栉孔扇贝（Chlamys farreri）BAC文库的构建及其基因组特征分析
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摘要
栉孔扇贝（Chlamys farreri）是我国北方沿海一种重要的经济贝类，在我国水产养殖产业中占有重要的位置。开展栉孔扇贝的基因组学研究，对于了解重要经济性状的遗传学基础，开发重要经济性状的遗传调控基因，从而指导品种改良和遗传育种具有重要意义。本研究首先构建了一个栉孔扇贝基因组学研究平台，此平台包含三个细菌人工染色体（BAC）文库，而后实现了文库的初步应用分析。与此同时，利用高通量测序技术将栉孔扇贝全基因组进行测序评估，并由此对基因组特征进行初步分析。本篇论文主要包括以下三部分内容：
1.栉孔扇贝BAC文库的构建及质量评价
本研究构建了栉孔扇贝三个BAC文库，分别为BamHI文库，HindIII文库和Sau3AI文库，其中BamHI文库包含34,560个克隆，平均插入片段为98Kb，未发现空载；HindIII文库包含132,480个克隆，平均插入片段为106Kb，空载率为1.67%；Sau3AI文库包含23,040个克隆，平均插入片段为53Kb，空载率为3.33%。三个文库总共由190,080个克隆组成，平均插入片段为98Kb，覆盖栉孔扇贝基因组的15.4倍。随机挑选BAC克隆100代的继代培养，没有发现插入片段的丢失或重排现象，说明所构建的BAC文库是稳定的。将所有克隆进行超级池及二级池的构建，筛选了栉孔扇贝3个基因以及遗传图谱上的7个微卫星标记，阳性克隆数在6-18之间，表明这三个文库可以在目的基因或标记的筛选，物理图谱构建以及大规模基因组测序过程中发挥重要作用。
2.栉孔扇贝BAC文库的应用
本研究选取栉孔扇贝8个免疫相关候选基因及17个与生长性状QTL连锁的SNP标记为研究对象，利用BAC文库的4D-PCR筛选系统对这25个位点进行筛选分析，每个位点筛选出3个阳性克隆，最终获得了75个含有目的基因或SNP标记的阳性克隆。这为日后栉孔扇贝免疫及生长性状相关基因的完整克隆奠定了基础。另外，还利用BAC-FISH技术对栉孔扇贝Toll样受体（TLR）信号通路中的关键基因（CfTLR, CfMyd88, CfTRAF6, CfNFκB和CfIκB）进行染色体定位。结果显示，包含这些基因的5个BAC克隆定位在染色体上的位置单一。进一步通过核型分析及共定位验证，这5个克隆被定位于5对不同的染色体上，表明TLR信号通路的5个关键基因在栉孔扇贝基因组中并不连锁。这些研究对探索栉孔扇贝免疫系统机理有着积极的作用，并有利于细胞染色体分析等工作的开展。
3.栉孔扇贝基因组勘探测序及特征分析
本研究采用全基因组鸟枪法策略，依托于Illumina solexa高通量测序平台对栉孔扇贝基因组进行勘探测序，获得了62.44Gb的有效数据，评估覆盖深度约为50X。对所有短序列组装结果显示contig N50为1,267bp，scaffold N50为1,517bp，获得scaffold总长约为870Mb。通过17-kmer分析估计栉孔扇贝基因组大小约为971Mb，并预测该测序个体的杂合度约为1.35%。对scaffold序列进行初步分析，估计其覆盖真实基因组的57.1%，覆盖转录组的88.7%。运用RepeatMasker软件进行扫描，获得了884,644个散在重复序列，主要分为四种类型：DNA转座子、SINE反转座子、LINE反转座子和LTR反转座子。其中，DNA转座子数目最多，其次是SINE反转座子、LINE反转座子和LTR反转座子。运用SciRoKo软件进行扫描，获得了134,887个微卫星序列，分为437种类型，其中单碱基重复的微卫星数目最多。这些结果的获得将有利于对栉孔扇贝基因组结构特征的探索，并为全基因组测序策略的选择及基因组学研究方向的确定提供重要的参考信息。
Zhikong scallop (Chlamys farreri) is one of the most important economicaquiculture bivalves in China, but study on its genome is underdeveloped. In thisstudy, we constructed three bacterial artificial chromosome (BAC) libraries ofC.farreri as platform for genomic research, and harvested positive BAC clonescontaining target genes, which are relevant to growth and immune function ofC.farreri. Additionally, we performed genome sequencing and comparative analysisof the C.farreri using next-generation sequencing technology, and thus made apreliminary assessment of the genome.
1. Construction and characterization of BAC libraries in C.farreri
We constructed three bacterial artificial chromosome (BAC) libraries forC.farreri. The BAC libraries were generated based on three restriction enzymes,BamHI, HindIII and Sau3AI, respectively. The BamHI library consists of34,560clones, with an average insert size of98kb and no insert-empty clones. The HindIIIlibrary consists of132,480clones, with an average size of106kb, but approximately1.67%of the clones don’t contain scallop nuclear DNA inserts. The Sau3AI libraryconsists of23,040clones, with an average size of53kb, approximately3.33%of theclones don’t contain DNA inserts. The combined libraries collectively contain a totalof190,080BAC clones with an average size of98kb, representing15.4x haploidgenome equivalents. BAC stability assays show that clones are stable in the bacterialhost during propagation. Libraries were screened with ten probes designed from DNAsequences of three genes and seven microsatellite markers. Positive clones wereidentified for each gene or microsatellite marker. These results suggest that the threescallop BAC libraries will serve as an invaluable resource for genetic studies onZhikong scallop such as gene cloning, physical mapping and even large-scale sequencing of the genome.
2. Application of C.farreri BAC libraries
We harvested positive BAC clones containing8candidate genes and17SNP sites,which are relevant to growth and immune function of C.farreri through4-dimensionalPCR screening system. Each gene or SNP gives three positive clones, and none ofthose was absent from the libraries. As a result,75positive BAC clones wereidentified, further verifying the genome coverage and utility of the library. Theseclones provide resources essential for research of molecular mechanismes underlyingthe growth and immune of C.farreri. Additionally, BAC clones containing fiveToll-like receptor (TLR) signaling pathway genes, including CfTLR, CfMyd88,CfTRAF6, CfNFκB and CfIκB, were physically mapped using fluorescence in situhybridisation (FISH) to C. farreri’s chromosomes. These BACs represented singlesignals respectively, and they were located on five non-homologous chromosomepairs through karyotypic analysis and cohybridization. The FISH mapping of thesekey immune genes in the Zhikong scallop will aid in the research of innate immunity,and assignment of interested genes to chromosomes.
3. Survey sequencing and analysis of the C.farreri genome
Using next-generation sequencing technology, we have successfully sequencedand de novo assembled a draft sequence of C.farreri. Illumina sequencing generated62.44Gb high quality reads, which covered the genome at50fold. The preliminaryassembly comprised870Mb, with a contig N50of1,267bp and a scaffold N50of1,517bp. The17-mer analysis suggested that the scallop genome was971Mb, andindicated high heterozygosity. The assembled scaffolds covered about57.1%of thewhole genome, and covered about88.7%of the transcriptome. Using RepeatMaskerto analyze all scaffold sequences, a total of884,644interspersed repetitive sequenceswere found, which were classified into4major types: DNA transposon, SINEretrotransposon, LINE retrotransposon and LTR retrotransposon. Among them, DNAtransposon was the most frequent type. SciRoKo analysis showed that a total of134,887simple sequence repeats (SSR) were found, among which monomeric repeat motifs were most abundant. In all, survey on the characterization of whole genomesequences in C.farreri will provide a useful resource for understanding the genomestructure, and allowing further evolutionary and biological complexity analysisof scallops.
