【作者】 李玉龙；

【导师】 孔晓瑜；

【作者基本信息】 中国海洋大学 ， 水生生物学， 2009， 博士

【摘要】 本文以中国沿海两种对虾类中国明对虾和日本囊对虾为研究对象，采用线粒体COI基因片段和控制区序列等标记技术开展了这两种虾类的群体遗传多样性研究，系统研究了这两种虾类的遗传多样性和群体遗传结构并探讨了其群体历史动态。另外，利用核基因ITS1序列和线粒体全长以及nd5假基因对对虾科虾类进行了系统分析，主要研究结果如下：1、为了检测中国明对虾的群体遗传结构合群体历史动态，我们对中国明对虾分布区内5个群体共93个个体的线粒体细胞色素氧化酶I亚基基因(COI)序列和控制区序列(CR)进行了测定和分析。在COI序列中，共检测到20个多态性核苷酸位点，定义了20种单倍型，其核苷酸多态度和单倍型多态度都很低(H=0．4816±0．0639，π=0．0846±0．0711％)。长993bp的CR序列中，共检测到84个多态性核苷酸位点，定义了68种单倍型，其单倍型多态度较高(0．95±0．03-0．99±0．02)，而核苷酸多态度中等或较低(0．66±0．36％-0．84±0．46％)。群体分化指数FST值(FST=-0．02343-0．02112)和分子变异分析(AMOVA)都表明不同群体间的中国明对虾还没有出现明显的遗传分化。IM的结果显示中国明对虾朝鲜半岛西海岸群和黄渤海沿岸群出现分化，用IM所估算的分化时间约为12，800(7，400-18，600)前。因此，中国明对虾不同地理群体间分化很小或不存在遗传分化的原因是由于分化的时间尚短，不足以在不同群体间形成明显的遗传结构。中性检验和核苷酸不配对分布都表明中国明对虾经历了群体扩张事件。中国明对虾较低的遗传多样性水平、简单的基因谱系结构、星状结构的单倍型网络关系图和检验显著的负的Tajima’D值都提示分布于黄渤海的中国明对虾群体可能经历过近期的瓶颈效应或奠基者效应。2、采用PCR技术扩增了日本囊对虾(Marsupenaeus japonicus)线粒体细胞色素氧化酶I亚基基因(COI)片段，PCR产物经测序得到858bp的核苷酸序列；分析了该片段的日本、浙江、福建、广东、广西5个自然群体85个个体的核苷酸序列多态性，共检测到106个多态性核苷酸位点、58种单倍型。结果表明，五个群体中以广西群体遗传多样性最高，日本群体最低；在日本囊对虾群体内存在两个明显的单倍型类群，这两个单倍型类群间的平均遗传距离为5．6％；分子变异分析(AMOVA)和群体间分化指数FST值均显示，不同自然分布区的日本囊对虾出现明显的遗传分化。中性检验和核苷酸不配对分布表明日本囊对虾两个单倍型类群都经历了更新世的群体扩张。3、对采自不同群体的5个中国明对虾个体的核糖体第一内转录间隔区(ITS1)序列特点进行分析，并利用GeneBank数据库中已有的对虾科(Penaeidae)ITS1同源序列对对虾科虾类进行系统分析，探讨ITS1序列在对虾科系统及演化中的应用。结果表明，中国明对虾ITS1序列在个体间和个体内都表现出长度多态性，序列长度范围为637-652bp，这种长度多态性主要是由于微卫星DNA简单重复序列的重复次数不同所造成。在中国明对虾ITS1序列中发现8个微卫星位点，根据目前已知的12种对虾的ITS1序列，发现某些微卫星位点只存于一种对虾中，如(CAGC)2-4只存在于中国明对虾中，(CGGA)4-9只存在于斑节对虾中，(GCGA)4只存在于短沟对虾中。利用ITS1序列对12种对虾进行的系统分析表明，12种对虾分为四个类群，同种的不同个体，同属的不同种各自聚支，与形态分类比较吻合。对虾科属间遗传距离范围为0．313-0．977，平均值为0．633，远高于用线粒体基因片段得出的属间的遗传距离，支持将原对虾属的6个亚属提升为属的观点。4、在扩增中国明对虾线粒体基因组全序列的过程中，我们发现疑似nd5基因片段的假基因，对该片段的特点和类型进行了分析，并以该片段为外群探讨了对虾科的系统关系。同时采用线粒体DNA全序列对十足目和对虾科的系统关系进行了研究。结果表明，用假基因为外群构建的对虾科的系统树与用线粒体蛋白编码基因核苷酸序列得出的系统树的拓扑结构一致，但不同于蛋白编码基因氨基酸序列得出的系统树的拓扑结构。要更多的了解对虾科的系统关系还需要更多的资料加以证实。更多还原

