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Fulltext Development of microsatellites in Labisia pumila (Myrsinaceae), an economically important Malaysian herb

Tnah LH, Lee CT, Lee SL, Ng CH, Ng KK

Appl Plant Sci, 2014 Jun;2(6).
PMID: 25202631 DOI: 10.3732/apps.1400019
Fulltext Isolation and characterization of microsatellite markers for Shorea platyclados (Dipterocarpaceae)

Ng CH, Lee CT, Lee SL, Tnah LH, Ng KK

Appl Plant Sci, 2013 Jul;1(7).
PMID: 25202566 DOI: 10.3732/apps.1200538
Fulltext Isolation and characterization of microsatellite loci in an endangered palm, Johannesteijsmannia lanceolata (Arecaceae)

Ang CC, Lee SL, Lee CT, Tnah LH, Zakaria RM, Ng CC

Am J Bot, 2011 May;98(5):e117-9.
PMID: 21613176 DOI: 10.3732/ajb.1000494

Microsatellite markers were developed for Johannesteijsmannia lanceolata to assess the genetic diversity and mating system of this alarmingly endangered species.
Fulltext Microsatellite markers of an important medicinal plant, Eurycoma longifolia (Simaroubaceae), for DNA profiling

Tnah LH, Lee CT, Lee SL, Ng KK, Ng CH, Hwang SS

Am J Bot, 2011 May;98(5):e130-2.
PMID: 21613180 DOI: 10.3732/ajb.1000469

Microsatellite markers of an important medicinal plant, Eurycoma longifolia (Simaroubaceae), were developed for DNA profiling and genetic diversity studies.
Fulltext Phylogeographical pattern and evolutionary history of an important Peninsular Malaysian timber species, Neobalanocarpus heimii (Dipterocarpaceae)

Tnah LH, Lee SL, Ng KK, Lee CT, Bhassu S, Othman RY

J Hered, 2013 Jan-Feb;104(1):115-26.
PMID: 23132907 DOI: 10.1093/jhered/ess076

Tectonic movements, climatic oscillations, and marine transgressions during the Cenozoic have had a dramatic effect on the biota of the tropical rain forest. This study aims to reveal the phylogeography and evolutionary history of a Peninsular Malaysian endemic tropical timber species, Neobalanocarpus heimii (Dipterocarpaceae). A total of 32 natural populations of N. heimii, with 8 samples from each population were investigated. Fifteen haplotypes were identified from five noncoding chloroplast DNA (cpDNA) regions. Overall, two major genealogical cpDNA lineages of N. heimii were elucidated: a widespread southern and a northern region. The species is predicted to have survived in multiple refugia during climatic oscillations: the northwestern region (R1), the northeastern region (R2), and the southern region (R3). These putative glacial refugia exhibited higher levels of genetic diversity, population differentiation, and the presence of unique haplotypes. Recolonization of refugia R1 and R2 could have first expanded into the northern region and migrated both northeastwards and northwestwards. Meanwhile, recolonization of N. heimii throughout the southern region could have commenced from refugia R3 and migrated toward the northeast and northwest, respectively. The populations of Tersang, Pasir Raja, and Rotan Tunggal exhibited remarkably high haplotype diversity, which could have been the contact zones that have received an admixture of gene pools from the northerly and also southerly regions. As a whole, the populations of N. heimii derived from glacial refugia and contact zones should be considered in the conservation strategies in order to safeguard the long-term survival of the species.
Genetic Diversity and Demographic History of an Upper Hill Dipterocarp (Shorea platyclados): Implications for Conservation

Ng CH, Lee SL, Tnah LH, Ng KKS, Lee CT, Diway B, et al.

J Hered, 2019 12 17;110(7):844-856.
PMID: 31554011 DOI: 10.1093/jhered/esz052

Southeast Asian rainforests at upper hill elevations are increasingly vulnerable to degradation because most lowland forest areas have been converted to different land uses. As such, understanding the genetics of upper hill species is becoming more crucial for their future management and conservation. Shorea platyclados is an important, widespread upper hill dipterocarp in Malaysia. To elucidate the genetic structure of S. platyclados and ultimately provide guidelines for a conservation strategy for this species, we carried out a comprehensive study of the genetic diversity and demographic history of S. platyclados. Twenty-seven populations of S. platyclados across its range in Malaysia were genotyped at 15 polymorphic microsatellite loci and sequenced at seven noncoding chloroplast DNA (cpDNA) regions. A total of 303 alleles were derived from the microsatellite loci, and 29 haplotypes were identified based on 2892 bp of concatenated cpDNA sequences. The populations showed moderately high genetic diversity (mean HE = 0.680 for microsatellite gene diversity and HT = 0.650 for total haplotype diversity) and low genetic differentiation (FST = 0.060). Bayesian clustering divided the studied populations into two groups corresponding to western and eastern Malaysia. Bottleneck analysis did not detect any recent bottleneck events. Extended Bayesian skyline analyses showed a model of constant size for the past population history of this species. Based on our findings, priority areas for in situ and ex situ conservation and a minimum population size are recommended for the sustainable utilization of S. platyclados.
A geographical traceability system for Merbau (Intsia palembanica Miq.), an important timber species from peninsular Malaysia

