Identification and expression analysis of the Broad-Complex core protein isoform 6 (BR-C Z6) gene in the giant tiger shrimp Penaeus monodon (Penaeidae: Decapoda).

Broad-Complex (BR-C) is an early ecdysone-responsive gene encoding a family of zinc-finger transcription factors that function during metamorphosis in insects. We identified two full-length cDNAs of BR-C Z6 in the giant tiger shrimp (Penaeus monodon). They were 2422 and 2060 bp in length, containing open reading frames of 1440 and 1443 bp, corresponding to polypeptides of 479 and 480 amino acids, respectively. Tissue distribution analysis indicated that PmBR-C Z6 was abundantly expressed in hemocytes and ovaries in juveniles. In broodstock, PmBR-C Z6 was constitutively expressed in all tissues examined, and the highest expression was observed in ovaries. The expression of PmBR-C Z6 in ovaries was significantly greater than in testes in both juveniles and broodstock of P. monodon. Quantitative real-time PCR indicated that the expression level of PmBR-C Z6 was significantly down-regulated in stages II and III of ovaries in intact wild broodstock and returned to the basal level in stage IV ovaries and after spawning. In eyestalk-ablated broodstock, PmBR-C Z6 was significantly up-regulated in stage IV (mature) ovaries. Moreover, the expression level of PmBR-C Z6 in vitellogenic, early cortical rod and mature (stages II-IV) ovaries of eyestalk-ablated broodstock was greater than that of the same ovarian stages in intact broodstock. In situ hybridization revealed that PmBR-C Z6 transcripts were localized in oogonia and cytoplasm of previtellogenic and vitellogenic oocytes of both wild intact and eyestalk-abated broodstock. The effects of 20-hydroxyecdysone on expression of PmBR-C Z6 were examined. The expression level of PmBR-C Z6 in ovaries of juvenile P. monodon was significantly increased at 168 h post-injection. Taken together, these findings indicate that PmBR-C Z6 plays an important role in ovarian development of P. monodon.

Genetic variation of Kaempferia (Zingiberaceae) in Thailand based on chloroplast DNA (psbA-trnH and petA-psbJ) sequences.

Genetic variation and species authentication of 71 Kaempferia accessions (representing 15 recognized, six new, and four unidentified species) found indigenously in Thailand were examined by determining chloroplast psbA-trnH and partial petA-psbJ spacer sequences. Ten closely related species (Boesenbergia rotunda, Gagnepainia godefroyi, G. thoreliana, Globba substrigosa, Smithatris myanmarensis, S. supraneanae, Scaphochlamys biloba, S. minutiflora, S. rubescens, and Stahlianthus sp) were also included. After sequence alignments, 1010 and 865 bp in length were obtained for the respective chloroplast DNA sequences. Intraspecific sequence variation was not observed in Kaempferia candida, K. angustifolia, K. laotica, K. galanga, K. pardi sp nov., K. bambusetorum sp nov., K. albomaculata sp nov., K. minuta sp nov., Kaempferia sp nov. 1, and G. thoreliana, for which more than one specimen was available. In contrast, intraspecific sequence polymorphisms were observed in various populations of K. fallax, K. filifolia, K. elegans, K. pulchra, K. rotunda, K. marginata, K. parviflora, K. larsenii, K. roscoeana, K. siamensis, and G. godefroyi. A strict consensus tree based on combined psbA-trnH and partial petA-psbJ sequences revealed four major groups of Kaempferia species. We suggest that the genus Kaempferia is a polyphyletic group, as K. candida was distantly related and did not group with other Kaempferia species. Polymorphic sites and indels of psbA-trnH and petA-psbJ can be used as DNA barcodes for species diagnosis of most Kaempferia and outgroup species. Nuclear DNA polymorphism should be examined to determine if there has been interspecific hybridization and chloroplast DNA introgression in these taxa.

Genetic population differentiation of the blue swimming crab Portunus pelagicus (Portunidae) in Thai waters revealed by RAPD analysis.

