Ability of trnH-psbA and trnL-tmF intergenic spacers to discriminate between closely related Paphiopedilum species

Yeoh, Keat Ai (2007) Ability of trnH-psbA and trnL-tmF intergenic spacers to discriminate between closely related Paphiopedilum species. Universiti Malaysia Sabah. (Unpublished)


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Two intergenic spacers, trnH-psbA and trnL-trnF of the chloroplast DNA (cpDNA) were used to detennine whether they can be used to distinguish between three closely related Paphiopedilum species namely P. rothschildianum, P. lowii and P. dayanum . Genomic DNA was extracted from the leaves of the three Paphiopedilum species without the use of phenol chloroform. Next, the two loci were amplified through PCR using two primers pairs (psbAF - trnHR and trnLF - trnFR). The amplified PCR products were ligated into the pCR 2.1 - TOPO vector, transformed using TOP 10 chemically competent cells. Then, plasmid mini preparation was performed and the plasmids obtained were sequenced. Data analysis was performed using the resulting sequences. A total of 29 indels mutations points with 95 indels were observed, accounting for tmH-psbA intergenic spacer length of P. rothschildianum, P. lowii and P. dayanum with a range of 924 to 1000 bp. In the trnL-trnF intergenic spacer, 11 indels mutation points with 48 indels have been discovered for the three Paphiopedilum species with a range of between 462 to 490 bp. The genetic similarity and differences between the Paphiopedilum species was examined by calculating the pairwise nucleotide differences that occurred in each intergenic region. The result showed that P. rothschildianum and P. lowii were more closely related compare to P. dayanum. Single nucleotide polymorphisms (SNP) were then determined and tmH-psbA intergenic spacer was found to provide 96 informative sites which indicate the variations that occur in this spacer region was higher compared to trnL-trnF spacer region which provide 48 informative sites. The phylogenies of the three species were also examined by constructing phylogenetic trees and confirmed by performing bootstrapping analysis. The results obtained indicate that trnL-trnF intergenic spacer is more reliable compared to the tmH-psbA intergenic region for distinguishing between the three Paphiopedilum species. However, more research should to be conducted on trnH-psbA intergenic spacer as it exhibit sufficient nucleotide variations which proved that it is also a potentially good marker for phylogenetics studies and DNA barcoding of other plants.

Item Type: Academic Exercise
Uncontrolled Keywords: Genomic DNA, Paphiopedilum, Single nucleotide polymorphisms, phylogenetic tree, bootstrapping analysis
Subjects: S Agriculture > SB Plant culture
Divisions: SCHOOL > School of Science and Technology
Date Deposited: 06 Mar 2014 08:31
Last Modified: 11 Oct 2017 05:57
URI: http://eprints.ums.edu.my/id/eprint/8183

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