Genome-wide association study of soybean (Glycine max [L.] Merr.) germplasm for dissecting the quantitative trait nucleotides and candidate genes underlying yield-related traits.

Soybean (Glycine max [L.] Merr.) is one of the most significant crops in the world in terms of oil and protein. Owing to the rising demand for soybean products, there is an increasing need for improved varieties for more productive farming. However, complex correlation patterns among quantitative traits along with genetic interactions pose a challenge for soybean breeding. Association studies play an important role in the identification of accession with useful alleles by locating genomic sites associated with the phenotype in germplasm collections. In the present study, a genome-wide association study was carried out for seven agronomic and yield-related traits. A field experiment was conducted in 2015/2016 at two locations that include 155 diverse soybean germplasm. These germplasms were genotyped using SoySNP50K Illumina Infinium Bead-Chip. A total of 51 markers were identified for node number, plant height, pods per plant, seeds per plant, seed weight per plant, hundred-grain weight, and total yield using a multi-locus linear mixed model (MLMM) in FarmCPU. Among these significant SNPs, 18 were putative novel QTNs, while 33 co-localized with previously reported QTLs. A total of 2,356 genes were found in 250 kb upstream and downstream of significant SNPs, of which 17 genes were functional and the rest were hypothetical proteins. These 17 candidate genes were located in the region of 14 QTNs, of which ss715580365, ss715608427, ss715632502, and ss715620131 are novel QTNs for PH, PPP, SDPP, and TY respectively. Four candidate genes, Glyma.01g199200, Glyma.10g065700, Glyma.18g297900, and Glyma.14g009900, were identified in the vicinity of these novel QTNs, which encode lsd one like 1, Ergosterol biosynthesis ERG4/ERG24 family, HEAT repeat-containing protein, and RbcX2, respectively. Although further experimental validation of these candidate genes is required, several appear to be involved in growth and developmental processes related to the respective agronomic traits when compared with their homologs in Arabidopsis thaliana. This study supports the usefulness of association studies and provides valuable data for functional markers and investigating candidate genes within a diverse germplasm collection in future breeding programs.

Assessment of halotolerant bacterial and fungal consortia for augmentation of wheat in saline soils.

Adaptations of green technologies to counter abiotic stress, including salinity for crops like wheat by using halotolerant microbes, is a promising approach. The current study investigated 17 salt-affected agroecological zones from the Punjab and Sindh provinces of Pakistan to explore the potential of indigenous microbial flora, with their multiple biochemical characteristics in addition to plant growth promoting (PGP) traits, for enhanced wheat production in saline areas. Initially, 297 isolated pure bacterial colonies were screened for salt tolerance, biochemical, and PGP traits. Three bacterial strains belonging to Pantoea spp. and Erwinia rhaphontici with possession of multiple characteristics were selected for the development of the halotolerant bacterial consortium. Inoculation of two local wheat varieties, Faisalabad 2008 and Galaxy 2013, with the consortium for in vitro seed germination assay and sand microcosm experiments exhibited significant improvement of selected plant growth parameters like germination percentage and root structure. Two previously reported PGP fungal strains of Trichoderma harzianum and T. viridae were also used as fungal consortium separately for pot experiments and field trials. The pot experiments exhibited a positive correlation of consortia with metabolic viz. catalase, peroxidase, and proline and agronomical parameters including shoot length, dry weight, number of spikes, spike length, and 100 grain weight. To evaluate their performance under natural environmental conditions, field trials were conducted at three salt-affected sites. Agronomical attributes including days of flowering and maturity, flag leaf weight, length and width, shoot length, number of spikes, spike length, spike weight, number of seeds spike-1, 1,000 grain weight, and plot yield indicated the efficiency of these microbes to enhance wheat growth. Concisely, the bacterial consortium showed better performance and Faisalabad 2008 was a more resistant variety as compared to Galaxy 2013. Initial promising results indicate that further extensive research on indigenous microbes might lead to the development of Pakistan's first saline-specific biofertilizers and sustainable eco-friendly agriculture practices.

Genetic Analyses of Seed Longevity in Capsicum annuum L. in Cold Storage Conditions.

