DNA barcoding in Cucumis spp

Abstract

The genus Cucumis L. has 52 species distributed mainly in Asia and Africa of which 13 belong to India. Cucumber (Cucumis sativus L.) and melon (C. melo L.) are among the most widely cultivated horticultural crops in the world. Both the species have progenitor populations in the Himalayan region and studies have suggested Asia as the ancestral area for the most recent common ancestor of melon and cucumber. Knowing the closest relatives is important for the ongoing efforts of breeders to improve cucumber and melon. The wild relatives of cultivated species form an essential gene pool for vigorous growth, productivity, resistance to biotic and abiotic stresses and nutritional quality. Many wild varieties and species belonging to the genus Cucumis L. have been reported from Asia, Africa and Australia. Nuclear or chloroplast gene sequence based phylogenetic analysis can reveal genetic relations between wild and cultivated genotypes. DNA barcoding is a novel system designed to provide rapid, accurate, and automatable species identification using short, standardized genomic regions as internal species tags. 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, 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. In plants DNA barcode markers like rbcL, matK, trnH-psbA, and ITS2 have been developed, tested, and used to address basic questions in systematics, ecology, evolutionary biology, and conservation. The chloroplast maturase K gene (matK) is one of the most rapidly evolving plastid coding regions and it consistently showed high levels of discrimination among angiosperm species. Although matK primers are reported to be having high discriminative power their universality is doubted. However, in recent studies done in Cucurbitaceae, matK gene was found to be highly successful in terms of both discriminatory power and universality. Present study is an attempt to understand the phylogenetic relationship between wild and cultivated genotypes of the genus Cucumis L. using matK coding sequence and to identify barcode targets for species identification. The study included accessions belonging to the two cultivated species and two wild species viz., C. metuliferus and C. maderaspatanus. Wild varieties of cultivated species like C. sativus var. hardwickii, C. melo var. conomon, C. melo var. callosus and C. melo var. momordica were also included in the analysis. Morphological characters of the different accessions were recorded as per the minimal descriptors developed by ECPGR working group on Cucurbits (2008). All the four species exhibited distinct morphological characteristics in their leaf, stem, tendril, flower and fruit. Early plant vigour and growth habit also differed between the species. Characters common to all species include stem pubescence, coiled and unbranched tendril, unified leaf margin and monecious yellow flowers. Total genomic DNA was isolated using CTAB method and subjected to PCR using matK primers. PCR generated specific amplicons of expected size 900 bp in all the samples. Gel purified PCR amplicons were subjected to paired end Sanger sequencing. Good quality sequences were generated for all accessions except C. melo var. callosus and C. melo var. momordica. Consensus sequences without PCR and sequencing induced errors were identified for each accession by aligning the sequences with reference sequences from Genbank database. The consensus sequences, tracefiles and specimen images were submitted to Barcode of Life Database (BOLD) and Process IDs were generated. The consensus sequence as well as their homologues from Genbank were subjected to global alignment using Multi Allignment using Fast Fourier Transformation (MAFFT) programme. The alignment produced a continuous stretch of 263 bases without any gaps which was used for identification of SNPs unique to each species. For different accessions of each species the sequences were identical without any variation. Unique SNPs were available for all the four species in a span of 92 bases within the region. In order to study the genetic relationship between wild and cultivated species phylogenetic analysis was performed using MEGAv11. All the accessions used for barcode identification were included. Tree was constructed by UPGMA method with 500 bootstrap replications. The phylogenetic tree consists of 2 main clades with bootstrap value 69. Clade I consist of C. sativus and C. maderaspatanus as sub clades Ia and Ib with bootstrap values 81 and 100 respectively. Clade II consist of C. metuliferus and C. melo as subclades IIa and IIb with bootstrap values 96 and 82 respectively. Pairwise genetic distance between accessions calculated by Kimura 2 Parameter model revealed that interspecific distance ranges between 0.77% - 1.15%. Although barcode gap as defined by BOLD cannot be established as interspecific distance is less than 2%, no overlap between intra and interspecific distances was noticed. Generally, in plants interspecific distances reported is very low for most barcode targets and it is suggested that varying bar code gaps should be fixed depending on the species.