Development of 16 polymorphic simple sequence repeat markers for Lycoris longituba from expressed sequence tags

Lycoris longituba is a tulip-like ornamental plant in China. We report on the data mining of L. longituba expressed sequence tags (ESTs) to generate simple sequence repeat (SSRs) markers. Eighteen EST-SSRs were isolated and validated for 32 individuals. These markers will be valuable for studying the genetic structure and diversity of populations for L. longituba.     

Major viral diseases of the black tiger prawn (Penaeus monodon) in Thailand

There are five different viruses which are currently being studied for their impact on commercial farming of the black tiger prawn (Penaeus monodon) in Thailand. Some of these viruses cause disease in other penaeid shrimp species and even other crustacean species. Some occur not only in cultivated shrimp in other Asian countries, but also in those from Australia and the western hemisphere. In descending order from greatest to least economic impact on the Thai shrimp industry, the five viruses are: white-spot baculovirus, yellow-head virus, hepatopancreatic parvo-like virus, infectious hypodermal and hematopoeitic necrosis virus and monodon baculovirus. The purpose of this review is to summarize recent work on these viruses and to suggest future directions of research that may be useful in the effort to develop a sustainable shrimp industry.     

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.     

Fifteen polymorphic simple sequence repeat markers from expressed sequence tags of Liriodendron tulipifera

We developed and evaluated simple sequence repeat (SSR) markers derived from expressed sequence tags (ESTs) of Liriodendron tulipifera. Characteristics of 15 EST‐SSR loci were investigated using 33 L. tulipifera individuals. The number of alleles per locus ranged from two to five. The expected and observed heterozygosities ranged from 0.216 to 0.751 and from 0.182 to 0.97, respectively. These loci were further tested for their cross‐species transferability to Liriodendron Chinense. Because of their high level of polymorphism and transferability, our 15 single‐locus EST‐SSR markers will be valuable tools for research on mating system, population genetics and systemic evolution of Liriodendron.     

Identification of abundant and informative microsatellites from shrimp (Penaeus monodon) genome

Microsatellites were isolated from P. monodon genomic libraries by direct sequencing of recombinant clones without probe screening. Forty-nine out of 83 clones sequenced contained 99 microsatellite arrays of three or more repeats. When five or more and ten or more repeats were considered, 28 and 14 microsatellites were detected, respectively. The 99 microsatellites were classified as perfect (75%), imperfect (6%), compound perfect (3%) and compound imperfect (16%). The abundance of di-, tri-, tetra- and hexanucleotide repeats were 67%, 20%, 9% and 3%, respectively. The dinucleotide repeats included 36 (CT)n, 31 (GT)n, 17(AT)n and 3 (CG)n. One octanucleotide repeat (ATTTATTC)5 was found within a large repeat sequence. Optimal annealing temperatures were determined for PCR using 11 primer sets encompassing 15 microsatellites. Ten primer sets provided successful amplifications with allele sizes generally ranging from 139 to 410 bp. All these primers amplified polymorphic loci with PIC values ranging from 0.63 to 0.96. Two primer sets amplified additional bands which can easily be distinguished from the bands of the main locus. Three out of 10 P. monodon microsatellites also amplified alleles in P. vannamei. The abundance and informative nature of P. monodon microsatellites and their potential for cross-species amplification make them useful for genetic studies.     

Alteration of egg activation by protease inhibitors in the black tiger shrimp,Penaeus monodon

In penaeoid shrimp, contact of spawned eggs with seawater induces egg activation. However, little is known about the factors that influence egg activation in Penaeus monodon. Therefore, the main objective of the present study was to determine whether shrimp-produced proteases that are released in seawater are essential for egg activation. Female shrimp were allowed to spawn in artificial seawater containing protease inhibitors. It was shown that 4-amidinophenylmethanesulfonyl fluoride hydrochloride (APMSF) and soybean trypsin inhibitor (SBTI) inhibited egg activation. High doses of APMSF and SBTI induced only 1–2% complete egg activation. Moreover, when the APMSF- and SBTI- treated eggs were subsequently washed, egg activation did not resume. In contrast, other protease inhibitors, pepstatin A, E-64, and ethylene glycol tetraacetic acid, did not inhibit egg activation, as evident by approximately 98% complete activation. Our results suggest that serine proteases, which are most likely trypsin-like proteases, released in seawater may be involved in egg activation of P. monodon.     

