Genome-wide association mapping reveals key genomic regions for physiological and yield-related traits under salinity stress in wheat (Triticum aestivum L.).

A genome-wide association study (GWAS) was conducted using six different multi-locus GWAS models and 35K SNP array to demarcate genomic regions underlying reproductive stage salinity tolerance. Marker-trait association analysis was performed for salt tolerance indices (STI) of 11 morpho-physiological traits, and the actual concentrations of Na+ and K+, and the Na+/K+ ratio in flag leaf. A total of 293 significantly associated quantitative trait nucleotides (QTNs) for 14 morpho-physiological traits were identified. Of these 293 QTNs, 12 major QTNs with R2 ≥ 10.0% were detected in three or more GWAS models. Novel major QTNs were identified for plant height, number of effective tillers, biomass, grain yield, thousand grain weight, Na+ and K+ content, and the Na+/K+ ratio in flag leaf. Moreover, 48 candidate genes were identified from the associated genomic regions. The QTNs identified in this study could potentially be targeted for improving salinity tolerance in wheat.

Comparative RNA-Seq analysis unfolds a complex regulatory network imparting yellow mosaic disease resistance in mungbean [Vigna radiata (L.) R. Wilczek].

Yellow Mosaic Disease (YMD) in mungbean [Vigna radiata (L.) R. Wilczek] is one of the most damaging diseases in Asia. In the northern part of India, the YMD is caused by Mungbean Yellow Mosaic India Virus (MYMIV), while in southern India this is caused by Mungbean Yellow Mosaic Virus (MYMV). The molecular mechanism of YMD resistance in mungbean remains largely unknown. In this study, RNA-seq analysis was conducted between a resistant (PMR-1) and a susceptible (Pusa Vishal) mungbean genotype under infected and control conditions to understand the regulatory network operating between mungbean-YMV. Overall, 76.8 million raw reads could be generated in different treatment combinations, while mapping rate per library to the reference genome varied from 86.78% to 93.35%. The resistance to MYMIV showed a very complicated gene network, which begins with the production of general PAMPs (pathogen-associated molecular patterns), then activation of various signaling cascades like kinases, jasmonic acid (JA) and brassinosteroid (BR), and finally the expression of specific genes (like PR-proteins, virus resistance and R-gene proteins) leading to resistance response. The function of WRKY, NAC and MYB transcription factors in imparting the resistance against MYMIV could be established. The string analysis also revealed the role of proteins involved in kinase, viral movement and phytoene synthase activity in imparting YMD resistance. A set of novel stress-related EST-SSRs are also identified from the RNA-Seq data which may be used to find the linked genes/QTLs with the YMD resistance. Also, 11 defence-related transcripts could be validated through quantitative real-time PCR analysis. The identified gene networks have led to an insight about the defence mechanism operating against MYMIV infection in mungbean which will be of immense use to manage the YMD resistance in mungbean.

Insights into the Host-Pathogen Interaction Pathways through RNA-Seq Analysis of Lens culinaris Medik. in Response to Rhizoctonia bataticola Infection.

Dry root rot (Rhizoctonia bataticola) is an important disease of lentils (Lens culinaris Medik.).To gain an insight into the molecular aspects of host-pathogen interactions, the RNA-seq approach was used in lentils following inoculation with R.bataticola. The RNA-Seq has generated >450 million high-quality reads (HQRs) and nearly 96.97% were properly aligned to the reference genome. Very high similarity in FPKM (fragments per kilobase of exon per million mapped fragments) values (R > 0.9) among biological replicates showed the consistency of the RNA-Seq results. The study revealed various DEGs (differentially expressed genes) that were associated with changes in phenolic compounds, transcription factors (TFs), antioxidants, receptor kinases, hormone signals which corresponded to the cell wall modification enzymes, defense-related metabolites, and jasmonic acid (JA)/ethylene (ET) pathways. Gene ontology (GO) categorization also showed similar kinds of significantly enriched similar GO terms. Interestingly, of the total unigenes (42,606), 12,648 got assembled and showed significant hit with Rhizoctonia species. String analysis also revealed the role of various disease responsive proteins viz., LRR family proteins, LRR-RLKs, protein kinases, etc. in the host-pathogen interaction. Insilico validation analysis was performed using Genevestigator® and DEGs belonging to six major defense-response groups viz., defense-related enzymes, disease responsive genes, hormones, kinases, PR (pathogenesis related) proteins, and TFs were validated. For the first time some key miRNA targets viz. miR156, miR159, miR167, miR169, and miR482 were identified from the studied transcriptome, which may have some vital role in Rhizoctonia-based responses in lentils. The study has revealed the molecular mechanisms of the lentil/R.bataticola interactions and also provided a theoretical approach for the development of lentil genotypes resistant to R.bataticola.

