| Sakehead is a traditional high-quality fish in China,mainly for snakehead(Channa argus),snakehead(Channa maculata)and hybrid snakehead.Hybrid snakehead is the progeny obtained from hybrid snakehead,with fast growth,high survival rate,and easy to feed puffed grain feed.In recent years,with the gradual promotion of hybrid snakehead breeding across the country,improved seed breeding has become a key issue for the healthy and sustainable development of the all-male hybrid snakehead breeding industry.Microsatellite molecular marker technology can be applied to the population genetic diversity analysis of snakehead and family paternity test,providing a powerful tool for superior seed breeding.In this study,we conducted artificial insemination and established two groups,developed common microsatellite markers and constructed multiple PCR system,conducted genetic diversity analysis and parental identification,compared genetic linkage map constructed using simplified genome sequencing,and developed SNP markers.The specific research content includes the following three aspects:1.Polymorphism microsatellite primer screening and multiplex PCR system constructionBased on the whole genome sequence of snakehead and snakehead,60 pairs of microsatellite primers were designed,PCR amplification of DNA of 9 and 3 parents,28 primers with clear and specific bands were identified by agarose gel electrophoresis,ABI3730,20 microsatellite primers with good polymorphism and appropriate allele size to facilitate multiple PCR amplification and typing.The 20 pairs of primers were grouped according to the amplification size and annealing temperature,and the number of multiplex PCR system was constructed.The selected microsatellite primers were validated in a random 30-tail hybrid progeny,Can be successfully amplified,The genetic parameters are: 181 alleles were detected at 20 microsatellite loci,Allele number(Na)is between 5 and 12,The mean allelic number was 9.05;Polymorphic information content(PIC)is between 0.589-0.871,The mean value was 0.741,Of these,20 microsatellite loci with polymorphic information content PIC> 0.5,It is highly polymorphic;Observe heterozygosity(Ho)between 0-1,The average value was 0.746;Expected heterozygosity(He)is between0.652-0.898,The mean value was 0.778.It shows that the multiple PCR system was successfully constructed.2.Genetic diversity analysis and paternity testFamily identification and genetic diversity analysis were according to the multiple PCR system.In the hybrid family of supermale snakehead and Shandong mother,a total of 300 alleles were detected,and the average number of alleles was 15,among which the number of site Opc036 genes was 23,while the least site Opc003 was 10.The average observed heterozygosity was 0.6595,the average expected heterozygosity was 0.7972,and the polymorphism information content was0.551-0.882,both with high polymorphism(P≥0.5)and 0.7775,indicating that the experimental population was at high polymorphism(P≥0.5),95.4% and 100%.In the hybrid family of supersnakehead and Hunan mother,a total of 283 alleles were detected,and the average number of alleles was 14.15,among which 22 Opc044 genes were 22,and the least Opc039 was 8.The average observed heterozygosity was0.5938,mean expected heterozygosity was 0.7389,polymorphism information content of 0.177-0.916,site Opc001 PIC of 0.177,site Opc039 PIC of 0.208,other sites PIC greater than 0.5,average polymorphism information content of 0.718,high polymorphism at 100%.In addition,by comparing the growth data of different families,we found that snakehead from Shandong has more growth advantages as a hybrid offspring with the same parent.Under the same parent,the average body weight of offspring of Shandong snakehead was 1840±343.36,2107.17±329.70,2000±303.81,and 1748.82211.04±1,1750±430.89 and 2000±303.81,respectively,reaching a significant level(P<0.01).This indicates that snakehead from Shandong has a great growth advantage as a hybrid offspring produced by the mother.In conclusion,the multiplex PCR system constructed in this study can be used as paternity testing with high accuracy.Comparing the growth data from the two families:when the parent is the same,the parent parent.3.Construction of genetic linkage map and development of growth-related SNP markersUsing 180 full sibling F1 offspring constructed from female parents,a high-density genetic linkage map was constructed using 2b-RAD simplified genome sequencing.The consensus map contains 6352 SNP markers and 2143.67 c M,with an average plot distance of 0.34 c M and map coverage of 99.3%.For QTL mapping analysis based on this map,QTL at 9 chromosome levels was obtained with an explained variation rate of 9.7%-11.9%.Growth-related single-nucleotide polymorphism(SNP)markers were screened in nine QTL and genotyped by flight mass spectrometry.The mean observed heterozygosity(HO)for SNP markers was0.418,the expected heterozygosity(He)0.414,and the polymorphic information content(PIC)0.313,indicating that the polymorphism level of the pedigree constructed families in this study was moderate.The genetic linkage map of snakehead constructed in this study laid the foundation for screening and digging into growth-related functional genes,and the SNP markers can be used for the breeding of the parental population. |