1.栉孔扇贝BAC文库的构建及质量评价
本研究构建了栉孔扇贝三个BAC文库，分别为BamHI文库，HindIII文库和Sau3AI文库，其中BamHI文库包含34,560个克隆，平均插入片段为98Kb，未发现空载；HindIII文库包含132,480个克隆，平均插入片段为106Kb，空载率为1.67%；Sau3AI文库包含23,040个克隆，平均插入片段为53Kb，空载率为3.33%。三个文库总共由190,080个克隆组成，平均插入片段为98Kb，覆盖栉孔扇贝基因组的15.4倍。随机挑选BAC克隆100代的继代培养，没有发现插入片段的丢失或重排现象，说明所构建的BAC文库是稳定的。将所有克隆进行超级池及二级池的构建，筛选了栉孔扇贝3个基因以及遗传图谱上的7个微卫星标记，阳性克隆数在6-18之间，表明这三个文库可以在目的基因或标记的筛选，物理图谱构建以及大规模基因组测序过程中发挥重要作用。
2.栉孔扇贝BAC文库的应用
本研究选取栉孔扇贝8个免疫相关候选基因及17个与生长性状QTL连锁的SNP标记为研究对象，利用BAC文库的4D-PCR筛选系统对这25个位点进行筛选分析，每个位点筛选出3个阳性克隆，最终获得了75个含有目的基因或SNP标记的阳性克隆。这为日后栉孔扇贝免疫及生长性状相关基因的完整克隆奠定了基础。另外，还利用BAC-FISH技术对栉孔扇贝Toll样受体（TLR）信号通路中的关键基因（CfTLR, CfMyd88, CfTRAF6, CfNFκB和CfIκB）进行染色体定位。结果显示，包含这些基因的5个BAC克隆定位在染色体上的位置单一。进一步通过核型分析及共定位验证，这5个克隆被定位于5对不同的染色体上，表明TLR信号通路的5个关键基因在栉孔扇贝基因组中并不连锁。这些研究对探索栉孔扇贝免疫系统机理有着积极的作用，并有利于细胞染色体分析等工作的开展。
3.栉孔扇贝基因组勘探测序及特征分析
本研究采用全基因组鸟枪法策略，依托于Illumina solexa高通量测序平台对栉孔扇贝基因组进行勘探测序，获得了62.44Gb的有效数据，评估覆盖深度约为50X。对所有短序列组装结果显示contig N50为1,267bp，scaffold N50为1,517bp，获得scaffold总长约为870Mb。通过17-kmer分析估计栉孔扇贝基因组大小约为971Mb，并预测该测序个体的杂合度约为1.35%。对scaffold序列进行初步分析，估计其覆盖真实基因组的57.1%，覆盖转录组的88.7%。运用RepeatMasker软件进行扫描，获得了884,644个散在重复序列，主要分为四种类型：DNA转座子、SINE反转座子、LINE反转座子和LTR反转座子。其中，DNA转座子数目最多，其次是SINE反转座子、LINE反转座子和LTR反转座子。运用SciRoKo软件进行扫描，获得了134,887个微卫星序列，分为437种类型，其中单碱基重复的微卫星数目最多。这些结果的获得将有利于对栉孔扇贝基因组结构特征的探索，并为全基因组测序策略的选择及基因组学研究方向的确定提供重要的参考信息。
Zhikong scallop (Chlamys farreri) is one of the most important economicaquiculture bivalves in China, but study on its genome is underdeveloped. In thisstudy, we constructed three bacterial artificial chromosome (BAC) libraries ofC.farreri as platform for genomic research, and harvested positive BAC clonescontaining target genes, which are relevant to growth and immune function ofC.farreri. Additionally, we performed genome sequencing and comparative analysisof the C.farreri using next-generation sequencing technology, and thus made apreliminary assessment of the genome.
1. Construction and characterization of BAC libraries in C.farreri
We constructed three bacterial artificial chromosome (BAC) libraries forC.farreri. The BAC libraries were generated based on three restriction enzymes,BamHI, HindIII and Sau3AI, respectively. The BamHI library consists of34,560clones, with an average insert size of98kb and no insert-empty clones. The HindIIIlibrary consists of132,480clones, with an average size of106kb, but approximately1.67%of the clones don’t contain scallop nuclear DNA inserts. The Sau3AI libraryconsists of23,040clones, with an average size of53kb, approximately3.33%of theclones don’t contain DNA inserts. The combined libraries collectively contain a totalof190,080BAC clones with an average size of98kb, representing15.4x haploidgenome equivalents. BAC stability assays show that clones are stable in the bacterialhost during propagation. Libraries were screened with ten probes designed from DNAsequences of three genes and seven microsatellite markers. Positive clones wereidentified for each gene or microsatellite marker. These results suggest that the threescallop BAC libraries will serve as an invaluable resource for genetic studies onZhikong scallop such as gene cloning, physical mapping and even large-scale sequencing of the genome.
2. Application of C.farreri BAC libraries
We harvested positive BAC clones containing8candidate genes and17SNP sites,which are relevant to growth and immune function of C.farreri through4-dimensionalPCR screening system. Each gene or SNP gives three positive clones, and none ofthose was absent from the libraries. As a result,75positive BAC clones wereidentified, further verifying the genome coverage and utility of the library. Theseclones provide resources essential for research of molecular mechanismes underlyingthe growth and immune of C.farreri. Additionally, BAC clones containing fiveToll-like receptor (TLR) signaling pathway genes, including CfTLR, CfMyd88,CfTRAF6, CfNFκB and CfIκB, were physically mapped using fluorescence in situhybridisation (FISH) to C. farreri’s chromosomes. These BACs represented singlesignals respectively, and they were located on five non-homologous chromosomepairs through karyotypic analysis and cohybridization. The FISH mapping of thesekey immune genes in the Zhikong scallop will aid in the research of innate immunity,and assignment of interested genes to chromosomes.
3. Survey sequencing and analysis of the C.farreri genome
Using next-generation sequencing technology, we have successfully sequencedand de novo assembled a draft sequence of C.farreri. Illumina sequencing generated62.44Gb high quality reads, which covered the genome at50fold. The preliminaryassembly comprised870Mb, with a contig N50of1,267bp and a scaffold N50of1,517bp. The17-mer analysis suggested that the scallop genome was971Mb, andindicated high heterozygosity. The assembled scaffolds covered about57.1%of thewhole genome, and covered about88.7%of the transcriptome. Using RepeatMaskerto analyze all scaffold sequences, a total of884,644interspersed repetitive sequenceswere found, which were classified into4major types: DNA transposon, SINEretrotransposon, LINE retrotransposon and LTR retrotransposon. Among them, DNAtransposon was the most frequent type. SciRoKo analysis showed that a total of134,887simple sequence repeats (SSR) were found, among which monomeric repeat motifs were most abundant. In all, survey on the characterization of whole genomesequences in C.farreri will provide a useful resource for understanding the genomestructure, and allowing further evolutionary and biological complexity analysisof scallops.
引文
1. Alan Coulson, John Sulston, Sydney Brenner, et al. Toward a physical map of the genome ofthe nematode Caenorhabditis elegans. Pnas,1986,83(20):7821-7825.
2. Anderson C. Genome shortcut leads to problems. Science,1993,259(5102):1684-1687.
3. Birren B, Green E, Klapholz S, et al. Cloning Systems(3), In Genome Analysis: A laboratorymanual. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press,1997,242-295.
4. Boulos C, Harry B, Michel C. Efficient cloning of plant genomes into bacterial artificialchromosome (BAC) libraries with larger and more uniform insert size. Plant biotechnologyJournal,2004,2:181-188.
5. Budiman MA, Mao L, Wood TC, et al. A deep-coverage tomato BAC library and prospectstoward development of an STC framework for genome sequencing. Genome Res,2000,10:129-136.
6. Chartier FL, Keer JT, Sutcliffe MJ, et al. Construction of a mouse yeast artificialchromosome library in a recombination-deficient strain of yeast. Nature Genetics,1992,1:132-136.
7. Check E, Abbott A, Powell K. Mouse genome: the real deal. Nature,2002,420:457
8. Choi S, Creelman RA, Mullet JE, et al. Construction and characterization of a bacterialartificial chromosome library of Arabidopsis thaliana. Plant Molecular Biology Reporter,1995,13:124-128.
9. Clarke L, Carbon J. A colony bank containing synthetic Col E1hybrid plasmidsrepresentative of the entire E.coli genome. Cell,1976,9:91.
10. Cohen S N, Chang A C, Boyer HW, et al. Construction of biologically functional bacterialplasmids in vitro. Proceedings of the National Academy of Sciences of the United States ofAmerica,1973,70(11):3240-3244.
11. Collins J, Hohn B. Cosmids: a type of plasmid gene-cloning vector that is packageable invitro in bacteriophage lambda heads. Proc Natl Acad Sci USA,1978,75(9):4242-4246
12. Daelen RA, Jonkers JJ, Zabel P. Preparation of megabase-sized tomato DNA and separationof large restriction fragments by field inversion gel electrophoresis (FIGE). Plant Mol Biol,1989,12(3):341-342.
13. Deng D, Zhao G, Xuan J, et al. Construction and Characterization of a Bacterial ArtificialChromosome Library of Marine Macroalga Porphyra yezoensis (Rhodophyta). PlainMolecular Biology Reporter,2004,22(11):375-386..
14. Edwards KJ, Thompson H, Edwards D, et al. Construction and characterization of a yeastartificial chromosome library containing three haploid maize genome equivalents. PlantMolecular Biology,1992,19(2):299-308.
15. El-Assal SE, Alonso-Blanco C, Peeters AJ, et al. A QTL for flowering timein Arabidopsis reveals a novel allele of CRY2. Nature Genetics,2001,29:435-440.
