Genome-wide survey and analysis of microsatellites in the Pacific oyster genome: abundance,distribution, and potential for marker development

Microsatellites are a ubiquitous component of the eukaryote genome and constitute one of the most popular sources of molecular markers for genetic studies. However, no data are currently available regarding microsatellites across the entire genome in oysters, despite their importance to the aquaculture industry. We present the fi rst genome-wide investigation of microsatellites in the Pacifi c oyster Crassostrea gigas by analysis of the complete genome, resequencing, and expression data. The Pacifi c oyster genome is rich in microsatellites. A total of 604 653 repeats were identifi ed, in average of one locus per 815 base pairs(bp). A total of 12 836 genes had coding repeats, and 7 332 were expressed normally, including genes with a wide range of molecular functions. Compared with 20 different species of animals, microsatellites in the oyster genome typically exhibited 1) an intermediate overall frequency; 2) relatively uniform contents of(A)n and(C)n repeats and abundant long(C)n repeats(≥24 bp); 3) large average length of(AG)n repeats; and 4) scarcity of trinucleotide repeats. The microsatellite-fl anking regions exhibited a high degree of polymorphism with a heterozygosity rate of around 2.0%, but there was no correlation between heterozygosity and microsatellite abundance. A total of 19 462 polymorphic microsatellites were discovered, and dinucleotide repeats were the most active, with over 26% of loci found to harbor allelic variations. In all, 7 451 loci with high potential for marker development were identifi ed. Better knowledge of the microsatellites in the oyster genome will provide information for the future design of a wide range of molecular markers and contribute to further advancements in the fi eld of oyster genetics, particularly for molecular-based selection and breeding.     

Habitat values for artificial oyster (Crassostrea ariakensis) reefs compared with natural shallow-water habitats in Changjiang River estuary

studies have compared oyster reefs with adjacent natural shallow-water habitats.Here the resident benthic macroinvertebrate communities in an artificial oyster(Crassostrea ariakensis) reef and in adjacent natural estuarine shallow-water habitats(salt marsh,intertidal mudflat,and subtidal soft bottom) in the Changjiang(Yangtze) River estuary were described.The mean total densities and biomass,Margalef’s species richness,Pielou’s evenness and Shannon-Weaver biodiversity indices of the resident benthic macroinvertebrate communities differed significantly among the habitats.Significantly higher densities and biomass of benthic macroinvertebrates occurred in the oyster reef compared with the other three habitats.Ordination plots showed a clear separation in benthic macroinvertebrate communities among the four habitat types.The results demonstrated that the artificial oyster reef supported distinct and unique benthic communities,playing an important role in the complex estuarine habitat by supplying prey resources and contributing to biodiversity.In addition,the results suggested that the oyster reef had been restored successfully.     

Iodothyronine deiodinase gene analysis of the Pacific oyster Crassostrea gigas reveals possible conservation of thyroid hormone feedback regulation mechanism in mollusks

Iodothyronine deiodinase catalyzes the initiation and termination of thyroid hormones(THs) effects, and plays a central role in the regulation of thyroid hormone level in vertebrates. In non-chordate invertebrates, only one deiodinase has been identified in the scallop C hlamys farreri. Here, two deiodinases were cloned in the Pacific oyster C rassostrea gigas( Cg Dx and C g Dy). The characteristic in-frame TGA codons and selenocysteine insertion sequence elements in the oyster deiodinase c DNAs supported the activity of them. Furthermore, seven orthologs of deiodinases were found by a tblastn search in the mollusk Lottia gigantea and the annelid C apitella teleta. A phylogenetic analysis revealed that the deiodinase gene originated from an common ancestor and a clade-specific gene duplication occurred independently during the differentiation of the mollusk, annelid, and vertebrate lineages. The distinct spatiotemporal expression patterns implied functional divergence of the two deiodinases. The expression of C g Dx and Cg Dy was influenced by L-thyroxine T4, and putative thyroid hormone responsive elements were found in their promoters, which suggested that the oyster deiodinases were feedback regulated by TH. Epinephrine stimulated the expression level of C g Dx and Cg Dy, suggesting an interaction effect between different hormones. This study provides the first evidence for the existence of a conserved TH feedback regulation mechanism in mollusks, providing insights into TH evolution.     

