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. 2011 Sep 2;12:78. doi: 10.1186/1471-2156-12-78

MHC polymorphism and disease resistance to vibrio anguillarum in 8 families of half-smooth tongue sole (Cynoglossus semilaevis)

Min Du 1,2,3, Song-lin Chen 1,✉, Yan-hong Liu 3, Yang Liu 1, Jing-feng Yang 1
PMCID: PMC3199252  PMID: 21888646

Abstract

Background

Genes in the major histocompatibility complex (MHC) have a critical role in both the innate and adaptive immune responses because of their involvement in presenting foreign peptides to T cells. However, the nature has remained largely unknown.

Results

We examined the genetic variation in MHC class IIB in half-smooth tongue sole (Cynoglossus semilaevis) after challenge with vibrio anguillarum. Two thousand and four hundred fry from 12 half-smooth tongue sole families were challenged with Vibrio anguillarum. To determine any association between alleles and resistance or susceptibility to V. anguillarum, 160 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). Eighty-eight 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 < 0.01, 0.01, 0.01, 0.01, 0.01), 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 < 0.01, 0.05, 0.05 and p = 0.064), respectively.

Conclusions

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.

Keywords: Cynoglossus semilaevis, Vibrio anguillarum, polymorphism, MHC IIB, susceptibility, resistance

Background

Major histocompatibility complex (MHC) molecules play a critical role in both innate and adaptive immunity by presenting foreign peptides to T cells in vertebrate organisms, and have been considered candidate molecular markers of an association between polymorphisms and resistance/susceptibility to diseases [1]. A combination of balanced and directional selection is thought to be responsible for allelic variation of MHC genes in vertebrate populations, because pathogen pressure varies at different times and locations [2]. Two classes of MHC are found in fish, MHC class I and class II molecules. The genes encode glycoproteins which bind peptides for the presentation of self and non-self peptides to T-cell receptors (TCR) [3].

The MHC class II molecules are symmetrical heterodimers, consisting of one alpha chain and one beta chain, with non-covalent contacts in which the alpha1 and beta1 domains form a peptide-binding region (PBR). In mammals, MHC class II genes are constitutively expressed in antigen-presenting cells such as macrophages, B cells, monocytes and dendritic cells, and have direct functional relevance in the immune response. Class I antigens are expressed in all somatic cells [1,4,5]. In teleosts, class I and class II genes were found to reside on different linkage groups [6-8]. Many MHC genes have been isolated, characterized expressed and analyzed in at least 30 different fish species over the last twenty years [9-14]. Multiple loci and a considerable number of alleles at each given locus were found in the classical MHC genes. The peptide-binding region (PBR) contains the highest level of polymorphisms in the MHC genes [15-29]. Certain MHC alleles of the class II genes linked to viral and bacterial diseases have been reported in some species [30-37]. The link between disease susceptibility/resistance and MHC polymorphism is crucial for detecting MHC alleles related to resistance in marine aquaculture species for molecular marker-assisted selective breeding programs [38].

Half-smooth tongue sole (Cynoglossus semilaevis) is widely cultured throughout the coastal areas of North China [39]. However, viral and bacterial diseases frequently occur in this cultured fish, and losses due to infectious disease limit the profitability and the extent of the development of the aquaculture [40,41]. One pathogen which is a significant threat to half-smooth tongue sole is Vibrio anguillarum [42]. Antibiotics have partially solved problem, but antibiotic residues in fish, environmental pollution and antibiotic resistance are questions about which grave concerns remain [43]. Therefore, the selective breeding of tongue sole with disease resistance, basing on molecular techniques which can enhance the resistance to specific pathogens, may be a good approach to solving these problems.

The half-smooth tongue sole MHC class IIB cDNA sequence and cDNA polymorphisms have been reported [40]. However, the polymorphisms at the DNA level and the link between specific alleles and resistance to V. anguillarum have not been elucidated yet. In the present study, we investigated the single nucleotide polymorphism (SNP) sites and polymorphisms in MHC II B exon2, and the association between certain alleles and disease resistance or susceptibility to Vibrio anguillarum, across 8 families of half-smooth tongue sole.

Methods

Fish and rearing

Eighteen full-sib families were established as reported [44], using a method for producing strains with a high growth rate and disease resistance. Male parents came from wild populations while female parents came from farming populations. Fertilized ova were hatched and reared at the breeding station at Minbo aquatic Co., Ltd. Located in Laizhou city, Shandong province, China. Each family was kept in a separate tank. The fry were fed a commercial diet using a standard feeding regimen [45].

