【作者】 武祥伟；

【导师】 王志勇；

【作者基本信息】 集美大学 ， 水产养殖， 2011， 硕士

【摘要】 本文建立了大黄鱼与黄姑鱼杂交鱼苗的基因组DNA扩增技术,以及通用序列加尾与荧光素标记的微卫星分型技术。利用所建立的技术,以大黄鱼♀×黄姑鱼♂和大黄鱼♂×黄姑鱼♀杂交初孵仔鱼为材料构建微卫星标记连锁图谱,同时利用诱导的杂交三倍体研究了32个大黄鱼Ⅰ型微卫星标记与着丝粒的遗传距离。主要结果如下:1.为实现利用Gel-Scan 3000遗传分析仪准确、自动地记录微卫星标记电泳产物并节省成本,研究了以添加荧光标记的M13引物为通用引物的微卫星标记分析技术。其PCR体系包含3条引物:5’端加上M13通用序列的正向引物、反向引物与M13引物(5’端标记一种荧光素),正向与反向引物用量之比为1:10,且正向引物与M13引物用量之和等于反向引物用量时扩增效果最好。研究确定了适用于大黄鱼已有微卫星标记的PCR反应体系、热循环条件,以及多重上样方法。与普通PCR及银染显色技术相比,该方法具有高通量、低成本以及分析精确等优点。2.鉴于大黄鱼与黄姑鱼杂交能获得拥有父本和母本单倍体基因组且形态正常的杂交仔鱼,但仔鱼不能开口摄食和成长,DNA数量不足以用于遗传图谱构建,因此研究了杂交仔鱼的DNA扩增技术。基因组DNA以TaqⅠ内切酶酶切,酶切片段与TaqⅠ寡核苷酸接头连接,然后以该接头序列为引物用高保真DNA聚合酶对连接产物进行PCR扩增。采用这种方法,可使基因组DNA增加约2.5×107倍,大部分扩增片段集中在500-1500 bp之间,用作大黄鱼微卫星标记连锁图谱构建的PCR反应模板效果良好,从而解决了用初孵仔鱼构建遗传图谱DNA模板不足的问题。3.采用纯粹的大黄鱼家系构建遗传图谱,由于亲本间的遗传差异不大,在很多微卫星位点上都缺乏作图信息,构建高密度图谱有困难。因此本文尝试利用大黄鱼与黄姑鱼杂交子代进行大黄鱼遗传图谱构建,以期能够对利用纯粹的大黄鱼家系构建的SSR标记图谱进行加密。共使用144尾大黄鱼♀×黄姑鱼♂家系、96尾黄姑鱼♀×大黄鱼♂家系的初孵仔鱼,主要选择在纯粹的大黄鱼家系中缺乏作图信息的位点,加上部分已在本实验室构建的大黄鱼SSR标记图谱上定位的位点作为锚定标记,共检测了221个标记,正反交家系中具作图信息的标记分别有81个与61个,能定位到雌雄图谱中的标记分别有43个与30个,分别构成15个与11个连锁群。构成的雌性图谱总长度529.17 cM,预期长度1138.96 cM,图谱覆盖率为46.4%;雄性图谱总长度327.56 cM,预期长度614.48 cM,图谱覆盖率为53.3%。雌雄图谱中偏离1:1分离比的位点分别有5个与7个,占总标记的6.1%与11.7%。32个微卫星标记可在黄姑鱼基因组中扩增出清晰的条带,其中14个具有多态性。4.进行大黄鱼与黄姑鱼杂交,通过抑制受精卵第二极体排出,构建了2个三倍体家系;利用这2个家系研究了32个大黄鱼Ⅰ型微卫星标记与着丝粒间遗传距离。其中21个位点的重组率大于2/3,说明这些位点处于与着丝粒距离较远的位置,位点LYC1737与着丝粒重组率达0.979,显示其可能位于远离着丝粒的染色体的端部区域;LYC3211与着丝粒重组率为0,表明位置紧邻着丝粒。更多还原

