| Molecular assisted breeding is a more and more practical techniques for breeding. During theprocess of breeding practices, marker-assisted selection can effectively improve the efficiency ofbreeding and the important growth traits. Double haploid is a good breeding material because of itshomozygosity. In this research, Doubled haploids population of Japanese flounder were produced fromone female by the method of mitotic gynogenesis and165offsprings was obtained. Sperm of red seabream was choosed as a stimulator for embryogenesis in gynogenesis. We constructed a linkage map ofJapanese flounder using574SSR markers based on a DH population and identify QTLs of growth andmajor skeletal traits. DH females can be used to. In the study, the genetic similarity of clone lines andheterozygous clone lines which were produce by DH females were analyzed by microsatellite markers.The results are as follows:(1) In the study, a total of15,268Japanese flounder EST sequences were obtained from publicdatabase. After analysis, we found that a total of917EST sequences contained SSRs. The occurrencefrequency of SSRs was12.6%and the distribution density was1/5.7kb. The dominant repeat motifs ofSSRs were dinucleotide and trinucleotide which were63.5%and24.3%respectively. The motif repeattimes of six were predominant, and the frequency was24.0%. According to the flanking sequence ofEST-SSRs, we designed484pairs of primers. After verification by the genome DNA of Japaneseflounder family, a total of357pairs of effective primers were received and the the proportion of whichwas73.8%. Research results can provided the basis for developing EST-SSR from public ESTdatabases in Japanese flounder and some allied species. And it also could provide effective molecularmarkers for the subsequent study of the construction of genetic linkage maps.(2) We constructed a genetic linkage map of Japanese flounder using165doubled haploid Japaneseflounder. Linkage analysis was determined using Joinmap4.0, and the final analysis was performed onclusters joined with a LOD>4.0. The map contained446SSR and128EST-SSR markers. It spans1214.7cM of genome in24linkage groups. The average genetic distance (cM/marker) between two lociwas2.2cM. The largest linkage group is LG1which consisted of45marker loci covering55.5cM andthe smallest was a linkage group consisting of2marker loci covering only1.2cM in linkage group2.An average distance of each linkage group concentrated between the0.88cM~8.72cM. It is the firstmolecular genetic linkage map of Japanese flounder based on DH population and is fundamental to the continuous and cooperative research in genetic mapping of important traits and marker assistedselection.(3) The map locations and effects of quantitative trait loci (QTL) were estimated for thirteengrowth-related traits using MQM mapping with MapQTL4.0software. The traits contained body weight,total Length, body length, head Length, caudal peduncle length, dorsal fin length, pelvic fin length, eyespacing, caudal length, caudal width, body height, head width, caudal peduncle width. The resultshowed that, a total of46QTLs explained phenotypic variation from3.7%to17.5%were distributedinto13linkage groups. Three QTLs effected on body weight which could explain phenotypic variationfrom7.3%到15.0%. Total Length related QTLs were four and the QTL with the largest LOD (3.8)explained12.2%of the phenotypic variation. Among three QTLs which controlling Head length, theQTL with the largest LOD (2.81) explained7.1%of the phenotypic variation. The QTLs controled bodyheight explained from4.5%to9.0%of the phenotypic variation. Four QTLs controlled dorsal fin baselength, three controlled pelvic fin length, three QTLs controlled eye spacing, seven controlled caudalpeduncle length, four controlled caudal peduncle width. Only one QTL were found controlling caudallength, three QTLs controlled caudal width. The result also showed that the QTLs controlled full length,body length, head height, caudal peduncle width and caudal length were mapped to the same positionand the QTLs controlled body length and caudal length co-mapping. The QTLs related to body weightand body height, QTLs controlled dorsal fin length and pelvic fin length were mapped to the sameposition respectively.(4) Skeleton development level is a good indication for maturity evaluation which were mainlyinfluenced by genetic factors. The studies on mapping QTL for skeletal traits are very important inexploring questions about the evolution of the system as well as in biomedical research on the geneticsof bone growth. Six skeletal traits were measured for all DHs by radiographs of skeleton were obtainedby X-ray equipment. Twenty-one QTLs were detected on14linkage groups and totally account for asmall proportion of phenotypic variation (5.4to17.3%). The number of QTLs mapping on LG5andLG8were both eight which is the most ones. Most of QTLs detected distribute on LG5, LG8, LG9andLG20, in which, some QTLs perform the pleiotropy.(5) Four clone lines were carried out from doubled haploid individuals of Japanese flounderparalichthys olivaceus by the method of meiotic gynogenesis, which their homozygous maternal parentswas produced by mitotic gynogenesis. The clone lines were analyzed by24microsatellites loci with thefamily of the second generation of meiotic gynogenetic Japanese flounder (Mei-G2) and normal diploid(ND). The microsatellites loci covered all24linkage group and located on the distal end away fromcentromeres of chromosomes for their high probability of recombinant. The result showed that thegenetic similarity indexes were0.6359among progeny individuals. Genetic similarity indexes of Mei-G2were very close to1.0000but still can’t reach completely identical between individuals. In clone lines, genotypes of the progenies were identical with their maternal parents. The genetic similarityindexes between maternal parents and progenies were all1.0000which were same as that amongprogeny individuals. Both the maternal parents and progenies were all homozygous in these24microsatellites loci. In heterozygous clone lines, the genetic similarity indexes among the individualswere also1.0000which proved the consistency of their genome. And in the study, we also discussed therelationship between the number of heterozygous loci and growth traits in Japanese flounder byheterozygous clone lines. |
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