Analysis Of Genetic Background And WSSV Resistant Characteristics Of Litopenaeus Vannamei Based On Microsatellite Markers

Litopenaeus vannamei has become the highest yield shrimp species in the world due to its strong adaptability,rapid growth rate and strong resistance to stress.The main populations of L.vannamei in China are offspring of imported broodstocks?SIS,PRIMO,Zhengda,etc.?,new domestic self-bred varieties,and second or third generation of self-bred seedlings.The genetic background of populations of L.vannamei in China is complex.At the same time,L.vannamei aquaculture is affected by many pathogens(such as IHHNV,TSV,VP_AHPND,YHV,IMNV,WSSV,etc.),among which WSSV is still one of the most harmful pathogens.Its acute onset and high mortality greatly restrict the development of L.vannamei aquaculture industry.Based on the current situation of L.vannamei aquaculture in China,in this paper the microsatellite molecular markers were used to identify the paternity,analyze the genetic diversity and WSSV resistance of introduced population of L.vannamei and the seedlings of different commercial brands in China.The results could provide basic reference data for the breeding of excellent varieties of L.vannamei.The main results of this study are as follows:1.Parentage identification using microsatellite markersThe female parents and offspring?396 tails?from 36 families of the introduced of L.vannamei were used as experimental materials.Direct amplification PCR technology was established for microsatellite genotyping and parentage identification.The results showed that the total number of alleles in the eight microsatellite loci was 108,the average number of alleles per locus was 13.5,the average PIC value was 0.706,the average observe heterozygosity?H_o?was 0.657,the average expected heterozygosity?H_e?was 0.731,the combined exclusion probability?first parent?reached 99.49%,the combined exclusion probability?second parent?reached 99.98%,and the combined exclusion probability?parent pair?reached 99.99%.The simulated maternal pairing rate was 94.40%.There were 360 offspring from 36 families.308 offspring were paired successfully.The actual maternal pairing rate was 85.56%.There were 10 offspring in each family.In average,8.56 offspring were paired successfully.The results showed that 8 microsatellite loci had better identification efficiency,and the direct amplification PCR technique could be applied to the parentage identification of L.vannamei.2.Genetic background analysis of L.vannamei based on microsatellite markersThe genetic diversity and genetic relationship of six commercial brands of L.vannamei?180 individuals?were analyzed using 8 pairs of microsatellite primers.The results showed that the average observed heterozygosity?H_o?of eight microsatellite loci was 0.486.The average expected heterozygosity?H_e?was 0.745.The average polymorphic information content?PIC?of 87.5%?7/8?loci was above 0.5.Five loci?62.5%?were deviated significantly from the HWE?P<0.05?.The molecular variance analysis?AMOVA?showed that only 12%of the genetic variation was among brands,24%of the genetic variation was from individuals within brands,the rest was among brands.According to the F_st value analysis,26.67%of the variances were non-differentiated,while 73.33%of the variances were moderately differentiated.From the UPGMA cluster diagram,six brands of L.vannamei were clustered into two distinct branches:Guangzhou P and Guangzhou Z were clustered into one branch,Dongying M,HuanghuaR,Hainan Z and Hainan S were clustered into the other branch.According to the comprehensive experimental data,the genetic diversity richness of each brand ranged from high to low,respectively:Huanghua R>Guangzhou Z>Guangzhou P>Hainan Z>Dongying M>Hainan S.The results showed the genetic background of some commercial seedlings in China,and provided basic data for the construction of basic population of L.vannamei.3.Comparison of resistance to white spot syndrome virus?WSSV?and prelimi nary study on microsatellite loci related to WSSV resistance in commercial see dlings of L.vannameiIn this experiment,six domestic commercial brands of L.vannamei?1409 tails?,were used as materials.The artificial single-tailed,quantitative feeding WSSV infection test was carried out to compare the anti-WSSV ability of each brand of L.vannamei.The results were compared with that in the second part of the study to analyze the relationship between genetic diversity and disease resistance.At the same time,microsatellite marker correlation analysis was performed to obtain anti-WSSV-related microsatellite loci.The results showed that the survival time of P brand of Guangzhou L.vannamei was the longest,which was significantly longer than that of other brands.In the same period,the cumulative mortality rate was the lowest,and the distribution of death was mostly concentrated in the middle and late stages of the experiment.26shrimps survived in the end of the experiment.All the other brands of L.vannamei died during the experimental period.The difference of WSSV-resistance of the other four brands was not significant.The WSSV-resistance ability of each brand shrimp ranked from strong to weak as follow:Guangzhou P>Huanghua R>Guangzhou Z>Hainan Z>Hainan S>Dongying M.The results showed that the L.vannamei with high genetic diversity has better disease resistance.Five microsatellite loci were moderately related to WSSV-resistance.The related alleles were TuMXLv7.56-332,TuMLv9.90-292,TuMXLv7.121-243,Lv12-127 and M1103-388.There was no microsatellite loci was highly associated with WSSV-resistance.Further study should be carried out to explore microsatellite loci highly associated with WSSV-resistance,which could be useful in the breeding of WSSV-resistance L.vannamei.