【作者】 胡丽萍；

【导师】 包振民；

【作者基本信息】 中国海洋大学 ， 海洋生物学， 2013， 博士

【摘要】 1．栉孔扇贝染色体识别技术的建立本研究应用荧光原位杂交（Fluorescence in situ hybridization，FISH）技术，通过开发染色体特异分子标记，首次实现了栉孔扇贝所有染色体的识别，并在此基础上构建了栉孔扇贝的染色体图谱，首次对栉孔扇贝微卫星遗传连锁图谱和染色体图谱进行了初步整合。主要结果如下：（1）通过三维两步PCR筛选系统，选取分布于栉孔扇贝微卫星遗传连锁图谱19个连锁群上的42个微卫星标记对本实验室构建的栉孔扇贝BAC文库进行克隆筛选，应用FISH技术对筛选到的阳性克隆进行染色体定位，最终成功实现32个含有微卫星标记BAC克隆的定位。其中30个标记克隆可在染色体上产生稳定、单一的荧光信号，另外2个克隆的定位信号出现在多条染色体上。30个具有单一染色体定位的标记在连锁群上的分布为：12个连锁群上分别被成功定位2个标记；6个连锁群上分别被成功定位1个标记。对位于同一连锁群的标记采用双色FISH技术进行共杂交检验及核型分析，结果显示：位于同一连锁群且被定位于同一染色体上的标记有16个，分别位于8个连锁群上；位于同一连锁群但被定位于不同染色体上的标记有8个，分别位于4个连锁群上。通过对位于不同连锁群上标记间的共杂交，结果表明：较小的连锁群LG16和LG18可以分别与较大的连锁群LG6和LG8整合到一起。（2）应用FISH技术，从96个BAC克隆和27个fosmid克隆中筛选获得可产生染色体单一信号位点的克隆标记69个。凭借多重双色FISH，从中筛选出15个具有无/低背景的克隆标记，构建了一套可用于区分所有形态相似的亚中部和亚端部着丝粒染色体的染色体特异标记。在所有染色体被识别的基础上，通过FISH共杂交及核型分析技术，构建了一张包含70个标记且覆盖19条染色体的栉孔扇贝染色体定位模式图。该图谱包含58个BAC克隆，11个fosmid克隆以及1个5S rRNA基因序列。其中58个BAC克隆中包括30个含有SSR标记信息，2个含有基因相关信息，和26个随机筛选克隆；11个fosmid克隆中包括7个含有重复序列信息和4个含有不同基因序列信息的克隆。栉孔扇贝每条染色体上的特异位点标记数目从1个到8个不等，平均为3.7个。该图谱将会在栉孔扇贝全基因组序列拼接，图位克隆，QTL定位等多个研究方面发挥重要作用。2．重复序列在几种扇贝染色体上的比较定位分析（1）C0t-1DNA的比较定位：将来自栉孔扇贝的C0t-1DNA通过FISH技术分别杂交到栉孔扇贝，虾夷扇贝以及海湾扇贝的中期分裂相染色体上，结果显示：C0t-1DNA在三种扇贝的所有染色体上均有分布，但信号的分布密度和强度有明显差异。在栉孔扇贝绝大多数染色体的着丝粒，近着丝粒，以及近端粒位置丛生有密集且非常明亮的荧光信号。在虾夷扇贝中，仅在少数染色体的着丝粒及靠近着丝粒的长臂区域观察到有较强的丛生明亮信号。而在海湾扇贝中，几乎没有这种密集明亮信号的分布。这种全基因组范围重复序列的比较定位分析表明，相比栉孔扇贝与海湾扇贝，栉孔扇贝和虾夷扇贝的重复DNA序列具有相对更高的同源性，该两种扇贝可能具有更近的亲缘关系。另外，由于C0t-1DNA在栉孔扇贝的同源染色体上呈现出相似的荧光信号带型，而在非同源染色体上表现较明显的差异，据此我们对栉孔扇贝进行了较为准确的核型分析。（2）重复序列-rDNA的染色体定位：对华贵栉孔扇贝和紫扇贝的核糖体基因进行染色体的FISH定位，结果显示：在华贵栉孔扇贝中，18S-28S rDNA具有一个信号位点，位于最大的1对中部着丝粒染色体的着丝粒位置；5S rDNA产生两个信号位点，分别位于两对端部着丝粒染色体的长臂中间和长臂端部位置。在紫扇贝中，18S-28S rDNA拥有多个信号位点，分别位于6～7对亚端部或端部着丝粒染色体的短臂上；5S rDNA具有两个信号位点，它们位于同一对端部（或亚端部）着丝粒染色体长臂中间的邻近位置。对两种rDNA的共杂交结果显示：两种扇贝的18S-28S rDNA和5S rDNA均位于不同的染色体上。结合华贵栉孔扇贝的核型及其18S-28S rDNA的定位结果，推测该扇贝染色体可能在进化过程中发生过罗伯逊融合。而紫扇贝多个18S-28S rDNA位点的产生则可能是在进化过程中发生了染色体的非相互易位。两种扇贝的两种rDNA均不在相同的染色体上，说明在进化过程中载有核糖体DNA的染色体可能发生过断裂或（和）易位。3．扇贝科多个物种的分子系统进化分析通过克隆测序获得了7个扇贝物种（海湾扇贝、紫扇贝、平濑掌扇贝、褶纹肋扇贝、新加坡掌扇贝、大西洋深水扇贝、太平洋花扇贝）的核糖体转录间隔区（ITS）序列，序列分析表明，7种扇贝的ITS区域总长从685bp（大西洋深水扇贝）到732bp（太平洋花扇贝）不等，GC含量从46.7%（紫扇贝）到52.7%（褶纹肋扇贝）不等。所有物种的5.8S rDNA区域长度均为157bp，且GC含量值相比ITS1和ITS2区域的都要高；所有测序扇贝的ITS1和ITS2长度均相当，且GC含量均为ITS2高于ITS1。结合GenBank中已发表扇贝物种的ITS序列，对在全球不同海域分布的21个扇贝物种进行了系统发生关系的研究。在根据ITS1和ITS2结合序列构建的NJ分子系统树中，大西洋深水扇贝单独成为一枝，其余所有扇贝物种形成两个大的分枝。其中一枝包括紫扇贝、太平洋花扇贝、海湾扇贝、日月贝、欧洲大扇贝、女王扇贝、美丽环扇贝、褶纹肋扇贝和Decatopectenradula共9个扇贝物种，其中同属海湾扇贝属的紫扇贝、太平洋花扇贝和海湾扇贝具有很近的亲缘关系，尤其是紫扇贝和太平洋花扇贝之间的遗传距离与紫扇贝种内遗传距离甚至存在重叠，表明该两种扇贝可能属于亚种的关系；另外的一枝包括了栉孔扇贝、虾夷扇贝、Semipallium fulvicostata、6种Mimachlamys属扇贝（M. varia、C. distorta、M. pyxidatus、华贵栉孔扇贝、M. senatoria、M. sp. TN-2006）和2种掌扇贝属扇贝（平濑掌扇贝和新加坡掌扇贝）。MP系统树与NJ树的聚类结果基本相似，但也有一些差异，尤其是对大西洋深水扇贝的聚类结果。本研究对扇贝物种的系统分析结果与根据双壳贝类形态学的分类结果以及根据线粒体基因的系统发生学研究结果基本一致。以上分析结果不仅使我们对不同扇贝间的亲缘关系远近有更清晰的认识，还可以为扇贝不同近缘物种间的杂交育种尝试提供理论指导。4．紫扇贝和海湾扇贝杂交子代的细胞与分子遗传学分析紫扇贝与海湾扇贝已成功获得杂交，且其杂交子代表现出明显的杂种优势。为了更好的理解该杂种优势产生的遗传基础，本研究采用GISH，AFLP，SSR，DNA测序等细胞和分子生物学手段对这两种扇贝正反交子代的基因组组成和变异进行了遗传学分析。主要结果如下：（1）对紫扇贝和海湾扇贝的正反杂交子代幼虫进行GISH检测，结果表明绝大多数的子代基因组中包含32条染色体，且其中一半可被紫扇贝的基因组探针标记上，另一半可被海湾扇贝的基因组探针标记上，因而表明该杂交子代分别继承了两亲本各一套的染色体，是真正精卵结合水平的杂交种。另外，实验中还检测到有少数的染色体丢失及异源多倍体的分裂相。（2）采用AFLP，SSR及DNA测序技术对正反杂交子代成体扇贝的遗传组成和变异进行研究。ITS序列扩增和AFLP分析结果均表明：杂交子代继承了来自双亲的绝大多数核遗传物质，充分确定了该杂交成体扇贝是真正的杂交种。但在杂种的ITS序列分析中还检测到了两亲本组合型的ITS变异中间体，AFLP分析中也检测到了少数AFLP位点的变异，这些均表明子代对双亲遗传物质的继承并不是父本和母本遗传物质的简单叠加，而是在继承双亲特异位点的同时，还有少量位点的变异。另外，群体遗传分析数据表明，杂交子代群体的遗传相似度降低，杂合度水平升高，杂种群体的遗传多样性增加，在遗传关系上正反杂交子代略偏向于各自的母本。16S rDNA序列分析表明，正反杂交子代中的该基因序列分别与其母本中的序列同源，揭示了线粒体基因在该杂交扇贝中是遵循母性遗传的。以上结果将对正确认识该两种扇贝的杂交甚至其它海洋贝类杂交育种和杂种优势的利用有重要意义。更多还原