Seed longevity is the most important trait in the genebank management system. No seed can remain infinitely viable. There are 1241 accessions of Capsicum annuum L. available at the German Federal ex situ genebank at IPK Gatersleben. C. annuum (Capsicum) is the most economically important species of the genus Capsicum. So far, there is no report that has addressed the genetic basis of seed longevity in Capsicum. Here, we convened a total of 1152 Capsicum accessions that were deposited in Gatersleben over forty years (from 1976 to 2017) and assessed their longevity by analyzing the standard germination percentage after 5-40 years of storage at -15/-18 °C. These data were used to determine the genetic causes of seed longevity, along with 23,462 single nucleotide polymorphism (SNP) markers covering all of the 12 Capsicum chromosomes. Using the association-mapping approach, we identified a total of 224 marker trait associations (MTAs) (34, 25, 31, 35, 39, 7, 21 and 32 MTAs after 5-, 10-, 15-, 20-, 25-, 30-, 35- and 40-year storage intervals) on all the Capsicum chromosomes. Several candidate genes were identified using the blast analysis of SNPs, and these candidate genes are discussed.

Estimation of genetic diversity using seed storage protein (SSP) profiling in wild and cultivated species of Cicer L.

BACKGROUND: The narrow genetic diversity of chickpea is a serious impediment to modern cultivar creation. Seed storage proteins (SSPs) are stable and have minimal or no degradation when subjected to isolation and SDS-PAGE. METHODS AND RESULTS: We have characterized SSPs of 436 chickpea genotypes, belonging to nine annual Cicer species, originated from 47 countries by SDS-PAGE and determined the extent of genetic diversity in chickpea through clustering. Based on scoring, a total of 44 bands (10 to 170 kDa) were identified, which were all polymorphic. The least appeared protein bands were 11, 160 and 170 kDa where band of 11 and 160 kDa was present exclusively in wild type. Five bands were present in < 10% of genotypes. Bands appeared in 200-300 genotypes were suggested less polymorphic, on contrary bands present in 10-150 genotypes were suggested more polymorphic. Polymorphism of protein bands in context to their potential functions reported in literature were explored and suggested that the glubulins were most and glutelins were least abundant, whereas albumins with their known role in stress tolerance can be used as marker in chickpea breeding. Cluster analysis produced 14 clusters, interestingly three clusters contained only Pakistani genotypes and thus Pakistani genotypes appeared as a separate entity from the rest of the genotypes. CONCLUSION: Our results indicate that SDS-PAGE of SSPs is a powerful technique in determining the genetic diversity plus it is easily adaptable, due to its cost effectiveness in comparison to other genomics tools.

A GBS-based genome-wide association study reveals the genetic basis of salinity tolerance at the seedling stage in bread wheat (Triticum aestivum L.).

High salinity levels affect 20% of the cultivated area and 9%-34% of the irrigated agricultural land worldwide, ultimately leading to yield losses of crops. The current study evaluated seven salt tolerance-related traits at the seedling stage in a set of 138 pre-breeding lines (PBLs) and identified 63 highly significant marker-trait associations (MTAs) linked to salt tolerance. Different candidate genes were identified in in silico analysis, many of which were involved in various stress conditions in plants, including glycine-rich cell wall structural protein 1-like, metacaspase-1, glyceraldehyde-3-phosphate dehydrogenase GAPA1, and plastidial GAPA1. Some of these genes coded for structural protein and participated in cell wall structure, some were linked to programmed cell death, and others were reported to show abiotic stress response roles in wheat and other plants. In addition, using the Multi-Trait Genotype-Ideotype Distance Index (MGIDI) protocol, the best-performing lines under salt stress were identified. The SNPs identified in this study and the genotypes with favorable alleles provide an excellent source to impart salt tolerance in wheat.

A GBS-Based GWAS Analysis of Leaf and Stripe Rust Resistance in Diverse Pre-Breeding Germplasm of Bread Wheat (Triticum aestivum L.).

Yellow (YR) and leaf (LR) rusts caused by Puccinia striiformis f. sp. tritici (Pst) and Puccinia triticina, respectively, are of utmost importance to wheat producers because of their qualitative and quantitative effect on yield. The search for new loci resistant to both rusts is an ongoing challenge faced by plant breeders and pathologists. Our investigation was conducted on a subset of 168 pre-breeding lines (PBLs) to identify the resistant germplasm against the prevalent local races of LR and YR under field conditions followed by its genetic mapping. Our analysis revealed a range of phenotypic responses towards both rusts. We identified 28 wheat lines with immune response and 85 resistant wheat genotypes against LR, whereas there were only eight immune and 52 resistant genotypes against YR. A GWAS (genome-wide association study) identified 190 marker-trait associations (MTAs), where 120 were specific to LR and 70 were specific to YR. These MTAs were confined to 86 quantitative trait loci (QTLs), where 50 QTLs carried MTAs associated with only LR, 29 QTLs carried MTAs associated with YR, and seven QTLs carried MTAs associated with both LR and YR. Possible candidate genes at the site of these QTLs are discussed. Overall, 70 PBLs carried all seven LR/YR QTLs. Furthermore, there were five PBLs with less than five scores for both LR and YR carrying positive alleles of all seven YR/LR QTLs, which are fit to be included in a breeding program for rust resistance induction.