Challenge test

For the challenge test, 200 individuals of each family (12 out of 18 families were large enough to be included), ten months old, were intraperitoneally injected with a 0.2 ml bacterial suspension of approximately 10,000,000 cells of V. anguillarum, while 16 individuals were injected with 0.9% saline as control [15]. Each fry weighed approximately 12-15 grams. The fry of each family were kept in a 1 m3 single tank with a fresh seawater supply at 23°C. This challenge experiment was performed twice and lasted for approximately two weeks. Mortality was recorded every day and the fin clips of all the fish were collected and preserved in absolute ethanol until use. The gross signs of fish mortality were based on a previous reporting method [42].

Sampling and DNA isolation

To identify whether MHC IIB exon2 alleles are associated with resistance or susceptibility to V. anguillarum, fin samples from each family of half-smooth tongue sole were collected and recorded from the first 20 to die and the last survivors at the time the bacterial challenge was terminated and preserved in absolute ethanol until use. High-resistance families (HR) with a survival rate (SR) > 59.45% and susceptible families or low-resistance families (LR) with a SR < 26.73% were selected from the challenge trials. The numbers fish which died or survived after the infection recorded for each family (Additional file 1).

Genomic DNA was isolated from the dorsal or caudal fin samples of 20 individuals per family (from the 4LR and 4HR families) using the phenol-chloroform method as described by Chen et al. [46]. The quality and concentration of DNA were assessed by agarose gel electrophoresis and then measured with a GENEQUANT Pro (Pharmacia Biotech Ltd.) RNA/DNA spectrophotometer. Finally, DNA was adjusted to 100 ng/μl and stored at -20°C.

Primer design and Polymerase Chain Reaction (PCR)

A pair of gene-specific primers was used for the PCR amplification of the MHC II B gene: hMPN12 (5'-CTCTCTTCTCTTCCTCCTCAC-3') and hMPC12 (5'-ACA CTCACCTGATTTAGCCA-3'). They were designed according to reported half-smooth tongue sole MHC II B cDNA sequences [40]. The primer pair was used to amplify part of exon1, and all of intron1 and exon2 from half-smooth tongue sole using a Polymerase Chain Reaction technique. A 25 μl PCR reaction mixture contained 1 μl of template DNA, 2.5 μl of 10×Taq polymerase buffer (TransGen Biotech), 1.5 mM MgCl2, 0.2 mM dNTP mix, 0.2 μM of the forward and reverse primers, and 1 unit of Taq polymerase (TransGen Biotech). The amplifications were performed on a Peltier Thermal Cycler (PTC-200). A Molecular Imager Gel Doc XR system (Bio-rad) was used to determine the PCR products by electrophoresis on a 1% agarose gel.

Cloning and sequencing

The PCR products were resolved by electrophoresis on 1.5% agarose gels. The fragments of interest were excised and purified with the QIAEX II gel extraction kit (Qiagen). The purified fragments were cloned into a PBS-T vector (Takara) according to the standard PBS-T vector protocol (Takara) and then transformed into TOP 10 Escherichia coli competent cells (TransGen Biotech). Forward and reverse M13 primers were used to screen for positive clones via PCR. Ten positive clones from the upper purified fragments were sequenced with an ABI 3730 automated sequencer using the M13+/- primer.

Genotyping, sequence analysis and statistical tests analysis

Sequence data were analyzed using DNASTAR 5.0 and DNAMAN software. The alignment was performed with MEGA4.0 [47]. The rate of synonymous substitution (dS) and non-synonymous substitution (dN) was calculated accord with an earlier report [47] using MEGA4.0 software. DAMBE and DnaSP5.0 software packages were used to analyze the polymorphisms [48]. Statistical analysis was carried out with SPSS13.0. Differences in the allelic frequency were verified using Fisher's exact test and the significance level [49] was determined for every individual (n = 160) and each family (n = 8).

The new alleles were designated Cyse-DBB*0101 to Cyse-DBB*6601 on the basis of the rules reported by Davies et al. [50]. Cyse refers to Cynoglossus semilaevis, D to class II, the first B to an uncharacterized family and the second B to β chain-encoding genes. In the first four digits after the asterisk, the first two digits refer to the major type (alleles that differ by at least five amino acid substitutions), while the last two digits refer to the subtype (alleles that differ by less than five amino acid substitutions within a single major type) [51,52].