Genetic diversity and population differentiation of the blue swimming crab, Portunus pelagicus, in Thailand were analyzed by RAPD analysis. One hundred and twelve RAPD fragments were generated from 109 individuals of P. pelagicus using OPA02, OPA14, OPB10, UBC122, and UBC158 primers. The percentage of polymorphic bands in each geographic sample and that of each primer across overall samples were 72.7-85.0 and 92.0-100%, respectively. Large numbers of polymorphic bands found in the RAPD analysis suggested high genetic diversity of Thai P. pelagicus. The mean genetic distance between samples across all primers was 0.0929-0.2471. Significant geographic heterogeneity was observed across samples overall and between all pairs of geographic samples (P < 0.01 for theta and P < 0.0001 for the exact test), indicating strong genetic differentiation of P. pelagicus in Thai waters, despite its high potential of dispersal. Limited gene flow levels (0.44-1.19 individuals per generation) of Thai P. pelagicus were also observed. A fine scale level of differentiation suggested that P. pelagicus from each geographic sample in Thai waters should be regarded as a separate genetic population and treated as a different exploited stock.

Genetic diversity of wild and domesticated stocks of Thai abalone, Haliotis asinina (Haliotidae), analyzed by single-strand conformational polymorphism of AFLP-derived markers.

Amplified fragment length polymorphism (AFLP) analysis was carried out on representative individuals of wild Haliotis asinina using 64 primer combinations. Nine polymorphic AFLPs were cloned and sequenced. Sequence-specific primers were designed from six AFLP-derived fragments. Three sequence-characterized amplified region (SCAR) markers (HaSCAR(320), HaSCAR(295), HaSCAR(327)) were selected for genotyping of 8-month-old domesticated stocks of H. asinina cultured separately at Sichang Marine Science Research and Training Station (N = 95) and at a hatchery in Trang province (N = 40) using single-strand conformational polymorphism analysis. Genotypes of wild abalone originating from Talibong Island (N = 25), Cambodia (N = 22), and the P(0) progeny established from Samet Island founders (N = 20) were also investigated. Significant genetic differentiation (P<0.0001 for the exact test and F(ST) = 0.8759-0.8919, P<0.001) between abalone from the Gulf of Thailand (Cambodia and Samet Island--east) and the Andaman Sea (Talibong Island--west) were observed. This demonstrated the strong biogeographic structure of H. asinina in Thai waters. Non-overlapping composite genotypes for wild abalone from different coastal regions allow us to determine founder contributions in domesticated abalone stocks. Almost all Sichang Marine Science Research and Training Station and the Trang province hatchery stocks exhibited the east coast genotypes (97% of the 135 samples). We suggest that abalone from the east coast population have better survival rates under cultivated conditions than those from the west coast population.

Development of a species-diagnostic marker and its application for population genetics studies of the stingless bee Trigona collina in Thailand.

A molecular maker for authenticating species origin of the stingless bee (Trigona collina) was developed. Initially, amplified fragment length polymorphism analysis was made of 11 stingless bee species using 64 primer combinations. A 316-bp band found only in T. collina was cloned and sequenced. A primer pair (CUTc1-F/R) was designed and tested for species-specificity in 15 stingless bee species (239 nests). The expected 259-bp fragment was consistently amplified in all T. collina individuals (134/134 nests, 100%). Cross-species amplification was observed in T. pagdeni (43/51 nests; 84.3%), but not in other species. SSCP analysis of CUTc1 unambiguously differentiated T. collina from T. pagdeni. CUTc1 generated three genotypes in Thai T. collina (134 nests). An AA (259/259 bp) genotype was found in all stingless bees from the north (21 nests) and northeast (32 nests), and 23/28 nests from the Central region, whereas a BB (253/253 bp) genotype was observed in most samples from peninsular Thailand (42/53 nests). Heterozygotes exhibiting the AB (253/259 bp) genotype were observed in 5 of 28 nests from Prachuap Khiri Khan located slightly above the Kra ecotone and 11 of 53 nests originated further south of the Kra ecotone. Genotype distribution patterns of CUTc1 clearly indicated intraspecific population differentiation of Thai T. collina.