Isolation and characterization of 23 polymorphic microsatellite markers for diversity and stock analysis in tiger shrimp (Penaeus monodon)

The tiger shrimp (Penaeus monodon) is an important marine crustacean in terms of biological diversity and aquaculture resource. The shrimp is widespread across the Indo‐Pacific region and shows apparent genetic differentiation among geographical populations. It is common practice to transport female brooders between different countries to seed the shrimp farms, posing potential problems of unwanted population admixture. We developed 23 polymorphic microsatellites for P. monodon (average H_E = 0.936) and these microsatellites were applicable for studying population differentiation, identifying valid stocks and tagging nonindigenous farmed shrimps.     

Molecular cloning of the black tiger shrimp (Penaeus monodon) elongation factor 2 (EF-2): sequence analysis and its expression on the ovarian maturation stage

The techniques of homology cloning and anchored PCR were used to clone the elongation factor 2 (EF-2) gene from black tiger shrimp (Penaeus monodon). The full length cDNA of black tiger shrimp EF-2 (btsEF-2) contained a 5' untranslated region (UTR) of 73 bp, an ORF of 2541 bp encoding a polypeptide of 846 amino acids with an estimated molecular mass of 95 kDa, and a 3( UTR of 112 bp. The searches for protein sequence similarities with BLAST analysis indicated that the deduced amino acid sequence of btsEF-2 was homological to the EF-2 of other species and even the mammalians. The conserved signature sequence of EF-2 gene family, GTPase effector domain and ADP-ribosylation domain were found in the btsEF-2 deduced amino acid sequence. The temporal expressions of gene in the different ovarian stages were measured by real time PCR. The mRNA expressions of the gene were constitutively expressed in ovary and different during the maturation stages. The result indicated that EF-2 gene was constitutively expressed and could play a critical role in the ovarian maturation stage.     

Diversity and characterization of oxytetracycline-resistant bacteria associated with non-native species, white-leg shrimp (Litopenaeus vannamei), and native species, black tiger shrimp (Penaeus monodon), intensively cultured in Thailand

Aims: This study aimed at surveying prevalence of oxytetracycline (OTC)‐resistant bacteria in the white‐leg shrimp Litopenaeus vannamei, and the black tiger shrimp Penaeus monodon, intensively cultured in Thailand. We investigated the phylogenetic diversity of the bacterial isolates, as well as the minimum inhibitory concentration (MIC) of OTC, the occurrence of major OTC‐resistant genes and multiple‐antibiotic resistance in the isolates. Methods and Results: Shrimps were collected from culture ponds, and the homogenates of whole bodies were plated on tryptic soy agar supplemented with or without OTC. Percentages of OTC‐resistant bacteria were 0·3–52·1% in white‐leg samples and 0·008–22·3% in black tiger samples. Analyses of 16S rDNA sequences indicated that most OTC‐resistant isolates were closely related to Aeromonas spp. and Lactococcus garvieae. MICs of OTC were 4–128 μg ml^?1 in the OTC‐resistant aeromonads and 128–256 μg ml^?1 in OTC‐resistant L. garvieae. OTC resistance was found to be conferred by the genes tet(A), tet(C), tet(D), tet(E), tet(M) and tet(S), detected either singly or in pairs. No resistance to ceftazidime, imipenem or chloramphenicol was observed in any isolate. Conclusions: Both species of shrimp are associated with OTC‐resistant bacteria, occasionally at high densities exceeding 10⁶ cfu g^?1. The associated bacteria, predominantly Lactococcus and Aeromonas genera, are potential pathogens and are reservoirs of a variety of OTC‐resistant genes. Significance and Impact of the Study: Cultured shrimps can be vehicle to carry OTC‐resistant bacteria to domestic and foreign consumers via the food chain. Very low populations of OTC‐resistant bacteria observed in the several ponds suggest that levels of the resistant bacteria are artificially high and should be reduced in farmed shrimps.     

