Nuclear magnetic resonance-based chemical signature and nutritional discrimination of Macrotyloma uniflorum accessions from the alpine Himalayas.

BACKGROUND: Macrotyloma uniflorum (horsegram) is cultivated in the alpine Himalayas for its nutritional benefits. The chemical diversity of M. uniflorum has been explored by several research groups, but none has reported the chemical signature to monitor the identity and quality. To overcome quality control problems and provide more accurate chemotype information, proton nuclear magnetic resonance (1 H-NMR)-based chemical signature and tentative metabotype discrimination was studied. RESULTS: Metabolites were identified by characteristic chemical shifts and their correlations in one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR) experiments. The identifications were confirmed by reference standards, with in-house databases, and with public databases. From the analysis of seed samples from eight locations, NMR detected 46 diversified metabolites, including 18 amino acids, nine carbohydrates, three vitamins, seven flavonoids, and nine miscellaneous molecules (fatty acids/choline/phenolics/organic acids/sterols). These metabolites generated a single snapshot chemical signature for M. uniflorum. Greater content of identified metabolites was found at high elevations of 1829 m (55.78 g kg-1 ) followed by 1000-1150 m (19.30-17.23 g kg-1 ) and 1150-1450 m (7.33-13.41 g kg-1 ). Principal component analysis of samples revealed that chemotypes grouped into three distinct clusters. 'Cluster-3' was positively correlated with metabolites and found the best nutritionally enriched chemotype. CONCLUSIONS: A proton nuclear magnetic resonance-based M. uniflorum chemical signature was generated for the first time. The environmental conditions of 1829 m were most suitable for obtaining nutritionally enriched M. uniflorum crops. Overall, this information could help in the conservation, quality control, product development, and agriculture inventions of M. uniflorum. © 2021 Society of Chemical Industry.

Development of transcriptome-wide SSR markers for genetic diversity and structure analysis in Macrotyloma uniflorum (Lam.) Verdc.

Horsegram is an important drought resistant pulse crop from Fabaceae and can be easily grown in dry lands with no irrigation facilities. However, it remained neglected since long and has been considered as orphan legume which requires immediate attention for its improvement and for the development of new promising varieties in future. In the present study, 7352 simple sequence repeat (SSR) markers were developed from the transcriptome data and 150 SSR were randomly synthesized for validation and diversity analysis in a panel of 58 horsegram genotypes. The synthesized primers included all types of repeats spanning direpeats to hexarepeats. Of the validated SSR markers, 33 markers were polymorphic and produced 40 loci which were used to analyze the genetic diversity and structure of horsegram. In total, 130 alleles were produced in a range of 2-9 alleles with maximum alleles produced by primer HTSSR 155. Expected heterozygosity (He) ranged from 0.03 to 1.00 and observed heterozygosity (Ho) ranged from 0.13 to 0.81. Polymorphism information content value ranged from 0.065 to 0.78. Dendrogram based on UPGMA and principal component analysis showed four groups of the 58 genotypes of horsegram. Structure analysis showed three genetic stocks for the analyzed germplasm. Thus, the developed SSRs can be useful in future population genetics analysis, molecular breeding studies and mapping works in horsegram germplasm as well as in related legume species.

Analysis of an intraspecific RIL population uncovers genomic segments harbouring multiple QTL for seed relevant traits in lentil (Lens culinaris L.).

Improving seed related traits remains key objective in lentil breeding. In recent years, genomic resources have shown great promise to accelerate crop improvement. However, limited genomic resources in lentil greatly restrict the use of genomics assisted breeding. The present investigation aims to build an intraspecific genetic linkage map and identify the QTL associated with important seed relevant traits using 94 recombinant inbreds (WA 8649090 × Precoz). A total of 288 polymorphic DNA markers including simple sequence repeat (SSR), inter simple sequence repeat (ISSR) and random amplified polymorphic DNA (RAPD) were assayed on mapping population. The resultant genetic linkage map comprised 220 loci spanning 604.2 cM of the lentil genome, with average inter-marker distance of 2.74 cM. QTL mapping in this RIL population uncovered a total of 18 QTL encompassing nine major and nine minor QTL. All major QTL were detected for seed related traits viz., seed diameter (SD), seed thickness (ST), seed weight (SW) and seed plumpness (SP) across two locations. A considerable proportion of the phenotypic variation (PV) was accounted to these QTL. For instance, one major QTL on LG5 controlling SW (QTL 15) explained 50% PV in one location, while the same QTL accounted for 34.18% PV in other location. Importantly, the genomic region containing multiple QTL for different seed traits was mapped to a 17-cM region on LG5. The genomic region harbouring QTL for multiple traits opens up exciting opportunities for genomics assisted improvement of lentil.

