Application Of Molecular Markers In The Selection Breeding Of Heat Resistant Japanese Flounder

In this study, The population structure of Japanese flounder (Paralichthys olivaceus) inthe Yellow Sea (Qingdao population, QD) and East China Sea (Zhoushan population, ZS)was analyzed using amplified fragment length polymorphism (AFLP), microsatellite andcytochrome c oxidase subunit I (COⅠ) gene sequencing. Understand the geneticinformation of the two wild populations to provide reference for parental choice ofselection breeding of heat-resistant flounders. The studied Japanese flounders (Paralichthysolivaceus) were divided into two categories: thermal-tolerance stock andthermal-susceptible stock by exposing in the heated water (32℃). AFLP and microsatellitetechnology were used to screen the molecular markers correlated to thermal-tolerance andanalyze the genetic diversity of thermal-tolerance stock and thermal-susceptible stock ofJapanese flounder. The polymorphisms in HSP70gene were analyized and the associationbetween gene polymorphisms and thermal tolerance susceptibility/resistance wasinvestigated by examining the distributions of polymorphisms in six loci of susceptible andresistant stocks using matrix-assisted laser desorption/ionization time-of-flight massspectrometry (MALDI-TOF MS) analysis. The aims to look for the potential markers weresignificantly correlated with thermal tolerance of flounders, which could be helpful for theselection breeding of heat-resistant flounders.A total of390bands were produced from100individuals using10primer combinationsand341(87.44%) of these bands were polymorphic. There were279and204polymorphicbands in the Qingdao and Zhoushan populations, respectively. The percentage ofpolymorphic loci (P), Nei’s genetic diversity (H) and Shannon’s information index (I)values were higher in the Qingdao population (P=72.85%, H=0.243and I=0.364) thanthose in the Zhoushan population (P=56.35%, H=0.189and I=0.284). The geneticdiversity reduction in the Zhoushan population may be attributed to fishing pressure andhabitat loss in this area. A total of68different alleles were observed over10microsatelliteloci. The total number of alleles per locus ranged2～9, and the number of genotypes perlocus ranged3～45, respectively. The observed heterozygosity (Ho) and expectedheterozygosity (He) in QD were0.733and0.779, while those in ZS were0.708and0.783,respectively. Significant departures from the Hardy-Weinberg equilibrium were observed in14out of20cases in the two populations. Based on the COⅠ sequencing analysis, a totalof25polymorphic sites were examined, and15haplotypes were identified in the2populations. The haplotype diversity (h) and nucleotide diversity (π) values in the Qingdao population were0.746±0.0728and0.00334±0.00181, respectively. The correspondingvalues in the Zhoushan population were0.712±0.0470and0.00318±0.00212. The AFLP(Fst=0.195, P <0.001), microsatellite (Fst=0.0487, P <0.001) and mtDNA (Fst=0.128,P <0.001) data revealed significant genetic differentiation between the two populations.The present study discussed the factors that may result in genetic differentiation betweenthe populations in the Yellow and East China Seas. Japanese flounder is an importantcommercial species in China, however, the genetic background of natural populations inChina seas was still limited which hampered the fishery management and aquaculturedevelopment programs of this species.The studied Japanese flounders (Paralichthys olivaceus) were divided into twocategories: thermal-tolerance stock and thermal-susceptible stock by exposing in heatedwater (32℃). Amplified fragment length polymorphism (AFLP) and microsatellitetechnology were employed to screen the molecular markers correlated to thermal-toleranceand analyze the genetic diversity of thermal-tolerance stock and thermal-susceptible stockof Japanese flounder. Eighty-one AFLP primer combinations and forty-two microsatelliteprimers were used to screen molecular markers correlated to thermal tolerance of Japaneseflounder. AFLP and microsatellite loci which occurred in different genotype frequencywere analyzed using χ2-test to examine the correlation between the molecular markers andthermal tolerance in Japanese flounder. The results showed that the frequency of AFLP lociA3was significantly higher in thermal-susceptible stock than that in thermal-tolerancestock, which indicated that this loci was negatively correlated with thermal tolerance andthe parameter of correlation was-0.453(P <0.01). Also, the frequencies of4AFLP lociwere signicantly higher in thermal-tolerance stock than those in thermal-susceptible stock,which indicated that the4loci were correlated with thermal tolerance (P <0.01). Based onmicrosatellite analysis, the loci S1and S2amplified by Po25A and205TUF showednegatively correlated with thermal tolerance, and the parameters of correlations were-0.408(P <0.01) and-0.398(P <0.01), respectively. In addition, the genetic diversity ofthermal-tolerance stock and thermal-susceptible stock were analyzed using eight AFLPprimer combinations and six microsatellite primers. The results showed high geneticvariations in the two stocks, and the genetic diversity of the two stocks was comparable.HSP70gene was amplified from susceptible and resistant founders, and16positiveclones from four susceptible flounders and four resistant flounders were sequenced. Thefull-length sequence of HSP70gene was about of4000bp, including5’ utr rntranslation region about of830bp, seven introns and eight exons by comparing the results of HSP70gene sequence with cDNA sequence of NCBI. Six ins-del polymorphisms and fifty-oneSNPs were found in the HSP70gene sequence, three ins-del polymorphisms and nineteenSNPs were found in the amplified5’ utr rntranslation intron region, three ins-delpolymorphisms and twenty SNPs were found in the amplified introns region, and twelveSNPs were found in the amplified exons region, which are synonymous mutations and can’tcause the change of amino acids. The association between gene polymorphisms and thermaltolerance susceptibility/resistance was investigated by examining the distributions ofpolymorphisms in six loci (snp1: Q--697C-A, snp2: Q--587C-G, snp3: Q--324TTTins-del, snp4: I2-67T-A, snp5: E4-524C-T and snp6: I4-73ins-del) of susceptible andresistant stocks. The six polymorphisms loci were screened by matrix-assisted laserdesorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis. TheHardy–Weinberg equilibrium for genotypic frequencies was analyzed with thegoodness-of-fit χ2-test. The genotypic frequency of each allele was in HWE (P>0.01) atlocus Q--697, Q--587, Q--324, I2-67, E4-524and I4-73. At locus Q--587, the G allelefrequency was significantly different distribution between resistant stock and susceptiblestock (P=0.028). Genotype frequency of Q--587G/G was significantly different betweenresistant stock and susceptible stock (P=0.0050). At locus I2-67, the C/A allele frequencywas significantly different distribution between resistant stock and susceptible stock (P=0.0001and0.0001). Genotype frequencies of I2-67A/A, I2-67T/T and I2-67A/T weresignificantly different between resistant stock and susceptible stock (P=0.0020,0.0010and0.0020). Genotype A/C, C/C and C/T were only a little distribution in the resistantstock, no distribution in the susceptible stock. Those results indicated that thepolymorphism of loci Q--587and I2-67was associated with thermal tolerance of flounders,which would be a potential gene marker associated with enhanced thermal tolerance, andcould be perhaps applied in future molecular selection program of Japanese flounder.