美国红鱼生长激素基因的克隆及其遗传进化分析

采用PCR方法从美国红鱼肝脏中扩增出美国红鱼生长激素基因。该基因序列长为1 497 bp,与5个已报道的鲈亚目鱼类生长激素基因结构比较发现,他们的外显子和内含子之比均为6∶5,而且相对应的6个外显子大小一致。用Clustal X软件对美国红鱼和其他13种鲈形目鱼类生长激素基因序列进行比对,选取同源性较高的551 bp的编码区序列构建NJ和MP系统进化树,结果发现由其构建的MP和NJ树与根据形态特征构建的系统发育树基本一致,特别是在鲈形目鱼类科间及科以上分类阶元的系统发育研究方面比较一致。研究结果表明鱼类生长激素基因序列能够较真实反映近缘鱼类之间的关系,是进行鲈形目鱼类系统进化分析的较好遗传标志。     

大黄鱼TRIM25基因克隆和表达分析

三重基序蛋白25 (Tripartite motif-containing protein 25, TRIM25)属于E3泛素连接酶家族, 在先天免疫反应中发挥重要作用。为研究TRIM25基因在大黄鱼(Larimichthys crocea)先天抗病毒免疫反应中的作用, 研究鉴定并克隆大黄鱼TRIM25基因(命名为LcTRIM25)。LcTRIM25基因编码序列2097 bp (GenBank登录号: MK327541), 编码698个氨基酸。蛋白结构域预测发现LcTRIM25包括保守的RING结构域、B-box2结构域、Coiled-coil结构域和可变的C末端PRY/SPRY结构域。多序列比对以及系统进化树分析表明LcTRIM25基因与斜带石斑鱼同源性高, 与哺乳动物、爬行动物、两栖动物和鸟类同源性相对低, 这说明不同物种受到来自环境不同的选择压力, 导致进化程度不同。应用实时荧光定量PCR方法分析大黄鱼TRIM25基因的表达水平。结果分析发现LcTRIM25基因在健康大黄鱼的9个组织中均有广泛表达, 且在肝脏中表达量最高, 在心脏中表达量最低。在poly(I:C)刺激后, 在外周血、头肾、脾脏和肝脏中LcTRIM25基因表达量迅速且明显上调, 均出现上升达到峰值后下降的趋势。LcTRIM25基因表达量在头肾和脾脏中6h达到最高表达量, 在肝脏中12h达到峰值, 外周血中在24h达到最高表达量。上述结果表明, 不同组织中LcTRIM25基因表达模式具有差异性。研究结果推测大黄鱼TRIM25基因参与抗病毒免疫反应且发挥十分关键的作用, 为进一步了解大黄鱼抗病毒免疫机制提供理论基础。     

Microsatellite–Centromere Mapping in Large Yellow Croaker (<Emphasis Type="Italic">Pseudosciaena crocea</Emphasis>) Using Gynogenetic Diploid Families

The large yellow croaker (Pseudosciaena crocea) is an economically important marine fish in China. Inheritance of 22 heterozygous microsatellite loci was examined in normal crossed diploid families and meio-gynogenetic families in P. crocea. Two gynogenetic families were produced via inhibition of the second polar body in eggs fertilized with UV-irradiated sperm. The ratio of gynogenesis was proven to be 100% and 96.9% in the two families, respectively. Of the 22 examined loci, 4 showed a segregation distortion in both control and gynogenetic families. Microsatellite–centromere (M–C) map distances were examined using 18 loci with normal Mendelian segregation. Estimated recombination rates ranged between 0 and 1.0 under the assumption of complete interference. High recombinant frequencies between heterozygous markers and the centromere were found in large yellow croaker, as in other teleosts. The average recombination frequency was 0.586. Ten loci showed high M–C recombination with frequency greater than 0.67. M–C distances provide useful information for gene mapping in large yellow croaker.     