Sequence comparison of partial cytochrome b genes of two Coilia species

Sequence variation of partial cytochrome b genes between two Coilia species, C. ectenes and C. mystus, was investigated. Of the 402 nucleotides, twenty-seven (6.72%) are polymorphic and all are synonymous substitutions. At the third positions of genetic condon of cytochrome b gene, the two species show an extreme anti-G bias (<4%) and a pronounced bias towards A and C (>68%). There is no amino acid sequence divergence between the partial cytochrome b genes of the two species, indicating a close genetic relationship between them. The k-2p genetic distance of partial cytochrome b segment of the two species is 0.072, suggesting that the species were separated 3.6 Ma ago, in the middle Pliocene. Our result reveals that the cytochrome b gene is an appropriate marker for studies of population genetic structures and phylogeographic patterns of the two species.     

Sequence Comparison of Partial Cytochrome b Genes of Two Coilia species

1Introduction Japanese grenadier anchovy,Coilia ectenes Jordanet Seale(1905),is distributed in the northwest Pacif-ic.The congener,Osbeck's grenadier anchovy,Coil-ia mystus(Linnaeus,1758),is widely distributed inthe west Pacific.Both species inhabit coastal watersand estuaries and can reach up to rivers.The twospecies are important contributors to the coastal fish-eries of China(Zheng et al.,2003).Many studies on the fishery biology of the twospecies have been conducted(Zheng et al.,2003)…     

The complete mitochondrial genome of Parachiloglanis hodgarti and its phylogenetic position within Sisoridae

The complete mitogenome of Parachiloglanis hodgarti was sequenced and characterized, which is the first mitogenome of the genus Parachiloglanis within Sisoridae. The mitogenome is 16 511-bp long and included 13 protein-coding genes, 22 transfer RNAs, two ribosomal RNAs, and one control region. The genome composition was A+T biased (58.64%) and exhibited a positive AT-skew (0.095) and a negative GC-skew (?0.283). Compared with other Sisoridae fishes, the characteristics of nucleotide skews, codon usage, and amino acid usage of the P. hodgarti mitogenome are more similar to those of non-Glyptosternoid fishes. Furthermore, the phylogenetic trees show that Sisoridae fishes fall into four major clades and P. hodgarti (Clade I) is basal to the Sisoridae family, forming a sister clade to the Glyptosternoids (Clade IV). The topological structures constructed in this study raised doubts over the traditional classification system. These results will help better understand the features of the P. hodgarti mitogenome and provide a reference for further phylogenetic research on Sisoridae species.

     

大珠母贝主要污损及寄居生物的研究

研究雷州半岛自然生活条件、养殖条件下的野生贝和全人工养殖条件下的养殖贝主要污损及寄居生物。结果表明:大珠母贝主要污损及寄居生物共46种,其中自然生活条件下野生贝的污损及寄居生物有24种,养殖条件下野生贝的污损与寄居生物42种,养殖贝的污损及寄居生物有31种;根据附着或寄居部位可将大珠母贝污损及寄居生物可分为4种类型:贝壳表面附生型、贝壳层寄居型、贝壳内侧寄居型、外套腔寄居型;海绵、缨鳃虫、龙介沙蚕、长鳃树蛰虫为贝壳表面附生型主要种类,砗磲江珧虾为外套腔寄居型种类,对大珠母贝危害较小;石蛏、开腹蛤、厥目革囊星虫、有盾海扇虫、才女虫、钻穿裂虫为贝壳层寄居型的主要种类;吻蛇稚虫为贝壳内侧寄居型,对大珠母贝危害较大。自然生活条件下野生贝主要危害种类为石蛏、开腹蛤、厥目革囊星虫、才女虫、钻穿裂虫,养殖条件下野生贝主要危害种类有石蛏、开腹蛤、厥目革囊星虫、有盾海扇虫、吻蛇稚虫、才女虫、钻穿裂虫;养殖贝受寄居生物危害较小。     

A new identification method for five species of oysters in genus Crassostrea from China based on high-resolution melting analysis

