DNA barcoding in momordica spp.
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Date
2014
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Centre for plant biotechnology and molecular biology, College of horticulture, Vellanikkara
Abstract
The genus Momordica comprises of 59 species, among which 7 are of Indian origin. Though the vegetables belonging to this genus are nutritionally rich with medicinal properties, their taxonomy remains confusive. The botanical names are often used incorrectly and interchangeably. Different taxonomic classification approaches have resulted in controversies about the number of species that exist and the phylogenetic relationships among these species. This situation necessitates an accurate, sensitive and simple alternate for the traditional taxonomy. DNA barcoding is a novel system designed to provide rapid, accurate, and automatable species identification using short, standardized genomic regions as internal species tags. DNA barcoding is based on the variation on the sequences identified genomic regions, which can distinguish individuals of a species because genetic variation between species exceeds that within species. Species identification through barcoding is usually achieved by the retrieval of a short DNA sequence i.e. the „barcode‟ from a standard part of the genome (i.e. a specific gene region either from chloroplast, mitochondria or nuclear genome). The barcode sequence from each unknown specimen is then compared with a library of reference barcode sequences derived from individuals of known identity. The study entitled “DNA barcoding in Momordica spp.” was done at the Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara. The objective of this study was to develop the barcodes for the 7 Momordica spp. which were found in India. For this study, 25 accessions of seven Momordica species M. charantia, M. balsamina, M. dioica, M. sahyadrica, M. cochinchinensis, M. subangulata subsp. renigera and M. cymbalaria and two accession of Luffa has been taken. Total genomic DNA isolated using CTAB method (Rogers and Bendich, 1994) was subjected to PCR assay using various combinations of universal barcode primers for three loci matK, ITS2 and trnH-psbA. As ITS2 and trnH-psbA gave the bands of 600 and 300bp, respectively, and which are not sufficient to develop the complete barcodes, these loci were not used in the study. Thus the bands generated using the matK primers from all the 25 Momordica and 2 Luffa were used for sequencing. Phylogenetic analysis using ClustalW has discriminated the various species under Momordica, except dioica and sahyadrica. Kimura 2 Parameter (K2P) model using MEGA software. A wide level of molecular diversity detected with both the method which shows the high level of genetic variation among the species of 25 accessions of Momordica of Indian origin and Momordica and Luffa. Barcoding gap, a position in the sequence at which unique nucleotide is present in all the members of a particular species, was assessed for all the Momordica species and the Luffa, and these gaps were used to generate the barcodes for that species. From this study, barcodes were successfully generated for M. charantia, M. subangulata, M. cochinchinensis, M. cymbalaria, M. balsamina and Luffa acutangula. The BLAST analysis had shown that matK is 95 per cent efficient for species discrimination in Momordica. From the base sequence of matK generated from this study, barcoding primers were designed for Momordica and were successful in all the 25 accessions. The matK barcodes developed in this study could be successfully used to solve the taxonomic confusion in Momordica.
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