Isolation, Characterization and Genetic Linkage Mapping of Microsatellite Markers in Mystus Nemurus

The river catfish (Mystus nemurus) is of great potential importance as an alternative fish protein source, notably in Malaysia and Thailand. The seed supply is seasonal and inability to reproduce in captivity is a hindrance to mass produce this species. Molecular breeding and genetic mapping with the use of DNA based markers have paved ways in overcoming conventional breeding to improve important economic traits. The main objectives of this study were to isolate and develop microsatellite markers for Mystus nemurus, and apply these markers on genetic linkage map for this species. Two methodologies were used to isolate the microsatellite loci in M. nemurus, namely Random Amplified Hybridizing Microsatellites (RAHMs) and 5' anchored Polymerase Chain Reaction (5' anchored PCR). A total of 236 repeat sequences were identified. Of these, 13 were developed fiom RAHMs and 223 were developed from 5' anchored PCR.A total of 177 primer pairs were developed. Of these 64 have been screened for polymorphisms on 90 unrelated fishes collected from six locations (Kedah, Selangor, Perak, Johor, Terengganu and Sarawak), where each location consisted of 15 individuals. Forty four primer pairs were found to be polymorphic. The highest heterozygous marker was MnBp5-1-12a (0.871 4) while the lowest heterozygosity was observed in MnBp8-1-25a (0.0000). A dendrogram was constructed from the 44 polymorphic loci. An additional 34 polymorphic markers, previously generated, were combined in order to construct another dendrogram, and it was found that the dendrograrn stabilized at 50 polymorphic microsatellite loci. Induced breeding was carried out to generate two known fish family groupings. The first family grouping was generated from the parents chosen randomly from the Terengganu broodstock. One hundred progeny were produced. The second family grouping was generated from the male chosen from Terengganu population and the female chosen from Pahang population. Fifty progenies were used for the analysis of Mendelian segregation and linkage mapping. A total of 70 microsatellite markers isolated for this species were screened and evaluated for the Mendelian Inheritance ratio on the two fish families, mentioned of which 31 showed segregation in either one of the families. These markers were analyzed with contingency chi-square analysis. Of these, 13 pair markers were grouped into 8 groups. Strategy "Pseudo-testcross" was applied in the linkage mapping analysis. Analysis of linkage using LOD score was carried out and a total of 7 linkage groups were generated from the 2 fish families under the LOD of 1.2. Additional analysis of LOD was performed on the family data of 44 dominant markers done previously. Three linkage groups were obtained at the LOD of 1.2 and fractionated into 6 groups when the LOD score was increased to 2.0. This is the first map generated for M. nemurus which provides information on genetic linkage for this species. It is a stepping-stone for carrying out more intensive genetic mapping and QTL analysis on this species in the future. Eventually, marker assisted selection (MAS) or isolation of economically important genes could be made possible and these would benefit the Malaysian aquaculture industr

Development of rapid screening method for the diagnostic of fragile x syndrome using real time PCR

Fragile X Syndrome (FXS) is the most prevalent inherited cause of mental retardation. The prevalence of FXS in males and females are approximately 1 in 4000 and I in 8000 respectively. It is caused by CGG repeat instability in the FMRl gene, located on chromosome Xq27.3. Normal individuals have CGG repeats ranging from 5 to 53. In premulation carriers, the CGG repeats range from 60 to 200 and shall be more than 200 repeats tor ti.Jil mutation patients. FXS patients have variable clinical features and because of that, an accurate clinical 'diagnosis is always a problem. Currently, Cytogenetic, PCR and Southern Blot Techniques are widely used for diagnosis of FXS. Here we report a pair of brothers suspected to be FXS patients with similar clinical features. However, the cytogenetic result for younger brother did not show fragile site at Xq27.3 of the X chromosome while molecular result was confirmatory for FXS. Conversely, the elder brother showed confirmatory results for Fragile X mutation in both cytogenetic and molecular analysis. We therefore conclude that cytogenetic analysis alone Cftnnot be dependable for the confirmatory diagnosis ofFXS

