Exploring Potential of Pearl Millet Germplasm Association Panel for Association Mapping of Drought Tolerance Traits

A pearl millet inbred germplasm association panel (PMiGAP) comprising 250 inbred lines, representative of cultivated germplasm from Africa and Asia, elite improved open-pollinated cultivars, hybrid parental inbreds and inbred mapping population parents, was recently established. This study presents the first report of genetic diversity in PMiGAP and its exploitation for association mapping of drought tolerance traits. For diversity and genetic structure analysis, PMiGAP was genotyped with 37 SSR and CISP markers representing all seven linkage groups. For association analysis, it was phenotyped for yield and yield components and morpho-physiological traits under both well-watered and drought conditions, and genotyped with SNPs and InDels from seventeen genes underlying a major validated drought tolerance (DT) QTL. The average gene diversity in PMiGAP was 0.54. The STRUCTURE analysis revealed six subpopulations within PMiGAP. Significant associations were obtained for 22 SNPs and 3 InDels from 13 genes under different treatments. Seven SNPs associations from 5 genes were common under irrigated and one of the drought stress treatments. Most significantly, an important SNP in putative acetyl CoA carboxylase gene showed constitutive association with grain yield, grain harvest index and panicle yield under all treatments. An InDel in putative chlorophyll a/b binding protein gene was significantly associated with both stay-green and grain yield traits under drought stress. This can be used as a functional marker for selecting high yielding genotypes with ‘stay green’ phenotype under drought stress. The present study identified useful marker-trait associations of important agronomics traits under irrigated and drought stress conditions with genes underlying a major validated DT-QTL in pearl millet. Results suggest that PMiGAP is a useful panel for association mapping. Expression patterns of genes also shed light on some physiological mechanisms underlying pearl millet drought tolerance.     

Identification of functional candidate genes for drought tolerance in rice

Drought tolerance (DT) in rice is known to be controlled by many quantitative trait loci (QTLs) and involved differential expression of large numbers of genes, but linking QTLs with their underlying genes remains the most challenging issue in plant molecular biology. To shed some light on this issue, differential gene expression in response to PEG simulated drought in 3 unique genetic materials (a lowland rice, IR64 and its derived line, PD86 which has 11 introgressed DT QTLs, and a upland rice IRAT109) was investigated using a PCR-based subtractive hybridization strategy. More than 300 unique subtracted cDNA sequences, covering genes of diverse cellular activities and functions, were identified and confirmed by semi-quantitative and quantitative RT-PCR. Detailed bioinformatics analyses of the data revealed two interesting results. First, the levels and mechanisms of DT of the three rice lines were associated with the number and types of differentially expressed genes, suggesting different DT mechanisms in rice are controlled by different sets of genes and different metabolic pathways, and most differentially expressed genes under drought were able to contribute to DT. Second, there appeared a high correspondence in genomic location between DT QTLs and clusters of differentially expressed genes in rice, suggesting some DT QTLs may represent clusters of co-regulated and functionally related genes. Thus, differential gene expression analyses using genetically characterized materials can provide additional insights into the molecular basis of QTLs and convergent evidence to shortlist the candidate genes for target QTLs. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Bin-Ying Fu and Jian-Hua Xiong are contributed to this work equally.     

Drought tolerance genes in rice

Quantitative trait loci (QTLs) for drought tolerance (DT) can be readily identified in available databases and in this paper, these QTLs were summarized in the form of a consensus map. An in silico strategy was then deployed to mine for candidate genes associated with DT QTLs using rice dbEST and rice genome databases. DT QTLs on rice chromosomes 1, 2, 4, 8, and 9 were selected to test the method. The result showed candidate genes associated with DT could be readily identified.     

