Molecular Identification and Phylogenetic Analysis of African Nightshade Using Its2 DNA Barcoding

Abstract

African nightshades are several species of plants that are consumed as leaf vegetables and herbs due to their nutritional value and potential medicinal properties. However, many African nightshade species have similar morphological characteristics, which complicate correct segregation and identification of targeted species for specific use. Therefore, the aim of this study was to perform identification and discrimination among species in African nightshade cultivated in Kenya using internal transcribed spacer 2 (ITS2) DNA barcode region followed by the ITS2 RNA secondary structure predictions. A total of 17 samples of African nightshade were collected as seeds from major growing regions of Kenya, planted in the greenhouse and young leaf samples from 2-week-old plants were used for genomic DNA extraction. The quality of the isolated DNA was ascertained using gel electrophoresis method. The PCR amplicons were used for bi-directional Sanger sequencing and the obtained sequences were edited using BioEdit and MEGA 11. The ITS2 sequences were aligned with Muscle, genetic distances calculated using MEGA 11 based on the Kimura 2-Parameter (K2P) and DNA polymorphism neutrality test was carried out using DnaSP version 6.12.03. The RNA secondary structure predictions were performed using the nucleotide sequences from ITS2 against the rRNA database of RNAfold Webserver v2.4.18. The PCR amplification efficiency and the success rate of sequencing of the ITS2 barcode region were 88.24% and 100%, respectively. The ITS2 barcode region showed higher interspecific variation and discriminatory power by accurately delineating the samples into five species of African nightshade. The ITS2 barcode region distinguished accurately 100% of monophyletic species using both BLAST and phylogenetic tree-based methods. Based on phylogenetic analysis, ITS2 region correctly identified all the accessions of African nightshade complex and were discriminated as Solanum nigrum, S. villosum, S. physalifolium, S. scabrum and S. americanum. Phylogenetic analysis of ITS2 barcode region grouped the 15 African nightshade samples into five genetically distinct clades representing the five different species. Nucleotide polymorphism computed a Tajima D value of -1.59715, while the nucleotide diversity was 0.01645 less than the conventional value of 0.05. According to the obtained sequence characteristics, inter and intraspecific K2P genetic distances and a neighbor-joining algorithm, relatively smaller variations occurred both at the species and genotypic levels. The findings from this study demonstrate that ITS2 had the capacity to identify, differentiate and infer phylogenetic relationships of the different species in the African nightshade. In addition, the RNA secondary structure information can be applied to design species-specific primers for identifying ANS samples/varieties. Based on the findings, there is need for the establishment of a database for all the species of African nightshade cultivated in Kenya.