Understanding the role of copy number (CNV) in the development of left ventricular hypertrophy in hypertension / Khalid Yusoff and Hoh Boon Peng

Left ventricular hypertrophy (LVH) is an independent risk factor for the development of heart failure, coronary heart disease and stroke. It develops as a result of hemodynamic overload, for instance, hypertension. Blood pressure is an important determinant of LVH, and a significant proportion of patients with essential hypertension develops this complication. However, this condition varies in a wide range of phenotypes, and studies had shown that patients with LVH may have near-normal blood pressure, suggesting that development of LVH may be due to an independent genetic factor from hypertension. LVH can be reversed with anti-hypertensive (anti-HT) agents. Angiotensinogen receptor blocker like losartan has been shown to improve the reversal effect. However, it is unknown whether using this anti-HT agent alone would be useful in preventing LVH. Hence, identifying HT patients with the risk of LVH may allow this hypothesis to be tested, and if successful, would lead to the prevention, treatment and improvement of prognosis of LVH. We recently carried out a genome-wide scan of copy number variation (CNV) on a group of hyptertensive LVH patients and observed a gain of copy number in AGTRII gene in a group of patients. We attempted to further investigate the role of CNV of this gene in the pathogenesis of LVH. However, we observed no significant contribution of this CNV in the disease complication. Therefore in this study, we attempted to investigate the contributions of rare CNV in the susceptibility of hypertensive LVH, and subsequently to predict the putative molecular pathways in the pathogenesis of LVH

Segregation and genetic linkage analyses of river catfish, Mystus nemurus, based on microsatellite markers

The river catfish Mystus nemurus is an important fresh water species for aquaculture in Malaysia. We report the first genetic linkage map of M. nemurus based on segregation analysis and a linkage map using newly developed microsatellite markers of M. nemurus. A total of 70 of the newly developed polymorphic DNA microsatellite markers were analyzed on pedigrees generated using a pseudo-testcross strategy from 2 mapping families. In the first mapping family, 100 offspring were produced from randomly selected dams of the same populations; dams of the second family were selected from 2 different populations, and this family had 50 offspring. Thirty-one of the 70 markers segregated according to the Mendelian segregation ratio. Linkage analysis revealed that 17 microsatellite markers belonging to 7 linkage groups were obtained at a logarithm of the odds score of 1.2 spanning 584 cM by the Kosambi mapping function, whereas the other 14 remained unlinked. The results from this study will act as primer to a more extensive genetic mapping study aimed towards identifying genetic loci involved in determining economically important traits

Mendelian inheritance of microsatellite markers in Southeast Asian River Catfish, Mystus nemurus

Eight previously designed polymorphic microsatellite loci, namely, Mnc434a, Mnc65b, Mnc441, MnBp5-1-02b, MnBp5-1-30b, MnBp5-2-02b, MnBp8-4-43a, and MnBp8-4-43b, were used to examine their modes ofsegregation in Mystus nemurus family study. Randomly selected broodstocks from Terengganu population were used to generate F 1 population. DNA was isolated from tissue samples of the parents, whilst the whole one-month-old F , 1 progeny was used to isolate the DNA. Chi-square analysis was performed to test the significance of the results obtained. It was shown that all the eight microsatellite loci examined segregated close to Mendelian inheritance ratios, which would be useful in generating a genetic linkage map for this species

FCGR3B gene copy number variation and host susceptibility to vascular leakage in Dengue Hemorrhagic Fever / Hoh Boon Peng , Sazaly Abu Bakar and Rafidah Hanim Shueb