Results

To analyze disease resistance among 12 half-smooth tongue sole families

The first specific mortality appeared after 16 h due to an ip injection of V. anguillarum, and the challenge test lasted two weeks, at which time the overall accumulated mortality reached 42.24%. The survival rate among the 12 test families ranged from 15% to79.25%, which was determined on the basis of each family. Here, we selected four high-resistance and four low-resistance families to ascertain whether MHC IIB exon2 alleles were associated with resistance to V. anguillarum among the 12 families of half-smooth tongue sole. The mean prevalence of survival of the four high-resistance families was 59.45%, while that of the four low-resistance families was considerably less at 26.73%.

To elucidate sequence polymorphism within exon2 of MHC IIB gene in 8 half-smooth tongue sole families

Eighty individuals from the four high-resistance families and eighty individuals from the four low-resistance families were used in the present study (Additional file 1). Nine to twelve positive clones per individual were sequenced and 1618 sequences were obtained. A fragment of 397 bp was obtained in reference to the complete half-smooth tongue sole MHC IIB cDNA sequence [40] and intron-exon boundary GT-AG rule. This fragment of 397 bp contains a part of exon1 (35 bp), the entire intron1 (84 bp, containing a 12 bp CA repeat sequence) and the entire exon2 of MHC IIB. A fragment of 270 bp containing the complete exon2 which encodes the β1 domain of the MHC IIB gene was also analyzed. The results indicated 88 different sequences, in which 88 novel alleles were designated (Table 1) belonging to 57 major allele types, following established allele nomenclature method [49,50].

Table 1.

Alleles and Genbank Accession Number of half-smooth tongue sole MHC class II exon2 gene

Allele GenBank
Accession
No.		 Allele GenBank
Accession
No.		 Allele GenBank
Accession
No.
Cyse-DBB*0101 GU194838 Cyse-DBB*2401 GU194876 Cyse-DBB*4601 GU194918
Cyse-DBB*0102 GU194839 Cyse-DBB*2501 GU194877 Cyse-DBB*4602 GU194919
Cyse-DBB*0201 GU194840 Cyse-DBB*2601 GU194878 Cyse-DBB*4701 GU194921
Cyse-DBB*0202 GU194841 Cyse-DBB*2602 GU194879 Cyse-DBB*4801 GU194922
Cyse-DBB*0301 GU194842 Cyse-DBB*2603 GU194880 Cyse-DBB*4802 GU194923
Cyse-DBB*0401 GU194843 Cyse-DBB*2801 GU194882 Cyse-DBB*4803 GU194924
Cyse-DBB*0701 GU194847 Cyse-DBB*2802 GU194883 Cyse-DBB*5002 GU194927
Cyse-DBB*0801 GU194848 Cyse-DBB*2803 GU194884 Cyse-DBB*5003 GU194928
Cyse-DBB*0901 GU194850 Cyse-DBB*2901 GU194886 Cyse-DBB*5101 GU194929
Cyse-DBB*1001 GU194851 Cyse-DBB*3002 GU194888 Cyse-DBB*5202 GU194932
Cyse-DBB*1002 GU194852 Cyse-DBB*3101 GU194889 Cyse-DBB*5401 GU194934
Cyse-DBB*1003 GU194853 Cyse-DBB*3102 GU194890 Cyse-DBB*5501 GU194935
