Chin Hong Ng

Isolation and characterization of microsatellite markers for an important tropical tree, Aquilaria malaccensis (Thymelaeaceae)

PREMISE OF THE STUDY 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. METHODS AND RESULTS Seventeen polymorphic microsatellite markers were developed for A. malaccensis using an enrichment protocol. The markers were screened on 24 samples from a natural population. The number of alleles ranged from two to 11, and the observed heterozygosity ranged from 0.042 to 0.957. No significant deviation from Hardy-Weinberg equilibrium was detected after conservative Bonferroni correction. CONCLUSIONS This is the first report on the development of microsatellite markers in A. malaccensis. The markers will be used to establish a DNA profiling database and to estimate the genetic diversity and population genetic structure of the species.

Expressed sequence tag–simple sequence repeats isolated from Shorea leprosula and their transferability to 36 species within the Dipterocarpaceae

Simple sequence repeats (SSRs) derived from expressed sequence tags (ESTs) are valuable markers because they represent transcribed regions and often transferable to related taxa. Here, we report the development and characterization of EST–SSRs from Shorea leprosula. Fifty‐four sequences containing SSRs were identified in 2003 unigenes assembled from 3159 ESTs. Twenty‐four EST–SSRs were developed, of which four gave multiple amplifications, five were found to be monomorphic and 15 showed polymorphism, with allele numbers ranging from two to 17 in a single Pasoh Forest Reserve population of 24 individuals. The observed and expected heterozygosities ranged from 0.05 to 0.91 and from 0.16 to 0.93, respectively. Cross‐species transferability of the 15 loci to 36 species within Dipterocarpaceae revealed between four and 14 loci that gave positive amplification and 10 loci were found to be transferable to more than 15 species.

Forensic timber identification: a case study of a CITES listed species, Gonystylus bancanus (Thymelaeaceae)

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 individuals 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.

Genome size variation and evolution in Dipterocarpaceae

Background: Dipterocarpaceae is a pantropical tree family that plays an important role in our understanding of the ecology of Asian tropical rain forests. However, genome sizes for members of the Dipterocarpaceae are still poorly known. Aims: To report the genome size of 115 dipterocarp species and examine the variation and evolution of genome size in this family. Methods: Genome size was estimated using flow cytometry. Both the rpoB and trnL intron were sequenced to uncover the evolution of genome size within a phylogenetic framework. Results: The 1C genome size varied between 0.267 and 0.705 pg in Shorea hemsleyana and Shorea ovalis, respectively, a 2.64-fold variation across the family. Most dipterocarps are characterised by very small genomes with a mean 1C value of 0.416 pg (sd = 0.075) and five polyploids are recorded. The ancestral genome size for dipterocarps was reconstructed as 1Cx = 0.481 pg (95% CI = 0.433–0.534). Conclusions: Genome size variation in dipterocarps was characterised by very small values with a narrow range. Overall, genome size reduction from the ancestral state is a general trend in Dipterocarpaceae.

Microsatellite markers of an important medicinal plant, Eurycoma longifolia (Simaroubaceae), for DNA profiling

PREMISE OF THE STUDY Microsatellite markers of an important medicinal plant, Eurycoma longifolia (Simaroubaceae), were developed for DNA profiling and genetic diversity studies. METHODS AND RESULTS Eighteen polymorphic microsatellite loci were developed for E. longifolia. The primers were designed from a genomic library enriched for dinucleotide (CT) repeats and screened on 32 samples from a natural population. The number of alleles detected per locus ranged from four to 16, while the observed heterozygosity ranged from 0.097 to 0.938. No significant deviation from Hardy-Weinberg equilibrium was detected in all the 18 loci, and no linkage disequilibrium was found between these loci after conservative Bonferroni correction. CONCLUSIONS The 18 microsatellite markers of E. longifolia are highly polymorphic and informative. These markers would serve as an important tool for DNA profiling and genetic diversity studies.

Mating system and seed variation of Acacia hybrid (A. mangium × A. auriculiformis)

The mating system and seed variation of Acacia hybrid (A. mangium × A. auriculiformis) were studied using allozymes and random amplified polymorphic DNA (RAPD) markers, respectively. Multi-locus outcrossing rate estimations indicated that the hybrid was predominantly outcrossed (mean±s.e. tm = 0.86±0.01). Seed variation was investigated using 35 polymorphic RAPD fragments. An analysis of molecular variance (AMOVA) revealed the highest genetic variation among seeds within a pod (66%–70%), followed by among pods within inflorescence (29%–37%), and the least variation among inflorescences within tree (<1%). In addition, two to four RAPD profiles could be detected among seeds within pod. Therefore, the results suggest that a maximum of four seeds per pod could be sampled for the establishment of a mapping population for further studies.

Isolation and Characterization of Microsatellite Markers for Shorea platyclados (Dipterocarpaceae)

Premise of the study: Microsatellite markers were isolated and characterized in Shorea platyclados (Dipterocarpaceae) for DNA profiling and genetic diversity assessment of this tropical timber species. Methods and Results: Fifteen polymorphic microsatellite loci were developed and characterized in S. platyclados using a genomic library enriched for dinucleotide (CT) repeats. The primers amplified dinucleotide repeats with 3–14 alleles per locus across four natural populations. The observed and expected heterozygosities ranged from 0.292 to 1.000 and from 0.301 to 0.894, respectively. No significant deviation from Hardy–Weinberg equilibrium was detected in the 15 loci. Four loci pairs displayed linkage disequilibrium. Conclusions: These highly polymorphic markers are adequate for DNA profiling and studies of population genetics in S. platyclados.

Geographic origin and individual assignment of Shorea platyclados (Dipterocarpaceae) for forensic identification

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.

Isolation and characterization of 16 microsatellite markers in Intsia palembanica, a high-value tropical hardwood species

AbstractIntsia palembanica is a high-value tropical hardwood species of the family Leguminosae. A total of 16 microsatellite markers were developed for this important timber species using an enrichment approach. These markers were screened on 24 samples from a natural population and subsequently tested for reproducibility, ease of scoring and locus-specificity, across five populations. The observed and expected heterozygosities ranged from 0.026 to 0.976 and 0.026 to 0.928, respectively, while the mean number of alleles per locus per population was 8.73. These microsatellite markers are informative and will be useful for conservation genetic study, individual identification and timber tracking.

Development of Microsatellites in Labisia pumila (Myrsinaceae), an Economically Important Malaysian Herb

Premise of the study: The exploitation of Labisia pumila for commercial demand is gradually increasing. It is therefore important that conservation is prioritized to ensure sustainable utilization. We developed microsatellites for L. pumila var. alata and evaluated their polymorphism across var. alata, var. pumila, and var. lanceolata. Methods and Results: Ten polymorphic microsatellites of L. pumila were developed using the magnetic bead hybridization selection approach. A total of 84, 48, and 66 alleles were observed in L. pumila var. alata, var. pumila, and var. lanceolata, respectively. The species is likely a tetraploid, with the majority of the loci exhibiting up to four alleles per individual. Conclusions: This is the first report on the development of microsatellites in L. pumila. The microsatellites will provide a good basis for investigating the population genetics of the species and will serve as a useful tool for DNA profiling.