Development, Characterization And Application Of EST-SNP Markers In Zhikong Scallop (Chlamys Farreri)

Zhikong scallop (Chlamys farreri) is one of the most economic mariculturespecies in China. Cultivating strains with good quality, high yield, fast-growth,resistance to diseases and stress are the basis for sustainable development ofaquaculture of this species, since various problems have risen recent years, such asfrequenct brokeout of disease, mass mortalities, individual miniaturization andso on. Marker-assist selection (MAS) and molecular marker based functionalresearch provided theories and effective approaches for the development ofnew strains. Here in this study, we validated and characterized a panel ofEST-SNPs and studied their applications in genetic breeding of C. farreri.1. Establishment of EST-SNP genotyping technology, validation andcharacterization of EST-SNPs in C. farreriIn this study, using two mistaches at the3’ end to block the probe, we modifiedHRM unlabelled probe technology, optimizated the screeing process, and establisheda high sensitivity, low-cost genotyping sestem for EST-SNP validation. With thissystem, the first massive EST-SNP development of C. farreri wrer conducted.5,630candidate loci were chosen for validation, of which1,696proved to bepolymorphism. Evaluated with48individuals from Qingdao population, all thepolymorphic loci had two alleles with the minor allele frequency ranged between0.010and0.500. The expected and observed heterozygosities ranged from0.0625to0.7447and from0.0998to0.5046, respectively. No significant linkage disequilibriawere detected and102loci exhibited significant departures from Hardy-Weinbergequilibrium. These novel polymorphic loci presented here will facilitatethe followingresearches, such as linkage map construction, functional genetic analysis and QTL mapping. And based on the results here, contig size of≥4sequences with the minorsequence allele at least twice and minor allele frequency above20%, were highlyrecommended as candidate sites for validation test when similar researches wereconducted.2. Inter-species transferibility of EST-SNPIn this study, the first transferability test of SNP between scallops was reported.Thirty-four EST-SNPs developed from C. farreri were chosen to test theirtransferability in in other five bivalve scallops in China: Noble Scallop (Chlamysnobilis), bay scallop (Argopectens irradias), moon scallop (Amusium pleuronectes),yesso scallop (Patinopecten yessoensis) and purple scallop (Argopecten purpuratus).And19maintained polymorphism in at least one relative species, of which two lociexihibited good transferability in four of the five scallops tested. The results heresuggested the good transferability of EST-SNPs, especially those synonymoussubstitutions, while the nonsynonymous substitutions were kind of species-specific.3. Scanning of shell height related genes in C. farreriIn order to search out phenotype-determing genes and provide theoretical basisand pratical experieces, we selected the individuals from two wild populations withmaximum or minimum shell height, and genotyped with1,696EST-SNPs. Allelefrequencies of six loci were different between the two extreme phenotype groups.Allele frequencies of another six loci did not significantly differ but the genotypefrequencies were significantly different between the two extreme phenotype groups.The scan results laid the foundation for research of shell height-determining genes andshell height-oriented breeding scheme.4. Construciton of EST-SNP linkage map of a male C. farreriA linkage map of a male C. farreri was constructed with three full-sib families, aparental half-sib family and285EST-SNP markers. The total length of the map was 2,025.4cM and the genome coverage was about87.1%. The maximum intervalbetween markers was25.8cM, while the minimum interval was0.0cM, with anaverage interval of7.70cM. The linkage group length was between22.3cM and234.5cM with an average of106.6cM. The number of mapped markers ranged from3to44with an average of15.0. Preliminary QTL mapping were conducted for thegrowth traits of C. farreri, and a total of16QTLs affecting shell height, shell length,shell width, weight and weight of adductor muscle were located. These QTL were on5linkage groups explaining4.0%–22.2%of the trait variation. This is the first SNPgenetic linkage map of C. farreri, and it will greatly facilitate QTL mapping,positional cloning and gene mapping research.