Genetic and phytochemical diversity analysis in Bunium persicum populations of north-western Himalaya.

The present study explores morphological, genetic and phytochemical composition of Bunium persicum populations belonging to high altitudinal areas of Indian Himalayan region. In total, 23 morphological traits (13 quantitative and 10 qualitative traits) and 32 Random Amplified Polymorphic DNA primers were employed to infer the population structure of the species. Of the fourteen populations, five genetically diverse populations were analyzed for phytochemical diversity. Among morphological traits, inflorescence, seed and branch traits were most significant in detecting variation. At molecular level, primers TIBMBA-06 and OPR-16 were found most polymorphic with respect to Polymorphism Information Content and Marker Index values. Dendrogram grouped all populations into two major clusters while population from Shong region out grouped separately showing its distantness from all other populations. STRUCTURE analysis was done by using Bayesian model, which characterised all populations into four clusters and some degree of admixture was also observed within individuals. Shong population showed distinct genetic makeup as also suggested by dendrogram. Phytochemical analysis showed the presence of 55 components, of which, 2-methyl-3-phenyl propanal, benzeneacetic acid, 1-phellandrene, γ-terpene, α-terpinolene, Δ0.3-carene and sabinene were major components in its essential oils. The present study revealed high genetic and phytochemical diversity in B. persicum accessions from north-western Himalayan regions. Specifically, accessions from Saptal regions were having higher quantity of essential oils and can be selected for cultivation to meet the commercial demand to some extent. Further, the diversity information provided herein can be useful in management and improvement of this species through future breeding programmes.

Mapping of Genomic Regions for Biochemical and Physiological Parameters Contributing Towards Drought Tolerance in Horsegram (Macrotyloma uniflorum (Lam.) Verdc.).

Horsegram (Macrotyloma uniflorum (Lam.) Verdc.) is a drought hardy legume which can be grown in varied soil and temperature regimes. Though it has numerous, nutritive and medicinal benefits, it still lags behind other legumes in terms of genomic resources and genetic improvement. This crop is mostly cultivated on marginal and drought-prone area; thus, genetics of drought stress tolerance can be understood by studying the various drought parameters. To get insight, quantitative trait loci for drought-tolerant traits were identified using an intraspecific mapping population of 162 F8 recombinant inbred lines derived from a cross between HPKM249 and HPK4. The linkage map already developed was used along with the phenotypic data for biochemical and physiological parameters to identify genomic regions which are linked to drought tolerance. In the study, a total of seven QTLs were identified for ten different drought-related traits. One QTL for malondialdehyde content on linkage group 2, two QTLs for root length on linkage groups 3 and 9, one QTL each for proline and chlorophyll content under drought stress on linkage group 4, and one QTL each for root dry weight and root fresh weight on linkage group 5 were identified using composite interval mapping. The identified QTLs will be utilized in marker-assisted breeding and increase our understanding on the physiology of drought stress tolerance.

Comprehensive transcriptomic study on horse gram (Macrotyloma uniflorum): De novo assembly, functional characterization and comparative analysis in relation to drought stress.

BACKGROUND: Drought tolerance is an attribute maintained in plants by cross-talk between multiple and cascading metabolic pathways. Without a sequenced genome available for horse gram, it is difficult to comprehend such complex networks and intercalated genes associated with drought tolerance of horse gram (Macrotyloma uniflorum). Therefore, de novo transcriptome discovery and associated analyses was done for this highly drought tolerant yet under exploited legume to decipher its genetic makeup. RESULTS: Eight samples comprising of shoot and root tissues of two horse gram genotypes (drought-sensitive; M-191 and drought-tolerant; M-249) were used for comparison under control and polyethylene glycol-induced drought stress conditions. Using Illumina sequencing technology, a total of 229,297,896 paired end read pairs were generated and utilized for de novo assembly of horse gram. Significant BLAST hits were obtained for 26,045 transcripts while, 3,558 transcripts had no hits but contained important conserved domains. A total of 21,887 unigenes were identified. SSRs containing sequences covered 16.25% of the transcriptome with predominant tri- and mono-nucleotides (43%). The total GC content of the transcriptome was found to be 43.44%. Under Gene Ontology response to stimulus, DNA binding and catalytic activity was highly expressed during drought stress conditions. Serine/threonine protein kinase was found to dominate in Enzyme Classification while pathways belonging to ribosome metabolism followed by plant pathogen interaction and plant hormone signal transduction were predominant in Kyoto Encyclopedia of Genes and Genomes analysis. Independent search on plant metabolic network pathways suggested valine degradation, gluconeogenesis and purine nucleotide degradation to be highly influenced under drought stress in horse gram. Transcription factors belonging to NAC, MYB-related, and WRKY families were found highly represented under drought stress. qRT-PCR validated the expression profile for 9 out of 10 genes analyzed in response to drought stress. CONCLUSIONS: De novo transcriptome discovery and analysis has generated enormous information over horse gram genomics. The genes and pathways identified suggest efficient regulation leading to active adaptation as a basal defense response against drought stress by horse gram. The knowledge generated can be further utilized for exploring other underexploited plants for stress responsive genes and improving plant tolerance.

