Generation and analysis of 10 000 ESTs from the half‐smooth tongue sole Cynoglossus semilaevis and identification of microsatellite and SNP markers

Three normalized cDNA libraries were constructed, two of which were constructed from reproductive tissues ovary and testis, and the other one from pooled immune tissues including head kidney, intestine, liver and spleen. A total of 10 542 clones were sequenced generating 10 128 expressed sequence tags (ESTs). Cluster analysis indicated a total of 5808 unique sequences including 1712 contigs and 4096 singletons. A total of 4249 (73%) of the unique ESTs had significant hits to the non‐redundant protein database, 2253 of which were annotated using Gene Ontology (GO) terms. A total of 311 microsatellites (with 246 having sufficient flanking sequences for primer design) and 6294 putative SNPs were identified. These genome resources provide the material basis for future microarray development, marker validation and genetic linkage and QTL analysis.     

Cathepsin B of Cynoglossus semilaevis: identification, expression, and activity analysis

Cathepsin B (EC 3.4.22.1) is a member of the papain family cysteine protease and in mammals is known to be involved in protein degradation and other biological functions. However, very little is known about the function of cathepsin B in fish. In this study, we identified and analyzed a cathepsin B homologue (CsCatB) from tongue sole (Cynoglossus semilaevis, Pleuronectiformes), an economic fish species cultured in China. CsCatB is composed of 322 amino acid residues and shares 70-81.3% overall sequence identities with its counterpart in teleosts and humans. CsCatB possesses typical cathepsin B structural features including the propeptide region and the papain family cysteine protease domain, the latter containing the four catalytic residues (Q101, C107, H277, and N297) that are conserved in lower and higher vertebrates. Quantitative real time RT-PCR analysis showed that CsCatB expression occurred in multiple tissues and was positively regulated by bacterial infection and by immunization with a subunit vaccine. Recombinant CsCatB purified from Escherichia coli exhibited apparent protease activity, which was optimal at 35 °C and pH 5.5. In contrast, a mutant CsCatB bearing glutamic acid substitution at H277 was dramatically reduced in proteolytic activity. These results indicate that CsCatB is a biologically active protease that is likely to be involved in host immune response during bacterial infection and vaccination.     

半滑舌鳎仔鱼的摄食与生长

在水温23.4-24.0℃、盐度33.00、pH7.78-8.02的培养条件下,半滑舌鳎初孵仔鱼全长为3.55±0.161mm(n=60),依照公式4/3π.R/2.(r/2)2计算其卵黄囊体积为0.606±0.106mm3(n=60)。1日龄仔鱼,全长为4.99±0.211mm(n=10),卵黄囊体积较初孵仔鱼缩小近10倍,约为0.066±0.008mm3(n=10);2日龄,仔鱼的巡游模式建立,仔鱼全长为5.61±0.069mm,卵黄囊体积为0.030±0.002mm3(n=10),口初开;2.5日龄,口完全裂开,口裂0.24±0.024mm(n=10),仔鱼进入摄食期;3日龄,仔鱼的外源性摄食关系初步建立;4日龄,仔鱼的摄食率达100%,完成了内源性营养向外源性营养的转换;5日龄,仔鱼的卵黄完全被吸收,仅剩聚成一团的小油球,仔鱼的混合营养期持续2.5天时间;21日龄,稚鱼全长为12.96±0.611mm(n=11),仍有40%的个体残余的油球还没有完全被吸收,其体积仅为0.0000005±0.000003mm3(n=11)。仔鱼发育过程中,其长度的生长存在内源性营养阶段的快速生长、混合营养阶段的慢速生长以及饥饿期间的负生长三个生长期相,平均增长率为0.45mm/d,依照TL=aD3 bD2 cD d方程式对仔鱼的全长与日龄进行回归,其生长模型为TL(mm)=0.0026D3-0.0704D2 0.7993D 3.55(R2=0.9811,n=324)。仔鱼耐受饥饿的时间临界点发生在孵化后第10天(即9日龄)。仔鱼具有摄食能力的时间约6天,不可逆转饥饿期的时间约3天。残余的油球较长时间的存在,相对地延长了仔鱼混合营养期的时间,对仔鱼的发育、生长和存活有着至关重要的作用。5-20日龄的个体都具有胸角这个明显的形态学特征,只是饥饿个体和不可逆转饥饿期个体的胸角比摄食个体更为明显和尖锐,胸角不能作为区分健康仔鱼和饥饿仔鱼的形态学依据之一[动物学报51(6)1023-1033,2005]。     

