Nine simple sequence repeat (SSR) markers were developed from Shorea curtisii using two different methods. One SSR locus was isolated by the commonly used method of screening by colony hybridization, and the other eight loci were isolated by a vectorette PCR method. Primer pairs were designed based on the sequences of all these SSR loci. Analysis of 40 individuals of S. curtisii from natural forest in Malaysia revealed that all SSR loci were polymorphic. Four SSR markers, Shc01, Shc04, Shc07 and Shc09, were highly polymorphic. We have also tested the applicability of these SSR printers to other species of Dipterocarpaceae using PCR amplification. Because the flanking region sequences of the S. curtisii SSRs were well conserved within this family, the SSR primers for S. curtisii can be applied to almost all species of Dipterocarpaceae.
Random amplification of polymorphic DNA (RAPD) was used to analyse genomic DNA from virgin females and males of Brugia malayi, with a view to identifying sex-specific differences predicted by an XX/XY system of chromosomal sex determination. A product of 2338 bp, amplified with the arbitrary primer 5' GTTGCGATCC 3', was obtained exclusively from males. Primers based on the sequence of this product amplified a DNA fragment of the expected size from each of two independent isolates of B. malayi (from Malaysia and Indonesia) by PCR. No reaction product was obtained from the closely related species Brugia pahangi. In a genetic cross between B. malayi males and B. pahangi females, F1 hybrid microfilariae were PCR-positive, indicating that the locus is paternally-inherited. Southern blotting demonstrated that the target sequence resides in the high molecular weight fraction of genomic DNA, confirming that it is of chromosomal, rather than mitochondrial, origin. Sequencing of the locus revealed significant similarity with members of a family of reverse transcriptase-like genes in Caenorhabditis elegans. In-frame stops indicate that the gene is non-functional, but multiple bands of hybridisation in Southern blots suggest that the RT sequence may be the relic of a transposable element. Multiple repeats of the dinucleotide AT occurred in another region of the sequence. These varied in number between the two isolates of B. malayi in the manner of a microsatellite, surprisingly the first to be described from the B. malayi genome. Because of its association with the Y chromosome, we have given the locus the acronym TOY (Tag On Y). Identification of this chromosome-specific marker confirms the XX/XY heterogametic karyotype in B. malayi and opens the way to elucidation of the role of Y in sex determination.
Pollen flow and population genetic structure among 30 potentially flowering individuals of Neobalanocarpus heimii, a tropical emergent tree, were investigated in a lowland tropical rainforest of Malaysia using microsatellite polymorphism. The 248 offspring in the vicinity of five reproductive trees of the 30 potentially flowering trees were used in paternity analysis for pollen-flow study. Four primer pairs, developed in different species of dipterocarps, were adopted to detect microsatellite polymorphism. Based upon microsatellite polymorphism, pollen flow and seed migration were detected. Pollen-flow events of more than 400 m were observed directly, based on paternity analysis in the study plot. The estimated average mating distance of the five reproductive trees was 524 m. This result suggests that reproduction of this species is mediated by a long-distance pollinator. The haplotypes of some offspring were not compatible with the nearest reproductive tree. Thus, the results suggest that some seeds are dispersed by a seed dispersal vector. Investigation of genetic structure showed significant and negative correlation of genetic relatedness and spatial distances between the 30 potentially flowering trees, but this correlation was weak. We suggest that long-distance gene flow and seed migration are responsible for the poorly developed genetic structure of this species.