Ng CH, Ng KKS, Lee SL, Tnah LH, Lee CT, Zakaria NF

Forensic Sci Int Genet, 2020 01;44:102188.
PMID: 31648150 DOI: 10.1016/j.fsigen.2019.102188

To inform product users about the origin of timber, the implementation of a traceability system is necessary for the forestry industry. In this study, we developed a comprehensive genetic database for the important tropical timber species Merbau, Intsia palembanica, to trace its geographic origin within peninsular Malaysia. A total of 1373 individual trees representing 39 geographically distinct populations of I. palembanica were sampled throughout peninsular Malaysia. We analyzed the samples using a combination of four chloroplast DNA (cpDNA) markers and 14 short tandem repeat (STR) markers to establish both cpDNA haplotype and STR allele frequency databases. A haplotype map was generated through cpDNA sequencing for population identification, resulting in six unique haplotypes based on 10 informative intraspecifically variable sites. Subsequently, an STR allele frequency database was developed from 14 STRs allowing individual identification. Bayesian cluster analysis divided the individuals into two genetic clusters corresponding to the northern and southern regions of peninsular Malaysia. Tests of conservativeness showed that the databases were conservative after the adjustment of the θ values to 0.2000 and 0.2900 for the northern (f = 0.0163) and southern (f = 0.0285) regions, respectively. Using self-assignment tests, we observed that individuals were correctly assigned to populations at rates of 40.54-94.12% and to the identified regions at rates of 79.80-80.62%. Both the cpDNA and STR markers appear to be useful for tracking Merbau timber originating from peninsular Malaysia. The use of these forensic tools in addition to the existing paper-based timber tracking system will help to verify the legality of the origin of I. palembanica and to combat illegal logging issues associated with the species.
Fulltext Geographic origin and individual assignment of Shorea platyclados (Dipterocarpaceae) for forensic identification

Ng CH, Lee SL, Tnah LH, Ng KKS, Lee CT, Diway B, et al.

PLoS One, 2017;12(4):e0176158.
PMID: 28430826 DOI: 10.1371/journal.pone.0176158

The development of timber tracking methods based on genetic markers can provide scientific evidence to verify the origin of timber products and fulfill the growing requirement for sustainable forestry practices. In this study, the origin of an important Dark Red Meranti wood, Shorea platyclados, was studied by using the combination of seven chloroplast DNA and 15 short tandem repeats (STRs) markers. A total of 27 natural populations of S. platyclados were sampled throughout Malaysia to establish population level and individual level identification databases. A haplotype map was generated from chloroplast DNA sequencing for population identification, resulting in 29 multilocus haplotypes, based on 39 informative intraspecific variable sites. Subsequently, a DNA profiling database was developed from 15 STRs allowing for individual identification in Malaysia. Cluster analysis divided the 27 populations into two genetic clusters, corresponding to the region of Eastern and Western Malaysia. The conservativeness tests showed that the Malaysia database is conservative after removal of bias from population subdivision and sampling effects. Independent self-assignment tests correctly assigned individuals to the database in an overall 60.60-94.95% of cases for identified populations, and in 98.99-99.23% of cases for identified regions. Both the chloroplast DNA database and the STRs appear to be useful for tracking timber originating in Malaysia. Hence, this DNA-based method could serve as an effective addition tool to the existing forensic timber identification system for ensuring the sustainably management of this species into the future.
DNA barcode identification of cultivated and wild tropical fruit species