Identification and expressional analysis of two cathepsins from half-smooth tongue sole (Cynoglossus semilaevis)

Cathepsins are a family of lysosomal proteases that play an important role in protein degradation, antigen presentation, apoptosis, and inflammation. Cathepsins are divided into three groups, i.e., cysteine protease, serine protease, and aspartic protease. Cathepsin D and cathepsin L, which are aspartic protease and cysteine protease respectively, have been identified in a number of teleosts; however, the immunological relevance of fish cathepsins is largely unknown. In this study, we cloned and analyzed the expression profiles of a cathepsin D (CsCatD) and a cathepsin L (CsCatL) homologs from half-smooth tongue sole (Cynoglossus semilaevis). CsCatD is composed of 396 amino acid residues and shares 67.6-88.4% overall sequence identities with fish and human cathepsin D. Structurally CsCatD possesses an aspartic endopeptidase domain, which contains two conserved aspartic acid residues that form the catalytic site. CsCatL is 336 residues in length and shares 64.7-90.2% overall sequence identities with fish and human cathepsin L. CsCatL has an N-terminal cathepsin propeptide inhibitor domain followed by a Papain family cysteine protease domain, the latter containing four conserved catalytic residues: Gln-133, Cys-139, His-279, and Asn-303. Recombinant CsCatL purified from Escherichia coli exhibited apparent protease activity. Quantitative real time RT-PCR analysis detected constitutive expression of CsCatD and CsCatL in multiple tissues, with the lowest level found in heart and the highest level found in liver. Experimental challenge of tongue sole with the bacterial pathogen Vibrio anguillarum and megalocytivirus caused significant inductions of both CsCatD and CsCatL expression in kidney and spleen in time-dependent manners. Immunization of the fish with a subunit vaccine also enhanced CsCatD and CsCatL expression in the first week post-vaccination. These results suggest involvement of CsCatD and CsCatL in host immune reactions to bacterial and viral infections and in the process of antigen-induced immune response.     

Gene cloning and expression analysis of IRF1 in half-smooth tongue sole (Cynoglossus semilaevis)

Interferon regulatory factor 1 (IRF1) was known to play key roles in antiviral defense in several species, and some other important biological processes. In this report, full length cDNA of IRF1 from Cynoglossus semilaevis (CsIRF1) was identified. It was of 1,455 bp, containing a 5′ UTR of 104 bp, a 3′ UTR of 541 bp with a poly (A) tail and an ORF of 810 bp encoding a putative protein of 269 amino acids. The putative CsIRF1protein contained one conserved IRF domain (1–113aa), and two low complexity regions (140–158aa and 230–242aa, respectively). Phylogenetic analysis showed that CsIRF1 was conserved in the teleost evolutionary branch, which was independent of mammalian, birds and amphibians. Additionally, CsIRF1 had the 96 % homology with marine fishes, while 66 % with freshwater fishes. The expression profiles of CsIRF1was analyzed by quantitative real-time PCR in healthy tissues and in immune tissues challenged with different pathogens [Vibrio anguillarum and Lymphocystis disease virus (LCDV)], respectively. CsIRF1 was widely expressed in healthy tissues of Cynoglossus semilaevis and with the highest expression in blood, as much as 19 times of that in liver. V. anguillarum and LCDV both induced the CsIRF1 gene expression distinctly in liver, with the peak value reached to 98-fold at 6 h and 25-fold at 24 h, respectively. The bacteria induced CsIRF1 suddenly up-expression in each detected tissues. However, at the initial stage of the challenge of virus LCDV, the CsIRF1 expression in blood and spleen were up regulated; on the contrary, its expression in liver and head kidney were down regulated, 0.3 and 0.4-fold 6 h post virus injection, respectively. These results suggested that CsIRF1 gene might involve in not only antiviral activity but also antibacterial procedure, indicating its vital role in Cynoglossus semilaevis innate defense system.     

