G-DIRT: a web server for identification and removal of duplicate germplasms based on identity-by-state analysis using single nucleotide polymorphism genotyping data.

Maintaining duplicate germplasms in genebanks hampers effective conservation and utilization of genebank resources. The redundant germplasm adds to the cost of germplasm conservation by requiring a large proportion of the genebank financial resources towards conservation rather than enriching the diversity. Besides, genome-wide-association analysis using an association panel with over-represented germplasms can be biased resulting in spurious marker-trait associations. The conventional methods of germplasm duplicate removal using passport information suffer from incomplete or missing passport information and data handling errors at various stages of germplasm enrichment. This limitation is less likely in the case of genotypic data. Therefore, we developed a web-based tool, Germplasm Duplicate Identification and Removal Tool (G-DIRT), which allows germplasm duplicate identification based on identity-by-state analysis using single-nucleotide polymorphism genotyping information along with pre-processing of genotypic data. A homozygous genotypic difference threshold of 0.1% for germplasm duplicates has been determined using tetraploid wheat genotypic data with 94.97% of accuracy. Based on the genotypic difference, the tool also builds a dendrogram that can visually depict the relationship between genotypes. To overcome the constraint of high-dimensional genotypic data, an offline version of G-DIRT in the interface of R has also been developed. The G-DIRT is expected to help genebank curators, breeders and other researchers across the world in identifying germplasm duplicates from the global genebank collections by only using the easily sharable genotypic data instead of physically exchanging the seeds or propagating materials. The web server will complement the existing methods of germplasm duplicate identification based on passport or phenotypic information being freely accessible at http://webtools.nbpgr.ernet.in/gdirt/.

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.

Multilocus functional characterization of indigenous and exotic inbreds for dgat1-2, fatb, ge2 and wri1a genes affecting kernel oil and fatty acid profile in maize.

Demand for maize oil is progressively increasing due to its diverse industrial applications, aside from its primary role in human nutrition and animal feed. Oil content and composition are two crucial determinants of maize oil in the international market. As kernel oil in maize is a complex quantitative trait, improving this trait presents a challenge for plant breeders and biotechnologists. Here, we characterized a set of 292 diverse maize inbreds of both indigenous and exotic origin by exploiting functional polymorphism of the dgat1-2, fatb, ge2, and wri1a genes governing kernel oil in maize. Genotyping using gene-based functional markers revealed a lower frequencies of dgat1-2 (0.15) and fatb (0.12) mutant alleles and a higher frequencies of wild-type alleles (Dgat1-2: 0.85; fatB: 0.88). The favorable wri1a allele was conserved across genotypes, while its wild-type allele (WRI1a) was not detected. In contrast, none of the genotypes possessed the ge2 favorable allele. The frequency of favorable alleles of both dgat1-2 and fatb decreased to 0.03 when considered together. Furthermore, pairwise protein-protein interactions among target gene products were conducted to understand the effect of one protein on another and their responses to kernel oil through functional enrichments. Thus, the identified maize genotypes with dgat1-2, fatb, and wri1a favourable alleles, along with insights gained through the protein-protein association network, serve as prominent and unique genetic resources for high-oil maize breeding programs. This is the first comprehensive report on the functional characterization of diverse genotypes at the molecular and protein levels.

Unlocking the potential of ancient hexaploid Indian dwarf wheat, Tritium sphaerococcum for grain quality improvement.

