裸体异鳔鳅(鱼它)人工繁殖与胚胎发育研究

为了解裸体异鳔鳅(鱼它)(Xenophysogobio nudicorpa)人工繁殖和胚胎发育规律,以从金沙江采集到的10尾雌鱼(平均体长17.5 cm,体质量22.3 g)、8尾雄鱼(平均体长15.6 cm,体质量17.5 g)作为亲本,分3批次开展了人工繁殖试验,并观察在水温(20.0±0.5)℃下该鱼的胚胎发育情况。试验结果显示：在水温(15.0±0.5)、(18.0±0.5)、(20.0±0.5)℃的条件下催产,催产剂(雌鱼第1针注射LHRH-A₃ 2μg/kg,第2针注射LHRH-A₃ 10μg/kg+HCG 1 200 IU/kg+PG 8 mg/kg,间距12 h;雄鱼不注射)的效应时间分别为21、15、14 h,受精率分别为8.5%、76.8%、74.5%。将受精卵在水温(15.0±0.5)℃条件下孵化288 h,大部分仔鱼未能正常出膜;当水温升至(18.0±0.5)℃,大部分仔鱼在孵化168 h后破膜而出,但仍有小部分无法正常出膜;当水温升至(20.0±0.5)℃时,仔鱼正常出膜,且出膜时间缩短至120 h。裸体异鳔鳅(鱼它...     

基于转录组测序的兴国红鲤微卫星标记筛选

采用Illumina高通量测序技术,对兴国红鲤(Cyprinus carpio var.singuonensis)垂体和性腺等组织进行转录组测序分析,筛选微卫星标记并分析其组成及特征。结果显示:共获得13 652个微卫星标记,对所得位点进行分类,单核苷酸重复类型占47.86%,二、三、四、五、六核苷酸重复类型所占比例分别为34.43%、16.32%、1.25%、0.12%以及0.02%。随机选取30个SSR位点进行PCR验证,有20对可以扩增出清晰稳定的条带。在24个兴国红鲤个体中对上述位点进行多态性分析,结果表明,具有多态性的微卫星引物为9对。不同位点得到的等位基因范围为2~4,平均等位基因数为3.111 1±0.993 8,观测杂合度(Ho)、期望杂合度(He)以及多态信息含量(PIC)平均值分别为0.597 2±0.233 2、0.539 7±0.178 0和0.467 2±0.172 1。9个微卫星位点中,有4个显著偏离哈代-温伯格平衡(Hardy-Weinberg equilibrium,HWE)(P0.05)。Illumina高通量测序提供了一种直观、高效开发微卫星标记的方法,所选兴国红鲤群体遗传多样性维持较好。     

基于高通量测序的虾夷扇贝基因组微卫星特征分析

为全面了解虾夷扇贝(Patinopecten yessoensis)微卫星分布频率和数量,深化对虾夷扇贝基因组的认识,本研究运用第二代高通量测序技术,进行虾夷扇贝简化基因组测序(RAD-seq),从基因组水平阐明虾夷扇贝基因组微卫星特征。结果显示,简化基因组测序共获得序列总长为92,551,435 bp,经过滤筛选,获得259,535个contig,其中,包含微卫星序列3618条,经引物设计共获得3460对微卫星引物。统计微卫星序列的重复类型,其中,三核苷酸重复单元数量最多(1587个,45.87%),其次是二核苷酸重复(1282个,37.05%),六核苷酸重复单元数量最少(20个,0.58%)。在三核苷酸重复中,以ATA重复类型所占比例最高(11.41%),共计181个。此外,虾夷扇贝同一重复类型的微卫星随着重复数增加其数量相对减少,而相同重复数的微卫星随着重复单元长度的增加其数量也呈下降趋势,可见微卫星长度与其数量呈负相关,表明长度较短的微卫星变异速率较快。本研究结果为认识虾夷扇贝基因组特征和在基因组水平开展种群遗传学研究提供了基础数据。     

基于线粒体控制区D-loop序列的内蒙古大鳍鼓鳔鳅群体遗传结构分析

为了解内蒙古地区大鳍鼓鳔鳅(Herzenstein)群体的遗传背景和分化情况,对该地区52个大鳍鼓鳔鳅样本的线粒体控制区D-loop序列进行了遗传多样性和遗传分化分析.结果显示,在947 bp的D-loop区序列中,碱基A、T、C、G的平均含量分别为34.5％、28.0％、21.3％、16.2％,表现出较强的AT偏好性...     

长牡蛎食物组成的高通量测序分析

为了更加细致地甄别滤食性贝类的食物组成,于2019年8月,以北方规模化典型养殖海湾——桑沟湾养殖的长牡蛎(Crassostrea gigas)为研究对象,运用Illumina高通量测序技术对长牡蛎的胃含物及所处养殖水体中的真核生物进行分析研究.结果显示,扩增18S rDNA V4区平均得到111,359个有效序列短片段...     

