| 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. |
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