Isolation and characterization of Toll-like receptor 9 in half-smooth tongue sole Cynoglossus semilaevis

Toll-like receptors (TLRs) are considered as key sensors to trigger the host's innate immune system and adaptive immune responses by recognizing various PAMPs and initiating signal transduction. TLR9, as a member of TLR family, mediates the recognition of unmethylated CpG dinucleotide motifs commonly found in both bacterial and viral genomes. In the current study, the TLR9 gene was isolated from one of flatfish species, half-smooth tongue sole (Cynoglossus semilaevis). In the 4588 bp genomic sequence, three exons, two introns, and 5′ UTR of 23 bp and 3′ UTR of 342 bp were identified. Putative amino acid sequence was 1062 residues long, including a typical conserved cytosolic Toll/interleukin-1 receptor (TIR) domain, 14 leucine-rich repeat (LRR) motifs, with greater than 60% identity to gilthead sea bream Sparus aurata and Japanese flounder Paralichthys olivaceus orthologs. Quantitative RT-PCR analysis indicated a broad expression of csTLR9, especially in spleen and gonads. No statistically significant changes were observed for csTLR9 mRNA levels in spleen and head kidney after inactive Vibrio anguillarum immunisation. In C. semilaevis ontogeny, the expression of csTLR9 appeared to be developmentally regulated. The presence of maternal TLR9 mRNA and the dramatic decrease of TLR9 expression at metamorphic stage indicated TLR9 might be involved in C. semilaevis development. Comparing sequence and expression profile of csTLR9 with mammalian and other piscine TLR9s suggested that the main function of TLR9 might be conserved across vertebrates, although species-specific features were present.     

Outlier SNP markers reveal fine‐scale genetic structuring across European hake populations (Merluccius merluccius)

Shallow population structure is generally reported for most marine fish and explained as a consequence of high dispersal, connectivity and large population size. Targeted gene analyses and more recently genome‐wide studies have challenged such view, suggesting that adaptive divergence might occur even when neutral markers provide genetic homogeneity across populations. Here, 381 SNPs located in transcribed regions were used to assess large‐ and fine‐scale population structure in the European hake (Merluccius merluccius), a widely distributed demersal species of high priority for the European fishery. Analysis of 850 individuals from 19 locations across the entire distribution range showed evidence for several outlier loci, with significantly higher resolving power. While 299 putatively neutral SNPs confirmed the genetic break between basins (F_CT = 0.016) and weak differentiation within basins, outlier loci revealed a dramatic divergence between Atlantic and Mediterranean populations (F_CT range 0.275–0.705) and fine‐scale significant population structure. Outlier loci separated North Sea and Northern Portugal populations from all other Atlantic samples and revealed a strong differentiation among Western, Central and Eastern Mediterranean geographical samples. Significant correlation of allele frequencies at outlier loci with seawater surface temperature and salinity supported the hypothesis that populations might be adapted to local conditions. Such evidence highlights the importance of integrating information from neutral and adaptive evolutionary patterns towards a better assessment of genetic diversity. Accordingly, the generated outlier SNP data could be used for tackling illegal practices in hake fishing and commercialization as well as to develop explicit spatial models for defining management units and stock boundaries.     

半滑舌鳎精子发生和精子形成的超微结构

用电子显微镜对半滑舌鳎(Cynoglossus semilaevis)精子发生的过程及精子的超微结构进行了观察。半滑舌鳎精巢属于小叶型,精小叶由各期生精细胞和支持细胞构成。半滑舌鳎的精子发生经历了初级精原细胞、次级精原细胞、初级精母细胞、次级精母细胞和精子细胞,再经过精子形成过程发育成为精子。初级精母细胞成熟分裂的前期Ⅰ,同源染色体经历了联会复合体形成和解聚的变化。在精子形成的过程中,精细胞大致经历了核质浓缩、线粒体迁移及鞭毛的发生等过程。核质浓缩时,精细胞核内位于植入窝周围的染色质首先由细颗粒状浓缩成粗大颗粒状,然后细胞核其他部位的染色质也逐渐浓缩成粗大颗粒状。这些已浓缩成粗大颗粒状的染色质再进一步浓缩为电子密度高的均匀状物质。随着核质的浓缩,核外膜与核内膜之间的间隙增大形成核膜间隙,核内一些没有参与染色质浓缩的物质通过出芽形成囊泡,先排入核膜间隙,然后再外排到细胞质中。核浓缩过程中细胞核的体积和表面积都大大缩小;鞭毛的形成与细胞核的浓缩是同步进行的,当一对中心粒移近细胞核时,核膜凹陷形成植入窝,其周围染色质浓缩的同时,远端中心粒(基体)逐渐向后产生轴丝。成熟精子无顶体,头细长,主要为核占据,核凹窝发达,线粒体4-5个环绕在鞭毛基部形成袖套,尾细长,具侧鳍,尾部轴丝为"9 2"结构。     

