Molecular Research in Rice.

Rice is the most important source of nutrition for approximately half of the human population [...].

Integration of QTL Mapping and Whole Genome Sequencing Identifies Candidate Genes for Alkalinity Tolerance in Rice (Oryza sativa).

Soil alkalinity is an important stressor that impairs crop growth and development, resulting in reduced crop productivity. Unlike salinity stress, research efforts to understand the mechanism of plant adaptation to alkaline stress is limited in rice, a major staple food for the world population. We evaluated a population of 193 recombinant inbred lines (RIL) developed from a cross between Cocodrie and N22 under alkaline stress at the seedling stage. Using a linkage map consisting of 4849 SNP markers, 42 additive QTLs were identified. There were seven genomic regions where two or more QTLs for multiple traits colocalized. Three important QTL clusters were targeted, and several candidate genes were identified based on high impact variants using whole genome sequences (WGS) of both parents and differential expression in response to alkalinity stress. These genes included two expressed protein genes, the glucan endo-1,3-beta-glucosidase precursor, F-box domain-containing proteins, double-stranded RNA-binding motif-containing protein, aquaporin protein, receptor kinase-like protein, semialdehyde hydrogenase, and NAD-binding domain-containing protein genes. Tolerance to alkaline stress in Cocodrie was most likely due to the low Na+/K+ ratio resulting from reduced accumulation of Na+ ions and higher accumulation of K+ in roots and shoots. Our study demonstrated the utility of integrating QTL mapping with WGS to identify the candidate genes in the QTL regions. The QTLs and candidate genes originating from the tolerant parent Cocodrie should be targeted for introgression to improve alkalinity tolerance in rice and to elucidate the molecular basis of alkali tolerance.

Whole-Genome Sequencing and RNA-Seq Reveal Differences in Genetic Mechanism for Flowering Response between Weedy Rice and Cultivated Rice.

Flowering is a key agronomic trait that influences adaptation and productivity. Previous studies have indicated the genetic complexity associated with the flowering response in a photoinsensitive weedy rice accession PSRR-1 despite the presence of a photosensitive allele of a key flowering gene Hd1. In this study, we used whole-genome and RNA sequencing data from both cultivated and weedy rice to add further insights. The de novo assembly of unaligned sequences predicted 225 genes, in which 45 were specific to PSRR-1, including two genes associated with flowering. Comparison of the variants in PSRR-1 with the 3K rice genome (RG) dataset identified unique variants within the heading date QTLs. Analyses of the RNA-Seq result under both short-day (SD) and long-day (LD) conditions revealed that many differentially expressed genes (DEGs) colocalized with the flowering QTLs, and some DEGs such as Hd1, OsMADS56, Hd3a, and RFT1 had unique variants in PSRR-1. Ehd1, Hd1, OsMADS15, and OsMADS56 showed different alternate splicing (AS) events between genotypes and day length conditions. OsMADS56 was expressed in PSRR-1 but not in Cypress under both LD and SD conditions. Based on variations in both sequence and expression, the unique flowering response in PSRR-1 may be due to the high-impact variants of flowering genes, and OsMADS56 is proposed as a key regulator for its day-neutral flowering response.

Comparative Transcriptomics of Rice Genotypes with Contrasting Responses to Nitrogen Stress Reveals Genes Influencing Nitrogen Uptake through the Regulation of Root Architecture.

The indiscriminate use of nitrogenous fertilizers continues unabated for commercial crop production, resulting in air and water pollution. The development of rice varieties with enhanced nitrogen use efficiency (NUE) will require a thorough understanding of the molecular basis of a plant's response to low nitrogen (N) availability. The global expression profiles of root tissues collected from low and high N treatments at different time points in two rice genotypes, Pokkali and Bengal, with contrasting responses to N stress and contrasting root architectures were examined. Overall, the number of differentially expressed genes (DEGs) in Pokkali (indica) was higher than in Bengal (japonica) during low N and early N recovery treatments. Most low N DEGs in both genotypes were downregulated whereas early N recovery DEGs were upregulated. Of these, 148 Pokkali-specific DEGs might contribute to Pokkali's advantage under N stress. These DEGs included transcription factors and transporters and were involved in stress responses, growth and development, regulation, and metabolism. Many DEGs are co-localized with quantitative trait loci (QTL) related to root growth and development, chlorate-resistance, and NUE. Our findings suggest that the superior growth performance of Pokkali under low N conditions could be due to the genetic differences in a diverse set of genes influencing N uptake through the regulation of root architecture.

