尼罗罗非鱼与萨罗罗非鱼及其正反杂交后代的微卫星分析

微卫星（microsatellite）技术是近年来广泛应用的DNA标记,具有重复性好、较易操作和共显性的特点。在硬骨鱼类中表现出高度多态性,已在家系鉴定、遗传作图、群体遗传分析、育种以及系统发生研究等多个领域得到应用。尼罗罗非鱼（Oreochromis niloticus）是我国主要淡水养殖鱼类,生长快,耐盐能力一般;萨罗罗非鱼（Sarotherodon melanotheron）耐盐能力强,适合咸淡水养殖,但生长较慢。[第一段]     

Relatedness structure estimated by microsatellites DNA and mitochondrial DNA polymerase chain reaction–restriction fragment length polymorphisms analyses in the wild population of kuruma prawn Penaeus japonicus

ABSTRACT: The genetic structure and variability of four wild populations of kuruma prawn in Japan were examined by estimating relatedness among individuals. The relatedness was estimated by five microsatellites (MS) DNA markers. Examination of relatedness showed that individuals were related significantly in Kumamoto and Kagoshima. In Kagoshima, some individuals showed full-sib level of relatedness. The analysis of mitochondrial (mt)DNA polymerase chain reaction–restriction fragment length polymorphisms (PCR-RFLP) was also performed, showing that the closely related individuals in Kagoshima tended to share a common haplotype. It is, therefore, supposed that there are many kins in Kumamoto and Kagoshima. However, the heterozygosities and allelic and genotypic frequencies in MS-DNA analysis were not significantly different among the localities. Moreover, the haplotype distributions of mtDNA in Kumamoto and Kagoshima were significantly different from other localities. Thus, it is suggested that no spatial differentiations occurred due to the geographic or historical effects between the localities and that there is the possibility of a mixture of hatchery populations in Kumamoto and Kagoshima.     

吉富罗非鱼与奥利亚罗非鱼完全双列杂交后代生长性能与肌肉营养成分的比较

以吉富罗非鱼(Oreochromis niloticus)与奥利亚罗非鱼(O.aureus)为繁育亲本,采用完全杂交进行配组,分别对吉富罗非鱼奥利亚罗非鱼纯繁与正反交后代生长性能和肌肉营养成分进行了比较,并且研究了脂代谢相关基因mRNA水平与肌肉脂肪含量的相关性。将初始规格基本一致的4组F1罗非鱼饲养100 d后,吉富罗非鱼纯繁组F1特定生长率最高(P0.05),吉富罗非鱼奥利亚正反交组合无显著差异(P0.05),奥利亚纯繁组F1特定生长率最低(P0.05)。同时,奥利亚纯繁组F1的饲料转化率、肝体比与内脏比指数显著高于其他实验组(P0.05)。各实验组水分、灰分与粗蛋白含量间无显著差异(P0.05),吉富纯繁组F1的粗脂肪含量显著高于其他组合(P0.05)。奥利亚纯繁组F1的必需氨基酸、呈味氨基酸与氨基酸总含量均显著低于其他实验组(P0.05),4组F1的肌肉必需氨基酸组成均符合FAO/WHO的标准。吉富纯繁组F1的饱和脂肪酸、单不饱和脂肪酸、多不饱和脂肪酸以及n-6多不饱和脂肪酸含量均显著高于正反交组合与奥利亚纯繁组F1(P0.05)。肌肉FAS、LPL、HSL和G6PD mRNA水平与脂肪含量的相关性分析表明,FAS与G6PD mRNA水平与肌肉脂肪含量呈负相关,相关系数(R2)分别为0.761 5(P0.01)和0.538 7(P0.05);LPL和HSL mRNA水平与肌肉脂肪含量呈正相关,相关系数分别为0.782 5(P0.01)和0.562 4(P0.05)。研究结果表明,奥利亚纯繁组F1的生长与肌肉营养成分明显劣于其他实验组,证明杂交能够提高后代的生长性能,改良肌肉品质,增加选育的综合效果。同时,吉富罗非鱼奥利亚正反交组合间LPL与G6PD mRNA水平间无显著差异(P0.05),增加肌肉脂肪含量有助于提高LPL和HSL mRNA表达水平。     

Sequence and polymerase chain reaction–restriction fragment length polymorphism analysis of the large subunit rRNA gene of bivalve: Simple and widely applicable technique for multiple species identification of bivalve larva

