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 extr...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.展开更多
Sandalwood (Santalum album L.) is the second most expensive wood in the world. There are approximately 16 species of sandalwood (S. album, S. spicatum, S. austrocaledonicum, S. yasi, S. lanceolatum, S. ellipticum, S. ...Sandalwood (Santalum album L.) is the second most expensive wood in the world. There are approximately 16 species of sandalwood (S. album, S. spicatum, S. austrocaledonicum, S. yasi, S. lanceolatum, S. ellipticum, S. macgregorii, S. insulare) occurring naturally throughout Australia, India, Indonesia, Papua New Guinea and the islands of the South Pacific. In India, S. album is found all over the country, with over 90% of the area in Karnataka, Tamil Nadu, Kerala, Andhra Pradesh and Telangana state. It is highly economic tropical tree species because of its scented heartwood and heartwood oil. Several causes have been attributed to the depletion of sandalwood population mainly amongst which theft is causing negative effect on the quality of species by constant removal of superior clones. The aim of this study was to determine the genetic diversity of S. album. For this, 177 genotypes of S. album from 14 populations of three states (Karnataka, Telangana state and Kerala) in southern India were selected. The genetic diversity and genetic structure were characterized through 25 SSR markers developed by cross amplification of different species of Sandalwood. Under this study, following genetic diversity parameters were estimated at individual level and population level;Number of alleles (Na) 9.107, Effective number of alleles (Ne) 7.56, Observed heterozygosity (Ho) 0.187, Expected heterozygosity (He) 0.861, Shannon information index (I) 2.03, F statistics 0.89, Polymorphic information content (PIC) 0.87 and Gene flow (Nm) 4.98. The estimates of gene flow among the populations of Kodada Telangana (Nm = 15.109);IWST Karnataka (Nm = 13.62) than across other geographical populations (Nm = 9.40). Analysis of molecular variance (AMOVA) revealed that 3% of the total variation was due to differences among populations and 97% due to differences within the populations. The genetic differentiation among populations (FST) 0.012 at p < 0.001 was significant. Structure clustering at Ks = 3 highlighted three distinct groups with the inferred clusters i. 0.369, ii. 0.304, iii. 0.327. Structure analysis revealed that genetic structure of Kerala and Karnataka and Telangana populations were admixtures and classified in three groups. In addition, all the accessions were clearly divided into three major clusters by UPGMA dendrogram which could be further divided into five sub groups. Principal component analysis (PCA) results showed the combined variation 82.2% of these markers. This study highlights the knowledge of genetic variation in sandalwood across the herd population of sandalwood in India. The highest range of polymorphism was detected with SSR markers developed from Osyris lanceolata compared to Santalum. austrocaledonicum, Santalum. insulare and Santalum. spicatum. This study would help in conservation of the Sandalwood populations with high profile of genetic diversity and selection of clones for genetic improvement program.展开更多
文摘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.
文摘Sandalwood (Santalum album L.) is the second most expensive wood in the world. There are approximately 16 species of sandalwood (S. album, S. spicatum, S. austrocaledonicum, S. yasi, S. lanceolatum, S. ellipticum, S. macgregorii, S. insulare) occurring naturally throughout Australia, India, Indonesia, Papua New Guinea and the islands of the South Pacific. In India, S. album is found all over the country, with over 90% of the area in Karnataka, Tamil Nadu, Kerala, Andhra Pradesh and Telangana state. It is highly economic tropical tree species because of its scented heartwood and heartwood oil. Several causes have been attributed to the depletion of sandalwood population mainly amongst which theft is causing negative effect on the quality of species by constant removal of superior clones. The aim of this study was to determine the genetic diversity of S. album. For this, 177 genotypes of S. album from 14 populations of three states (Karnataka, Telangana state and Kerala) in southern India were selected. The genetic diversity and genetic structure were characterized through 25 SSR markers developed by cross amplification of different species of Sandalwood. Under this study, following genetic diversity parameters were estimated at individual level and population level;Number of alleles (Na) 9.107, Effective number of alleles (Ne) 7.56, Observed heterozygosity (Ho) 0.187, Expected heterozygosity (He) 0.861, Shannon information index (I) 2.03, F statistics 0.89, Polymorphic information content (PIC) 0.87 and Gene flow (Nm) 4.98. The estimates of gene flow among the populations of Kodada Telangana (Nm = 15.109);IWST Karnataka (Nm = 13.62) than across other geographical populations (Nm = 9.40). Analysis of molecular variance (AMOVA) revealed that 3% of the total variation was due to differences among populations and 97% due to differences within the populations. The genetic differentiation among populations (FST) 0.012 at p < 0.001 was significant. Structure clustering at Ks = 3 highlighted three distinct groups with the inferred clusters i. 0.369, ii. 0.304, iii. 0.327. Structure analysis revealed that genetic structure of Kerala and Karnataka and Telangana populations were admixtures and classified in three groups. In addition, all the accessions were clearly divided into three major clusters by UPGMA dendrogram which could be further divided into five sub groups. Principal component analysis (PCA) results showed the combined variation 82.2% of these markers. This study highlights the knowledge of genetic variation in sandalwood across the herd population of sandalwood in India. The highest range of polymorphism was detected with SSR markers developed from Osyris lanceolata compared to Santalum. austrocaledonicum, Santalum. insulare and Santalum. spicatum. This study would help in conservation of the Sandalwood populations with high profile of genetic diversity and selection of clones for genetic improvement program.