A Novel Mutation of the NARROW LEAF 1 Gene Adversely Affects Plant Architecture in Rice (Oryza sativa L.).

Plant architecture is critical for enhancing the adaptability and productivity of crop plants. Mutants with an altered plant architecture allow researchers to elucidate the genetic network and the underlying mechanisms. In this study, we characterized a novel nal1 rice mutant with short height, small panicle, and narrow and thick deep green leaves that was identified from a cross between a rice cultivar and a weedy rice accession. Bulked segregant analysis coupled with genome re-sequencing and cosegregation analysis revealed that the overall mutant phenotype was caused by a 1395-bp deletion spanning over the last two exons including the transcriptional end site of the nal1 gene. This deletion resulted in chimeric transcripts involving nal1 and the adjacent gene, which were validated by a reference-guided assembly of transcripts followed by PCR amplification. A comparative transcriptome analysis of the mutant and the wild-type rice revealed 263 differentially expressed genes involved in cell division, cell expansion, photosynthesis, reproduction, and gibberellin (GA) and brassinosteroids (BR) signaling pathways, suggesting the important regulatory role of nal1. Our study indicated that nal1 controls plant architecture through the regulation of genes involved in the photosynthetic apparatus, cell cycle, and GA and BR signaling pathways.

Genetic Dissection of Seedling Stage Salinity Tolerance in Rice Using Introgression Lines of a Salt Tolerant Landrace Nona Bokra.

Salinity is an important abiotic stress affecting rice production worldwide. Development of salt tolerant varieties is the most feasible approach for improving rice productivity in salt affected soils. In rice, seedling stage salinity tolerance is crucial for better crop establishment. Quantitative trait loci (QTL) mapping using introgression lines (ILs) is useful for identification and simultaneous transfer of desirable alleles into elite genetic background. In the present study, 138 ILs derived from the cross between a high yielding elite salt susceptible japonica rice cultivar Jupiter and a salt tolerant indica landrace Nona Bokra were evaluated for salt tolerance at seedling stage in a hydroponics experiment and were genotyped using 126 simple sequence repeat markers. A total of 33 additive QTLs were detected by composite interval mapping for 8 morphophysiological traits. The phenotypic responses, genomic composition, and QTLs identified from the study indicated that Na/K ratio is the key factor for salinity tolerance. The mechanisms of tolerance might be due to homeostasis between Na+ and K+ or Na+ compartmentation. Gene ontology (GO) analysis revealed that significant GO terms in the selected QTL regions were associated with the genes/pathways involved in signaling, enzyme inhibition, and ion transport. Because majority of QTLs are with small effects, marker-assisted recurrent selection is proposed to accumulate favorable alleles for improving salt tolerance using the tolerant ILs identified in this study. The tolerant ILs also provide an opportunity for functional genomics studies to provide molecular insights into salt tolerance mechanisms in Nona Bokra.

Mapping of seed shattering loci provides insights into origin of weedy rice and rice domestication.

Seed shattering is an important trait that distinguishes crop cultivars from the wild and weedy species. The genetics of seed shattering was investigated in this study to provide insights into rice domestication and the evolution of weedy rice. Quantitative trait locus (QTL) analysis, conducted in 2 recombinant inbred populations involving 2 rice cultivars and a weedy rice accession of the southern United States, revealed 3-5 QTLs that controlled seed shattering with 38-45% of the total phenotypic variation. Two QTLs on chromosomes 4 and 10 were consistent in both populations. Both cultivar and weedy rice contributed alleles for increased seed shattering. Genetic backgrounds affected both QTL number and the magnitude of QTL effects. The major QTL qSH4 and a minor QTL qSH3 were validated in near-isogenic lines, with the former conferring a significantly higher degree of seed shattering than the latter. Although the major QTL qSH4 overlapped with the sh4, the presence of the nonshattering single nucleotide polymorphism allele in the weedy rice accession suggested involvement of a linked locus or an alternative molecular genetic mechanism. Overlapping of several QTLs with those from earlier studies indicated that weedy rice may have been derived from the wild species Oryza rufipogon. Natural hybridization of rice cultivars with the highly variable O. rufipogon present in different geographic regions might be responsible for the evolution of a wide range of phenotypic and genotypic variabilities seen in weedy rice populations worldwide.

