SSR Detection And QTL Analysis For Vibrio Harveyi Resistance In Cynoglossus Semilaevis

In recent years, the bacterium Vibrio harveyi has caused tremendous losses in the aquaculture industry of Cynoglossus semilaevis. Half smooth tongue sole（Cynoglossus semilaevis） has been deepley loved for its delicious tasteandrich nutritionsby consumers,and the economic value of which is extremely high. However, disease, such as ascites, lousy fin, and so on, outbreak frequently make Cynoglossus semilaevis factory farming industry inflicted heavy loss.Vibrio harveyi is one of Pathogenic bacteria for Cynoglossus semilaevis. Traditional selective breeding mode is combined with marker-assisted selection breeding, which are advantageous to the Cynoglossus semilaevis resistant strain breeding.Molecular marker-assisted selection（MAS）is a new breeding method using molecular means to screen out related markers which could scan out from the genome in finding the optimal parents. This study was conducted by utilizing Bulked Segregant Analysis（BSA） and Quantitative Trait Loci（QTL） mapping to screen disease-resistance markers and loci in a family challenged with V.harveyi in 2014. The main results are summarized as follows: 1、SSR markers detection for Vibrio harveyi resistance using Bulked Segregant in Cynoglossus semilaevis100 individuals were selected to form the family F1412（Nomenclature rule: F+ year + family number, survival rate 52.22%） challenged with V. harveyi and 169 microsatellite loci across all chromosomes. Firstly, constructed using 15 resistant individuals and 15 susceptible ones,two pools were scanned by the 169 SSR markers. We got two marker（scaffold479₂3523,scaffold7277₁163） amplified different bands. Secondly,the markerscaffold479₂3523 and scaffold7277₁163 were verified using 30 individuals used to construct gene pools, then we found that scaffold479₂3523 still showed obvious difference（P=0.003<0.05）, scaffold7277₁163 had no obvious difference（P=0.682>0.05）. Then a second verification using 100 individuals was performed to verified the difference ofscaffold479₂3523still exist（P=0.00002<0.01）. The results show thatscaffold479₂3523still had significant differences. Therefore, we concluded that the maker scaffold479₂3523may be closely associated with resistance to V. harveyiinfection. The marker were cloned and sequenced, the results of sequences confirmed that the sequence were fragments of scaffold479₂3523 published already and the homology were 98%. 2、QTL detection for Vibrio harveyi resistance in Cynoglossus semilaevisIn this study, nighty four individuals were then genotyped using all of the 37 SSR markers on LG18, on which the Scaffold479₂3523 was located. Three new linkage groups（LG18, LG18 F, and LG18M） were identified, respectively. Furthermore, two different analytical models were applied to perform single marker analysis and composite interval mapping（CIM） with different levels of significance in LG18, LG18 F,and LG18 M,respectively.Inmodel 1, three significant markers（scaffold4475₇1287,scx9-1,cyse80） and one very significant marker（scaffold080437） were determined, and meanwhile the resistance-related QTL qE-F1 was founded. The maker scaffold479₂3523 is the left marker of LG18 F, while the P value of Scaffold479₂3523 is 0.0516, getting close tosignificant difference in model 1. Model 2 obtained four significant markers（hncyse110, scaffold414₁9940, scaffold4475₇1287, cyse80）, one very significant marker（scaffold08043）, and two QTL including q E-M1 and qE-M2. The marker scaffold080437 both showed significant difference（P<0.001） in two different analytical models. These four markers（scaffold080437, scaffold479₂3523, scaffold4475₇1287, cyse80） may be closely associated with resistance to V. harveyi infection in Cynoglossus semilaevis. The qE-F1, which explained 87.36% of the phenotypic variance, contained the qE-M1 and q E-M2 of G18 M. Thus, the qE-F1 was considered as a major candidate region for V. Harveyi resistance.