ABSTRACT:   One of the biggest and long-standing difficulties in investigation of larval ecology in the field is species-level identification. In the present study, we developed polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) analysis based on the large subunit (LSU) rRNA gene (rDNA) D1/D2/D3 region for identification of multiple species of bivalve larvae using 14 species of bivalve collected from Maizuru Bay. The LSU rDNA D1/D2/D3 region of all analyzed species could be amplified by PCR using bvD1f/bvD3r primers, and RFLP analysis by Hae III digestion on the PCR products showed species-specific fragment patterns. Furthermore, this analysis applied to single bivalve larvae in Maizuru Bay revealed efficient amplification of the target region and the species-specific pattern from 80% of the larvae, 75% of which showed a pattern that matched a certain pattern of the adult bivalves. In addition, the analysis of inter- and intraspecies variation of the LSU rDNA D1/D2/D3 region using the sequence data of the genus Crassostrea from the DDBJ database showed high applicability of this RFLP analysis on closely related species. Because of the wide applicability and technical simplicity, this method can become the standard for the identification of bivalve larvae species once the sequence data of the LSU rDNA D1/D2/D3 region of many bivalve species have been accumulated.     

11种鲈形目鱼类的核糖体基因GC含量及其与硬骨鱼类的特征比较

核糖体基因为串联重复多拷贝的基因,包括3个编码基因(18S,5.8S,28S)和两个间隔区ITS1(internal transcribed spacer 1)和ITS2(internal transcribed spacer 2)。目前,对核糖体基因的相关报道主要集中在个体内不同拷贝间的多态特征,以及其作为分子标记在系统演化关系中的应用,GC含量作为一项非常重要的核苷酸序列指标,而鲜有报道。为了探讨鱼类的核糖体基因GC含量特征以及间隔区是否也存在GC平衡现象,本研究选择了鲈形目(Perciformes)5科11种鱼类5个片段的核糖体基因进行研究,包括尖吻鲈科(Latidae)、射水鱼科(Toxotidae)、军曹鱼科(Rachycentridae)、剑鱼科(Xiphiidae)、鲹科(Carangidae)。获得了1651个单克隆序列,通过分析并比较已有的其他硬骨鱼序列片段的GC含量变化特征,结果发现:本研究鱼类的18S的GC含量为52.6%~57.1%(平均54.6%),5.8S为55.6%~58.9%(平均57.4%),28S为64.2%~65.8%(平均64.6%),ITS1为56.5%~73.0%(平均65.0%),ITS2为62.3%~77.5%(平均69.1%)。编码区的GC含量相对较保守,变异范围较小,18S和5.8S变化范围明显小于间隔区,28S则位于间隔区的最低值和最高值之间。因此,我们发现硬骨鱼核糖体ITS高于60%的GC含量是该类群的一个特征,并且高GC含量的ITS1和ITS2序列中不存在明显的高GC富集区,其含量高低的变化与序列长度也没有相关性。本研究11种鱼类的ITS1和ITS2的GC含量在种内的相似性既有大于也有小于种间相同片段的相似性,因此GC平衡现象只存在部分种类中。本研究结果可为鱼类核糖体基因序列特征的进一步研究及利用提供科学依据。     

Phylogenetic analysis and species identification of popular shrimp species in southeast China using the first internally transcribed spacer of ribosomal DNA

The ribosomal DNA internally transcribed spacer 1 (ITS1) was investigated in the search for an appropriate genetic marker that was suitable for phylogenetic study and species identification of eight major exported shrimp species in southeast China. Using the selected primers, the amplified ITS1 sequences exhibited a high degree of length polymorphisms, ranging from 448 bp in Metapenaeopsis dalei to 1491 bp in Macrobrachium nipponense . Many microsatellite loci were found at the 5' end and in the middle region of ITS1, which seemed to be associated with intragenomic sequence variation among samples of the same species. This variation might obscure the phylogenetic relationship between some shrimp populations, but the separation of five Penaeus species was well supported. In combination with polymerase chain reaction-restriction fragment length polymerism methods analysis, ITS1 sequences from shrimp species belonging to different families and genera could also be easily discernable. The results suggested that ITS1 was highly variable among different shrimp groups and could be an appropriate marker for species identification and molecular systematic studies.     