DENV causes significantly more human disease than any other abovirus. Annually, an estimated of 50-100 million cases of severe dengue require hospitalization in which 500,000 resulted in DHF/DSS, with more than 20,000 death worldwide (WHO DengueNet report, 2005). Hence, it has now been recognized as a major expanding public health problem of the country. Dengue viruses cause a spectrum of illness ranging from asymptomatic infection or mild febrile illness to severe and fatal hemorrhagic disease. While majority experience uncomplicated Dengue Fever (DF), Dengue Hemorrhagic Fever (DHF) can present with severe clinical manifestations including transient vascular permeability resulting in plasma leakage (WHO, 1997). No specific treatment is available to date. Previous studies suggested the involvement of the events in the peripheral blood in association with the DENV disease severity, such as dengue viral replication, cytokine expression, and cellular activation / proliferation and robust host inflammatory immune response (Rothman, 2004). Antibody-dependent enhancement of viral replication is the most widely accepted explanation for the association between DHF and pre-existing antibody. However, it remains considerable uncertainty as to how virus-host interaction triggers the inflammatory response resulting in plasma leakage, the hallmark of DHF/DSS. FcGRII has been reported to play a role in pathogenesis of severe dengue infections (Loke et al., 2002; Littaua et al., 1990). It functions to mediate antibody enhancement in vitro by binding to virus-IgG complexes. A fundamental to any discussion of DENV pathogenesis is the association of secondary infection with heterologous serotypes with DHF/DSS. Antibody Dependent Enhancement (ADE) model during secondary infections, postulates that DENV specific antibodies either cross reactive antibodies, can interact with DENV without neutralizing the virus, and thereby requires FcG receptors to mediate entry of antibody coated DENV into cells (Clyde et al., 2006; Coffey et al, 2009). Therefore, this proposed study investigated and to characterized the CNV of FcGRII gene among the DF and DHF patients. Though association of FcGRII gene SNP polymorphism with dengue has been reported earlier (Loke et al, 2002), none investigated the copy number of this gene and its association to the susceptibility of plasma leakage

Illegitimacy and sibship assignments in oil palm (Elaeis guineensis Jacq.) half-sib families using single locus DNA microsatellite markers

Oil palm breeding has been progressing very well in Southeast Asia, especially in Malaysia and Indonesia. Despite this progress, there are still problems due to the difficulty of controlled crossing in oil palm. Contaminated/illegitimate progeny has appeared in some breeding programs; late and failure of detection by the traditional method causes a waste of time and labor. The use of molecular markers improves the integrity of breeding programs in perennial crops such as oil palm. Four half-sib families with a total of 200 progeny were used in this study. Thirty polymorphic single locus DNA microsatellites markers were typed to identify the illegitimate individuals and to obtain the correct parental and progeny assignments by using the CERVUS and COLONY programs. Three illegitimate palms (1.5 %) were found, and 16 loci proved to be sufficient for sibship assignments without parental genotypes by using the COLONY program. The pairwise-likelihood score (PLS) method was better for half-sib family assignments than the full likelihood (FL) method

Association between basal stem rot disease and simple sequence repeat markers in oil palm, Elaeis guineensis Jacq.

The oil palm is badly affected by basal stem rot (BSR) disease in Southeast Asia. BSR disease is caused by the fungus Ganoderma boninense, which is a major threat to oil palm compared with other Ganoderma spp. Molecular markers associated with BSR disease will accelerate the identification process of resistant breeding materials in screening of plants for tolerance to the disease at the nursery stage. In this study, 58 simple sequence repeat markers were utilized with three progeny types, namely, KA4G1, KA4G8, and KA14G8, to perform a comparative molecular mapping for association with BSR. A total of 319 alleles were identified with an average of 5.51 alleles per locus. Five markers, mEgCIR0793:180, mEgCIR0894:200, mEgCIR03295:210, mEgCIR3737:146 and mEgCIR3785:299 were found to be associated with Ganoderma disease with P values of 0.018, 0.033, 0.037, 0.034 and 0.037, respectively, in single progeny analysis. However, in pooled data (KA4G1, KA4G8 and KA14G8), only two alleles, mEgCIR0804:213 (P value = 0.001) and mEgCIR3292:183 (P value = 0.001), were found to be associated with Ganoderma disease. These analyses confirmed that progeny type KA4G1 was tolerant, whereas the other two were susceptible progeny types. These markers and KA4 progeny will be useful in future works on BSR disease resistance in oil palm

Analysis of five deep-sequenced trio-genomes of the Peninsular Malaysia Orang Asli and North Borneo populations