16. Feng Q, Moran JV, Kazazian HJ, et al. Human L1retrotransposon encodes a conservedendonuclease required for retrotransposition. Cell,1996,87(5):905-916.
17. Frijters A, Zhang Z, Van Damme M, et al. Construction of a bacterial artificial chromosomelibrary containing large EcoRI and HindIII genomic fragments of lettuce. TAG Theoreticaland Applied Genetics,1997,94(3):390-399.
18. Froschauer A, Korting C, Katagiri T, et al. Construction and initial analysis of bacterialartificial chromosome (BAC) contigs from the sex-determining region of the platyfishXiphophorus maculates. Gene,2002,295:247-254.
19. Fuhrmann DR, Krzywinski MI, Chiu R, et al. Marra MA Software for automated analysis ofDNA fingerprinting gels. Genome Res,2003,13:940–953.
20. Ganal MW. Isolation and analysis of high-molecular-weight DNA from plants, In:Nonmammalian Genomic Analysis: A Practical Guide. Academic Press Inc., San Diego,1996,61-73.
21. Gao Q, Song L, Ni D, et al. cDNA cloning and mRNA expression of heat shock protein90gene in the haemocytes of Zhikong scallop Chlamys farreri. Comparative Biochemistry andPhysiology. Part B: Biochemistry and Molecular Biology,2007,147(4):704-715.
22. Gingrich JC, Boehrer DM, Games JA, et al. Construction and characterization of humanchromosome2-specific cosmid,fosmid,and PAC clone libraries. Genomics,1996,32:65-74.
23. Grill E, Somerville C. Construction and characterization of a yeast artificial chromosomelibrary of Arabidopsis which is suitable for chromosome walking. Molecular and GeneralGenetics,1991,226(3):484-490.
24. Grosveld FG, Lund T, Murray EJ, et al. The construction of cosmid libraries which can beused to transform eukaryotic cells. Nucleic Acids Res,1982,10:6715-6732.
25. Guo H, Bao Z, Li J, et al. Molecular Characterization of TGF-β Type I Receptor Gene(Tgfbr1) in Chlamys farreri, and the Association of Allelic Variants with Growth Traits. PLosOne,2012,7(11): e51005.
26. Hamilton CM, Frary A, Xu Y, et al. Construction of tomato genomic DNA libraries in abinary-BAC (BIBAC) vector. Plant J,1999,18(2):223-229.
27. Hamilton CM. BIBAC technology: progress and prospects. Agbiotech News and Information,1998,10(1):23-28.
28. Hillier LW, Marth GT, Quinlan AR, et al. Whole-genome sequencing and variant discoveryin C.elegans. Nat Methods,2008,5:183–188.
29. Hohmann U, Jacobs G, Telgmann A, et al. A bacterial artificial chromosome (BAC) library ofsugar beet and a physical map of the region encompassing the bolting gene B. MolecularGenetics and Genomics,2003,269:126-136.
30. Hong YS, Hogan JR, Wang X, et al. Construction of a BAC library and generation of BACend sequence-tagged connectors for genome sequencing of the African malaria mosquitoAnopheles gambiae. Mol Genet Genomics,2003,268:720-728.
31. Hu X, Guo H, He Y, et al. Molecular characterization of myostatin gene from Zhikongscallop Chlamys farreri (Jones et Preston1904). Genes Genet Syst,2010,85:207-218.
32. Huan P, Zhang X, Li F, et al. Chromosomal localization and molecular marker developmentof the lipopolysaccharide and beta-1,3-glucan binding protein gene in the Zhikong scallopChlamys farreri (Jones et Preston)(Pectinoida, Pectinidae). Genetics and Molecular Biology,2010,33(1):36-43.
33. Ioannou PA, Amemiya CT, Garnes J, et al. A new bacteriophage P1-derived vector for thepropagation of large human DNA fragments. Nature Genet,1994,6:84-89.
34. Jiang G, Li J, Li L, et al. Development of44gene-based SNP markers in Zhikong scallop,Chlamys farreri. Conservation Genetics Resources,2011,3(4):659-663.
35. Kim UJ, Birren BW, Slepak T, et al. Construction and Characterization of a Human BacterialArtificial Chromosome Library. Genomics,1996,34(2):213-218.
36. Kim UJ, Shizuya H, de Jong PJ, et al. Stable propagation of cosmid sized human DNA insertsin an F factor based vector. Nucleic Acids Res,1992,20:1083-1085.
37. Klein CB and Costa M. DNA methylation,heterochromatin and epigenetic carcinogens.Mutation Research,1997,386:163-180.
38. Kleine M, Cai D, Hermann RG, et al. Physical mapping and cloning of a translocation insugar beet (Beta vulgaris L.) carrying a gene for nematode (Heterodera schachtii) resistancefrom B.procumbens. Theor Appl Genet,1995,90:399-406.
39. Larin Z, Monaco AP, Lehrach H. Yeast artificial chromosome libraries containing largeinserts from mouse and human DNA. Genetics,1991,88:4123-4127.
40. Laurent Schibler, Daniel Vaiman, Anne Oustry, et al. Cribiu A1Construction andextensivecharacterization of a goat Bacterial Artificial Chromosome library with threefoldgenome coverage. Mammalian Genome,1998,9:119–124.
41. Le Paslier MC, Pierce RJ, Merlin F, et al. Construction and characterization of aschistosoma mansoni bacterial artificial chromosome library. Genomics,2000,65:87-94.
42. Lee JM, Sonnhammer EL. Genomic gene clustering analysis of pathways in eukaryotes.Genome Res,2003,13:875–882.
43. Li L, Xiang J, Liu X, et al. Construction of AFLP-based genetic linkage map for Zhikongscallop, Chlamys farreri Jones et Preston and mapping of sex-linked markers. Aquaculture,2005,245(1-4):63-73.
44. Li R, Fan W, Tian G, et al. The sequence and de novo assembly of the giant panda genome.Nature,2010,463:311-317.
45. Liu YG, Liu HM, Qiu WH, et al. Development of new transformation-Competent artificialchromosome vectors and rice genomic libraries for efficient gene cloning. Gene,2002,282:247-255.
46. Liu YG, Shirano Y, Fukaki H, et al. Complementation of plant mutants with large genomicDNA fragments by a transformation-competent artificial chromosome vector acceleratepositional choning. Proceedings of the National Academy of Sciences of the United States ofAmerica,1999,96:6535-6540.
47. Luo MC, Thomas C, You FM., et al. High-throughput fingerprinting of bacterial artificialchromosomes using the snapshot labeling kit and sizing of restriction fragments by capillaryelectrophoresis. Genomics,2003,82:378-389.
48. Ma ZY, Song W, Peter JS, et al. Non-gridded library: A new approach for BAC (bacterialartificial chromosome) exploitation in hexaploid wheat (Triticum aestivum). Nucleic AcidsResearch,2000,28(24): e106.
49. Marra J. Analysis of diel variability in chlorophyll fluorescence. Journal of Marine Research55,1997,767-784.
50. Matsuda M, Kawato N, Asakawa S, et al. Construction of a BAC library derived from theinbred Hd-rR strain of the teleost fish, Oryzias latipes. Genes Genet,2001,76:61-63.
51. Meyerowitz EM, Guild GM, Prestidge LS, et al. A new high-capacity cosmid vector and itsuse. Gene,1980,11:271-282.
52. Murray NE, Murray K. Manipulation of restriction targets in phage λ to form receptorchromosomes for DNA fragments. Nature,1974,251(5475):476-481.
53. Mozo T, Fischer S, Shizuya H, et al. Construction and characterization of the IGFArabidopsis BAC library. Mol Gen Genet,1998,258:562-570.
54. Murray AW, Szostak JW. Construction of Chromosomes in Yeast. Nature,1983,305:189-193.
55. Murray NE, Murray K. Manipulation of restriction targets in phage lambda to form receptorchromosomes for DNA fragments. Nature,1974,251(5475):476-481.
56. Osoegawa K, Woon PY, Zhao B, et al. An improved approach for construction of bacterialartificial chromosome libraries. Genomics,1998,52:1-8.
57. Patocchi A, Vinatzer BA, Gianfranseschi L, et al. Construction of a550kb BAC contigspanning the genomic region containing the apple resistance gene Vf. Mol Gen Genet,1999,262:884–891.
58. Peterson DG, Tomkins JP, Frisch DA, et al. Construction of plant bacterial artificialchromosome (BAC) libraries: An illustrated guide. Journal of Agricultural Genomics,2000,5:www.nugr.org/research/jag.
59. Phillips RB, Amores A, Morasch MR, et al. Assignment of zebrafish genetic linkage groupsto chromosomes. Cytogenet Genome Res,2006,114:155-162.
