Isolation and characterization of microsatellite markers in Ruditapes philippinarum.

Ruditapes philippinarum is considered a commercially valuable species, which is commonly found in tidal flats along West Pacific coasts. In China, it is mainly distributed in the southeast sea. In this study, 16 novel microsatellite loci from the R. philippinarum genome were developed, using the protocol of fast isolation by amplified fragment length polymorphism of sequence containing repeats. Thirty-two wild-caught individuals were used to evaluate the degree of polymorphism of these markers. Our results show that there were 10 polymorphic loci and 6 monomorphic loci. The number of alleles per locus and the polymorphism information content ranged from 2 to 6 and from 0.199 to 0.751, respectively. The observed and expected heterozygosities varied from 0.1333 to 0.6207 and 0.1603 to 0.7412, respectively. Of all loci, only one locus was found to deviate significantly from Hardy-Weinberg equilibrium after Bonferroni correction. The loci identified here will provide useful information for future population genetic studies of R. philippinarum.

Isolation and characterization of eight novel microsatellite markers in Acanthopagrus schlegelii.

Acanthopagrus schlegelii is a warm temperate demersal fish, which inhabits the sediment substrate or rocky reefs in shallow seas. As this fish is a nutritionally endowed species with good palatability, it is a highly valuable commercial species for aquaculture and has a long historical standing in Western Pacific countries. Because the population of this fish is currently declining in China, studies and measures aimed at addressing this decline are needed. In this study, eight microsatellite markers were screened from 30 wild A. schlegelii fishes through the FIASCO method, whereby sequences containing repeats were obtained from amplified fragment length polymorphisms. The allelic number ranged from 3 to 5, with a mean number of 3.625. The average observed heterozygosity was 0.6290, ranging from 0.3214 to 0.8966, while the expected heterozygosity was 0.5435, ranging from 0.3452 to 0.6721. The value for polymorphism information content ranged from 0.313 to 0.666. These results show this population has moderate genetic variation and low genetic diversity. These novel polymorphic loci will be useful for future genetic studies of A. schlegelii.

Isolation and characterization of novel microsatellite markers in Mercenaria mercenaria.

Mercenaria mercenaria, also known as the hard clam, is widely distributed in the coastal waters of temperate and tropical areas in the Asian Pacific region. This species is widely popular in the international market, especially in the United States, Europe, and other Western countries, because of its high protein value, taste, and simple farming requirements. In this study, 17 novel microsatellite loci from the M. mercenaria genome were developed using the fast isolation by amplified fragment length polymorphism of sequences containing repeats protocol. Thirty-two wild individuals were used to evaluate the degree of polymorphism of these markers. Results indicated that there were 11 polymorphic loci and six monomorphic loci, and the number of alleles per locus and the polymorphism information content ranged from two to six and from 0.059 to 0.498, respectively. The observed and expected heterozygosity varied from 0.0625 to 0.5333 and 0.0615 to 0.4977, respectively. The Y1-4 locus deviated significantly from Hardy-Weinberg equilibrium (HWE) after Bonferroni correction was applied, while the other loci were in HWE. These loci will provide useful information for M. mercenaria population genetic studies.

Isolation and characterization of new microsatellite loci in Fenneropenaeus penicillatus.

Fenneropenaeus penicillatus is one of the major economic shrimp species in China. In this study, 14 novel microsatellite loci were developed using the fast isolation protocol with amplified fragment length polymorphism of sequences containing repeats (FIASCO). Polymorphisms were tested in 30 individuals from a single-wild population. The results showed that the number of alleles at each locus ranged from two to four, and the polymorphism information content varied from 0.314 to 0.692. The observed and expected heterozygosities ranged from 0.3343 to 0.6542 and from 0.3458 to 0.6657, respectively. Three loci deviated significantly from Hardy-Weinberg equilibrium after a Bonferroni correction was applied, while no deviations were detected in the other 11 loci. The new microsatellite loci identified in this study could be useful in future F. penicillatus population genetic, conservation research, population structure assessment, and linkage map construction studies.

Development of novel polymorphic microsatellite markers in Siganus fuscescens.