Genome-wide association mapping of yield components and drought tolerance-related traits in cotton

Drought causes serious yield losses in cotton production throughout the world. Association mapping allows identification and localization of the genes controlling drought-related traits which will be helpful in cotton breeding. In the present study, genetic diversity analysis and association mapping of yield and drought traits were performed on a panel of 99 upland cotton genotypes using 177 SSR (simple sequence repeat) markers. Yield parameters and drought tolerance-related traits were evaluated for two seasons under two watering regimes: water-stressed and well-watered. The traits included seed cotton yield (SCY), lint yield (LY), lint percentage (LP), water-use efficiency (WUE), yield potential (YP), yield reduction (YR), yield index (YI), drought sensitivity index (DSI), stress tolerance index (STI), harmonic mean (HM), and geometric mean productivity (GMP). The genotypes with the least change in seed cotton yield under drought stress were Zeta 2, Delcerro, Nazilli 87, and DAK 66/3 which were also the most water-use efficient cultivars. The average genetic diversity of the panel was 0.38. The linkage disequilibrium decayed relatively rapidly at 20–30 cM (r²?≥?0.5). We identified 30 different SSR markers associated with the traits. Fifteen and 23 SSR markers were linked to the traits under well-watered and water-stress conditions, respectively. To our knowledge, most of these quantitative yield and drought tolerance-associated loci were newly identified. The genetic diversity and association mapping results should facilitate the development of drought-tolerant cotton lines with high yield in molecular breeding programs.     

Grain yield responses to moisture regimes in a rice population: association among traits and genetic markers

Drought is a major constraint to rice (Oryza sativa L.) production in rainfed and poorly irrigated environments. Identifying genomic regions influencing the response of yield and its components to water deficits will aid our understanding of the genetic mechanism of drought tolerance (DT) of rice and the development of DT varieties. Grain yield (GY) and its components of a recombinant inbred population developed from a lowland rice and an upland rice were investigated under different water levels in 2003 and 2004 in a rainout DT screening facility. Correlation and path analysis indicated that spikelet fertility (SF) was particularly important for grain yield with direct effect (P=0.60) under drought stress, while spikelet number per panicle (SN) contributed the most to grain yield (P=0.41) under well-watered condition. A total of 32 quantitative trait loci (QTLs) for grain yield and its components were identified. The phenotypic variation explained by individual QTLs varied from 1.29% to 14.76%. Several main effect QTLs affecting SF, 1,000-grain weight (TGW), panicle number (PN), and SN were mapped to the same regions on chromosome 4 and 8. These QTLs were detected consistently across 2 years and under both water levels in this study. Several digenic interactions among yield components were also detected. The identification of genomic regions associated with GY and its components under stress will be useful to improve drought tolerance of rice by marker-aided approaches.G. H. Zou and H. W. Mei contribute equally to this work.     

Identification of Loci Associated with Drought Resistance Traits in Heterozygous Autotetraploid Alfalfa (Medicago sativa L.) Using Genome-Wide Association Studies with Genotyping by Sequencing

Drought resistance is an important breeding target for enhancing alfalfa productivity in arid and semi-arid regions. Identification of genes involved in drought tolerance will facilitate breeding for improving drought resistance and water use efficiency in alfalfa. Our objective was to use a diversity panel of alfalfa accessions comprised of 198 cultivars and landraces to identify genes involved in drought tolerance. The panel was selected from the USDA-ARS National Plant Germplasm System alfalfa collection and genotyped using genotyping by sequencing. A greenhouse procedure was used for phenotyping two important traits associated with drought tolerance: drought resistance index (DRI) and relative leaf water content (RWC). Marker-trait association identified nineteen and fifteen loci associated with DRI and RWC, respectively. Alignments of target sequences flanking to the resistance loci against the reference genome of M. truncatula revealed multiple chromosomal locations. Markers associated with DRI are located on all chromosomes while markers associated with RWC are located on chromosomes 1, 2, 3, 4, 5, 6 and 7. Co-localizations of significant markers between DRI and RWC were found on chromosomes 3, 5 and 7. Most loci associated with DRI in this work overlap with the reported QTLs associated with biomass under drought in alfalfa. Additional significant markers were targeted to several contigs with unknown chromosomal locations. BLAST search using their flanking sequences revealed homology to several annotated genes with functions in stress tolerance. With further validation, these markers may be used for marker-assisted breeding new alfalfa varieties with drought resistance and enhanced water use efficiency.     