60. Phillips RB, Zimmerman A, Noakes MA, et al. Physical and genetic mapping of the rainbowtrout major histocompatibility regions:evidence for duplication of the class I region.Immunogenetics,2003,55:561–569.
61. Qiu L, Song L, Xu W, et al. Molecular cloning and expression of a Toll receptor genehomologue from Zhikong Scallop,Chlamys farreri. Fish Shellfish Immunol,2007,22(5):451-466.
62. Qiu L, Song L, Yu Y, et al. Identification and characterization of a myeloid differentiationfactor88(MyD88) cDNA from Zhikong scallop, Chlamys farreri. Fish Shellfish Immunol,2007,23(3):614-623.
63. Ren C, Lee MK, Yan B, et al. A BAC-based physical map of the chicken genome. GenomeRes,2003,13:2754–2758.
64. Rodriguez MF, Gahr SA, RexroadIII CE, et al. A Polymerase Chain Reaction ScreeningMethod for Rapid Detection of Microsatellites in Bacterial Artificial Chromosomes. MarineBiotechnology,2006,8(4):346-350.
65. Royal A, Garapin A, Cami B, et al. The ovalbumin gene region:common features in theorganisation of threegenes expressed in chicken oviduct under hormonal control. Nature,1979,279:125-132.
66. Schelling BC, Schlapfer J, Billault A, et al. Construction of a canine Bacterial ArtificialChromosome library for screening with PCR. Anim Breed Genet,2002,119:400–401.
67. Shizuya H, Birren B, Kim UJ, et al. Cloning and Stable maintenance of300kilobase-pairfragments of human DNA in E.coli using an F-factor-based vector. Proc Natl Acad Sci,1992,89(18):8794-8797.
68. Stanke M, Diekhans M, Baertsch R, et al. Using native and syntenically mapped cDNAalignments to improve de novo gene finding. Bioinformatics,2008,24:637-644.
69. Sternberg N. Bacteriophage P1cloning system for the isolation, amplification, and recoveryof DNA fragments as large as100kilobase pairs. Proc Natl Acad Sci,1990,87(1):103–107.
70. Strong SJ, Ohta Y, Litman GW, et al. Marked improvement of PAC and BAC cloning isachieved using electroelution of pulsed-field gel-separated partial digests of genomic DNA.Nucleic Acids Res,1997,25:3959-3961.
71. Su J, Ni D, Song L, et al. Molecular cloning and characterization of a shorttypepeptidoglycan recognition protein(CfPGRP-S1) cDNA from Zhikong scallop Chlamys farreri.Fish Shellfish Immunol,2007,23(3):646-656.
72. Sulston J, Mallett F, Staden R, et al. Software for genome mapping by fingerprintingtechniques. Comput Appl Biosci,1988,4:125-132.
73. Suzuki K, Asakawa S, Iida M, et al. Construction and evaluation of a porcine bacterialartificial chromosome library. Anim Genet,2000,31:8–12.
74. TAGI. Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature,2000,408:796–815.
75. Umehara Y, Inagaki A, Tanoue H, et al. Construction and characterization of a rice YAClibrary for physical mapping. Molecular Breeding,1995,1:79-89.
76. Vaiman D, Billault A, Tabet-Aoul K, et al. Construction and characterization of a sheep BAClibrary of three genome equivalents. Mammalian Genome,1999,10:585–587.
77. Venter JC, Smith HO, Hood L. A new strategy for genome sequencing. Nature,1996,381:364-366.
78. Vinatzer BA, Zhang HB, Sansavini S. Construction and characterization of a bacterialartificial chromosome library of apple. Theoretical and Applied Genetics,1998,97:1183-1190.
79. Wang H, Song L, Li C, et al. Cloning and characterizationof a novel C-type lectin fromZhikong scallop Chlamys farreri. Mol Immunol,2007,44(5):722-731.
80. Wang L, Song L, Chang Y, et al. A preliminary genetic map of Zhikong scallop(Chlamysfarreri Jones et Preston1904). Aquaculture research,2005,36(7):643-653.
81. Wang L, Song L, Zhao J, et al. Expressed sequence tags from the zhikong scallop(Chlamysfarreri): Discovery and annotation of host-defense genes. Fish Shellfish Immun,2009,26(5):744-750.
82. Wang M, Yang J, Zhou Z, et al. A primitive Toll-like receptor signaling pathway in molluskZhikong scallop, Chlamys farreri. Developmental and Comparative Immunology,2011,35:511–520.
83. Wang S, Bao Z, Pan J, et al. AFLP linkage map of an intraspecific cross in Chlamys farreri.Journal of Shellfish Research,2004,23(2):491-500.
84. Wang S, Hou R, Bao Z, et al. Transcriptome sequencing of Zhikong scallop (Chlamys farreri)and comparative transcriptomic analysis with Yesso scallop (Patinopecten yessoensis). PLoSOne,2013,8(5):e63927.
85. Wang S, Meyer E, McKay J K, et al.2b-RAD:a simple and flexible method for genome-widegenotyping. Nature Methods,2012,9(8):808–810.
86. Wang Y, Xu Z, Pierce JC, et al. Characterization of Eastern oyster (Crassostrea virginicaGmelin) chromosomes by fluoresecence in situ hybridization with bacteriophage P1clones.Mar Biotechnol,2005,7:207–214.
87. Waterston RH, Lindblad-Toh K, Birney E, et al. Initial sequencing and comparative analysisof the mouse genome. Nature,2002,420:520–562.
88. Wegner. Clustering of Drosophila melanogaster immune genes in interplay withrecombination rate. PLos One,2008,3: e2835.
89. Wing RA, Rastogi VK, Zhang HB, et al. An improved method of plant megabase DNAisolation in agarose microbeads suitable for physical mapping and YAC cloning. Plant J,1993,4:893-898.
90. Woo SS, Jiang J, Gill BS, et al. Construction and characterization of a bacterial artificialchromosome library for Sorghum bicolor. Nucleic Acids Res,1994,22(23):4922-4931.
91. Woo SS, Rastogi VK, Zhang HB, et al. Isolation of megabase-size DNA from sorghum sandapplications for physical mapping and bacterial and yeast artificial chromosome libraryconstruction. Plant Mol Biol Rep,1995,13:82-94.
92. Wu TD, Watanabe CK. GMAP: a genomic mapping and alignment program for mRNA andEST sequences. Bioinformatics,2005,21(9):1859-1875.
93. Xu P, Wang S, Liu L, et al. Channel catfish BAC-end sequences for marker development andassessment of syntenic conservation with other fish species. Anim Genet,2006,37(4):321-326.
94. Yang H, Zhang F, Guo X.2000. Triploid and tetraploid Zhikong scallop, Chlamys farreriJones et Preston, produced by inhibiting polar body I. Marine Biotechnology New YorkSpringer Verlag,2000,2(5):466-475.
95. Yang JL, Wang QH, Deng DY, et al. Construction and characterization of a bacterial artificialchromosome library of maize inbred line77Ht2. Plant Molecular Biology Reporter,2003,21(2):159-169.
96. Yu J, Hu S, Wang J, et al. A draft sequence of the rice genome (Oryza sativa L. ssp. indica).Science,2002,296:79–92.
97. Zhan A, Bao Z, Hu X, et al. Isolation and characterization of150novel microsatellitemarkers for Zhikong scallop (Chlamys farreri). Mol Ecol Notes,2007,7(6):1015-1022.
98. Zhan A, Bao Z, Hu X,et al. Characterization of95novel microsatellite markers for Zhikongscallop Chlamys farreri using FIASCO-colony hybridization and EST database mining.Fisheries Science,2008,74(3):516-526.
99. Zhan A, Bao Z, Yao B, et al. Polymorphic microsatellite markers in the Zhikong scallopChlamys farreri. Mol Ecol Notes,2006,6(1):127-129.
100. Zhan A, Hu J, Hu X, et al. Construction of microsatellite-based linkage maps andidentification of size-related quantitative trait loci for Zhikong scallop(Chlamys farreri).Anim Genet,2009,40(6):821-831.
101. Zhang G, Fang X, Guo X, et al. The oyster genome reveals stress adaptation and complexityof shell formation. Nature,2012,490:49-54.
102. Zhang H. Construction and manipulation of large-insert bacterial clone libraries manual.Texas A&M University, Texas,2000.(http://hbz7.tamu.edu).
103. Zhang H, Zhao X, Ding X, et al. Preparation of megabase-size DNA from plant nuclei. PlantJ,1995,7:175-184.
104. Zhang H, Wang H, Wang L, et al. Cflec-4,amultidomain C-type lectin involved in immunedefense of Zhikong scallop, Chlamys farreri. Dev Comp Immunol,2009,33(6):780-788.