Rabbitfish, Siganus fuscescens, is widely distributed in the Indo-Pacific regions and eastern Mediterranean. Its dwelling place includes reef flats, coral reef regions, and seagrass meadows in tropical area and reef areas or shallow waters in locations at high latitudes. In the present study, 10 new polymorphic microsatellite markers were screened from 30 wild S. fuscescens individuals, using a method of fast isolation protocol and amplified fragment length polymorphism of sequences containing repeats. The number of polymorphic alleles per locus was 3 to 5 with a mean of 4.3, while the value of polymorphic information content ranged from 0.283 to 0.680. The values of the observed and expected heterozygosities were in the range 0.3333-0.8462 and 0.3011-0.7424, respectively. Deviation from Hardy-Weinberg equilibrium was not observed in this study. These polymorphic loci are expected to be effective in evaluating the genetic diversity, population structure, and gene flow and in determining the paternity in S. fuscescens, as well as for conservation management.

Isolation and characterization of microsatellite markers in Atrina vexillum Born.

Atrina vexillum Born is an economically valuable species, widely distributed in the coastal waters of temperate and tropical areas of the Asia Pacific region. Twenty one novel microsatellite loci were identified in the genome of A. vexillum Born using the protocol for fast isolation by amplified fragment length polymorphism of sequence containing repeats. Thirty-two wild type individuals were used to evaluate the degree of polymorphism of these markers. We identified 13 polymorphic and 8 monomorphic loci with the number of alleles per locus and the polymorphism information content ranging from 2 to 5 and 0.141 to 0.664, respectively. The observed and expected heterozygosity varied from 0.1250 to 0.7000 and 0.1223 to 0.6216, respectively. Two loci deviated significantly from Hardy-Weinberg equilibrium (HWE) after Bonferroni correction, whereas the other loci were in HWE. These loci are expected to provide useful information for population genetic studies of A. vexillum Born.

Screening and characterization of novel microsatellite loci in Lateolabrax japonicus.

Lateolabrax japonicus, an economically important species, is widely consumed in the offshore coasts of China, Korea, and Japan. We identified 10 new L. japonicus microsatellite markers, using a modified protocol of fast isolation by AFLP of sequences containing repeats. Thirty L. japonicus individuals were collected from Xiamen, China, to evaluate the degree of polymorphism. The number of identified alleles ranged from three to five. The polymorphism information content varied from 0.267 to 0.711, whereas the observed and expected heterozygosities ranged from 0.249 to 0.706 and 0.294 to 0.751, respectively. One of the 10 loci (L10) deviated from the Hardy-Weinberg equilibrium. These new microsatellite markers will provide a useful tool for the determination of population genetic structure and genetic diversity in L. japonicus.

Isolation and characterization of microsatellite loci in hybrid giant tiger grouper.

The hybrid giant tiger grouper is a fish that has considerable commercial value and has become increasingly important for aquaculture in South East Asia since 2008. In order to prevent any reduction in genetic diversity in hybrid grouper as a result of aquaculture, we have identified 21 microsatellite markers that can be used to estimate genetic variation in the fish population. The number of alleles at polymorphic microsatellite loci ranged from 2 to 7, and observed and expected heterozygosities varied from 0.0323 to 0.9643 and 0.0921 to 0.7174, respectively. Polymorphism information content values ranged from 0.088 to 0.737. Nineteen of the 21 loci were in Hardy-Weinberg equilibrium (P > 0.005) after application of the Bonferroni correction (k = 10), the exceptions being ZZLD35 (P < 0.005) and ZZLD36 (P < 0.001). No linkage disequilibrium was detected. These 21 microsatellite markers are potentially of great value for analyzing genetic diversity to provide essential information for sustainable management of these fish.

Isolation and characterization of new microsatellite markers in red tail prawn, Fenneropenaeus penicillatus, an endangered species in China.

Until recently, Fenneropenaeus penicillatus was considered a commercial shrimp species. However, in 2005, it was included on the Red List as an endangered species by the Chinese government. In this study, 19 new microsatellite markers in F. penicillatus were developed and tested in samples of 32 wild individuals from Nanao, China. Twelve loci were polymorphic and 7 were monomorphic. Of the 12 polymorphic loci, the number of alleles per locus ranged from 3 to 6, with an average of 4.42 alleles per locus. The polymorphism information content ranged from 0.302 to 0.670, with a mean of 0.4817. The observed and expected heterozygosities ranged from 0.2250 to 0.8889 and from 0.1111 to 0.7750, respectively. Significant deviations from Hardy-Weinberg equilibrium (HWE, adjusted P < 0.0042) after a Bonferroni correction were observed in 3 loci (NA-9, NA-57, and NA-64), whereas the other 9 loci were in HWE. These new microsatellite markers will be useful in further research on the population genetic structure of F. penicillatus.

