Hu Xiaoli

Genetic variation in two sea cucumber (Apostichopus japonicus) stocks revealed by ISSR markers

Sea cucumber Apostichopus japonicus samples were collected in Changdao, Penglai (PL), 27 individuals, and Lingshandao, Qingdao (QD), 30 individuals, in the Shandong Peninsula, China. Ten SSR primers were used to assess the genetic variation and relationship between and within the two stocks. Respectively, for each stock, the percentage of polymorphic bands was 85.2% and 86.9%; the gene diversity was 0.360 5 and 0.342 8; and the Shannon’s information index was 0.515 0 and 0.499 0. At species level, the percentage of polymorphic bands was 92.2%, the total gene diversity was 0.378 9 and the Shannon’s information index was 0.550 4. The coefficient of overall genetic differentiation and the genetic distances between the stocks were also calculated to be 0.073 0 and 0.079 6 using the POPGENE program. Results show that the genetic diversity of the two stocks is still large but the genetic distance between the two stocks is close. A dendrogram was constructed for the 57 individuals from the two stocks, showing that the genetic structure was unitary for PL stock but complex for QD stock.

An Efficient Procedure for Isolating Microsatellite DNAs from Sea Cucumber (Apostichopus japonicus)

The construction of enrichment library proves to be one of the efficient approaches for isolating microsatellites in this study. The genomic DNA of sea cucumber was digested with HaeIII and size-selected DNA fragments (250–700 bp) were ligated to an adaptor. Microsatellite-containing sequences were captured by using a combination of GA and CA probes, which were attached to a nylon membrane. The microsatellite enrichment library constructed in this study consisted of approximately 700 clones. Two hundred and thirty-two clones reacted positively after the library screening procedure. Of the 50 clones sequenced, all contained at least one microsatellite and one duplicate clone was found. Approximately 86% of the sequenced fragments permitted to design primers for sequence tagged microsatellite site (STMS).

Existence of microsatellites in expressed sequence tags of common carp (Cyprinus carpio L.) available in GenBank dbEST database

Common carp expressed sequence tags (ESTs) were analyzed for the existence of microsatellites, or simple sequence repeats (SSRs). In the NCBI dbEST database, a total of 10612 sequences were registered before December 31, 2004. A complete search of 2–6 nucleotide microsatellites resulted in the identification of 513 SSR-containing ESTs, accounting for 4.8% of the total. Cluster analysis indicated that 73 sequences of SSR-containing ESTs fell into 27 groups and the remaining 440 ESTs were indenpendent. A total of 467 unique SSR-containing ESTs were identified. These EST-SSRs contained a variety of simple sequence types, and di- and tri-nucleotide repeats were the most abundant, accounting for 42.1% and 27.9% of the whole, respectively. Of the dinucleotide repeats, CA/TG was the most abundant, followed by GA/TC. BLASTx search showed that 38.1% of the SSR loci could be associated with genes or proteins of known or unknown function. BLASTx searches of SSR-containing ESTs also showed high frequencies (98/179) of hits on zebrafish sequences.