The prophenoloxidase activating system is an important innate immune response against microbial infections in invertebrates. The major enzyme, phenoloxidase, is synthesized as an inactive precursor and its activation to an active enzyme is mediated by a cascade of clip domain serine proteinases. In this study, a cDNA encoding a prophenoloxidase activating enzyme-III from the giant freshwater prawn Macrobrachium rosenbergii, designated as MrProAE-III, was identified and characterized. The full-length cDNA contains an open reading frame of 1110 base pair (bp) encoding a predicted protein of 370 amino acids including an 22 amino acid signal peptide. The MrProAE-III protein exhibits a characteristic sequence structure of a long serine proteases-trypsin domain and an N- and C-terminal serine proteases-trypsin family histidine active sites, respectively, which together are the characteristics of the clip-serin proteases. Sequence analysis showed that MrProAE-III exhibited the highest amino acid sequence similarity (63%) to a ProAE-III from Atlantic blue crab, Callinectes sapidus. MrProAE-III mRNA and enzyme activity of MrProAE-III were detectable in all examined tissues, including hepatopancreas, hemocytes, pleopods, walking legs, eye stalk, gill, stomach, intestine, brain and muscle with the highest level of both in hepatopancreas. This is regulated after systemic infectious hypodermal and hematopoietic necrosis virus infection supporting that it is an immune-responsive gene. These results indicate that MrProAE-III functions in the proPO system and is an important component in the prawn immune system.
Trichoderma species are commercially applied as biocontrol agents against numerous plant pathogenic fungi due to their production of antifungal metabolites, competition for nutrients and space, and mycoparasitism. However, currently the identification of Trichoderma species from throughout the world based on micro-morphological descriptions is tedious and prone to error. The correct identification of Trichoderma species is important as several traits are species-specific. The Random Amplified Microsatellites (RAMS) analysis done using five primers in this study showed different degrees of the genetic similarity among 42 isolates of this genus. The genetic similarity values were found to be in the range of 12.50-85.11% based on a total of 76 bands scored in the Trichoderma isolates. Of these 76 bands, 96.05% were polymorphic, 3.95% were monomorphic and 16% were exclusive bands. Two bands (250 bp and 200 bp) produced by primer LR-5 and one band (250 bp) by primer P1A were present in all the Trichoderma isolates collected from healthy and infected oil palm plantation soils. Cluster analysis based on UPGMA of the RAMS marker data showed that T. harzianum, T. virens and T. longibrachiatum isolates were grouped into different clades and lineages. In this study we found that although T. aureoviride isolates were morphologically different when compared to T. harzianum isolates, the UPGMA cluster analysis showed that the majority isolates of T. aureoviride (seven from nine) were closely related to the isolates of T. harzianum.
In this study, we have reported a full length of small heat shock protein 37 (designated MrHSP37) gene, identified from the transcriptome database of freshwater prawn Macrobrachium rosenbergii. The complete gene sequence of the MrHSP37 is 2,425 base pairs in length, and encodes 338 amino acids. MrHSP37 contains a long heat shock protein family profile in the amino acid sequence between 205 and 288. The mRNA expressions of MrHSP37 in healthy and the infectious hypodermal and hematopoietic necrosis virus (IHHNV) challenged M. rosenbergii were examined using quantitative real time polymerase chain reaction (qRT-PCR). MrHSP37 is highly expressed in hepatopancreas and all the other tissues (walking leg, gills, muscle, stomach, haemocyte, intestine, pleopods, brain and eye stalk) of M. rosenbergii taken for analysis. The expression is strongly up-regulated after IHHNV challenge. To understand its biological activity, the recombinant MrHSP37 gene was constructed and expressed in Escherichia coli BL21 (DE3). The results of ATPase assay showed that the recombinant MrHSP37 protein exhibited apparent ATPase activity which increased with the concentration of the protein. And also the purified recombinant MrHSP37 protein was used for thermal aggregation assay (chaperone activity). It showed that the recombinant MrHSP37 protein is an active chaperone in this assay. Taken together, these results suggest that MrHSP37 is potentially involved in the immune responses against IHHNV challenge in M. rosenbergii.
