海带雌配子体细菌人工染色体(BAC)文库构建及特异分子标记邻近基因的图位克隆与序列  

Construction of a bacterial artificial chromosome(BAC)library of the female gametophytes of Saccharina japonica and map cloning and sequencing of genes neighboring with a female-specific marker FRML-1488

作　　者：刘鹏飞 谷俊刚 毕燕会 周志刚[2] LIU Pengfei;GU Jungang;BI Yanhui;ZHOU Zhigang(Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources,Ministry of Education,Shanghai Ocean University,Shanghai 201306,China;International Research Center for Marine Biosciences,Ministry of Science and Technology,Shanghai Ocean University,Shanghai 201306,China)

机构地区：[1]上海海洋大学,水产种质资源发掘与利用教育部重点实验室,上海201306 [2]上海海洋大学,国家海洋生物科学国际联合研究中心,上海201306:

出　　处：《水产学报》2021年第5期672-681,共10页Journal of Fisheries of China

基　　金：国家自然科学基金(41376136);国家“双一流”水产学科。

摘　　要：UV性别决定系统在一些低等生物生活史的单倍体阶段展现出特定的进化和遗传特性。实验构建了一个海带雌配子体基因组的细菌人工染色体(BAC)文库,结果显示,该文库包含31872个克隆,插入片段平均长为115 kb,覆盖6.57倍的海带基因组。利用海带雌配子体特异性的标记FRML-1488的序列为探针筛选BAC文库,获得4个阳性BAC克隆。随机挑选一个克隆(638-C12),通过Roche454第二代测序平台进行测序,并经过间隔序列的扩增、克隆和测序,了解到该BAC克隆的插入片段长为86996 bp。该序列位于海带基因组的Scaffold285上,占后者序列总长的22.4%。序列分析结果显示,在雌配子体特异性标记FRML-1488的上游存在大量的微卫星以及Copia逆转录转座子等重复序列。推测BAC克隆638-C12的插入片段可能与海带U染色体的性别决定区相关。本研究是BAC文库在海带性别相关序列图位克隆的首次应用报道,将有助于海带性别染色体结构的揭示及性别决定机制的解析。UV sex-chromosome system exhibits unique evolution and genetic features during the haploid stage of the life cycle in some algae and bryophytes.In this study,a bacterial artificial chromosome(BAC)library was constructed from the female gametophytes of Saccharina japonica.The library,contained a total of 31872 BAC clones with an average insert size of 115 kb as revealed by pulsed-field gel electrophoresis.It covered 6.57-fold of the estimated haploid genome equivalents.To evaluate the quality of this library,one pair of primers was designed on the basis a female-specific marker FRML-1488 to screen positive BAC clones harboring this specific marker by PCR amplification.Four positive clones were obtained,demonstrating that the possibility of selecting any gene from the library was 99.86%.A randomly selected BAC clone(638-C12)was sequenced by Roche454 pyrosequencing method.Total 10 contigs were assembled and the gaps were further filled by PCR amplification.Finally,a full-length fragment was assembled with the length of 86996 bp.It was located in Scaffold285 of the kelp genome by blast,and it accounted for 22.4%of this scaffold in length.This well-matching result pointed out that the insert in BAC clone 638-C12 was from the kelp genome.Sequence analysis showed that a large number of repetitive sequences including microsatellites,satellites and retrotransposons(Copia)were found upstream of the female-specific marker FRML-1488.Since sex-determining region(SDR)is characterized by high proportion of repetitive sequences associated with non-combination,the insert of BAC clone 638-C12 might be involved in the SDR of S.japonica U chromosome.This study is the first report on the application of BAC library in map-based cloning of sex-related sequences in S.japonica,which contributes to comprehensive understanding of sex chromosome structure and the sex determination mechanism of this kelp.

关 键 词：海带 雌配子体 细菌人工染色体(BAC)文库 分子标记 图位克隆 

分 类 号：S917.3[农业科学—水产科学]