Comparing patterns of genetic variation at multiple loci in the genome of a species can potentially identify loci which are under selection. The large number of polymorphic microsatellites in the malaria parasite Plasmodium falciparum are available markers to screen for selectively important loci. The Pfs48/45 gene on Chromosome 13 encodes an antigenic protein located on the surface of parasite gametes, which is a candidate for a transmission blocking vaccine. Here, genotypic data from 255 P. falciparum isolates are presented, which show that alleles and haplotypes of five single nucleotide polymorphisms (SNPs) in the Pfs48/45 gene are exceptionally skewed in frequency among different P. falciparum populations, compared with alleles at 11 microsatellite loci sampled widely from the parasite genome. Fixation indices measuring inter-population variance in allele frequencies (F(ST)) were in the order of four to seven times higher for Pfs48/45 than for the microsatellites, whether considered (i) among populations within Africa, or (ii) among different continents. Differing mutational processes at microsatellite and SNP loci could generally affect the population structure at these different types of loci, to an unknown extent which deserves further investigation. The highly contrasting population structure may also suggest divergent selection on the amino acid sequence of Pfs48/45 in different populations, which plausibly indicates a role for the protein in determining gamete recognition and compatibility.
In various biological studies, for example those in population genetics, conservation biology, forensic science, gene mapping, breed, strain and population characterization and identification, marker assisted selection and the identification of cryptic species complexes, codominant genetic markers play important roles. The information that can be gained from them are far superior than those from dominant markers like random amplified polymorphic DNA (RAPD), amplified fragment length polymorphisms (AFLP), direct amplification of length polymorphisms (DALP) and randomly amplified microsatellites (RAM) or inter simple sequence repeats (ISSR).
A total of 143 microsatellites were isolated from Mystus nemurus using a 5' anchored polymerase chain reaction technique or the random amplified hybridization microsatellite method, the first set of microsatellite markers developed for the Southeast Asian river catfish. Twenty polymorphic microsatellite loci were used as markers for population characterization of M. nemurus from six different geographical locations in Malaysia (Perak, Kedah, Johor, UPM, Sarawak and Terengganu). The number of alleles per locus ranged from 2 to 11 with 6.3 as the average number of alleles per locus. Characterization of the populations showed relatively high levels of genetic variation compared with previous studies using allozyme markers. The highest genetic similarity was found between Perak and Kedah, while the highest genetic distance was found between Terengganu and Kedah. The majority of clustering was in accordance with geographical locations and the histories of the populations. Microsatellite analysis indicated that the Sarawak population might be genetically closer to the Peninsular Malaysian populations than has been previously shown by other molecular marker studies.
Recent advances in neuro-oncology have revealed different pathways of molecular oncogenesis in malignant gliomas including loss of heterozygosity on chromosomal regions harboring tumor suppressor genes. In the present study, we performed polymerase chain reaction-loss of heterozygosity (PCR-LOH) analysis using microsatellite markers to identify loss of heterozygosity on chromosomes 10q, 9p, 17p and 13q in the Malays with malignant gliomas. Of 12 cases with allelic losses, seven (58.3%) cases showed LOH on chromosome 10q, three (25.0%) cases showed LOH on chromosome 9p, four (33.3%) cases showed LOH on chromosome 17p and two (16.7%) cases showed LOH on chromosome 13q. The cases include five (41.7%) cases of glioblastoma multiforme, three (25.0%) cases of anaplastic astrocytoma, three (25.0%) cases of anaplastic oligodendroglioma and one (8.3%) case of anaplastic ependymoma. Four cases showed loss of heterozygosity on more than one locus. Our findings showed that loss of heterozygosity on specific chromosomal regions contributes to the molecular pathway of glioma progression in Malay population. In addition, these data provide useful evidence of molecular genetic alterations of malignant glioma in South East Asian patients, particularly in the East Coast of Malaysia.
