Fab Advances in Fabaceae for Abiotic Stress Resilience: From 'Omics' to Artificial Intelligence.

Legumes are a better source of proteins and are richer in diverse micronutrients over the nutritional profile of widely consumed cereals. However, when exposed to a diverse range of abiotic stresses, their overall productivity and quality are hugely impacted. Our limited understanding of genetic determinants and novel variants associated with the abiotic stress response in food legume crops restricts its amelioration. Therefore, it is imperative to understand different molecular approaches in food legume crops that can be utilized in crop improvement programs to minimize the economic loss. 'Omics'-based molecular breeding provides better opportunities over conventional breeding for diversifying the natural germplasm together with improving yield and quality parameters. Due to molecular advancements, the technique is now equipped with novel 'omics' approaches such as ionomics, epigenomics, fluxomics, RNomics, glycomics, glycoproteomics, phosphoproteomics, lipidomics, regulomics, and secretomics. Pan-omics-which utilizes the molecular bases of the stress response to identify genes (genomics), mRNAs (transcriptomics), proteins (proteomics), and biomolecules (metabolomics) associated with stress regulation-has been widely used for abiotic stress amelioration in food legume crops. Integration of pan-omics with novel omics approaches will fast-track legume breeding programs. Moreover, artificial intelligence (AI)-based algorithms can be utilized for simulating crop yield under changing environments, which can help in predicting the genetic gain beforehand. Application of machine learning (ML) in quantitative trait loci (QTL) mining will further help in determining the genetic determinants of abiotic stress tolerance in pulses.

Molecular mapping of aluminium resistance loci based on root re-growth and Al-induced fluorescent signals (callose accumulation) in lentil (Lens culinaris Medikus).

Development of aluminium (Al) resistant genotypes through molecular breeding is a major approach for increasing seed yield under acidic conditions. There are no available reports on mapping of Al resistance loci and molecular breeding for Al resistant varieties in lentil. The present study reports a major quantitative trait loci (QTL) for Al resistance using simple sequence repeat (SSR) markers in F2 and F3 mapping populations derived from contrasting parents. Phenotypic response to Al was measured on the bases of root re-growth (RRG), fluorescent signals (callose accumulation) and Al contents in hydroponic assay. After screening 495 SSR markers to search polymorphism between two contrasting parents, 73 polymorphic markers were used for bulk segregation analysis. Two major QTLs were identified using seven trait linked markers, one each for fluorescent signals and RRG mapped on linkage group (LG) 1 under Al stress conditions in F2 mapping population of cross BM-4 × L-4602. One major QTL (qAlt_fs) was localised between PLC_88 and PBA_LC_373, covering 25.9 cM with adjacent marker PLC_88 at a distance of 0.4 cM. Another major QTL (qAlt_rrg) for RRG was in the marker interval of PBA_LC_1247 and PLC_51, covering a distance of 45.7 cM with nearest marker PBA_LC_1247 at a distance of 21.2 cM. Similarly, in F3 families of BM-4 × L-4602 and BM-4 × L-7903, LG-1 was extended to 285.9 and 216.4 cM respectively, having four newly developed genic-SSR markers. These QTLs had a logarithm of odd (LOD) value of 140.5 and 28.8 along with phenotypic variation of 52% and 11% for fluorescent signals and RRG respectively, whereas, qAlt_rrg had LOD of 36 and phenotypic variance of 25% in F3 population of BM-4 × L-4602. Two major QTLs identified in the present study can be further dissected for candidate gene discovery and development of molecular markers for breeding improved varieties with high Al resistance.

Transcriptome analysis of lentil (Lens culinaris Medikus) in response to seedling drought stress.

