DNA barcoding in genera benincasa and praecitrullus
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Date
2019
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Centre for Plant Biotechnology and Molecular Biology, College of Horticulture,Vellanikkara
Abstract
Benincasa is a monotypic genus with only one cultivated species hispida. In India, Benincasa exhibits rich diversity. There is substantial variation in vegetative traits and fruitcharacters and it is difficult to distinguish ashgourd genotypes based on their external morphology alone. In spite of having enormous morphological variability, all members at present are accommodated into asingle species. The taxonomic classification of Praecitrullus is still uncertain. The marker and pollen morphology studieshave revealed that P. fistulosusis more related to B. hispida, than compared to other cucurbit species. 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 characteristic variations on the sequences of identified genomic regions, which can distinguish individuals of a species. Species identification through barcoding is usually achieved by the retrieval of a short DNA sequence from a standard part of the genome (i.e. a specific gene region either from chloroplast, mitochondria or nuclear genome) and identifying the barcode gaps for each species. 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 genera Benincasa and Praecitrullus was done at Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, with the objectives to assess genetic diversity and interspecies relationship among Benincasa and Praecitrullus genotypes and to check the species status of Benincasa using barcodes. Twenty-six ashgourd and five tinda accessions were morphologically screened in the field using the standard descriptor and basedaccessions were clustered, based on morphological characters recorded. The ashgourd accessions varied widely for their morphological traits,fruit size varied between0.191 to 12.0 kg. Thefruit shape, varied from club, spindle, cylindrical, oblong to ridged shape. The growth habit varied from long viny to short viny.Serrated, deep serrated and double serrated leaf margin was observed. For stem pubescencevariations recorded were hispid, downy, tomentose, puberulent, pilose,villous and hirsute. The cross compatibility of the ash gourd accessions were also checked to establish the species status. All the accessions of ash gourd were cross compatiblein both the directions, showing that they belong to the same species. Based on the morphological data of ashgourd accessions nine cluster were generated. One representative accession from each cluster and three representative accessions of tinda selected for the molecular studies. Total genomic was isolated and subjected to PCR assay. The matK and ITS2 gave the bands of 950 bp and 799 bp respectively, which were sufficient to identify barcode gaps.The markers were sequenced from 9 genotypes of ashgourd and 3 tinda genotypes.The BLAST analysis had shown that matK and ITS2 both loci is 99 per cent efficient for species discrimination in Benincasa and Praecitrullus. Barcode gap, a position in the sequence at which a particular nucleotide shows characteristic variation in all the members of a particular species, was identified for all the members of Benincasa and Praecitrullus species. Barcode gaps were identified for tinda, in comparison with Benincasa for both the loci. The barcodes in tinda were observed in matK locus at 47, 127 and 232bp. Similarly, for the locus ITS2, the barcode gaps in tinda were identified at 162, 207, 278, 279 and 288 bp. However, within Benincasa, there were no such characteristic barcode gaps, indicating that the null hypothesis of monotypic status of Benincasa is true. Phylogenetic analysis using Clustal Omega showed the variation within Benincasa species and Praecitrullus species.The barcodes developed in this study could be successfully used to solve the genetic relatedness of Benincasa and Praecitrullus species.
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174600