养殖大黄鱼一个繁育群体亲本亲缘关系剖析

该文利用23个微卫星标记对103尾大黄鱼繁育亲本进行遗传多样性检测和亲缘关系重建分析,并构建繁育体系指导大黄鱼配组。遗传多样性检测显示,103尾亲鱼在23个座位共获得等位基因数134个,平均5.82个,总平均观察杂合度0.599 3,表明该繁育群体尚保持一定水平的遗传多样性。采用似然率和组合优化法统计模型重建的同胞关系不尽相同,但结果均证实了这些繁育亲本亲缘关系十分相近。配组比对分析结果显示,两种方法的配组方案一致性高达85%,最终选择组合优化法的分组方案指导大黄鱼配组繁育。     

大黄鱼ATG10基因的分子特征及其在抗病毒免疫中的功能

为了研究自噬相关基因ATG10(Autophagy related gene 10)在鱼类免疫应答中的功能, 研究克隆了大黄鱼(Larimichthys crocea)ATG10基因(LcATG10)的cDNA序列, 其开放阅读框全长747个核苷酸, 编码248个氨基酸的蛋白, 包含1个Autophagy_act_C结构域。系统进化分析显示, LcATG10和其他硬骨鱼类ATG10聚成一支, 与金头鲷和石斑鱼ATG10亲缘关系最近。LcATG10在所检测的正常大黄鱼各组织中都有表达。在病毒类似物poly (I:C)刺激后, 大黄鱼脾脏和头肾组织中LcATG10的表达水平显著上调, 分别在12h和6h达到峰值(9.4和5.9倍)。LcATG10在大黄鱼头肾细胞系(LYCK)、原代巨噬细胞、淋巴细胞和粒细胞中也均有表达, 在原代粒细胞中的表达量相对较高; 在poly (I:C)刺激后, 大黄鱼原代头肾粒细胞和LYCK细胞中LcATG10的表达水平显著上调。过表达LcATG10的鲤上皮瘤(Epithelioma papulosum cyprinid, EPC)细胞受鲤春病毒血症病毒(Spring viremia of carp virus, SVCV)感染48h后, 细胞病变效应(Cytopathic effects, CPEs)明显低于对照组; 细胞培养上清中SVCV病毒滴度为103.55 TCID₅₀/mL, 显著少于对照组; SVCV标志基因SVCV- G、SVCV-M和SVCV- P的表达水平也显著低于对照组, 分别是对照组的0.022、0.015和0.022倍。这些研究结果表明LcATG10在大黄鱼抗病毒免疫应答中发挥作用, 为深入研究自噬在大黄鱼抗病毒免疫中的分子机制奠定了基础。     

Molecular cloning and functional characterization of a RabGTPase in large yellow croaker (Pseudosciaena crocea)

The molecular mechanisms of the immune system against pathogens in large yellow croaker (Pseudosciaena crocea) are not well known, despite its economic importance as an aquaculture species. In this investigation, a Rab gene (named as LycRab gene) was obtained from this fish, which exhibited high homology with Rab8 of other species. It was expressed in Escherichia coli, and the specific antibody was raised using the purified fusion protein (GST-LycRab). The LycRab protein, containing characteristic signatures of Rab proteins with 5 GTP-binding domains, had GTP-binding activity. The LycRab gene was ubiquitously expressed in all analyzed tissues as revealed by Western blot, although expression levels varied from tissue to tissue. Real-time PCR revealed that the LycRab gene was up-regulated after immunization with poly I:C, formalin-inactive Gram-negative bacterium Vibrio parahaemolyticus or bacterial lipopolysaccharides (LPS), suggesting that LycRab protein might play an important role in large yellow croaker defense against pathogens infection. This discovery might contribute better understanding to the molecular events involved in fish immune responses.     