Genetic Variation and Association Analysis of the SSR Markers Linked to the Major Drought-Yield QTLs of Rice

Drought is one of the major abiotic stresses, which hampers the production of rice worldwide. Informative molecular markers are valuable tools for improving the drought tolerance in various varieties of rice. The present study was conducted to evaluate the informative simple sequence repeat (SSR) markers in a diverse set of rice genotypes. The genetic diversity analyses of the 83 studied rice genotypes were performed using 34 SSR markers closely linked to the major quantitative trait loci (QTLs) of grain yield under drought stress (qDTYs). In general, our results indicated high levels of polymorphism. In addition, we screened these rice genotypes at the reproductive stage under both drought stress and nonstressful conditions. The results of the regression analysis demonstrated a significant relationship between 11 SSR marker alleles and the plant paddy weight under stressful conditions. Under the nonstressful conditions, 16 SSR marker alleles showed a significant correlation with the plant paddy weight. Finally, four markers (RM279, RM231, RM166, and RM231) demonstrated a significant association with the plant paddy weight under both stressful and nonstressful conditions. These informative-associated alleles may be useful for improving the crop yield under both drought stress and nonstressful conditions in breeding programs.     

SNPs in stress-responsive rice genes: validation, genotyping, functional relevance and population structure

ABSTRACT: BACKGROUND: Single nucleotide polymorphism (SNP) validation and large-scale genotyping are required to maximize the use of DNA sequence variation and determine the functional relevance of candidate genes for complex stress tolerance traits through genetic association in rice. We used the bead array platform-based Illumina GoldenGate assay to validate and genotype SNPs in a select set of stress-responsive genes to understand their functional relevance and study the population structure in rice. RESULTS: Of the 384 putative SNPs assayed, we successfully validated and genotyped 362 (94.3%). Of these 325 (84.6%) showed polymorphism among the 91 rice genotypes examined. Physical distribution, degree of allele sharing, admixtures and introgression, and amino acid replacement of SNPs in 263 abiotic and 62 biotic stress-responsive genes provided clues for identification and targeted mapping of trait-associated genomic regions. We assessed the functional and adaptive significance of validated SNPs in a set of contrasting drought tolerant upland and sensitive lowland rice genotypes by correlating their allelic variation with amino acid sequence alterations in catalytic domains and three-dimensional secondary protein structure encoded by stress-responsive genes. We found a strong genetic association among SNPs in the nine stress-responsive genes with upland and lowland ecological adaptation. Higher nucleotide diversity was observed in indica accessions compared with other rice sub-populations based on different population genetic parameters. The inferred ancestry of 16% among rice genotypes was derived from admixed populations with the maximum between upland aus and wild Oryza species. CONCLUSIONS: SNPs validated in biotic and abiotic stress-responsive rice genes can be used in association analyses to identify candidate genes and develop functional markers for stress tolerance in rice.     

Physiological characterization and allelic diversity of selected drought tolerant traditional rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) landraces of Koraput,India

Water-deficit stress tolerance in rice is important for maintaining stable yield, especially under rain-fed ecosystem. After a thorough drought-tolerance screening of more than 130 rice genotypes from various regions of Koraput in our previous study, six rice landraces were selected for drought tolerance capacity. These six rice landraces were further used for detailed physiological and molecular assessment under control and simulated drought stress conditions. After imposing various levels of drought stress, leaf photosynthetic rate (P_N), photochemical efficiency of photosystem II (Fv/Fm), SPAD chlorophyll index, membrane stability index and relative water content were found comparable with the drought-tolerant check variety (N22). Compared to the drought-susceptible variety IR64, significant positive attributes and varietal differences were observed for all the above physiological parameters in drought-tolerant landraces. Genetic diversity among the studied rice landraces was assessed using 19 previously reported drought tolerance trait linked SSR markers. A total of 50 alleles with an average of 2.6 per locus were detected at the loci of the 19 markers across studied rice genotypes. The Nei’s genetic diversity (He) and the polymorphism information content (PIC) ranged from 0.0 to 0.767 and 0.0 to 0.718, respectively. Seven SSR loci, such as RM324, RM19367, RM72, RM246, RM3549, RM566 and RM515, showed the highest PIC values and are thus, useful in assessing the genetic diversity of studied rice lines for drought tolerance. Based on the result, two rice landraces (Pandkagura and Mugudi) showed the highest similarity index with tolerant check variety. However, three rice landraces (Kalajeera, Machhakanta and Haldichudi) are more diverse and showed highest genetic distance with N22. These landraces can be considered as the potential genetic resources for drought breeding program.     