【Abstract】 In this study,the genetic diversity and evolutionary demography ofFeneropenaeus chinensis and Marsupenaeus japonicus were studied by mtDNAmarkers.We discussed the role of Pleistocene ice ages and present environmentalfactors in shaping the phylogeographic pattern and genetic structures of the twospecies.In addition,ITS1,mt genome sequences and Numts were used as molecularmarkers for studying the phylogenetic relationships among species of penaeid shrimps.1、Genetic structure and demographic history ofFeneropenaeus chinensis wasexamined using sequence data of partial mitochondrial cytochrome oxidase I (COI)gene and the complete mitochondrial control region (CR).Samples of 93 individualswere collected from 5 localities over most of the species’ range in the Yellow Sea andthe Bohai Sea.For COI data,atotal of 20 nucleotide sites were detected variable,defining 20 haplotypes.The variation level was low,with H=0.4816±0.0639 and=0.0846±0.0711 (%),respectively.For CR data,there are 84 variable sites defining68 haplotypes.Haplotype diversity levels were very high (0.95±0.03-0.99±0.02),while that ofnucleotide diversitywere at moderate to low level (0.66±0.36%-0.84±0.46%).Analysis of molecular variance and the conventional population statisticFST revealed no significant genetic structure throughout the range of F.chinensis.However,Isolation with Migration (IM) coalescence results based on CR data suggestF.chinensis distributed along the coasts of northern China and Korean Peninsula(about 1000 km apart) diverged recently,and the estimated split time was 12,800 (7,400-18,600) years ago.Both mismatch distribution analyses and neutrality testsbased on COI data and CR data suggested a late Pleistocene or Holocene populationexpansion (12,100-28,500 years ago) for F.chinensis,which was consistent withthe geological period of formation of the Bohai Sea and the Yellow Sea.These results indicated that F.chinensis in the Bohai Sea and the Yellow Sea might have undergonea genetic bottleneck or founder event during the late Pleistocene globle glacial period.2、DNA sequencing is very useful for the genetic diversity analysis at molycular level.Mitochondrial COI gene fragments of kuruma shrimp Marsupenaeus japonicus wereamplified via polymerase chain reaction (PCR),the PCR products were sequenced for85 individuals,5 population of China.858 base-pair nucleotide sequences wereexamined and analyzed for genetic polymorphism.Resultant sequences data analysisshowed that all 85 sequences were grouped into 58 haplotypes and there were 106variable nucleotide positions in this gene fragment.The genetic diversity indexesincluding S (polymorphic sites),π(nucleotide diversity) and H (haplotype diversity)were calculated and the results indicated that Guangxi population showed morevariation than others and more differences to others.Based on the mitochondrial DNAsequence data,the shrimps were separated into two distinct clades.Analyses ofmolecular variance and the population statistic FST revealed significant geneticstructure.Both mismatch distribution analyses and neutrality tests based suggested aPleistocene population expansion for Marsupenaeus japonicus.3、The ribosomal DNA internal transcribed spacer 1 (ITS1) of Fenneropenaeuschinensis was cloned and sequenced.The objective of the present study is to estimatephylogenies and explore the feasibility of using ITS1 as a molecular marker forstudying the interspecific and intraspecific genetic variations among penaeid shrimps.Inter-and intraindividual polymorphisms of ITS 1 were clearly observed in F.chinensis.The length of the ITS1 ranged from 637 to 652bp,with a GC content of 66.9%.Repeats of microsatellite loci of ITS 1 plays an important role in length heteroplasmyat both between-and within-individual levels in this species.Eight microsatellite lociwere found in the ITS1 sequence of F.chinensis.According to existing data,somemicrosatellites were found in only one specie,(CAGC) 2-4 in F.chinensis,(GCGA)4in P.semisalcatus and (CGGA)4-9 in P.monodon.The DNA data of the penaeidshrimps were processed in neighbor-joining (NJ) methods to build phylogeneticrelationships within the family of Penaeidae.According to the NJ tree,differentindividuals of the same species,different species of the same genus were grouped together,in consistence with the morphological taxonomy.The genetic distancesamong different genus ranged from 0.313 to 0.977 and the average was 0.633,exceeding those estimated with COI and partial 16S rRNAgene sequences,and beingtherefore larger than the value among subgenera.The results of the present studyindicated that Fenneropenaeus,Litopenaeus,Farfanatepenaeus,Marsupenaeus andPenaeus are at a higher phylogenetic level than subgenus,which supports the opinionof the elevation of phylogenetic status of the four subgenera to genus level.Theseresults show that ITS 1 is highly divergent among different penaeid shrimps and couldbe an appropriate marker for molecular systematic studies at the species levels,although the presence of intragenomic variation needs to be taken into consideration.4、Mt genome sequences have provided useful markers for investigating populationgenetic structures,systematics and phylogenetics of organisms.In this study,mtgenome sequences and Numts were used as molecular markers for studying the phylogeneticrelationships among species of penaeid shrimps.Phylogenetic analyses based onconcatenated amino acid and nucleic acid sequences of the 13 mt protein-codinggenes strongly support the monophyly of the Decapoda and Penaeidae.However,relationships among species within the Penaeidae based on the 13 mt protein-codinggenes and Numts could not be resolved,and further studies were necessary to becarried out based.on the increasing number of mitochondrial genome sequences,inorder to better understand the relationships among species of Penaeidae.更多还原