【Abstract】 1. Establishment of chromosomal recognition technology for Chlamys farreriIn this study, using fluorescence in situ hybridization (FISH), we identified all19C.farreri chromosomes with the chromosome-specific markers for the first time,constructed a cytogenetic map for C. farreri, and initially accomplished theintegration of microsatellite-based genetic linkage map and cytogenetic map in C.farreri, The main results are as follows:(1) Through three-dimensional PCR screening system,42microsatellite markersfrom19linkage groups of Zhikong scallop were used to screen the BAC library. Thepositive clones were then tested by FISH. As a result,32BAC clones containingmicrosatellites were successfully mapped on the chromosomes. Among them,30clones were mapped to unique locus with or without C0t-1DNA and the other2represented multiple signals. The30SSR markers with unique chromosomal locuswere from18linkage groups. Of which,24microsatellites were from12linkages,2microsatellites each linkage,6microsatellites were from the other6linkage groups,1microsatellites each linkage. The microsatellites from the same linkage wereco-hybridized by two-color FISH. The results showed that16microsatellites from8linkages could be assigned to their consistent chromosomes, respectively, while thelinkage assignments of another8microsatellites from another4linkages wereinconsistent with their chromosomal assignments. The microsatellites from differentlinkage were co-hybridized by two-color FISH. And the results revealed that theminor LGs16and18could be assigned to the major LGs6and8, respectively.(2)96BAC clones and27fosmid clones were tested by FISH and a total of69ofthem could produce a single specific signal on the chromosomes with or without C0t-1DNA.15clones that could produce bright signals and no or low background wereisolated and made one set of chromosome-specific markers for distinguishing all thesubmetacentric and subtelocentric chromosomes with similar morphology by multipletwo-color FISH. On this basis, a cytogenetic map of C. farreri containing70markerswas constructed by cohybridization and karyotypic analysis. All the markers comprised58BAC clones,11fosmid clones and15S rDNA marker. Of which, the58BAC clones involved30microsatellites-anchored BAC clones,2genes-anchoredBAC clones and26random BAC clones; the11fosmid clones included7clonescontaining repetitive DNA and4containing gene sequences. The markers covered all19chromosomes of C. farreri, and there were1to8markers on differentchromosomes with3.7as an average number of markers. This map will facilitate thewhole genome sequencing and assembly, positional cloning and QTL mapping in C.farreri.2. Comparative chromosome mapping of repetitive sequences in severalscallops(1) Chromosomal mapping of C0t-1DNA: The chromosomes of C. farreri,Patinopecten yessoensis and Argopecten irradians were studied by FISH using C.farreri C0t-1DNA probes. The results showed that C0t-1DNA signals spread on allchromosomes in the three scallops, whereas signal density and intensity were differentstrikingly. Clustering