基于镜检和高通量测序技术的铜锈环棱螺胃含物分析

以养殖的铜锈环棱螺(Bellamya aeruginosa)为研究对象,采用镜检和高通量测序技术分析其胃含物情况。结果显示：铜锈环棱螺的摄食率为86.87%,其胃含物以浮游植物和碎屑为主,其中胃内主要的食物成分是小球藻(Chlorella)和裸藻(Euglena),后肠中则以隐藻和颤藻为主;铜锈环棱螺胃内微生物菌群主要由变形菌门、放线菌门、拟杆菌门、厚壁菌门等23门和盐单胞菌属(Halomonas)、乳球菌属(Lactococcus)、肉杆菌属(Carnobacterium)等88属组成,其中真菌类群主要由子囊菌门、担子菌门等5门和马拉色氏霉菌属(Malassezia)、曲霉菌属(Aspergillus)、被孢霉属(Mortierella)等50属组成。研究结果可为铜锈环棱螺饵料生物学和生态食物网研究提供基础资料,为开展其人工养殖提供基础生物学基础。     

基于高通量测序技术分析微冻凡纳滨对虾的微生物

文章以微冻贮藏的凡纳滨对虾(Litopenaeus vannamei)整虾、去头尾虾和虾仁的表面微生物作为研究对象,采用高通量测序技术鉴定不同贮藏时期样品菌群的组成及变化情况.结果显示:1)各组样品的操作分类单元(Operational taxonomic unit,OTU)随贮藏时间的推移呈下降趋势,贮藏4周后虾仁的...     

基于高通量测序的长江口中华绒螯蟹雌蟹食性分析

为探究长江中华绒螯蟹雌蟹抱卵前后的食性,在长江口东滩水域采集生殖洄游期中华绒螯蟹雌蟹,采用高通量测序技术,解析其抱卵前后的食物种类组成及其食性特征。试验结果显示,37个食物样品的高质量序列为53 288～90 598个。通过聚类分析共得到423个运算分类单元(OTU),筛选有效序列后,经比对获得的食物类别包括藻类、高等植物、鱼类、甲壳类、软体动物、轮虫、原生动物等。雌蟹抱卵前食物组成中占比较高的类别为高等植物、甲壳动物和鱼类;而在抱卵后食物组成中占比较高的种类为高等植物和藻类。抱卵前的Chao1指数和ACE丰富度指数显著大于抱卵后的,抱卵前、后的Shannon和Simpson多样性指数均无显著性差异。研究结果表明,中华绒螯蟹雌蟹摄食宽度较广,属于机会主义摄食者,亲蟹抱卵前后的食性具有一定的差异性。研究结果揭示了生殖洄游期中华绒螯蟹雌蟹的食物种类组成及雌蟹在抱卵前、后的食性变化特征,并为长江口中华绒螯蟹资源增殖和保护提供科学依据。高通量测序分析技术具有生物检测灵敏度较高的特点,可在一定程度上揭示水生动物的食物种类组成信息,但仍需与传统的胃含物形态学分析方法相结合,以便更准确全面地解析其食...     

Phytoplankton community structure and diversity in the indoor industrial aquaculture system for Litopenaeus vannamei revealed by high‐throughput sequencing and morphological identification

The explosive multiplication of phytoplankton caused by water eutrophication often occur in the intensive shrimp aquaculture. To comprehensively assess the diversity and community structure of phytoplankton in the waters of typical indoor industrial aquaculture system for Litopenaeus vannamei, a combination of high‐throughput sequencing and morphological identification methods were used in the present study. A total of 41 genera belong to nine phyla were detected by both methods. Chlorophyta, Cyanophyta and Bacillariophyta were found to be three dominant phyla. The high‐throughput sequencing revealed that green algae and cyanobacteria were the most dominant phytoplankton; however, diatoms were the first dominant phytoplankton by using the morphological identification. At the genus level, Picochlorum and Synechococcus were dominant, accounting for 20.94%–97.19% and 0.01%–52.81% of total phytoplankton, revealed by the high‐throughput sequencing. Therefore, more attention should be paid to their ecological impacts on the surrounding sea areas or potential toxicity to shrimp. Cyclotella was the most dominant genus revealed by the morphological identification. High‐throughput sequencing revealed a high diversity and small‐sized phytoplankton which were undetected by microscopy. Both methods provide similar information on the environmental drivers of phytoplankton community. NO₃^?, NH₄⁺, DIP, DSi, DON and DOP concentrations were the main factors influencing the phytoplankton community structure and diversity.     

