Studies On Genetic Characteristics And Immunologic Function Of Backcross Populations From The Ridgetail White Prawn Exopalaemon Carinicauda

The ridgetail white prawn Exopalaemon carinicauda belong to Crustacea,Decapoda,Palaemonidae,is a kind of small benthic shrimp.In recent years,the ridgetail white prawn has become the main characteristic aquaculture breed of the coastal area.As an important breeding method,backcross has been widely used because of its ability to change the individual traits and accelerate the cultivation of pure lines.In thisstudy,we conducted the researches of inherited characteristics and immunologic function of backcross populations from E.carinicauda.We developed 31 polymorphic microsatellite loci and 49 SNP loci,and these markers were used for analysis of genetic characteristic of 3 backcross families.Meanwhile,we cloned TRAF6 gene of E.carinicauda and detected TRAF6 gene expression profiles after infected by WSSV in wild populations and backcross families.1.Isolation of microsatellite markers and genetic characteristics of E.carinicauda backcross populations by microsatellite marker analysisIn order to isolate and characterize microsatellite markers from E.carinicauda,we developed 31 microsatellite loci from 269 pairs of microsatellite primers.And we detected them in 30 individuals from wild population.The number of alleles at each locus ranged from 2 to 9 with an average of 4.19,ECL03 and ECL27 have maximum number of alleles(9),the average of observed heterozygosity(Ho)was 0.5787,the average of expected heterozygosity(He)was 0.6535 and the average of PIC was 0.5821.According to the Botstein(1980)theory,25 of the 31 loci were highly polymorphic and 6 loci were moderately polymorphism.In order to analyze genetic effect of E.carinicauda backcross populations,25 microsatellite loci were chosen to detected the genetic characteristics ofthree backcrossfamiliesA,B,C constructed by our laboratory.The results showed that the average of Ne are 2.21,2.18 and 2.09,the average of Ho were0.3805,0.3703 and 0.2489,the average of Hewere 0.3629,0.3998 and 0.2503 and the average of PICwere 0.3257,0.3391 and 0.2585,respectively.Compared to the wild population,the genetic diversities of backcross populations were greatly reduced and belonged to moderate or low polymorphism.The three backcross families obtained62 alleles using 25 loci.Abackcross family got 56 alleles,B backcross family got 54 alleles and C backcross family got 51 alleles.These three backcross families have 39 common alleles.Six sites have been homozygous in all backcross populations(ECL10 in all A,B and C families).The average of homozygous rate was 53.86%,56.53% and 63.33%.Compared to the wild population 16.91%,the average of homozygousgreatly improved,which showed that the purification rate of gene was rapid and the backcross is suitable for selective breeding in E.carinicauda.2.Identification SNP markers correlated with growth of E.carinicauda and genetic characteristics of backcross populations by SNP marker analysisIn order to explore SNP markers correlated with growth of E.carinicauda,we have identified 11 gene segments associated with growth based on transcriptome data.7 gene segments were amplified based on comparative transcriptomics,and 4579 bp DNA fragments were amplified.SNP frequency was estimated to be 1.07/100 bp of DNA sequence,the conversion mutation rate was 78% and the transversion mutation rate was 22%.The conversion ratio was far greater than the transversion ratio to comply with the principle of “transition bias”.By direct sequencing,7 SNPs loci were successfully genotyped in wild populations and backcross pedigree;two SNPs were significantly correlated with growth traits(P<0.05).The multivariate analysis of variance showed that X2-261 was significantly closely related to body weight,body length,body width,and carapace width(P<0.01);9S-111 was significantly related to body weight(P<0.05).Genetic characteristics of E.carinicauda backcross populations were detected by SNP marker analysis.None of the SNP loci deviated significantly from Hardy-Weinberg equilibrium after a Bonferroni's correction(P ? 0.05).Based on the SNP polymorphism analysis found that all SNP sites did not deviate from the habowen balance(HWE).The observed heterozygosities of E.carinicauda SNPs was 0.1667-0.5667,and the expected heterozygosities was 0.3045-0.5079,indicating less information content than that from microsatellite markers.These SNPs will be helpful for pedigree analysis,association studies and marker-associated selection in E.carinicauda.Polymorphism evaluation of E.carinicauda backcross families by 7 SNP loci,we found that the range of heterozygosity(Ho)was 0.0333-0.2233,the range of expected heterozygosity(He)was 0.1244-0.3300;and the PIC values of the 7 loci were lower.The results proved that heterozygosity of alleles decreased,homozygous genotype increased and the results was similarity with those of the microsatellite.At the same time,we found that heterozygosity of the same locus in different backcross families were not consistent,which may be related to the parent's genotype of the backcross families.3.Cloning and expression of TRAF6 gene from E.carinicaudaWe cloned the TRAF6 cDNA sequence based on transcriptome library of E.carinicauda.The cDNA possessed an ORF of 2290 bp encoding a protein of 566 amino acids.The TRAF6 protein contains a RING-type zinc finger structure,two TRAF-type zinc finger structures,a coiled-coil domain and a MATH domain.BLAST comparison and phylogenetic analysis showed that TRAF6 belonged to a member of the TRAF6 family.The TRAF6 similarities of E.carinicauda with Penaeus chinensis and Litopenaeus vannamei were 73% and 72%,respectively.In addition,we detected the tissual expression profiles of TRAF6 in 10 tissues by real-time PCR,the expression was highest in hepatopancreas and lowest in the shell.These results suggested that TRAF6 might be involved in the regulation of various physiological functions of E.carinicauda.In order to study the immune characteristics of TRAF6 gene in E.carinicauda wild populations and backcross populations,TRAF6 gene expression were detected in hepatopancreas and hemocytes at different time points after WSSV stimulation.The results showed that the expression of TRAR6 gene in hepatopancreas and hemocytes showed a clear up-regulation trend,and the expression of TRAR6 gene in the hepatopancreas reached the highest level at 24 hafter WSSV stimulation;the expression in the hemocytes reached the highest value at 12 h after infection,.These results suggested that the TRAF6 gene of E.carinicauda played an important role in the immune defense.Compared the wild populations and backcross families,no significant differences found in the expression of the TRAF6 between them.It proved that there is no significant difference in immune function between wild populations and backcross populations.