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 was constructed from the female gametophytes of Saccharina japonica. The library, contained a total of 31 872 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 86 996 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.