Artificial Gynogenesis and Sex Determination in Half-Smooth Tongue Sole (Cynoglossus semilaevis)

Half-smooth tongue sole (Cynoglossus semilaevis) is an important cultured marine fish as well as a promising model fish for the study of sex determination mechanisms. In the present study, a protocol for artificial gynogenesis of half-smooth tongue sole was developed in order to identify the sex determination mechanism and to generate all-female stock. The optimal UV-irradiation dose for genetically inactivating sea perch spermatozoa was determined to be ≥30 mJ/cm². The optimal initiation time for cold shock of gynogenetic embryos was determined to be 5 min after fertilization, while the optimal temperature and treatment duration were determined to be 20–25 min at 5°C. Chromosomes from common diploids, gynogenetic haploids, and diploids were analyzed. WW chromosomes were discovered in some of the gynogenetic diploids. The microsatellite marker was applied to analyze gynogenetic diploid fry. Among the 30 gynogenetic diploid fry, 11 fry contained only one allele, while 19 contained two alleles, which had the same genotype as their mother. The female-specific DNA marker was observed in four individuals out of ten gynogenetic diploid fry. Ploidy analysis of 20 putative gynogenetic fry showed them all to be diploid. Thus, a protocol for the induction of artificial gynogenesis has been developed for the first time in half smooth tongue sole, and the sex determination mechanism in the tongue sole was determined to be female heterogametic with the ZW chromosome.     

A patched1 gene homologue participates in female differentiation of Cynoglossus semilaevis

Patched (Ptch) is a receptor in the hedgehog signaling pathway, essential for animal development. Our previous study showed that ptch1 gene participates in the maintenance of the male germline and spermatogenesis in Cynoglossus semilaevis (csptch1). In this study, we identified a patched1 gene homolog (csptch1 x1). The csptch1 x1 gene is 5761 bp long, with a 4638 bp coding sequence that encodes 1545 amino acids. The Csptch1 x1 protein has 12 transmembrane regions and sterol-sensing domains and is highly homologous to the csptch1 (91 amino acids difference). Expression pattern analysis showed that csptch1 x1 is expressed in eight different tissues of adult tongue sole, and the expression is significantly higher in tissues of female than that in male tissues. The expression pattern in developmental stages was also analyzed. csptch1 x1 could be detected at the 1-cell stage and was highly expressed at the blastocyst, somite, and blastopore closing stages, implying that it participates in cell differentiation. In ovarian development, the expression of csptch1 x1 was initiated at 20 days after hatching (dah) and was significantly high at 35–50 and 95–150 dah. In situ hybridization showed that csptch1 x1 was predominantly expressed in primordial germ cells, oocytes, and follicular cells, but the expression of the gene was lower in the testis. These results suggest that csptch1 x1 may be mainly involved in female differentiation and ovarian development, different from the role of csptch1 in spermatogenesis.     