Construction of integrated genetic linkage maps of the tiger shrimp (Penaeus monodon) using microsatellite and AFLP markers

The linkage maps of male and female tiger shrimp (P. monodon) were constructed based on 256 microsatellite and 85 amplified fragment length polymorphism (AFLP) markers. Microsatellite markers obtained from clone sequences of partial genomic libraries, tandem repeat sequences from databases and previous publications and fosmid end sequences were employed. Of 670 microsatellite and 158 AFLP markers tested for polymorphism, 341 (256 microsatellite and 85 AFLP markers) were used for genotyping with three F₁ mapping panels, each comprising two parents and more than 100 progeny. Chi‐square goodness‐of‐fit test (χ²) revealed that only 19 microsatellite and 28 AFLP markers showed a highly significant segregation distortion (P < 0.005). Linkage analysis with a LOD score of 4.5 revealed 43 and 46 linkage groups in male and female linkage maps respectively. The male map consisted of 176 microsatellite and 49 AFLP markers spaced every ～11.2 cM, with an observed genome length of 2033.4 cM. The female map consisted of 171 microsatellite and 36 AFLP markers spaced every ～13.8 cM, with an observed genome length of 2182 cM. Both maps shared 136 microsatellite markers, and the alignment between them indicated 38 homologous pairs of linkage groups including the linkage group representing the sex chromosome. The karyotype of P. monodon is also presented. The tentative assignment of the 44 pairs of P. monodon haploid chromosomes showed the composition of forty metacentric, one submetacentric and three acrocentric chromosomes. Our maps provided a solid foundation for gene and QTL mapping in the tiger shrimp.     

Development of polymorphic markers from expressed sequence tags of Manihot esculenta Crantz

In this study, 49 primers were designed from sequences containing di-, tri-, tetra-, penta- and hexanucleotide motifs with a minimum of four repeats and presence of motif size polymorphisms (insertion/deletion) from cassava (Manihot esculenta Crantz) expressed sequence tags deposited in public sequence database. Each locus was subsequently screened on 29 M. esculenta Crantz obtained from 15 different countries. Cross-amplification was tested with M. esculenta Crantz (ssp. flabellifolia) and four different Manihot species, M. chlorosticta, M. carthaginensis, M. filamentosa and M. tristis. Of these, nine loci showed polymorphic profiles within M. esculenta Crantz, which revealed two to four alleles per locus. The average unbiased and direct count heterozygosities were 0.4901 and 0.5674, respectively.     

Photobacterium damselae ssp. damselae associated with diseased black tiger shrimp Penaeus monodon Fabricius in India

AIM: To characterize and identify Photobacterium damselae ssp. damselae present in black gill diseased Penaeus monodon collected from east coast of India. METHODS AND RESULTS: Photobacterium damselae ssp. damselae was isolated from hepatopancreas, muscles and gills by using the thiosulfate citrate bile salts sucrose agar supplemented with 1.5% NaCl (TCBS-1) medium. A total of 32 Ph. damselae ssp. damselae isolates were studied together with two reference strains. The biochemical tests and analysis of ureC and 16S rRNA genes confirmed the phenotypic characterization of the isolates as Ph damselae ssp. damselae. Experimental infection studies revealed that the LD50 values of P. monodon and P. indicus ranged from 2x10(3) to 5x10(5) CFU per shrimp and from 4x10(2) to 2x10(4) CFU per shrimp, respectively. CONCLUSIONS: Photobacterium damselae ssp. damselae was found in the internal organs of P. monodon and it showed pathogenic to shrimp. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study on the Ph. damselae ssp. damselae present in the black gill diseased P. monodon in India and therefore might serve as a basis for future studies and diagnosis purpose to shrimp culturists.     

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.     

