A limited backcross procedure was utilized to introgress genes associated with grain quality traits from Oryza rufipogon (Accession No. IRGC 105491), a wild rice from Malaysia, to the cultivated rice O. sativa cv. MR219, a popular high yielding Malaysian rice cultivar. A set of 10 BC(2)F(7) progenies were selected based on the field performance and phenotypic appearance in BC(2)F(5) and BC(2)F(6) generations, which initially started with 266 progenies in the BC(2)F(2) generation. These 10 advanced breeding lines are similar to each other but differ in several important grain quality traits, which can be traced to O. rufipogon introgressions. Phenotyping and genotyping of BC(2)F(7) variants were considered for QTL analysis. The introgressed lines did not show any significant changes compared to the recurrent parent MR219 for the traits grain density and milled rice percentage. All 10 progenies showed significantly higher head rice percentages (70-88%) than the recurrent parent MR219. Variants G13 and G15 had higher amylose contents than MR219. All variants were analyzed using polymorphic SSR markers. Of the 34 SSR markers, only 18 showed introgression from O. rufipogon for chromosomes 1, 2, 3, 5, 6, 8, 10, and 11. Graphical genotypes were prepared for each variant, and association between the introgression regions and the traits that increased grain quality was visualized. Based on marker trait association, some of the QTLs are stable across environments and genetic backgrounds and could be used universally.
Seven single locus microsatellite markers were characterized in Malaysian giant freshwater prawn, Macrobrachium rosenbergii from an enriched genomic library Primer pairs were designed to flank the repeat sequences and the loci characterized for this species. The bands resulting from the PCR amplifications of these eight microsatellite loci were polymorphic with the number of alleles ranging from 8 to 26 alleles per locus, whereas the observed heterozygosity ranged from 0.0641 to 0.6564. These newly developed microsatellite markers should prove to be useful for population studies and in the management of genetic variations in broodstocks of freshwater prawn, M. rosenbergii.
Allele frequencies of 15 short tandem repeat (STR) loci, namely D5S818, D7S820, D13S317, D16S539, TH01, TPOX, Penta D, Penta E, D3S1358, D8S1179, D18S51, D21S11, CSF1PO, vWA, and FGA, were determined for 154 individuals from the Kadazan-Dusun tribe, an indigenous population of East Malaysia. All loci were amplified by polymerase chain reaction, using the Powerplex 16 system. Alleles were typed using a gene analyzer and the Genemapper ID software. Various statistical parameters were calculated and the combined power of discrimination for the 15 loci in the population was calculated as 0.999999999999999. These loci are thus, informative and can be used effectively in forensic and genetic studies of this indigenous population.
Malaysian arowana (dragonfish; Scleropages formosus) is an ancient osteoglossid fish from southeast Asia. Due to the high demand of the ornamental fish trade and because of habitat loss, the species is close to extinction. We isolated and characterized 10 polymorphic microsatellites of this species, using 5'-anchored PCR. The number of alleles at the 10 microsatellite loci ranged from 2 to 28, with a mean of 7.8/locus. The observed heterozygosity ranged from 0.03 to 0.93 (mean: 0.39), whereas the expected heterozygosity ranged from 0.03 to 0.94 (mean: 0.46). Seven microsatellites deviated from Hardy-Weinberg equilibrium, and three conformed to Hardy-Weinberg equilibrium and were in linkage equilibrium. These 10 novel microsatellites should facilitate studies of genetic diversity and population structure of arowana to help plan actions for the conservation of the indigenous Malaysian arowana.
The genotypes of black pepper are morphologically and genotypically highly diverse and carry all the cumulative variations inherited and maintained through generations. The present study describes the Simple Sequence Repeat (SSR) or microsatellite based assessment of genetic diversity among forty popular genotypes and four different species of black pepper in Southern region of India. For isolation of SSR primers, our earlier attempts with enrichment strategies like 'Triplex affinity capture' did not extract a single SSR primer due to close proximity of restriction sites to the SSR motif. Hence we developed a 'Sequential Reverse Genome Walking (SRGW)' strategy with better enrichment efficiency of 72% that generated seven new SSR primers. Genotyping precisely discriminated majority of genotypes which indicated that the SSR primers are very informative. A total of 62 alleles with an average of 15.5 alleles over 4 loci were identified. All the SSR primers showed an average Polymorphism Information Content (PIC) value of 0.85. The estimated average Shared Allele Frequency ranged between 1.57 and 20.12%. The PCA plot revealed four closely related individual groups and identified Karimunda, Wild pepper and a local landrace 'local b' as the most divergent genotypes. Cluster analysis exposed the genetic relatedness between hybrids and selections with other known cultivars. The introduction of black pepper from South India to Malaysia was emphasized from the observation of genetic similarity of Malaysian cultivar 'Kuching' with other indigenous popular cultivars. The study was first to portray the precise genetic relatedness among the major indigenous genotypes of black pepper.