Genetic architecture of aluminum tolerance in rice (Oryza sativa) determined through genome-wide association analysis and QTL mapping

Aluminum (Al) toxicity is a primary limitation to crop productivity on acid soils, and rice has been demonstrated to be significantly more Al tolerant than other cereal crops. However, the mechanisms of rice Al tolerance are largely unknown, and no genes underlying natural variation have been reported. We screened 383 diverse rice accessions, conducted a genome-wide association (GWA) study, and conducted QTL mapping in two bi-parental populations using three estimates of Al tolerance based on root growth. Subpopulation structure explained 57% of the phenotypic variation, and the mean Al tolerance in Japonica was twice that of Indica. Forty-eight regions associated with Al tolerance were identified by GWA analysis, most of which were subpopulation-specific. Four of these regions co-localized with a priori candidate genes, and two highly significant regions co-localized with previously identified QTLs. Three regions corresponding to induced Al-sensitive rice mutants (ART1, STAR2, Nrat1) were identified through bi-parental QTL mapping or GWA to be involved in natural variation for Al tolerance. Haplotype analysis around the Nrat1 gene identified susceptible and tolerant haplotypes explaining 40% of the Al tolerance variation within the aus subpopulation, and sequence analysis of Nrat1 identified a trio of non-synonymous mutations predictive of Al sensitivity in our diversity panel. GWA analysis discovered more phenotype-genotype associations and provided higher resolution, but QTL mapping identified critical rare and/or subpopulation-specific alleles not detected by GWA analysis. Mapping using Indica/Japonica populations identified QTLs associated with transgressive variation where alleles from a susceptible aus or indica parent enhanced Al tolerance in a tolerant Japonica background. This work supports the hypothesis that selectively introgressing alleles across subpopulations is an efficient approach for trait enhancement in plant breeding programs and demonstrates the fundamental importance of subpopulation in interpreting and manipulating the genetics of complex traits in rice.     

Drought tolerance in rice: morphological and molecular genetic consideration

Rice is one of the most important food crop drastically affected by drought in lowland rice ecosystem. Dissecting out the traits of importance and genomic regions influencing the response of drought tolerance and yield traits on grain yield will aid the breeders to know the genetic mechanism of drought tolerance of rice leads to the development of drought tolerant varieties. Grain yield and its components on drought situation of recombinant inbred population (IR 58821/IR 52561) were investigated under lowland managed stress situation in 2003 and 2004 by given importance to the relative water content. Water deficit resulted in significant effect on phenology and grain yield. Best lines were selected for further varietal development programme. Variability studies showed the traits viz., days to 70% relative water content, leaf rolling, leaf drying, harvest index, biomass yield and grain yield offer high scope for improvement for drought tolerance by way of simple selection technique. Correlation and path analysis indicated that, to harness high yielding combined with drought tolerance breeders should give selection pressure on relative water content, panicle length, grains per panicle, harvest index, biomass yield, root/shoot ratio and root length in positive direction, and low scores of leaf rolling, leaf drying and drought recovery rate. Analysis of quantitative trait loci for drought tolerance, yield and its components allowed the identification of 38 regions associated with both drought tolerant and yield traits. Out of these, 18 were closely linked with DNA markers could be used for marker assisted selection in breeding for drought tolerance in rice. Pleiotropism and G × E effects interaction were noticed in some of the traits. Parent IR 58821 contributed favorable alleles for the entire drought related and most of the yield component traits. Identification of traits of importance and their nature of relationship by morphological and molecular level under lowland condition will be useful to improve drought tolerance of rice.