The high phenotypic plasticity in the shell of oysters presents a challenge during taxonomic and phylogenetie studies of these economically important bivalves. However, because DNA can exhibit marked differences among morphologically similar species, DNA barcoding offers a potential means for oyster identification. We analyzed the complete sequences of the cytochrome oxidase subunit I （COI） of five common Crassostrea species in China （including Hong Kong oyster C. hongkongensis, Jinjiang oyster C. ariakensis, Portuguese oyster C. angulata, Kumamoto oyster C. sikamea, and Pacific oyster C. gigas） and screened for distinct fragments. Using these distinct fragments on a high-resolution melting analysis platform, we developed an identification method that does not rely on species-specific PCR or fragment length polymorphism and is efficient, reliable, and easy to visualize. Using a single pair of primers （Oyster- COI-1）, we were able to successfully distinguish among the five oyster species. This new method provides a simple and powerful tool for the identification of oyster species.     

Spatio-Temporal Variations in Co-Occurrence Patterns of Fish Communities in Haizhou Bay,China: Null Model Analysis

Co-occurrence pattern of fish species plays an important role in understanding the spatio-temporal structure and the stability of fish community. Species coexistence may vary with time and space. The co-occurrence patterns of fish species were examined using the C-score under fixed-fixed null model for fish communities in spring and autumn over different years in the Haizhou Bay, China. The results showed that fish assemblages in the whole bay had non-random patterns in spring and autumn over different years. However, the fish co-occurrence patterns were different for the northern and southern fish assemblages in spring and autumn. The northern fish assemblage showed structured pattern, whereas the southern assemblage were randomly assembled in spring. The co-occurrence patterns of fish communities were relatively stable over different years, and the number of significant species pairs in northern assemblage was more than that in the southern assemblage. Environmental heterogeneity played an important role in determining the distributions of fish species that formed significant species pairs, which might affect the co-occurrence patterns of northern and southern assemblages further in the Haizhou Bay.     

Impact of Colonization of an Invasive Species on Genetic Differentiation in New Environments: A Study on American Artemia franciscana(Crustacea: Anostraca) in the United Arab Emirates

Artemia franciscana, native to America, has recently colonized as non-indigenous population in Asia, Europe, North Africa, and Australia. We evaluated the effects of the colonization of A. franciscana on genetic differentiation in new environments in the United Arab Emirates(UAE). We used the COI marker to determine the genetic structure and origins of exotic populations in the UAE. Results confirmed the colonization of A. franciscana in two localities. Invasive populations of A. franciscana had significantly lower genetic variation than native populations in the Great Salt Lake and San Francisco Bay. Results showed that the studied populations could not have colonized directly from natural American habitats, and they possibly were from secondary introduction events of other non-indigenous populations. Genetic analysis yielded different demographic patterns for the studied invasive populations. The population in Al Wathba Wetland Reserve(AWWR) demonstrated demographic expansion, whereas in Godolphin Lakes(GL), it reached a demographic equilibrium. Neutrality tests showed an excess of recent and historical mutations in the COI gene pool of invasive AWWR Artemia in the new environment. The results suggest that different ecological conditions in new environments can exert selective pressures during the introduction of an exotic population, which can affect genetic variation.     

High Pressure Treatment Changes Spoilage Characteristics and Shelf Life of Pacific Oysters(Crassostrea gigas) During Refrigerated Storage

The effects of high pressure(HP) treatment on spoilage characteristic and shelf life extension of Pacific oysters(Crassostrea gigas) during refrigerated storage were studied.Results showed that HP treatment of 275 MPa for 3 min or 300 MPa for 2 min could achieve 100% full release of oyster adductor muscle,pressures higher than 350 MPa caused excessive release as the shells of oysters were broken,thus use of higher pressures should be cautious in oyster processing industry because of its adverse impact on the appearance of shells.HP treatment(300 MPa,2 min) was proper for the shucking of Pacific oyster(Crassostrea gigas) in China.This treatment caused no organoleptic disadvantage.Moreover,HP treatment resulted in obvious differences in biochemical spoilage indicators(p H,TVB-N and TBARS) changes and volatile compounds profile determined by electronic nose during storage.HP treatment(300 MPa,2 min) also led to a reduction of aerobic bacterial count(APC) by 1.27 log cycles.Furthermore,the APC values of oysters treated by HP were always lower than those of the control samples during storage.Based on the organoleptic,biochemical and microbiological indicators,shelf life of 6-8 d for control and 12 d for HP-treated oysters could be expected.HP treatment showed great potential in oyster processing and preservation.     