大黄鱼CCT基因的克隆及其表达量随稚鱼生长发育的变化

研究旨在克隆大黄鱼磷脂酰胆碱合成关键基因磷脂酰胆碱胞苷转移酶 (CCT)基因全长, 并检测其表达量随稚鱼生长发育的变化。利用同源克隆技术和RACE技术从大黄鱼肝脏中成功扩增出CCT的全长。同时应用real-time PCR法检测不同日龄大黄鱼稚鱼CCT的表达变化。序列分析表明, CCT全长2419 bp(Genbank登录号: KF006239.1), 包括273 bp 的5'端非编码区, 1107 bp的开放阅读框, 1010 bp的3'端非编码区,共编码369个氨基酸。系统进化树分析表明, 相比其他物种, 大黄鱼CCT基因与红鳍东方鲀的亲缘关系较近。定量结果表明, 孵化后, 大黄鱼仔稚鱼CCT的表达量随日龄的变化先显著升高, 在15日龄时达到最大值,随后显著下降并趋于平稳, CCT基因表达量的变化趋势与大黄鱼稚鱼消化系统的发育密切相关。     

EST analysis and identification of gonad-related genes from the normalized cDNA library of large yellow croaker,Larimichthys crocea

On grounds of the especially limited numbers of identified gonad-specific or gonad-related genes of large yellow croaker Larimichthys crocea which may represent a major obstacle for the study of gonad development and sex differentiation, we initiated a sequencing program of Expressed Sequence Tags (ESTs) in large yellow croaker. In this study, we firstly constructed a normalized gonad cDNA library using the combination of SMART technique and DSN treatment. The titer of amplified cDNA library was 4.8 × 10¹¹ and the percentage of unique cDNA sequences of the library was 82.49%. 2916 unique cDNAs were clustered from the 3535 high quality ESTs. Among the 1785 ESTs which had significant homology with known genes in the NCBI database, about 64 significant gonad-related genes were found, accounting for 3.59% of the total unique cDNAs. Specifically, the testis-specific LRR gene and testis-specific chromodomain Y-like protein gene were identified from fish for the first time. Six gonad-related microsatellite-containing ESTs were identified from the 129 ESTs containing 149 microsatellites. Expression patterns of 10 of these gonad-related gene homologues in ovaries and testes were examined by qRT-PCR. The results will be powerful resources for our further investigation to establish the molecular mechanisms of gonad development and sex differentiation in large yellow croaker.     

Genetic Mapping and QTL Analysis of Growth Traits in the Large Yellow Croaker Larimichthys crocea

Large yellow croaker (Larimichthys crocea) is an important maricultured species in China. A genetic linkage map of the large yellow croaker was constructed using type II microsatellites and expressed sequence tag (EST)-derived microsatellites in two half-sib families (two females and one male). A total of 289 microsatellite markers (contained 93 EST-SSRs) were integrated into 24 linkage groups, which agreed with the haploid chromosome number. The map spanned a length of 1,430.8 cm with an average interval of 5.4 cm, covering 83.9 % of the estimated genome size (1,704.8 cm). A total of seven quantitative trait locis (QTLs) were detected for growth traits on five linkage groups, including two 1 % and five 5 % chromosome-wide significant QTLs, and explained from 2.33 to 5.31 % of the trait variation. The identified QTLs can be applied in marker-assisted selection programs to improve the growth traits.     

Using all Gene Families Vastly Expands Data Available for Phylogenomic Inference

Traditionally, single-copy orthologs have been the gold standard in phylogenomics. Most phylogenomic studies identify putative single-copy orthologs using clustering approaches and retain families with a single sequence per species. This limits the amount of data available by excluding larger families. Recent advances have suggested several ways to include data from larger families. For instance, tree-based decomposition methods facilitate the extraction of orthologs from large families. Additionally, several methods for species tree inference are robust to the inclusion of paralogs and could use all of the data from larger families. Here, we explore the effects of using all families for phylogenetic inference by examining relationships among 26 primate species in detail and by analyzing five additional data sets. We compare single-copy families, orthologs extracted using tree-based decomposition approaches, and all families with all data. We explore several species tree inference methods, finding that identical trees are returned across nearly all subsets of the data and methods for primates. The relationships among Platyrrhini remain contentious; however, the species tree inference method matters more than the subset of data used. Using data from larger gene families drastically increases the number of genes available and leads to consistent estimates of branch lengths, nodal certainty and concordance, and inferences of introgression in primates. For the other data sets, topological inferences are consistent whether single-copy families or orthologs extracted using decomposition approaches are analyzed. Using larger gene families is a promising approach to include more data in phylogenomics without sacrificing accuracy, at least when high-quality genomes are available.     