Replicated analysis of the genetic architecture of quantitative traits in two wild great tit populations

Currently, there is much debate on the genetic architecture of quantitative traits in wild populations. Is trait variation influenced by many genes of small effect or by a few genes of major effect? Where is additive genetic variation located in the genome? Do the same loci cause similar phenotypic variation in different populations? Great tits (Parus major) have been studied extensively in long‐term studies across Europe and consequently are considered an ecological ‘model organism’. Recently, genomic resources have been developed for the great tit, including a custom SNP chip and genetic linkage map. In this study, we used a suite of approaches to investigate the genetic architecture of eight quantitative traits in two long‐term study populations of great tits—one in the Netherlands and the other in the United Kingdom. Overall, we found little evidence for the presence of genes of large effects in either population. Instead, traits appeared to be influenced by many genes of small effect, with conservative estimates of the number of contributing loci ranging from 31 to 310. Despite concordance between population‐specific heritabilities, we found no evidence for the presence of loci having similar effects in both populations. While population‐specific genetic architectures are possible, an undetected shared architecture cannot be rejected because of limited power to map loci of small and moderate effects. This study is one of few examples of genetic architecture analysis in replicated wild populations and highlights some of the challenges and limitations researchers will face when attempting similar molecular quantitative genetic studies in free‐living populations.     

Impact of cross-ancestry genetic architecture on GWASs in admixed populations

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Mapping the genetic architecture of complex traits in experimental populations

SUMMARY: Understanding how interactions among set of genes affect diverse phenotypes is having a greater impact on biomedical research, agriculture and evolutionary biology. Mapping and characterizing the isolated effects of single quantitative trait locus (QTL) is a first step, but we also need to assemble networks of QTLs and define non-additive interactions (epistasis) together with a host of potential environmental modulators. In this article, we present a full-QTL model with which to explore the genetic architecture of complex trait in multiple environments. Our model includes the effects of multiple QTLs, epistasis, QTL-by-environment interactions and epistasis-by-environment interactions. A new mapping strategy, including marker interval selection, detection of marker interval interactions and genome scans, is used to evaluate putative locations of multiple QTLs and their interactions. All the mapping procedures are performed in the framework of mixed linear model that are flexible to model environmental factors regardless of fix or random effects being assumed. An F-statistic based on Henderson method III is used for hypothesis tests. This method is less computationally greedy than corresponding likelihood ratio test. In each of the mapping procedures, permutation testing is exploited to control for genome-wide false positive rate, and model selection is used to reduce ghost peaks in F-statistic profile. Parameters of the full-QTL model are estimated using a Bayesian method via Gibbs sampling. Monte Carlo simulations help define the reliability and efficiency of the method. Two real-world phenotypes (BXD mouse olfactory bulb weight data and rice yield data) are used as exemplars to demonstrate our methods. AVAILABILITY: A software package is freely available at http://ibi.zju.edu.cn/software/qtlnetwork     

Estimation of genetic parameters for disease‐resistance traits in Cynoglossus semilaevis (Günther, 1873)

The objective of this study was to estimate genetic parameters for three traits based on 28 Cynoglossus semilaevis families approximately 6 months of age (at least 5 cm total length), including trait_1 (survival of 26 families, 3434 individuals in total subjected to challenge tests with Edwardsiella tarda), trait_2 (survival of 20 families, 2016 individuals in total subjected to challenge tests with Vibrio anguillarum) and trait_3 (survival of 27 families, 9340 individuals tagged at circa 180 days of age and reared in indoor ponds for circa another 5 months). The result showed that there were large differences in the survival of the families after challenge (11.11–65.31% for E. tarda and 9.18–70.54% for V. anguillarum). Additionally, the survival of families reared in indoor ponds was also different, varying from 21.00% to 73.67%. Heritabilities of the three traits varied from 0.14 to 0.26, as estimated by the linear model (LM) and the threshold model (TM). The trait_1 heritabilities (0.26 and 0.19 estimated by LM and TM) were higher than those of the others (0.20 and 0.23 estimated by LM, 0.14 and 0.19 estimated by TM). The estimates of heritabilities using LM were consistently higher than those of TM in this study. There were significant medium genetic correlations of 0.44 and 0.42 between trait_1 and trait_2 obtained from LM and TM (P < 0.05). However, very low and non‐significant genetic correlations of trait_1 and trait_3 (?0.10 for LM, ?0.05 for TM), as well as those of trait_2 and trait_3 (0.05 for LM, 0.04 for TM) were obtained. Therefore, indirect selection for trait_1 and trait_2 was effective, but almost ineffectual for trait_1 and trait_3 as well as trait_2 and trait_3. Otherwise, there was no significant difference in the predictive abilities of LM and TM. Two families resistant to both Edwardsiella tarda and Vibrio anguillarum were selected plus one family resistant to both Vibrio anguillarum and naturally infected by unknown pathogens through family selection. As there was very low and non‐significant genetic correlation of trait_3 and trait_1 as well as trait_2, superior strains are anticipated with the ability to resist two or more kinds of diseases, through the crossing of families selected for the three traits described above. The results support the hypothesis that genetic variation exists for disease survival, which could be used to design a breeding program for selecting strains of Cynoglossus semilaevis with high disease resistance.     