Species identification method for <Emphasis Type="Italic">Scombrops boops</Emphasis> and <Emphasis Type="Italic">Scombrops gilberti</Emphasis> based on polymerase chain reaction-restriction fragment length polymorphism analysis of mitochondrial DNA

ABSTRACT:   Gnomefish Scombrops boops and Scombrops gilberti are commercially important fishes in Japan, but these species are often confused in the markets because of their morphological similarity. To identify these two species, we performed nucleotide sequencing and restriction fragment length polymorphism (RFLP) analysis on 16S ribosomal RNA (rRNA) gene and the control region in mitochondrial DNA. Five and 12 nucleotide substitutions were observed between species in the 777-bp 16S rRNA gene and 471-bp control region, respectively. Diagnostic restriction sites for discriminating between S. boops and S. gilberti were found in the 16S rRNA gene, but not in the control region. Polymerase chain reaction (PCR)–RFLP analysis using two enzymes, Eco NI and Mva I, clearly discriminated between S. boops and S. gilberti identified by meristic characters. The PCR–RFLP analysis identified most of the 168 Scombrops young caught in the coastal waters of the Izu and Miura peninsulas as S. boops , suggesting that S. gilberti juveniles are rare in this area.     

散鳞镜鲤、团头鲂及其杂交F1肌肉营养成分的比较

采用生化分析方法对散鳞镜鲤、团头鲂及其杂交F1肌肉的营养成分进行了测定并比较。结果表明，交杂种F1肌肉中的水分含量为77．89％、灰分含量为5．52％，均低于亲本；脂肪含量为4．77％，高于团头鲂，低于散鳞镜鲤；F1的氨基酸含量为77．89％，低于双亲，但大多成分含量与散鳞镜鲤相当。在总体水平上，杂交种品质得到初步的改良。     

雅鲁藏布江拉萨裂腹鱼、异齿裂腹鱼及其自然杂交种的形态与COI基因条形码分析

2015年5月,在西藏雅鲁藏布江中游的桑日、朗县段水域开展鱼类调查,发现了1种疑似为拉萨裂腹鱼(Schizothorax waltoni)和异齿裂腹鱼(Schizothorax o’connori)的自然杂交种群体。形态学研究显示,该种的吻、口部、下颌、须等头部上的形态特征居于拉萨裂腹鱼和异齿裂腹鱼之间,并明显区别于双亲,且群体中个体的头部形态稳定一致,均具有典型的中间型特征,个体间没有明显差异。基于线粒体COI基因条形码分析显示,在杂交种群体共15个样本中,有4个与拉萨裂腹鱼为同源,其余11个与异齿裂腹鱼为同源,各自间的亲缘关系很近,即杂交种群体中既有属于拉萨裂腹鱼遗传基因的个体,也有属于异齿裂腹的遗传基因的个体。综合以上研究表明,拉萨裂腹鱼和异齿裂腹鱼在雅鲁藏布江中游的桑日等自然水域中能够自由杂交而产生后代,且两者都可以互为父母本,但以异齿裂腹鱼为母本产生的杂交种在群体中占多数,推测特殊的生态环境可能促使拉萨裂腹鱼和异齿裂腹鱼在自然水域中容易杂交。     

珠江口鱼类浮游生物种类组成与数量分布

江门位于珠江口西南部,其近海海域是多种小型鱼类的产卵场和育幼场。以珠江口江门海域春季采集到的鱼卵为研究对象,基于DNA条形码技术分析鉴定了鱼卵种类,描述了鱼卵的形态特征,并初步研究了其形态分类。共采获13种鱼卵,分属于3目10科,其中4种鉴定至种水平,分别为眶棘双边鱼 (Ambassis gymnocephalus)、缘边银鲈 (Gerres limbatus)、多鳞 (Sillago sihama) 和斑头舌鳎 (Cynoglossus puncticeps);8种鉴定至属水平,分别为沙丁鱼属、小公鱼属、棘鲷属、副叶鲹属、叫姑鱼属、属和舌鳎属;1种鉴定至科水平,为鲾科。结果发现,江门海域大部分鱼卵可通过形状与大小、卵周隙宽窄、油球数量和色素分布等形态特征进行分类;对于胚体发育良好的鱼卵,胚体上色素胞颜色、数量及分布也是种类鉴定的重要依据。然而相近种类难以通过单一发育阶段形态特征进行准确区分,更深入的形态学研究尚待开展。