Wild and ancient wheat are considered to be a rich source of nutrients and better stress tolerant, hence being re-considered for mainstreaming its cultivation by the farmers and bringing it back to the food basket. In the present study, thirty-four diverse accessions of Indian dwarf wheat, Triticum sphaerococcum conserved in the Indian National Genebank were evaluated for thirteen-grain quality parameters namely thousand-grain weight (TGW), hectolitre weight (HW), sedimentation value (Sed), grain hardness index (HI), protein (Pro), albumin (Alb), globulin (Glo), gliadin (Gli), glutenin (Glu), gluten, lysine (Lys), Fe2+ and Zn2+ content, and four antioxidant enzymes activities. Substantial variations were recorded for studied traits. TGW, HW, Sed, HI, Pro, Alb, Glo, Gli, Glu, Gluten, Lys, Fe2+, and Zn2+ varied from 26.50-45.55 g, 70.50-86.00 kg/hl, 24.00-38.00 ml, 40.49-104.90, 15.34-19.35%, 17.60-40.31 mg/g, 10.75-16.56 mg/g, 26.35-44.94 mg/g, 24.47-39.56 mg/g, 55.33-75.06 mg/g, 0.04-0.29%, 42.72-90.72 ppm, and 11.45-25.70 ppm, respectively. Among antioxidants, peroxidase (POX), catalase (CAT), glutathione reductase (GR), and superoxide dismutase (SOD) activity ranged from 0.06-0.60 unit/ml, 0.02-0.61 unit/ml, 0.11-2.26 unit/ml, and 0.14-0.97 unit/ml, respectively. Hardness Index was positively associated with Pro and Zn2+ content whereas Lys was negatively associated with gluten content. Likewise, gluten and Fe2+ content had a positive association with the major protein fraction i.e., Gli and Glu. Hierarchical cluster analysis grouped 34 accessions into four clusters and the major group had nine indigenous and eight exotic accessions. We also validated high GPC accessions and EC182958 (17.16%), EC187176 and EC182945 (16.16%), EC613057 (15.79%), IC634028 (15.72%) and IC533826 (15.01%) were confirmed with more than 15% GPC. Also, superior trait-specific accessions namely, EC187167, IC534021, EC613055, EC180066, and EC182959 for low gluten content and IC384530, EC313761, EC180063, IC397363, EC10494 for high iron content (>76.51) were identified that may be used in wheat quality improvement for nutritional security of mankind.

Phenotypic Characterization, Genetic Diversity Assessment in 6,778 Accessions of Barley (Hordeum vulgare L. ssp. vulgare) Germplasm Conserved in National Genebank of India and Development of a Core Set.

The entire collection of cultivated barley germplasm accessions conserved in the Indian National Genebank (INGB) was characterized for nine qualitative and 8 quantitative traits to assess the nature and magnitude of prevailing genetic variability and to develop a core set. A wide range of variability was observed for days to spike emergence (51-139 days), days to physiological maturity (100-152 days), plant height (45.96-171.32 cm), spike length (3.44-13.73 cm), grain number/spike (10.48-82.35), and 100-grain weight (1.20-6.86 g). Initially, seven independent core sets were derived using 3 core construction tools- MSTRAT, PowerCore, and Core Hunter 3 by employing the maximization method, heuristic sampling, and optimisation of average genetic distances, respectively. The core set-3 generated by Core Hunter 3 by simultaneous optimisation of diversity and representativeness, captured maximum genetic diversity of the whole collection as evident from the desirable genetic distance, variance difference percentage (VD; 87.5%), coincidence rate of range (CR; 94.27%) and variable rate of coefficient of variance (VR; 113.8%), which were more than threshold value of VD (80%), CR (80%), and VR (100%) required for good core collection. The coefficient of variation and Shannon-Weaver diversity indices were increased in the core set as compared with the whole collection. The low value of Kullback-Leibler distance (0.024-0.071) for all traits and quantile-quantile plots revealed a negligible difference between trait distribution patterns among the core set and entire assembly. Correlogram revealed that trait associations and their magnitude were conserved for most of the traits after sampling of the core set. The extraction of the INGB barley core set and identification of promising accessions for agronomically important traits in different genetic backgrounds will pave the way for expedited access to genetically diverse and agronomically important germplasm for barley breeding.

Nutritional Diversity in Native Germplasm of Maize Collected From Three Different Fragile Ecosystems of India.

Native germplasm resources are adapted to specific ecological niches. They have sustained over generations owing to the preference of local communities for their unique taste, the utility to particular dishes, and the low cost of cultivation. They may help eradicate malnutrition and act as a source for trait-linked genes. The present dataset comprises thirty-three native germplasm of maize collected from Rajasthan, Himachal Pradesh, and Andhra Pradesh states of India with an altitudinal variation of 386-2,028 m. They were evaluated for proximate composition, minerals, nutritional attributes, and antioxidant activity and compared with the standard values reported in the Indian Food Composition Table 2017 (IFCT2017). The nutritional profile showed moisture content in the range of 7.16-10.9%, ash 0.73-1.93%, crude protein 8.68-12.0%, crude fat 3.72-8.03%, dietary fiber 5.21-11.2%, and available carbohydrates 60.6-69.8%. Three accessions, namely, Malan 11 (7.06%), Malan 24 (7.20%), and Yellow Chamba Local 02 (8.03%) exhibited almost double the crude fat content as compared with the values notified in IFCT2017 (3.77). Total sugar content obtained was in the range of 5.00-11.3%, whereas the starch content was found between 50.9 and 64.9%. All the germplasm except Yellow Chamba Local reflected a higher protein content than reported values in IFCT2017 (8.80). Sathi, Safed Chamba Local, and Ragal Makka had nearly 12% protein content. Mineral malnutrition, mainly due to iron (Fe) deficiency, is a worldwide issue to science, humanity, and society. The mineral profile revealed that most germplasm had a higher iron content. Accessions with the iron content of nearly three times of IFCT2017 reported value were identified in germplasm belonging to three states. A negative relationship was observed between the altitude of the sample collection site and available carbohydrate content. In contrast, available carbohydrate showed inverse correlations with dietary fiber, protein, and fat content. The information generated in this study can be utilized to promote these germplasm as nutrifood, nutritional surveillance, labeling, and crop improvement programs.

