Combining Fourier-transform infrared spectroscopy and multivariate analysis for chemotyping of cell wall composition in Mungbean (Vigna radiata (L.) Wizcek).

BACKGROUND: Dissection of complex plant cell wall structures demands a sensitive and quantitative method. FTIR is used regularly as a screening method to identify specific linkages in cell walls. However, quantification and assigning spectral bands to particular cell wall components is still a major challenge, specifically in crop species. In this study, we addressed these challenges using ATR-FTIR spectroscopy as it is a high throughput, cost-effective and non-destructive approach to understand the plant cell wall composition. This method was validated by analysing different varieties of mungbean which is one of the most important legume crops grown widely in Asia. RESULTS: Using standards and extraction of a specific component of cell wall components, we assigned 1050-1060 cm-1 and 1390-1420 cm-1 wavenumbers that can be widely used to quantify cellulose and lignin, respectively, in Arabidopsis, Populus, rice and mungbean. Also, using KBr as a diluent, we established a method that can relatively quantify the cellulose and lignin composition among different tissue types of the above species. We further used this method to quantify cellulose and lignin in field-grown mungbean genotypes. The ATR-FTIR-based study revealed the cellulose content variation ranges from 27.9% to 52.3%, and the lignin content variation ranges from 13.7% to 31.6% in mungbean genotypes. CONCLUSION: Multivariate analysis of FT-IR data revealed differences in total cell wall (600-2000 cm-1), cellulose (1000-1100 cm-1) and lignin (1390-1420 cm-1) among leaf and stem of four plant species. Overall, our data suggested that ATR-FTIR can be used for the relative quantification of lignin and cellulose in different plant species. This method was successfully applied for rapid screening of cell wall composition in mungbean stem, and similarly, it can be used for screening other crops or tree species.

Modifying lignin composition and xylan O-acetylation induces changes in cell wall composition, extractability, and digestibility.

BACKGROUND: Lignin and xylan are important determinants of cell wall structure and lignocellulosic biomass digestibility. Genetic manipulations that individually modify either lignin or xylan structure improve polysaccharide digestibility. However, the effects of their simultaneous modifications have not been explored in a similar context. Here, both individual and combinatorial modification in xylan and lignin was studied by analysing the effect on plant cell wall properties, biotic stress responses and integrity sensing. RESULTS: Arabidopsis plant co-harbouring mutation in FERULATE 5-HYDROXYLASE (F5H) and overexpressing Aspergillus niger acetyl xylan esterase (35S:AnAXE1) were generated and displayed normal growth attributes with intact xylem architecture. This fah1-2/35S:AnAXE1 cross was named as hyper G lignin and hypoacetylated (HrGHypAc) line. The HrGHypAc plants showed increased crystalline cellulose content with enhanced digestibility after chemical and enzymatic pre-treatment. Moreover, both parents and HrGHypAc without and after pre-treating with glucuronyl esterase and alpha glucuronidase exhibited an increase in xylose release after xylanase digestion as compared to wild type. The de-pectinated fraction in HrGHypAc displayed elevated levels of xylan and cellulose. Furthermore, the transcriptomic analysis revealed differential expression in cell wall biosynthetic, transcription factors and wall-associated kinases genes implying the role of lignin and xylan modification on cellular regulatory processes. CONCLUSIONS: Simultaneous modification in xylan and lignin enhances cellulose content with improved saccharification efficiency. These modifications loosen cell wall complexity and hence resulted in enhanced xylose and xylobiose release with or without pretreatment after xylanase digestion in both parent and HrGHypAc. This study also revealed that the disruption of xylan and lignin structure is possible without compromising either growth and development or defense responses against Pseudomonas syringae infection.

Identification of quantitative trait loci (QTLs) regulating leaf SPAD value and trichome density in mungbean (Vigna radiata L.) using genotyping-by-sequencing (GBS) approach.