Impact of temporal changes and host factors on the genetic structure of a population of Opisthorchis viverrini sensu lato in Khon Kaen Province (Thailand).

The population genetics of 317 individual Opisthorchis viverrini from Khon Kaen Province Thailand, from 4 different years and 4 cyprinid fish species was examined using multilocus enzyme electrophoresis of enolase (Enol), phosphoglucomutase (Pgm) and triose phosphate isomerase (Tpi). Allele and genotype frequencies for Enol and Pgm were consistent irrespective of year or host species. No heterozygote deficiency was detected for Enol. Significant heterozygote deficiencies were detected in 3 of 4 years for Pgm. For Tpi, allele frequencies of the most common allele and genotype frequency varied between years and among individuals from different host species. Heterozygote deficiencies for Tpi were detected in 2 years. No significant heterozygous deficiencies were detected among O. virerrini from different fish species in 2005, except at Pgm and Tpi from Puntioplites protozsron. There was no statistical significance in pairwise FST values between O. viverrini from Cyclocheilichthys armatus in different years or different host species in 2005. Significant departures from Hardy-Weinberg expectations and a high rate of gene flow in a population of O. viverrini are discussed in terms of self- and cross-fertilisation, natural selection, non-random mating, the Wahlund effect, presence of null alleles, intensity of infection, biology and ecology of their intermediate cyprinid hosts.

Microsatellite and mitochondrial haplotype diversity reveals population differentiation in the tiger shrimp (Penaeus monodon) in the Indo-Pacific region.

The black tiger shrimp (Penaeus monodon) is an ecologically and economically important penaeid species and is widely distributed in the Indo-Pacific region. Here we investigated the genetic diversity of P. monodon (n = 355) from eight geographical regions by genotyping at 10 microsatellite loci. The average observed heterozygosity at various loci ranged from 0.638 to 0.743, indicating a high level of genetic variability in this region. Significant departures from Hardy-Weinberg equilibrium caused by heterozygote deficiency were recorded for most loci and populations. Pairwise F(ST) and R(ST) values revealed genetic differentiation among the populations. Evidence from the assignment test showed that the populations in the West Indian Ocean were unique, whereas other populations examined were partially admixed. In addition, the non-metric multidimensional scaling analysis indicated the presence of three geographic groups in the Indo-Pacific region, i.e. the African populations, a population from western Thailand and the remaining populations as a whole. We also sequenced and analysed the mitochondrial control region (mtCR) in these shrimp stocks to determine whether the nuclear and mitochondrial genomes show a similar pattern of genetic differentiation. A total of 262 haplotypes were identified, and nucleotide divergence among haplotypes ranged from 0.2% to 16.3%. Haplotype diversity was high in all populations, with a range from 0.969 to 1. Phylogenetic analysis using the mtCR data revealed that the West Indian Ocean populations were genetically differentiated from the West Pacific populations, consistent with the microsatellite data. These results should have implications for aquaculture management and conservation of aquatic diversity.

Genetic differentiation of the giant honey bee (Apis dorsata) in Thailand analyzed by mitochondrial genes and microsatellites.

Genetic diversity and population differentiation of the giant honey bee (Apis dorsata) in Thailand were examined. Six PCR-RFLP mitotypes were generated from digestion of the COI-COII, Cytb-tRNA(ser), ATPase6-8, and lrRNA genes with Dra I and Hin fI. Low genetic diversity (h=0.074, pi=0.032%) and a lack of genetic population differentiation between A. dorsata originating from geographically different regions were observed from mtDNA polymorphisms (P > 0.05). In contrast, microsatellite (A14, A24, and A88) polymorphisms revealed a relatively high level of genetic diversity in A. dorsata (H (o)=0.68-0.74, average number of alleles per locus=6.0-9.0). Both A24 and A88 indicated significant population differentiation between bees from the north-to-central region (north, northeast, and central regions), peninsular Thailand, and Samui Island.

Development of polymorphic expressed sequence tag-derived microsatellites for the extension of the genetic linkage map of the black tiger shrimp (Penaeus monodon).