Screening and characterization of novel polymorphic microsatellite markers from sea cucumber Holothuria leucospilota.

The sea cucumber Holothuria leucospilota has high me-dicinal value and rich nutritional edible value, and thus is a commercially important aquatic product in China. Microsatellite loci were developed and screened using a fast isolation protocol and amplified fragment length polymorphism of sequences containing repeats. In this study, 16 novel polymorphic microsatellite markers in H. leucospilota were identified, and the relevant genetic variability index was assessed using 30 individu-als from a wild population. The polymorphic information content ranged from 0.183 to 0.668, and the number of alleles per locus varied from 3 to 5. The observed and expected heterozygosities were 0.0370-0.5000 and 0.0776-0.6250, respectively. With the exception of 3 loci (Y1-15, Y11-1, and Y28), the polymorphic loci were in Hardy-Weinberg equilibrium (P > 0.003125). These polymorphic microsatellite loci will contribute to studies of genetic diversity, the research of population structure, and the design of conservation strategies for H. leucospilota.

Identification and characterization of rhizosphere fungal strain MF-91 antagonistic to rice blast and sheath blight pathogens.

AIM: To examine the inhibition effects of rhizosphere fungal strain MF-91 on the rice blast pathogen Magnaporthe grisea and sheath blight pathogen Rhizoctonia solani. METHODS AND RESULTS: Rhizosphere fungal strain MF-91 and its metabolites suppressed the in vitro mycelial growth of R. solani. The inhibitory effect of the metabolites was affected by incubation temperature, lighting time, initial pH and incubation time of rhizosphere fungal strain MF-91. The in vitro mycelial growth of M. grisea was insignificantly inhibited by rhizosphere fungal strain MF-91 and its metabolites. The metabolites of rhizosphere fungal strain MF-91 significantly inhibited the conidial germination and appressorium formation of M. grisea. Moreover, the metabolites reduced the disease index of rice sheath blight by 35·02% in a greenhouse and 57·81% in a field as well as reduced the disease index of rice blast by 66·07% in a field. Rhizosphere fungal strain MF-91 was identified as Chaetomium aureum based on the morphological observation, the analysis of 18S ribosomal DNA internal transcribed spacer sequence and its physiological characteristics, such as the optimal medium, temperature and initial pH for mycelial growth and sporulation production. CONCLUSIONS: Rhizosphere fungus C. aureum is effective in the biocontrolling of rice blast pathogen M. grisea and sheath blight pathogen R. solani both in in vitro and in vivo conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is the first to show that rhizosphere fungus C. aureum is a potential fungicide against rice blast and sheath blight pathogens.

First Report of Anthracnose of Digitaria sanguinalis Caused by Colletotrichum hanaui in China.