Quantitative trait loci (QTL) mapping is used for the precise localization of genomic regions regulating various traits in plants. Two major QTLs regulating Soil Plant Analysis Development (SPAD) value (qSPAD-7-1) and trichome density (qTric-7-2) in mungbean were identified using recombinant inbred line (RIL) populations (PMR-1×Pusa Baisakhi) on chromosome 7. Functional analysis of QTL region identified 35 candidate genes for SPAD value (16 No) and trichome (19 No) traits. The candidate genes regulating trichome density on the dorsal leaf surface of the mungbean include VRADI07G24840, VRADI07G17780, and VRADI07G15650, which encodes for ZFP6, TFs bHLH DNA-binding superfamily protein, and MYB102, respectively. Also, candidate genes having vital roles in chlorophyll biosynthesis are VRADIO7G29860, VRADIO7G29450, and VRADIO7G28520, which encodes for s-adenosyl-L-methionine, FTSHI1 protein, and CRS2-associated factor, respectively. The findings unfolded the opportunity for the development of customized genotypes having high SPAD value and high trichome density having a possible role in yield and mungbean yellow vein mosaic India virus (MYMIV) resistance in mungbean.

Xylobiose treatment triggers a defense-related response and alters cell wall composition.

Plant cell wall-derived oligosaccharides, i.e., damage-associated molecular patterns (DAMPs), could be generated after pathogen attack or during normal plant development, perceived by cell wall receptors, and can alter immunity and cell wall composition. Therefore, we hypothesised that xylo-oligosaccharides (XOS) could act as an elicitor and trigger immune responses. To test this, we treated Arabidopsis with xylobiose (XB) and investigated different parameters. XB-treatment significantly triggered the generation of reactive oxygen species (ROS), activated MAPK protein phosphorylation, and induced callose deposition. The combination of XB (DAMP) and flg22 a microbe-associated molecular pattern (MAMP) further enhanced ROS response and gene expression of PTI marker genes. RNA sequencing analysis revealed that more genes were differentially regulated after 30 min compared to 24 h XB-treated leaves, which correlated with ROS response. Increased xylosidase activity and soluble xylose level after 30 min and 3 h of XB-treatment were observed which might have weakened the DAMP response. However, an increase in total cell wall sugar and a decrease in uronic acid level was observed at both 30 min and 24 h. Additionally, arabinose, rhamnose, and xylose levels were increased in 30 min, and glucose was increased in 24 h compared to mock-treated leaves. The level of jasmonic acid, abscisic acid, auxin, and cytokinin were also affected after XB treatment. Overall, our data revealed that the shortest XOS can act as a DAMP, which triggers the PTI response and alters cell wall composition and hormone level.

Morpho-biochemical characterization of a RIL population for seed parameters and identification of candidate genes regulating seed size trait in lentil (Lens culinaris Medik.).

The seed size and shape in lentil (Lens culinaris Medik.) are important quality traits as these influences the milled grain yield, cooking time, and market class of the grains. Linkage analysis was done for seed size in a RIL (F5:6) population derived by crossing L830 (20.9 g/1000 seeds) with L4602 (42.13 g/1000 seeds) which consisted of 188 lines (15.0 to 40.5 g/1000 seeds). Parental polymorphism survey using 394 SSRs identified 31 polymorphic primers, which were used for the bulked segregant analysis (BSA). Marker PBALC449 differentiated the parents and small seed size bulk only, whereas large seeded bulk or the individual plants constituting the large-seeded bulk could not be differentiated. Single plant analysis identified only six recombinant and 13 heterozygotes, of 93 small-seeded RILs (<24.0 g/1000 seed). This clearly showed that the small seed size trait is very strongly regulated by the locus near PBLAC449; whereas, large seed size trait seems governed by more than one locus. The PCR amplified products from the PBLAC449 marker (149bp from L4602 and 131bp from L830) were cloned, sequenced and BLAST searched using the lentil reference genome and was found amplified from chromosome 03. Afterward, the nearby region on chromosome 3 was searched, and a few candidate genes like ubiquitin carboxyl-terminal hydrolase, E3 ubiquitin ligase, TIFY-like protein, and hexosyltransferase having a role in seed size determination were identified. Validation study in another RIL mapping population which is differing for seed size, showed a number of SNPs and InDels among these genes when studied using whole genome resequencing (WGRS) approach. Biochemical parameters like cellulose, lignin, and xylose content showed no significant differences between parents and the extreme RILs, at maturity. Various seed morphological traits like area, length, width, compactness, volume, perimeter, etc., when measured using VideometerLab 4.0 showed significant differences for the parents and RILs. The results have ultimately helped in better understanding the region regulating the seed size trait in genomically less explored crops like lentils.