Improving the quality of the non-climacteric fruit, pineapple, is possible with information on the expression of genes that occur during the process of fruit ripening. This can be made known though the generation of partial mRNA transcript sequences known as expressed sequence tags (ESTs). ESTs are useful not only for gene discovery but also function as a resource for the identification of molecular markers, such as simple sequence repeats (SSRs). This paper reports on firstly, the construction of a normalized library of the mature green pineapple fruit and secondly, the mining of EST-SSRs markers using the newly obtained pineapple ESTs as well as publically available pineapple ESTs deposited in GenBank. Sequencing of the clones from the EST library resulted in 282 good sequences. Assembly of sequences generated 168 unique transcripts (UTs) consisting of 34 contigs and 134 singletons with an average length of ≈500 bp. Annotation of the UTs categorized the known proteins transcripts into the three ontologies as: molecular function (34.88%), biological process (38.43%), and cellular component (26.69%). Approximately 7% (416) of the pineapple ESTs contained SSRs with an abundance of trinucleotide SSRs (48.3%) being identified. This was followed by dinucleotide and tetranucleotide SSRs with frequency of 46 and 57%, respectively. From these EST-containing SSRs, 355 (85.3%) matched to known proteins while 133 contained flanking regions for primer design. Both the ESTs were sequenced and the mined EST-SSRs will be useful in the understanding of non-climacteric ripening and the screening of biomarkers linked to fruit quality traits.
Infectious hypodermal and haematopoietic necrosis virus (IHHNV) has been detected widely in penaeid culture facilities in Asia and the Americas. IHHNV infection on sub-adult and postlarvae of the giant freshwater prawn, Macrobrachium rosenbergii which had caused up to 80% mortalities was first reported in Southeast Taiwan in 2006. In Malaysia, although, there has been no report on IHHNV infections in M. rosenbergii, preliminary work suggests that there is an urgent need to setup a screening protocol for IHHNV for both wild and cultured populations. In this study, polymerase chain reaction based screening was carried out on 30 randomly sampled berried wild M. rosenbergii before and after spawning. All samples did not showed any sign of IHHNV infection. However, the results showed that 20% of the samples were IHHNV positive. Sequence analysis of the amplified band using NCBI-BLAST showed that the putative IHHNV sequence had 98% nucleotide sequence (388 bp) identity with the IHHNV isolate AC-05-005 non-structural protein 1 gene and seven other IHHNV strains in the data bank further affirming the suggestion on the presence of IHHNV in wild freshwater prawn populations in Malaysia.
MicroRNAs (miRNAs) are small RNAs (sRNAs) with approximately 21-24 nucleotides in length. They regulate the expression of target genes through the mechanism of RNA silencing. Conventional isolation and cloning of miRNAs methods are usually technical demanding and inefficient. These limitations include the requirement for high amounts of starting total RNA, inefficient ligation of linkers, high amount of PCR artifacts and bias in the formation of short miRNA-concatamers. Here we describe in detail a method that uses 80 μg of total RNA as the starting material. Enhancement of the ligation of sRNAs and linkers with the use of polyethylene glycol (PEG8000) was described. PCR artifacts from the amplification of reverse-transcribed sRNAs were greatly decreased by using lower concentrations of primers and reducing the number of amplification cycles. Large concatamers with up to 1 kb in size with around 20 sRNAs/concatamer were obtained by using an optimized reaction condition. This protocol provide researchers with a rapid, efficient and cost-effective method for the construction of miRNA profiles from plant tissues containing low amounts of total RNA, such as fruit flesh and senescent leaves.
White tail disease (WTD) is a serious viral disease in the hatcheries and nursery ponds of Macrobrachium rosenbergii in many parts of the world. A new disease similar to WTD was observed in larvae and post larvae of M. rosenbergii cultured in Malaysia. In the present study, RT-PCR assay was used to detect the causative agents of WTD, M. rosenbergii nodavirus (MrNV) and extra small virus (XSV) using specific primers for MrNV RNA2 and XSV. The results showed the presence of MrNV in the samples with or without signs of WTD. However, XSV was only detected in some of the MrNV-positive samples. Phylogenetic analysis showed that the RNA2 of our Malaysian isolates were significantly different from the other isolates. Histopathological studies revealed myofiber degeneration of the tail muscles and liquefactive myopathy in the infected prawns. This was the first report on the occurrence of MrNV in the Malaysian freshwater prawn.
