DNA barcoding of endangered peninsular Malaysian slipper orchids, the genus Paphiopedilum
Paphiopedilum is a genus of the orchid family that is highly prized for its beautiful and long lasting flowers. Due to its attractiveness, many plants have been collected from the wild to meet the demands of the market and thus reduced the populations till the point of endangerment. Thus, all Pap...
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| Format: | Thesis |
| Language: | English |
| Published: |
2018
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| Subjects: | |
| Online Access: | http://psasir.upm.edu.my/id/eprint/83679/1/FS%202019%2023%20-ir.pdf http://psasir.upm.edu.my/id/eprint/83679/ |
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| Summary: | Paphiopedilum is a genus of the orchid family that is highly prized for its
beautiful and long lasting flowers. Due to its attractiveness, many plants have
been collected from the wild to meet the demands of the market and thus
reduced the populations till the point of endangerment. Thus, all Paphiopedilum
are protected by CITES Appendix I. Even though they produce beautiful and
distinctive flowers, the vegetative form of this genus, however, lacks sufficient
distinguishing features that enable species discrimination, creating a problem
for accurate species identification for biodiversity inventory and in regulating
the control of its trade. DNA barcoding uses standardized regions of DNA that
can supplement the current taxonomic identification data. Five species have
been identified to occur in Peninsular Malaysia and IUCN Redlist categorized
all species as Endangered. Some of the threats recognized to affect
Paphiopedilum survival in the wild are poaching, deforestation and logging in
Malaysia. The samples were collected in localities throughout Peninsular
Malaysia. To produce species-specific barcodes, DNA extraction was
performed using the CTAB method. Selected barcode regions were amplified
using PCR and the protocols were optimized specifically for all barcodes
chosen. Amplified products were sequenced using Sanger bi-directional
sequencing method. rbcL and matK has the highest sequence quality and
followed by trnH-psbA and ITS. Sequences obtained were edited, aligned and
its effectiveness was measured by using several approaches: presence of
monophyletic groups in Neighbour-Joining trees, barcoding gap (global and
local) and BLASTn sequence similarity. matK showed the highest species
resolution with 100% correct match in NCBI BLASTn database followed by ITS
(52.9%), trnH-psbA (26.3%) and lastly rbcL (23.5%). All barcodes were correct
at the genus level. Phylogenetic trees were constructed using the Neighbourjoining
method with Kimura-2-parameter metric and supported by 1000
bootstrap replicates in MEGA 6.0. Trees constructed using matK and ITS barcodes grouped similar species into a clade and congruent with currently
accepted taxonomy while those constructed with rbcL and trnH-psbA were
unable to resolve the differences between the species. Differences between
barcode efficiency are attributed to differing molecular rate of evolution of the
individual loci. Global barcoding gap exists only in matK sequences but could
be the result of sampling bias and may not be a reliable indicator for species
delimitation. Local barcoding gap exists for all species studied using matK
sequences except between P. barbatum and P. callosum var. sublaeve. The
shared similarities and uneven sampling between both species may contribute
to the lack of local barcoding gap. Based on the findings of the study, matK is
the most suitable barcode for identifying Paphiopedilum species in Peninsular
Malaysia. DNA barcoding presents an effective and simple method for species
level identification for Paphiopedilum species. |
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