大黄鱼连续两代雌核发育群体的微卫星标记分析

通过对大黄鱼(Pseudosciaena crocea)异质雌核发育一代群体(meio-G1)与二代群体(meio-G2)微卫星位点的纯合度进行分析,研究异质雌核发育对大黄鱼基因纯化的效率。结果显示:meio-G1和meio-G215个微卫星座位的平均纯合度分别为0.661和0.803,纯合位点比例最高个体分别为0.867(13/15)和0.933(14/15),两个群体内个体间的平均相似系数分别为0.5903和0.8672,最高分别达0.9286和1.0(遗传距离为0.0741和0),远高于两性交配繁殖群体(平均纯合度0.376,平均相似系数0.4687,个体间最小遗传距离0.2288);其中meio-G2群体有7个位点(46.7%)已经完全纯合固定,并与普通养殖群体产生较明显的遗传分化;表明人工诱导异质雌核发育可大大加速大黄鱼大多数基因位点的纯合,是快速建立高纯品系的有效手段。但不同位点的纯合度差异很大,部分位点在异质雌核发育后代中迅速纯合,在meio-G1中就达到很高的纯合度,而有些位点则在meio-G1和meio-G2中仍保持很高的杂合度;meio-G1和meio-G2群体中不同个体纯合位点比例差异也很大。研究培育的雌核发育群体为大黄鱼进一步选育提供了良好的遗传材料。       

Perils of paralogy: using HSP70 genes for inferring organismal phylogenies

Conserved genes have found their way into the mainstream of molecular systematics. Many of these genes are members of multigene families. A difficulty with using single genes of multigene families for phylogenetic inference is that genes from one species may be paralogous to those from another taxon. We focus attention on this problem using heat shock 70 (HSP70) genes. Using polymerase chain reaction techniques with genomic DNA, we isolated and sequenced 123 distinct sequences from 12 species of sharks. Phylogenetic analysis indicated that the sequences cluster with constituitively expressed cytoplasmic heat shock-like genes. Three highly divergent gene clades were sampled. A number of similar sequences were sampled from each species within each distinct gene clade. Comparison of published species trees with an HSP70 gene tree inferred using Bayesian phylogenetic analysis revealed several cases of gene duplication and differential sorting of gene lineages within this group of sharks. Gene tree parsimony based on the objective criteria of duplication and losses showed that previously published hypotheses of species relationships and two novel hypothesis based on Bayesian phylogenetics were concordant with the history of HSP70 gene duplication and loss. By contrast, two published hypotheses based on morphological data were not significantly different from the null hypothesis of a random association between species relatedness and the HSP70 gene tree. These results suggest that gene tree parsimony using data from multigene families can be used for inferring species relationships or testing published alternative hypotheses. More importantly, the results suggest that systematic studies relying on phylogenetic inferences from HSP70 genes may by plagued by unrecognized paralogy of sampled genes. Our results underscore the distinction between gene and species trees and highlight an underappreciated source of discordance between gene trees and organismal phylogeny, i.e., unrecognized paralogy of sampled genes.     