Comparative transcriptome analysis, unfolding the pathways regulating the seed-size trait in cultivated lentil (Lens culinaris Medik.).

Market class, cooking time, quality, and milled grain yield are largely influenced by the seed size and shape of the lentil (Lens culinaris Medik.); thus, they are considered to be important quality traits. To unfold the pathways regulating seed size in lentils, a transcriptomic approach was performed using large-seeded (L4602) and small-seeded (L830) genotypes. The study has generated nearly 375 million high-quality reads, of which 98.70% were properly aligned to the reference genome. Among biological replicates, very high similarity in fragments per kilobase of exon per million mapped fragments values (R > 0.9) showed the consistency of RNA-seq results. Various differentially expressed genes associated mainly with the hormone signaling and cell division pathways, transcription factors, kinases, etc. were identified as having a role in cell expansion and seed growth. A total of 106,996 unigenes were used for differential expression (DE) analysis. String analysis identified various modules having certain key proteins like Ser/Thr protein kinase, seed storage protein, DNA-binding protein, microtubule-associated protein, etc. In addition, some growth and cell division-related micro-RNAs like miR3457 (cell wall formation), miR1440 (cell proliferation and cell cycles), and miR1533 (biosynthesis of plant hormones) were identified as having a role in seed size determination. Using RNA-seq data, 5254 EST-SSR primers were generated as a source for future studies aiming for the identification of linked markers. In silico validation using Genevestigator® was done for the Ser/Thr protein kinase, ethylene response factor, and Myb transcription factor genes. It is of interest that the xyloglucan endotransglucosylase gene was found differentially regulated, suggesting their role during seed development; however, at maturity, no significant differences were recorded for various cell wall parameters including cellulose, lignin, and xylose content. This is the first report on lentils that has unfolded the key seed size regulating pathways and unveiled a theoretical way for the development of lentil genotypes having customized seed sizes.

Yield optimization, microbial load analysis, and sensory evaluation of mungbean (Vigna radiata L.), lentil (Lens culinaris subsp. culinaris), and Indian mustard (Brassica juncea L.) microgreens grown under greenhouse conditions.

Microgreens have been used for raw consumption and are generally viewed as healthy food. This study aimed to optimize the yield parameters, shelf life, sensory evaluation and characterization of total aerobic bacteria (TAB), yeast and mold (Y&M), Escherichia coli, Salmonella spp., and Listeria spp. incidence in mungbean (Vigna radiata (L.) Wilczek), lentil (Lens culinaris Medikus subsp. culinaris), and Indian mustard (Brassica juncea (L.) Czern & Coss.) microgreens. In mungbean and lentil, seeding-density of three seed/cm2, while in Indian mustard, eight seed/cm2 were recorded as optimum. The optimal time to harvest mungbean, Indian mustard, and lentil microgreens were found as 7th, 8th, and 9th day after sowing, respectively. Interestingly, seed size was found highly correlated with the overall yield in both mungbeans (r2 = .73) and lentils (r2 = .78), whereas no such relationship has been recorded for Indian mustard microgreens. The target pathogenic bacteria such as Salmonella spp. and Listeria spp. were not detected; while TAB, Y&M, Shigella spp., and E. coli were recorded well within the limit to cause any human illness in the studied microgreens. Washing with double distilled water for two minutes has shown some reduction in the overall microbial load of these microgreens. The results provided evidence that microgreens if grown and stored properly, are generally safe for human consumption. This is the first study from India on the safety of mungbean, lentils, and Indian mustard microgreens.

Transcranial lateral perforating gunshot injury through skull base presenting without residual damage: A fortunate survivor.

We reported a case of a 32 years old male presenting with a perforating gunshot injury in craniocerebral region 3 h after the assault. The bullet entered above the right zygomatic arch, travelling through the coronal plane, and exited from the left zygomatic arch. The patient was fully conscious at presentation and developed facial nerve palsy during his hospital stay. Non-contrast CT scan of the head revealed fractures of the right orbit, bilateral maxilla, bilateral pterygoid plates, ethmoid air cells, vomer and left zygoma, and without any cerebral damage. He was treated conservatively and the facial palsy was resolved. The patient survived without any complications. Such case has not been described in the available literature till date.

