Isolation Of Microsatellites And Mapping QTLs Related To Body Weight,Eye Diameter And Eye Cross In Cyprinidae

Enrichment by magnetic beads is a kind of method for isolation of microsatellite DNAs.Microsatellites of grass carp (Ctenopharyngodon idellus) were isolated with this method in this experiment.One thousand three hundred and seventy-eight positive clones were obtained through the second screening by hybridization with [γ-32P]ATP labeled probes.Eight hundred and fourty-six microsatellites were isolated from 705 positive clones.There were 632(74.7%)perfect motifs,114(13.48%)imperfect motifs and 100(11.82%)compound motifs in these sequences.One hundred and thirty seven primer pairs were designed according to the flanking sequences,and 100 pairs were synthesized.The results showed that 35 primer(s35%)showed polymorphism in the wild population from Hanjiang River,and 20 microsatellite markers were assessed genetic diversity.A total of 212 different alleles were found.The average observed heterozygosity(Ho)was 0.6811,the expected heterozygosity(He)was 0.7975,and the polymorphism information content(PIC)was 0.7777,respectively.A genetic linkage map has been constructed for the common carp (Cyprinus carpio L.).217 microsatellite markers and 336 SNPs markers were selected to analyze the genomic DNA of 68 individuals derived from the group of F2 hybrids mirror carp(Cyprinus carpio L.).Genetic linkage maps were constructed using MapQTL 4.0 software, in which 507(186SSRs,321SNPs)of markers were arranged into 62 linkage groups and 46 were unlinked with any other groups.The linkage map covered 2805.85 cM with an average distance of 6.31 cM.The biggest linkage groups for the 11st group comprised of 19 marker loci and covered 119.5 cM with an average distance of 6.64 cM.There are five the smallest linkage groups respectively 21, 52, 53, 61, 62 and the linkage map covered is only about 1.5 cM.Quantitative traits loci(QTLs)associated with body weight,eye diameter,and eye cross were identified by composite interval mapping of the software MapQTL 4.0.The results show:(1)Fourteen QTLs were identified for body weight on nine linkage groupsAmong the existence of fourteen QTLs controlling body weight in mirror carp the maximum LOD score was found to be 4.46(BW-5-1)and the minimum score was found to be 2.25(BW-1-1).The variances explained by these QTLs ranged from 14.10 % to 45.50%,besides,nine major QTLs explained over 20% of the phenotypic variations.(2) Ten QTLs were identified for eye diameter on nine linkage groups.Among the existence of ten QTLs controlling eye diameter in mirror carp the maximum LOD score was found to be 2.28(EL-37-2)and the minimum score was found to be 2.22(EL-11-1).The variances explained by these QTLs ranged from 15.83 % to 34.93%,besides,four major QTLs explained over 20% of the phenotypic variations.(3) Ten QTLs were identified for eye cross on eight linkage groups.Among the existence of ten QTLs controlling eye cross in mirror carp the maximum LOD score was found to be 3.66(WBL-1-2)and the minimum score was found to be 2.16(WBL-35-1).The variances explained by these QTLs ranged from 13.50 % to 26.60%,besides,four major QTLs explained over 20% of the phenotypic variations.For further identification,SNPs markers were used to screen the protein and nucleotide database using bioinformatics tools in order to find the homologies.Take less than or equal to 1e - 05 expectations of gene annotation and the results show:high sequence similarities of SNP1480,SNP1103, SNP1067,SNP1067 markers were observed with the protein sequence of ACADM protein, Alpha 2 pancreatic amylase,Apoeb protein,Gapdh protein of zebrafish,respectively.ACADM protein is the specificity of chain (C4 - C12) acyl coenzyme A dehydrogenase protein,catalytic mitochondrial fatty acids beta oxidation way the first step.Alpha 2 pancreatic amylase hydrolysis oligosaccharides and polysaccharide the Alpha 1,4- indican key, which is an important enzyme in the process of glucose metabolism in the organisms.Apoeb protein involved in lipid transportation and lipoprotein metabolism.Gapdh protein as glycerin aldehyde - 3 - phosphoric acid dehydrogenase protein, participate in glycolysis reaction.This study constructs a relatively saturated linkage map,and to the common carp body weight,eye diameter,eye spacing testing, aim at for QTLs carp molecular marker assisted breeding provided theoretical basis.