| The pearl oyster, Pinctada martensii (P. fucata or P. fucata martensii) is the primary species for production of marine pearl with high commercial and productive values and it covers over90%of the country’s pearl production in China. However, recently the pearl oyster farming industry in China faces several problems (e.g. habitat destruction, decline of farming and breeding) and the yield of pearl decrease significantly. To solve those problems, it is necessary to study the genetic diversity, genetic resource conservation, genetic improvement and marker-assisted selection to breed species with excellent economic trait.In this study, we developed EST-SSR and EST-SNP markers from EST database of P. martensii and characterized them in populations, meanwhile analyzed their correlation with growth and nacreous secretion traits. Our goal is to find functional genes which are significantly correlated with growth and nacreous secretion traits and provide a reference for QTL mapping and marker-assisted breeding. The results are as following:1. Two hundred and ninety nine microsatellite-containing EST sequences were found from EST database through Misa software. Of them,115(38.46%) contained more than one SSR. Among those456EST-SSRs,153(33.55%) were compound SSRs that had more than one repeat type. The most common repeat types were dinucleotide (147,32.24%) and trinucleotide repeats (119,26.10%), followed by tetranucleotide (101,22.15%), pentanucleotide (15,3.29%), hexanucleotide repeats (19,4.17%), and55(12.06%) were other nucleotide repeats.2. Sixty eighte primer pairs were designed using software Primer3and47(69.12%) primer pairs amplified successfully. Among them,36(52.94%) were polymorphic, with the size of product ranging from108-435bp.3. The genetic analysis in wild population and hatchery population in P. martensii showed the average FST was0.123. For every locus, the FST ranged from-0.006(PamtE13) to0.473(PamtEOl). The number of alleles ranged from3to11for wild population (mean6.0) and4to8for hatchery population (mean5.0), respectively. For wild population, the expected heterozygosity ranged from0.50to0.88and the observed heterozygosity ranged from0.10to0.86. For hatchery population, the expected heterozygosity ranged from0.38to0.85and the observed heterozygosity ranged from0.10to0.91.10of the17loci (58.82%) were in Hardy-Weinberg equilibrium (P<0.05after Bonferroni correction) in wild population, while in hatchery population,11of the17loci (64.71%) were in. The departure from HWE expectations at all loci can be explained by the presence of null alleles.4. Seventeen EST-SSR markers were segregated in the full-sib family. Six (35.29%) showed significant deviation from Mendelian ratios after Bonferroni correction (P<0.0029). Null alleles were inferred at four loci (23.53%) in the full-sib family.5. A GLM procedure of SPSS software was used to analyze the effects of these microsatellites on morphological traits of P. martensii in the full-sib offspring and96individuals of the hatchery population, meanwhile the multiple comparison (LSD) of different genotypes between individuals was detected. The results showed that in full-sib offspring, PmartE05was significantly correlated with total weight, PmartE35was significantly correlated with shell width and total weight and PmartE64was significantly correlated with nacre thickness. In hatchery population, PmartE05was significantly correlated with shell height. Although other loci didn’t show significantly correlated with morphological traits, different genotypes between individuals showed significantly difference. We can find genotypes which were advantageous for growth through multiple comparison. By GenBank BlastX,4(PmartE01, Pmart13, Pmart17and PmartE37) of30SSR-containing ESTs (13.3%) were found to have significant (at e-value<1.00E-10) homology to known genes or predicted proteins from other organisms.6. Eleven candidate EST-SNPs were identified by EST sequence. We designed11SNP primer pairs through Primer Premier5.0and7(63.64%) primer pairs were polymorphic. To determine the type of SNP, open reading frames (ORF) were inferred by ORF finder.4SNPs (57.14%) were synonymous,2SNPs (28.57%) were non-synonymous and1SNP were intronic.7. Seven EST-SNPs amplified in three wild populations. The results showed there were two alleles in7SNPs and minor allele rate ranged from0.052to0.500, the expected heterozygosity ranged from0.13to0.52, the observed heterozygosity ranged from0.07to0.74, the average expected heterozygosity was0.38and the average observed heterozygosity was0.43. Actb-106was significant (P<0.05after Bonferroni correction) deviation from HWE in India wild population. She-7-269and col4a4-121were significant (P<0.05after Bonferroni correction) deviation from HWE in Sanya wild population. Ctsl1-434was significant (P<0.05after Bonferroni correction) deviation from HWE in Nanshan and Sanya wild populations. Other loci were in Hardy-Weinberg equilibrium. Three markers were segregated in the full-sib family and all loci fit to to the expected Mendelian ratios (P>0.05after Bonferroni correction). The results showed that those SNPs could be useful for the population genetic analysis of P. martensii.EST-SSR and EST-SNP markers that were identified in this study will be useful for molecular genetic analysis, germplasm identification and wild resource conservation. The functional genes and genotypes which were significantly correlated with growth and nacreous secretion traits might be useful for the QTL mapping and marker assisted selection for important growth traits. |
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