Multi-locus genome-wide association studies reveal novel genomic regions associated with vegetative stage salt tolerance in bread wheat (Triticum aestivum L.).

Soil salinity is one of the typical abiotic stresses affecting sustainability of wheat production worldwide. In the present study, we performed a 35 K SNP genotyping assay on association panel of 135 diverse wheat genotypes evaluated for vegetative stage tolerance in hydroponics. Association analyses using five multi-locus GWAS models revealed 42 reliable QTNs for 10 salt tolerance associated traits. Among these 42 reliable QTNs, 9, 17 and 16 QTNs were associated with physiological, biomass and shoot ionic traits respectively. Novel major QTNs were identified for chlorophyll content, shoot fresh weight, seedling total biomass, Na+ and K+ concentration and Na+/K+ ratio in shoots. Further, 10 major QTNs showed significant effect on the corresponding salt tolerance traits. Gene ontology analysis of the associated genomic regions identified 58 candidate genes. The information generated in this study will be of potential value for improvement of salt tolerance of wheat cultivars using marker assisted selection.

Identification and evolutionary analysis of polycistronic miRNA clusters in domesticated and wild wheat.

MicroRNAs are ~22 nucleotide long non-coding RNAs that regulate gene expression at posttranscriptional level. Genome-wide analysis was performed to identify polycistronic miRNAs from wheat. Total 89 polycistronic miRNAs were identified in bread wheat which were distributed on three component sub-genomes (A = 26, B = 33 and D = 30). Except some, most of the identified polycistronic miRNAs were also present in other cultivated and wild wheat species. Expression of 11 identified polycistronic miRNAs could be validated using previously assembled transcriptomes, RNA-seq/s-RNA seq data of cultivated and wild wheats and RT-PCR. Polycistronic miRNAs orthologs were also localized on rice and Brachypodium genomes. As a case study, we also analyzed molecular evolution of miR395 family polycistrons in wheat. Both tandem and segmental duplications contributed to expansion of miR395 family polycistrons. Our findings provide a comprehensive view on wheat polycitronic miRNAs that will enable their in-depth functional analysis in the future.

Identification, analysis and development of salt responsive candidate gene based SSR markers in wheat.

BACKGROUND: Salinity severely limits wheat production in many parts of the world. Development of salt tolerant varieties represents the most practical option for enhancing wheat production from these areas. Application of marker assisted selection may assist in fast tracking development of salt tolerant wheat varieties. However, SSR markers available in the public domain are not specifically targeted to functional regions of wheat genome, therefore large numbers of these need to be analysed for identification of markers associated with traits of interest. With the availability of a fully annotated wheat genome assembly, it is possible to develop SSR markers specifically targeted to genic regions. We performed extensive analysis to identify candidate gene based SSRs and assessed their utility in characterizing molecular diversity in a panel of wheat genotypes. RESULTS: Our analysis revealed, 161 SSR motifs in 94 salt tolerance candidate genes of wheat. These SSR motifs were nearly equally distributed on the three wheat sub-genomes; 29.8% in A, 35.7% in B and 34.4% in D sub-genome. The maximum number of SSR motifs was present in exons (31.1%) followed by promoters (29.8%), 5'UTRs (21.1%), introns (14.3%) and 3'UTRs (3.7%). Out of the 65 candidate gene based SSR markers selected for validation, 30 were found polymorphic based on initial screening and employed for characterizing genetic diversity in a panel of wheat genotypes including salt tolerant and susceptible lines. These markers generated an average of 2.83 alleles/locus. Phylogenetic analysis revealed four clusters. Salt susceptible genotypes were mainly represented in clusters I and III, whereas high and moderate salt tolerant genotypes were distributed in the remaining two clusters. Population structure analysis revealed two sub-populations, sub-population 1 contained the majority of salt tolerant whereas sub-population 2 contained majority of susceptible genotypes. Moreover, we observed reasonably higher transferability of SSR markers to related wheat species. CONCLUSION: We have developed salt responsive gene based SSRs in wheat for the first time. These were highly useful in unravelling functional diversity among wheat genotypes with varying responses to salt stress. The identified gene based SSR markers will be valuable genomic resources for genetic/association mapping of salinity tolerance in wheat.