Tnah LH, Lee SL, Lee CT, Ng KKS, Ng CH, Zawiah N

3 Biotech, 2024 Jan;14(1):7.
PMID: 38074292 DOI: 10.1007/s13205-023-03848-w

With the rapid growth of the fruit industry worldwide, it is important to assess adulteration to ensure the authenticity and the safety of fruit products. The DNA barcoding approach offers a quick and accurate way of identifying and authenticating species. In this study, we developed reference DNA barcodes (rbcL, ITS2, and trnH-psbA) for 70 cultivated and wild tropical fruit species, representing 43 genera and 26 families. In terms of species recoverability, rbcL has a greater recoverability (100%) than ITS2 (95.7%) and trnH-psbA (88.6%). We evaluated the performance of these barcodes in species discrimination using similarity BLAST, phylogenetic tree, and barcoding gap analyses. The efficiency of rbcL, ITS2, and trnH-psbA in discriminating species was 80%, 100%, and 93.6%, respectively. We employed a multigene-tiered approach for species identification, with the rbcL region used for primary differentiation and ITS2 or trnH-psbA used for secondary differentiation. The two-locus barcodes rbcL + ITS2 and rbcL + trnH-psbA demonstrated robustness, achieving species discrimination rates of 100% and 94.3% respectively. Beyond the conventional species identification method based on plant morphology, the developed reference barcodes will aid the fruit agroindustry and trade, by making fruit-based product authentication possible.
SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-023-03848-w.
Fulltext Isolation and characterization of microsatellite markers for an important tropical tree, Aquilaria malaccensis (Thymelaeaceae)

Tnah LH, Lee CT, Lee SL, Ng KK, Ng CH, Nurul-Farhanah Z, et al.

Am J Bot, 2012 Nov;99(11):e431-3.
PMID: 23108468 DOI: 10.3732/ajb.1200165

Aggressive collections and trade activities in recent decades have resulted in heavy pressure on the natural stands of Aquilaria malaccensis and concerns over its long-term survival potential. To aid DNA profiling and assessment of its genetic diversity, microsatellite markers were developed for the species.
DNA databases of a CITES listed species Aquilaria malaccensis (Thymelaeaceae) as the tracking tools for forensic identification and chain of custody certification

Lee SL, Zakaria NF, Tnah LH, Ng CH, Ng KKS, Lee CT, et al.

Forensic Sci Int Genet, 2022 Mar;57:102658.
PMID: 34998185 DOI: 10.1016/j.fsigen.2021.102658

Aquilaria malaccensis (Thymelaeaceae) is the main source of high-grade agarwood in Southeast Asia. Aggressive collections and trade activities over the past decades have put great pressure on the natural stands and raised concerns over the long-term survival potential of A. malaccensis. Tracking and authentication of agarwood require method with a high degree of accuracy. Therefore, this study aimed to develop DNA databases of A. malaccensis as the tracking tools at species, population and individual levels for forensic identification and chain of custody certification. Using two cpDNA (rbcL and matK) and an rDNA (ITS2) markers, species identification database of Aquilaria was developed to distinguish A. malaccensis from A. hirta, A. microcarpa, A. beccariana, A. crassna, A. sinensis and A. rostrata. In addition, based on 35 populations of A. malaccensis throughout Peninsular Malaysia, cpDNA haplotype and STR allele frequency databases were developed for population and individual identification. A haplotype distribution map based on 29 haplotypes derived from seven cpDNA showed that the A. malaccensis in Peninsular Malaysia can be associated to Kedah-Perak and Kelantan-Johor regions. Similarly, genetic relatedness and Bayesian clustering analyses based on 10 STR markers also divided the 35 populations into two main genetic clusters, corresponding to Kedah-Perak and Kelantan-Johor regions. The STR allele frequency databases were established and characterized according to these two regions. To determine the performance of the STR allele frequency databases for population identification, independent self-assignment tests showed that the percentage of individuals correctly assigned into the origin population was 93.88% in Kedah-Perak and 90.29% in Kelantan-Johor. For the STR allele frequency databases to be used for individual identification, conservativeness tests showed that the θ should be adjusted to 0.250 and 0.200 in the Kedah-Perak and Kelantan-Johor databases, respectively. To ensure consistency in allele calling for the dinucleotide repeat loci across different electrophoretic platforms or laboratories, allelic ladders have been developed for the 10 STR loci. Two case studies are presented of how these databases were used to track A. malaccensis to the origin population and stump. These databases are ready to be used to provide admissible forensic evidence for legal proceedings against the illegal harvesters of agarwood and for agarwood certification to meet the consumer country regulations.
Forensic timber identification: a case study of a CITES listed species, Gonystylus bancanus (Thymelaeaceae)

Ng KKS, Lee SL, Tnah LH, Nurul-Farhanah Z, Ng CH, Lee CT, et al.