Indian Wheat Genomics Initiative for Harnessing the Potential of Wheat Germplasm Resources for Breeding Disease-Resistant, Nutrient-Dense, and Climate-Resilient Cultivars.

Wheat is one of the major staple cereal food crops in India. However, most of the wheat-growing areas experience several biotic and abiotic stresses, resulting in poor quality grains and reduced yield. To ensure food security for the growing population in India, there is a compelling need to explore the untapped genetic diversity available in gene banks for the development of stress-resistant/tolerant cultivars. The improvement of any crop lies in exploring and harnessing the genetic diversity available in its genetic resources in the form of cultivated varieties, landraces, wild relatives, and related genera. A huge collection of wheat genetic resources is conserved in various gene banks across the globe. Molecular and phenotypic characterization followed by documentation of conserved genetic resources is a prerequisite for germplasm utilization in crop improvement. The National Genebank of India has an extensive and diverse collection of wheat germplasm, comprising Indian wheat landraces, primitive cultivars, breeding lines, and collection from other countries. The conserved germplasm can contribute immensely to the development of wheat cultivars with high levels of biotic and abiotic stress tolerance. Breeding wheat varieties that can give high yields under different stress environments has not made much headway due to high genotypes and environmental interaction, non-availability of truly resistant/tolerant germplasm, and non-availability of reliable markers linked with the QTL having a significant impact on resistance/tolerance. The development of new breeding technologies like genomic selection (GS), which takes into account the G × E interaction, will facilitate crop improvement through enhanced climate resilience, by combining biotic and abiotic stress resistance/tolerance and maximizing yield potential. In this review article, we have summarized different constraints being faced by Indian wheat-breeding programs, challenges in addressing biotic and abiotic stresses, and improving quality and nutrition. Efforts have been made to highlight the wealth of Indian wheat genetic resources available in our National Genebank and their evaluation for the identification of trait-specific germplasm. Promising genotypes to develop varieties of important targeted traits and the development of different genomics resources have also been highlighted.

Identification of Novel Broad-Spectrum Leaf Rust Resistance Sources from Khapli Wheat Landraces.

Wheat leaf rust caused by Puccinia triticina Eriks is an important disease that causes yield losses of up to 40% in susceptible varieties. Tetraploid emmer wheat (T. turgidum ssp. Dicoccum), commonly called Khapli wheat in India, is known to have evolved from wild emmer (Triticum turgidum var. dicoccoides), and harbors a good number of leaf rust resistance genes. In the present study, we are reporting on the screening of one hundred and twenty-three dicoccum wheat germplasm accessions against the leaf rust pathotype 77-5. Among these, an average of 45.50% of the germplasms were resistant, 46.74% were susceptible, and 8.53% had mesothetic reactions. Further, selected germplasm lines with accession numbers IC138898, IC47022, IC535116, IC535133, IC535139, IC551396, and IC534144 showed high level of resistance against the eighteen prevalent pathotypes. The infection type varied from ";", ";N", ";N1" to ";NC". PCR-based analysis of the resistant dicoccum lines with SSR marker gwm508 linked to the Lr53 gene, a leaf rust resistance gene effective against all the prevalent pathotypes of leaf rust in India and identified from a T. turgidum var. dicoccoides germplasm, indicated that Lr53 is not present in the selected accessions. Moreover, we have also generated 35K SNP genotyping data of seven lines and the susceptible control, Mandsaur Local, to study their relationships. The GDIRT tool based on homozygous genotypic differences revealed that the seven genotypes are unique to each other and may carry different resistance genes for leaf rust.

