An Effective Wood DNA Extraction Protocol for Three Economic Important Timber Species of India

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 forenHow to cite this paper: Fatima, T., Srivastava, A., Hanur, V.S. and Srinivasa Rao, M. (2018) An Effective Wood DNA Extraction Protocol for Three Economic Important Timber Species of India. American Journal of Plant Sciences, 9, 139-149. https://doi.org/10.4236/ajps.2018.92012 Received: October 24, 2017 Accepted: January 16, 2018 Published: January 19, 2018 Copyright © 2018 by authors and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY 4.0). http://creativecommons.org/licenses/by/4.0/ Open Access

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 foren-How to cite this paper: Fatima, T., Srivastava, A., Hanur, V.S. and Srinivasa Rao, M.

Introduction
Preservation of endangered species is an indispensible part of accomplishment the objective of the Convention on Biological Diversity 2020 on cultivating the prominence of global biodiversity [1]. The first critical phase in protecting and managing threatened species is correct identification and delimitation of the target species [2]. Identification of plant species traditionally relies on morphological characters of especially leaf, flowers and fruits, which for trees can be time consuming to access and only present during parts of the year. Accurate identification in species-rich or taxonomically complex groups also typically requires genomic DNA that is not always available, especially in tropical forest samples [3] [4]. Teak (Tectona grandis f; verbanaceae), Black Rosewood (Dalbergia latifolia f; Fabaceae) Ben Teak (Lagerstroemia lanceolata f; Lytheraceae) are incredibly significant economic timber species in tropical countries chiefly in India, Indonesia, Myanmar and Burma. These three genuses contain many valuable timber species threatened by illegal logging and deforestation, but knowledge on distribution and threats is often limited and accurate species identification difficult. Illegal logging and associated trade are the cause of many economic and ecological problems both in timber producer and timber consumer countries [5]. Although many legal instruments have been established to combat illegal logging and trade of illegally sourced timber, practical controls mechanisms to identify the tree species and geographic origin of wood and wood products are still lacking [6] [7]. The action of illegal logging crimes is hampered by a lack of available forensic timber identification tools and time of harvest for both screening of suspect material and definitive identification of illegally sourced wood, which were scam by forest department. Processed timber products such as decking, flooring and furniture are subjected to drying, engineering and treatment processes that degrade the DNA present in the wood, just as in ancient samples [8]. Extraction of whole genomic DNA from fresh tissues is routine in studies of tropical forest species [9] [10]. DNA extraction from wood (Sapwood and heartwood) is quite difficult due to presence of higher quantity of secondary metabolites phenolic and lignin compounds and the concentration of leaf DNA is higher than wood DNA [11] [12]. There are number of protocols for DNA extraction from various plant and animal species and tissues published, but these protocols are the best for leaf and soft tissues, while mature tree samples (core tissues) often narrows down the scope of DNA extraction [13]. Genotypes the tracing of timber origin, and species can be identified based on an investigation of wood if suitable DNA content and high amount of pure DNA is available [14]. The aims for this study were to optimize the total genomic DNA extraction protocol by means of the standardized the modified CTAB wood (heartwood and sapwood) DNA extraction protocol [15] [16] and the comparison between the modified CTAB protocol, CTAB protocol and Plant DNA extraction kits (Nucleopore) protocols. This modified protocol made the intact wood DNA isolation that facilitates the important forensic timber species effort.

Sampling
Collection of leaves and core samples from naturally grown populations of

Storage
Leaf and core samples were put into plastic cover and immediately placed into an ice storage box. After arrival to the laboratory the wood samples that had

Sample Preparation
Before DNA extraction, core samples were cleaned and washed with distilled water and kept it in a fresh autoclaved glass bottles with distilled water for about 72 -96 h and repeatedly changed the water at every 12 h to avoid the fungal or any other contaminations. Samples were cut into two Sections 1. Sapwood 2.
Heartwood, cut the samples separately of an approx. 3 -5 μM thickness was prepared from each samples using paper cutter or sliding microtome to produce small chips of wood samples. Kept the chipped slices for drying 5 -10 min before imperilling to for CTAB based protocols for genomic DNA extraction which comprised of CTAB method [17], DNAsure plant mini kit (Nucleopore) [18], and CTAB method (protocol 3) developed at IWST laboratory. Protocol 1 are executed earlier while the protocol 2 are kit manufacturer based and all are a routine based protocol published in number of papers so here only the modified standardised developed i.e., protocol 3 (modified CTAB) has been described below.

Method
Around 500 mg. samples (sapwood, heartwood and leaf separately)were taken into chilled autoclaved mortar pestle and added 250 mg. PVPP then rapidly ground it into a fine powder using liquid nitrogen. After grinding added pre-wormed at 65˚C extraction buffer and vigorously shaken the tubes in vortex for few seconds and retained it at 65˚C pre-heated water bath for 2 1/2 h with periodic shuddering. Taken away the reaction tubes from water bath and retained it at room temperature to cool it for 30 min.