105. Zhang H, Wu C. BACs as tools for genome sequencing. Plant Physiol Biochem,2001,39:195-209.
106. Zhang L, Bao Z, Cheng J, et al. Fosmid library construction and initial analysis of endsequences in Zhikong scallop(Chlamys farreri). Marine Biotechnology,2007,6:606-612.
107. Zhang L, Bao Z, Wang S, et al. FISH mapping and identification of Zhikong scallop(Chlamysfarreri) chromosomes. Mar Biotechnol,2008,10(2):151-157.
108. Zhang X, Zhao C, Huang C, et al. A BAC-base physcal map of Zhikong scallop (Chlamlysfarreri Jones et Preston). Plos One,2011,6(11): e27612.
109. Zhang Y, Zhang X, Scheuring CF, et al. Construction and characterization of two bacterialartificial chromosome libraries of Zhikong scallop, Chlamys farreri Jones et Preston,andidentification of BAC clones containing the genes involved in its innate immune system.Marine Biotechnology,2008,10(4):358-365.
110. Zhao Cui, Zhang Tongwu, Zhang Xiaojun, et al. Sequencing and analysis of four BAC clonescontaining innate immune genes from the Zhikong scallop (Chlamys farreri). Gene,2012,502:9-15.
111. Zimmer R, Verrinder Gibbins AM. Construction and characterization of a large-fragmentchicken bacterial artificial chromosome library. Genomics,1997,42:217-226.
112.巴恒星.中国梅花鹿全基因组初步组装、分析及单核苷酸多态性研究:[博士学位论文].北京:中国农业科学院,2012.
113.高焕.中国对虾基因组串联重复序列分析及其分子标记的开发与应用:[博士学位论文].北京:中国科学院研究生院,2006.
114.高晓明,陈艳玲,刘贯山,等.绒毛状烟草BAC文库的构建.中国烟草科学,2012-06,33(3):68-71.
115.关媛,陈琪,潘俊松,等.黄瓜(Cucumis sativus L.) BAC文库的构建及连锁群特异克隆的分离.自然科学进展,2008,18(2):211-215.
116.郭熙志,刘耀光,罗达.以可转化人工染色体(transformation competentartificialchromosome, TAC)载体为基础的百脉根基因组文库的构建及筛选.植物生理与分子生物学报,2004,30(2):234-238.
117.郇聘.利用荧光原位杂交技术对中国明对虾和栉孔扇贝若干重要基因定位的研究:[博士学位论文].北京:中国科学院研究生院,2010.
118.侯睿.虾夷扇贝(Patinopecten yessoensis)基因组结构特征与进化基因组学分析:[博士学位论文].青岛:中国海洋大学，2012.
119.蒋增杰.桑沟湾栉孔扇贝大规模死亡之原因分析.齐鲁渔业,2004,21(8):10-11.
120.李纪勤,栉孔扇贝（Chlamys farreri）EST-SNP的开发及其应用:[博士学位论文].青岛:中国海洋大学.2012.
121.梁成真,张锐,郭三堆.染色体步移技术研究进展.生物技术通报,2009,10:75-82.
122.马芳,王静思.我国完成首个鱼类基因组测序项目.中国水产,2010,09.
123.牛建峰,高胜寒,骆迎峰,等.条斑紫菜低覆盖度基因组草图分析.海洋科学,2011,35(06):76-81.
124.潘英,李琪,于瑞海,等.栉孔扇贝人工雌核发育的细胞学观察.水产学报,2004,28(06):616-622.
125.彭开蔓,张洪斌,张启发.优良水稻品种明恢63BAC文库的构建.植物学报,1998,40(12):1108-1114.
126.商文聪,赵柏淞,陈亮,等.电刺激诱导栉孔扇贝雌核发育的相关参数的筛选.中国海洋大学学报.2012,42(10):65-70.
127.尚毅,马璐琳,亓增军,等.噬菌体cDNA文库筛选方法的改良.生物学杂志,2010,27(5):91-93.
128.宋平根,李素文.流式细胞术的原理和应用.北京师范大学出版社,1992,259:1-8
129.宋微波,王崇明,王秀华,等.栉孔扇贝大规模死亡的病原研究新进展.海洋科学,2001,25(12):23-26.
130.苏建国,宋林生,胥炜,等.栉孔扇贝脂多糖葡聚糖结合蛋白（LGBP）基因的克隆及其特征分析.高技术通讯,2004,14(3):87-91.
131.王崇明,刘英杰,王秀华,等.栉孔扇贝急性病毒性坏死症（AVND）病理学观察与分析.海洋水产研究,2007,28(4):1-8.
132.王继华,杨茹冰,李晶.荧光原位杂交在微生物学中的应用及进展.哈尔滨师范大学自然科学学报,2006,22(5):84-88.
133.王江波,潘东明,潘腾飞,等.中国水仙BAC文库构建的研究.中国农学通报,2012,28(10):194-197.
134.王梅林,郑家声,余海.栉孔扇贝Chlamys farreri (Jones&Preston,1904)染色体核型.青岛海洋大学学报,1990,20(1):81-85.
135.王省芬,马骏,马峙英,等.高纤维强力棉花种质系苏远7235BAC文库的构建.棉花学报,2006,18(4):200-203.
136.王运涛,相建海.栉孔扇贝大规模死亡的原因探讨.海洋与湖沼,1999,30(6):770-774.
137.魏朝明,孔光耀,廉振民,等.蜜蜂全基因组中微卫星的丰度及其分布,昆虫知识,2007,44(04):501-504.
138.吴龙涛,宋林生,胥炜,等.栉孔扇贝热休克蛋白70基因cDNA的克隆与分析.高技术通讯,2003,13(11):75-79.
139.吴乃虎.基因工程原理（下册）.北京:科学出版社,2003.535.
140.徐俊,包振民,任晓亮,等.栉孔扇贝DAPI带型和PI带型研究.中国海洋大学学报(自然科学版),2011,41(4):77-89.
141.张福绥,杨红生.栉孔扇贝大规模死亡问题的对策与应急措施.海洋科学,1999,(2):38-42.
142.张巨永,黄盛丰,王蔚,等.白氏文昌鱼单个体基因组BAC文库的构建.遗传,2010,32(1):67-72.
143.张俊成,孔秀英,吴佳洁,等.小麦BAC shotgun测序文库的构建.麦类作物学报,2007,27(3):374-377.
144.张琳琳,魏朝明,廉振民,等.赤拟谷盗全基因组和EST中微卫星的丰度.昆虫知识,2008,45(1):38-42.
145.张玲玲.栉孔扇贝(Chlamys farreri) fosmid文库的构建及基因组结构特征分析:[博士学位论文].青岛:中国海洋大学学报,2008.
146.张榭令,王晓梅,王少政,等.改进栉孔扇贝(Chlamys farreri)养殖技术研究.现代农业科技,2007,(1):111-113.
147.张秀英.栉孔扇贝（Chlamys farreri）BAC末端序列中SSR和SNP分子标记的开发及初步应用研究:[硕士学位论文].北京:中国科学院研究生院,2012.
148.张园园,方肇勤,潘志强,等.气虚小鼠脾脏B细胞受体信号通路基因差异表达特征.辽宁中医杂志,2012,39(11):2315-2318.
149.赵翠.对虾和扇贝BAC基因组文库的发掘和应用研究:[博士学位论文].北京:中国科学院研究生院,2012.
2. Anderson C. Genome shortcut leads to problems. Science,1993,259(5102):1684-1687.
3. Birren B, Green E, Klapholz S, et al. Cloning Systems(3), In Genome Analysis: A laboratorymanual. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press,1997,242-295.
4. Boulos C, Harry B, Michel C. Efficient cloning of plant genomes into bacterial artificialchromosome (BAC) libraries with larger and more uniform insert size. Plant biotechnologyJournal,2004,2:181-188.
5. Budiman MA, Mao L, Wood TC, et al. A deep-coverage tomato BAC library and prospectstoward development of an STC framework for genome sequencing. Genome Res,2000,10:129-136.
6. Chartier FL, Keer JT, Sutcliffe MJ, et al. Construction of a mouse yeast artificialchromosome library in a recombination-deficient strain of yeast. Nature Genetics,1992,1:132-136.
7. Check E, Abbott A, Powell K. Mouse genome: the real deal. Nature,2002,420:457
8. Choi S, Creelman RA, Mullet JE, et al. Construction and characterization of a bacterialartificial chromosome library of Arabidopsis thaliana. Plant Molecular Biology Reporter,1995,13:124-128.
9. Clarke L, Carbon J. A colony bank containing synthetic Col E1hybrid plasmidsrepresentative of the entire E.coli genome. Cell,1976,9:91.
