Identification And Bioinformatic Analysis Of Single Nucleotide Polymorphism (SNP) Loci Related To Growth Trait In Turbot (Scophthalmus Maximus L.)

Turbot(Scophthalmus maximus L.) is a famous flounder native to the coast of Europe. They became the main economic fish in north of china since introduced from Britain in 1992, as its fast growth, sweet-meated, easy to tame and characteristics of resistance. However, turbot aquaculture industry appeared some problems with the constant improvement level of factory farming in recent years. The phenomenon of inbreeding depression and decreased growth rates has emerged because of the limited original stock and nonstandard breeding operation, which restricts the sustainable development of the turbot breeding seriously. Therefore, it is urgent to perform the genetic selective breeding to improve the important and good growth traits of turbot. With the application and development of DNA marker technology, the combination between traditional breeding method and molecular-marker assisted selection(MAS) breeding have promoted the process of new variety breeding in fishes. Single nucleotide polymorphism(SNP) has been widely used in animal and plant genetics or breeding programs because its advantage such as high density, good genetic stability and easy to automated analysis.In order to further identify single nucleotide polymorphism(SNP) associated with growth traits of turbot(Scophthalmus maximus L.),the time of flight mass spectrometer(TOF-MS)was used to genotyping four turbot families in total of 160 individuals for 100 SNP locus, and the association were analyzed between the SNPs and the growth traits(body weight and body length),which was based on the QTL mapping results in turbot. Additionally, genetic diversity of four families were performed, the genetic differentiation and the relationship between turbot families were analyzed based on the SNP genotypes. At last, we cloned the candidate gene related to turbot growth traits—NOTCH1, which the SNP86 located in, using the RT-PCR technology. With the method of real time fluorescent quantitative PCR, the expression patterns of NOTCH1 gene in different organizations during the two months and five months juvenile turbot were determined in the present study. The main results are as follows:1. The time of flight mass spectrometer(TOF-MS)was used to genotyping four turbot families for 100 SNP locus, and the association were analyzed between the SNPs and the growth traits using the general linear model statistics. The results showed that 9 SNPs were identified to be associated with the growth traits(P<0.05). Among these SNPs, four locus were associated with body weight and seven were associated with body length significantly(P<0.05). SNP51 and SNP111 were identified to be associated with both body weight and body length(P<0.05). The genetic parameters showed that the observed heterozygosity(Ho) ranged from 0.126 to 0.719(mean=0.387) and expected heterozygosity(He) estimates from 0.118 to 0.501(mean=0.338). The average polymorphism information content(PIC) was 0.267, indicating that the 9 locus were reasonably informative. Five of the nine SNP loci deviated significantly from Hardy–Weinberg equilibrium(HWE). Strong linkage disequilibrium occurred among the five SNPs in the LG18 according to the LD analysis results. Moreover, haplotype CTTGT and CTTGA were two important types as they accounted for 26.8% and 25.9% of total haplotypes, respectively. Finally, the alignment results showed that COL11A1 gene and NOTCH1 gene may be related to the growth traits. However, further studies are needed to explore their function in the turbot development. These findings will boost molecular markers research related to growth traits and provide theoretical basis to the marker assisted selection(MAS) and the next gene location.2. The genetic diversity of four turbot families were analyzed based on the SNP genotype. The results showed that eleven SNP loci were no polymorphism among the 88 successfully genotyped SNP loci, which indicated that the proportion of polymorphic loci was 87.5%. The average number of alleles of each family was between 1.6477 to 1.7614, which the NO.3 turbot family shared the maximum effective alleles number(1.4796). Four the average heterozygosity, observed heterozygosity were greater than expected heterozygosity of all families indicating that excess heterozygosity occurred in the families and the population retained a high level of genetic diversity. Polymorphism information content analysis results showed that average PIC of all families is less than 0.25, belong to low polymorphism. Comprehensive comparison, 3 is the highest level of diversity. A bigger genetic differentiation occurred within the populations than among the populations, as indicated by the POPGENE analysis, which suggested that 82.48% genetic differentiation occurred within the populations. Cluster analysis based on genetic distances using UPGMA method and the results showed that pedigree NO.2 and NO.4 shared the nearest relationship, and then cluster with NO.3. Above all, the genetic diversity of turbot families retained a high level and had significant genetic differentiation among the population. Additionally, there was no significant inbreeding within the families. The results are consistent with the idea of avoiding the inbreeding in the progress of the turbot pedigree breeding.3. In the present study, the sequence of the candidate gene related to growth trait—NOTCH1 were cloned using the RT-PCR technology and the length of cDNA was 544 bp which contain the SNP86. The homology comparison results through BLASTX program showed that cloned fragments were similar to those in other fishes such as zebrafish, half smooth tongue sole, rainbow trout and so on. The proportion of gene homology was higher than 95%. In addition, the expression patterns of NOTCH1 gene in different tissues during the two months and five months juvenile fish were determined with the method of real time fluorescent quantitative PCR. The results showed that the NOTCH1 gene mRNA were highly expressed in heart and eye, and moderately expressed in the liver, spleen and other tissues. There was significant variation in the expression pattern of NOTCH1 gene mRNA in the tissues during the two months and five months juvenile turbot. According to the research of the NOTCH genes in other fishes, temporal expression patterns of NOTCH1 gene can be studied during early development of turbot in the next research. This would further explore the relationship between the gene function and growth development. These results would benefit for the gene location and selection related to turbot growth traits, and promote the molecular-marker assisted selection(MAS) breeding process of turbot.