Double-digest restriction-associated DNA sequencing-based genotyping and its applications in sesame germplasm management.

Sesame (Sesamum indicum L.) is an ancient oilseed crop belonging to the family Pedaliaceae and a globally cultivated crop for its use as oil and food. In this study, 2496 sesame accessions, being conserved at the National Genebank of ICAR-National Bureau of Plant Genetic Resources (NBPGR), were genotyped using genomics-assisted double-digest restriction-associated DNA sequencing (ddRAD-seq) approach. A total of 64,910 filtered single-nucleotide polymorphisms (SNPs) were utilized to assess the genome-scale diversity. Applications of this genome-scale information (reduced representation using restriction enzymes) are demonstrated through the development of a molecular core collection (CC) representing maximal SNP diversity. This information is also applied in developing a mid-density panel (MDP) comprising 2515 hyper-variable SNPs, representing almost equally the genic and non-genic regions. The sesame CC comprising 384 accessions, a representative set of accessions with maximal diversity, was identified using multiple criteria such as k-mer (subsequence of length "k" in a sequence read) diversity, observed heterozygosity, CoreHunter3, GenoCore, and genetic differentiation. The coreset constituted around 15% of the total accessions studied, and this small subset had captured >60% SNP diversity of the entire population. In the coreset, the admixture analysis shows reduced genetic complexity, increased nucleotide diversity (π), and is geographically distributed without any repetitiveness in the CC germplasm. Within the CC, India-originated accessions exhibit higher diversity (as expected based on the center of diversity concept), than those accessions that were procured from various other countries. The identified CC set and the MDP will be a valuable resource for genomics-assisted accelerated sesame improvement program.

Genome-wide microsatellites in amaranth: development, characterization, and cross-species transferability.

Amaranth (Amaranthus spp.) belonging to Amaranthaceae, is known as "the crop of the future" because of its incredible nutritional quality. Amaranthus spp. (> 70) have a huge diversity in terms of their plant morphology, production and nutritional quality; however, these species are not well characterized at molecular level due to unavailability of robust and reproducible molecular markers, which is essential for crop improvement programs. In the present study, 13,051 genome-wide microsatellite motifs were identified and subsequently utilized for marker development using A. hypochondriacus (L.) genome (JPXE01.1). Out of those, 1538 motifs were found with flanking sequences suitable for primer designing. Among designed primers, 225 were utilized for validation of which 119 (52.89%) primers were amplified. Cross-species transferability and evolutionary relatedness among ten species of Amaranthus (A. hypochondriacus, A. caudatus, A. retroflexus, A. cruentus, A. tricolor, A. lividus, A. hybridus, A. viridis, A. edulis, and A. dubius) were also studied using 45 microsatellite motifs. The maximum (86.67%) and minimum (28.89%) cross-species transferability were observed in A. caudatus and A. dubius, respectively, that indicated high variability present across the Amaranthus spp. Total 97 alleles were detected among 10 species of Amaranthus. The averages of major allele frequency, gene diversity, heterozygosity and PIC were 0.733, 0.347, 0.06, and 0.291, respectively. Nei's genetic dissimilarity coefficients ranged from 0.0625 (between A. tricolor and A. hybridus) to 0.7918 (between A. viridis and A. lividus). The phylogenetic tree grouped ten species into three major clusters. Genome-wide development of microsatellite markers and their transferability revealed relationships among amaranth species which ultimately can be useful for species identification, DNA fingerprinting, and QTLs/gene(s) identification. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02930-5.

Genetic diversity and population structure analysis of Asian and African aromatic rice (Oryza sativa L.) genotypes.

Rice germplasms collected from different regions could be used as valuable resources for the future breeding programme. For the utilization of such collections, knowledge about the level and distribution of genetic diversity among these collections will facilitate the breeder. In this study, we report the phenotypic correlation, biochemical quality parameters and population genetic analysis of 35 rice accessions including 34 aromatic rice from different countries and a nonaromatic, Nagina 22, a well-known drought resistance variety. Further biochemical quality analysis, gel consistency test, molecular diversity analysis with 55 simple sequence repeat markers, population structure analysis and pair wise FST analysis were also conducted to assess the genetic diversity. The collected rice genotypes showed significant variability in different agronomic traits, i.e. spikelet per panicle, branches per panicle etc. Results obtained from the above tests demonstrated the importance of regional genetic studies for understanding the diversification of aromatic rice in Asian and African rice.