Response to selection for fast growth in the second generation of Pacific oyster (Crassostrea gigas)

Mass selection for fast growth was conducted in three Pacific oyster (Crassostrea gigas) stocks from China, Japan and Korea using previously established lines (CS1, JS1, and KS1). To determine whether continuous progress can be achieved by selection for growth, the progeny of three second-generation Pacific oyster lines was selected for shell height and evaluated via a 400-day farming experiment. When harvested at the end of the experiment, the selected crosses of CS2, JS2, and KS2 lines grew by 9.2%, 10.2% and 9.6% larger than the control crosses, respectively. During grow-out stage, the genetic gain of three selected lines was (10.2 ± 1.4)%, (10.4 ± 0.3)%, and (8.4 ± 1.6)%, respectively; and the corresponding realized heritability was 0.457 ± 0.143, 0.312 ± 0.071 and 0.332 ± 0.009, respectively. These results indicated that the selection for fast growth achieved steady progress in the second generation of oyster. Our work provides supportive evidence for the continuity of the Pacific oyster selective breeding program.     

Spatial patterns and storage composition of woody debris in a natural secondary forest dominated by <Emphasis Type="Italic">Pinus tabulaeformis</Emphasis> on Loess Plateau,China

Woody debris (WD) is an important part of natural Pinus tabulaeformis mixed stands, and it affects the forest ecosystem stability and development. The WD spatial patterns are especially important structural characteristics that can provide insights into forest dynamics. In this paper, the WD storage, WD spatial patterns and WD associations among the main species were examined in the natural secondary forest on Loess Plateau in northwest China. Data were collected in a 1 ha (100 m × 100 m) permanent plot, and all the trees with a diameter at breast height of more than 3 cm were measured and stem-mapped. Ripley’s K functions from the spatial-point-pattern-analysis method were used to analyze the spatial distribution and associations. The results showed that: (1) The total storage of WD was 10.73 t/ha, fallen wood was the main source of WD, and the majority diameters were greater than 20 cm, and in intermediate levels of decay; (2) The overall spatial pattern was closely related to the spatial scale, which exhibited an aggregated pattern on a small scale, and a random pattern on a large scale. The spatial patterns of coarse woody debris also gradually transitioned from an aggregated pattern in fine scales to a random pattern in broader spatial scales, which matched the overall spatial pattern. The spatial intensity was gradually decreased with the increasing diameters, and increased with the decomposition classes; (3) The WD of Pinus tabulaeformis species was negatively associated with Betula platyphylla and Populus davidiana on a small scale but positively associated with these species on a large scale. The spatial pattern and interspecies relations were the results of long-term interactions between the natural secondary forest community and the surrounding natural environment. These findings would provide a scientific basis for the sustainable management and protection of natural secondary forest ecosystems on Loess Plateau.     

Sequence analysis and quantitative detection of Norwalk-like viruses in cultured oysters of China

We isolated 4 Norwalk-like viruses (NLVs) contaminated oysters from 33 Chinese oysters collected from local commer-cial sources of Shandong Province. After amplification of the RNA-dependent RNA polymerase (RdRp) region of NLVs genomes with RT-PCR, the open reading frame 1 (ORF1) of the RdRp was sequenced and subjected to multiple-sequence alignment. The re-suits showed that NLVs in the four isolates belong to genogroup Ⅱ. The sequence comparison showed that the similarity between four Chinese oyster isolates were higher than 99.0%, which indicated that NLVs prevalent in close areas have high homogeneity in genome sequences. In addition, the most conserved sequences between diverse NLVs were used to design primers and TaqMan probes, then the real-time quantitative PCR assay was performed. According to the standard curve of GII NLVs, the original amounts (copies) of NLVs in positive patient's fecal isolate, positive Japanese oyster isolate, and the Chinese oyster isolate were 8.9×108, 1.25×108 and 4.7×101 respectively. The detecting limit of NLVs was 1×101 copies. This study will be helpful for routine diagnosis of NLVs pathogens in foods and thus for avoiding food poisoning in the future.     