Growth and Antioxidant Responses in Iron-Biofortified Lentil under Cadmium Stress.

Cadmium (Cd) is a hazardous heavy metal, toxic to our ecosystem even at low concentrations. Cd stress negatively affects plant growth and development by triggering oxidative stress. Limited information is available on the role of iron (Fe) in ameliorating Cd stress tolerance in legumes. This study assessed the effect of Cd stress in two lentil (Lens culinaris Medik.) varieties differing in seed Fe concentration (L4717 (Fe-biofortified) and JL3) under controlled conditions. Six biochemical traits, five growth parameters, and Cd uptake were recorded at the seedling stage (21 days after sowing) in the studied genotypes grown under controlled conditions at two levels (100 µM and 200 µM) of cadmium chloride (CdCl2). The studied traits revealed significant genotype, treatment, and genotype × treatment interactions. Cd-induced oxidative damage led to the accumulation of hydrogen peroxide (H2O2) and malondialdehyde in both genotypes. JL3 accumulated 77.1% more H2O2 and 75% more lipid peroxidation products than L4717 at the high Cd level. Antioxidant enzyme activities increased in response to Cd stress, with significant genotype, treatment, and genotype × treatment interactions (p < 0.01). L4717 had remarkably higher catalase (40.5%), peroxidase (43.9%), superoxide dismutase (31.7%), and glutathione reductase (47.3%) activities than JL3 under high Cd conditions. In addition, L4717 sustained better growth in terms of fresh weight and dry weight than JL3 under stress. JL3 exhibited high Cd uptake (14.87 mg g-1 fresh weight) compared to L4717 (7.32 mg g-1 fresh weight). The study concluded that the Fe-biofortified lentil genotype L4717 exhibited Cd tolerance by inciting an efficient antioxidative response to Cd toxicity. Further studies are required to elucidate the possibility of seed Fe content as a surrogacy trait for Cd tolerance.

Mining of Indian wheat germplasm collection for adult plant resistance to leaf rust.

Leaf rust (Puccinia triticina Eriks.) is a fungal disease of wheat (Triticum spp.), which causes considerable yield loss. Adult plant resistance (APR) is one of the most sustainable approaches to control leaf rust. In this study, field-testing was carried out across ten different locations, followed by molecular screening, to detect the presence of APR genes, Lr34+, Lr46+, Lr67+ and Lr68 in Indian wheat germplasm. In field screening, 190 wheat accessions were selected from 6,319 accessions based on leaf tip necrosis (LTN), disease severity and the average coefficient of infection. Molecular screening revealed that 73% of the accessions possessed known APR genes either as single or as a combination of two or three genes. The occurrence of increased LTN intensity, decreased leaf rust severity and greater expression of APR genes were more in relatively cooler locations. In 52 lines, although the presence of the APR genes was not detected, it still displayed high levels of resistance. Furthermore, 49 accessions possessing either two or three APR genes were evaluated for stability across locations for grain yield. It emerged that eight accessions had wider adaptability. Resistance based on APR genes, in the background of high yielding cultivars, is expected to provide a high level of race non-specific resistance, which is durable.

Correction: Evaluation of 19,460 Wheat Accessions Conserved in the Indian National Genebank to Identify New Sources of Resistance to Rust and Spot Blotch Diseases.

[This corrects the article DOI: 10.1371/journal.pone.0167702.].

Evaluation of 19,460 Wheat Accessions Conserved in the Indian National Genebank to Identify New Sources of Resistance to Rust and Spot Blotch Diseases.

A comprehensive germplasm evaluation study of wheat accessions conserved in the Indian National Genebank was conducted to identify sources of rust and spot blotch resistance. Genebank accessions comprising three species of wheat-Triticum aestivum, T. durum and T. dicoccum were screened sequentially at multiple disease hotspots, during the 2011-14 crop seasons, carrying only resistant accessions to the next step of evaluation. Wheat accessions which were found to be resistant in the field were then assayed for seedling resistance and profiled using molecular markers. In the primary evaluation, 19,460 accessions were screened at Wellington (Tamil Nadu), a hotspot for wheat rusts. We identified 4925 accessions to be resistant and these were further evaluated at Gurdaspur (Punjab), a hotspot for stripe rust and at Cooch Behar (West Bengal), a hotspot for spot blotch. The second round evaluation identified 498 accessions potentially resistant to multiple rusts and 868 accessions potentially resistant to spot blotch. Evaluation of rust resistant accessions for seedling resistance against seven virulent pathotypes of three rusts under artificial epiphytotic conditions identified 137 accessions potentially resistant to multiple rusts. Molecular analysis to identify different combinations of genetic loci imparting resistance to leaf rust, stem rust, stripe rust and spot blotch using linked molecular markers, identified 45 wheat accessions containing known resistance genes against all three rusts as well as a QTL for spot blotch resistance. The resistant germplasm accessions, particularly against stripe rust, identified in this study can be excellent potential candidates to be employed for breeding resistance into the background of high yielding wheat cultivars through conventional or molecular breeding approaches, and are expected to contribute toward food security at national and global levels.

