphism of genes underlying the quantitative trait loci (QTL). A population of recombinant inbred lines (RILs) derived from cross between salt-tolerant variety CSR 27 and salt-sensitive variety MI 48 was used to map QTL for salt ion concentrations in different tissues and salt stress susceptibility index (SSI) for spikelet fertility, grain weight, and grain yield. Eight significant QTL intervals were mapped on chromosomes 1, 8, and 12 for the salt ion concentrations and a QTL controlling SSI for spikelet fertility was co-located in one of these intervals on chromosome 8. However, there were total 2,681 genes in these QTL intervals, making it difficult to pinpoint the genes responsible for the functional differences for the traits. Similarly, transcriptome profiling of the seedlings of tolerant and sensitive parents grown under control and salt-stress conditions showed 798 and 2,407 differentially expressed gene probes, respectively. By analyzing pools of RNA extracted from ten each of extremely tolerant and extremely sensitive RILs to normalize the background noise, the number of differentially expressed genes under salt stress was drastically reduced to 30 only. Two of these genes, an integral transmembrane protein DUF6 and a cation chloride cotransporter, were not only co-located in the QTL intervals but also showed the expected distortion of allele frequencies in the extreme tolerant and sensitive RILs, and therefore are suitable for future validation studies and development of functional markers for salt tolerance in rice to facilitate marker-assisted breeding.

Betaine is an important osmoprotectant, synthesized by many plants in response to abiotic stresses. Almost all known biosynthetic pathways of betaine are two-step oxidations of choline. Recently, a biosynthetic pathway of betaine from glycine, catalyzed by two N-methyltransferase enzymes, was found. Here, the potential role of N-methyltransferase genes for betaine synthesis was examined in a freshwater cyanobacterium, Synechococcus sp. PCC 7942, and in Arabidopsis plants. It was found that the coexpression of N-methyltransferase genes in Synechococcus caused accumulation of a significant amount of betaine and conferred salt tolerance to a freshwater cyanobacterium sufficient for it to become capable of growth in seawater. Arabidopsis plants expressing N-methyltransferase genes also accumulated betaine to a high level in roots, stems, leaves, and flowers and improved seed yield under stress conditions. Betaine levels were higher than those produced by choline-oxidizing enzymes. These results demonstrate the usefulness of glycine N-methyltransferase genes for the improvement of abiotic stress tolerance in crop plants.cyanobacteria ͉ methyltransferase ͉ osmoprotectant ͉ stress resistance T oday, Ϸ20% of the world's cultivated land and nearly half of all irrigated lands are affected by high salinity (1). High salinity causes ion imbalance and hyperosmotic stress in plants. Organisms that thrive in hypersaline environments possess specific mechanisms for the adjustment of their internal osmotic status. One such mechanism is the ability to accumulate low-molecular-weight organic-compatible solutes such as sugars, some amino acids, and quaternary ammonium compounds (2-4). Glycine betaine (N,N,Ntrimethylglycine, hereafter betaine) is a major osmolyte (2-4). Another mechanism for adaptation to high salinity is the exclusion of the Na ϩ ion from sodium-sensitive sites (5). Genetic engineering techniques have been applied to improve the salt tolerance of plants (6-13). Considerable success has been demonstrated by manipulating the Na ϩ ͞H ϩ antiporter genes (6-8). By contrast, the genetic engineering of betaine synthesis has been hampered by low accumulation levels of betaine (9-13). Most known biosynthetic pathways of betaine include a two-step oxidation of choline: choline 3 betaine aldehyde 3 betaine. The first step is catalyzed by choline monooxygenase (CMO) in plants (14), choline dehydrogenase (CDH) in animals and bacteria (15,16), and choline oxidase in some bacteria (11,17). The second step is catalyzed by NAD ϩ -dependent betaine aldehyde dehydrogenase in all organisms (15,18,19), although in some bacteria, CDH and choline oxidase also catalyze the second step (15-17). Hitherto, all attempts at betaine synthesis have been carried out by using choline-oxidizing enzymes (9-13). The supply and transport of betaine precursors such as choline, ethanolamine, and serine to plastids may be of importance, because these precursors have been suggested to be limiting (12,13).Recently, we showed that a halotolerant cyanobacter...

Single nucleotide polymorphism (SNP) is the most abundant DNA sequence variation present in plant genomes. Here, we report the design and validation of a unique genic-SNP genotyping chip for genetic and evolutionary studies as well as molecular breeding applications in rice. The chip incorporates 50,051 SNPs from 18,980 different genes spanning 12 rice chromosomes, including 3,710 single-copy (SC) genes conserved between wheat and rice, 14,959 SC genes unique to rice, 194 agronomically important cloned rice genes and 117 multi-copy rice genes. Assays with this chip showed high success rate and reproducibility because of the SC gene based array with no sequence redundancy and cross-hybridisation problems. The usefulness of the chip in genetic diversity and phylogenetic studies of cultivated and wild rice germplasm was demonstrated. Furthermore, its efficacy was validated for analysing background recovery in improved mega rice varieties with submergence tolerance developed through marker-assisted backcross breeding.

