Genome skimming-based STMS marker discovery and its validation in temperate hill bamboo Drepanostachyum falcatum.

Drepanostachyum falcatum (Nees) Keng f. is one of the most widely distributed shrubby bamboo species in the temperate region of northwest (NW) Himalayas. Along with the other three temperate bamboo species, namely Yushania anceps, Thamnocalamus spathiflorus and Himalayacalamus falconeri, commonly called as 'ringal', and utilized for making various articles of household and commercial purpose by local artisans. Despite huge ecological and socio-economic importance, they are least studied and lacks baseline genetic information. In this study, ~10 Gb genome sequence data with 70.68 million reads were generated for D. falcatum, through genome skimming approach based on high throughput next-generation sequencing technology with Illumina protocol. The high-quality reads were de novo assembled into 31,997 contigs, which comprised 1943 microsatellite repeats. The dinucleotide and trinucleotide repeats were most abundantly distributed in the genome with 52.95 and 41.17%, respectively. Depending on the sufficient flanking sequence, only 1123 repeats were successfully tagged with primer pairs and these sites were designated as sequence-tagged microsatellite (STMS) markers. Further, a subset of 106 STMS markers were validated through PCR amplification; 77 marker loci were successfully amplified, and 48 of these showed polymorphism. Same set of marker loci were also tested for their cross-amplification in other three temperate bamboo species of the NW Himalayas, which revealed good level of transferability (27-48%) but lesser polymorphism (4-12%). In addition, the genomewide in silico cross-amplification revealed poor cross-transferability in other bamboo taxa representing four different phylogenetic lineages, namely Phyllostachys edulis (10.2%), Bonia amplexicaulis (3.03%), Guadua angustifolia (1.60%), Olyra latifolia (0.89%) and Raddia guianensis (0.36%). Ten polymorphic markers were further used to estimate the measures of genetic diversity in two natural populations, which revealed high genetic diversity (polymorphic information content, PIC = 0.889; expected heterozygosity, He = 0.756) and low genetic differentiation (FST=0.061; Nm = 5.445). To the best of our knowledge, this is one of the pioneer studies carried out for the development of genomic STMS markers through genome skimming approach in Indian bamboo species. The marker information generated here is novel and of paramount importance for future genetic studies in D. falcatum as well as other temperate bamboo species through cross-transferability.

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.