Genome-Wide Association Analysis for Phosphorus Use Efficiency Traits in Mungbean (Vigna radiata L. Wilczek) Using Genotyping by Sequencing Approach.

Mungbean (Vigna radiata L. Wilczek) is an annual grain legume crop affected by low availability of phosphorus. Phosphorus deficiency mainly affects the growth and development of plants along with changes in root morphology and increase in root-to-shoot ratio. Deciphering the genetic basis of phosphorus use efficiency (PUE) traits can benefit our understanding of mungbean tolerance to low-phosphorus condition. To address this issue, 144 diverse mungbean genotypes were evaluated for 12 PUE traits under hydroponics with optimum- and low-phosphorus levels. The broad sense heritability of traits ranged from 0.63 to 0.92 and 0.58 to 0.92 under optimum- and low-phosphorus conditions, respectively. This study, reports for the first time such a large number of genome wide Single nucleotide polymorphisms (SNPs) (76,160) in mungbean. Further, genome wide association study was conducted using 55,634 SNPs obtained by genotyping-by-sequencing method. The results indicated that total 136 SNPs shared by both GLM and MLM models were associated with tested PUE traits under different phosphorus regimes. We have identified SNPs with highest p value (-log10(p)) for some traits like, TLA and RDW with p value (-log10(p)) of more than 6.0 at LP/OP and OP condition. We have identified nine SNPs (three for TLA and six for RDW trait) which was found to be present in chromosomes 8, 4, and 7. One SNP present in Vradi07g06230 gene contains zinc finger CCCH domain. In total, 71 protein coding genes were identified, of which 13 genes were found to be putative candidate genes controlling PUE by regulating nutrient uptake and root architectural development pathways in mungbean. Moreover, we identified three potential candidate genes VRADI11G08340, VRADI01G05520, and VRADI04G10750 with missense SNPs in coding sequence region, which results in significant variation in protein structure at tertiary level. The identified SNPs and candidate genes provide the essential information for genetic studies and marker-assisted breeding program for improving low-phosphorus tolerance in mungbean.

Risk of extreme events in delta environment: A case study of the Mahanadi delta.

Anthropogenic climate change is considered as one of the greatest environmental, social and economic threats to the future world. Low lying deltas all over the world are increasingly subjected to multidimensional risk of sea level rise, cyclone, surges and salinisation. The life and livelihood of the communities of such deltas are endangered due to climate change acting as risk multiplier. The Mahanadi delta in the state of Odisha, India is one of the such populous deltas with estimated 8 million population in 2011 with a density of 613 persons/km2. Over the past decades, it experienced major climatic threats in the form of cyclone, surge inundation and flooding with variable intensities and impacts along and across the coast. The present research assessed the risk of climatic extreme events and their variability in the delta, with an intention of mitigation or adaptation to possible impacts in specific region. Synthetic Aperture Radar (SAR) data and daily rainfall data were used to extract flood inundation. Tropical Cyclone Risk Model (TCRM) along with surge decay function was used to estimate cyclonic wind speed and surge inundation and risk indices were computed using fuzzy logic based approach. The result shows that in the coastal districts, risk of severe cyclones rank above the heavy floods. Agriculture, the main livelihood of these districts (71%) is impacted adversely making the delta community vulnerable to such extreme events. Kendrapara followed by Bhadrak and Jagasinghpur districts appear to be most risk prone segment in the delta making the northern part comparatively more risk prone where focused mitigation and adaptation actions are needed.

Threats to coastal communities of Mahanadi delta due to imminent consequences of erosion - Present and near future.

Coastal erosion is a natural hazard which causes significant loss to properties as well as coastal habitats. Coastal districts of Mahanadi delta, one of the most populated deltas of the Indian subcontinent, are suffering from the ill effects of coastal erosion. An important amount of assets is being lost every year along with forced migration of huge portions of coastal communities due to erosion. An attempt has been made in this study to predict the future coastline of the Mahanadi Delta based on historical trends. Historical coastlines of the delta have been extracted using semi-automated Tasselled Cap technique from the LANDSAT satellite imageries of the year 1990, 1995, 2000, 2006 and 2010. Using Digital Shoreline Assessment System (DSAS) tool of USGS, the trend of the coastline has been assessed in the form of End Point Rate (EPR) and Linear Regression Rate (LRR). A hybrid methodology has been adopted using statistical (EPR) and trigonometric functions to predict the future positions of the coastlines of the years 2020, 2035 and 2050. The result showed that most of the coastline (≈65%) is facing erosion at present. The predicted outcome shows that by the end of year 2050 the erosion scenario will worsen which in turn would lead to very high erosion risk for 30% of the total coastal mouzas (small administrative blocks). This study revealed the coastal erosion trend of Mahanadi delta and based on the predicted coastlines it can be inferred that the coastal communities in near future would be facing substantial threat due to erosion particularly in areas surrounding Puri (a renowned tourist pilgrimage) and Paradwip (one of the busiest ports and harbours of the country).