Analyses of the spatial distribution pattern, spatial genetic structure and of genetic diversity were carried out in two tropical tree species with contrasting breeding systems and different ploidy levels using a 50-ha demographic plot in a lowland dipterocarp forest in Peninsular Malaysia. Shorea leprosula is a diploid and predominantly outcrossed species, whereas S. ovalis ssp. sericea is an autotetraploid species with apomictic mode of reproduction. Genetic diversity parameters estimated for S. leprosula using microsatellite were consistently higher than using allozyme. In comparisons with S. leprosula and other tropical tree species, S. ovalis ssp. sericea also displayed relatively high levels of genetic diversity. This might be explained by the lower pressure of genetic drift due to tetrasomic inheritance, and for autotetraploids each locus can accommodate up to four different alleles and this allows maintenance of more alleles at individual loci. The observed high levels of genetic diversity in S. ovalis ssp. sericea can also be due to a random retention of more heterogeneous individuals in the past, and the apomictic mode of reproduction might be an evolutionary strategy, which allows the species to maintain high levels of genetic diversity. The spatial distribution pattern analyses of both species showed significant levels of aggregation at small and medium but random distribution at the big diameter-class. The decrease in magnitude of spatial aggregation from small- to large-diameter classes might be due to compensatory mortality during recruitment and survival under competitive thinning process. Spatial genetic structure analyses for both species revealed significant spatial genetic structure for short distances in all the three diameter-classes. The magnitude of spatial genetic structure in both species was observed to be decreasing from smaller- to larger-diameter classes. The high spatial genetic structuring observed in S. ovalis ssp. sericea at the small-diameter class is due primarily to limited seed dispersal and apomictic mode of reproduction. The similar observation in S. leprosula, however, can be explained by limited seed and pollen dispersal, which supports further the fact that the species is pollinated by weak fliers, mainly of Thrips and Megalurothrips in the lowland dipterocarp forest.
We investigated the fine-scale genetic structure of three tropical-rainforest trees, Hopea dryobalanoides, Shorea parvifolia and S. acuminata (Dipterocarpaceae), in Peninsular Malaysia, all of which cooccurred within a 6-ha plot in Pasoh Forest Reserve. A significant genetic structure was found in H. dryobalanoides, weaker (but still significant) genetic structure in S. parvifolia and nonsignificant structure in S. acuminata. Seeds of all three species are wind dispersed, and their flowers are thought to be insect pollinated. The most obvious difference among these species is their height: S. parvifolia and S. acuminata are canopy species, whereas H. dryobalanoides is a subcanopy species. Clear differences were also found among these species in their range of seed dispersal, which depends on the height of the release point; so taller trees disperse their seed more extensively. The estimates of seed dispersal area were consistent with the degree of genetic structure found in the three species. Therefore, tree height probably had a strong influence on the fine-scale genetic structure of the three species.
Microsatellite primers are often developed in one species and used to assess neutral variability in related species. Such analyses may be confounded by ascertainment bias (i.e. a decline in amplification success and allelic variability with increasing genetic distance from the source of the microsatellites). In addition, other factors, such as the size of the microsatellite, whether it consists of perfect or interrupted tandem repeats, and whether it is autosomal or X-linked, can affect variation. To test the relative importance of these factors on microsatellite variation, we examine patterns of amplification and allelic diversity in 52 microsatellite loci amplified from five individuals in each of six populations of Cyrtodiopsis stalk-eyed flies that range from 2.2 % to 11.2% mitochondrial DNA sequence divergence from the population used for microsatellite development. We find that amplification success and most measures of allelic diversity declined with genetic distance from the source population, in some cases an order of magnitude faster than in birds or mammals. The median and range of the repeat array length did not decline with genetic distance. In addition, for loci on the X chromosome, we find evidence of lower observed heterozygosity compared with loci on autosomes. The differences in variability between X-linked and autosomal loci are not adequately explained by differences in effective population sizes of the chromosomes. We suggest, instead, that periodic selection events associated with X-chromosome meiotic drive, which is present in many of these populations, reduces X-linked variation.
The genetic make-up of five populations of Oreochromis spp. was examined by microsatellite analysis. Eleven polymorphic microsatellite loci showed significant departures from the Hardy-Weinberg equilibrium. The mean heterozygosity ranged from 0.6280 to 0.7040 for each population. The genetic distance values showed a clear separation between O. niloticus and O. mossambicus. The differentiation of the O. niloticus populations was then tested with various genetic measures, which are based on both the Infinite Allele and the Stepwise Mutation models. All these measures grouped the populations similarly.