In this study, microsatellite markers were developed for the genetic linkage mapping and breeding program of the black tiger shrimp Penaeus monodon. A total of 997 unique microsatellite-containing expressed sequence tags (ESTs) were identified from 10 100 EST sequences in the P. monodon EST database. AT-rich microsatellite types were predominant in the EST sequences. Homology searching by the blastn and blastx programs revealed that these 997 ESTs represented 8.6% known gene products, 27.8% hypothetical proteins and 63.6% unknown gene products. Characterization of 50 markers on a panel of 35-48 unrelated shrimp indicated an average number of alleles of 12.6 and an average polymorphic information content of 0.723. These EST microsatellite markers along with 208 other markers (185 amplified fragment length polymorphisms, one exon-primed intron-crossing, six single strand conformation polymorphisms, one single nucleotide polymorphism, 13 non-EST-associated microsatellites and two EST-associated microsatellites) were analysed across the international P. monodon mapping family. A total of 144 new markers were added to the P. monodon maps, including 36 of the microsatellite-containing ESTs. The current P. monodon male and female linkage maps have 47 and 36 linkage groups respectively with coverage across half the P. monodon genome.

Molecular taxonomy of cupped oysters (Crassostrea, Saccostrea, and Striostrea) in Thailand based on COI, 16S, and 18S rDNA polymorphism.

Genetic diversity of oysters Crassostrea belcheri (Sowerby, 1871), C. iredalei (Faustino, 1932), Saccostrea cucullata (Born, 1778), S. forskali (Gmelin, 1791), and Striostrea (Parastriostrea) mytiloides (Lamarck, 1819) (Ostreoida, Mollusca) was analyzed by polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) of 16S ribosomal DNA with AcsI, AluI, DdeI, DraI, RsaI, and TaqI, 18S ribosomal DNA with HinfI, and cytochrome oxidase subunit I with AcsI, DdeI and MboI. A total of 54 composite haplotypes were observed. Species-diagnostic markers were specifically found in C. belcheri, C. iredalei, and S. cucullata, but not in S. forskali and Striostrea mytiloides, which shared common composite haplotypes. Neighbor-joining trees constructed from genetic distances between pairs of composite haplotypes and species indicated large genetic differences between Crassostrea and Saccostrea (including Striostrea mytiloides), but closer relationships were observed within each genus. Four groups of unidentified oysters (Crassostrea sp. and Saccostrea sp. groups 1, 2, and 3) were also genetically analyzed. Fixed RFLP markers were found in Crassostrea sp. and Saccostrea sp. group 2, but not in Saccostrea sp. groups 1 and 3. Phylogenetic and genetic heterogeneity analyses indicated that Crassostrea sp. and Saccostrea sp. group 2 should be considered as newly unidentified oyster species in Thailand.

Population structure of tropical abalone (Haliotis asinina) in coastal waters of Thailand determined using microsatellite markers.

Three partial genomic libraries were constructed from genomic DNA of the tropical abalone (Haliotis asinina) that was digested with AluI, vortexed/sonicated, and digested with mixed enzyme (AluI, HincII, and RsaI). The libraries yielded 0.02%, 0.42%, and 1.46% positive microsatellite-containing clones, respectively. Eleven clones each of perfect, imperfect, and compound microsatellites were isolated. Ten primer pairs (CUHas1-CUHas10) were analyzed to evaluate their polymorphic level. The numbers of alleles per locus, observed heterozygosity (H0), and expected heterozygosity (He) ranged from 3 to 26 alleles, and varied between 0.27 and 0.85 and between 0.24 and 0.93, respectively. Three microsatellite loci (CUHas2, CUHas3, and CUHas8) were further used for examination of genetic diversity and differentiation of natural H. asinina in coastal waters of Thailand. Genetic variabilities in terms of the effective number of alleles (n(e)), H0, and He were higher in 2 samples from the Gulf of Thailand (n(e)=9.37, 7.66; H0=0.62, 0.78; and He=0.87, 0.86) than those of one sample (n(e)=6.04; H0=0.58; and He=0.62) derived from the Andaman Sea. Assessment of genetic heterogeneity, including allele frequency comparison and pairwise F(ST) analysis, indicated interpopulational differentiation, between natural H. asinina from the Gulf of Thailand and that from the Andaman Sea (P<0.0001).