基于集成模型的小黄鱼越冬群体适宜生境及其环境影响因素

小黄鱼是中韩渔业共同利用鱼种,其跨界洄游习性限制了对越冬场范围的调查和评估,导致对越冬群体适宜栖息地分布缺乏了解。本研究基于越冬期我国自然海域的物种分布点位数据和5个环境数据,运用8个物种分布模型(SDM)分析了小黄鱼越冬场分布范围,采用5折交叉验证,利用受试者工作特征曲线下面积(AUC)评价模型预测性能,并通过加权集成方法构建综合生境模型预测越冬场核心分布位置。结果表明: 出现/未出现数据模型预测准确度普遍高于仅出现模型;在出现/未出现数据模型中,机器学习方法预测准确度高于经典回归模型,支持向量模型(SVM)准确度最高(AUC=0.85),广义线性模型(GLM)准确度最低(AUC=0.73)。集成模型AUC较单一独立模型的准确度有所提升,表明集成模型能有效降低单一独立模型所带来的不确定性,提高模型预测准确度。变量重要性分析结果显示,盐度和温度是决定小黄鱼越冬场地理分布的重要因素,适宜分布区集中在黄海南部外海、东海北部外海和浙江省沿岸海域,而黄海南部沿岸海域和东海中南部外海为不适宜越冬区。研究结果为预测小黄鱼潜在越冬场提供了理论基础,可支撑越冬场渔业资源的空间规划和可持续利用。     

大黄鱼与小黄鱼细胞色素b基因全序列的比较分析

对大黄鱼、小黄鱼线粒体细胞色素b基因进行了PCR扩增及序列测定,得到1140bp的全序列。大黄鱼和小黄鱼的碱基组成相似,前者T、C、A、G含量分别为28.4%、33.0%、23.2%和15.4%,A+T含量为51.6%;后者T、C、A、G含量分别为26.7%、34.1%、23.8%和15.4%,A+T含量为50.5%。大、小黄鱼cytb基因中三联体密码子中碱基的使用频率很相似,第一位较均一,第二位富含T,第三位富含C。大小黄鱼cytb基因存在明显差异,序列相似性仅为88.95%;两序列间具有126个差异位点;碱基转换/颠换率为3.1,碱基替换多发生在密码子第三位;碱基转换中C\T显著高于A\G,表现出转换偏歧。     

Rapid construction of genome map for large yellow croaker (Larimichthys crocea) by the whole-genome mapping in BioNano Genomics Irys system

Background

Large yellow croaker (Larimichthys crocea) is an important commercial fish in China and East-Asia. The annual product of the species from the aqua-farming industry is about 90 thousand tons. In spite of its economic importance, genetic studies of economic traits and genomic selections of the species are hindered by the lack of genomic resources. Specifically, a whole-genome physical map of large yellow croaker is still missing. The traditional BAC-based fingerprint method is extremely time- and labour-consuming. Here we report the first genome map construction using the high-throughput whole-genome mapping technique by nanochannel arrays in BioNano Genomics Irys system.

Results

For an optimal marker density of ~10 per 100 kb, the nicking endonuclease Nt.BspQ1 was chosen for the genome map generation. 645,305 DNA molecules with a total length of ~112 Gb were labelled and detected, covering more than 160X of the large yellow croaker genome. Employing IrysView package and signature patterns in raw DNA molecules, a whole-genome map of large yellow croaker was assembled into 686 maps with a total length of 727 Mb, which was consistent with the estimated genome size. The N50 length of the whole-genome map, including 126 maps, was up to 1.7 Mb. The excellent hybrid alignment with large yellow croaker draft genome validated the consensus genome map assembly and highlighted a promising application of whole-genome mapping on draft genome sequence super-scaffolding. The genome map data of large yellow croaker are accessible on lycgenomics.jmu.edu.cn/pm.

Conclusion

Using the state-of-the-art whole-genome mapping technique in Irys system, the first whole-genome map for large yellow croaker has been constructed and thus highly facilitates the ongoing genomic and evolutionary studies for the species. To our knowledge, this is the first public report on genome map construction by the whole-genome mapping for aquatic-organisms. Our study demonstrates a promising application of the whole-genome mapping on genome maps construction for other non-model organisms in a fast and reliable manner.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1871-z) contains supplementary material, which is available to authorized users.     

