An improved and effective DNA extraction protocol for Pyracantha crenulata with optimal PCR reliability

Abstract

There are many challenges involved in obtaining high-quality DNA from tissues/cells, especially when using plants as the raw material. Three different DNA extraction protocols were employed in order to isolate high quality of genomic DNA from Pyracantha crenulata leaves. P. crenulata is a complex, versatile, and evergreen shrub species in Rosaceae family which is ecologically, economically and culturally important. This species produces valuable antioxidants like polyphenols, polysaccharides and secondary metabolites which interfere with DNA extraction thus making molecular marker based studies difficult in this species. This study aimed to develop a simple, rapid, cost-effective and highly efficient protocol for P. crenulata leaves rich in salts and phenols. The results demonstrated that modified CTAB (double buffer) protocol is very effective in overcoming the challenges that could impede next-generation sequencing analysis. The yield of the extracted DNA was excellent ranging from 368 to 410 ng/μL DNA with A260/280 ratio ranging from 1.80 to 1.84. The extracted DNA was amenable to PCR amplification making it suitable for DNA-based molecular marker studies. None procedures have been published for the purpose up this species, therefore, double buffer based extraction protocol is reported for the first time in P. crenulata and might prove useful in other species of family Rosa-ceae. This study describes an effective DNA isolation method that is appropriate for frequent population genetic screening. In conclusion, the double buffer protocol showed the most efficient and effective approach for extracting DNA from phenol rich, fresh, mature, and dry leaves.