BACKGROUND: Drought stress is one of the most harmful abiotic stresses in crop plants. As a moderately drought tolerant crop, lentil is a major crop in rainfed areas and a suitable candidate for drought stress tolerance research work. Screening for drought tolerance stress under hydroponic conditions at seedling stage with air exposure is an efficient technique to select genotypes with contrasting traits. Transcriptome analysis provides valuable resources, especially for lentil, as here the information on complete genome sequence is not available. Hence, the present studies were carried out. RESULTS: This study was undertaken to understand the biochemical mechanisms and transcriptome changes involved in imparting adaptation to drought stress at seedling stage in drought-tolerant (PDL-2) and drought-sensitive (JL-3) cultivars. Among different physiological and biochemical parameters, a significant increase was recorded in proline, glycine betaine contents and activities of SOD, APX and GPX in PDL-2 compared to JL-3while chlorophyll, RWC and catalase activity decreased significantly in JL-3. Transcriptome changes between the PDL-2 and JL-3 under drought stress were evaluated using Illumina HiSeq 2500 platform. Total number of bases ranged from 5.1 to 6.7 Gb. Sequence analysis of control and drought treated cDNA libraries of PDL-2 and JL-3 produced 74032, 75500, 78328 and 81523 contigs, respectively with respective N50 value of 2011, 2008, 2000 and 1991. Differential gene expression of drought treated genotypes along with their controls revealed a total of 11,435 upregulated and 6,934 downregulated transcripts. For functional classification of DEGs, KEGG pathway annotation analysis extracted a total of 413 GO annotation terms where 176 were within molecular process, 128 in cellular and 109 in biological process groups. CONCLUSION: The transcriptional profiles provide a foundation for deciphering the underlying mechanism for drought tolerance in lentil. Transcriptional regulation, signal transduction and secondary metabolism in two genotypes revealed significant differences at seedling stage under severe drought. Our finding suggests role of candidate genes for improving drought tolerance in lentil.

Mechanistic Association of Quantitative Trait Locus with Malate Secretion in Lentil (Lens culinaris Medikus) Seedlings under Aluminium Stress.

Aluminium (Al) toxicity acts as a major delimiting factor in the productivity of many crops including lentil. To alleviate its effect, plants have evolved with Al exclusion and inclusion mechanisms. The former involves the exudation of organic acid to restrict the entry of Al3+ to the root cells while latter involves detoxification of entered Al3+ by organic acids. Al-induced secretion of organic acids from roots is a well-documented mechanism that chelates and neutralizes Al3+ toxicity. In this study, F6 recombinant inbred lines (RILs) derived from a cross between L-7903 (Al-resistant) and BM-4 (Al-sensitive) were phenotyped to assess variation in secretion levels of malate and was combined with genotypic data obtained from 10 Al-resistance linked simple sequence repeat (SSRs) markers. A major quantitative trait loci (QTL) was mapped for malate (qAlt_ma) secretion with a logarithm of odd (LOD) value of 7.7 and phenotypic variation of 60.2%.Validated SSRs associated with this major QTL will be useful in marker assisted selection programmes for improving Al resistance in lentil.

Comparative Inter- and IntraSpecies Transcriptomics Revealed Key Differential Pathways Associated With Aluminium Stress Tolerance in Lentil.

