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

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31 872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86 996 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。

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

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 of female gametophytes Saccharina japonica was constructed. The library, containing a total of 31,872 BAC clones with an average insert size of 115 kb, covering 6.57-fold of the estimated haploid genome equivalents. The specific primers of a female-specific marker FRML-1488 were to screen those BAC clones by PCR amplification. In total four positive clones were obtained, randomly selected a BAC clone (638-C12) and sequenced by Roche454 method. Finally, the insert fragment was assembled with the length of 86,996 bp, which was located in Scaffold285 and accounted for 22.4% of the length of this scaffold. Sequence analysis results showed that a large number of repetitive sequences including microsatellites and retrotransposons (Copia) were found upstream of the female-specific marker FRML-1488. Above all, the insert of BAC clone 638-C12 may be related to the U chromosome sex determining region of Saccharina japonica. This study report of BAC library for Saccharina japonica and sex-linked map-based cloning for the first time, contributing to a comprehensive understanding of sex chromosome structure and the sex determination mechanism of this kelp.