The Research On Bacterial Artificial Chromosome (BAC) Library Construction Of Sea Cucumber, Apostichopus Japonicus

Sea cucumber, Apostichopus japonicus is one of the most important economicaquaculture species in China. Previous studies mainly focused on these researchfields such as aquaculture ecology, physiology, nutrition, ethology and etc., study onits genomics is relatively dropped behind. Large-insert bacterial artificialchromosome (BAC) library is indispensable tools for genomics research. A Mbo Ilibrary has been constructed based on optimization of several key technologies inthe BAC library construction process in this study in order to clarify the structureand function of genome, gene mapping, map-based cloning the important functiongenes, QTL positioning, and ultimately achieve whole-genome sequencing.1、Genome size determination of sea cucumber, A. japonicusThe genome size (C-value) of sea cucumber, A. japonicus was measured throughflow cytometry in15individuals, using erythrocyte DNA content of chicken, Gallusdomesticus (2.5pg/2C) as internal standard. The result indicated that the C-value of A.japonicus is0.84±0.02pg, equivalent to821.5±19.56Mb.2、Construction and identification of the BAC library of sea cucumber, A.japonicusThe BAC library of sea cucumber was constructed with Mbo I, using oneindividual. Genomic BAC library provides an essential platform for genomicresearch, and its quality is directly related to the follow-up research and utilization.Therefore, it must be evaluated to determine if suitable for further study. The librarycontains7,206clones, which average insert-DNA size is approximate to103kb inlength and covers0.9fold of the haploid genome of A. japonicus after identification.The reservoir storage capacity has been increasing because genomic coverage hasclosely relationship with high probability for finding the target gene. Meanwhile,5BAC clones genetic stability detection showed that the inserted DNA（>100kb）has no rearrangements and losses even reproduced for at least100cell generations.Though the coverage and the insert size of the BAC library could not serve theneeds of genetic screening, mapping and sequencing, however, a whole technologysystem has been established in sea cucumber. It provided a crucial technical supportfor the genomic research of this species.