The most economically important form of aquaculture is fish farming, which is an industry that accounts for an ever increasing share of world fishery production. Molecular markers can be used to enhance the productivity of the aquaculture and fish industries to meet the increasing demand. Molecular markers can be identified via a DNA test regardless of the developmental stage, age or environmental challenges experienced by the organism. The application of 16s and cytochrome b markers has enabled rapid progress in investigations of genetic variability and inbreeding, parentage assignments, species and strain identification and the construction of high resolution genetic linkage maps for aquaculture fisheries. In this review, the advantages of principles and potential power tools of 16s and cytochrome b markers are discussed. Main findings in term of trend, aspects and debates on the reviewed issue made from the model of aquatic species for the benefit of aquaculture genomics and aquaculture genetics research are discussed. The concepts in this review are illustrated with various research examples and results that relate theory to reality and provide a strong review of the current status of these biotechnology topics.
Teleosts are useful vertebrate model species for understanding copper toxicity due to the dual entry route for copper intake via the gills and intestine. In this present study, we utilized the differential display reverse transcription-polymerase chain reaction to isolate potential novel hepatic genes induced by sublethal copper exposure in the freshwater swordtail fish, Xiphophorus helleri. Full length cloning of a cDNA fragment induced by copper exposure to 1 μg/ml during 24 h resulted in the positive identification of a hepatic ribosomal protein L19 (RPL19) gene. Further characterization of this gene revealed that its transcriptional expression was dependent on dosage and time of copper exposure. This study describes for the first time the involvement of RPL19 in copper toxicity, probably as a result of increase in ribosome synthesis rate to support activities such as cellular protein translation, transcriptional activation and mRNA stabilization during sublethal copper exposure.
Horseshoe crabs are said to be declining worldwide. However, there is still no published report on the status of horseshoe crabs in Malaysia. Thus, we report here eight informative microsatellite markers that were developed using the 5'-anchored ISSR-PCR enrichment procedure to diagnose the population genetic structure of the mangrove horseshoe crab, Carcinoscorpius rotundicauda from Peninsular Malaysia. This set of markers was tested on 127 samples and showed polymorphism in this species. Hence they should be useful in future essential population genetic studies of these living fossils in the Southeast Asian region.
Green microalga Ankistrodesmus convolutus Corda is a fast growing alga which produces appreciable amount of carotenoids and polyunsaturated fatty acids. To our knowledge, this is the first report on the construction of cDNA library and preliminary analysis of ESTs for this species. The titers of the primary and amplified cDNA libraries were 1.1×10(6) and 6.0×10(9) pfu/ml respectively. The percentage of recombinants was 97% in the primary library and a total of 337 out of 415 original cDNA clones selected randomly contained inserts ranging from 600 to 1,500 bps. A total of 201 individual ESTs with sizes ranging from 390 to 1,038 bps were then analyzed and the BLASTX score revealed that 35.8% of the sequences were classified as strong match, 38.3% as nominal and 25.9% as weak match. Among the ESTs with known putative function, 21.4% of them were found to be related to gene expression, 14.4% ESTs to photosynthesis, 10.9% ESTs to metabolism, 5.5% ESTs to miscellaneous, 2.0% to stress response, and the remaining 45.8% were classified as novel genes. Analysis of ESTs described in this paper can be an effective approach to isolate and characterize new genes from A. convolutus and thus the sequences obtained represented a significant contribution to the extensive database of sequences from green microalgae.