Genetic networks underlying salinity tolerance in wheat uncovered with genome-wide analyses and selective sweeps.

KEY MESSAGE: A genetic framework underpinning salinity tolerance at reproductive stage was revealed by genome-wide SNP markers and major adaptability genes in synthetic-derived wheats, and trait-associated loci were used to predict phenotypes. Using wild relatives of crops to identify genes related to improved productivity and resilience to climate extremes is a prioritized area of crop genetic improvement. High salinity is a widespread crop production constraint, and development of salt-tolerant cultivars is a sustainable solution. We evaluated a panel of 294 wheat accessions comprising synthetic-derived wheat lines (SYN-DERs) and modern bread wheat advanced lines under control and high salinity conditions at two locations. The GWAS analysis revealed a quantitative genetic framework of more than 200 loci with minor effect underlying salinity tolerance at reproductive stage. The significant trait-associated SNPs were used to predict phenotypes using a GBLUP model, and the prediction accuracy (r2) ranged between 0.57 and 0.74. The r2 values for flag leaf weight, days to flowering, biomass, and number of spikes per plant were all above 0.70, validating the phenotypic effects of the loci discovered in this study. Furthermore, the germplasm sets were compared to identify selection sweeps associated with salt tolerance loci in SYN-DERs. Six loci associated with salinity tolerance were found to be differentially selected in the SYN-DERs (12.4 Mb on chromosome (chr)1B, 7.1 Mb on chr2A, 11.2 Mb on chr2D, 200 Mb on chr3D, 600 Mb on chr6B, and 700.9 Mb on chr7B). A total of 228 reported markers and genes, including 17 well-characterized genes, were uncovered using GWAS and EigenGWAS. A linkage disequilibrium (LD) block on chr5A, including the Vrn-A1 gene at 575 Mb and its homeologs on chr5D, were strongly associated with multiple yield-related traits and flowering time under salinity stress conditions. The diversity panel was screened with more than 68 kompetitive allele-specific PCR (KASP) markers of functional genes in wheat, and the pleiotropic effects of superior alleles of Rht-1, TaGASR-A1, and TaCwi-A1 were revealed under salinity stress. To effectively utilize the extensive genetic information obtained from the GWAS analysis, a genetic interaction network was constructed to reveal correlations among the investigated traits. The genetic network data combined with GWAS, selective sweeps, and the functional gene survey provided a quantitative genetic framework for identifying differentially retained loci associated with salinity tolerance in wheat.

Genetic Insight Into the Insect Resistance in Bread Wheat Exploiting the Untapped Natural Diversity.

Climate change is an undeniable threat to sustainable wheat production in the future as an increased temperature will significantly increase grain loss due to the increased number of generations per season of multivoltine species that are detrimental to plants. Among insects, orange wheat blossom midge (OWBM), yellow wheat blossom midge (YWBM), saddle gall midge (SGM), thrips, and frit fly (FF) are important wheat pests in the European environments, which can be managed by the development of resistant cultivars. This involves the identification, confirmation, and incorporation of insect resistance sources into new high-yielding cultivars. We used two diverse and unrelated wheat [winter wheat (WW) and spring wheat (SW)] panels to associate single-nucleotide polymorphism (SNP) markers with the mentioned pests using the tools of association mapping. All in all, a total of 645 and 123 significant associations were detected in WW and SW, respectively, which were confined to 246 quantitative trait loci. Many candidate genes were identified using the BLAST analysis of the sequences of associated SNPs. Some of them are involved in controlling the physical structures of plants such as stomatal immunity and closure, cuticular wax in leaf blade, whereas others are involved in the production of certain enzymes in response to biotic and abiotic stresses. To our knowledge, this is the first detailed investigation that deals with YWBM, SGM, thrips, and FF resistance genetics using the natural variation in wheat. The reported germplasm is also readily available to breeders across the world that can make rational decisions to breed for the pest resilience of their interest by including the resistant genotypes being reported.

Genetic basis of some physiological traits and yield in early and late sowing conditions in bread wheat (Triticum aestivum L.).