Cyse-DBB*1201 GU194855 Cyse-DBB*3201 GU194891 Cyse-DBB*5601 GU194936
Cyse-DBB*1301 GU194856 Cyse-DBB*3301 GU194892 Cyse-DBB*5602 GU194937
Cyse-DBB*1402 GU194858 Cyse-DBB*3302 GU194893 Cyse-DBB*5604 GU194939
Cyse-DBB*1403 GU194859 Cyse-DBB*3401 GU194896 Cyse-DBB*5701 GU194940
Cyse-DBB*1501 GU194860 Cyse-DBB*3501 GU194897 Cyse-DBB*5801 GU194941
Cyse-DBB*1601 GU194861 Cyse-DBB*3701 GU194902 Cyse-DBB*5901 GU194942
Cyse-DBB*1602 GU194862 Cyse-DBB*3702 GU194903 Cyse-DBB*5902 GU194943
Cyse-DBB*1701 GU194864 Cyse-DBB*3901 GU194905 Cyse-DBB*6001 GU194944
Cyse-DBB*1702 GU194865 Cyse-DBB*4001 GU194906 Cyse-DBB*6002 GU194945
Cyse-DBB*1703 GU194866 Cyse-DBB*4002 GU194907 Cyse-DBB*6102 GU194947
Cyse-DBB*1801 GU194867 Cyse-DBB*4101 GU194910 Cyse-DBB*6201 GU194948
Cyse-DBB*2002 GU194870 Cyse-DBB*4201 GU194911 Cyse-DBB*6301 GU194949
Cyse-DBB*2101 GU194871 Cyse-DBB*4301 GU194912 Cyse-DBB*6401 GU194950
Cyse-DBB*2201 GU194872 Cyse-DBB*4302 GU194913 Cyse-DBB*6402 GU194951
Cyse-DBB*2202 GU194873 Cyse-DBB*4402 GU194915 Cyse-DBB*6403 GU194952
Cyse-DBB*2203 GU194874 Cyse-DBB*4501 GU194916 Cyse-DBB*6404 GU194954
Cyse-DBB*2301 GU194875 Cyse-DBB*4502 GU194917 Cyse-DBB*6501 GU194955
Cyse-DBB*6601 GU194956
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Gaps were not found in the full alignment of the 270 bp exon2 of the MHC IIB gene. A putative 90 amino acid peptide was based on a sequence alignment with the half-smooth tongue sole MHC II B cDNA sequence [40]. Among the 270 nucleotides, 72 regions and 121(44.8%) nucleotide positions were variable. The numbers of two-nucleotide mutation, three-nucleotide mutation and four-nucleotide mutation were 24, 11 and 1, respectively (Table 2). At the SNP sites, there were two kinds of nucleotide substitutions, i. e. transition (Table 2, Serial No. 1, 7, 11, 13, 18, 23, 28, 29, 32, 33, 35, 42, 43, 44, 46, 49, 52, 53, 54, 60 and 69) and transversion (Table 2, Serial No. 20, 21, 25, 59). Three kinds of mutation per site (Table 2, Serial No. 2, 4, 6, 9, 14, 15, 16, 22, 26, 30, 31, 36, 37, 41, 51, 56, 58, 61, 62, 63, 65, 67, 68 and 71) which revealed the mutation hotspots. 36 out of 72 mutation regions were multi-nucleotide co-mutations, ranging from two to five nucleotides per region. The SNP sites were located in a tight region from position 9 to 29 (Table 2), so this were most of the mutation hotspots of MHC exon2 herein must be located. The frequency ratio ranged from 0.989:0.011 (Table 2, Serial No.1, 23, 32, 49, 59 and 60) to 0.557:0.443 (Table 2, Serial No.7). No frame-shift mutation was observed in these sequences. The peptide binding regions in half-smooth tongue sole MHC II B were based on the corresponding peptide binding region identified in humans [53].