10. Cohen S N, Chang A C, Boyer HW, et al. Construction of biologically functional bacterialplasmids in vitro. Proceedings of the National Academy of Sciences of the United States ofAmerica,1973,70(11):3240-3244.
11. Collins J, Hohn B. Cosmids: a type of plasmid gene-cloning vector that is packageable invitro in bacteriophage lambda heads. Proc Natl Acad Sci USA,1978,75(9):4242-4246
12. Daelen RA, Jonkers JJ, Zabel P. Preparation of megabase-sized tomato DNA and separationof large restriction fragments by field inversion gel electrophoresis (FIGE). Plant Mol Biol,1989,12(3):341-342.
13. Deng D, Zhao G, Xuan J, et al. Construction and Characterization of a Bacterial ArtificialChromosome Library of Marine Macroalga Porphyra yezoensis (Rhodophyta). PlainMolecular Biology Reporter,2004,22(11):375-386..
14. Edwards KJ, Thompson H, Edwards D, et al. Construction and characterization of a yeastartificial chromosome library containing three haploid maize genome equivalents. PlantMolecular Biology,1992,19(2):299-308.
15. El-Assal SE, Alonso-Blanco C, Peeters AJ, et al. A QTL for flowering timein Arabidopsis reveals a novel allele of CRY2. Nature Genetics,2001,29:435-440.
16. Feng Q, Moran JV, Kazazian HJ, et al. Human L1retrotransposon encodes a conservedendonuclease required for retrotransposition. Cell,1996,87(5):905-916.
17. Frijters A, Zhang Z, Van Damme M, et al. Construction of a bacterial artificial chromosomelibrary containing large EcoRI and HindIII genomic fragments of lettuce. TAG Theoreticaland Applied Genetics,1997,94(3):390-399.
18. Froschauer A, Korting C, Katagiri T, et al. Construction and initial analysis of bacterialartificial chromosome (BAC) contigs from the sex-determining region of the platyfishXiphophorus maculates. Gene,2002,295:247-254.
19. Fuhrmann DR, Krzywinski MI, Chiu R, et al. Marra MA Software for automated analysis ofDNA fingerprinting gels. Genome Res,2003,13:940–953.
20. Ganal MW. Isolation and analysis of high-molecular-weight DNA from plants, In:Nonmammalian Genomic Analysis: A Practical Guide. Academic Press Inc., San Diego,1996,61-73.
21. Gao Q, Song L, Ni D, et al. cDNA cloning and mRNA expression of heat shock protein90gene in the haemocytes of Zhikong scallop Chlamys farreri. Comparative Biochemistry andPhysiology. Part B: Biochemistry and Molecular Biology,2007,147(4):704-715.
22. Gingrich JC, Boehrer DM, Games JA, et al. Construction and characterization of humanchromosome2-specific cosmid,fosmid,and PAC clone libraries. Genomics,1996,32:65-74.
23. Grill E, Somerville C. Construction and characterization of a yeast artificial chromosomelibrary of Arabidopsis which is suitable for chromosome walking. Molecular and GeneralGenetics,1991,226(3):484-490.
24. Grosveld FG, Lund T, Murray EJ, et al. The construction of cosmid libraries which can beused to transform eukaryotic cells. Nucleic Acids Res,1982,10:6715-6732.
25. Guo H, Bao Z, Li J, et al. Molecular Characterization of TGF-β Type I Receptor Gene(Tgfbr1) in Chlamys farreri, and the Association of Allelic Variants with Growth Traits. PLosOne,2012,7(11): e51005.
26. Hamilton CM, Frary A, Xu Y, et al. Construction of tomato genomic DNA libraries in abinary-BAC (BIBAC) vector. Plant J,1999,18(2):223-229.
27. Hamilton CM. BIBAC technology: progress and prospects. Agbiotech News and Information,1998,10(1):23-28.
28. Hillier LW, Marth GT, Quinlan AR, et al. Whole-genome sequencing and variant discoveryin C.elegans. Nat Methods,2008,5:183–188.
29. Hohmann U, Jacobs G, Telgmann A, et al. A bacterial artificial chromosome (BAC) library ofsugar beet and a physical map of the region encompassing the bolting gene B. MolecularGenetics and Genomics,2003,269:126-136.
30. Hong YS, Hogan JR, Wang X, et al. Construction of a BAC library and generation of BACend sequence-tagged connectors for genome sequencing of the African malaria mosquitoAnopheles gambiae. Mol Genet Genomics,2003,268:720-728.
31. Hu X, Guo H, He Y, et al. Molecular characterization of myostatin gene from Zhikongscallop Chlamys farreri (Jones et Preston1904). Genes Genet Syst,2010,85:207-218.
32. Huan P, Zhang X, Li F, et al. Chromosomal localization and molecular marker developmentof the lipopolysaccharide and beta-1,3-glucan binding protein gene in the Zhikong scallopChlamys farreri (Jones et Preston)(Pectinoida, Pectinidae). Genetics and Molecular Biology,2010,33(1):36-43.
33. Ioannou PA, Amemiya CT, Garnes J, et al. A new bacteriophage P1-derived vector for thepropagation of large human DNA fragments. Nature Genet,1994,6:84-89.
34. Jiang G, Li J, Li L, et al. Development of44gene-based SNP markers in Zhikong scallop,Chlamys farreri. Conservation Genetics Resources,2011,3(4):659-663.
35. Kim UJ, Birren BW, Slepak T, et al. Construction and Characterization of a Human BacterialArtificial Chromosome Library. Genomics,1996,34(2):213-218.
36. Kim UJ, Shizuya H, de Jong PJ, et al. Stable propagation of cosmid sized human DNA insertsin an F factor based vector. Nucleic Acids Res,1992,20:1083-1085.
37. Klein CB and Costa M. DNA methylation,heterochromatin and epigenetic carcinogens.Mutation Research,1997,386:163-180.
38. Kleine M, Cai D, Hermann RG, et al. Physical mapping and cloning of a translocation insugar beet (Beta vulgaris L.) carrying a gene for nematode (Heterodera schachtii) resistancefrom B.procumbens. Theor Appl Genet,1995,90:399-406.
39. Larin Z, Monaco AP, Lehrach H. Yeast artificial chromosome libraries containing largeinserts from mouse and human DNA. Genetics,1991,88:4123-4127.
40. Laurent Schibler, Daniel Vaiman, Anne Oustry, et al. Cribiu A1Construction andextensivecharacterization of a goat Bacterial Artificial Chromosome library with threefoldgenome coverage. Mammalian Genome,1998,9:119–124.
41. Le Paslier MC, Pierce RJ, Merlin F, et al. Construction and characterization of aschistosoma mansoni bacterial artificial chromosome library. Genomics,2000,65:87-94.
42. Lee JM, Sonnhammer EL. Genomic gene clustering analysis of pathways in eukaryotes.Genome Res,2003,13:875–882.
43. Li L, Xiang J, Liu X, et al. Construction of AFLP-based genetic linkage map for Zhikongscallop, Chlamys farreri Jones et Preston and mapping of sex-linked markers. Aquaculture,2005,245(1-4):63-73.
44. Li R, Fan W, Tian G, et al. The sequence and de novo assembly of the giant panda genome.Nature,2010,463:311-317.
45. Liu YG, Liu HM, Qiu WH, et al. Development of new transformation-Competent artificialchromosome vectors and rice genomic libraries for efficient gene cloning. Gene,2002,282:247-255.
46. Liu YG, Shirano Y, Fukaki H, et al. Complementation of plant mutants with large genomicDNA fragments by a transformation-competent artificial chromosome vector acceleratepositional choning. Proceedings of the National Academy of Sciences of the United States ofAmerica,1999,96:6535-6540.
47. Luo MC, Thomas C, You FM., et al. High-throughput fingerprinting of bacterial artificialchromosomes using the snapshot labeling kit and sizing of restriction fragments by capillaryelectrophoresis. Genomics,2003,82:378-389.
48. Ma ZY, Song W, Peter JS, et al. Non-gridded library: A new approach for BAC (bacterialartificial chromosome) exploitation in hexaploid wheat (Triticum aestivum). Nucleic AcidsResearch,2000,28(24): e106.
49. Marra J. Analysis of diel variability in chlorophyll fluorescence. Journal of Marine Research55,1997,767-784.
50. Matsuda M, Kawato N, Asakawa S, et al. Construction of a BAC library derived from theinbred Hd-rR strain of the teleost fish, Oryzias latipes. Genes Genet,2001,76:61-63.
51. Meyerowitz EM, Guild GM, Prestidge LS, et al. A new high-capacity cosmid vector and itsuse. Gene,1980,11:271-282.
52. Murray NE, Murray K. Manipulation of restriction targets in phage λ to form receptorchromosomes for DNA fragments. Nature,1974,251(5475):476-481.