Metabolite profiling and protein quantification to a large library of 96 horsegram (Macrotyloma uniflorum) germplasm.

The present study was framed for the assessment of metabolic diversity of 96 diverse horsegram lines derived from 700 germplasm accessions of Pan India. The nutritional component of horsegram germplasm was studied using nuclear magnetic resonance spectroscopy (1H NMR) and micro-Kjeldal method. Differential range of protein content was found 13-40% in the selected panel. The related wild species Macrotyloma sar-gharwalensis contained highest protein content (40%), and the lowest protein content (13%) was found in IC-120837 and TCR-1439. Seed based 1H NMR spectroscopy of horsegram discovered 45 different metabolites (17 amino acids, 7 flavonoids, 10 organic acids/phenolic acids/sugar alcohols/steroids, 7 carbohydrades/sugars, and 4 vitamins). The number of total metabolites quantified among diverse horsegram panel ranged from 25-44. The lowest metabolites number was 26 in PLKU-38, while, IC-280031 and IC-139356 lines showed the presence of highest number of metabolites (44). Lines IC-280031 and IC-139356 were found most nutritive amongst selected panel of horsegram germplasm by containing maximum number of quantifiable metabolites during 1H NMR spectroscopy study. Further, the NMR based data of 96 germplasms was subjected for statistical analysis (PCA, Matrix plot, stacked charts) that revealed the similarities and variations among the whole germplasm. The methionine, sucrose, maltose, riboflavin and myricetin were observed as differential chemo-markers that help to differentiate the horsegram lines of selected panel. This information will further aid in the selection of nutritionally efficient cultivars for proteomics and genomics studies and these lines can be used as nutraceutical food for the prevention of various health ailments including kidney stone.

Elucidation of drought tolerance potential of horsegram (Macrotyloma uniflorum Var.) germplasm using genome wide association studies.

Horsegram [Macrotyloma uniflorum Lam (Verdc.)] is an undervalued and under studied legume though is a good source of proteins, carbohydrates and energy. Drought is an abiotic stress that effects plant development and ecosystem sustainability. Drought is expected to become more common in the future as a result of climate change. Horsegram is known to withstand drought, salt and heavy metal stress. In the past few decades application of genome-wide association studies (GWAS) to explore complex traits has risen in popularity. Considering the above mentioned factors drought tolerance ability of horsegram germplasm was investigated in 96 diverse horsegram lines with GWAS by exploring 20241 SNPs. Highest number of SNPs were found to be located in intergenic regions (43.8%) followed by intronic SNPs (21.6%). In this investigation three drought tolerant representing parameters were selected for QTL identification. In the present study, we identified different SNPs associated with QTLs governing these traits, which involved in drought stress response of horsegram plant. Seven QTLs were found to be associated with relative water content in horsegram whereas for root volume and root length 4 and 8 QTLs were found respectively. By using horsegram database of Kazusa DNA research institute Japan, we identify the genes present on these marker sites which were found to be involved in many biochemical pathways related to plant abiotic stresses. Many of these genes were previously characterized and few uncharacterized genes were also found controlling these traits. These findings will help in identifying new mechanisms responsible for plant drought stress tolerance in future.

Identification of QTLs Linked to Phenological and Morphological Traits in RILs Population of Horsegram (Macrotyloma uniflorum).

Horsegram [Macrotyloma uniflorum (Lam.) Verdc.] is an important legume but understudied in terms of its genetic improvement. Genetic information on various phenological and morphological traits may help in the utilization of new genes for breeding in horsegram and thus affect agronomic practices and crop yield. A total of 162 recombinant inbred lines derived from intraspecific crosses between HPKM249 × HPK4 was used to construct a genetic linkage map and to identify quantitative trait loci (QTLs) associated with phenological and morphological traits. Of the total 2011 molecular markers, which were screened on parental lines for polymorphism survey, 493 markers were found to be polymorphic and used for genotyping of recombinant inbred line population. Out of 493 polymorphic markers, 295 were mapped on ten linkage groups at LOD 3.5 spanning a total distance of 1,541.7 cM with an average distance between markers of 5.20 cM. Phenotypic data of two years at two different locations were used to identify QTLs by composite interval mapping A total of four QTLs (LOD ≥2.5) for phenological traits (days to 50% flowering, reproductive period and days to maturity) and seven QTLs (LOD ≥2.5) for morphological traits (plant height, primary branches and secondary branches) were detected across different environments. The phenotypic variation explained by QTLs ranged from 6.36 to 47.53%. The present study will help to augment scanty genomic information in this orphan crop that would provide genomics tools to breeders for its genetic enhancement through molecular-assisted selection.