Genetic dissection of seed-iron and zinc concentrations in chickpea.

The SNP-based high-resolution QTL mapping mapped eight major genomic regions harbouring robust QTLs governing seed-Fe and Zn concentrations (39.4% combined phenotypic variation explained/PVE) on six chromosomes of an intra-specific high-density genetic linkage map (1.56 cM map-density). 24620 SNPs discovered from genome-wide GBS (genotyping-by-sequencing) and 13 known cloned Fe and Zn contents-related chickpea gene-orthologs were genotyped in a structured population of 92 sequenced desi and kabuli accessions. The large-scale 16591 SNP genotyping- and phenotyping-based GWAS (genome-wide association study) identified 16 genomic loci/genes associated (29% combined PVE) with seed-Fe and Zn concentrations. Of these, 11 trait-associated SNPs in the genes linked tightly with eight QTLs were validated by QTL mapping. The seed-specific expression, including pronounced differential-regulation of 16 trait-associated genes particularly in accessions/mapping individuals with contrasting level of seed-Fe and Zn contents was apparent. Collectively, the aforementioned rapid integrated genomic strategy led to delineate novel functional non-synonymous and regulatory SNP allelic-variants from 16 known/candidate genes, including three strong trait-associated genes (encoding late embryogenesis abundant and yellow stripe-like 1 protein, and vacuolar protein sorting-associated protein) and eight major QTLs regulating seed-Fe and Zn concentrations in chickpea. These essential inputs thus have potential to be deployed in marker-assisted genetic enhancement for developing nutritionally-rich iron/zinc-biofortified chickpea cultivars.

Genome-Wide Scans for Delineation of Candidate Genes Regulating Seed-Protein Content in Chickpea.

Identification of potential genes/alleles governing complex seed-protein content (SPC) is essential in marker-assisted breeding for quality trait improvement of chickpea. Henceforth, the present study utilized an integrated genomics-assisted breeding strategy encompassing trait association analysis, selective genotyping in traditional bi-parental mapping population and differential expression profiling for the first-time to understand the complex genetic architecture of quantitative SPC trait in chickpea. For GWAS (genome-wide association study), high-throughput genotyping information of 16376 genome-based SNPs (single nucleotide polymorphism) discovered from a structured population of 336 sequenced desi and kabuli accessions [with 150-200 kb LD (linkage disequilibrium) decay] was utilized. This led to identification of seven most effective genomic loci (genes) associated [10-20% with 41% combined PVE (phenotypic variation explained)] with SPC trait in chickpea. Regardless of the diverse desi and kabuli genetic backgrounds, a comparable level of association potential of the identified seven genomic loci with SPC trait was observed. Five SPC-associated genes were validated successfully in parental accessions and homozygous individuals of an intra-specific desi RIL (recombinant inbred line) mapping population (ICC 12299 × ICC 4958) by selective genotyping. The seed-specific expression, including differential up-regulation (>four fold) of six SPC-associated genes particularly in accessions, parents and homozygous individuals of the aforementioned mapping population with a high level of contrasting SPC (21-22%) was evident. Collectively, the integrated genomic approach delineated diverse naturally occurring novel functional SNP allelic variants in six potential candidate genes regulating SPC trait in chickpea. Of these, a non-synonymous SNP allele-carrying zinc finger transcription factor gene exhibiting strong association with SPC trait was found to be the most promising in chickpea. The informative functionally relevant molecular tags scaled-down essentially have potential to accelerate marker-assisted genetic improvement by developing nutritionally rich chickpea cultivars with enhanced SPC.

Identification of candidate genes for dissecting complex branch number trait in chickpea.