53. Mozo T, Fischer S, Shizuya H, et al. Construction and characterization of the IGFArabidopsis BAC library. Mol Gen Genet,1998,258:562-570.
54. Murray AW, Szostak JW. Construction of Chromosomes in Yeast. Nature,1983,305:189-193.
55. Murray NE, Murray K. Manipulation of restriction targets in phage lambda to form receptorchromosomes for DNA fragments. Nature,1974,251(5475):476-481.
56. Osoegawa K, Woon PY, Zhao B, et al. An improved approach for construction of bacterialartificial chromosome libraries. Genomics,1998,52:1-8.
57. Patocchi A, Vinatzer BA, Gianfranseschi L, et al. Construction of a550kb BAC contigspanning the genomic region containing the apple resistance gene Vf. Mol Gen Genet,1999,262:884–891.
58. Peterson DG, Tomkins JP, Frisch DA, et al. Construction of plant bacterial artificialchromosome (BAC) libraries: An illustrated guide. Journal of Agricultural Genomics,2000,5:www.nugr.org/research/jag.
59. Phillips RB, Amores A, Morasch MR, et al. Assignment of zebrafish genetic linkage groupsto chromosomes. Cytogenet Genome Res,2006,114:155-162.
60. Phillips RB, Zimmerman A, Noakes MA, et al. Physical and genetic mapping of the rainbowtrout major histocompatibility regions:evidence for duplication of the class I region.Immunogenetics,2003,55:561–569.
61. Qiu L, Song L, Xu W, et al. Molecular cloning and expression of a Toll receptor genehomologue from Zhikong Scallop,Chlamys farreri. Fish Shellfish Immunol,2007,22(5):451-466.
62. Qiu L, Song L, Yu Y, et al. Identification and characterization of a myeloid differentiationfactor88(MyD88) cDNA from Zhikong scallop, Chlamys farreri. Fish Shellfish Immunol,2007,23(3):614-623.
63. Ren C, Lee MK, Yan B, et al. A BAC-based physical map of the chicken genome. GenomeRes,2003,13:2754–2758.
64. Rodriguez MF, Gahr SA, RexroadIII CE, et al. A Polymerase Chain Reaction ScreeningMethod for Rapid Detection of Microsatellites in Bacterial Artificial Chromosomes. MarineBiotechnology,2006,8(4):346-350.
65. Royal A, Garapin A, Cami B, et al. The ovalbumin gene region:common features in theorganisation of threegenes expressed in chicken oviduct under hormonal control. Nature,1979,279:125-132.
66. Schelling BC, Schlapfer J, Billault A, et al. Construction of a canine Bacterial ArtificialChromosome library for screening with PCR. Anim Breed Genet,2002,119:400–401.
67. Shizuya H, Birren B, Kim UJ, et al. Cloning and Stable maintenance of300kilobase-pairfragments of human DNA in E.coli using an F-factor-based vector. Proc Natl Acad Sci,1992,89(18):8794-8797.
68. Stanke M, Diekhans M, Baertsch R, et al. Using native and syntenically mapped cDNAalignments to improve de novo gene finding. Bioinformatics,2008,24:637-644.
69. Sternberg N. Bacteriophage P1cloning system for the isolation, amplification, and recoveryof DNA fragments as large as100kilobase pairs. Proc Natl Acad Sci,1990,87(1):103–107.
70. Strong SJ, Ohta Y, Litman GW, et al. Marked improvement of PAC and BAC cloning isachieved using electroelution of pulsed-field gel-separated partial digests of genomic DNA.Nucleic Acids Res,1997,25:3959-3961.
71. Su J, Ni D, Song L, et al. Molecular cloning and characterization of a shorttypepeptidoglycan recognition protein(CfPGRP-S1) cDNA from Zhikong scallop Chlamys farreri.Fish Shellfish Immunol,2007,23(3):646-656.
72. Sulston J, Mallett F, Staden R, et al. Software for genome mapping by fingerprintingtechniques. Comput Appl Biosci,1988,4:125-132.
73. Suzuki K, Asakawa S, Iida M, et al. Construction and evaluation of a porcine bacterialartificial chromosome library. Anim Genet,2000,31:8–12.
74. TAGI. Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature,2000,408:796–815.
75. Umehara Y, Inagaki A, Tanoue H, et al. Construction and characterization of a rice YAClibrary for physical mapping. Molecular Breeding,1995,1:79-89.
76. Vaiman D, Billault A, Tabet-Aoul K, et al. Construction and characterization of a sheep BAClibrary of three genome equivalents. Mammalian Genome,1999,10:585–587.
77. Venter JC, Smith HO, Hood L. A new strategy for genome sequencing. Nature,1996,381:364-366.
78. Vinatzer BA, Zhang HB, Sansavini S. Construction and characterization of a bacterialartificial chromosome library of apple. Theoretical and Applied Genetics,1998,97:1183-1190.
79. Wang H, Song L, Li C, et al. Cloning and characterizationof a novel C-type lectin fromZhikong scallop Chlamys farreri. Mol Immunol,2007,44(5):722-731.
80. Wang L, Song L, Chang Y, et al. A preliminary genetic map of Zhikong scallop(Chlamysfarreri Jones et Preston1904). Aquaculture research,2005,36(7):643-653.
81. Wang L, Song L, Zhao J, et al. Expressed sequence tags from the zhikong scallop(Chlamysfarreri): Discovery and annotation of host-defense genes. Fish Shellfish Immun,2009,26(5):744-750.
82. Wang M, Yang J, Zhou Z, et al. A primitive Toll-like receptor signaling pathway in molluskZhikong scallop, Chlamys farreri. Developmental and Comparative Immunology,2011,35:511–520.
83. Wang S, Bao Z, Pan J, et al. AFLP linkage map of an intraspecific cross in Chlamys farreri.Journal of Shellfish Research,2004,23(2):491-500.
84. Wang S, Hou R, Bao Z, et al. Transcriptome sequencing of Zhikong scallop (Chlamys farreri)and comparative transcriptomic analysis with Yesso scallop (Patinopecten yessoensis). PLoSOne,2013,8(5):e63927.
85. Wang S, Meyer E, McKay J K, et al.2b-RAD:a simple and flexible method for genome-widegenotyping. Nature Methods,2012,9(8):808–810.
86. Wang Y, Xu Z, Pierce JC, et al. Characterization of Eastern oyster (Crassostrea virginicaGmelin) chromosomes by fluoresecence in situ hybridization with bacteriophage P1clones.Mar Biotechnol,2005,7:207–214.
87. Waterston RH, Lindblad-Toh K, Birney E, et al. Initial sequencing and comparative analysisof the mouse genome. Nature,2002,420:520–562.
88. Wegner. Clustering of Drosophila melanogaster immune genes in interplay withrecombination rate. PLos One,2008,3: e2835.
89. Wing RA, Rastogi VK, Zhang HB, et al. An improved method of plant megabase DNAisolation in agarose microbeads suitable for physical mapping and YAC cloning. Plant J,1993,4:893-898.
90. Woo SS, Jiang J, Gill BS, et al. Construction and characterization of a bacterial artificialchromosome library for Sorghum bicolor. Nucleic Acids Res,1994,22(23):4922-4931.
91. Woo SS, Rastogi VK, Zhang HB, et al. Isolation of megabase-size DNA from sorghum sandapplications for physical mapping and bacterial and yeast artificial chromosome libraryconstruction. Plant Mol Biol Rep,1995,13:82-94.
92. Wu TD, Watanabe CK. GMAP: a genomic mapping and alignment program for mRNA andEST sequences. Bioinformatics,2005,21(9):1859-1875.
93. Xu P, Wang S, Liu L, et al. Channel catfish BAC-end sequences for marker development andassessment of syntenic conservation with other fish species. Anim Genet,2006,37(4):321-326.
94. Yang H, Zhang F, Guo X.2000. Triploid and tetraploid Zhikong scallop, Chlamys farreriJones et Preston, produced by inhibiting polar body I. Marine Biotechnology New YorkSpringer Verlag,2000,2(5):466-475.
95. Yang JL, Wang QH, Deng DY, et al. Construction and characterization of a bacterial artificialchromosome library of maize inbred line77Ht2. Plant Molecular Biology Reporter,2003,21(2):159-169.
96. Yu J, Hu S, Wang J, et al. A draft sequence of the rice genome (Oryza sativa L. ssp. indica).Science,2002,296:79–92.
97. Zhan A, Bao Z, Hu X, et al. Isolation and characterization of150novel microsatellitemarkers for Zhikong scallop (Chlamys farreri). Mol Ecol Notes,2007,7(6):1015-1022.