An initial study on gene cloning and characterization of unicellular green microalga Ankistrodesmus convolutus was carried out to isolate and characterize the full-length cDNA of ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (RbcS) as a first step towards elucidating the structure of A. convolutus RbcS gene. The full-length of A. convolutus RbcS cDNA (AcRbcS) contained 28 bp of 5' untranslated region (UTR), 225 bp of 3' non-coding region, and an open reading frame of 165 amino acids consisting of a chloroplast transit peptide with 24 amino acids and a mature protein of 141 amino acids. The amino acid sequence has high identity to those of other green algae RbcS genes. The AcRbcS contained a few conserved domains including protein kinase C phosphorylation site, tyrosine kinase phosphorylation site and N-myristoylation sites. The AcRbcS was successfully expressed in Escherichia coli and a ~21 kDa of anticipated protein band was observed on SDS-PAGE. From the phylogenetic analysis of RbcS protein sequences, it was found that the RbcS of A. convolutus has closer genetic relationship with green microalgae species compared to those of green seaweed and green macroalgae species. Southern hybridization analysis revealed that the AcRbcS is a member of a small multigene family comprising of two to six members in A. convolutus genome. Under different illumination conditions, RT-PCR analysis showed that AcRbcS transcription was reduced in the dark, and drastically recovered in the light condition. Results presented in this paper established a good foundation for further study on the photosynthetic process of A. convolutus and other green algae species where little information is known on Rubisco small subunit.
Vanda Mimi Palmer (VMP) is a highly sought as fragrant-orchid hybrid in Malaysia. It is economically important in cosmetic and beauty industries and also a famous potted ornamental plant. To date, no work on fragrance-related genes of vandaceous orchids has been reported from other research groups although the analysis of floral fragrance or volatiles have been extensively studied. An expressed sequence tag (EST) resource was developed for VMP principally to mine any potential fragrance-related expressed sequence tag-simple sequence repeat (EST-SSR) for future development as markers in the identification of fragrant vandaceous orchids endemic to Malaysia. Clustering, annotation and assembling of the ESTs identified 1,196 unigenes which defined 966 singletons and 230 contigs. The VMP dbEST was functionally classified by gene ontology (GO) into three groups: molecular functions (51.2%), cellular components (16.4%) and biological processes (24.6%) while the remaining 7.8% showed no hits with GO identifier. A total of 112 EST-SSR (9.4%) was mined on which at least five units of di-, tri-, tetra-, penta-, or hexa-nucleotide repeats were predicted. The di-nucleotide motif repeats appeared to be the most frequent repeats among the detected SSRs with the AT/TA types as the most abundant among the dimerics, while AAG/TTC, AGA/TCT-type were the most frequent trimerics. The mined EST-SSR is believed to be useful in the development of EST-SSR markers that is applicable in the screening and characterization of fragrance-related transcripts in closely related species.
Rice blast is one of the major fungal diseases that badly reduce rice production in Asia including Malaysia. There is not much information on identification of QTLs as well as linked markers and their association with blast resistance within local rice cultivars. In order to understanding of the genetic control of blast in the F3 families from indica rice cross Pongsu seribu2/Mahsuri, an analysis of quantitative trait loci against one of the highly virulent Malaysian rice blast isolate Magnaporthe oryzae, P5.0 was carried out. Result indicated that partial resistance to this pathotype observed in the present study was controlled by multiple loci or different QTLs. In QTL analysis in F3 progeny fifteen QTLs on chromosomes 1, 2, 3, 5, 6, 11 and 12 for resistance to blast nursery tests was identified. Three of detected QTLs (qRBr-6.1, qRBr-11.4, and qRBr-12.1) had significant threshold (LOD >3) and approved by both IM and CIM methods. Twelve suggestive QTLs, qRBr-1.2, qRBr-2.1, qRBr-4.1, qRBr-5.1, qRBr-6.2, qRBr-6.3, qRBr-8.1, qRBr-10.1, qRBr-10.2, qRBr-11.1, qRBr-11.2 and qRBr-11.3) with Logarithmic of Odds (LOD) <3.0 or LRS <15) were distributed on chromosomes 1, 2, 4, 5, 6, 8, 10, and 11. Most of the QTLs detected using single isolate had the resistant alleles from Pongsu seribu 2 which involved in the resistance in the greenhouse. We found that QTLs detected for deferent traits for the using isolate were frequently located in similar genomic regions. Inheritance study showed among F3 lines resistance segregated in the expected ratio of 15: 1 for resistant to susceptible. The average score for blast resistance measured in the green house was 3.15, 1.98 and 29.95 % for three traits, BLD, BLT and % DLA, respectively.