The present study exploited integrated genomics-assisted breeding strategy for genetic dissection of complex branch number quantitative trait in chickpea. Candidate gene-based association analysis in a branch number association panel was performed by utilizing the genotyping data of 401 SNP allelic variants mined from 27 known cloned branch number gene orthologs of chickpea. The genome-wide association study (GWAS) integrating both genome-wide GBS- (4556 SNPs) and candidate gene-based genotyping information of 4957 SNPs in a structured population of 60 sequenced desi and kabuli accessions (with 350-400 kb LD decay), detected 11 significant genomic loci (genes) associated (41% combined PVE) with branch number in chickpea. Of these, seven branch number-associated genes were further validated successfully in two inter (ICC 4958 × ICC 17160)- and intra (ICC 12299 × ICC 8261)-specific mapping populations. The axillary meristem and shoot apical meristem-specific expression, including differential up- and down-regulation (4-5 fold) of the validated seven branch number-associated genes especially in high branch number as compared to the low branch number-containing parental accessions and homozygous individuals of two aforesaid mapping populations was apparent. Collectively, this combinatorial genomic approach delineated diverse naturally occurring novel functional SNP allelic variants in seven potential known/candidate genes [PIN1 (PIN-FORMED protein 1), TB1 (teosinte branched 1), BA1/LAX1 (BARREN STALK1/LIKE AUXIN1), GRAS8 (gibberellic acid insensitive/GAI, Repressor of ga13/RGA and Scarecrow8/SCR8), ERF (ethylene-responsive element-binding factor), MAX2 (more axillary growth 2) and lipase] governing chickpea branch number. The useful information generated from this study have potential to expedite marker-assisted genetic enhancement by developing high-yielding cultivars with more number of productive (pods and seeds) branches in chickpea.

mQTL-seq delineates functionally relevant candidate gene harbouring a major QTL regulating pod number in chickpea.

The present study used a whole-genome, NGS resequencing-based mQTL-seq (multiple QTL-seq) strategy in two inter-specific mapping populations (Pusa 1103 × ILWC 46 and Pusa 256 × ILWC 46) to scan the major genomic region(s) underlying QTL(s) governing pod number trait in chickpea. Essentially, the whole-genome resequencing of low and high pod number-containing parental accessions and homozygous individuals (constituting bulks) from each of these two mapping populations discovered >8 million high-quality homozygous SNPs with respect to the reference kabuli chickpea. The functional significance of the physically mapped SNPs was apparent from the identified 2,264 non-synonymous and 23,550 regulatory SNPs, with 8-10% of these SNPs-carrying genes corresponding to transcription factors and disease resistance-related proteins. The utilization of these mined SNPs in Δ (SNP index)-led QTL-seq analysis and their correlation between two mapping populations based on mQTL-seq, narrowed down two (Caq(a)PN4.1: 867.8 kb and Caq(a)PN4.2: 1.8 Mb) major genomic regions harbouring robust pod number QTLs into the high-resolution short QTL intervals (Caq(b)PN4.1: 637.5 kb and Caq(b)PN4.2: 1.28 Mb) on chickpea chromosome 4. The integration of mQTL-seq-derived one novel robust QTL with QTL region-specific association analysis delineated the regulatory (C/T) and coding (C/A) SNPs-containing one pentatricopeptide repeat (PPR) gene at a major QTL region regulating pod number in chickpea. This target gene exhibited anther, mature pollen and pod-specific expression, including pronounced higher up-regulated (∼3.5-folds) transcript expression in high pod number-containing parental accessions and homozygous individuals of two mapping populations especially during pollen and pod development. The proposed mQTL-seq-driven combinatorial strategy has profound efficacy in rapid genome-wide scanning of potential candidate gene(s) underlying trait-associated high-resolution robust QTL(s), thereby expediting genomics-assisted breeding and genetic enhancement of crop plants, including chickpea.

A Genome-wide Combinatorial Strategy Dissects Complex Genetic Architecture of Seed Coat Color in Chickpea.

