Molecular identification and evaluation of genetic diversity of Kenyan Dioscorea species using universal DNA barcodes of rbcL and matK regions.

Abstract

Yams (Dioscorea species) are tuber crops that are grown in tropical regions of Africa, the Caribbean, South America, Asia and South Pacific islands. In Kenya, yams are grown in western, coastal and central highland regions as an important food security crop. The crop has also gained commercial interest due to the potent medicinal/pharmacological properties as well as industrial use. However, many Dioscorea species have similar morphology and easily confused and consequently their phylogenetic relationships is poorly understood, which hinders their full utilization and genetic conservation. Therefore, the aim of this study was to identify and evaluate the genetic diversity of Dioscorea species cultivated in Kenya using ribulose 1, 5-bisphosphate carboxylase/oxygenase (rbcL) and Maturase K (matK) barcoding markers. Leaf/tuber samples of 22 yam accessions were collected from farmers’ fields and the National Genebank of Kenya (GBK) followed by DNA extraction and PCR amplifications using rbcL and matK barcoding markers. The purified PCR products were bidirectionally sequenced and sequences generated assembled in Geneious Prime 2019.2. Sequences alignment was conducted on Muscle version 3.8., and subsequent analysis done using PhyML package implemented in Geneious prime 2019.2, BEAST version 1.10, DnaSP version 6.12.03 and Arlequin 3.5.2.2. Phylogenetic analysis of rbcL and matK sequences revealed four strongly supported distinct species that included Dioscorea bulbifera, Dioscorea alata, Dioscorea minutiflora and Dioscorea cayenensis. The yam accessions explored were maximally differentiated into two distinct clades. The specific clade of each of the yam accession was informed by its geographic location and species. DNA polymorphism in the yam species exhibited both synonymous and non-synonymous mutation. RbcL sequences had nucleotide diversity and average number of nucleotide differences of 0.00392 and 2.668, respectively. MatK gene sequences had a nucleotide diversity of 0.00632 and average number of nucleotide differences of 4.87273. There were seven haplotypes within the rbcL gene with a diversity index of 0.800 and variation of 0.00374. There were four haplotypes within the matK gene with a diversity index of 0.745 and variation of 0.00956. Tajima neutrality indicated that rbcL gene of the yam accessions is under sweep selection while matK gene is under balanced selection suggesting that the population is growing. The findings from this study demonstrate that rbcL and matK barcoding markers can identify and differentiate Dioscorea species as well as establish their phylogenetic relationships. These results provide a scientific basis for molecular identification and discrimination of Dioscorea species which is useful in the efficient utilization of the genetic resources as well as in designing their breeding strategies.