| In this study, we examine the genetic variation in the major histocompatibility complex (MHC) classⅡB gene in turbot (Scophthalmus maximus), through a challenge with a virulent bacterial pathogen. One hundred fry from each of six families were infected by an intraperitoneal (ip) injection with Edwardsiella tarda suspension, the family mortality was from 28% to 83.33%. Five survivor and five non-survivor individuals per family were used for amplifying complete exon 2 and intron 1 of MHC classⅡB genes using the clone-sequence method. Thirty-eight sequences which were obtained from 60 individuals showed 37 alleles and 25 alleles was the first found in this study, the latter belonging to 16 allele major types and eight alleles were used to examine association between alleles and resistance/susceptibility to disease. Five alleles presented in an individual denoted that there was a minimum of three loci or copies in turbot MHC classⅡB gene. The rates of non-synonymous substitution (dN) were 2.298 and 1.58 times higher than that of synonymous substitution (dS) in the peptide-binding regions (PBR) and non-PBR in whole families, respectively, which suggested balancing selection was performed in the exon 2 of the MHC classⅡA gene of turbot. One allele, Scma-DBB1*02 was significantly more prevalent in survivors stock than in non-survivors stock (P=0.001). This might be associated with resistance to bacteria; while the other allele, Scma-DBB1*10 was significantly more prevalent in non-survivors stock than in survivors stock (P=0.021), which might be associated with susceptibility to bacteria.Genes of the MHC are characterized by extremely high levels of polymorphisms in cell surface glycoprotein classⅠandⅡmolecules. In a previous study, the Paol-DAB*4301 allele was found to be associated with resistance to Vibrio anguillarum in flounder (Paralichthys olivaceus) and to present in the high resistance families 0768 and 0751 in generation1(G1). In order to determine the genetic variation of the Paol-DAB*4301 allele in the offspring, 700 fry from 7 families of Japanese flounder challenged with V. anguillarum were studied, and different mortality rates were found in those families. Five to ten surviving and dead fry from each of the seven families were selected to study the MHC class II B exon2 gene with Polymorphism Chain Reaction (PCR) and a direct sequence method. 116 different exon2 sequences were found and 116 different alleles were identified, while a minimum of four loci were revealed in the MHC class II B exon2 gene. The alleles present in the surviving and dead individuals in each of the seven families had different frequencies, although the frequency was not significant. The ratio (dN/dS) of non-synonymous substitution (dN) to synonymous substitutions (dS) in the peptide-binding region (PBR) of the MHC class IIB gene was 6.234, which indicate that balancing selection is acting on the MHC class IIB genes. One allele, Paol-DAB*4301, was shared by family 101, 104, 92 and 102. Another allele, Paol-DAB*0801, was also shared by family 41. The MHC IIB alleles were thus being passed on to their progeny. These two alleles were not discovered in family 5, which was the most resistant family and had the lowest heterozygosity (55%) of the MHC class IIB gene in generation2 (G2). In family 101, an 11.2% frequency was found for Paol-DAB*4301 in the surviving individuals and a 4.8% frequency in the dead and this difference was significant (p=0.001). In family 104, the Paol-DAB*4601 allele, which was first identified in this study, was significantly more frequent in surviving (13.6%) than dead individuals (6.5%, p=0.001). In family41, the Paol-DAB*3803 allele was significantly more frequent in surviving (17.1%) than dead individuals (5.7%, p=0.009). In family 5, the Paol-DAB*4101 allele was significantly more frequent in surviving (26%) than dead individuals (8%, p=0.01). It is therefore suggested that the alleles Paol-DAB*4301, Paol-DAB*4601, Paol-DAB*4302, Paol-DAB*3803 and Paol-DAB*4101 are associated with resistance to V.anguillarum in flounder.In this study, we measured genetic variation and balancing selection at MHC IIB loci in 10 families of half-smooth tongue sole through Polymorphism Chain Reaction and direct sequence method. We cloned and sequenced a 397bp fragment of MHC class IIB exon2. Amplified fragment spanned partial exon1, complete intron1 and exon2. Five clones per individual and five fry per family in ten families of half-smooth tongue sole were selected for MHC IIB sequence analysis. 