Mining legume germplasm for genetic gains: An Indian perspective.

Legumes play a significant role in food and nutritional security and contribute to environmental sustainability. Although legumes are highly beneficial crops, it has not yet been possible to enhance their yield and production to a satisfactory level. Amid a rising population and low yield levels, per capita average legume consumption in India has fallen by 71% over the last 50 years, and this has led to protein-related malnutrition in a large segment of the Indian population, especially women and children. Several factors have hindered attempts to achieve yield enhancement in grain legumes, including biotic and abiotic pressures, a lack of good ideotypes, less amenability to mechanization, poorer responsiveness to fertilizer input, and a poor genetic base. Therefore, there is a need to mine the approximately 0.4 million ex situ collections of legumes that are being conserved in gene banks globally for identification of ideal donors for various traits. The Indian National Gene Bank conserves over 63,000 accessions of legumes belonging to 61 species. Recent initiatives have been undertaken in consortia mode with the aim of unlocking the genetic potential of ex situ collections and conducting large-scale germplasm characterization and evaluation analyses. We assume that large-scale phenotyping integrated with omics-based science will aid the identification of target traits and their use to enhance genetic gains. Additionally, in cases where the genetic base of major legumes is narrow, wild relatives have been evaluated, and these are being exploited through pre-breeding. Thus far, >200 accessions of various legumes have been registered as unique donors for various traits of interest.

Quantitative assessment of sediment delivery and retention in four watersheds in the Godavari River Basin, India, using InVEST model - an aquatic ecosystem services perspective.

Sediment export and retention are important ecosystem processes in any landscape causing soil erosion and sediment loading in waterways consequently affecting the health of aquatic habitats downstream. The present study quantifies sediment export and retention in four watersheds, viz., Hivra, Satrapur, Konta, and Jagdalpur in the Godavari River Basin, India, using Integrated Valuation of Ecosystem Services and Trade-offs (InVEST) ecosystem service modelling tool. The  results revealed that the sediment export yields ranged from 0.75 (Hivra) to 2.77 t/ha/year (Jagdalpur). The mean deviation between modelled values and observed sediment export yield was - 11.11%, which indicated good prediction by the model. The sediment retention ranged from 16.04 (Hivra) to 101.52 t/ha/year (Konta). Most sediment export and retention occurred on cropland or shrubland land use land cover types in all four watersheds. For decision making on soil conservation, soil loss tolerance limits have been established for these watersheds. For aquatic habitats, sediment concentration is considered more important than the total annual sediment export, since water turbidity is an important determinant of water quality, and the aquatic lives therein. Therefore, the temporal distribution of rainfall and corresponding sediment export becomes important, since these two factors determine the sediment concentration as well as turbidity in the waterbody. In current study, "Precipitation Concentration Index adjusted Sediment Export Yield Index" was developed to account for the effects of the temporal rainfall distribution and its impact on sediment export. The index for four watersheds was quantified (Hivra > Satrapur > Konta > Jagdalpur), which is concordant to the turbidity values reported by respective gauge stations. Thus, the proposed index can efficiently capture the impact of temporal rainfall distribution on sediment export, and consequently its effect on water turbidity. The study revealed the potential of InVEST model to quantify the sediment export and retention in the watersheds studied. Together with the proposed index, it would help the policy makers in making informed decisions for planning conservation strategies for aquatic biodiversity.

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.

Identification of novel resistant sources for ascochyta blight (Ascochyta rabiei) in chickpea.