Table 2.

Distribution of SNP sites within exon2 of MHC IIB allelic sequences of half-smooth tongue sole

Serial
number		 Position Base
type		 Allele no.
(n = 88)		 Frequency Serial
number		 Position Base
type		 Allele no.
(n = 88)		 Frequency
1 6 T 87 0.989 39 104-106 ATC 51 0.580
C 1 0.011 ATT 1 0.011
2 9-11 CTA 52 0.591 ATG 1 0.011
GTA 1 0.011 CAG 35 0.398
GAG 35 0.398 40 109-111 TCG 84 0.955
3 12 C 17 0.193 TCA 2 0.023
T 34 0.386 CCG 1 0.011
A 7 0.080 TTG 1 0.011
G 30 0.341 41 124-126 GGA 49 0.557
4 13 A 82 0.932 AGA 12 0.136
T 5 0.057 GAG 27 0.307
G 1 0.011 42 130 A 56 0.636
5 14-15 AT 30 0.341 T 32 0.364
AC 1 0.011 43 143 C 2 0.023
TT 55 0.625 T 86 0.977
CT 2 0.023 44 148-149 AT 49 0.557
6 16 C 32 0.364 TA 8 0.091
T 20 0.227 TT 31 0.352
A 36 0.409 45 156-157 CC 1 0.011
7 18 G 39 0.443 TA 24 0.273
A 49 0.557 TC 63 0.716
8 19 T 33 0.375 46 163 C 87 0.989
G 20 0.227 T 1 0.011
C 30 0.341 47 168-169 AG 71 0.807
A 5 0.057 GC 17 0.193
9 20 G 46 0.523 48 170-172 ATG 75 0.852
A 34 0.386 ATT 9 0.102
C 8 0.091 TTG 3 0.034
10 21-23 ACA 53 0.602 ACT 1 0.011
GTG 35 0.398 49 174 G 1 0.011
11 24 G 74 0.841 A 87 0.989
A 14 0.159 50 177-178 GA 86 0.977
12 25 A 35 0.398 AA 1 0.011
G 31 0.352 GG 1 0.011
T 19 0.216 51 181-183 GTC 80 0.909
C 3 0.034 ATC 7 0.080
13 26 T 5 0.057 GAA 1 0.011
C 83 0.943 52 193 C 24 0.273
14 28-29 CC 52 0.591 T 64 0.727
CA 1 0.011 53 196 A 62 0.705
GA 35 0.398 G 26 0.295
15 32-33 CG 35 0.398 54 198 A 69 0.784
TG 1 0.011 G 19 0.216
TC 52 0.591 55 199-200 GG 40 0.455
16 38-39 CA 52 0.591 GT 18 0.205
CG 1 0.011 TG 8 0.091
TA 35 0.398 CG 22 0.25
17 40-41 AC 51 0.580 56 205 A 67 0.761
GC 1 0.011 C 20 0.227
AT 35 0.398 G 1 0.011
CT 1 0.011 57 207-208 GA 62 0.705
18 44 C 36 0.409 AC 3 0.034
T 52 0.591 GG 8 0.091
19 47-49 AAA 52 0.591 GC 5 0.057
AAG 1 0.011 58 210-211 AA 86 0.977
TAA 19 0.216 GA 1 0.011
TGA 16 0.182 AT 1 0.011
20 51 G 53 0.602 59 218 G 87 0.989
C 35 0.398 T 1 0.011
21 53 G 36 0.409 60 220 A 87 0.989
C 52 0.591 G 1 0.011
22 55-56 AC 69 0.784 61 226-227 TG 47 0.534
AT 18 0.205 TA 40 0.455
GC 1 0.011 CG 1 0.011
23 58 A 87 0.989 62 228 A 50 0.568
G 1 0.011 C 29 0.330
24 63-64 GC 51 0.580 T 9 0.102
GT 1 0.011 63 229 A 82 0.932
GA 1 0.011 T 2 0.023
CA 35 0.398 G 4 0.045
25 67 A 68 0.773 64 231-234 AAC 61 0.693
T 20 0.227 ACT 2 0.022
26 72 C 13 0.148 CAC 20 0.227
G 40 0.455 AGC 5 0.057
T 35 0.398 65 237-238 GG 79 0.898
27 74 C 40 0.455 GA 5 0.057
G 48 0.545 AG 4 0.045
28 78 C 38 0.432 66 240-241 AA 10 0.114
T 50 0.568 AT 54 0.614
29 80 C 35 0.398 CT 23 0.261
T 53 0.602 GT 1 0.011
30 82-83 AC 52 0.591 67 242-243 TG 46 0.523
AT 35 0.398 TT 19 0.216
GT 1 0.011 GG 23 0.261
31 84-85 TT 30 0.341 68 245-246 CT 68 0.773
CT 23 0.261 CA 19 0.216
TA 35 0.398 AT 1 0.011
32 87 A 87 0.989 69 248 T 2 0.023
G 1 0.011 C 86 0.977
33 90 A 86 0.977 70 250-253 ACCA 10 0.114
G 2 0.023 ACGC 64 0.727
34 92-94 ACT 52 0.591 AGCC 1 0.011
GAG 36 0.409 GGAC 12 0.136
35 96 G 35 0.398 ACGG 1 0.011
A 53 0.602 71 254-256 TGC 70 0.796
36 98-99 GA 42 0.477 TGG 12 0.136
GT 11 0.125 GTT 6 0.068
AT 35 0.398 72 256-258 TT 12 0.136
37 100 C 1 0.011 TC 63 0.716
A 38 0.432 CC 2 0.023
T 49 0.557 TG 11 0.125
38 101-102 CA 34 0.386
CG 16 0.182
GA 3 0.034
TG 31 0.352
TA 4 0.046
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The variable positions of the PBR comprised 20 (87%) out of 23 and the polymorphic nucleotide PBR sites were 40 (57.97%) of 69. In the putative peptide-binding region, the ratio of non-synonymous (dN) substitution (0.261) was 1.7 times higher than that of synonymous (dS) substitution (0.153). The rates of dN and dS in the non-PBR were 0.087 and 0.159, respectively. All of the sequences were used to calculate these rates. The rate of ds in the non-PBR(0.159) was slightly higher than that of dS in the PBR(0.153), and dN in the PBR (0.261) occurred at a significantly higher rate than that in the non-PBR (0.087), but dS in the PBR (0.153) was a little lower than that in the non-PBR (0.159) (Table 3).

Table 3.