The importance of plant secondary metabolites for both mankind and the plant itself has long been established. However, despite extensive research on plant secondary metabolites, plant secondary metabolism and its regulation still remained poorly characterized. In this present study, cDNA-amplified fragment length polymorphism (cDNA-AFLP) transcript profiling was applied to generate the expression profiles of Polygonum minus in response to salicylic acid (SA) and methyl jasmonate (MeJA) elicitations. This study reveals two different sets of genes induced by SA and MeJA, respectively where stress-related genes were proved to lead to the expression of genes involved in plant secondary metabolite biosynthetic pathways. A total of 98 transcript-derived fragments (TDFs) were up-regulated, including 46 from SA-treated and 52 from MeJA-treated samples. The cDNA-AFLP transcripts generated using 64 different Mse1/Taq1 primer combinations showed that treatments with SA and MeJA induced genes mostly involved in scavenging reactive oxygen species, including zeaxanthin epoxidase, cytosolic ascorbate peroxidase 1 and peroxidase. Of these stress-related genes, 15 % of other annotated TDFs are involved mainly in secondary metabolic processes where among these, two genes encoding (+)-delta cadinene synthase and cinnamoyl-CoA reductase were highlighted.
Blast disease caused by the fungal pathogen Magnaporthe oryzae is the most severe diseases of rice. Using classical plant breeding techniques, breeders have developed a number of blast resistant cultivars adapted to different rice growing regions worldwide. However, the rice industry remains threatened by blast disease due to the instability of blast fungus. Recent advances in rice genomics provide additional tools for plant breeders to improve rice production systems that would be environmentally friendly. This article outlines the application of conventional breeding, tissue culture and DNA-based markers that are used for accelerating the development of blast resistant rice cultivars. The best way for controlling the disease is to incorporate both qualitative and quantitative genes in resistant variety. Through conventional and molecular breeding many blast-resistant varieties have been developed. Conventional breeding for disease resistance is tedious, time consuming and mostly dependent on environment as compare to molecular breeding particularly marker assisted selection, which is easier, highly efficient and precise. For effective management of blast disease, breeding work should be focused on utilizing the broad spectrum of resistance genes and pyramiding genes and quantitative trait loci. Marker assisted selection provides potential solution to some of the problems that conventional breeding cannot resolve. In recent years, blast resistant genes have introgressed into Luhui 17, G46B, Zhenshan 97B, Jin 23B, CO39, IR50, Pusa1602 and Pusa1603 lines through marker assisted selection. Introduction of exotic genes for resistance induced the occurrence of new races of blast fungus, therefore breeding work should be concentrated in local resistance genes. This review focuses on the conventional breeding to the latest molecular progress in blast disease resistance in rice. This update information will be helpful guidance for rice breeders to develop durable blast resistant rice variety through marker assisted selection.
The mRNA differential display method was utilized to study the differential expression and regulation of genes in two species of oil palm, the commercially grown variety Elaeis guineensis, var. tenera and the South American species, Elaeis oleifera. We demonstrated the differential expression of genes in the mesocarp and kernel at the week of active oil synthesis (15 week after anthesis) during fruit development as compare to the roots and leaves and the isolation of tissue-specific and species-specific cDNA clones. A total of eight specific cDNA clones were isolated and their specificities were confirmed by Northern hybridization and classified into three groups. Group one contains four clones (KT3, KT4, KT5 and KT6) that are kernel-specific for E. guineensis, tenera and E. oleifera. The second group represents clone FST1, which is mesocarp and kernel-specific for E. guineensis, tenera and E. oleifera. The third group represents clones MLT1, MLT2 and MLO1 that are mesocarp and leaf-specific. Northern analysis showed that their expressions were developmentally regulated. Nucleotide sequencing and homology search in GenBank data revealed that clones KT3 and KT4 encode for the same maturation protein PM3. While clones MLT1 and MLT2 encode for S-ribonuclease binding protein and fibrillin, respectively. The other clones (KT5, KT6, FST1 and MLO1) did not display any significant homology to any known protein.