Overexpression of an adenosine diphosphate-ribosylation factor gene from the halophytic grass Spartina alterniflora confers salinity and drought tolerance in transgenic Arabidopsis.

Adenosine diphosphate-ribosylation factors (ARFs) are small guanine nucleotide-binding proteins that play an important role in intracellular protein trafficking necessary for undertaking multiple physiological functions in plant growth and developmental processes. However, little is known about the mechanism of ARF functioning at the molecular level, as well as its involvement in abiotic stress tolerance. In this study, we demonstrated the direct involvement of an ARF gene SaARF from a grass halophyte Spartina alterniflora in abiotic stress adaptation for the first time. SaARF, which encodes a protein with predicted molecular mass of 21 kDa, revealed highest identity with ARF of Oryza sativa. The SaARF gene is transcriptionally regulated by salt, drought, cold, and ABA in the leaves and roots of S. alterniflora. Arabidopsis plants overexpressing SaARF showed improved seed germination and survival of seedlings under salinity stress. Similarly, SaARF transgenic Arabidopsis plants were more tolerant to drought stress, compared to wild-type plants, by maintaining chlorophyll synthesis, increasing osmolyte synthesis, and stabilizing membrane integrity. Oxidative damage due to moisture stress in transgenic Arabidopsis was also reduced possibly by activating antioxidant genes, AtSOD1 and AtCAT. Our results suggest that enhanced drought and salinity tolerance conferred by the SaARF gene may be due to its role in mediating multiple abiotic stress tolerance mechanisms.

Phytochemical Profiling and Bioactive Potential of Grape Seed Extract in Enhancing Salinity Tolerance of Vicia faba.

Salinity stress poses a significant threat to crop productivity worldwide, necessitating effective mitigation strategies. This study investigated the phytochemical composition and potential of grape seed extract (GSE) to mitigate salinity stress effects on faba bean plants. GC-MS analysis revealed several bioactive components in GSE, predominantly fatty acids. GSE was rich in essential nutrients and possessed a high antioxidant capacity. After 14 days of germination, GSE was applied as a foliar spray at different concentrations (0, 2, 4, 6, and 8 g/L) to mitigate the negative effects of salt stress (150 mM NaCl) on faba bean plants. Foliar application of 2-8 g/L GSE significantly enhanced growth parameters such as shoot length, root length, fresh weight, and dry weight of salt-stressed bean plants compared to the control. The Fv/Fm ratio, indicating photosynthetic activity, also improved with GSE treatment under salinity stress compared to the control. GSE effectively alleviated the oxidative stress induced by salinity, reducing malondialdehyde, hydrogen peroxide, praline, and glycine betaine levels. Total soluble proteins, amino acids, and sugars were enhanced in GSE-treated, salt-stressed plants. GSE treatment under salinity stress modulated the total antioxidant capacity, antioxidant responses, and enzyme activities such as peroxidase, ascorbate peroxidase, and polyphenol oxidase compared to salt-stressed plants. Gene expression analysis revealed GSE (6 g/L) upregulated photosynthesis (chlorophyll a/b-binding protein of LHCII type 1-like (Lhcb1) and ribulose bisphosphate carboxylase large chain-like (RbcL)) and carbohydrate metabolism (cell wall invertase I (CWINV1) genes) while downregulating stress response genes (ornithine aminotransferase (OAT) and ethylene-responsive transcription factor 1 (ERF1)) in salt-stressed bean plants. The study demonstrates GSE's usefulness in mitigating salinity stress effects on bean plants by modulating growth, physiology, and gene expression patterns, highlighting its potential as a natural approach to enhance salt tolerance.