Soil salinity is a major constraint to rice production in large inland and coastal areas around the world. Modern high yielding rice varieties are particularly sensitive to high salt stress. There are salt tolerant landraces and traditional varieties of rice but with limited information on genomic regions (QTLs) and genes responsible for their tolerance. Here we describe a method for rapid identification of QTLs for reproductive stage salt tolerance in rice using bulked segregant analysis (BSA) of bi-parental recombinant inbred lines (RIL). The number of RILs required for the creation of two bulks with extreme phenotypes was optimized to be thirty each. The parents and bulks were genotyped using a 50K SNP chip to identify genomic regions showing homogeneity for contrasting alleles of polymorphic SNPs in the two bulks. The method was applied to ‘CSR11/MI48’ RILs segregating for reproductive stage salt tolerance. Genotyping of the parents and RIL bulks, made on the basis of salt sensitivity index for grain yield, revealed 6,068 polymorphic SNPs and 21 QTL regions showing homogeneity of contrasting alleles in the two bulks. The method was validated further with ‘CSR27/MI48’ RILs used earlier for mapping salt tolerance QTLs using low-density SSR markers. BSA with 50K SNP chip revealed 5,021 polymorphic loci and 34 QTL regions. This not only confirmed the location of previously mapped QTLs but also identified several new QTLs, and provided a rapid way to scan the whole genome for mapping QTLs for complex agronomic traits in rice.

Phosphate (Pi) is pivotal for plant growth and development. Pi deficiency triggers local and systemically regulated adaptive responses in Arabidopsis thaliana. Inhibition of primary root growth (PRG) and retarded development of lateral roots (LRs) are typical local Pi deficiency-mediated responses of the root system. Expression of Pi starvation-responsive (PSR) genes is regulated systemically. Here, we report the differential influence of iron (Fe) availability on local and systemic sensing of Pi by Arabidopsis. P-Fe- condition disrupted local Pi sensing, resulting in an elongated primary root (PR). Altered Fe homeostasis in the lpsi mutant with aberration in local Pi sensing provided circumstantial evidence towards the role of Fe in the maintenance of Pi homeostasis. Reporter gene assays, expression analysis of auxin-responsive genes (ARGs) and root phenotyping of the arf7arf19 mutant demonstrated the role of Fe availability on local Pi deficiency-mediated LR development. In addition, Fe availability also exerted a significant influence on PSR genes belonging to different functional categories. Together, these results demonstrated a substantial influence of Fe availability on Pi deficiency-mediated responses of ontogenetically distinct traits of the root system and PSR genes. The study also provided evidence of cross-talk between Pi, Fe and Zn, highlighting a complex tripartite interaction amongst them for maintaining Pi homeostasis.

Pusa Basmati 1121 (PB1121), an elite Basmati rice cultivar is vulnerable to salinity at seedling stage. A study was undertaken to impart seedling-stage salt tolerance into PB1121 by transferring a quantitative trait locus (QTL), Saltol, using FL478 as donor, through marker assisted backcrossing. Sequence tagged microsatellite site (STMS) marker RM 3412, tightly linked to Saltol was used for foreground selection. Background recovery was estimated using 90 genome-wide STMS markers. Systematic phenotypic selection helped in accelerated recovery of recurrent parent phenome (RPP). A set of 51 BC3F2 lines homozygous for Saltol were advanced to develop four improved near isogenic lines (NILs) of PB1121 with seedling stage salt tolerance. The background genome recovery in the NILs ranged from 93.3 to 99.4%. The improved NILs were either similar or better than the recurrent parent PB1121 for yield, grain and cooking quality and duration. Biochemical analyses revealed significant variation in shoot and root Na+ and K+ concentrations. Correlation between shoot and root Na+ concentration was stronger than that between root and shoot K+ concentration. The effect of QTL integration into the NILs was studied through expression profiling of OsHKT1;5, one of the genes present in the Saltol region. The NILs had significantly higher OsHKT1;5 expression than the recurrent parent PB1121, but lower than FL478 on salt exposure validating the successful introgression of Saltol in the NILs. This was also confirmed under agronomic evaluation, wherein the NILs showed greater salt tolerance at seedling stage. One of the NILs, Pusa1734-8-3-3 (NIL3) showed comparable yield and cooking quality to the recurrent parent PB1121, with high field level seedling stage salinity tolerance and shorter duration. This is the first report of successful introgression of Saltol into a Basmati rice cultivar.

BackgroundRice is one of the most important crops for global food security but its productivity is adversely affected by salt stress prevalent in about 30 % of the cultivated land. For developing salt-tolerant rice varieties through conventional breeding or biotechnological interventions, there is an urgent need to identify natural allelic variants that may confer salt tolerance. Here, 299 wild rice accessions collected from different agro-climatic regions of India were evaluated during growth under salt stress. Of these 95 representative accessions were sequenced for members of HKT ion transporter family genes by employing Ion Torrent PGM sequencing platform.ResultsHaplotype analysis revealed haplotypes H5 and H1 of HKT1;5 and HKT2;3, respectively associated with high salinity tolerance. This is the first study of allele mining of eight members of HKT gene family from Indian wild rice reporting a salt tolerant allele of HKT2;3. HKT1;5 also showed a salt tolerant allele from wild rice. Phylogenetic analysis based on the nucleotide sequences showed different grouping of the HKT family genes as compared to the prevailing protein sequence based classification.ConclusionsThe salt tolerant alleles of the HKT genes from wild rice may be introgressed into modern high yielding cultivars to widen the existing gene pool and enhance rice production in the salt affected areas.Electronic supplementary materialThe online version of this article (doi:10.1186/s12284-016-0083-8) contains supplementary material, which is available to authorized users.

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