Rapid seed DNA extraction for species identification and diversity analysis of Pumpkin
Paper Details
Rapid seed DNA extraction for species identification and diversity analysis of Pumpkin
Abstract
Cucurbita moschata an economically important species of the family Cucurbitaceae, shows high variability in fruit characteristics. A standardized DNA isolation protocol has been developed from dried pumpkin seeds for polymerase chain reaction for species identification and diversity analysis. Higher concentration of polysaccharides and polyphenols in pumpkin seeds interferes with DNA during its isolation resulting in no PCR products. Good quality DNA, with no coloured pigments and contaminants was isolated from sun dried pumpkin seeds with modified CTAB buffer protocol without using liquid nitrogen. The average DNA concentration obtained was 63.9μg/gm with purity ranging between 1.66 to 1.85. The isolated DNA was successfully amplified using barcoding (rbcL), RAPD and SSR primers. The quantity and quality of the DNA isolated by this method was high enough to perform more than 150 PCR reactions. The species identify for Cucurbita moschata was also confirmed through sequencing and NCBI BLASTn analysis of bracoding primer (rbcL) product using isolated DNA. On the basis of UPGMA analysis, 14 pumpkin genotypes were categorized into two broad clusters. Broad cluster I and II comprised of one genotype (NEHUP8) and 13 independent genotypes respectively. The major cluster-I comprised of two genotypes viz. NEHUP1 and NEHUP14 with a genetic similarity percent of 0.58 approximately. The major cluster-II was sub divided into three minor clusters. This modified DNA isolation protocol may be adequate for isolating high-molecular weight DNA from other cucurbitaceous species containing large amounts of secondary metabolites.
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