Isolation Of Microsatellite Markers In Scallop And Their Application In The Species Identification

Scallop, as important commercial shellfish, is widely distributed in China. The marker-assisted selection (MAS) provides the fundamental theories and applied experiences for cultivating good strains with the performances of disease-resistance and fast growth for the sustainable development of scallop fishery. Microsatellite DNA markers, with many advantages, are playing important part in the genetic analysis of scallop. In the present study, we developed microsatellite markers for four main economic scallop species (Chlamys farreri, Argopecten irradians, Chlamys nobilis, Patinopecten yessoensis) and established the species-specific microsatellite markers for four species to identify commercial adductor muscles and larvae.(1)Isolation of microsatellite markers for Chlamys farreri.By searching NCBI dbGSS of Zhikong scallop, a total of 3648 sequences were generated. The sequences were mined for tri- and tetranucleotide microsatellites with software Repeat Reporter Version1.5. Twenty-four polymorphic tri- and tetranucleotide microsatellites were isolated from a fosmid library of Chlamys farreri. The number of alleles was between 2 and 9. The Ho and He ranged from 0.000 to 0.7500 and 0.1866 to 0.8495,respectively. No significant linkage disequilibrium between pairs of loci was observed and 12 loci showed significant departure from the HWE. These markers are expected to provide more useful resource for the research of population genetics of Chlamys farreri.(2)Isolation of microsatellite markers from EST database for Argopecten irradians.By screening the EST database of Bay scallop,193 microsatellite sequences were obtained from 7060 EST sequences,about 2.73% in whole database. Among the microsatellite sequences, the number of dinucleotide microsatellites >the tri- >the penta- >the tetra- >the hexa-. (AT) n in dinucleotide repeats was the most common, and then (AG)n and (AC) n. Finally, 20 polymorphic microsatellites were developed from the EST database of Argopecten irradians. 90 alleles were clearly amplified. The number of alleles per locus was between 2 and 9. The Ho and He ranged from 0.0667-0.8000 and 0.0961-0.8253, respectively.(3)Isolation of microsatellite markers for Chlamys nobilis.Microsatellite enrichment library of noble scallop was constructed, which was digested by RsaI. The primers flanking 22 microsatellites isolated from the genomic library enriched for (CA)n and (GA)n were designed in the noble scallop Chlamys nobilis. Ten primer pairs provided clear and polymorphic amplification products. Based on characterization with 48 individuals, the number of alleles ranged from 3 to 6. The values of Ho and He varied from 0 to 0.88 and 0.29 to 0.76, respectively. These markers are therefore potentially useful for studies of the population structure of the species.(4)Isolation of microsatellite markers for Patinopecten yessoensis.As the method above, twelve polymorphic and informative microsatellite markers were isolated and characterized for the Patinopecten yessoensis. We characterized these loci by genotyping 48 individuals, the number of alleles ranged from 2 to 8, and the values of Ho and He varied from 0 to 0.8333 and 0.2546 to 0.8231, respectively.(5)The affirmation of species-specific microsatellite markers and their sensitivity detection.72 microsatellite markers for the four scallop species were subjected to the transferability analysis, and to ensure the accuracy of identification, 8 loci displaying a high degree of specificity to a certain species and no amplification in any other species at any annealing temperature were chosen as the species-specific microsatellite markers for the identification. Based on further genotyping of 48-50 individuals for stability tests, two primer pairs for each species were selected as the species-specific microsatellite markers for identification. By sensitivity detection, we found that this panel of microsatellites had high sensitivity, 2% and 1%.(6)Identification of scallop adductor muscles and larvae using species-specific microsatellite markersSeven batches of muscles (the dried, canned and frozen) were selected for identification. A total of six batches composed of a single species, however, the dried sample numbered as DM3 exhibited diagnostic peaks in the regions of Chlamys farreri and Argopecten irradians. The adductor muscles of DM3 were individually amplified, and 4 and 16 individuals of Chlamys farreri and Argopecten irradians were checked out, respectively. It was therefore indicated that about 20% of Chlamys farreri and 80% of Argopecten irradians in this sample. No Chlamys nobilis was detected in all of the muscle samples.The results of identification of larvae indicated that all 10 batches of larvae showed one band or peak value in PAGE or DHPLC analysis, which meant the number of intermixed samples should be less than 1%. 4 batches of larvae, 72 individuals per batch were randomly selected for PCR identification, and 306 of 336 (91.1%) were successfully amplified, which demonstrated that all larvae were produced in one species. Based on the result above, we suppose that this species-specific microsatellite marker system would play important role in the identification of these four scallop species. Moreover, the results in this study gave the experimental evidences for the potential use of species-specific microsatellite markers for species identification in the market management and larvae.