98. Zhan A, Bao Z, Hu X,et al. Characterization of95novel microsatellite markers for Zhikongscallop Chlamys farreri using FIASCO-colony hybridization and EST database mining.Fisheries Science,2008,74(3):516-526.
99. Zhan A, Bao Z, Yao B, et al. Polymorphic microsatellite markers in the Zhikong scallopChlamys farreri. Mol Ecol Notes,2006,6(1):127-129.
100. Zhan A, Hu J, Hu X, et al. Construction of microsatellite-based linkage maps andidentification of size-related quantitative trait loci for Zhikong scallop(Chlamys farreri).Anim Genet,2009,40(6):821-831.
101. Zhang G, Fang X, Guo X, et al. The oyster genome reveals stress adaptation and complexityof shell formation. Nature,2012,490:49-54.
102. Zhang H. Construction and manipulation of large-insert bacterial clone libraries manual.Texas A&M University, Texas,2000.(http://hbz7.tamu.edu).
103. Zhang H, Zhao X, Ding X, et al. Preparation of megabase-size DNA from plant nuclei. PlantJ,1995,7:175-184.
104. Zhang H, Wang H, Wang L, et al. Cflec-4,amultidomain C-type lectin involved in immunedefense of Zhikong scallop, Chlamys farreri. Dev Comp Immunol,2009,33(6):780-788.
105. Zhang H, Wu C. BACs as tools for genome sequencing. Plant Physiol Biochem,2001,39:195-209.
106. Zhang L, Bao Z, Cheng J, et al. Fosmid library construction and initial analysis of endsequences in Zhikong scallop(Chlamys farreri). Marine Biotechnology,2007,6:606-612.
107. Zhang L, Bao Z, Wang S, et al. FISH mapping and identification of Zhikong scallop(Chlamysfarreri) chromosomes. Mar Biotechnol,2008,10(2):151-157.
108. Zhang X, Zhao C, Huang C, et al. A BAC-base physcal map of Zhikong scallop (Chlamlysfarreri Jones et Preston). Plos One,2011,6(11): e27612.
109. Zhang Y, Zhang X, Scheuring CF, et al. Construction and characterization of two bacterialartificial chromosome libraries of Zhikong scallop, Chlamys farreri Jones et Preston,andidentification of BAC clones containing the genes involved in its innate immune system.Marine Biotechnology,2008,10(4):358-365.
110. Zhao Cui, Zhang Tongwu, Zhang Xiaojun, et al. Sequencing and analysis of four BAC clonescontaining innate immune genes from the Zhikong scallop (Chlamys farreri). Gene,2012,502:9-15.
111. Zimmer R, Verrinder Gibbins AM. Construction and characterization of a large-fragmentchicken bacterial artificial chromosome library. Genomics,1997,42:217-226.
112.巴恒星.中国梅花鹿全基因组初步组装、分析及单核苷酸多态性研究:[博士学位论文].北京:中国农业科学院,2012.
113.高焕.中国对虾基因组串联重复序列分析及其分子标记的开发与应用:[博士学位论文].北京:中国科学院研究生院,2006.
114.高晓明,陈艳玲,刘贯山,等.绒毛状烟草BAC文库的构建.中国烟草科学,2012-06,33(3):68-71.
115.关媛,陈琪,潘俊松,等.黄瓜(Cucumis sativus L.) BAC文库的构建及连锁群特异克隆的分离.自然科学进展,2008,18(2):211-215.
116.郭熙志,刘耀光,罗达.以可转化人工染色体(transformation competentartificialchromosome, TAC)载体为基础的百脉根基因组文库的构建及筛选.植物生理与分子生物学报,2004,30(2):234-238.
117.郇聘.利用荧光原位杂交技术对中国明对虾和栉孔扇贝若干重要基因定位的研究:[博士学位论文].北京:中国科学院研究生院,2010.
118.侯睿.虾夷扇贝(Patinopecten yessoensis)基因组结构特征与进化基因组学分析:[博士学位论文].青岛:中国海洋大学，2012.
119.蒋增杰.桑沟湾栉孔扇贝大规模死亡之原因分析.齐鲁渔业,2004,21(8):10-11.
120.李纪勤,栉孔扇贝（Chlamys farreri）EST-SNP的开发及其应用:[博士学位论文].青岛:中国海洋大学.2012.
121.梁成真,张锐,郭三堆.染色体步移技术研究进展.生物技术通报,2009,10:75-82.
122.马芳,王静思.我国完成首个鱼类基因组测序项目.中国水产,2010,09.
123.牛建峰,高胜寒,骆迎峰,等.条斑紫菜低覆盖度基因组草图分析.海洋科学,2011,35(06):76-81.
124.潘英,李琪,于瑞海,等.栉孔扇贝人工雌核发育的细胞学观察.水产学报,2004,28(06):616-622.
125.彭开蔓,张洪斌,张启发.优良水稻品种明恢63BAC文库的构建.植物学报,1998,40(12):1108-1114.
126.商文聪,赵柏淞,陈亮,等.电刺激诱导栉孔扇贝雌核发育的相关参数的筛选.中国海洋大学学报.2012,42(10):65-70.
127.尚毅,马璐琳,亓增军,等.噬菌体cDNA文库筛选方法的改良.生物学杂志,2010,27(5):91-93.
128.宋平根,李素文.流式细胞术的原理和应用.北京师范大学出版社,1992,259:1-8
129.宋微波,王崇明,王秀华,等.栉孔扇贝大规模死亡的病原研究新进展.海洋科学,2001,25(12):23-26.
130.苏建国,宋林生,胥炜,等.栉孔扇贝脂多糖葡聚糖结合蛋白（LGBP）基因的克隆及其特征分析.高技术通讯,2004,14(3):87-91.
131.王崇明,刘英杰,王秀华,等.栉孔扇贝急性病毒性坏死症（AVND）病理学观察与分析.海洋水产研究,2007,28(4):1-8.
132.王继华,杨茹冰,李晶.荧光原位杂交在微生物学中的应用及进展.哈尔滨师范大学自然科学学报,2006,22(5):84-88.
133.王江波,潘东明,潘腾飞,等.中国水仙BAC文库构建的研究.中国农学通报,2012,28(10):194-197.
134.王梅林,郑家声,余海.栉孔扇贝Chlamys farreri (Jones&Preston,1904)染色体核型.青岛海洋大学学报,1990,20(1):81-85.
135.王省芬,马骏,马峙英,等.高纤维强力棉花种质系苏远7235BAC文库的构建.棉花学报,2006,18(4):200-203.
136.王运涛,相建海.栉孔扇贝大规模死亡的原因探讨.海洋与湖沼,1999,30(6):770-774.
137.魏朝明,孔光耀,廉振民,等.蜜蜂全基因组中微卫星的丰度及其分布,昆虫知识,2007,44(04):501-504.
138.吴龙涛,宋林生,胥炜,等.栉孔扇贝热休克蛋白70基因cDNA的克隆与分析.高技术通讯,2003,13(11):75-79.
139.吴乃虎.基因工程原理（下册）.北京:科学出版社,2003.535.
140.徐俊,包振民,任晓亮,等.栉孔扇贝DAPI带型和PI带型研究.中国海洋大学学报(自然科学版),2011,41(4):77-89.
141.张福绥,杨红生.栉孔扇贝大规模死亡问题的对策与应急措施.海洋科学,1999,(2):38-42.
142.张巨永,黄盛丰,王蔚,等.白氏文昌鱼单个体基因组BAC文库的构建.遗传,2010,32(1):67-72.
143.张俊成,孔秀英,吴佳洁,等.小麦BAC shotgun测序文库的构建.麦类作物学报,2007,27(3):374-377.
144.张琳琳,魏朝明,廉振民,等.赤拟谷盗全基因组和EST中微卫星的丰度.昆虫知识,2008,45(1):38-42.
145.张玲玲.栉孔扇贝(Chlamys farreri) fosmid文库的构建及基因组结构特征分析:[博士学位论文].青岛:中国海洋大学学报,2008.
146.张榭令,王晓梅,王少政,等.改进栉孔扇贝(Chlamys farreri)养殖技术研究.现代农业科技,2007,(1):111-113.
147.张秀英.栉孔扇贝（Chlamys farreri）BAC末端序列中SSR和SNP分子标记的开发及初步应用研究:[硕士学位论文].北京:中国科学院研究生院,2012.
148.张园园,方肇勤,潘志强,等.气虚小鼠脾脏B细胞受体信号通路基因差异表达特征.辽宁中医杂志,2012,39(11):2315-2318.
149.赵翠.对虾和扇贝BAC基因组文库的发掘和应用研究:[博士学位论文].北京:中国科学院研究生院,2012.