Synonymous (dS) and nonsynonymous (dN) substitution rate in the putative peptides binding region (PBR) and non-peptides binding region (non-PBR) among half-smooth tongue sole alleles

Region No. of
codons		 dN(SE) dS(SE) dN/dS
PBR 23 0.261 ± 0.033 0.153 ± 0.052 1.70
Non-PBR 67 0.087 ± 0.016 0.159 ± 0.034 0.547
Total 90 0.132 ± 0.017 0.157 ± 0.027 0.841
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The per site nucleotide diversity Pi (p) was 0.13785, and per the site Theta-W value of the 88 sequences was 0.08876. Ninety-six out of the 121 variable sites were parsimony informative sites. The haplotype diversity (H) and the average number of nucleotide differences (k) were 1 and 37.220, respectively. DnaSP5.0 software was used to calculate these polymorphic values. The exon2 sequence of MHC IIB indicated high nucleotide diversity in the 8 families of tongue sole. Figure 1 shows the spatial distribution of the nucleotide diversity. Two peaks appeared at the downstream and upstream of exon2 of the MHC IIB sequences, respectively, while the Theta-W value in the middle region was lower.

Figure 1.

Figure 1

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The nucleotide diversity within exon2 sequences of MHC IIB genes at the 88 alleles denoted by Theta-W. Sliding window length: 100; step size: 10.

To identify association between the MHC IIB alleles and disease resistance/susceptibility to V. anguillarum in half-smooth tongue sole

Additional file 2 shows the number of alleles per individual and the comparative individual number. An average ten clones per individual were sequenced, and 2 to 7 alleles per individual were discovered, which inferred the existence of at least seven alleles and four loci of the MHC IIB gene, in accordance with the reports of Xu et al. [40]. Among the 8 families examined, only 2.5% of the individuals were homozygous (all families were heterozygous) for exon2 of the MHC class IIB gene of tongue sole. Eighty-eight sequences resulted in eighty-eight different MHC IIB exon2 alleles deduced from 160 individuals. The distribution of the alleles was unequal. Certain alleles had a low frequency and were excluded from allele distribution analysis between the HR and LR families. Thirteen alleles were used for distribution analysis (Figure 2). The alleles Cyse-DBB*3301, Cyse-DBB*4701, Cyse-DBB*6801 and Cyse-DBB*5901 were more prevalent in individuals from the HR families (P = 0.005, 0.018, 0.018 and 0.064, respectively n = 160 individuals), while Cyse-DBB*6501, Cyse-DBB*4002, Cyse-DBB*6102, Cyse-DBB*5601 and Cyse-DBB*2801 were more prevalent in individuals from low-resistance families, as shown by chi-square test (P < 0.01, 0.01, 0.01, 0.01, 0.01 respectively n = 160 individuals). Some alleles were not significantly different in the HR and LR families, such as Cyse-DBB*0101(P = 0.247), Cyse-DBB*1601(P = 0.107), Cyse-DBB*4602 (P = 0.117) and Cyse-DBB*5003 alleles (P = 0.159). Here we (deduced) show that Cyse-DBB*3301, Cyse-DBB*4701, Cyse-DBB*6801 and Cyse-DBB*5901 were associated with resistance, while Cyse-DBB*6501, Cyse-DBB*4002, Cyse-DBB*6102, Cyse-DBB*5601 and Cyse-DBB*2801 were associated with susceptibility to V. anguillarum in half-smooth tongue sole. Alignment of the 13 deduced MHC IIB amino acid sequences (Figure 3) indicated that no specific single amino acid substitution was evidently involved in the resistance or susceptibility, as there was no specific amino acid substitution difference between the HR families and LR families.

Figure 2.

Figure 2

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Sequence polymorphism analysis within exon 2 of MHCIIB gene. (Asterisks indicate the correlative amino acid that combines the antigen).

Figure 3.

Figure 3

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Distribution of MHC class IIB alleles in high-resistance families individuals (white bars) and low-resistance families individuals (black bars) of half-smooth tongue sole. *Asterisks denote P < 0.05. ** denote P < 0.01.

4. Discussion

It is well known that MHC genes are vital components of both the innate and adaptive immune system. They present foreign peptides to T cells. Cloning and cDNA polymorphism of the MHC II B gene has been discussed [40]. In the present study, partial sequences of the MHC class IIB gene in different families of half-smooth tongue sole were isolated, then molecular polymorphisms as well as the link between alleles and resistance/susceptibility to V. anguillarum were analyzed.