Genome-Wide Association Study Identified Candidate Genes for Alkalinity Tolerance in Rice.

Alkalinity stress is a major hindrance to enhancing rice production globally due to its damaging effect on plants' growth and development compared with salinity stress. However, understanding of the physiological and molecular mechanisms of alkalinity tolerance is limited. Therefore, a panel of indica and japonica rice genotypes was evaluated for alkalinity tolerance at the seedling stage in a genome-wide association study to identify tolerant genotypes and candidate genes. Principal component analysis revealed that traits such as alkalinity tolerance score, shoot dry weight, and shoot fresh weight had the highest contribution to variations in tolerance, while shoot Na+ concentration, shoot Na+:K+ ratio, and root-to-shoot ratio had moderate contributions. Phenotypic clustering and population structure analysis grouped the genotypes into five subgroups. Several salt-susceptible genotypes such as IR29, Cocodrie, and Cheniere placed in the highly tolerant cluster suggesting different underlying tolerance mechanisms for salinity and alkalinity tolerance. Twenty-nine significant SNPs associated with alkalinity tolerance were identified. In addition to three alkalinity tolerance QTLs, qSNK4, qSNC9, and qSKC10, which co-localized with the earlier reported QTLs, a novel QTL, qSNC7, was identified. Six candidate genes that were differentially expressed between tolerant and susceptible genotypes were selected: LOC_Os04g50090 (Helix-loop-helix DNA-binding protein), LOC_Os08g23440 (amino acid permease family protein), LOC_Os09g32972 (MYB protein), LOC_Os08g25480 (Cytochrome P450), LOC_Os08g25390 (Bifunctional homoserine dehydrogenase), and LOC_Os09g38340 (C2H2 zinc finger protein). The genomic and genetic resources such as tolerant genotypes and candidate genes would be valuable for investigating the alkalinity tolerance mechanisms and for marker-assisted pyramiding of the favorable alleles for improving alkalinity tolerance at the seedling stage in rice.

Comparison of the Morpho-Physiological and Molecular Responses to Salinity and Alkalinity Stresses in Rice.

Rice is a major food crop that has a critical role in ensuring food security for the global population. However, major abiotic stresses such as salinity and alkalinity pose a major threat to rice farming worldwide. Compared with salinity stress, there is limited progress in elucidating the molecular mechanisms associated with alkalinity tolerance in rice. Since both stresses coexist in coastal and arid regions, unraveling of the underlying molecular mechanisms will help the breeding of high-yielding stress-tolerant rice varieties for these areas. This study examined the morpho-physiological and molecular response of four rice genotypes to both salinity and alkalinity stresses. Geumgangbyeo was highly tolerant and Mermentau was the least tolerant to both stresses, while Pokkali and Bengal were tolerant to only salinity and alkalinity stress, respectively. A set of salinity and alkalinity stress-responsive genes showed differential expression in the above rice genotypes under both stress conditions. The expression patterns were consistent with the observed morphological responses in these rice genotypes, suggesting the potential role of these genes in regulating tolerance to these abiotic stresses. Overall, this study suggested that divergence in response to alkalinity and salinity stresses among rice genotypes could be due to different molecular mechanisms conferring tolerance to each stress. In addition to providing a basis for further investigations into differentiating the molecular bases underlying tolerance, this study also emphasizes the possibilities of developing climate-resilient rice varieties using donors that are tolerant to both abiotic stresses.

Overexpression of a nascent polypeptide associated complex gene (SaßNAC) of Spartina alterniflora improves tolerance to salinity and drought in transgenic Arabidopsis.

Salinity and drought are the most important environmental constraints limiting crop growth and productivity. Here, we have characterized a gene 'SaßNAC' encoding the ß subunit of nascent polypeptide associated complex from a halophyte Spartina alterniflora and investigated its role toward abiotic stress regulation. Expression of SaßNAC was differentially regulated by abiotic stresses, including salinity, drought, cold, and ABA in leaves and roots of S. alterniflora. Constitutive over-expression of SaßNAC in Arabidopsis exhibited normal growth under non-stress conditions but enhanced tolerance to salt and drought stresses. Transgenic SaßNAC Arabidopsis retained more chlorophyll, proline, and showed improved ionic homeostasis with less damage under stress conditions compared to WT plants. This is a first report to demonstrate the involvement of ßNAC in imparting abiotic stress tolerance which might be due to protection of the newly synthesized polypeptides involved in various stress tolerance mechanisms from abiotic stress induced damage and inhibition of cell death in plant.