Hairy crabgrass (Digitaria sanguinalis (L.) Scop.) is a troublesome weed in most agricultural crops worldwide. Considerable efforts are made to limit the invasiveness and impact of crabgrass on crop productivity, including evaluation of fungi as biocontrol agents (3). In September 2005, a severe disease was observed on crabgrass plants in Zhejiang Province. Leaves and stems of the affected plant showed small, water-soaked, brownish spots that rapidly turned into longitudinal elliptic or spindle-shaped lesions, 6.5 to 8 × 22 to 24 mm, with a brown outer edge and a gray sunken central area. Coalescence of large lesions gave rise to extensive rotting and necrosis, and the stems were broken when the lesions encircled. Acervuli with brown setae and falcate single-celled spores, typical of some Colletotrichum species (2), formed on the lesions at this late stage. One fungal isolate (Col-68) was obtained from symptomatic tissues on potato dextrose agar that led to white-to-gray appressed mycelium growth with orange conidial masses at 28°C in darkness. Setae were septate, dark brown, rounded and sometimes lobed at base, 32.0 to 116.5 × 3.2 to 6.0 µm, with apices acute. Hyphae were septate, hyaline, 1.0 to 6.5 µm, and sometimes guttulate. Conidia were falcate or fusiform, apices acute or obtuse, and 8.16 to 26.37 × 2.9 to 9.2 µm with an average of 18.15 × 5.65 µm. Hyphopodial appressoria were smooth, globose to prolate, ovoid or obovoid with obtuse or cylindrical apices, edges entire, and 4.17 to 14.25 × 3.77 to 8.94 µm with an average of 7.0 × 6.9 µm. The pathogen was initially identified as a Colletotrichum species based on morphology. Suspensions of 3-day-old spores collected from potato dextrose liquid cultures (106 conidia per ml) were used to spray inoculate (15 ml per pot) three 9-cm-diameter pots of crabgrass seedlings at the three- to four-leaf growth stage. Another three pots of healthy crabgrass were simultaneously sprayed with sterilized distilled water without conidia, which served as noninoculated checks. The seedlings were kept at 25 to 28°C for 24 h under a polyethylene sheet cover in the greenhouse. Symptoms that developed in all inoculated seedlings were identical to those observed on the affected crabgrass in the field, meanwhile the seedlings inoculated with sterilized water had no significant symptoms, and the reisolated strain had the same characteristics as the original isolate. To diagnose the pathogen to the species level, three isolates were tested and an approximately 580-bp DNA amplicon of this isolate was amplified using the primers ITS1/ITS4. The sequence (GenBank Accession No. GQ456160) had 98% sequence identity with the sequences of Colletotrichum hanaui (GenBank Accession Nos. EU554101and EU554124), which is supported by phylogenetic analysis with bootstrap support. On the basis of the morphological, pathological characteristics, and phylogenetic tree, the isolated strain was identified as C. hanaui (1). To our knowledge, this is the first confirmed report of anthracnose of D. sanguinalis caused by newly described C. hanaui in China. References: (1) J. A. Crouch et al. Mycologia, 101:717, 2009. (2) B. C. Sutton. The Coelomycetes. CAB International Publishing, New York, 1980. (3) Y. Z. Zhu and S. Qiang. Chin. J. Biol. Control 20:206, 2004.

Effect of Schisandra chinensis extract and Ginkgo biloba extract on the pharmacokinetics of talinolol in healthy volunteers.

The authors investigated the effect of herbal medicine Schisandra chinensis extract (SchE) and Ginkgo biloba extract (GBE) on the oral pharmacokinetics of P-glycoprotein substrate talinolol in humans. Twelve healthy male volunteers took a single 100-mg oral dose of talinolol either alone or after pretreatment with 300 mg SchE twice daily or with 120 mg GBE three times daily for 14 days. On day 14, a single 100-mg oral dose of talinolol was administered. Plasma concentrations of talinolol from zero to 24 h were measured by high-performance liquid chromatography. SchE increased the area under the curve (AUC)(0-24) of talinolol by 47% (90% confidence interval (CI), 18-84%; p = 0.010), and GBE by 21% (90% CI = 11-32%; p = 0.002). The C(max) of talinolol increased by 51% (90% CI = 21-89%; p = 0.007) with SchE treatment and by 33% (90% CI = 18-51%; p = 0.002) with GBE treatment, respectively. The t(1/2) of talinolol increased by 7% (90% CI = -4% to 19%; p = 0.320) with SchE treatment and by 11% (90% CI = -12% to 38%; p = 0.436) with GBE treatment, respectively. The results suggest that both SchE and GBE significantly inhibited P-glycoprotein in humans. Patients receiving either SchE or GBE may require dose adjustments when treated with drugs primarily transported by P-glycoprotein.

Systematic review with meta-analysis: thiopurines decrease the risk of colorectal neoplasia in patients with inflammatory bowel disease.

