Genetic Test Of Breeding Populations And Identification Of Growth-associated SNPs In The Clam Meretrix Meretrix

The clam Meretrix meretrix is one of the most important commercial mollusks inChina. With the development of its artificial culture, the demand for quality seedresources increasingly urgent. High yield and benefit have been the focus of itsaquculture, which requires us to raise improved seeds with high-yield, resistance andgenetic stability. In the present study, with the microsatellite markers and thetraditional methods of selection and hybridization, we conducted genetic test fordifferent populations of M. meretrix. And we further developed SNP markersassociated with growth traits. These are expected to provide basic data and theory forbreeding new strains and promote the sustainable and healthy development of clamaquaculture.A natural population of M. meretrix was used to found a number of families, fromwhich three (P1, P2, P3) were chosen to set a complete3×3diallel cross comprisingnine crosses. Eight microsatellite markers were used for parentage analysis in the ninecrosses. Estimation of parental contributions to offspring showed that thecontributions of different sires or dams from one family to offspring had greatdifference and the contributions of the same parent to offspring in different crosseswere markedly unequal. Effective parent number (Ne) was calculated based onparental contribution. It was found that there was a significant reduction of Nein allthe crosses. The Ne/N ratio for the nine crosses ranged from0.58to0.86. In addition,the genetic diversity of all the parents was some degree higher than that of theirrespective offspring in most crosses.Three outbred crosses with high survival rate and three inbred crosses wererespectively chosen from the nine crosses to establish a control group and an inbredgroup. After evaluated by eight microsatellite markers, genetic variability of the inbred group diminished relative to the control group, as evidenced by drop of19.4%,20.8%and9.3%on average number of alleles, observed and expected heterozygosity,respectively. The inbred line had a higher average FISvalue than the control group andnatural population. The estimated inbreeding depression coefficients for shell length,shell height, shell width and whole body weight at12months post-fertilization were0.803,0.747,0.826and2.073per10%increase in FIS. Investigation of familyP1and its progeny cross P1×P1indicated that a serious genetic drift occurred in thebreeding. Simulation results showed that a large Necould control reduction ofheterozygosity in a closed population.Progeny of the nine crosses were reared in two environments, indoor ponds andopen-air ponds. After cultured for12months, the growth data were measured and ageneral linear model was used for analysis of variance. The results indicated highlysignificant cross effects, environment effects and the interaction of cross byenvironment. Analysis of combining ability showed that P2was the top combineramong the three parental families for growth traits, the cross of P1and P3had thehighest specific combing ability (SCA) and there were significant reciprocal effects.In addition, Mid-parent heterosis (MPH) was estimated for the six crossbredcombinations in individual environment and over environments. The results showedthat about half of the crossbred combinations had favorable MPH (>1%).Based on EST database of M. meretrix and bioinformatic methods, a batch ofEST-SNPs were predicted, and then validated by pooled sequencing. A total of16confirmed SNPs were genotyped with SNaPshot assay between a fast-growingpopulation and a control population. A marker-trait association analysis identifiedseven SNPs (SNPg3, SNPg4, SNPg5, SNPg6, SNPg8, SNPg9and SNPg16)significantly associated with growth (P <0.05). After bidirectional selectivegenotyping in an independent population, three SNPs (SNPg5, SNPg6and SNPg9) ofthe seven markers were further confirmed to be associated (P <0.05). This is the firstreport on identification of a batch of growth-associated SNPs in M. meretrix, whichprovides valuable information for marker-assisted selection for this species. Based on two populations with significant difference in growth, association studyof SNPs in important functional genes with the growth traits of M. meretrix was made.A long-chain fatty acyl-CoA ligase gene (MmeACSL1) was cloned in M. meretrix,with a full-length cDNA of1876bp encoding475amino acids. Examination ofMmeACSL1gene expression under different nutrient conditions by Real-time PCRshowed that the expression of MmeACSL1gene under fasting condition had asignificant increase compared with that under feeding condition (P <0.05), whichsuggested that MmeACSL1was involved in energy metabolism of M. meretrix. Bymeans of bioinformatic prediction and direct sequencing, two exon SNPs and sixintron SNPs were detected in this gene. A marker-trait association analysis identifiedfive SNPs (mmACSLe-2, mmACSLi-2, mmACSLi-3, mmACSLi-5and mmACSLi-4)significantly associated with growth (P <0.05), and haplotypes comprising these fiveSNPs were also significantly growth-associated (P <0.05), which implied theimportant role of MmeACSL1in energy metabolism and its association with thegrowth of M. meretrix.A cyclin-dependent kinase (CDK) gene was cloned in M. meretrix, with afull-length cDNA of1854bp, which encoded390amino acids. This gene showed thehighest homology with CDK10in mammals, so it was named MmeCDK10. Directsequencing of this gene detected five exon SNPs and five intron SNPs, from whichfour exon SNPs (mmCDKe-1, mmCDKe-3, mmCDKe-4and mmCDKe-5) and oneintron SNP (mmCDKi-1) were found to be significantly associated with growth traits(P <0.05), by a marker-trait association analysis between two populations withsignificant difference in growt, which suggested that MmeCDK10was involved in thegrowth of this clam.