A stress inducible SUMO conjugating enzyme gene (SaSce9) from a grass halophyte Spartina alterniflora enhances salinity and drought stress tolerance in Arabidopsis.

BACKGROUND: SUMO (Small Ubiquitin related Modifier) conjugation is a post translational regulatory process found in all eukaryotes, mediated by SUMO activating enzyme, SUMO conjugating enzyme, and SUMO ligase for the attachment of SUMO to its target protein. Although the mechanism for regulation of SUMO conjugation pathway genes under abiotic stress has been studied to certain extent, the role of SUMO conjugating enzyme in improving abiotic stress tolerance to plant is largely unexplored. Here, we have characterized a SUMO conjugating enzyme gene 'SaSce9' from a halophytic grass Spartina alterniflora and investigated its role in imparting abiotic stress tolerance. RESULTS: SaSce9 gene encodes for a polypeptide of 162 amino acids with a molecular weight of ~18 kD and isoelectric point 8.43. Amino acid sequence comparisons of SaSce9 with its orthologs from other plant species showed high degree (~85-93%) of structural conservation among each other. Complementation analysis using yeast SCE mutant, Ubc9, revealed functional conservation of SaSce9 between yeast and S. alterniflora. SaSce9 transcript was inducible by salinity, drought, cold, and exogenously supplied ABA both in leaves and roots of S. alterniflora. Constitutive overexpression of SaSce9 in Arabidopsis through Agrobacterium mediated transformation improved salinity and drought tolerance of Arabidopsis. SaSce9 overexpressing Arabidopsis plants retained more chlorophyll and proline both under salinity and drought stress. SaSce9 transgenic plants accumulated lower levels of reactive oxygen under salinity stress. Expression analysis of stress responsive genes in SaSce9 Arabidopsis plants revealed the increased expression of antioxidant genes, AtSOD and AtCAT, ion antiporter genes, AtNHX1 and AtSOS1, a gene involved in proline biosynthesis, AtP5CS, and a gene involved in ABA dependent signaling pathway, AtRD22. CONCLUSIONS: These results highlight the prospect of improving abiotic stress tolerance in plants through genetic engineering of the sumoylation pathway. The study provides evidence that the overexpression of SaSce9 in plant can improve salinity and drought stress tolerance by protecting the plant through scavenging of ROS, accumulation of an osmolyte, proline, and expression of stress responsive genes. In addition, this study demonstrates the potential of the halophyte grass S. alterniflora as a reservoir of abiotic stress related genes for crop improvement.

Genetic Dissection of Alkalinity Tolerance at the Seedling Stage in Rice (Oryza sativa) Using a High-Resolution Linkage Map.

Although both salinity and alkalinity result from accumulation of soluble salts in soil, high pH and ionic imbalance make alkaline stress more harmful to plants. This study aimed to provide molecular insights into the alkalinity tolerance using a recombinant inbred line (RIL) population developed from a cross between Cocodrie and Dular with contrasting response to alkalinity stress. Forty-six additive QTLs for nine morpho-physiological traits were mapped on to a linkage map of 4679 SNPs under alkalinity stress at the seedling stage and seven major-effect QTLs were for alkalinity tolerance scoring, Na+ and K+ concentrations and Na+:K+ ratio. The candidate genes were identified based on the comparison of the impacts of variants of genes present in five QTL intervals using the whole genome sequences of both parents. Differential expression of no apical meristem protein, cysteine protease precursor, retrotransposon protein, OsWAK28, MYB transcription factor, protein kinase, ubiquitin-carboxyl protein, and NAD binding protein genes in parents indicated their role in response to alkali stress. Our study suggests that the genetic basis of tolerance to alkalinity stress is most likely different from that of salinity stress. Introgression and validation of the QTLs and genes can be useful for improving alkalinity tolerance in rice at the seedling stage and advancing understanding of the molecular genetic basis of alkalinity stress adaptation.

