Identification of growth-related molecular markers in the tropical abalone Haliotis asinina

Abstract

Growth-related DNA markers of abalone Haliotis asinina were identified by AFLP analysis. A total of 64 primer combinations was primarily screened with bulked genomic DNA of the first sample set, including fast growing (FG1, N = 10) and slow growing (SG1, N = 10) H. asinina. Forty candidate fast growing and 43 candidate slow growing related markers from 44 informative primers were further tested with both the first and the second sample set (FG2, N =10; SG2, N = 10). Only 2 candidate fast-growing and 7 candidate slow-growing related markers were found. To confirm the candidate markers, the third sample set (FG3, N = 10; SG3, N = 10) was tested but no growth-related markers was found. Then, 112 primer combinations were further used. As a result, one candidate of fast-growing related AFLP marker (225 bp) was found from primers Eco RI[subscript +3] 3 and Mse I[subscript +3] 12 (FGE3M12). However, the sequence of FGE3M13 did not show significant matching to the sequences previously deposited in the GenBank (E-value cut off of 10[superscript 4]). SCAR marker was developed from candidate growth-related markers. Primers were designed for PCR amplification in genomic DNA of fast growing (N = 9) and slow growing (N = 9) H. asinina. The primers generated the expected amplicon in both fast growing and slow growing H. asinina. Therefore, the single nucleotide polymorphism (SNP) was further determined by SSCP analysis. The result showed that the SCAR marker was polymorphic, but not growth linked. RAP-PCR analysis was carried out to isolate various types of expression markers in fast growing and slow growing H. asinina. Two slow-growing related expression markers were identified, SGRAP1 and SGRAP2 (210 bp and 260 bp respectively), through screening with 43 primer combinations. The two markers were generated from primers UBC101 and UBC191. SGRAP1 fragment showed a significant match to ADP/ATP carrier protein (E-value=10[superscript 17]), while the SGRAP2 fragment was homologue to inosine triphosphatase (E-value=10[superscript 7]). RT-PCR analysis was carried out using primers designed based on sequence of SGRAP1 and SGRAP2 markers. The analysis showed that the level of expression of SGRAP1 in slow-growing H. asinina was statistically significant than that of the fast-growing ones (p<0.05) and the level of expression of SGRAP2 in slow-growing H. asinina was obviously higher than that of the fast-growing ones. For the identification of insulin-related peptides homologue in H. asinina, using degenerated primers designed from insulin like peptides of the gastropod mollusks, Lymnaea stagnalis and Aplysia californica, the homologue in H. asinina was not identified