The rise in human population necessitates the use of all available tools to enhance wheat productivity. In this regard, pre-breeding has mobilized novel underutilized genetic variation into breeding programs. However, this germplasm needs to be characterized for its efficient utilization. This investigation was initiated to evaluate the early and late sown wheat pre-breeding germplasm for physiology- and yield-related traits and to associate the mapped SNPs using association mapping approach. Our results indicate that the germplasm performed better in early sowing in comparison to late planting where grain yield (Yd) was found positively correlated with water use efficiency (WUE), heading time, and chlorophyll contents (Chl). We discovered a total of 210 associations involving 155 SNPs. Taking into consideration either early or late sowing and the mean values, only 12 marker traits were associated with trait germination, plant height, stomatal conductance, transpiration rate, Chl, carotenoids, and Yd. Our correlations and mapping results indicate that higher WUE along with Chl can be targeted as indirect physiological markers to enhance wheat yield.

A comparative metabolomic study on desi and kabuli chickpea (Cicer arietinum L.) genotypes under rainfed and irrigated field conditions.

Chickpea is considered among the most important leguminous crops in the world. However, in recent years drought conditions and/or limited availability of water have significantly reduced the production of chickpea. The current study was aimed to understand the legume stress response at the metabolic level for the determination of chickpea genotypes which can resist yield losses and could be cultivated with limited water availability. Here, we have analyzed two genotypes of chickpea, desi and kabuli under rainfed condition using a GC-MS based untargeted metabolomics approach. Results revealed significant differences in several metabolite features including oxalic acid, threonic acid, inositol, maltose and L-proline between studied groups. Accumulation of plant osmoprotectants such as L-proline, sugars and sugar alcohols was higher in desi genotype than kabuli genotype of chickpea when grown under the rainfed condition. Metabolic pathway analysis suggests that the inositol phosphate metabolism was involved in plant defense mechanisms against the limited water availability.

Mapping of QTLs Associated with Yield and Yield Related Traits in Durum Wheat (Triticum durum Desf.) Under Irrigated and Drought Conditions.

Global durum wheat consumption (Triticum durum Desf.) is ahead of its production. One reason for this is abiotic stress, e.g., drought. Breeding for resistance to drought is complicated by the lack of fast, reproducible screening techniques and the inability to routinely create defined and repeatable water stress conditions. Here, we report the first analysis of dissection of yield and yield-related traits in durum wheat in Pakistan, seeking to elucidate the genetic components of yield and agronomic traits. Analysis of several traits revealed a total of 221 (160 with logarithm of odds (LOD) > 2 ≤ 3 and 61 with LOD > 3) quantitative trait loci (QTLs) distributed on all fourteen durum wheat chromosomes, of which 109 (78 with LOD > 2 ≤ 3 and 31 with LOD > 3) were observed in 2016-17 (S1) and 112 (82 with LOD > 2 ≤ 3 and 30 with LOD > 3) were observed in 2017-18 (S2). Allelic profiles of yield QTLs on chromosome 2A and 7B indicate that allele A of Xgwm895 and allele B of Xbarc276 can enhance the Yd up to 6.16% in control and 5.27% under drought. Moreover, if combined, a yield gain of up to 11% would be possible.

A Comparative Study for Assessing the Drought-Tolerance of Chickpea Under Varying Natural Growth Environments.

This study was planned with the purpose of evaluating the drought tolerance of advanced breeding lines of chickpea in natural field conditions. Two methods were employed to impose field conditions; the first: simulating drought stress by growing chickpea genotypes at five rainfed areas, with Faisalabad as the non-stressed control environment; and the second: planting chickpea genotypes in spring to simulate a drought stress environment, with winter-sowing serving as the non-stressed environment. Additive main effects and multiplicative interaction (AMMI) and generalized linear models (GLM) models were both found to be equally effective in extracting main effects in the rainfed experiment. Results demonstrated that environment influenced seed yield, number of primary and secondary branches, number of pods, and number of seeds most predominantly; however, genotype was the main source of variation in 100 seed weight and plant height. The GGE biplot showed that Faisalabad, Kallur Kot, and Bhakkar were contributing the most in the GEI, respectively, while Bahawalpur, Bhawana, and Karor were relatively stable environments, respectively. Faisalabad was the most, and Bhakkar the least productive in terms of seed yield. The best genotypes to grow in non-stressed environments were CH39/08, CH40/09, and CH15/11, whereas CH28/07 and CH39/08 were found suitable for both conditions. CH55/09 displayed the best performance in stress conditions only. The AMMI stability and drought-tolerance indices enabled us to select genotypes with differential performance in both conditions. It is therefore concluded that the spring-sown experiment revealed a high-grade drought stress imposition on plants, and that the genotypes selected by both methods shared quite similar rankings, and also that manually computed drought-tolerance indices are also comparable for usage for better genotypic selections. This study could provide sufficient evidence for using the aforementioned as drought-tolerance evaluation methods, especially for countries and research organizations who have limited resources and funding for conducting multilocation trials, and performing sophisticated analyses on expensive software.