Among the 72 mutated regions in the complete sequence of MHC IIB exon2, 36 regions were multi-nucleotide co-mutations, which indicate inter-allelic recombination took place in these regions. Moreover, no deletion, insertion or stop codon was observed, indicating that all of these alleles were functional genes. The frequency ratio of substituted nucleotides per mutation region was not equally distributed, which suggests that different regions might have different impact.

The rate of non-synonymous substitutions to synonymous substitutions (dN/dS) in the PBR and non-PBR of MHC IIB exon2 of half-smooth tongue sole was studied (Table 3). The dN/dS ratio was higher in the PBR than non-PBR, which corresponds with the results reported in other species [43,54-56]. The dN/dS ratio in exon2 was higher than 1. The location of the PBR sites in the MHC genes of fish was not yet defined, therefore PBR sites were identified using the model of Brown et al. [53] to define HLA-DRB, It was also in accordance with a previous application by Xu et al. [38] for half-smooth tongue sole. The 23 positions were used as PBR sites for in-depth study: 3, 5, 7, 25, 27, 29, 34, 35, 44, 53, 57, 58, 62, 65, 67, 71, 74, 77, 78, 82, 83, 85 and 86 (Figure 3).

It is possible that the PBR sites in fish do not exactly correspond to those in humans [57]. In mammals, MHC polymorphisms are maintained over long periods of time by balanced selection or positive selection at the non-synonymous sites specifying the PBR of the MHC [7]. The ratio between non-synonymous and synonymous substitutions in PBR sites of MHC IIB exon2 genes is greater than 1 (Table 3), as would be expected if the locus were evolving under a condition of balanced selection [58]. The number of alleles per individual ranged from 1 to 5, which showed that at least three loci existed per individual, a result is in accordance with previous studies [22,28,40]. Polymorphism of the 88 alleles in the 160 individuals was higher in half-smooth tongue sole than in Atlantic salmon [57,59] and cyprinid fish [54], and each family had 25-38 alleles. A few hypotheses have been put forward to interpret the abundant polymorphism of the MHC genes, including overdominant selection or heterozygous advantage [60], negative frequency-dependent selection [61,62] and balanced selection [24]. Pathogen-driven selection [26,60] is reported to be contributing to MHC gene diversity through both frequency-dependent selection and heterozygote advantage (over-dominance) [15]. In the present study, the high rate of dN/dS score and high levels of polymorphism which occurred in half-smooth tongue sole revealed that balanced selection is responsible for presence in the PBR domain of the MHC class IIB exon2 gene. This results in the high polymorphism levels in MHC IIB genes in half-smooth tongue sole. Due to the polymorphic nature of MHC genes, certain alleles/haplotypes may be associated with increased disease resistance. In the present study, the distinct distribution pattern of the alleles exhibited a relationship between MHC class IIB alleles and resistance/susceptibility to V. anguillarum in half-smooth tongue sole.

The Cyse-DBB*3301, Cyse-DBB*4701 and Cyse-DBB*6801 alleles which was found in three families, and the Cyse-DBB*5901 allele in two families, were markedly more frequent in HR families (13.75%, 11.25%, 11.25%, 8.75% respectively) than in LR families (1.25%, 1.25%, 1.25%, 1.25%, respectively). This suggests an association of the V. anguillarum disease resistance alleles in half-smooth tongue sole. The Cyse-DBB*6501, Cyse-DBB*4002 and Cyse-DBB*5601 alleles were found in two LR families (35%, 33.75% and 16.25% respectively) and one HR family (1.25%, 1.25% and 1.25%, respectively), while the Cyse-DBB*6102 allele was found in three LR families (27.5%) and one HR family (1.25%), Cyse-DBB*2801 was found in two LR families (15%) and two HR families (2.5%), which might be associated with susceptibility to V. anguillarum in half-smooth tongue sole. In the present study, statistical analysis was used to reveal the associations between the alleles and resistance or susceptibility to V. anguillarum in half-smooth tongue sole. The observed link between alleles Cyse-DBB*3301, Cyse-DBB*4701, Cyse-DBB*6801, Cyse-DBB*5901, Cyse-DBB*6501, Cyse-DBB*4002, Cyse-DBB*6102, Cyse-DBB*5601 and Cyse-DBB*2801 and resistance/susceptibility to V. anguillarum supported the hypothesis that frequency-dependent selection is crucial for the maintenance of MHC variation [63]. This experimental result was in accord with reports in Atlantic salmon [64] and flounder [38]. However, it was not possible to identify a single allele which appeared in all HR families or all LR families. This might indicate the importance of multiple polymorphisms. One MHC haplotype has been reported to be significantly associated with resistance to Marek's disease in chickens [65], and MHC polymorphism was significantly associated with both juvenile survival and resistance to nematode parasites was also reported in Soay sheep [31].