The study identified 9045 high-quality SNPs employing both genome-wide GBS- and candidate gene-based SNP genotyping assays in 172, including 93 cultivated (desi and kabuli) and 79 wild chickpea accessions. The GWAS in a structured population of 93 sequenced accessions detected 15 major genomic loci exhibiting significant association with seed coat color. Five seed color-associated major genomic loci underlying robust QTLs mapped on a high-density intra-specific genetic linkage map were validated by QTL mapping. The integration of association and QTL mapping with gene haplotype-specific LD mapping and transcript profiling identified novel allelic variants (non-synonymous SNPs) and haplotypes in a MATE secondary transporter gene regulating light/yellow brown and beige seed coat color differentiation in chickpea. The down-regulation and decreased transcript expression of beige seed coat color-associated MATE gene haplotype was correlated with reduced proanthocyanidins accumulation in the mature seed coats of beige than light/yellow brown seed colored desi and kabuli accessions for their coloration/pigmentation. This seed color-regulating MATE gene revealed strong purifying selection pressure primarily in LB/YB seed colored desi and wild Cicer reticulatum accessions compared with the BE seed colored kabuli accessions. The functionally relevant molecular tags identified have potential to decipher the complex transcriptional regulatory gene function of seed coat coloration and for understanding the selective sweep-based seed color trait evolutionary pattern in cultivated and wild accessions during chickpea domestication. The genome-wide integrated approach employed will expedite marker-assisted genetic enhancement for developing cultivars with desirable seed coat color types in chickpea.

Development of genome-wide informative simple sequence repeat markers for large-scale genotyping applications in chickpea and development of web resource.

Development of informative polymorphic simple sequence repeat (SSR) markers at a genome-wide scale is essential for efficient large-scale genotyping applications. We identified genome-wide 1835 SSRs showing polymorphism between desi and kabuli chickpea. A total of 1470 polymorphic SSR markers from diverse coding and non-coding regions of the chickpea genome were developed. These physically mapped SSR markers exhibited robust amplification efficiency (73.9%) and high intra- and inter-specific polymorphic potential (63.5%), thereby suggesting their immense use in various genomics-assisted breeding applications. The SSR markers particularly derived from intergenic and intronic sequences revealed high polymorphic potential. Using the mapped SSR markers, a wider functional molecular diversity (16-94%, mean: 68%), and parentage- and cultivar-specific admixed domestication pattern and phylogenetic relationships in a structured population of desi and kabuli chickpea genotypes was evident. The intra-specific polymorphism (47.6%) and functional molecular diversity (65%) potential of polymorphic SSR markers developed in our study is much higher than that of previous documentations. Finally, we have developed a user-friendly web resource, Chickpea Microsatellite Database (CMsDB; http://www.nipgr.res.in/CMsDB.html), which provides public access to the data and results reported in this study. The developed informative SSR markers can serve as a resource for various genotyping applications, including genetic enhancement studies in chickpea.

CNMS: The preferred genic markers for comparative genomic, molecular phylogenetic, functional genetic diversity and differential gene regulatory expression analyses in chickpea.

The intra/inter-genomic comparative mapping-based phylogenetic footprinting identified 5 paralogous and 656 orthologous genome-wide CNMS markers in the upstream sequences of chickpea genes. These CNMS markers revealed a high-degree of gene-based syntenic relationship between chickpea and Medicago genomes while minimum between chickpea and Vitis genomes. The time of divergence and duplication estimated using CNMS markers highlight the expected phylogenetic relationships between chickpea and six dicot (legume) species as well as occurrence of ancient genome (approximately 53 Mya) with small-scale recent segmental (approximately 10 Mya) duplication events in chickpea. A wider level of functional molecular diversity (14 to 88 percent) and admixed population genetic structure was detected among desi, kabuli and wild genotypes by genic CNMS markers at a genome-wide scale suggesting their utility in large-scale genetic analysis in chickpea. The subfunctionalization at the cis-regulatory element region and TFBS (transcription factor binding site) motif levels in the upstream sequences of CNMS marker-associated orthologous genes than the paralogues was predominant. Functional constraint might have considerable effect on these CNMScontaining regulatory elements controlling consistent orthologous gene expression in dicots. A rapid subfunctionalization based on diverge differential expression of paralogous CNMS marker-associated genes particularly those that underwent recent small-scale segmental duplication events in chickpea was apparent. The differential regulation of expression and subfunctionalization potential of Ultra CNMS marker-associated genes suggest their utility in deciphering the complex gene regulatory function as well as identification and targeted mapping of potential genes/QTLs governing vital agronomic traits in chickpea. The gene-based CNMS markers with desirable inherent genetic attributes like higher degree of comparative genome mapping, functional genetic diversity and differential gene regulatory expression potential can significantly propel the genomics-assisted chickpea crop improvement.