60 sequences were discovered among 50 individuals which revealed 60 alleles, 28 of these alleles were the first found in this study and submitted Genbank. The homology similarity was 89.36% among sixty sequences. 5 distinct sequences were presented in each of six individuals which denoted there were three loci at least in half-smooth tongue sole MHC class IIB genes. Non-synonymous (dN) substitution is significantly higher than synonymous (dS) in peptide-binging region (PBR) of MHC IIB gene in nine of ten half-smooth tongue sole families.Genes in the MHC have a crucial role in both the innate and adaptive immune responses because of their involvement in presenting foreign peptides to T cells. We examined the genetic variation in (the) MHC class IIB in half-smooth tongue sole (Cynoglossus semilaevis) after challenge with bacteria. 2400 fry from 12 half-smooth tongue sole families were challenged with V. anguillarum. To determine any association between alleles and resistance or susceptibility to V. anguillarum, one hundred and sixty individuals from four high-resistance (HR, <40.55% mortality) families and four low-resistance (LR, >73.27% mortality) families were selected for MHC IIB exon2 gene sequence analysis. The MHC IIB exon2 genes of tongue sole displayed a high level of polymorphism and were discovered at least four loci. Meanwhile, the dN/dS [the ratio of non-synonymous (dN) substitutions to synonymous (dS) substitutions] in the peptide-binding region (PBR) was higher than that in the non-peptide-binding region (non-PBR). 88 alleles were discovered among 160 individuals, and 13 out of 88 alleles were used to analyze the distribution pattern between the resistant and susceptible families. Certain alleles presented in HR and LR with a different frequency, while other alleles were discovered in only the HR or LR families, not both. Five alleles, Cyse-DBB*6501, Cyse-DBB*4002, Cyse-DBB*6102, Cyse-DBB*5601 and Cyse-DBB*2801, were found to be associated with susceptibility to V. anguillarum with a frequency of 1.25%, 1.25%, 1.25%, 1.25% and 2.5% in the HR families, and 35%, 33.75%, 27.5%, 16.25% , 15% in the LR families (p=0.000, 0.000, 0.000, 0.001, 0.009), respectively. Four alleles, Cyse-DBB*3301, Cyse-DBB*4701, Cyse-DBB*6801 and Cyse-DBB*5901, were found to be associated with resistance to V. anguillarum, with a frequency of 13.75%, 11.25%, 11.25%, 8.75% in the HR families and 1.25%, 1.25%, 1.25%, 1.25% and 1.25% in the LR families (p=0.005, 0.018, 0.018 and 0.064), respectively. Elucidation of the role of MHC II B genes in half-smooth tongue sole should prove to be helpful to the in-depth development of marker-assisted selective breeding in half-smooth tongue sole.Half-smooth tongue-sole is distributed along the coast of china to the Yellow Sea, Bohai Sea. It is a cultured marine fish species which is high valued because of its delicious, tender meat and nutritious. However, diseases of the farmed fish have taken place constantly and yield losses due to epidemic diseases restrict earnings and progress of fishery. To develop new strain with enhanced disease-resistance and fast growth is an important task in tongue sole aquaculture. In the present study, wild population from Bohai Sea and cultured population were used as basic populations. 18 families of half smooth tongue sole were established. Growth comparison indicated that significant differences were observed in growth rate among the 18 families. Among which 2 fast-growth families (family 15 and 16), 4 secondly fast-growth families (family 6, 7, 16 and 27), and 12 common growth families (family 1, 3, 4, 10, 12, 14, 19, 24, 27, 28, 33 and 34) were obtained. 12 families were chose for pathogenic bacteria, V. anguillarum, challenge experiments which showed that one family (family 2) had strong disease resistance with the survival rate of 79%, 2 families (family 12 and 14) had secondly strong disease resistance with the survival rate of 50-60%, 6 families (family 3, 6, 7, 10, 16 and 19) had common disease resistance with the survival rate of 35-50%, and 3 families (family15, 27, and 30 ) had low disease resistance with the survival rate of less than 35%. |
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