Genetic diversity and molecular markers of the tropical abalone (Haliotis asinina) in Thailand.

Genetic diversity of abalone in Thailand, Haliotis asinina, H. ovina, and H. varia, was analyzed by polymerase chain reaction (PCR) of 18S and 16S rDNAs, with randomly amplified polymorphic DNA (RAPD) and restriction fragment length polymorphism (RFLP). Species-specific RAPD markers were found in each abalone species. Restriction analysis of 18S (nuclear) ribosomal DNA with Alu I, Taq I, and Hae III and 16S (mitochondrial) rDNA with Bam HI, Eco RI, Hae III, and Alu I gave 12 and 13 digestion patterns, respectively. A total of 49 composite haplotypes were found. A dendogram obtained by the unweighted pair-group method with arithmetic mean, constructed from divergence between pairs of composite haplotypes, revealed reproductively isolated gene pools of these abalone and indicated that H. asinina and H. ovina are genetically closer than H. varia. When H. varia was discovered owing to small sample sizes, geographic heterogeneity analysis and FST estimate indicated clear genetic differentiation between H. ovina originating from the Andaman Sea (west) and the Gulf of Thailand (east, P<0.0001), whereas partial differentiation was observed between the Philippines and the remaining H. asinina samples (P<0.0021). The amplified 16S rDNAs of individuals representing composite haplotypes found in this study were cloned and sequenced. A neighbor-joining tree constructed from sequence divergence of 16S rDNA accurately allocated those sequences according to species origins of abalone. Species-specific PCR based on 16S rDNA polymorphism was successfully developed in H. asinina and H. varia but not in H. ovina.

Molecular genetic identification tools for three commercially cultured oysters (Crassostrea belcheri, Crassostrea iredalei, and Saccostrea cucullata) in Thailand.

Molecular genetic keys for identification of 3 commercially cultured oysters (Crassostrea belcheri, Crassostrea iredalei, and Saccostrea cucullata) in Thailand were developed based on restriction analysis of 18S ribosomal DNA and cytochrome oxidase subunit I (COI). Digestion of the amplified 18S rDNA with Hinf I unambiguously differentiated Crassostrea oysters from Saccostrea oysters and Striostrea (Parastriostrea) mytiloides. In addition, species-specific restriction fragment length polymorphism patterns of C. belcheri, C. iredalei, and S. cucullata were consistently observed when the gel-eluted COI was digested with Mbo I and Dde I. Thirty composite haplotypes were observed across all individuals. Species-specific composite haplotypes were found in C. belcheri (AAAA and AAAB), C. iredalei (AABC and AABU), and S. cucullata (BBCD and BBCE), respectively. The most common composite haplotype of COI in C. belcheri (AAAA), C. iredalei (AABC), and S. cucullata (BBCD) was amplified, cloned, and sequenced. Detection of C. belcheri and C. iredalei based on polymerase chain reaction was further developed using more specific primers (HCO2198 and R372) followed by digestion of a 372-bp product with Mbo I.

Genetic heterogeneity of the giant tiger shrimp (Penaeus monodon) in Thailand revealed by RAPD and mitochondrial DNA RFLP analyses.