半滑舌鳎幼鱼消化酶同功酶谱初步研究

以半滑舌鳎幼鱼为研究材料,利用聚丙烯酰胺非变性电泳和同功酶活性染色对半滑舌鳎消化酶进行了初步分离。蛋白酶采用明胶原位消化法,肝脏蛋白酶检测出3条酶带,其中1条强带,2条弱带;后肠蛋白酶出现了2条酶带,其中有1条强带,1条弱带,并且与肝脏蛋白酶的前2条酶带分别在同一分子量水平上;前肠和中肠蛋白酶均只出现了1条弱带,且在同一分子量水平上。半滑舌鳎肠道各部分的淀粉酶带都在相似分子量附近出现了一条弱酶带,而肝脏在不同的分子量水平出现了1条酶带。肝脏、前肠和后肠脂肪酶均出现了4条酶带,其中,肝脏和前肠均分别有1条强带,1条中强带,2条弱带;后肠脂肪酶则有1条中强带,3条弱带;中肠脂肪酶出现了3条弱带。肝脏和肠道各部分脂肪酶的第一条酶带都在同一分子量水平上,而且活性相似。     

半滑舌鳎侧线器官和无眼侧皮肤表面的特殊结构

为了解决半滑舌鳎的摄食难题,探讨其摄食机理,本文采用光镜和扫描电镜手段对半滑舌鳎有眼侧侧线管和无眼侧皮肤表面的特殊结构进行了研究。结果表明(1)有眼侧:半滑舌鳎侧线孔圆形,孔径与所在部位侧线管径相同,孔上并连有一胶质管,这种特殊结构既可以提高管道内感觉器官(管道神经丘)的敏感性,又可阻止外界异物进入侧线管内部,具有保护作用;半滑舌鳎口腔附近的侧线管管径及侧线孔孔径较其它部位大,侧线孔密度高,认为口腔附近侧线管道内的感觉器官(管道神经丘)的敏感性较其它部位高,在鱼类捕食行为中具有重要作用;(2)无眼侧:躯干部表面覆盖圆鳞,头部皮肤无鳞,表面被覆相互连结的黏液管,形成黏液管皮肤;极其发达的黏液管构成管状黏液分泌系统。扫描电镜观察发现,在头部黏液管皮肤表面镶嵌着一种乳头状突起(Pailla),其典型特征是在表面被覆一盾牌状结构,一般多个簇生在一起,很少单独存在,其分布密度是从吻端向内逐渐减少,组织切片显示内部结构周边是套细胞,中央是感觉细胞,具一柄或两柄。根据其外部形态和内部结构,作者推测,这可能是半滑舌鳎特有的一种触觉器官,并在其摄食行为中起重要作用。半滑舌鳎极其发达的黏液分泌系统对于裸露的乳头状突起(Pailla)具有相当重要的保护作用。     

Development and characterization of 60 novel EST-SSR markers in half-smooth tongue sole Cynoglossus semilaevis

Sixty novel simple sequence repeat (SSR) markers were developed from expressed sequence tags (EST) of half-smooth tongue sole Cynoglossus semilaevis exploited in the laboratory. The number of alleles, observed and expected heterozygosity per locus ranged from two to 16, from 0·0833 to 1·0000 and from 0·0816 to 0·913, respectively. Of these SSRs, 20 had significant homology to known genes by BLASTx (basic local alignment search tool x) search. For cross-species amplification, there are 53 positive amplifications in Japanese flounder Paralichthys olivaceus with 12 polymorphic loci and 51 positive amplifications in Senegalese sole Solea senegalensis with 11 polymorphic loci. These new EST-SSR markers will be useful for genetic studies and genome mapping of C. semilaevis and its closely related fishes.