Development of simple sequence repeat markers in Pyropia yezoensis (Bangiales,Rhodophyta) by high‐throughput sequencing technology

Pyropia yezoensis is one of the most important economical seaweeds across the world and major efforts are underway to increase the production. However, its genome is relatively unexplored. In this study, genome sequence of wild‐type strain LS was determined through paired‐end sequencing of small‐size fractionated genomic library. In total, 283,606 contigs were assembled and 20,620 SSR‐containing sequences were identified. Most of the SSRs contained tri‐ and di‐nucleotide motifs (95.42% and 2.34% respectively), of which GCC was the most abundant (15.7%). Furthermore, 1,253 pairs of primer sets were designed and 124 of them were selected randomly for validation. The results showed that 120 pairs were successful in PCR, and the remaining four failed to generate PCR product at various conditions. Among the 120 pairs of which the PCR products were subsequently sequenced by Sanger sequencing, 104 pairs amplified SSRs with the same motif and repeat times, three pairs with the same motif but different repeat times and 13 pairs with different motifs. Moreover, 21 primer pairs amplified polymorphic products between LS and an improved strain HT, and tri‐nucleotide SSRs showed higher polymorphism frequency when compared to di‐nucleotide SSRs. These SSR markers may enrich the current molecular resources in P. yezoensis.     

Effects of a probiotic (Bacillus subtilis FY99‐01) on the bacterial community structure and composition of shrimp (Litopenaeus vannamei,Boone) culture water assessed by denaturing gradient gel electrophoresis and high‐throughput sequencing

In China, many commercial probiotics are added as water additives into shrimp culture ponds with no sediment. In these ponds, probiotics should have great effects on the bacterial community of culture water at different culture stages. However, the specific effects have not been clarified yet. In this study, the effects of applying the probiotic Bacillus subtilis FY99‐01 on the bacterial community of shrimp (Litopenaeus vannamei) culture water at different culture stages were determined by denaturing gradient gel electrophoresis and high‐throughput sequencing. The water quality parameters, shrimp production, survival rate and feed conversion ratio (FCR) were also analysed. The results showed that application of probiotics improved the water quality by reducing the levels of pH, nitrite and soluble reactive phosphorus, whereas there was no significant effect on shrimp production, survival rate and FCR. Application of probiotics brought no obvious effect on bacterial α diversity while had effect on bacterial β diversity. In addition, probiotics brought different effects on the bacterial community structure at different culture stages, and the effects are stronger in the early and middle phase than in the late phase. Sequencing results demonstrated that probiotics could increase the abundance of Flavobacteria, whereas decrease the abundance of α‐Proteobacteria in the late phase of culture. Besides, principle component analysis results indicated that using probiotics also could increase the abundance of beneficial microalgae (Bacillariophyta and Chlorophyta) and decrease the abundance of Vibrionaceae.     

西南大西洋阿根廷滑柔鱼遗传多样性的微卫星分析

西南大西洋阿根廷滑柔鱼(Illex argentinus)是重要的经济大洋性头足类,其种群结构较为复杂。本研究采用8个微卫星标记,对西南大西洋阿根廷滑柔鱼群体的遗传多样性进行了检测。8个基因座位上,共检测出172个等位基因,每个基因座位检测到10～30个等位基因不等。8个位点的平均观测等位基因数为21.500 0,平均有效等位基因数为12.074 1。各位点的观测杂合度(Ho)变化范围为0.300 0～0.775 0,期望杂合度(He)为0.484 2～0.977 0,平均观测杂合度为0.504 6,平均期望杂合度为0.873 4。8个微卫星位点的多态信息含量(PIC)平均值为0.853 6。以卡方检验估计Hardy-Weinberg平衡,发现8个基因座位均不符合Hardy-Weinberg平衡。Fst分析显示该群体无明显的遗传分化。研究结果揭示:西南大西洋阿根廷滑柔鱼种群显示了高水平的微卫星多态性,该海域阿根廷滑柔鱼群体的遗传多态性水平在经历了近十年的密集捕捞压力后没有明显的变化,同时证明微卫星分子标记是研究该物种自然种群的有利工具。     