brighter signals presented in the centromeric and telomericregions of most C. farreri chromosomes, and in the centromeric or pericentromericregions of several P. yessoensis chromosomes. Comparative analysis of the mappingindicated a relatively higher homology in the repetitive DNA sequences of thegenome between C. farreri and P. yessoensis than that between C. farreri and A.irradians. In addition, FISH showed that the distribution of C0t-1DNA clusteringsignals in C. farreri displayed completely similar signal bands between homologouschromosomes. Based on the C0t-1DNA fluorescent bands, a more exact karyotype ofC. farreri has been obtained.(2) Chromosomal mapping of repeated sequences-rDNA: Chromosomal localizationof major (18S-28S) and minor (5S) ribosomal RNA genes were studied in two speciesof Pectinidae, Mimachlamys nobilis and Argopecten purpuratus using FISHtechnology. In M. nobilis,18S-28S rDNA had one locus and was mapped to thecentromere of the biggest pair of metacentric chromosomes.5S rDNA produced twosignal loci, and they were located on the long arms of one telomeric chromosome andthe telomeric regions of the long arms of another telomeric chromosome, respectively.In A. purpuratus,18S-28S rDNA had many loci, located on the short arms of six toseven subtelomeric/telomeric chromosomes, and the5S rDNA was found two loci,mapped on the long arm of one telomeric (subtelomeric) chromosome. The results ofco-hybridization of these two rDNAs showed that18S-28S rDNA and5S rDNA were lcated on different chromosomes in M. nobilis, and the same as in A. purpuratus. Thekaryotype and location of18S-28S rDNA in M. nobili might be explained byRobertsonian fusion. Many18S-28S rDNA loci in A. purpuratus may result from thenon-reciprocal chromosomal translocation in evolution. Chromosomal breakage or(and) translocation may contribute to the different chromosomal location of tworDNA families in these two scallop species.3. Molecular phylogenetic analysis of PectinidaeThe internal transcribed spacer (ITS) region of the ribosomal DNA from7scallopspecies (A. irradians, A. purpuratus, A. ventricosus, Volachlamys hirasei, V.singaporinus, Decatopecten plica and Placopecten magellanicus) was PCR amplifiedand sequenced. The size of total ITS region ranged from685bp (P. magellanicus) to732bp (A. ventricosus) and GC content ranged from46.7%(A. purpuratus) to52.7%(D. plica). The size of5.8S rDNA in all7species was157bp and the GC content of5.8S rDNA was higher than those of ITS1or ITS2region. ITS1and ITS2possessedsimilar length, and the GC content of ITS2was higher than that of ITS1in all thesescallop species. Combined with the published ITS sequences of the other14scallopspecies, the molecular phylogenetic tree of total21scallop species was constructed bythe neighbor-joining (NJ) and maximum parsimony (MP) methods using ITS1andITS2sequences. NJ tree revealed that P. magellanicus formed an independent clam, A.irradians, A. purpuratus, A. ventricosus, Amusium pleuronectes, Pecten maximus,Aequipecten opercularis, Annachlamys macassarensis, D. plica and D. radulagrouped in one big clade, and the other11scallop species (C. farreri, C. distorta, M.nobilis, M. sp. TN-2006, M. senatoria, M. varia, P. yessoensis, Minnivolva pyxidatus,Semipallium