Aluminium stress causes plant growth retardation and engenders productivity loss under acidic soil conditions. This study accentuates morpho-physiological and molecular bases of aluminium (Al) tolerance within and between wild (ILWL-15) and cultivated (L-4602 and BM-4) lentil species. Morpho-physiological studies revealed better cyto-morphology of tolerant genotypes over sensitive under Al3+ stress conditions. Mitotic lesions were observed in root cells under these conditions. Transcriptome analysis under Al3+ stress revealed 30,158 specifically up-regulated genes in different comparison groups showing contigs between 15,305 and 18,861 bp. In tolerant genotypes, top up-regulated differentially expressed genes (DEGs) were found to be involved in organic acid synthesis and exudation, production of antioxidants, callose synthesis, protein degradation, and phytohormone- and calcium-mediated signalling under stress conditions. DEGs associated with epigenetic regulation and Al3+ sequestration inside vacuole were specifically upregulated in wild and cultivars, respectively. Based on assembled unigenes, an average of 6,645.7 simple sequence repeats (SSRs) and 14,953.7 high-quality single nucleotide polymorphisms (SNPs) were spotted. By quantitative real-time polymerase chain reaction (qRT-PCR), 12 selected genes were validated. Gene ontology (GO) annotation revealed a total of 8,757 GO terms in three categories, viz., molecular, biological, and cellular processes. Kyoto Encyclopaedia of Genes and Genomes pathway scanning also revealed another probable pathway pertaining to metacaspase-1,-4, and -9 for programmed cell death under Al-stress conditions. This investigation reveals key inter- and intraspecies metabolic pathways associated with Al-stress tolerance in lentil species that can be utilised in designing future breeding programmes to improve lentil and related species towards Al3+ stress.

Evaluation of cultivated and wild genotypes of Lens species under alkalinity stress and their molecular collocation using microsatellite markers.

In this study, 285 lentil genotypes were phenotyped under hydroponic and alkaline field conditions. Significant genotypic variation for alkalinity stress was observed among the six Lens species screened hydroponically and in the field having pH up to 9.1. The crucial parameters, like whole Na+ and K+ contents and the Na+/K+ ratio at 40 mM NaHCO3 were found significantly correlated with seedling survivability under hydroponics (r = -0.95, r = 0.93 and -0.97). Genotypes, ranked on the bases of seed yield, restricted uptake of Na+ with thick pith area, increased vascular bundles, less H2O2 production and low Na+/K+ ratio, were found important physio-anatomical traits for alkalinity stress tolerance. The proper regulation of Na+ uptake was found for maintaining higher K+. This relationship is probably the main factor responsible for a better mechanism for tolerance to high pH up to 9.1 in tolerant breeding lines PDL-1 and PSL-9 (cultivars) and ILWL-15, ILWL-192 and ILWL-20 (wild accessions). Based on UPGMA dendrogram, all the genotypes were clustered into four diverse groups. DMRT was implied within the group to differentiate genotypes based on phenotypic response under alkalinity stress. These results can be utilized for selecting diverse parents for developing alkalinity tolerant genotypes.

Correction: Discerning morpho-anatomical, physiological and molecular multiformity in cultivated and wild genotypes of lentil with reconciliation to salinity stress.

[This corrects the article DOI: 10.1371/journal.pone.0177465.].

Molecular Assortment of Lens Species with Different Adaptations to Drought Conditions Using SSR Markers.

The success of drought tolerance breeding programs can be enhanced through molecular assortment of germplasm. This study was designed to characterize molecular diversity within and between Lens species with different adaptations to drought stress conditions using SSR markers. Drought stress was applied at seedling stage to study the effects on morpho-physiological traits under controlled condition, where tolerant cultivars and wilds showed 12.8-27.6% and 9.5-23.2% reduction in seed yield per plant respectively. When juxtaposed to field conditions, the tolerant cultivars (PDL-1 and PDL-2) and wild (ILWL-314 and ILWL-436) accessions showed 10.5-26.5% and 7.5%-15.6% reduction in seed yield per plant, respectively under rain-fed conditions. The reductions in seed yield in the two tolerant cultivars and wilds under severe drought condition were 48-49% and 30.5-45.3% respectively. A set of 258 alleles were identified among 278 genotypes using 35 SSR markers. Genetic diversity and polymorphism information contents varied between 0.321-0.854 and 0.299-0.836, with mean value of 0.682 and 0.643, respectively. All the genotypes were clustered into 11 groups based on SSR markers. Tolerant genotypes were grouped in cluster 6 while sensitive ones were mainly grouped into cluster 7. Wild accessions were separated from cultivars on the basis of both population structure and cluster analysis. Cluster analysis has further grouped the wild accessions on the basis of species and sub-species into 5 clusters. Physiological and morphological characters under drought stress were significantly (P = 0.05) different among microsatellite clusters. These findings suggest that drought adaptation is variable among wild and cultivated genotypes. Also, genotypes from contrasting clusters can be selected for hybridization which could help in evolution of better segregants for improving drought tolerance in lentil.