Genetic diversity of the giant tiger shrimp (Penaeus monodon) collected from 5 areas, Chumphon and Trat (Gulf of Thailand), and Phangnga, Satun, and Trang (Andaman Sea), was examined by randomly amplified polymorphic DNA (RAPD) and mitochondrial DNA (16S ribosomal DNA and an intergenic COI-COII) polymorphism. A total of 53 polymorphic fragments from UBC299, UBC273, and UBC268 was consistently scored across all samples. From the respective primers 26, 32, and 30 genotypes were generated. A 260-bp RAPD fragment generated by the primer UBC268 was specifically observed in 95.8% of Trat P. monodon, suggesting that this RAPD could be used as a marker for comparing phenotypic performance of P. monodon from Trat and other geographic samples. In addition, 37 mtDNA composite haplotypes were observed from restriction analysis of the same P. monodon samples. High haplotype diversity (0.855) and nucleotide diversity (3.328%) of Thai P. monodon were observed. Population differentiation of P. monodon between the Andaman Sea and Gulf of Thailand was clearly illustrated by both techniques (P <.0001). Nevertheless, contradictory results on patterns of differentiation were observed between P. monodon within the Gulf of Thailand. Analysis of nuclear DNA polymorphism (RAPD) indicated a genetically significant difference between Chumphon and Trat ( P <.0001), whereas mtDNA polymorphism did not show differentiation between these samples (P =.0497). Under the presumption of selective neutrality of these markers, biased female gene flow between Trat and Chumphon P. monodon may exist and be responsible for an anomalous differentiation pattern between these geographic samples.

Genetic diversity and molecular markers of cupped oysters (genera Crassostrea, Caccostrea, and Striostrea) in Thailand revealed by randomly amplified polymorphic DNA analysis.

Genetic diversity and species-diagnostic markers of 5 oysters in Thailand, Crassostrea belcheri (Sowerby, 1871), Crassostrea iredalei (Faustino, 1932), Saccostrea cucullata (Born, 1778), Saccostrea forskali (Gmelin, 1791), and Striostrea Parastriostrea) mytiloides (Lamarck, 1819), were investigated by randomly amplified polymorphic DNA (RAPD) analysis. In a total, 135, 127, and 108 genotypes were observed from primers OPA09, OPB01, and OPB08 (Operon Technologies Inc., kits A and B), and 131 and 122 genotypes from primers UBC210 and UBC220 (University of British Columbia), respectively. Two hundred fifty-four reproducible and polymorphic fragments (200-2500 bp in length) were generated across the 5 investigated species. The average number of bands per primer varied between 12.4 and 32.2. The percentage of polymorphic bands within Crassostrea (53.23%-77.67%) was lower than that within Saccostrea and Striostrea oysters (86.21%-99.36%). Nine, species-specific markers were found in C. belcheri, 4 in C. iredalei, and 2 in S. cucullata. The mean of a ratio between the number of genotypes generated by each primer and the number of investigated specimens of C. belcheri (0.58) was lower than that of the remaining species (0.90-1.00). Genetic distances between pairs of oyster samples were between 0.105 and 0.811. A neighbor-joining tree indicated distant relationships between Crassostrea and Saccostrea oysters, but closer relationships were observed between the latter and Striostrea mytiloides.

Microsatellite Polymorphism and the Population Structure of the Black Tiger Shrimp (Penaeus monodon) in Thailand.

Genetic variation and differentiation of Thai Penaeus monodon from five geographic locations (Chumphon, Trad, Phangnga, Satun, and Trang) were investigated using five microsatellite loci (CUPmo18, Di25, Di27, CSCUPmo1, and CSCUPmo2). The number of alleles across the five loci ranged from 19 to 30, and heterozygosities ranged from 0.49 to 0.95. The mean number of alleles and effective number of alleles per locus were 21.0 to 26.6 and 13.1 to 20.4, respectively. The average heterozygosity across all investigated samples was 0.78, indicating high genetic diversity in this species. Geographic heterogeneity analysis of the results from two of the loci, CUPmo18 and Di25, showed significant differences among the Gulf of Thailand (Trad and Chumphon) but not the Andaman samples. Comparison between regions revealed significant heterogeneity of the Andaman and Trad P. monodon (P <.001), whereas those from Chumphon and the Andaman were genetically similar (P >.05). Significant genetic differentiation was consistently observed between the Andaman-Trad samples (F(ST) = 0.0101, P <.0001) and the Chumphon-Trad samples (F(ST) = 0.0101, P <.0001). On the basis of our analyses, the investigated samples from five geographic locations were allocated to three distinct populations composed of the Andaman Sea (A), Chumphon (B), and Trad (C).