GNSS单站授时系统性偏差分析

从单站授时基本观测方程出发,对引起系统性偏差的原因进行研究,结合具体实验分析卫星钟差和接收机硬件延迟对授时结果的影响。实验结果表明,卫星钟差引起的系统性偏差随时间变化,对所有测站影响相同;接收机硬件延迟会对授时结果造成系统性偏差影响,不同测站的系统性偏差不同,其大小取决于各自接收机硬件延迟的偏差,在进行授时及时间同步时应当加以改正;接收机硬件延迟不影响授时结果频率的稳定度,在进行频率传递时无需改正。     

Genetic structure and demographic histories of two sympatric Culter species in eastern China

Geographic isolation is a key factor in shaping the genetic structure of many fish species.Two sympatric species,Culter alburnus and C.mongolicus,are economically important fish that are widely distributed in China and have been recently used as new aquaculture species.We used the mitochondrial DNA control region(CR) as a marker to investigate the genetic structure of the two species.Phylogenetic analysis revealed two major lineages(Lineages I and II) that were highly consistent with geographical patterns for C.alburnus and C.mongolicus.Based on genetic distance,the Zhujiang(Pearl) River Basin(ZRB) populations potentially represented a cryptic subspecies,which might be differentiated as the result of strict geographic isolation,an earlier diverged event,or peripheral areas.Genetic diversity analysis suggested that the Changjiang(Yangtze) River Basin(CRB) populations are located at,or near the core region of their origination,not only due to the larger population size at the CRB,but also their habitat diversity and suitability for its survival,and the genetic diversity dif ferences among basin populations were significant for the two species.Moreover,demographic analysis indicated that the two Culter species and most populations had undergone a period of population expansion during warm interglacial periods.However,the C.alburnus population of Huaihe River Basin(Weishan Lake) exhibited different patterns during the interglacial period,which may due to the latest diverged time of HRB and Weishan Lake located at the permafrost.Notably,the ZRB(in Songtao Reservoir) C.mongolicus population showed no genetic diversity and had a unique haplotype,which could be treated as a special gene pool for species conservation.In summary,geographic isolation is most likely responsible for the two Culter species distribution patterns.     

Molecular identification of Prorocentrum （Dinophyceae） species

A fragment of a large sub-unit ribosomal DNA (LrDNA) of 12 strains of Prorocentrum species was amplified by polymerase chain reaction (PCR). The PCR products were digested by 3 restriction endonucleases (Cfo Ⅰ, Hae Ⅲ, and RSA Ⅰ) and then resolved in agarose gels. Results show that different species had different RFLP patterns, except for P arcuatum (ME 131), which had the same pattern to P. micans (ME 160 and 04).The same fragment of 19 strains of the genus was also amplified and subjected to denaturing gradient gel electrophoresis (DGGE). 11 different patterns were resolved. Different cultures of a same species had the same pattern. The results of RFLP and DGGE analyses showed that eight newly isolated epibenthic Prorocentrum species were different from each other, and also from other cultured ones examined in this study. P arcuatum(MEI32) could not be differentiated from P. micans (MEI60 and 04), it was probably mis-identified, since they are quite different morphologically. P. redfieldii (MEI38) could also not be distinguished form P. triestinium(MEI32), it should be regarded as a synonym ofP. triestinium. Unexpectedly, a restriction site was found in P.micans, compared with previous sequence data.     

3种江珧同工酶遗传标记

通过对江珧科的栉江珧、旗江珧及二色裂江珧三物种的 5种组织 (消化盲囊、肾组织、外套膜、后闭壳肌和鳃组织 )进行垂直板状聚丙烯酰胺凝胶电泳分离技术 ,研究江珧科的 SOD、EST同工酶酶谱 ,结果表明 ,不同种间的个体其酶谱表型有稳定的差异 ,同属的种间差异小于不同属的种间差异 ,即酶谱的差异程度与形态分类学中的亲缘关系相关。可利用其酶谱表型具有种特异性作为一种蛋白分子标记 ,应用于江珧物种尤其是经济品种的鉴定上