Control of pathogenic Vibrio spp. by Bacillus subtilis BT23, a possible probiotic treatment for black tiger shrimp Penaeus monodon

AIMS: The present study evaluated the in vitro and in vivo antagonistic effect of Bacillus against the pathogenic vibrios. METHODS AND RESULTS: Cell-free extracts of Bacillus subtilis BT23 showed greater inhibitory effects against the growth of Vibrio harveyi isolated by agar antagonism assay from Penaeus monodon with black gill disease. The probiotic effect of Bacillus was tested by exposing shrimp to B. subtilis BT23 at a density of 106-108 cfu ml-1 for 6 d before a challenge with V. harveyi at 103-104 cfu ml-1 for 1 h infection. The combined results of long- and short-term probiotic treatment of B. subtilis BT23 showed a 90% reduction in accumulated mortality. CONCLUSIONS: This study reports that pathogenic vibrios were controlled by Bacillus under in vitro and in vivo conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: Results indicated that probiotic treatment offers a promising alternative to the use of antibiotics in shrimp aquaculture.     

High-density linkage maps and sex-linked markers for the black tiger shrimp (Penaeus monodon)

We report on the construction of sex-specific high-density linkage maps and identification of sex-linked markers for the black tiger shrimp (Penaeus monodon). Overall, we identified 44 male and 43 female linkage groups (2n = 88) from the analysis of 2,306 AFLP markers segregating in three full-sib families, covering 2,378 and 2,362 cM, respectively. Twenty-one putatively homologous linkage groups, including the sex-linkage groups, were identified between the female and male linkage maps. Six sex-linked AFLP marker alleles were inherited from female parents in the three families, suggesting that the P. monodon adopts a WZ-ZZ sex-determining system. Two sex-linked AFLP markers, one of which we converted into an allele-specific assay, confirmed their association with sex in a panel of 52 genetically unrelated animals.     

Identification and characterization of simple sequence repeat markers from a glandular Origanum vulgare expressed sequence tag

Oregano (Origanum vulgare) and marjoram (Origanum majorana) are two sensorial distinct spices within the genus Origanum (Lamiaceae). Simple sequence repeat (SSR) markers were developed from expressed sequence tags (ESTs) of essential oil glands of O. vulgare. Thirteen EST-SSR loci were evaluated using 20 individual plants of O. vulgare and 19 plants of Origanum majorana. The number of alleles per locus ranged from one to four. All loci developed from O. vulgare successfully cross-amplified in O. majorana.     

Study of simple sequence repeat (SSR) markers from wheat expressed sequence tags (ESTs)

The increasing availability of expressed sequence tags (ESTs) in wheat (Triticum aestivum) and related cereals provides a valuable resource of non-anonymous DNA molecular markers. We examined 170,746 wheat ESTs from the public (International Triticeae EST Cooperative) and Génoplante databases, previously clustered in contigs, for the presence of di- to hexanucleotide simple sequence repeats (SSRs). Analysis of 46,510 contigs identified 3,530 SSRs, which represented 7.5% of the total number of contigs. Only 74% of the sequences allowed primer pairs to be designed, 70% led to an amplification product, mainly of a high quality (68%), and 53% exhibited polymorphism for at least one cultivar among the eight tested. Even though dinucleotide SSRs were less represented than trinucleotide SSRs (15.5% versus 66.5%, respectively), the former showed a much higher polymorphism level (83% versus 46%). The effect of the number and type of repeats is also discussed. The development of new EST-SSRs markers will have important implications for the genetic analysis and exploitation of the genetic resources of wheat and related species and will provide a more direct estimate of functional diversity.     

Tissue-Specific Expressed Sequence Tags from the Black Tiger Shrimp Penaeus monodon

Expressed sequence tag data were generated from complementary DNA libraries created from cephalothorax, eyestalk, and pleopod tissue of the black tiger shrimp (Penaeus monodon). Significant database matches were found for 48 of 83 nuclear genes sequenced from the cephalothorax library, 22 of 55 nuclear genes from the eyestalk library, and 6 of 13 nuclear genes from the pleopod library. The putative identities of these genes reflected the expected tissue specificity. For example, genes for digestive enzymes were identified from the cephalothorax library and genes involved in the visual and neuroendocrine system from the eyestalk library. A few sequences matched anonymous EST or genomic sequences, and others contained mini-satellite or microsatellite repeat sequences. The remainder, 31 from the cephalothorax library, 25 from the eyestalk library, and 5 from the pleopod library, were sequences of high nucleotide complexity with no matches in any database searched and thus may represent novel genes. Received February 12, 1999; accepted April 13, 1999