Optimization of DNA Fingerprinting Protocols for Diospyros melanoxylon Roxb. (Kendu) Using ISSR, RAPD and SCoT Markers to Enable Genetic Diversity Assessment

Abstract

The present investigation aimed to establish and optimize molecular marker-based protocol for germplasm samples enabling future assessment of genetic diversity of D. melanoxylon Roxb. natural tree populations exist in different forest regions in Odisha. In addition, high-quality genomic DNA isolation protocol was established from the leaves. Three different marker systems, such as inter simple sequence repeat (ISSR), random amplified polymorphic DNA (RAPD), and start codon targeted polymorphism (SCoT) were screened with kendu genomic DNA, to optimize and obtain clear, scorable and reproducible DNA fingerprinting profiles through polymerase chain reaction (PCR). Twenty-seven- ISSRs, 15 RAPDs and 9 SCoT markers were considered for the study. Out of 27-ISSR markers, 17-markers gave scorable alleles.  In contrast, all RAPD and SCoT markers responded and gave clear and scorable alleles. The ISSR markers (AC)₈T, (GA)₈YT, (GA)₈T, (AC)₈C, and CAGCGACAAG demonstrated strong amplification performance, collectively covered 45.8% of the total alleles (96). In contrast, SCoT markers SCoT23, SCoT40, SCoT9, SCoT12, SCoT17, and SCoT34 provided a wider genomic representation, accounting for 77.1% of the total allele (70) coverage. Meanwhile, the RAPD markers CCGCCTAGTC, ACCTGGACAC, GACCGCTTGT, AGCCAGCGAA, CACACTCCAG, CCGCCCAAAC, and CACCTTTCCC amplified 59% of the total alleles (105). The established DNA isolation and PCR optimized protocols for different marker systems are highly useful in providing a reliable molecular framework for future studies on genetic diversity assessment thereby implementing appropriate conservation strategies for kendu natural forest populations.