Giant grouper (Epinephelus lanceolatus) is a commercially important species, but its wild population has recently been classified as vulnerable. This species has significant potential for use in aquaculture, though a greater understanding of population genetics is necessary for selective breeding programs to minimize kinship for genetically healthy individuals. High-throughput pyrosequencing of genomic DNA was used to identify and characterize novel tetra- and trinucleotide microsatellite markers in giant grouper from Sabah, Malaysia. In total, of 62,763 sequences containing simple sequence repeats (SSRs) were obtained, and 78 SSR loci were selected to possibly contain tetra- and trinucleotide repeats. Of these loci, 16 had tetra- and 8 had trinucleotide repeats, all of which exhibited polymorphisms within easily genotyped regions. A total of 143 alleles were identified with an average of 5.94 alleles per locus, with mean observed and expected heterozygosities of 0.648 and 0.620, respectively. Among of them, 15 microsatellite markers were identified without null alleles and with Hardy-Weinberg equilibrium. These alleles showed a combined non-exclusion probability of 0.01138. The probability of individual identification (PID) value combined with in descending order 12 microsatellite markers was 0.00008, which strongly suggests that the use of the microsatellite markers developed in this study in various combinations would result in a high resolution method for parentage analysis and individual identification. These markers could be used to establish a broodstock management program for giant grouper and to provide a foundation for genetic studies such as population structure, parentage analysis, and kinship selection.
The marine clam Lutraria rhynchaena is gaining popularity as an aquaculture species in Asia. Lutraria populations are present in the wild throughout Vietnam and several stocks have been established and translocated for breeding and aquaculture grow-out purposes. In this study, we demonstrate the feasibility of utilising Illumina next-generation sequencing technology to streamline the identification and genotyping of microsatellite loci from this clam species. Based on an initial partial genome scan, 48 microsatellite markers with similar melting temperatures were identified and characterised. The 12 most suitable polymorphic loci were then genotyped using 51 individuals from a population in Quang Ninh Province, North Vietnam. Genetic variation was low (mean number of alleles per locus = 2.6; mean expected heterozygosity = 0.41). Two loci showed significant deviation from Hardy-Weinberg equilibrium (HWE) and the presence of null alleles, but there was no evidence of linkage disequilibrium among loci. Three additional populations were screened (n = 7-36) to test the geographic utility of the 12 loci, which revealed 100 % successful genotyping in two populations from central Vietnam (Nha Trang). However, a second population from north Vietnam (Co To) could not be successfully genotyped and morphological evidence and mitochondrial variation suggests that this population represents a cryptic species of Lutraria. Comparisons of the Qang Ninh and Nha Trang populations, excluding the 2 loci out of HWE, revealed statistically significant allelic variation at 4 loci. We reported the first microsatellite loci set for the marine clam Lutraria rhynchaena and demonstrated its potential in differentiating clam populations. Additionally, a cryptic species population of Lutraria rhynchaena was identified during initial loci development, underscoring the overlooked diversity of marine clam species in Vietnam and the need to genetically characterise population representatives prior to microsatellite development. The rapid identification and validation of microsatellite loci using next-generation sequencing technology warrant its integration into future microsatellite loci development for key aquaculture species in Vietnam and more generally, aquaculture countries in the South East Asia region.
Human diploid fibroblasts (HDFs) cultured in vitro have limited capacity to proliferate after population doubling is repeated several times, and they enter into a state known as replicative senescence or cellular senescence. This study aimed to investigate the effect of Chlorella vulgaris on the replicative senescence of HDFs by determining the expression of senescence-associated genes. Young and senescent HDFs were divided into untreated control and C. vulgaris-treated groups. A senescence-associated gene transcription analysis was carried out with qRT-PCR. Treatment of young HDFs with C. vulgaris reduced the expression of SOD1, CAT and CCS (p