Genome survey sequencing-based SSR marker development and their validation in Dendrocalamus longispathus.

Bamboo is an important genetic resource in India, supporting rural livelihood and industries. Unfortunately, most Indian bamboo taxa are devoid of basic genomic or marker information required to comprehend the genetic processes for further conservation and management. In this study, we perform genome survey sequencing for development of de novo genomic SSRs in Dendrocalamus longispathus, a socioeconomically important bamboo species of northeast India. Using Illumina platform, 69.49 million raw reads were generated and assembled into 1,145,321 contig with GC content 43% and N50 1228 bp. In total, 46,984 microsatellite repeats were mined-out wherein di-nucleotide repeats were most abundant (54.71%) followed by mono- (31.91%) and tri-repeats (9.85%). Overall, AT-rich repeats were predominant in the genome, but GC-rich motifs were more frequent in tri-repeats. Afterwards, 21,596 SSR loci were successfully tagged with the primer pairs, and a subset of 50 were validated through polymerase chain reaction amplification. Of these, 36 SSR loci were successfully amplified, and 16 demonstrated polymorphism. Using 13 polymorphic SSRs, a moderate level of gene diversity (He = 0.480; Ar = 3.52) was recorded in the analysed populations of D. longispathus. Despite the high gene flow (Nm = 4.928) and low genetic differentiation (FST = 0.119), severe inbreeding (FIS = 0.407) was detected. Further, genetic clustering and STRUCTURE analysis revealed that the entire genetic variability is captured under two major gene pools. Conclusively, we present a comprehensive set of novel SSR markers in D. longispathus as well as other taxa of tropical woody bamboos.

Genome skimming-based simple sequence repeat (SSR) marker discovery and characterization in Grevillea robusta.

Proteaceae, a largely southern hemisphere family consisting of 80 genera distributed in Australia and southern Africa as its centres of greatest diversity, also extends well in northern and southern America. Under this family, Grevillea robusta is a fast-growing species got popularity in farm and avenue plantations. Despite the ecological and economic importance, the species has not yet been investigated for its genetic improvement and genome-based studies. Only a few molecular markers are available for the species or its close relatives, which hinders  genomic and population genetics studies. Genetic markers have been intensively applied for the main strategies in breeding programs, especially for the economically important traits. Hence, it is of utmost priority to develop genomic database resources and species-specific markers for studying quantitative genetics in G. robusta. Given this, the present study aimed to develop de novo genome sequencing, robust microsatellites markers, sequence annotation and their validation in different stands of G. robusta in northern India. Library preparation and sequencing were carried out using Illumina paired-end sequencing technology. Approximately, ten gigabases (Gb) sequence data with 70.87 million raw reads assembled into 425,923 contigs (read mapped to 76.48%) comprising 455 Mb genome size (23 × coverage) generated through genome skimming approach. In total, 9421 simple sequence repeat (SSR) primer pairs were successfully designed from 13,335 microsatellite repeats. Afterward, a subset of 161 primer pairs was randomly selected, synthesized and validated. All the tested primers showed successful amplification but only 13 showed polymorphisms. The polymorphic SSRs were further used to estimate the measures of genetic diversity in 12 genotypes each from the states of Punjab, Haryana, Himachal Pradesh and Uttarakhand. Importantly, the average number of alleles (Na), observed heterozygosity (Ho), expected heterozygosity (He), and the polymorphism information content (PIC) were recorded as 2.69, 0.356, 0.557 and 0.388, respectively. The availability of sequence information and newly developed SSR markers could potentially be used in various genetic analyses and improvements through molecular breeding strategies for G. robusta. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-01035-w.

Chloroplast-based DNA barcode analysis indicates high discriminatory potential of matK locus in Himalayan temperate bamboos.

The study was conducted to evaluate the discriminatory potential of selected chloroplast-based DNA barcode regions for identifying and resolving phylogeny of the Indian bamboos. Among 11 chloroplast markers screened, only four, namely matK, rbcL, psbK-I and rps16-trnQ showed successful amplification in 88 genotypes of 30 Indian bamboo taxa under Bambuseae and Arundinarieae tribes. A total of 244 sequences were generated for the four chloroplast regions. Tree-based analysis demonstrated that none of the tested regions successfully discriminated the taxa under Bambuseae tribe. Importantly, our highly concerned Himalayan temperate bamboo species under Arundinarieae tribe, were successfully discriminated by matK locus with high bootstrap support (>60%). Sequence comparisons revealed that the discriminatory power demonstrated by matK region actually lies in the few unique fixed nucleotides (UFNs) despite the overall DNA polymorphism. Although, rps16-trnQ region was found to be the most polymorphic and revealed high genetic divergence among different taxonomic levels, it could not successfully discriminated the taxa with strong statistical support. In a taxonomically difficult plant group like bamboos, whose genome is relatively more complex and has a slow rate of molecular evolution, it is difficult to get a universal marker. Further, highly variable barcode regions utilized in other species may not be informative, and thus, the development of DNA barcodes for different taxonomic levels, such as lineages or tribes could be a viable approach.

First de novo genome specific development, characterization and validation of simple sequence repeat (SSR) markers in Genus Salvadora.

Salvadoraceae constitutes ecologically imperative desert families of 3 genera-Azima, Dobera and Salvadora. Under genus Salvadora of this family, S. oleoides is a keystone species of socio-economic and medicinal value. This species naturally grows in the arid zones but currently experiencing severe fragmentation due to land use change and reduced regeneration, which may have resulted in the depletion of genetic diversity. Hence, it is up-most important to develop genomic resources for studying the population genetics in S. oleoides. This study aims to develop robust microsatellites markers, which were not yet reported in genus Salvodora due to lack of genome sequence information. We developed novel microsatellites markers in S. oleoides using Illumina paired-end sequencing technology. In total, 14,552 simple sequence repeat (SSR) markers were successfully designed from 21,055 microsatellite repeats detected in the 13 Gb raw sequence data. Afterwards, a subset of 101 SSRs were randomly selected and validated, 94 primers were successfully amplified and 34 showed polymorphisms. These SSRs were used to estimate the measures of genetic diversity in three natural populations of state Rajasthan and Gujarat. Importantly, average number of alleles (Na), observed heterozygosity (Ho), expected heterozygosity (He), and polymorphism information content (PIC) were recorded as 2.4, 0.529, 0.357, and 0.326, respectively. Furthermore, 15 primers were evaluated in S. persica for cross-transferability, and all were successfully amplified but only eight showed polymorphisms. This study has been conducted first time for S. oleoides and pioneer among the native species of arid-zone in India.

Peptide Combination Generator: a Tool for Generating Peptide Combinations.

Peptides are used as reagents both for basic research and diagnostic purposes. Therefore, there is a need for novel methods for the design of peptide molecules with a particular specific physiochemical profile. The properties of the peptides are governed by the nature of amino acids constituting the peptide. There is a lack of a web server or tools which could predict all the possible combinations of the peptides generated because of the combinations of amino acids based on the physiochemical properties. We have developed a peptide combination generator (PepCoGen), a web server for generating all the possible combinations of peptides by varying the amino acids having similar physiochemical properties at a particular position. It also predicts other properties of the peptides including molecular weight, charge, solubility, hydrophobic plot, and isoelectric point, and random three-dimensional models for each generated combination.