Molecular and Morpho-Agronomical Characterization of Root Architecture at Seedling and Reproductive Stages for Drought Tolerance in Wheat.

Water availability is a major limiting factor for wheat (Triticum aestivum L.) production in rain-fed agricultural systems worldwide. Root architecture is important for water and nutrition acquisition for all crops, including wheat. A set of 158 diverse wheat genotypes of Australian (72) and Indian (86) origin were studied for morpho-agronomical traits in field under irrigated and drought stress conditions during 2010-11 and 2011-12.Out of these 31 Indian wheat genotypes comprising 28 hexaploid (Triticum aestivum L.) and 3 tetraploid (T. durum) were characterized for root traits at reproductive stage in polyvinyl chloride (PVC) pipes. Roots of drought tolerant genotypes grew upto137cm (C306) as compared to sensitive one of 63cm with a mean value of 94.8cm. Root architecture traits of four drought tolerant (C306, HW2004, HD2888 and NI5439) and drought sensitive (HD2877, HD2012, HD2851 and MACS2496) genotypes were also observed at 6 and 9 days old seedling stage. The genotypes did not show any significant variation for root traits except for longer coleoptiles and shoot and higher absorptive surface area in drought tolerant genotypes. The visible evaluation of root images using WinRhizo Tron root scanner of drought tolerant genotype HW2004 indicated compact root system with longer depth while drought sensitive genotype HD2877 exhibited higher horizontal root spread and less depth at reproductive stage. Thirty SSR markers were used to study genetic variation which ranged from 0.12 to 0.77 with an average value of 0.57. The genotypes were categorized into three subgroups as highly tolerant, sensitive, moderately sensitive and tolerant as intermediate group based on UPGMA cluster, STRUCTURE and principal coordinate analyses. The genotypic clustering was positively correlated to grouping based on root and morpho-agronomical traits. The genetic variability identified in current study demonstrated these traits can be used to improve drought tolerance and association mapping.

Molecular Scanning and Morpho-Physiological Dissection of Component Mechanism in Lens Species in Response to Aluminium Stress.

Aluminium (Al) stress was imposed on 285 lentil genotypes at seedling stage under hydroponics to study its effects on morpho-physiological traits where resistant cultigens and wilds showed minimum reduction in root and shoot length and maximum root re-growth (RRG) after staining. Molecular assortment based on 46 simple sequence repeat (SSR) markers clustered the genotypes into 11 groups, where wilds were separated from the cultigens. Genetic diversity and polymorphism information content (PIC) varied between 0.148-0.775 and 0.140-0.739, respectively. Breeding lines which were found to be most resistant (L-7903, L-4602); sensitive cultivars (BM-4, L-4147) and wilds ILWL-185 (resistant), ILWL-436 (sensitive) were grouped into different clusters. These genotypes were also separated on the basis of population structure and Jaccard's similarity index and analysed to study Al resistance mechanism through determination of different attributes like localization of Al and callose, lipid peroxidation, secretion of organic acids and production of antioxidant enzymes. In contrast to sensitive genotypes, in resistant ones most of the Al was localized in the epidermal cells, where its movement to apoplastic region was restricted due to release of citrate and malate. Under acidic field conditions, resistant genotypes produced maximum seed yield/plant as compared to sensitive genotypes at two different locations i.e. Imphal, Manipur, India and Basar, Arunanchal Pradesh, India during 2012-13, 2013-14 and 2014-15. These findings suggest that Al stress adaptation in lentil is through exclusion mechanism and hybridization between the contrasting genotypes from distinct clusters can help in development of resistant varieties.