微卫星标记分析大鳞鲃养殖群体的遗传结构及生长性状

大鳞鲃作为水产引进种,受建群数量较小的影响其遗传资源相对有限,因此在育种实践中有效地保护和利用现有的基因资源显得尤为重要。实验用24个微卫星标记分析了大鳞鲃养殖群体的遗传结构,在随机采样的96个个体中共检测到74个等位基因,各标记等位基因数为2～5个,片段大小为109～367 bp,有效等位基因数(Ne)为1.144 5～4.626 4,观测杂合度(Ho)为0.135 4～1.000 0,期望杂合度(He)为0.126 9～0.788 1,标记的多态信息含量(PIC)为0.118 3～0.749 0,平均为0.428 1。统计结果显示,大鳞鲃养殖群体处于中度多态水平;经χ2检验估计Hardy-Weinberg平衡,结果表明,大鳞鲃养殖群体处于平衡状态,但有8个微卫星标记显著偏离了平衡。利用SPSS 19.0的GLM模型分析24个微卫星标记与体质量、体长、体高和体厚的相关性,结果表明,B1、B20、B45、B51、B59、BC38与4项生长性状均具有显著相关性,B26与体质量、体高和体厚具有显著相关性,BC3与体质量和体长具有显著相关性,进而使用Duncan氏多重比较找到了每个微卫星标记具有生长性状优势的基因型。     

4 个斧文蛤群体微卫星标记的遗传多样性分析

为研究斧文蛤(Meretrix lamarckii)不同地理群体(浙江苍南群体、福建长乐群体、福建宁德群体以及广东汕头群体)的遗传结构和系统发生关系,采用13对微卫星分子标记,对4个不同地理群体进行了分析。结果显示,13对引物共检测出63个等位基因,每个位点等位基因数(Na)2~7个,平均每个位点观测等位基因数(Na)为4.87;有效等位基因数(Ne)为1.927~2.591;平均观测杂合度(Ho)为0.437~0.562;平均期望杂合度(He)为0.446~0.549;平均多态信息含量(PIC)为0.383~0.490;Hardy-Weinberg平衡检验表明,4个群体的大部分微卫星位点都偏离平衡状态(P0.05);UPMGA聚类分析表明,浙江苍南群体与福建宁德群体聚为一支,福建长乐群体和广东汕头群体聚为一支;群体间遗传变异指数Fst为0.230 9,表明4个斧文蛤群体间的遗传变异为23.09%。研究表明,斧文蛤4个不同地理群体遗传多样性处于中等多态水平;4个群体的遗传距离与它们实际的地理分布情况基本一致;4个地理群体间的变异较大,遗传分化水平较高。该研究为斧文蛤种质资源的有效保存、管理和恢复提供了理论依据。     

高通量测序法分析两株益生菌对凡纳滨对虾肠道菌群结构的影响

为分析荚膜红细菌和蜡样芽孢杆菌2株益生菌对凡纳滨对虾肠道菌群结构的影响,本实验对凡纳滨对虾进行为期30 d的养殖饲喂实验,饲喂后期利用高通量测序凡纳滨对虾肠道微生物的16S rDNA V4区,来分析不同益生菌饲喂后凡纳滨对虾肠道菌群的结构特征,并结合对虾体质量增加率和攻毒后累计死亡率的宏观指标来进行分析。结果显示：①空白组（CK）、荚膜红细菌组（Rc）和蜡样芽孢杆菌组（Bc）样品OTU范围为374~506,其中CK组对虾肠道菌群OTU数量最低,饲喂益生菌后的2组对虾肠道菌群中OTU数量相对较高;②在门分类水平上,3组的变形菌门数均为最多,CK组主要为变形菌门和少量拟杆菌门,Bc组主要有变形菌门、拟杆菌门、无壁细菌门、厚壁细菌门和假单胞菌,Rc组主要是变形菌门、拟杆菌门、厚壁菌门、酸杆菌门、放线细菌、梭杆菌门和蓝细菌;③稀释曲线和Shannon指数结果可见,CK组样品物种丰度和复杂度最低,且Bc组样品丰度和复杂度相对较高;④PCA分析发现,Rc组和CK组样品微生物组成较为接近,结合宏观对虾体质量增加率和攻毒后累计死亡率的结果分析,可见相较于荚膜红细菌,蜡样芽孢杆菌对凡纳滨对虾肠道微生物菌群影响更显著,且益生效果更佳。研究表明,饲喂益生菌可以扩增对虾肠道微生物菌群丰度,并能抑制弧菌属等有害菌群的生长,提高对虾体质量增加率并降低死亡率,从而达到益生效果,其中以饲喂蜡样芽孢杆菌菌株效果更佳。     

微卫星DNA鉴别克隆香鱼

实验应用微卫星DNA检测技术，分析了I至Ⅶ组共42尾香鱼的Pal-1*和Pal-2*两个基因座位的基因型。结果显示Ⅱ、Ⅲ、Ⅳ和Ⅶ组共22尾在两个基因座位上均为纯合，是基因型完全相同的克隆鱼，并且Ⅶ组l0尾为人工诱导成功的雌核发育二倍体，而I为混入本克隆的其它类型的香鱼；V组9尾为I与海产香鱼的子代；Ⅵ组10尾为Ⅱ或Ⅲ与海产香鱼的子代。