Genetic Diversity and Species-Diagnostic Markers of Mud Crabs (Genus Scylla) in Eastern Thailand Determined by RAPD Analysis.

Genetic diversity of three mud crab species, Scylla serrata (Forskål), S. oceanica (Dana), and S. tranquebarica (Fabricius), collected from two locations in eastern Thailand (Chanthaburi and Trat) was examined by randomly amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR). Ninety-one reproducible RAPD fragments, generated by UBC456, UBC457, and YNZ22, were polymorphic. The percentage of polymorphic bands within populations ranged from 47.92% to 77.59%. Species-specific RAPD markers were also observed and used to construct a molecular diagnostic key in these taxa. Large genetic differences between species were found (D(ij) = 0.425 to 0.751), whereas those between populations within each species were much lower (D(ij) = 0.171 to 0.199). The neighbor-joining tree based on genetic distances among pairs of individuals indicated three distinct groups, corresponding to S. serrata, S. oceanica, and S. tranquebarica. No genotypes were shared among these three species. This suggests the absence of genetic exchanges between sympatric mud crab species in eastern Thailand. Therefore, mud crabs in this area should be recognized as three different species rather than a single panmictic species exhibiting different morphs.

Development of Species-Specific Markers of the Tropical Oyster (Crassostrea belcheri) in Thailand.

Randomly amplified polymorphic DNA (RAPD) analysis was used to identify species-specific markers of 5 oyster species in Thailand: Crassostrea belcheri, Crassostrea iredalei, Saccostrea cucullata, Saccostrea forskali, and Striostrea (Parastriostrea) mytiloides. Species-specific markers were found in C. belcheri, C. iredalei, and S. cucullata but not in S. forskali and S. mytiloides. Three C. belcheri-specific RAPD fragments were cloned and sequenced. A primer set was designed from each of the recombinant clones (pPACB1, pPACB2, and pPACB3). The polymerase chain reaction products showed expected sizes of 536, 600, and 500 bp, respectively, with the sensitivity of detection approximately 30 pg of C. belcheri total DNA template. The specificity of pPACB1 was examined against 135 individuals of indigenous oyster species in Thailand and against outgroup references S. commercialis (N = 12) and Perna viridis (N = 12). Results indicated the species-specific nature of primers developed from pPACB1. This primer set can be used for broodstock selection and determination of C. belcheri larvae to assist the selective breeding program for this commercially important species.

Mitochondrial DNA Diversity in Three Populations of the Giant Tiger Shrimp Penaeus monodon.

: Mitochondrial DNA restriction fragment length polymorphism (mtDNA-RFLP) was utilized for determination of genetic variation and population structure in Penaeus monodon collected from Satun (the Andaman Sea) and Surat and Trat (the Gulf of Thailand). Twenty-eight composite haplotypes were generated from 52 restriction profiles of P. monodon mtDNA digested with 11 restriction endonucleases. The size of the entire P. monodon mitochondrial genome was estimated to be 15.913 +/- 0.177 kb. The average haplotype diversity in P. monodon was 0.864, whereas the mean nucleotide diversity within populations was 2.51%, 2.22%, and 1.91% for Satun, Trat, and Surat, respectively. Geographic heterogeneity analysis indicated population differentiation between P. monodon from the Andaman Sea and P. monodon from the Gulf of Thailand (p <.0001). On the basis of the high genetic diversity level of P. monodon in Thailand, the Satun and Trat P. monodon populations from the west and east of the pennisula were selected to be founder stocks in our selective breeding program.

A preliminary study of ribosomal DNA polymorphism in the tiger shrimp, Penaeus monodon.

The preliminary study of rDNA polymorphisms in P. monodon showed that inter- and intraindividual polymorphisms of rDNA were clearly observed in this species. Individual-specific rDNA restriction patterns were observed when digested with BamHI and SacI. The intergenic spacers (IGS) region of P. monodon rDNA plays an important role in length heteroplasmy at both between- and within-individual levels in this species.