【Abstract】 Whole genome amplification technique, universal sequence tailed and fluorescence labeled microsatellite analysis method by the universal M13 primer were established for the distant hybridization fingerprints of Larimichthys crocea×Nibea albiflora in this study. Those methods were employed to construct the SSR genetic linkage maps by distant hybridized fry of Larimichthys crocea (♀)×Nibea albiflora (♂) and Larimichthys crocea (♂)×Nibea albiflora (♀) separately. Additonally, the genetic distance between centromere and 32 typeⅠmicrosatellites were analyzed by inducing hybrid triploid lines of Larimichthys crocea. The major results were presented as following.1. M13-tailed microsatellite analysis method was developed by the fluorescent labeling universal M13 primer, which facilated to collect the genotyping datas of microsatellites automatically and accurately. The PCR system included three primers: forward tailed primer by M13 universal sequence, reverse primer, and labeled M13 universal primer by fluorescence dye. The desired PCR result could be obtained when the proportion of forward primer and reverse primer was 1:10, and the amount of the forward primer and M13 primer equaled to reverse primer. In this study, the PCR reaction system, thermal cycling procedure, and multi-loading method for PCR products had been optimized for the developed microsatellites. Comparing with the common PCR and silver-staining methods, our approach had the advantages of high-throughput, low-cost, and being accurate analysis.2. Although the normal distant hybridized fry which processed the parental haploid genomes and normal morphology could be created by the distant hybridization between Larimichthys crocea and Nibea albiflora, they could not ingest exogenous food and growed normally. The quality of their gomomic DNA, thus, was unsufficient for constructing of genetic linkage maps. Therefore, whole genome amplification was developed in this study for the amplifying genomic DNA of distant hybridized fry. The endonuclease TaqⅠwas used to digeste the genomic DNA, then the oligounclotide adaptor was ligated to the DNA fragments, and finally the PCR amplification was performed for the ligation products by the primer from the adaptor sequence and the high fidelity DNA polymerase. Genomic DNA could be multiplied about 2.5×107 folds, and the most amplified DNA fragments fall the range of 500 bp-1500 bp. Desried PCR amplification could be attained when the amplified gemomic DNA was used as PCR template. Whole genome amplification provided an aviliable solution for the unsufficient quality of genomic DNA when using fingerprints to construct genetic linkage maps.3. It was very difficulty to construct high density genetic maps by families of large yellow croaker alone, because there were a few genetic variances among parents, resulting in the lacks of genetic linkage information for many microsatellites. We, thus, attempted to use the distant hybridized fry of Larimichthys crocea and Nibea albiflora to construct the genetic maps for large yellow croaker, with the expecting to increase the markers’dencity in genetic maps. 144 and 96 F1 progenies from two reciprocal hybrid lines of Larimichthys crocea and Nibea albiflora were used. The major resources of microsatellites were those that lack linkage information in families of Larimichthys crocea, and those that had been mapped on the genetic maps of Larimichthys crocea with regarding as anchor markers. 221 microsatellites were tested, and there were 81 and 61 microsatellites of processing linkage information in reciprocal crossing families, while the female map and male map were consisted of 43 and 30 loci, with assembling into 15 and 11 linkage groups separately. The total length of two maps were 529.17 cM and 327.56 cM, with the expected length of 1138.96 cM and 614.48 cM, respectively. The coverage rates of two maps, hence, were 46.4% and 53.3% separately. Additionly, specific amplified products could be obtained by 32 microsatellites in Nibea albiflora, and 14 markers were polymorphic.4. When distant hybridization between Larimichthys crocea and Nibea albiflora, the releasing of secondary polar body was inhabited to found two hybrid triploid lines. The distance between centromere and 32 typeⅠmicrosatellites were analyzed by this triploid lines for Larimichthys crocea. The value of recombination rate for 21 markers was larger than 2/3, inllustrating there were long distance between centromere and those microsatellites. The recombination rate value between LYC1737 and centromere was 0.979, presenting that LYC1737 was possible at the terminal region of chromosome; while the value was 0 for LYC3211, displaying that LYC3211 was likely to be very close to the centromere.更多还原