Comprehensive nutritional profiling and activity directed identification of lead antioxidant, antilithiatic agent from Macrotyloma uniflorum (Lam.) Verdc.

Macrotyloma uniflorum (Lam.) Verdc. (family Fabaceae) is traditionally important pulse used for kidney stone degradation, cold,cough, asthma and in bronchial issues. It is well reported for the antilithiatic activity and nutritional profiling but no attempt has been made to find out the mechanism and nutrient responsible for the activity in its seed decoction. Therefore, lead high altitude variety responsible for the highest nutrient content and antilithiatic activity was selected for the study. Boiled spray dried extract (M1), lead fractionation (Spray dried residual water fraction: M2), and crude powder extract (M3) were nutritionally profiled using proximate composition analysis, Atomic absorption spectroscopy (minerals and trace elements), Ion exchange chromatography (carbohydrate analysis) and NMR spectroscopy (polar and non polar metabolite analysis). These extracts were also screened for the antilithiatic activity and found significantly important. M1 showed more activity than M3. Further, boiled extract was fractionated with different polarity of solvents (hexane, chloroform, n-butanol, residual water extract). All fractions and reference standard (cystone) were screened for the antilithiatic activity. M2 showed highest activity followed by ethyl acteate while hexane and chloroform did not show any activity in comparison to standards. Thus, lead fraction and market reference standard (cystone) were validated for the presence of nutritional components. Moreover, the selected nutritional components were individually and in combinations screened for the activity which revealed that kidney stone degradation was due to the synergetic action of components than the individual. The results clearly pave a way that Macrotyloma uniflorum is best for combating with malnutrition and kidney stone related problems.

Assessment of calcium oxalate crystal inhibition potential, antioxidant activity and amino acid profiling in horse gram (Macrotyloma uniflorum): high altitude farmer's varieties.

Seeds of Macrotyloma uniflorum collected from different Himalayan regions of Himachal Pradesh, India were screened for inhibition potential of calcium oxalate crystals along with their antioxidant and amino acid profiling. Aqueous, ethanol, 50% ethanol extracts were tested using agar-gel overlay, nucleation, and artificial urine methods in comparison to marketed products. The aqueous extract of Sundernagar and Rampur seeds showed highest inhibition potential (9.0 ± 0.81 mm) than alcohol and hydroalcohol. Sundernagar seeds (aqueous extract) also showed maximum inhibition (45 ± 1.2 and 24 ± 0.88%) in nucleation and artificial urine assay, respectively. The Tris-buffer (pH. 8.0) extract of dry seeds showed more inhibition with low protein than high protein germinated seeds. The findings revealed that horse gram protein is not alone responsible for CaOx-crystals inhibition but its secondary metabolites may also contribute. Among all samples, buffer and aqueous extracts showed maximum activity, followed by hydroalcohol and alcohol. Further, TLC and UPLC method were used to monitor the presence of amino acids in aqueous and Cystone extracts. Sundernagar seeds contain 11 amino acids with additional arginine, aspargine, and methionine. Moreover, higher antioxidant potential was observed in seeds of high altitude, which relates the factors of altitudinal variation in secondary metabolism of plants. Therefore, this study will help in the development of economical and more reliable plant based nutraceuticals for kidney stone problems and related health issues.

Identification of new cytotypes of Valeriana jatamansi Jones, 1970 (Valerianaceae) from North-Western Himalayan region of India.

Valeriana jatamansi, a medicinally important species of the family Valerianaceae, has been cytologically studied in different geographical areas of North-Western Himalayan region of India. The tetraploid cytotype with chromosome numbers 2n=32 is in conformity with the earlier reports of the species from different parts of the world. An octoploid cytotype (2n=64) makes a new addition for the species on a worldwide basis, whereas the diploid cytotype (2n=16) is new to India have been reported for the first time in India. These cytotypes (2n=16, 32, 64) show significant variations with respect to morphology as well as geographical distribution in the Western Indian Himalayas. Further, anomalous populations have been marked with meiotic abnormalities in the form of cytomixis, chromosomal stickiness, unoriented bivalents, formation of laggards and bridges resulting in abnormal microsporogenesis, and production of heterogeneous-sized fertile pollen grains along with reduced pollen fertility.