Whole genome sequence analysis of rice genotypes with contrasting response to salinity stress.

Salinity is a major abiotic constraint for rice farming. Abundant natural variability exists in rice germplasm for salt tolerance traits. Since few studies focused on the genome level variation in rice genotypes with contrasting response to salt stress, genomic resequencing in diverse genetic materials is needed to elucidate the molecular basis of salt tolerance mechanisms. The whole genome sequences of two salt tolerant (Pokkali and Nona Bokra) and three salt sensitive (Bengal, Cocodrie, and IR64) rice genotypes were analyzed. A total of 413 million reads were generated with a mean genome coverage of 93% and mean sequencing depth of 18X. Analysis of the DNA polymorphisms revealed that 2347 nonsynonymous SNPs and 51 frameshift mutations could differentiate the salt tolerant from the salt sensitive genotypes. The integration of genome-wide polymorphism information with the QTL mapping and expression profiling data led to identification of 396 differentially expressed genes with large effect variants in the coding regions. These genes were involved in multiple salt tolerance mechanisms, such as ion transport, oxidative stress tolerance, signal transduction, and transcriptional regulation. The genome-wide DNA polymorphisms and the promising candidate genes identified in this study represent a valuable resource for molecular breeding of salt tolerant rice varieties.

Heat stress alters the expression of salt stress induced genes in smooth cordgrass (Spartina alterniflora L.).

To understand the tolerance mechanism associated with high temperature stress in a halophyte smooth cordgrass (Spartina alterniflora), a few selected salt-induced genes including transcription factors were studied for their transcript abundance. The mRNA analysis of eight genes and nine transcription factors known to be involved in various salt tolerance mechanisms revealed temporal and tissue-dependent variation in their expression under high temperature stress. Differential response of genes under heat and salt stress (reported earlier) indicated different mode of action in the metabolic pathway in response to different environmental cues, and a few common genes responsive to multiple stresses showed temporal and tissue-dependent variation in their expression. This study demonstrates that S. alterniflora could be a potential source of candidate genes conferring tolerance against high temperature in addition to salt tolerance for crop improvement.

Genetic Diversity, Population Structure, and Marker-Trait Association for Drought Tolerance in US Rice Germplasm.

Drought is a major constraint in some rice-growing areas of the United States. Its impact is most severe at the reproductive stage resulting in low grain yield. Therefore, assessment of genetic and phenotypic variation for drought tolerance in US rice germplasm is necessary to accelerate the breeding effort. Evaluation of 205 US rice genotypes for drought tolerance at the reproductive stage revealed tolerant response in rice genotypes Bengal, Jupiter, Cypress, Jazzman, Caffey, and Trenasse. Harvest index and fresh shoot weight were identified as important traits to explain the majority of variability among the genotypes under drought tolerance. Genotyping with 80 SSR markers indicated a low level of genetic diversity in US germplasm. Population structure analysis grouped the genotypes into eight clusters. The genotypes from California, Louisiana, and Arkansas formed distinct subgroups. Texas genotypes were similar to those from Louisiana and Arkansas. Marker-trait association analysis showed significant association of RM570 and RM351 with grain yield, spikelet fertility, and harvest index whereas shoot dry weight showed association with RM302 and RM461. The drought-tolerant genotypes identified in this study and the SSR markers associated with drought tolerance attributes will be helpful for development of improved drought-tolerant rice varieties through marker assisted selection.

Meta-Analysis of Quantitative Trait Loci Associated with Seedling-Stage Salt Tolerance in Rice (Oryza sativa L.).