BACKGROUND: Patients with inflammatory bowel disease (IBD) have a high risk of developing colorectal neoplasia. AIM: To investigate whether thiopurines can decrease the risk of developing colorectal neoplasia in patients with ulcerative colitis (UC) or Crohn's disease (CD). METHODS: We conducted a meta-analysis of 24 observational studies involving 76,999 participants to evaluate the risks of developing colorectal neoplasia in IBD patients receiving thiopurine treatment. Pooled odds ratios (ORs) and 95% confidence intervals (CIs) for the risks of colorectal neoplasia were calculated using a random-effects model. RESULTS: The overall pooled estimate revealed a protective effect of thiopurine use on colorectal neoplasia in patients with IBD (OR = 0.63, 95% CI 0.46-0.86). The effect was significant in UC patients (OR = 0.67, 95% CI 0.45-0.98), but was not significant in CD patients (OR = 1.06, 95% CI 0.54-2.09). Thiopurines exposure significantly decreased the risk of colorectal cancer (CRC) (OR = 0.65, 95% CI 0.45-0.96) and advanced colorectal neoplasia (CRC and/or high-grade dysplasia) (OR = 0.62, 95% CI 0.44-0.89), but did not decrease the risk of dysplasia alone (OR = 0.90, 95% CI 0.37-2.21). Tendencies towards the protective effect of thiopurines were distinct in clinic-based studies (OR = 0.59, 95% CI 0.42-0.82) and case-control studies (OR = 0.40, 95% CI 0.26-0.62), but not in population-based studies (OR = 0.95, 95% CI 0.55-1.62) and cohort studies (OR = 0.98, 95% CI 0.81-1.18). Interestingly, studies conducted in Europe (OR = 0.48, 95% CI 0.31-0.77), rather than in North America (OR = 0.91, 95% CI 0.67-1.24), showed the protective effect of thiopurines. CONCLUSIONS: This meta-analysis revealed an antineoplastic effect of thiopurines on colorectal neoplasia in patients with IBD, particularly amongst patients with UC.

The contribution of polymorphism in the alcohol dehydrogenase beta subunit to alcohol sensitivity in a Japanese population.

In humans, ingested alcohol is mainly metabolized by the combination of class I alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH). In Orientals, there are highly frequent polymorphisms both in the class I ADH beta subunit (ADH2) and in the low Km ALDH (ALDH2). We characterized the three genotypes of ALDH2 in a Japanese population. In the present study, we evaluated the effects of the ADH2 polymorphism in the same population (424 males and 100 females) controlling for the effects of the ALDH2 polymorphism. In the ALDH2(1)/ALDH2(2) group, the frequency of facial flushing with one glass of beer was significantly higher in the ADH2(1)/ADH2(2) and ADH2(2)/ADH2(2) genotype than in the ADH2(1)/ADH2(1) genotype. Likewise, the proportion of persons with positive results for ethanol-induced cutaneous erythema differed significantly depending on the ADH2 genotype in both the ALDH2(1)/ALDH2(1) and ALDH2(1)/ALDH2(2) genotypes. However, drinking habits were not significantly associated with the ADH2 genotype, suggesting that the ADH2 genotype influences the metabolism of ethanol only in the peripheral tissues.

Association between atopic asthma and a coding variant of Fc epsilon RI beta in a Japanese population.

A genetic association study was performed with coding variants of Fc epsilon RI beta in relation to atopic and non-atopic asthma in a Japanese population (n = 400). A coding variant of Gly237Glu in exon 7 of Fc epsilon RI beta gene showed association with atopic asthma (OR = 3.00, chi 2 = 5.10, p < 0.03), but not with non-atopic asthma; this was seen particularly in childhood asthma (OR = 3.92, chi 2 = 8.66, p < 0.005). This variant is also associated with very high total serum IgE levels (> mean + 3 SD, OR = 8.56, chi 2 = 46.2, p < 0.0001), but not any allergen specific IgE. However, Leu181lle, another variant of Fc epsilon RI beta related to atopy in British and Australian populations, was not found in this Japanese population. These results suggest that variants of Fc epsilon RI beta may be an important genetic cause of the atopic asthma.

Association between variants of mast cell chymase gene and serum IgE levels in eczema.

851 school children aged at 12-13 years including 145 with eczema were tested for genetic association to a mast cell chymase (MCC) genetic variant. MCC genotypes showed a strong association with eczema, but not with asthma and rhinitis. This association is strongest in eczematous children with lower serum total IgE levels. Independent of IgE responsiveness when total serum IgE of less than 500 IU/ml, MCC variants may play an important role in inflammatory skin disorders.