A genome-scale integrated approach aids in genetic dissection of complex flowering time trait in chickpea.

A combinatorial approach of candidate gene-based association analysis and genome-wide association study (GWAS) integrated with QTL mapping, differential gene expression profiling and molecular haplotyping was deployed in the present study for quantitative dissection of complex flowering time trait in chickpea. Candidate gene-based association mapping in a flowering time association panel (92 diverse desi and kabuli accessions) was performed by employing the genotyping information of 5724 SNPs discovered from 82 known flowering chickpea gene orthologs of Arabidopsis and legumes as well as 832 gene-encoding transcripts that are differentially expressed during flower development in chickpea. GWAS using both genome-wide GBS- and candidate gene-based genotyping data of 30,129 SNPs in a structured population of 92 sequenced accessions (with 200-250 kb LD decay) detected eight maximum effect genomic SNP loci (genes) associated (34% combined PVE) with flowering time. Six flowering time-associated major genomic loci harbouring five robust QTLs mapped on a high-resolution intra-specific genetic linkage map were validated (11.6-27.3% PVE at 5.4-11.7 LOD) further by traditional QTL mapping. The flower-specific expression, including differential up- and down-regulation (>three folds) of eight flowering time-associated genes (including six genes validated by QTL mapping) especially in early flowering than late flowering contrasting chickpea accessions/mapping individuals during flower development was evident. The gene haplotype-based LD mapping discovered diverse novel natural allelic variants and haplotypes in eight genes with high trait association potential (41% combined PVE) for flowering time differentiation in cultivated and wild chickpea. Taken together, eight potential known/candidate flowering time-regulating genes [efl1 (early flowering 1), FLD (Flowering locus D), GI (GIGANTEA), Myb (Myeloblastosis), SFH3 (SEC14-like 3), bZIP (basic-leucine zipper), bHLH (basic helix-loop-helix) and SBP (SQUAMOSA promoter binding protein)], including novel markers, QTLs, alleles and haplotypes delineated by aforesaid genome-wide integrated approach have potential for marker-assisted genetic improvement and unravelling the domestication pattern of flowering time in chickpea.

Genome-wide insertion-deletion (InDel) marker discovery and genotyping for genomics-assisted breeding applications in chickpea.

We developed 21,499 genome-wide insertion-deletion (InDel) markers (2- to 54-bp in silico fragment length polymorphism) by comparing the genomic sequences of four (desi, kabuli and wild C. reticulatum) chickpea [Cicer arietinum (L.)] accessions. InDel markers showing 2- to 6-bp fragment length polymorphism among accessions were abundant (76.8%) in the chickpea genome. The physically mapped 7,643 and 13,856 markers on eight chromosomes and unanchored scaffolds, respectively, were structurally and functionally annotated. The 4,506 coding (23% large-effect frameshift mutations) and regulatory InDel markers were identified from 3,228 genes (representing 11.7% of total 27,571 desi genes), suggesting their functional relevance for trait association/genetic mapping. High amplification (97%) and intra-specific polymorphic (60-83%) potential and wider genetic diversity (15-89%) were detected by genome-wide 6,254 InDel markers among desi, kabuli and wild accessions using even a simpler cost-effective agarose gel-based assay. This signifies added advantages of this user-friendly genetic marker system for manifold large-scale genotyping applications in laboratories with limited infrastructure and resources. Utilizing 6,254 InDel markers-based high-density (inter-marker distance: 0.212 cM) inter-specific genetic linkage map (ICC 4958 × ICC 17160) of chickpea as a reference, three major genomic regions harboring six flowering and maturity time robust QTLs (16.4-27.5% phenotypic variation explained, 8.1-11.5 logarithm of odds) were identified. Integration of genetic and physical maps at these target QTL intervals mapped on three chromosomes delineated five InDel markers-containing candidate genes tightly linked to the QTLs governing flowering and maturity time in chickpea. Taken together, our study demonstrated the practical utility of developing and high-throughput genotyping of such beneficial InDel markers at a genome-wide scale to expedite genomics-assisted breeding applications in chickpea.