Loss of heterozygosity (LOH) on several loci and mutations on PTEN tumor suppressor gene (10q23.3) occur frequently in sporadic gliomas. We have performed polymerase chain reaction (PCR)-LOH analysis using microsatellite markers and single-stranded conformational polymorphism (SSCP) analysis to determine the incidence of allelic losses on chromosome 10q, 9p, 17p and 13q and mutations of exons 5, 6 and 8 of the PTEN gene in malignant gliomas. Twelve of 23 (52.2%) malignant glioma cases showed allelic losses whereas 7 of 23, (30.4%) samples showed aberrant band patterns and mutations of the PTEN gene. Four of these cases showed LOH on 10q23 and mutations of the PTEN gene. The data on LOH indicated the involvement of different genes in gliomagenesis whereas mutations of the PTEN gene indicated the role of PTEN tumor suppressor gene in the progression of glioma in Malay population.
We examined differences in pollen dispersal efficiency between 2 years in terms of both spatial dispersal range and genetic relatedness of pollen in a tropical emergent tree, Dipterocarpus tempehes. The species was pollinated by the giant honeybee (Apis dorsata) in a year of intensive community-level mass-flowering or general flowering (1996), but by several species of moths in a year of less-intensive general flowering (1998). We carried out paternity analysis based on six DNA microsatellite markers on a total of 277 mature trees forming four spatially distinct subpopulations in a 70 ha area, and 147 and 188 2-year-old seedlings originating from seeds produced in 1996 and 1998 (cohorts 96 and 98, respectively). Outcrossing rates (0.93 and 0.96 for cohorts 96 and 98, respectively) did not differ between years. Mean dispersal distances (222 and 192 m) were not significantly different between the 2 years but marginally more biased to long distance in 1996. The mean relatedness among cross-pollinated seedlings sharing the same mothers in cohort 96 was lower than that in cohort 98. This can be attributed to the two facts that the proportion of intersubpopulations pollen flow among cross-pollination events was marginally higher in cohort 96 (44%) than in cohort 98 (33%), and that mature trees within the same subpopulations are genetically more related to each other than those between different subpopulations. We conclude that D. tempehes maintained effective pollen dispersal in terms of outcrossing rate and pollen dispersal distance in spite of the large difference in foraging characteristics between two types of pollinators. In terms of pollen relatedness, however, a slight difference was suggested between years in the level of biparental inbreeding.
We investigated the genetic structure within and among Bornean orang-utans (Pongo pygmaeus) in forest fragments of the Lower Kinabatangan flood plain in Sabah, Malaysia. DNA was extracted from hair and faecal samples for 200 wild individuals collected during boat surveys on the Kinabatangan River. Fourteen microsatellite loci were used to characterize patterns of genetic diversity. We found that genetic diversity was high in the set of samples (mean H(E) = 0.74) and that genetic differentiation was significant between the samples (average F(ST) = 0.04, P < 0.001) with F(ST) values ranging from low (0.01) to moderately large (0.12) values. Pairwise F(ST) values were significantly higher across the Kinabatangan River than between samples from the same river side, thereby confirming the role of the river as a natural barrier to gene flow. The correlation between genetic and geographical distance was tested by means of a series of Mantel tests based on different measures of geographical distance. We used a Bayesian method to estimate immigration rates. The results indicate that migration is unlikely across the river but cannot be completely ruled out because of the limited F(ST) values. Assignment tests confirm the overall picture that gene flow is limited across the river. We found that migration between samples from the same side of the river had a high probability indicating that orang-utans used to move relatively freely between neighbouring areas. This strongly suggests that there is a need to maintain migration between isolated forest fragments. This could be done by restoring forest corridors alongside the river banks and between patches.
