Development and characterization of EST-SSR markers from Scapharca broughtonii and their transferability in Scapharca subcrenata and Tegillarca granosa.

Twenty-five novel EST-derived simple sequence repeat (EST-SSR) markers were developed in the ark shell Scapharca broughtonii. Polymorphisms of these EST-SSR markers were evaluated in 48 wild individuals collected from Shidao, Shandong Province, China. A total of 202 alleles were detected at 25 loci. The numbers of alleles per locus ranged from 4 to 14, with an average of 8.08. The observed and expected heterozygosities varied from 0.2917 to 1.000 and from 0.3570 to 0.9002, respectively. After sequential Bonferroni correction for multiple tests, only one locus was found to deviate from Hardy-Weinberg equilibrium. Twenty-five EST-SSR markers showed a high rate of across-species transferability (100%) in Scapharca subcrenata and a low rate of across-genus transferability (20%) in Tegillarca granosa. These EST-SSRs will be helpful for QTL mapping, molecular breeding and investigation of population genetic diversity in ark shell S. broughtonii and other Scapharca species.

Molecular characterization and expression of a novel big defensin (Sb-BDef1) from ark shell, Scapharca broughtonii.

Big defensins, endogenous cysteine-rich antimicrobial peptides (AMPs) with antimicrobial activity and immunomodulatory property, play crucial roles in host defense against various microbial pathogens. A novel big defensin (Sb-BDef1) of ark shell Scapharca broughtonii was identified by expressed sequence tag (EST) and RACE techniques. The Sb-BDef1 cDNA contained an open reading frame (ORF) of 336-bp encoding a polypeptide of 111 amino acids with a putative signal peptide of 21 amino acid residues, followed by a putative propeptide of 11 residues and a putative mature peptide of 79 residues. The mature peptide shared the common features of big defensins, including a high hydrophobic residues region (59%) in the N-terminus, a defensin domain in the C-terminus, which perfectly corresponds to the six conserved disulfide-bonded cysteine residues involved in the formation of the internal disulfide bridges (C1-C5, C2-C4 and C3-C6) in all big defensins from mollusk, horseshoe crab and amphioxus. Quantitative real-time PCR analysis revealed that the expression of Sb-BDef1 transcript was detected in all the tissues examined from normal ark shells, and the temporal expression of Sb-BDef1 mRNA was remarkably up-regulated at 8, 16 h in hemocytes, and at 16, 24 h in hepatopancreas after Vibrio anguillarum-challenge, respectively. These results suggested that Sb-BDef1 was a constitutive and inducible acute-phase protein and should be involved in immune response of Gram-negative microbial infection in ark shell S. broughtonii.