DNA barcoding in Abelmoschus spp.(Malvaceae)

Abstract

The genus Abelmoschus, a member of the Malvaceae family, is reported to have 14 species worldwide. Of these, twelve viz., A. esculentus (L.) Moench, A. moschatus Medik., A. ficulneus (Linn.) Wight & Arn., A. manihot (L.) Medik., A. tetraphyllus (Roxb. ex Hornem.) Borss., A. crinitus Wall., A. tuberculatus Pal & Singh, A. caillei (A. Chev.) Stevels, A. angulosus Wall. ex Wight & Arn., A. enbeepeegeearensis, A. palianus and A. odishae, are reported in India, either as native or introduced species. The A. esculentus and A. callei are cultivated for edible pods whereas A. manihot is primarily grown for its consumable leaves, and A. moschatus is valued for the muskscented seeds. Taxonomic classification and species identification in this genus is often challenging due to their polyploid nature, morphological plasticity and intermediate crossability, all resulted through long cultivation and global dispersion. Accurate identification of species is extremely important in crop breeding programmes. DNA barcoding accurately differentiates the closely related taxa based on the nucleotide polymorphisms characteristically conserved for each in the candidate locus. The matK is one of the most extensively studied loci in plants, and has been shown efficient in many horticultural crops including Momordica, Luffa, and Mangifera. This study, "DNA barcoding in Abelmoschus spp. (Malvaceae)" was conducted at Centre for Plant Biotechnology and Molecular Biology, Kerala Agricultural University, and ICAR-National Bureau of Plant Genetic Resources, Regional Station, Thrissur, with the objective of generation of characteristic barcodes for different species in genus Abelmoschus using identified chloroplast genomic locus/ loci. The study included eighteen Abelmoschus accessions representing six species: A. esculentus, A. caillei, A. tetraphyllus, A. tuberculatus, A. moschatus, and A. enbeepeegeearensis. Morphological traits of each accession were also studied in detail to support the species identifications. The accessions were raised in the open field at ICAR-NBPGR, Regional Station, Thrissur during from mid-August 2023 to February 2024. Morphological characterization of six Abelmoschus species, using seventeen traits, revealed diversity in leaf area, pod length, trichome density, seed characteristics, and epicalyx persistence. These traits, assessed using IBPGR descriptors, provided insights into species differentiation and relationships within the genus. Among the species, A. caillei had the largest leaf area, while A. enbeepeegeearensis had the smallest leaves. The A. esculentus and A. caillei produced larger pods, contrasting with A. tetraphyllus and A. tuberculatus, which had shorter pods of less than 7 cm length. Trichome characteristics varied: A. esculentus and A. caillei had pubescent fruit trichomes, while they were short and prickly in A. moschatus, A. tuberculatus, and A. tetraphyllus, and soft and medium in A. enbeepeegeearensis. Seed morphology was distinctive in A. tuberculatus due to trichomes. The epicalyx was non-persistent in A. esculentus, A. caillei, and A. tetraphyllus, adding further differentiation. DNA obtained through manual extraction protocols has failed to amplify the matK gene in PCR reactions and hence good quality DNA was extracted from the tender leaves and seeds using commercial kit (CTAB plant DNA extraction kit). The matK gene from all the accessions was PCR amplified using a universal primer combination designed in this study. PCR products were cleaned up and paired-end sequenced on Sanger platform, sequence contigs generated using CAP3, submitted to the Barcode of Life Data System (BOLD) and barcodes were generated. Alignment of the sequences using MAFFT has shown species-specific nucleotide polymorphisms, with unique variations observed in A. enbeepeegeearensis and A. tetraphyllus. In A. enbeepeegeearensis, all the accessions had specific base substitutions at two positions: adenine (A) replacing thymine (T) at position 54, and cytosine (C) substituting for adenine (A) at position 289. Similarly, A. tetraphyllus exhibited a consistent variation at position 154, where C was replaced with A. These unique polymorphisms in these species are keys to precisely identify them, highlighting their genetic distinction, contributing valuable data to support future taxonomic and conservation efforts. To identify the unique barcodes to differentiate the remaining species of Abelmoschus, alternate chloroplast genes have to be studied. This is the first study on DNA barcoding in this genus and the findings have contributed to its taxonomic resolution. The findings underscore the utility of combining morphological and DNA barcoding methods for accurate species identification and conservation planning.