Lack of genetic structure in endangered large yellow croaker Larimichthys crocea from China inferred from mitochondrial control region sequence data

The large yellow croaker Larimichthys crocea is an important commercial marine fish species in China. However, information about the population structure of this species is limited. In the present study, mitochondrial DNA (mtDNA) control region was sequenced from four populations of the yellow croaker in the southern Yellow Sea, East China Sea and South China Sea to investigate the genetic structure of this species. A total of 54 haplotypes were detected among 62 individuals of large yellow croaker. High levels of population genetic diversity were observed. Among the four populations, the haplotype diversity was between 0.9895 ± 0.0193 (Xiamen) and 1.0000 ± 0.0524 (Lvsi, Zhoushan). The nucleotide diversity ranged from 0.0208 ± 0.0108 (Xuwen) to 0.0246 ± 0.0138 (Lvsi). The results of AMOVA detected no significant differences among populations. The conventional F_ST statistics were negative and insignificant values. These indicated lack of population genetic structure throughout the Yellow Sea, East China Sea and South China Sea, and random mixing of individuals among the samples. Biological characters of large yellow croaker and lack of physical barrier in the studied area could be the reasons for lack of genetic structure in this species.     

Genetic divergence and historical demography in the endangered large yellow croaker revealed by mtDNA

Due to high level of fishing, many marine fish species are in danger of becoming extinct. The large yellow croaker, Larimichthys crocea, was once an important commercial marine species in China. At present, this croaker is seldom captured in the wild. In this work, mitochondrial DNA data was obtained from five populations of the yellow croaker from the Yellow Sea and the East China Sea to investigate their genetic divergence and demographic history. Results of phylogenetic analysis suggest two genetic lineages for this croaker, although the divergence was relatively shallow. This might reflect the effects of glacial isolation on population structure. Furthermore, our Bayesian based approach showed that the effective population size for both genetic lineages experienced recent sharp decline. This study provides new insights into the genetic divergence and historical demography of this croaker. Strict measures must be taken to protect this species.     

大黄鱼HIF-1α基因的克隆鉴定与表达分析

为研究低氧诱导因子1α(Hypoxia-inducible factor-1α, HIF-1α)在鱼类免疫应答中的作用, 研究克隆得到了大黄鱼(Larimichthys crocea)HIF-1α基因(LcHIF-1α)的cDNA序列, 其开放阅读框全长2256个核苷酸, 编码一个751个氨基酸的蛋白。通过氨基酸序列比对发现, LcHIF-1α蛋白具有5个典型的功能结构域, 包括1个helix-loop-helix(HLH)结构域, 2个PAS结构域, 1个DNA结合结构域(HIF-1)和1个羧基端反式激活结构域(HIF-1a_CTAD)。系统进化分析显示, LcHIF-1α和其他硬骨鱼类HIF-1α聚成一支, 与两栖类、鸟类和哺乳类HIF-1α的亲缘关系相对较远。序列分析结果表明, LcHIF-1α具有保守的特征结构域, 预示着其功能可能与哺乳动物HIF-1α类似。荧光定量PCR结果显示, LcHIF-1α在所检测的健康大黄鱼各个组织中都有表达, 在溶藻弧菌(Vibrio alginolyticus)感染后, 大黄鱼脾脏和头肾组织中的LcHIF-1α表达水平显著上调, 达到峰值时分别上升了6.8倍和2.9倍。此外, LcHIF-1α在大黄鱼的3种免疫细胞(中性粒细胞、巨噬细胞和淋巴细胞)中也都有表达, 在中性粒细胞和巨噬细胞中的表达量相对较高; 在脂多糖(LPS)诱导后, 中性粒细胞和巨噬细胞中LcHIF-1α的表达量显著增加, 峰值分别是对照组的2.6倍和1.8倍, 这些结果表明LcHIF-1α可能参与调控大黄鱼抗细菌免疫应答。