The diamondback moth, Plutella xylostella, is renowned for developing resistance to insecticides and causing significant economic damage to Brassica vegetable crops throughout the world. Yet despite its economic importance, little is known about the population structure and movement patterns of this pest both at local and regional scales. In Australia, the movement patterns and insecticide resistance status of P. xylostella infesting canola, vegetables, forage brassicas and weeds have fundamental implications for the management of this pest. Here we use six polymorphic microsatellite loci to investigate population structure and gene flow in Australian populations of P. xylostella. Samples of P. xylostella from New Zealand, Malaysia, Indonesia and Kenya were also scored at these loci. We found no evidence of population structure within Australia, with most populations having low inbreeding coefficients and in Hardy-Weinberg equilibrium. In addition, a sample from the North Island of New Zealand was indistinguishable from the Australian samples. However, large genetic differences were found between the Australia/New Zealand samples and samples from Kenya, Malaysia and Indonesia. There was no relationship between genetic distance and geographic distance among Australian and New Zealand samples. Two of the loci were found to have null alleles, the frequency of which was increased in the populations outside the Australia/New Zealand region. We discuss these results with reference to insecticide resistance management strategies for P. xylostella in Australia.
The extent to which response to environmental change is mediated by species-specific ecology is an important aspect of the population histories of tropical taxa. During the Pleistocene glacial cycles and associated sea level fluctuations, the Sunda region in Southeast Asia experienced concurrent changes in landmass area and the ratio of forest to open habitat, providing an ideal setting to test the expectation that habitat associations played an important role in determining species' response to the opportunity for geographic expansion. We used mitochondrial control region sequences and six microsatellite loci to compare the phylogeographic structure and demographic histories of four broadly sympatric species of Old World fruit bats in the genus, Cynopterus. Two forest-associated species and two open-habitat generalists were sampled along a latitudinal transect in Singapore, peninsular Malaysia, and southern Thailand. Contrary to expectations based on habitat associations, the geographic scale of population structure was not concordant across ecologically similar species. We found evidence for long and relatively stable demographic history in one forest and one open-habitat species, and inferred non-coincident demographic expansions in the second forest and open-habitat species. Thus, while these results indicate that Pleistocene climate change did not have a single effect on population structure across species, a correlation between habitat association and response to environmental change was supported in only two of four species. We conclude that interactions between multiple factors, including historical and contemporary environmental change, species-specific ecology and interspecific interactions, have shaped the recent evolutionary histories of Cynopterus fruit bats in Southeast Asia.
Anopheles (Cellia) maculatus Theobald is a major malaria vector in southern Thailand and peninsular Malaysia, and previous population genetic studies suggested that mountain ranges act as barriers to gene flow. In this study, we examine the genetic variance among 12 collections of natural populations in southern Thailand by analyzing 7 microsatellite loci. Based on analysis of molecular variance (AMOVA), three geographic populations of An. maculatus are suggested. The southern population exists in western Thailand north of 12 degrees north latitude. Mosquitoes to the south fall into two genetic populations: 1) the middle southern collections located on the west side of the Phuket mountain range between 8 degrees and 10 degrees north latitude, and 2) the southern collections located on the east of the Phuket mountain range located between approximately 6.5 degrees and 11.5 degrees north latitude. AMOVA revealed significant genetic differentiation between northern and middle southern and southern populations. The middle southern population was moderately differentiated from the southern population. Furthermore, gene flow was restricted between proximal collections located on different sides of the Phuket mountain range. Collections separated by 50 km exhibited restriction of gene flow when separated by geographic barriers, whereas greater gene flow was evident among collections 650 km apart but without geographic barriers.
Paroxysmal nocturnal haemoglobinuria (PNH) is an acquired haemopoietic stem cell disorder arising from somatic mutation of the X-linked PIG-A gene which leads to deficiency of the glycosylphosphatidylinositol (GP1) membrane anchor proteins such as CD 59 (MIRL: membrane inhibitor of reactive lysis) and CD 55 (DAF: decay accelerating factor). Allogeneic peripheral blood stem cell transplant (PBSCT) is a curative mode of treatment in symptomatic PNH patients. Assessment of donor chimerism for PBSCT can be performed by various methods including short tandem repeat loci (STR) and variable number of tandem repeats (VNTR). Flow cytometry, which is much cheaper and faster, also can be used to assess engraftment in patients with PNH. Engrafted patients will show the presence of CD 55 and CD 59 on their red cells and white cells. We describe here the usefulness of flow cytometry in the assessment of donor chimerism following allogeneic PBSCT, in a case of PNH.