Forensic Sci Int Genet, 2016 07;23:197-209.
PMID: 27213560 DOI: 10.1016/j.fsigen.2016.05.002

Illegal logging and smuggling of Gonystylus bancanus (Thymelaeaceae) poses a serious threat to this fragile valuable peat swamp timber species. Using G. bancanus as a case study, DNA markers were used to develop identification databases at the species, population and individual level. The species level database for Gonystylus comprised of an rDNA (ITS2) and two cpDNA (trnH-psbA and trnL) markers based on a 20 Gonystylus species database. When concatenated, taxonomic species recognition was achieved with a resolution of 90% (18 out of the 20 species). In addition, based on 17 natural populations of G. bancanus throughout West (Peninsular Malaysia) and East (Sabah and Sarawak) Malaysia, population and individual identification databases were developed using cpDNA and STR markers respectively. A haplotype distribution map for Malaysia was generated using six cpDNA markers, resulting in 12 unique multilocus haplotypes, from 24 informative intraspecific variable sites. These unique haplotypes suggest a clear genetic structuring of West and East regions. A simulation procedure based on the composition of the samples was used to test whether a suspected sample conformed to a given regional origin. Overall, the observed type I and II errors of the databases showed good concordance with the predicted 5% threshold which indicates that the databases were useful in revealing provenance and establishing conformity of samples from West and East Malaysia. Sixteen STRs were used to develop the DNA profiling databases for individual identification. Bayesian clustering analyses divided the 17 populations into two main genetic clusters, corresponding to the regions of West and East Malaysia. Population substructuring (K=2) was observed within each region. After removal of bias resulting from sampling effects and population subdivision, conservativeness tests showed that the West and East Malaysia databases were conservative. This suggests that both databases can be used independently for random match probability estimation within respective regions. The reliability of the databases was further determined by independent self-assignment tests based on the likelihood of each individual's multilocus genotype occurring in each identified population, genetic cluster and region with an average percentage of correctly assigned individuals of 54.80%, 99.60% and 100% respectively. Thus, after appropriate validation, the genetic identification databases developed for G. bancanus in this study could support forensic applications and help safeguard this valuable species into the future.
Fulltext Low genetic diversity indicating the threatened status of Rhizophora apiculata (Rhizophoraceae) in Malaysia: declined evolution meets habitat destruction

Azman A, Ng KK, Ng CH, Lee CT, Tnah LH, Zakaria NF, et al.

Sci Rep, 2020 11 05;10(1):19112.
PMID: 33154411 DOI: 10.1038/s41598-020-76092-4

Worldwide, many mangrove species are experiencing significant population declines, including Rhizophora apiculata, which is one of the most widespread and economically important species in tropical Asia. In Malaysia, there has been an alarming decline in R. apiculata populations driven primarily by anthropogenic activities. However, the lack of genetic and demographic information on this species has hampered local efforts to conserve it. To address these gaps, we generated novel genetic information for R. apiculata, based on 1,120 samples collected from 39 natural populations in Peninsular Malaysia. We investigated its genetic diversity and genetic structure with 19 transcriptome and three nuclear microsatellite markers. Our analyses revealed a low genetic diversity (mean He: 0.352) with significant genetic differentiation (FST: 0.315) among populations of R. apiculata. Approximately two-third of the populations showed significant excess of homozygotes, indicating persistent inbreeding which might be due to the decrease in population size or fragmentation. From the cluster analyses, the populations investigated were divided into two distinct clusters, comprising the west and east coasts of Peninsular Malaysia. The western cluster was further divided into two sub-clusters with one of the sub-clusters showing strong admixture pattern that harbours high levels of genetic diversity, thus deserving high priority for conservation.
Fulltext The genome of Shorea leprosula (Dipterocarpaceae) highlights the ecological relevance of drought in aseasonal tropical rainforests

Ng KKS, Kobayashi MJ, Fawcett JA, Hatakeyama M, Paape T, Ng CH, et al.

Commun Biol, 2021 Oct 07;4(1):1166.
PMID: 34620991 DOI: 10.1038/s42003-021-02682-1

Hyperdiverse tropical rainforests, such as the aseasonal forests in Southeast Asia, are supported by high annual rainfall. Its canopy is dominated by the species-rich tree family of Dipterocarpaceae (Asian dipterocarps), which has both ecological (e.g., supports flora and fauna) and economical (e.g., timber production) importance. Recent ecological studies suggested that rare irregular drought events may be an environmental stress and signal for the tropical trees. We assembled the genome of a widespread but near threatened dipterocarp, Shorea leprosula, and analyzed the transcriptome sequences of ten dipterocarp species representing seven genera. Comparative genomic and molecular dating analyses suggested a whole-genome duplication close to the Cretaceous-Paleogene extinction event followed by the diversification of major dipterocarp lineages (i.e. Dipterocarpoideae). Interestingly, the retained duplicated genes were enriched for genes upregulated by no-irrigation treatment. These findings provide molecular support for the relevance of drought for tropical trees despite the lack of an annual dry season.

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