The population structure of Probarbus jullieni from Malaysia and Thailand stocks was based on seven microsatellite primers and truss network measurements. Truss morphometric measurements were made on Temoleh, Probarbus jullieni to demonstrate the degree of speciation that can be induced by both biotic and abiotic conditions and contribute to the definition of different stocks of Probarbus sp. At the momment no relevant information on stock definition has been produced recently concerning Probarbus spp., which is now in IUCN threatened red list. We also summarize the possible discriminant morphological characteristics that shows differentiation between Malaysia and Thailand stocks. We also compare the levels of morphology and genetic differences for Malaysian stocks throughout one year of sampling to determine whether sampling season and possible sexual dimorphism can be detected in this fishes. A total of 25 different alleles were found across the two populations by the seven microsatellites, of which 21 and 19 alleles were detected in Pahang, Malaysia and Thailand, respectively At the population level, the mean number of alleles of Pahang (3.4991) per locus was higher than that (3.1665) of Thailand. From both molecular and morphometric measurements showed that there were two distinct populations. However the differences between these two populations showed that they belong to the same species with least degree of separation
Seven single locus dinucleotide microsatellite markers were developed to characterize an economically important sport fish and food fish in Malaysia and in Southeast Asia. They were obtained by using a rapid method namely the 5' anchored PCR enrichment protocol. The specific primers were designed to flank the repeat sequences and these were subsequently used to characterize 120 unrelated fish from Malaysia and 30 fishes from Indonesia. The number of alleles per locus ranged from 2 (SYKVJ1-11) to 6 (SYKVJ1-4) while the levels of heterozygosity ranged from 0.0472 (SYKVJ1-11) to 0.7745 (SYKVJ1-2).
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.
Coloured rice genotypes have greater nutritious value and consumer demand for these varieties is now greater than ever. The documentation of these genotypes is important for the improvement of the rice plant. In this study, 42 coloured rice genotypes were selected for determination of their genetic divergence using 25 simple sequence repeat (SSR) primers and 15 agro-morphological traits. Twenty-one out of the 25 SSR primers showed distinct, reproducible polymorphism. A dendrogram constructed using the SSR primers clustered the 42 coloured rice genotypes into 7 groups. Further, principle component analysis showed 75.28% of total variations were explained by the first-three components. All agro-morphological traits showed significant difference at the (p≤0.05) and (p≤0.01) levels. From the dendrogram constructed using the agro-morphological traits, all the genotypes were clustered into four distinct groups. Pearson's correlation coefficient showed that among the 15 agro-morphological traits, the yield contributing factor had positive correlation with the number of tillers, number of panicles, and panicle length. The heritability of the 15 traits ranged from 17.68 to 99.69%. Yield per plant and harvest index showed the highest value for both heritability and genetic advance. The information on the molecular and agro-morphological traits can be used in rice breeding programmes to improve nutritional value and produce higher yields.
Genetic variability and differences in wild striped snakehead Channa striata from Malaysia were analysed by genotyping nine novel nuclear microsatellite loci. Analysis revealed moderate-to-high genetic diversity in most of the populations, indicative of large effective population sizes. The highly diversified populations are admixed populations and, therefore, can be recommended as potential candidates for selective breeding and conservation since they each contain most of the alleles found in their particular region. Three homogenous groups of the wild populations were identified, apparently separated by effective barriers, in accordance with contemporary drainage patterns. The highest population pairwise FST found between members of the same group reflects the ancient population connectivity; yet prolonged geographical isolation resulted in adaptation of alleles to local contemporary environmental change. A significant relationship between genetic distance and geographical isolation was observed (r = 0·644, P < 0·01). Anthropogenic perturbations indicated apparent genetic proximity between distant populations.
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.
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.
