Development of genetic markers for increasing production efficiency of the tropical abalone Haliotis asinina in Thailand

Abstract

Species-specific markers were developed in the tropical abalone (Haliotis asinina, H. ovina and H. varia) found in Thai waters. RAPD analysis generated 10, 3 and 2 candidate species-specific markers in H. asinina, H. ovina, and H. varia, respectively. All RAPD fragments were cloned and sequenced. Twenty pairs of primers were designed and preliminary tested. Specificity of five primer sets (CUHA2, CUHA12, CUHA13, CUHO3 and CUHV1) was further examined against a large sample size (N = 216). Results indicated species-specific nature of all except CUHO3. Sensitivity of detection was approximately 100 pg, 100 pg, 500 pg and 20 pg of genomic DNA of the target species for CUHA2, CUHA12, CUHA13 and CUHV1, respectively. Identification of species-origin of ethanol-preserved, dried and boiled H. asinina specimens were successfully carried out. SCAR markers developed in this study can be used for quality control of various forms of H. asinina products from Thailand. Sex-related markers of H. asinina were identified and characterized by AFLP, EST, subtractive cDNA, RT-PCR and RACE-PCR approaches. Seven candidate female-specific and seven male-specific AFLP fragments were identified from screening 214 AFLP primer combinations against 4-bulked genomic DNA of male and female H. asinina. Six and five SCAR markers were developed from those fragments and tested with genomic DNA of male and female abalone. Results indicated the positive amplification product in both male and female H. asinina. Further analysis using SSCP indicated polymorphic patterns/fragments in most of the markers (HaMale1, 5, and 6 and HaFemale2, 3, 4, and 5) but they were not sex-linked. In addition, normal and subtractive cDNA libraries from ovaries and testes of H. asinina were established. A total of 588 randomly selected clones (200 and 118 transcripts for normal and 110 and 160 transcripts for subtractive libraries from ovaries and testes, respectively) were unidirectional sequenced. Results indicated that 109 (54.5%) and 73 (61.9%) of normal cDNA libraries from H. asinina ovaries and testes significantly match with known genes in the GenBank (E value <10[superscript -4]). Of these, vitelline coat proteins (VCPs; 40 clones, 20.0%) were predominant in the former library, but sperm lysin (9 clones, 7.6%) was the most abundant transcript in the latter library. For subtractive cDNA library, 71 clones (64.5%) and 56 clones (35.0%) of ovary (forward subtraction) and testis (reverse subtraction) libraries were known transcripts. Using RT-PCR, sex-specific expression of VCP1, VCP3, VCP7, VCP49, VCP75, and VTG-1 and axonemal p66.0, tektin A1, sperm lysin, FP, and DMRT1 was found in adult females and males of H. asinina, respectively. TektinA1, FP, sperm lysin, VCP1, VCP2, VCP3, VCP7, VCP49, and VCP75 were expressed in 2, 3, 5-month-old juvenile abalone whereas axonemal p66.0, DMRT1 and VTG-1 were not expressed in those stages. Expression of sex-related transcripts was semi-quantitatively estimated. The relative expression level of axonemal p66.0, tektinA1 and DMRT1 in testes was significantly different between adults having stage 1 and stages 2, 3, and 4 of testicular development (p < 0.05). Likewise, expression levels of VCP1, VCP2, VCP3, VCP7, VCP49 and VCP75 in ovaries were significantly different between adults having stages 1 and 3 of ovarian development (p < 0.05). No significant expression levels of sperm lysin and FP in testes and VTG-1 in ovaries were observed at all stages of testicular and ovarian development. RACE-PCR was carried out for characterization of full-length cDNA of tektinA1 (2166 bp, 1350 bp ORF), axonemal p66.0 (2038 bp, 1683 bp ORF), and DMRT1 (1727 bp, 732 bp ORF). An EST containing a DM domain (1727 bp) and its variant (1351 bp) without the DM domain were also successfully isolated and characterized. These transcripts were specifically expressed in testes of adult H. asinina.