DNA Quantification
The yield of extracted DNA was quantified by nanodrop at 260/280 nm wavelength [20]. The purity of DNA was checked by running the samples on 0.8% agarose gel in 1X TAE buffer (8.0 pH) stain containing 4 µL of 0.3% Ethidium Bromide and visualized on UV transilluminator gel doc (Syngene version 7.0) to get the DNA bands.

DNA analysis through ISSR and Universal SSR (RbcL & TrnH) Markers
Ten ISSR primers were screened for analysis [20] [21] out of those four primers was amplified and showing high polymorphism in all three species. UBC834 (AG) 8 YT, UBC874 (CCT) 4 , UBC848(CA) 8 RG, UBC857 (AC) 8 YG) ISSR primers amplified. The universal SSR markers rbcL (5'-AACACCAGCTTTRAATCCAA-3') and MatK (5'-GGGTTGCTAACTCAATGGTAGAG-3') primers [22] were used to validate the quality of extracted wood DNA. In this study, basically particular universal SSR primers which were used to carry out research in barcoding of timbers, which would be useful in illegal logging and tracing the timber origin. DNA amplification was carried out in 13 µL reaction volume containing genomic DNA 1.5 µL (30 ng/µL), 10 mM 2 µL primers, 1.5 µL PCR buffer, 1.5 µL dNTPs, 1.5 µL MgCl 2 , 0.3 µL (3 U/µL) Taq polymerase (Bangalore genie) and 4.2 µLRNase freedouble distilled water (Sigma aldrich). Amplification cycle consist of an initial 3 min denaturation at 94˚C, 30 cycles for 30 sec at 50˚C, 1 min 72˚C and final extension step for 10 min at 72˚C. The amplified product loaded with 5 µL loading buffer were size fractionated by electrophoresis on a 1.5% for ISSR and 2.0% SSR agarose gel with 0.3% Ethidium bromide and visualized on UV transilluminator to determined the amplified clear bands to validated the DNA quality and suitability for PCR reactions.

Results
Isolation of DNA from CTAB method and using plant DNA extraction kits were American Journal of Plant Sciences unable to extract DNA from wood tissues. Hence we developed a new modified protocol by modifying the CTAB protocol [23] by enhancing the incubation time, CTAB concentration, high concentration of proteinase and RNase treatment and most important keeping the sample in water to release the resilience of cells by process of osmosis. The ratio of extraction buffer and sample was 500 mg: 5 mL. The additional washing with C: I (24:1) helped to removal of polysaccharides and strong protein contamination. Table 1 represented the quantity and quality of leaf, sapwood and heartwood tissues with all four protocols, which were used to standardize the protocol 3 to yield high genomic DNA with all selected timber species. For comparative purposes ( Figure 2) DNA extraction of leaves, sapwood and heartwood tissues the graph of nanodrop were representing the quantity and qualitative analysis of all three selected species. It was summarized in which shows that the yield of DNA from sapwood from 0.26 -0.244 µg/µL in heartwood from 0.186 -0.166 µg/µL and in leaf samples from 1.30 -1.511 µg/µL according to spectrophotometer measurements with respect to purity from 1.7 -1.8. In general, a higher quantity of DNA could be obtained using modified CTAB protocol (Figure 3). The quantity of DNA from Dried leaf samples and from wood samples was 56 % higher than the CTAB and other DNA kit protocol. In Figure 4(a) and Figure 4(b) were showing the DNA bands obtained by protocol 1 i.e., CTAB method and protocol 2,3 i.e., DNA isolation kits which is basically used for industrial purpose and specific for plant genomic DNA isolation. The bands were not clear and the pellet was dark brownish in color with undissolved PCR inhibitors (carbohydrate and proteins) contaminations. Figure 4(c) and Figure 4(d) was presenting the PCR amplification of DNA extracted through protocols 1,2 but in

Conclusion
The present analysis clearly established the need for different tree species of appropriate DNA isolation methods for timber species. A single method may not be appropriate for extraction of DNA with good quantity and purity from all species but this method could be executed as standard method for isolation of wood DNA from D. latifolia, L. lanceolata and T. grandisor similar perennial timber species containing rich polysaccharides and defined here is hasty, uncertain and steady permitting the handling of large number of trials with easy routine. Previous studies [24] relying on relatively fresh samples but by using this developed protocol we could able to isolate pure DNA from mature dried wood samples of timbers or perennial tree species. Through Figure 4 the final validation of qualitative DNA was proved that this protocol would be useful for each species of wood and mature dried or even disintegrating leaves of timber species. The isolated DNA as a result of using standardized protocol of various wood samples (fresh, dried, old and mature cores) was tested in PCR amplification for ISSR and ISSR profiling with selected primers to validate the protocol.