We investigated 12 X-chromosomal short tandem repeat (STR) polymorphisms in 283 unrelated Malay individuals (160 males and 123 females) living in and around Kuala Lumpur using the Investigator Argus X-12 kit. Heterozygosity among the present 12 X-STRs showed a distribution of from 55.3 to 93.5 %. The diversity values of the haplotypes constructed using four closely linked groups were all higher than 0.9865. A comparison of allelic frequency in each system and haplotype variation indicated that the nature of these X-STRs in the Malay population differed from that in East Asian, European, or African populations. Several microvariant alleles found in the Malay population were characterized and compared with known sequence data. The present data may be helpful in forensic casework such as personal identification and kinship testing in the Malay population in Malaysia.
Curculin, a sweet protein found in Curculigo latifolia fruit has great potential for the pharmaceutical industry. This protein interestingly has been found to have both sweet taste and taste-modifying capacities comparable with other natural sweeteners. According to our knowledge this is the first reported case on the isolation of microsatellite loci in this genus. Hence, the current development of microsatellite markers for C. latifolia will facilitate future population genetic studies and breeding programs for this valuable plant. In this study 11 microsatellite markers were developed using 3' and 5' ISSR markers. The primers were tested on 27 accessions from all states of Peninsular Malaysia. The number of alleles per locus ranged from three to seven, with allele size ranging from 141 to 306 bp. The observed and expected heterozygosity ranged between 0.00-0.65 and 0.38-0.79, respectively. The polymorphic information content ranged from 0.35 to 0.74 and the Shannon's information index ranged from 0.82 to 1.57. These developed polymorphic microsatellites were used for constructing a dendrogram by unweighted pair group method with arithmetic mean cluster analysis using the Dice's similarity coefficient. Accessions association according to their geographical origin was observed. Based on characteristics of isolated microsatellites for C. latifolia accessions all genotype can be distinguished using these 11 microsatellite markers. These polymorphic markers could also be applied to studies on uniformity determination and somaclonal variation of tissue culture plantlets, varieties identification, genetic diversity, analysis of phylogenetic relationship, genetic linkage maps and quantitative trait loci in C. latifolia.
A total of 19 polymorphic microsatellite loci were used to analyse levels of genetic variation for 10 populations of Perna viridis L. collected from all over peninsular Malaysia. The populations involved in this study included Pulau Aman in Penang, Tanjung Rhu in Kedah, Bagan Tiang in Perak, Pulau Ketam in Selangor, Muar, Parit Jawa, Pantai Lido and Kampung Pasir Puteh in Johore, and Kuala Pontian and Nenasi in Pahang state. The number of alleles per locus ranged from two to seven, with an average of 3.1. Heterozygote deficiencies were observed across all the 10 populations. Characterization of the populations revealed that local populations of P. viridis in peninsular Malaysia were genetically similar enough to be used as a biomonitoring agent for heavy metal contamination in the Straits of Malacca. Cluster analysis grouped the P. viridis populations according to their geographical distributions with the exception of Parit Jawa. The analysis also revealed that P. viridis from the northern parts of peninsular Malaysia were found to be the most distant populations among the populations of mussels investigated and P. viridis from the eastern part of peninsular Malaysia were closer to the central and southern populations than to the northern populations.
Msb069 primer pairs encompassed region is believed to be associated with a quantitative trait loci (QTL) of dorsal fin length in subgenus Poecilia. However, detailed investigation on Msb069 which originated from Xiphophorus on subgenus Poecilia remains unexplored. In this study, full sequence of Msb069 was characterized by sequencing bioinformatics analysis and gene expression. The sequence analysis of Msb069 primer pairs encompassed region on three species of Poecilia revealed higher number of microsatellite tandem repeats in Poecilia latipinna (ATG 16 ) compared to P. sphenops (ATG 13-14 ). There is no notable pattern of ATGtandem repeats discovered in the hybrids. The full sequence of Msb069 is 734 bp in length and showed a 233 bp conserved region between Xiphophorus and Poecilia. BLAST search performed on this sequence revealed no significant similarities. Nonquantitative RT-PCR exhibited the presence of Msb069 transcripts in three different tissues in subgenus Poecilia. Meanwhile, quantitative RTPCR expression on two different tissues showed relatively higher expression of Msb069 transcript in P. latipinna dorsal fin tissues in both male and female fishes, suggesting a repressive function of this transcript with respect to dorsal fin length. However the exact gene expression event of Msb069 is still unknown and requires further investigation.
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.