Molecular Profiling and Phylogenetic Clustering of [Vigna Unquictdata (L.) Walp.] Gene Pools from the Gene Bank of Kenya and Ecotypes Based On Putative DNA Barcode Loci And intergenic Spacers

Abstract

Thenovelty of mtDNA gene loci, COl in DNA barcoding is undisputed. This is attributed to its standardized sequencing segment of the cytochrome c oxidase-l gene with the necessary universality and variability making it a generally acceptable .locus. mtDNA is robust with threshold for species delineation, not subject to drastic length variation, frequent mononucleotide repeats or microinversions. However, low nucleotide substitutionrate of mtDNA in plants precludes its use making the search for alternative barcoderegions necessary. The putative loci of the plastid region conserved in plant target lineages can achieve rapid, reproducible, and molecular inferencing yet to be explored in biodiversitystudies in Kenya. Four are portions of coding genes [matK, rbeL, rpoB, and rpoCl),. and noncoding spacers (atpF-atpfi tmll-psiiA, and psbK-psbL]. Currently, there existsno universal barcode for plants and while various combinations have been proposed, there exists no consensus; the lack thereof impedes progress in getting a suitable DNA barcode for molecular and phylogenetic inferencing of GeneBank repositories. The genetic pool of these repositories is not adequately characterized, remains scanty and undocumented.DNA was obtained from 54 accessions from Kenyan GeneBank followed by DNA extraction, purification and quantification using DNA Nanodrop, PCR and sequencing. The feasibility and overall utility of each locus was tested singly and in combination to establish a locus that could have applicability in delineating phylogeographically diverse genotypes. Statistical analyses were based on genetic parameters including analyses of intra- and infra- specific genetic divergence based on K2P distance matrices on a scale of 0.001 distance units; Wilcoxon signed rank tests and coalescenceanalyses to delineate independent genetic clades. Results indicate that: matK, trnH-psbA, psbK-psbL, and rbeL are informative in delineating intra and infraspecific distances.rbeL had the highest identification efficiency [50%]. DNA barcoding gaps were evaluatedby comparing intra and infraspecific divergences. Median and Wilcoxon Two- Sample Tests were used to evaluate distributions. Overall, MLA was more informative than SLA. The trnH ysbA generated five distinct and well differentiated clusters. The study revealed 38 haplotypes circulating among Kenyan Cowpea. The atpF_atpH recorded the highest SNPs [2240] and infraspecific frequencies of SNPs 1100 bp. It is notable that rbcl. recorded the highest heterozygous sites. Results indicate that matK »trnH-psbA {P<O.OOOl]. The combinations matK+psbK-psbL and matK+ ysbKpsbL+ _atpF-atpH were informative to {matK+trnH-psbA+atpF--atpH+psbK-psbLJ [p<O.000]. However, the four loci were more parsimonious than the three loci: matK + trnH-psbA + atpli-atpll, P<O.OOOl. Overall, the current study reveals low genetic diversity among Kenyan cowpea but high %GC content as reason for adaptability to diverse phylogeographic settings. It would appear therefore that rbeL is the most informative. Pairwise FST values for population differentiation were insignificant indicating low genetic diversity. The study clearly demonstrates the overall utility of plastid markers in molecular and phylogenetic inferencing of Kenyan GeneBank repositories and informs the scanty body of knowledge on the novelty of DNA barcoding as a tool in cataloguing and phylogenetic inferring at molecular level and acknowledges its use in future research on genomics