Microbial keratinases: industrial enzymes with waste management potential.

Proteases are ubiquitous enzymes that occur in various biological systems ranging from microorganisms to higher organisms. Microbial proteases are largely utilized in various established industrial processes. Despite their numerous industrial applications, they are not efficient in hydrolysis of recalcitrant, protein-rich keratinous wastes which result in environmental pollution and health hazards. This paved the way for the search of keratinolytic microorganisms having the ability to hydrolyze "hard to degrade" keratinous wastes. This new class of proteases is known as "keratinases". Due to their specificity, keratinases have an advantage over normal proteases and have replaced them in many industrial applications, such as nematicidal agents, nitrogenous fertilizer production from keratinous waste, animal feed and biofuel production. Keratinases have also replaced the normal proteases in the leather industry and detergent additive application due to their better performance. They have also been proved efficient in prion protein degradation. Above all, one of the major hurdles of enzyme industrial applications (cost effective production) can be achieved by using keratinous waste biomass, such as chicken feathers and hairs as fermentation substrate. Use of these low cost waste materials serves dual purposes: to reduce the fermentation cost for enzyme production as well as reducing the environmental waste load. The advent of keratinases has given new direction for waste management with industrial applications giving rise to green technology for sustainable development.

Genome-wide association mapping of salinity tolerance in rice (Oryza sativa).

Salinity tolerance in rice is highly desirable to sustain production in areas rendered saline due to various reasons. It is a complex quantitative trait having different components, which can be dissected effectively by genome-wide association study (GWAS). Here, we implemented GWAS to identify loci controlling salinity tolerance in rice. A custom-designed array based on 6,000 single nucleotide polymorphisms (SNPs) in as many stress-responsive genes, distributed at an average physical interval of <100 kb on 12 rice chromosomes, was used to genotype 220 rice accessions using Infinium high-throughput assay. Genetic association was analysed with 12 different traits recorded on these accessions under field conditions at reproductive stage. We identified 20 SNPs (loci) significantly associated with Na(+)/K(+) ratio, and 44 SNPs with other traits observed under stress condition. The loci identified for various salinity indices through GWAS explained 5-18% of the phenotypic variance. The region harbouring Saltol, a major quantitative trait loci (QTLs) on chromosome 1 in rice, which is known to control salinity tolerance at seedling stage, was detected as a major association with Na(+)/K(+) ratio measured at reproductive stage in our study. In addition to Saltol, we also found GWAS peaks representing new QTLs on chromosomes 4, 6 and 7. The current association mapping panel contained mostly indica accessions that can serve as source of novel salt tolerance genes and alleles. The gene-based SNP array used in this study was found cost-effective and efficient in unveiling genomic regions/candidate genes regulating salinity stress tolerance in rice.

Use of degenerate primers in rapid generation of microsatellite markers in Panicum maximum.

Guineagrass (Panicum maximum Jacq.) is an important forage grass of tropical and semi-tropical regions, largely apomictic and predominantly exist in tetraploid form. For molecular breeding work, it is prerequisite to develop and design molecular markers for characterization of genotypes, development of linkage map and marker assisted selection. Hence, it is an important researchable issue to develop molecular markers in those crops where such information is scanty. Among many molecular markers, microsatellites or simple sequence repeat (SSR) markers are preferred markers in plant breeding. Degenerate primers bearing simple sequence repeat as anchor motifs can be utilized in rapid development of SSR markers; however selection of suitable degenerate primers is a prerequisite for such procedure so that SSR enriched genomic library can be made rapidly. In the present study seven degenerated primers namely KKVRVRV(AG)10, KKVRVRV(GGT)5, KKVRVRV(CT)10, KKVRVRV(AAT)6, KKVRVRV(GTG)6, KKVRVRV(GACA)5, and KKVRVRV(CAA)6 were used in amplification of Panicum maximum genomic DNA. Primers with repeat motifs (GGT)5 and (AAT)6 have not reacted whereas (AG)10, (GACA)5 and (CAA)6 highly informative as they have generated many DNA fragments ranging from 250 to 1600 bps as revealed from the results obtained with restriction digestion of recombinant plasmids. Primer with (CT)10 anchor repeat, amplified fragments of high molecular weight where as (GTG)6 primer generated only six bands with low concentration indicating less suitability of these primerin SSR markers development in P maximum.