Chickpea (Cicer arietinum L.) is the second largest pulse crop grown worldwide and ascochyta blight caused by Ascochyta rabiei (Pass.) Labr. is the most devastating disease of the crop in all chickpea growing areas across the continents. The pathogen A. rabiei is highly variable. The resistant sources available are not sufficient and new sources needs to be identified from time to time as resistance breakdown in existing chickpea varieties is very frequent due to fast evolution of new pathotypes of the pathogen. Therefore, this work was undertaken to evaluate the existing chickpea germplasm diversity conserved in Indian National Genebank against the disease under artificial epiphytotic conditions. An artificial standard inoculation procedure was followed for uniform spread of the pathogen. During the last five winter seasons from 2014-15 to 2018-19, a total of 1,970 accessions have been screened against the disease and promising accessions were identified and validated. Screening has resulted in identification of some promising chickpea accessions such as IC275447, IC117744, EC267301, IC248147 and EC220109 which have shown the disease resistance (disease severity score ≤3) in multiple seasons and locations. Promising accessions can serve as the potential donors in chickpea improvement programs. The frequency of resistant and moderately resistant type accessions was comparatively higher in accessions originated from Southwest Asian countries particularly Iran and Syria than the accessions originated from Indian sub-continent. Further large scale screening of chickpea germplasm originated from Southwest Asia may result in identifying new resistant sources for the disease.

Comparative Agronomic Performance and Reaction to Fusarium wilt of Lens culinaris × L. orientalis and L. culinaris × L. ervoides derivatives.

The development of transgressive phenotype in the segregating populations has been speculated to contribute to niche divergence of hybrid lineages, which occurs most frequently at larger genetic distances. Wild Lens species are considered to be more resistant against major biotic and abiotic stresses than that of the cultivated species. In the present study, we assessed the comparative agronomic performance of lentil (Lens culinaris subsp. culinaris) inter-sub-specific (L. culinaris subsp. orientalis) and interspecific (L. ervoides) derivatives, also discussed its probable basis of occurrence. The F3, F4, and F5 inter sub-specific and interspecific populations of ILL8006 × ILWL62 and ILL10829 × ILWL30, respectively revealed a substantial range of variation for majority of agro-morphological traits as reflected by the range, mean and coefficient of variation. A high level of fruitful heterosis was also observed in F3 and F4 progeny for important traits of interest. Phenotypic coefficient of variation (PCV) was higher in magnitude than genotypic coefficient of variation (GCV) in all generations for several quantitative characters. The results showed high heritability estimates for majority of traits in conjunction with low to high genetic advance in F3 and F4 generations. Further, F5 progeny of ILL10829 × ILWL30, manifested resistant disease reaction for fifteen recombinant inbred lines (RILs) against (Fusarium oxysporum f. sp. lentis (Vasd. Srin.) Gord.). The multilocation agronomic evaluation of both crosses showed better results for earliness, desirable seed yield and Fusarium wilt resistance under two agro-ecological regions of north-western India. These better performing recombinants of ILL8006 × ILWL62 and ILL10829 × ILWL30 can be advanced for further genetic improvement and developing high yielding disease resistant cultivars of lentil.

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.

AFLP fingerprinting of tartary buckwheat accessions (Fagopyrum tataricum) displaying rutin content variation.

In light of the economic importance of buckwheat as well as existence of enormous accessions of Fagopyrum species in the Himalayan regions of India, the characterization of tartary buckwheat for rutin content variation vis-à-vis DNA fingerprinting was undertaken so as to identify fingerprint profiles unique to high rutin content accessions. Rutin content analysis in mature seeds of 195 accessions of Fagopyrum tataricum showed a wide range of variation (6 µg/mg to 30 µg/mg D.W.) with most of the accessions (81%) containing 10-16 µg/mg of rutin followed by 14% accessions with significantly higher rutin content (17 µg/mg to 30 µg/mg) and 5% accessions with low rutin content (≤10 µg/mg). AFLP fingerprinting of 18 accessions having high (≥17 µg/mg) and low rutin content (≤10 µg/mg) with 19 EcoRI/MseI primer combinations yielded 136 polymorphic fragments out of total 907. The hierarchical and model-based cluster analyses of AFLP data strongly suggested that the 18 populations of F. tataricum were clustered into two separate groups. The high and low rutin content accessions were clustered into two separate groups based on AFLP fingerprinting. The AFLP fingerprints associated with high rutin content accessions of F. tataricum are expected to be useful for evaluation, conservation and genetic improvement of buckwheat.