Soil and water salinity is one of the major abiotic stresses that reduce growth and productivity in major food crops including rice. The lack of congruence of salt tolerance quantitative trait loci (QTLs) in multiple genetic backgrounds and multiple environments is a major hindrance for undertaking marker-assisted selection (MAS). A genome-wide meta-analysis of QTLs controlling seedling-stage salt tolerance was conducted in rice using QTL information from 12 studies. Using a consensus map, 11 meta-QTLs for three traits with smaller confidence intervals were localized on chromosomes 1 and 2. The phenotypic variance of 3 meta-QTLs was ≥20%. Based on phenotyping of 56 diverse genotypes and breeding lines, six salt-tolerant genotypes (Bharathy, I Kung Ban 4-2 Mutant, Langmanbi, Fatehpur 3, CT-329, and IARI 5823) were identified. The perusal of the meta-QTL regions revealed several candidate genes associated with salt-tolerance attributes. The lack of association between meta-QTL linked markers and the level of salt tolerance could be due to the low resolution of meta-QTL regions and the genetic complexity of salt tolerance. The meta-QTLs identified in this study will be useful not only for MAS and pyramiding, but will also accelerate the fine mapping and cloning of candidate genes associated with salt-tolerance mechanisms in rice.

Transgene Pyramiding of Salt Responsive Protein 3-1 (SaSRP3-1) and SaVHAc1 From Spartina alterniflora L. Enhances Salt Tolerance in Rice.

The transgenic technology using a single gene has been widely used for crop improvement. But the transgenic pyramiding of multiple genes, a promising alternative especially for enhancing complexly inherited abiotic stress tolerance, has received little attention. Here, we developed and evaluated transgenic rice lines with a single Salt Responsive Protein 3-1 (SaSRP3-1) gene as well as pyramids with two-genes SaSRP3-1 and Vacuolar H+-ATPase subunit c1 (SaVHAc1) derived from a halophyte grass Spartina alterniflora L. for salt tolerance at seedling, vegetative, and reproductive stages. The overexpression of this novel gene SaSRP3-1 resulted in significantly better growth of E. coli with the recombinant plasmid under 600 mM NaCl stress condition compared with the control. During early seedling and vegetative stages, the single gene and pyramided transgenic rice plants showed enhanced tolerance to salt stress with minimal wilting and drying symptoms, improved shoot and root growth, and significantly higher chlorophyll content, relative water content, and K+/Na+ ratio than the control plants. The salt stress screening during reproductive stage revealed that the transgenic plants with single gene and pyramids had better grain filling, whereas the pyramided plants showed significantly higher grain yield and higher grain weight compared to control plants. Our study demonstrated transgenic pyramiding as a viable approach to achieve higher level of salt tolerance in crop plants.

Genetic analysis of yield and agronomic traits under reproductive-stage drought stress in rice using a high-resolution linkage map.

Drought stress at the reproductive stage of rice crop leads to a huge loss in grain yield. Identification and introgression of large effect drought tolerant QTLs are necessary to develop drought-tolerant rice varieties. Compared to the high-density linkage maps, widely spaced markers lead to the identification of QTLs with large confidence intervals which are difficult to incorporate in a breeding program. A previously generated genotyping-by-sequencing (GBS) based linkage map consisting of 4748 SNP markers was used to map QTLs in Cocodrie × N-22 recombinant inbred line (RIL) population. Twenty-one QTLs were discovered for days to flowering (DTF), plant height (PH), leaf rolling score (LRS), plant dry matter content (DM), spikelet fertility (SF), grain yield (GY), yield index (YI), and harvest index (HI) under drought stress. A major QTL qPH1.38 was identified in a narrow confidence interval on chromosome 1. The QTLs, qDTF3.01 and qPH1.38, overlapped with the previously identified QTL qDTY1.1 and Hd9, respectively. Another large-effect QTL qLRS1.37 was identified close to the sd1 locus on chromosome 1. A grain yield QTL qGY1.42 located on chromosome 1 contained only 4 candidate genes. There was no overlapping of QTLs for the root traits and the yield attributes. The important candidate genes present within the large effect QTL regions are MYB transcription factors, no apical meristem protein (NAC), potassium channel protein, nuclear matrix protein1, and chlorophyll A-B binding protein. The large effect QTLs (qDTF3.01, qPH1.38, and qLRS1.37) and a novel grain yield QTL qGYS1.42 can be used to incorporate in elite breeding lines to develop drought-tolerant rice varieties.