A link between MHC polymorphism and resistance/susceptibility to disease in fish has been reported. Kjøglum et al. [5] demonstrated that fish with the genotypes UBA*0201/UBA*030 and DAA*0201/*0201 were the most resistant to infectious anaemia in Atlantic salmon, while fish with the genotypes UBA*0601/*080, DAA*0501/*0501 and UBA*0201/*030, DAA*0301/*0501 were the most susceptible, based on an analysis of the combined MHC class I and class II A genotypes. It is reported [15] that the allele combinations DAA*0201-*0201 and DAA*0301-*0301 displayed a significantly lower prevalence of death in homozygous fish than in Atlantic salmon containing one copy or no copy of the allele in Aeromonas salmonicida-challenged Atlantic salmon.

The Sasa-DAA-3'UTR 239 allele [36] was shown to be significantly associated with a decrease in the severity of amoebic gill disease in Atlantic salmon. It was also reported [66] that Sasa-B-04, at the non-classical class I locus, was highly associated with resistance to infectious hematopoietic necrosis in Atlantic salmon. The alleles Paol-DAB*4301 and Paol-DAB*1601 were shown to be associated with resistance and susceptibility to V. anguillarum in flounder [38].

In this study in half-smooth tongue sole, the alleles Cyse-DBB*3301, Cyse-DBB*4701, Cyse-DBB*6801 and Cyse-DBB*5901 were found to be associated with resistance while the Cyse-DBB*6501, Cyse-DBB*4002, Cyse-DBB*6102, Cyse-DBB*5601 and Cyse-DBB*2801 alleles were associated with susceptibility to V. anguillarum. Associations of MHC with resistance or susceptibility to specific pathogens can also be derived through linkage disequilibrium with a resistance or susceptibility locus or gene, and may not be due to the MHC gene itself [55,67-69].

Conclusions

It can not ruled out that another linked gene, individual genetic background and different strains or populations may to some extent have caused the observed link, but here the Cyse-DBB*3301, Cyse-DBB*4701, Cyse-DBB*6801 and Cyse-DBB*5901 alleles were associated with resistance to V. anguillarum, while the Cyse-DBB*6501, Cyse-DBB*4002, Cyse-DBB*6102, Cyse-DBB*5601 and Cyse-DBB*2801 alleles were associated with susceptibility to V. anguillarum in half-smooth tongue sole. Further studies are needed to confirm the association between MHC class IIB exon2 gene with resistance to V. anguillarum in half-smooth tongue sole.

Authors' contributions

Professor SLC and MD designed of the study. MD carried out the molecular genetic studies, participated in the sequence alignment and wrote the final drafts of the manuscript. Professor SLC and YHL provided academic advising of this study. YL participated in the manuscript revision. MD and JFY were in charge of fish breeding. All authors read and approved the final manuscript.

Supplementary Material

Additional file 1

Results of the infection with bacterial. Results of the infection with bacterial is presented. Numbers of high-resistance (HR, survivor rate(SR) > 59.45% when infected with the bacterium Vibrio anguillarum) and low-resistance (LR, SR < 26.73%)families of Cynoglossus semilaevis from which dead, surviving individuals were sampled.

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Additional file 2

The individual ID and corresponding number of allele. We presented the number of alleles per individual of half-smooth tongue sole and its corresponding individual number.

Click here for file (27KB, DOC)

Contributor Information

Min Du, Email: du2005min@126.com.

Song-lin Chen, Email: chensl@ysfri.ac.cn.

Yan-hong Liu, Email: kidliu1968@126.com.

Yang Liu, Email: littlexueyang@163.com.

Jing-feng Yang, Email: yjfchina@gmail.com.

Acknowledgements

This work was supported by grants from 973 National Major Basic Research Program of China (2010CB126303) and Taishan scholar project Fund of Shan-dong province, China and Agriculture Science and Technology fund projects of China (2009GB23260436).

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Additional file 1

Results of the infection with bacterial. Results of the infection with bacterial is presented. Numbers of high-resistance (HR, survivor rate(SR) > 59.45% when infected with the bacterium Vibrio anguillarum) and low-resistance (LR, SR < 26.73%)families of Cynoglossus semilaevis from which dead, surviving individuals were sampled.

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Additional file 2

The individual ID and corresponding number of allele. We presented the number of alleles per individual of half-smooth tongue sole and its corresponding individual number.

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