TITLE:
An Effective Wood DNA Extraction Protocol for Three Economic Important Timber Species of India
AUTHORS:
Tanzeem Fatima, Ashutosh Srivastava, Vageeshbabu S. Hanur, M. Srinivasa Rao
KEYWORDS:
Wood Genomic DNA, Plant DNA Kits, Timber, ISSR and SSR Markers
JOURNAL NAME:
American Journal of Plant Sciences,
Vol.9 No.2,
January
19,
2018
ABSTRACT: Extraction of DNA from fresh
tissues is routine in studies of tropical forest species, but DNA extraction
from wood is considered as difficult due to its highly degraded nature and
adequate quality of genomic DNA extraction is essential for molecular studies.
Very few studies have validated the potential for isolating DNA from dried wood
(Heartwood and Sapwood). Wood genomic DNA extraction is difficult from mature
timber (Teak (Tectona grandis f;
verbanaceae), Black Rosewood (Dalbergia
latifolia f; Fabaceae) Ben Teak (Lagerstroemia
lanceolata f; Lytheraceae) tissues due to presence of high quantity of
secondary metabolites polyphenols, tannins and terpenoids and protein
inhibitors. Mostly in laboratories DNA extraction kits are available but by
using kits, DNA yield is very low and it is quite expensive too. We have
standardized and validated the DNA extraction through manual protocol which is
applicable for Bark, Sapwood and Heartwood samples of tree species which
contains huge amount of inflexible tissues and fibers. The quality of the DNA
was tested by spectrophotometer, gel electrophoresis and PCR (ISSR and SSR)
amplification. An avrage DNA yield for heartwood ranges from 0.186 - 0.166 μg/μL and sapwood was ranges from
0.26 - 0.244 μg/μL. Modification
of CTAB method was by addition of polyvinylpyrrolidone (PVP) appx 0.25%,
variation in Rnase concentration, proteinase treatment with different
concentration and incubation time. In order to evaluate the
standardized wood genomic DNA extraction protocol, we compared it with the
mature leaf and core samples (heartwood and sapwood) of the same timber
species. The outcome was also quantified and proved by means of polymerase
chain reaction analysis by using ISSR and SSR microsatellite markers conducted
with isolated pure DNAs. This modified protocol made increased yield and purity of wood total
genomic DNA and facilitate the important application of forensic timber species
effort.