Indian plant germplasm on the global platter: an analysis.

Food security is a global concern amongst scientists, researchers and policy makers. No country is self-sufficient to address food security issues independently as almost all countries are inter-dependent for availability of plant genetic resources (PGR) in their national crop improvement programmes. Consultative Group of International Agricultural Research (CGIAR; in short CG) centres play an important role in conserving and distributing PGR through their genebanks. CG genebanks assembled the germplasm through collecting missions and acquisition the same from national genebanks of other countries. Using the Genesys Global Portal on Plant Genetic Resources, the World Information and Early Warning System (WIEWS) on Plant Genetic Resources for Food and Agriculture and other relevant databases, we analysed the conservation status of Indian-origin PGR accessions (both cultivated and wild forms possessed by India) in CG genebanks and other national genebanks, including the United States Department of Agriculture (USDA) genebanks, which can be considered as an indicator of Indian contribution to the global germplasm collection. A total of 28,027,770 accessions are being conserved world-wide by 446 organizations represented in Genesys; of these, 3.78% (100,607) are Indian-origin accessions. Similarly, 62,920 Indian-origin accessions (8.73%) have been conserved in CG genebanks which are accessible to the global research community for utilization in their respective crop improvement programmes. A total of 60 genebanks including 11 CG genebanks have deposited 824,625 accessions of PGR in the Svalbard Global Seed Vault (SGSV) as safety duplicates; the average number of accessions deposited by each genebank is 13,744, and amongst them there are 66,339 Indian-origin accessions. In principle, India has contributed 4.85 times the number of germplasm accessions to SGSV, in comparison to the mean value (13,744) of any individual genebank including CG genebanks. More importantly, about 50% of the Indian-origin accessions deposited in SGSV are traditional varieties or landraces with defined traits which form the backbone of any crop gene pool. This paper is also attempting to correlate the global data on Indian-origin germplasm with the national germplasm export profile. The analysis from this paper is discussed with the perspective of possible implications in the access and benefit sharing regime of both the International Treaty on Plant Genetic Resources for Food and Agriculture and the newly enforced Nagoya Protocol under the Convention on Biological Diversity.

An analysis of the persistence and potency of film-coated seed protectant as influenced by various storage parameters.

BACKGROUND: An efficient delivery system for seed-protectant chemicals is needed in light of several disadvantages of conventional seed treatment methods. This study evaluates the efficacy of film-coat application in maintaining the persistence and potency of imidacloprid on Lycopersicon esculentum (L.) Mill. seeds after simultaneous storage under ambient and regulated environment in paper and aluminium packages. RESULTS: High-performance liquid chromatography (HPLC) revealed 0.135 mg kg(-1) of herbage material to be the threshold value beyond which absolute control was obtained, and with film coating the latter was achieved even with half-dosage seed treatment, irrespective of the storage condition. The technique provided early protection to the crop and also nullified the deleterious effects of ambient storage on the persistence and potency of the pesticide. CONCLUSION: Film coating enabled superior pesticide dosage as well as higher biological efficacy to be achieved. Hence, in addition to being an ecofriendly alternative, the technique would be a more economically viable option for storage of treated seeds.

Application of potassium-titanyl-phosphate (KTP) laser in the excision of pyriform fossa hemangioma.

Pyriform fossa hemangioma, especially of the cavernous type, is a rare case and very few such lesions have been encountered in general otolaryngological practice. We report such a lesion in a 36-year-old, middle-aged woman presenting with complaint of foreign body sensation in the throat. Examination revealed a hemangioma in the right pyriform fossa that was successfully managed using KTP-532 laser and bipolar cautery.