BackgroundRecent advances in genomic technologies have facilitated genome-wide investigation of human genetic variations. However, most efforts have focused on the major populations, yet trio genomes of indigenous populations from Southeast Asia have been under-investigated.ResultsWe analyzed the whole-genome deep sequencing data (30x) of five native trios from Peninsular Malaysia and North Borneo, and characterized the genomic variants, including single nucleotide variants (SNVs), small insertions and deletions (indels) and copy number variants (CNVs). We discovered approximately 6.9 million SNVs, 1.2 million indels, and 9000 CNVs in the 15 samples, of which 2.7% SNVs, 2.3% indels and 22% CNVs were novel, implying the insufficient coverage of population diversity in existing databases. We identified a higher proportion of novel variants in the Orang Asli (OA) samples, i.e., the indigenous people from Peninsular Malaysia, than that of the North Bornean (NB) samples, likely due to more complex demographic history and long-time isolation of the OA groups. We used the pedigree information to identify de novo variants and estimated the autosomal mutation rates to be 0.81x10(-8) - 1.33x10(-8), 1.0x10(-9) - 2.9x10(-9), and 0.001 per site per generation for SNVs, indels, and CNVs, respectively. The trio-genomes also allowed for haplotype phasing with high accuracy, which serves as references to the future genomic studies of OA and NB populations. In addition, high-frequency inherited CNVs specific to OA or NB were identified. One example is a 50-kb duplication in DEFA1B detected only in the Negrito trios, implying plausible effects on host defense against the exposure of diverse microbial in tropical rainforest environment of these hunter-gatherers. The CNVs shared between OA and NB groups were much fewer than those specific to each group. Nevertheless, we identified a 142-kb duplication in AMY1A in all the 15 samples, and this gene is associated with the high-starch diet. Moreover, novel insertions shared with archaic hominids were identified in our samples.ConclusionOur study presents a full catalogue of the genome variants of the native Malaysian populations, which is a complement of the genome diversity in Southeast Asians. It implies specific population history of the native inhabitants, and demonstrated the necessity of more genome sequencing efforts on the multi-ethnic native groups of Malaysia and Southeast Asia

Genetic polymorphism and natural selection in the C-terminal 42 kDa region of merozoite surface protein-1 (MSP-1) among Plasmodium knowlesi samples from Malaysia

Background: The merozoite surface protein-1 (MSP-1) gene encodes for a leading malaria vaccine candidate antigen. However, its extensive polymorphic nature represents a major obstacle to the development of a protective vaccine. Previously, a pilot study was carried out to explore the sequence variation of the C-terminal 42 kDa fragment within P. knowlesi MSP-1 gene (PkMSP-142) based on 12 clinical samples; however, further study on an adequate sample size is vital in estimating the genetic diversity of the parasite population. Methods: In the present study, we included a larger sample size of P. knowlesi (83 samples) covering eight states of Malaysia to determine the genetic polymorphism, natural selection and haplotype groups of the gene fragment coding PkMSP-142. The region flanking PkMSP-142 was amplified by PCR and directly sequenced. Genetic diversity, haplotype diversity, population genetic differentiation and natural selection were determined in order to study the polymorphic characteristic of PkMSP-142. Results: A high level of genetic diversity (Hd = 0.970 ± 0.007; л = 0.01079 ± 0.00033) was observed among the 83 P. knowlesi samples, confirming the extensive genetic polymorphism exhibited among the P. knowlesi population found in Malaysia. A total of 18 distinct haplotypes with 17 amino acid changes were identified, whereby 15 were new haplotypes. High population differentiation values were observed within samples from Peninsular Malaysia and Malaysian Borneo. The 42 kDa fragments of P. knowlesi from Malaysian Borneo were found to be acting on balancing selection whilst purifying selection was suggested to act on isolates from Peninsular Malaysia. The separation of PkMSP-142 haplotypes into two main groups based on geographical separation has further supported the existence of two distinct P. knowlesi lineages. Conclusions: A high level of genetic diversity was observed among PkMSP-142 in Malaysia, whereby most of the polymorphisms were found within the 33 kDa region. Taken together, these data will be useful in order to understand the nature of P. knowlesi population in Malaysia as well as the design and development of a MSP-142 based knowlesi malaria vaccin