Expression of flavonoid biosynthesis genes vis-à-vis rutin content variation in different growth stages of Fagopyrum species.

Buckwheat is one of the field crops with the highest concentration of rutin, an important flavonoid of medicinal value. Two species of buckwheat, Fagopyrum esculentum and Fagopyrum tataricum, are the major sources of rutin. Seeds of latter contain 40-50× higher rutin compared to the former. The physiological and molecular bases of rutin content variation between Fagopyrum species are not known. The current study investigated the differences in rutin content in seeds and in other tissues and growth stages of two Fagopyrum species, and also correlated those differences with the expression of flavonoid pathway genes. The analysis of rutin content dynamics at different growth stages, S1-S9 (from seed germination to mature seed formation) of Fagopyrum species revealed that rutin content was higher during seedling stages of F. tataricum (3.5 to 4.6-fold) compared to F. esculentum and then increased exponentially from stages S3 to S6 (different leaf maturing stages and inflorescence) of F. esculentum, whereas it fluctuated in F. tataricum. The rutin content was highest in the inflorescence stage (S6) of both species, with a relatively higher biosynthesis and accumulation during post-flowering stages of F. tataricum compared to F. esculentum. The expression of flavonoid pathway genes, through qRT-PCR, in different growth stages vis-à-vis rutin content variation showed differential expression for four genes, PAL, CHS, CHI and FLS with the amounts of transcripts relatively higher in F. tataricum compared to F. esculentum, thereby, correlating these genes with the biosynthesis and accumulation of rutin. The expression of PAL was highest, 7.69 and 8.96-fold in Stages 2 (seedling stage) and 9 (fully developed seeds) of F. tataricum compared to F. esculentum, respectively. The expression of the CHS gene correlated with the rutin content because it was highest in the flowers (S6) and fully developed seeds (S9) of both Fagopyrum species, with relatively higher transcript amounts (2.13 and 3.19-fold, respectively) in F. tataricum (IC-329457) compared to F. esculentum (IC-540858). This study provides useful information on molecular and physiological dynamics of rutin biosynthesis and accumulation in Fagopyrum species and the correlation of expression of flavonoid biosynthesis genes with the rutin content can be useful in planning for genetic improvement.

Genome-wide distribution and organization of microsatellites in plants: an insight into marker development in Brachypodium.

Plant genomes are complex and contain large amounts of repetitive DNA including microsatellites that are distributed across entire genomes. Whole genome sequences of several monocot and dicot plants that are available in the public domain provide an opportunity to study the origin, distribution and evolution of microsatellites, and also facilitate the development of new molecular markers. In the present investigation, a genome-wide analysis of microsatellite distribution in monocots (Brachypodium, sorghum and rice) and dicots (Arabidopsis, Medicago and Populus) was performed. A total of 797,863 simple sequence repeats (SSRs) were identified in the whole genome sequences of six plant species. Characterization of these SSRs revealed that mono-nucleotide repeats were the most abundant repeats, and that the frequency of repeats decreased with increase in motif length both in monocots and dicots. However, the frequency of SSRs was higher in dicots than in monocots both for nuclear and chloroplast genomes. Interestingly, GC-rich repeats were the dominant repeats only in monocots, with the majority of them being present in the coding region. These coding GC-rich repeats were found to be involved in different biological processes, predominantly binding activities. In addition, a set of 22,879 SSR markers that were validated by e-PCR were developed and mapped on different chromosomes in Brachypodium for the first time, with a frequency of 101 SSR markers per Mb. Experimental validation of 55 markers showed successful amplification of 80% SSR markers in 16 Brachypodium accessions. An online database 'BraMi' (Brachypodium microsatellite markers) of these genome-wide SSR markers was developed and made available in the public domain. The observed differential patterns of SSR marker distribution would be useful for studying microsatellite evolution in a monocot-dicot system. SSR markers developed in this study would be helpful for genomic studies in Brachypodium and related grass species, especially for the map based cloning of the candidate gene(s).