fulvicostata, V. hirasei and V. singaporinus) grouped in another big clade.Three Argopecten species (A. irradians, A. purpuratus and A. ventricosus) showedclose relationship, even the distance between A. purpuratus and A. ventricosusoverlapped with the distance within A. purpuratus, evidenced that A. purpuratus andA. ventricosus might belong to the subspecies. The MP tree has a similar topologywith the NJ tree but a little difference, of which, the biggest was the cluster of P.magellanicus. Results in this study are nearly consistent with the conclusions inferredfrom the morphological classifications of bivalve and the phylogenetic relationshipsinferred from mitochondrial genes. These results provide new insights into therelationships among scallop species and contribute to the crossbreeding attemptbetween different related scallop species. 4. Cytogenetic and molecular analysis of the hybridsThe Peruvian scallop (A. purpuratus) has been successfully hybridized with the bayscallop (A. i. irradians), and the F1hybrids of these two scallops exhibited a largeincrease in production traits. To understand the genetic basis of this heterosis, thecytogenetic and molecular analysis technologies, such as GISH, AFLP, SSR, DNAsequencing, were applied to investigate the genomic characterization of the hybrids.The main results are as follows:(1) GISH was employed to detect different chromosome components and identifydonor chromatin in the hybrids. The results showed that most of the hybrid progeniespossessed half set of chromosomes (n=16), which could be labeled with the genomicDNA probes of one parent, and the other half set of chromosomes that could belabeled with the genomic DNA probes of the other parent. It thus confirmed thehybrid status of the F1progenies from cross of the two scallop species. However,small portion of aneuploids and allopolyploids were also observed in the hybridprogenies.(2) The genetic composition and variation of the adult hybrids were investigatedthrough AFLP, SSR and DNA sequencing. AFLP analysis and the amplification ofITS region both indicated that the hybrids inherited most genetic materials of theirparents and confirmed their hybrid identity. However, some ITS recombinant variantswere detected through ITS sequencing and AFLP loci alterations were also found byAFLP analysis in the hybrid genomes. These suggested that the inherited geneticmarkers in hybrids was not a simple combination of parental specific markers, butshowed some variations. Moreover, the genetic diversities of the hybrids and parentswere analyzed with AFLP and SSR markers. The results showed that the geneticsimilarities were lower and the heterozygosities were higher in the hybrids than thosein their parents, which suggested the genetic diversities of the hybrids increased. Thegenetic distance between hybrids and their maternal parents was slightly smaller thanthat between hybrids and paternal parents. Sequence analysis of mitochondrial16SrDNA showed that the hybrids possessed sequences that were identical to the16SrDNA of the female parents, proving matrilineal inheritance of mitochondrial genes inthe hybrid scallops. All these results may contribute to understanding of inter-specifichybridization and heterosis in marine shellfish species.更多还原