MHC polymorphism and disease resistance to Vibrio anguillarum in 12 selective Japanese flounder (Paralichthys olivaceus) families

Fish & Shellfish Immunology (2008) 25, 213e221

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MHC polymorphism and disease resistance to Vibrio anguillarum in 12 selective Japanese flounder (Paralichthys olivaceus) families Tian-jun Xu a,b, Song-lin Chen a,*, Xiang-shan Ji a, Yong-sheng Tian a a

Key Lab for Sustainable Utilization of Marine Fisheries Resources, Ministry of Agriculture, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Nanjing Road 106, 266071 Qingdao, China b College of Aqua-life Science and technology, Shanghai Ocean University, Shanghai 200090, China Received 22 February 2008; revised 16 May 2008; accepted 20 May 2008 Available online 25 May 2008

KEYWORDS Paralichthys olivaceus; Major histocompatibility complex; Vibrio anguillarum; Disease resistance

Abstract Genetic variation in the major histocompatibility complex (MHC) class IIB was tested in Japanese flounder (Paralichthys olivaceus) for survival after challenge with bacterial infection. The material consisted of 6000 Japanese flounder from 60 families challenged with Vibrio anguillarum, which causes significantly different mortality in flounder families. Five individuals from each of six high-resistance (HR) and six low-resistance (LR) families were screened for their MHC class IIB genotypes using sequence analysis. High polymorphism of MHC IIB gene and at least three loci were discovered in Japanese flounder and the rate of dN occurred at a significantly higher frequency than that of dS in PBR. Among 60 individuals, 76 alleles were discovered and 15 alleles were used to study associations between alleles and resistance to disease. We found highly significant associations between resistance towards infectious disease caused by V. anguillarum and MHC class IIB polymorphism in Japanese flounder. Some alleles appeared in both HR and LR families, while some alleles were only discovered in HR or LR families. One allele, Paol-DAB*4301, was significantly more prevalent in HR families than in LR families (P Z 0.023). Paol-DAB*0601, Paol-DAB*0801, Paol-DAB*2001, Paol-DAB*3803 were discovered in two HR families with high frequency. One allele, Paol-DAB*1601, was discovered in three LR families. The steady heredity of MHC class IIB alleles was observed, and the family having Paol-DAB*4301 alleles was confirmed with higher resistance to V. anguillarum. This study confirmed the association between alleles of MHC class IIB gene and disease resistance, and also detected some alleles which might be correlated with high bacterial infection resistance. The disease resistance-related MHC markers could be used for molecular marker-assisted selective breeding in the flounder. ª 2008 Elsevier Ltd. All rights reserved.

* Corresponding author. Tel.: þ86 532 85844606; fax: þ86 532 85811514. E-mail address: [email protected] (S.-lin Chen). 1050-4648/$ - see front matter ª 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2008.05.007

214

Introduction Japanese flounder (Paralichthys olivaceus) is one of the most important marine aquaculture species and widely cultured throughout the coastal areas of North China due to its good taste and nutrition value. Because of the frequent outbreaks of viral and bacterial diseases in cultured flounder, the production of the fish has not increased in the last decade in spite of extensive fishery management efforts [1]. Vibrio anguillarum is of particular concern due to its ubiquitous presence and is a significant threat to commercial production in marine aquaculture [2]. The use of antibiotics has partially solved the problem of bacterial diseases, but has raised concerns regarding antibiotic residues in fish, environmental pollution, and antibiotic resistance development [3]. Hence, an important approach to disease prevention is to culture strains of fish with enhanced resistance to some major diseases. Selective breeding of flounder with disease resistance has been conducted in China. However, molecular marker-assisted selective breeding is still lacking because it demands analysis of genetic characteristics and QTL markers of selected strains using molecular techniques. MHC is one of the most studied regions of the genome in mammalian, non-mammalian species and fish species known to date, and has been considered a candidate for molecular marker association between polymorphism and resistance/susceptibility to diseases. MHC genes are crucial elements of both innate and adaptive immunity in vertebrate organisms. Allelic variation of MHC genes within vertebrate populations is thought to derive from a combination of balancing and directional selection because pathogen pressure varies in space and time [4]. There are two classes of MHC in vertebrates, MHC class I and class II genes encode cell-surface molecules responsible for binding foreign peptides for the presentation of self and non-self peptides to T-cell receptors (TCR). The MHC class I molecule consisting of one alpha chain and b2-microglobulin, present foreign peptide products by the degradation of intracellular pathogens to cytotoxic CD8þ T cells [5]. The MHC class II molecule, consisting of one alpha chain and one beta chain, present foreign peptides derived from extracellular pathogens and present them to helper CD4þ T cells [6]. In mammals, class I antigens are expressed in all somatic cells, and class II antigens are expressed on antigen presenting cells. In Japanese flounder, MHC IIa and IIb have been characterized [5]. Surprisingly, unlike mammals, class I gene and class II gene were found to reside on different linkage groups in teleosts [7e9]. Many genes of MHC have been isolated and characterized in fish species since Hashimoto et al. reported the first MHC genes in the common carp [10]. The classical MHC genes represent some of the most polymorphic genes, with multiple loci and a considerable number of alleles at each given locus [11]. Generally, class Ia and class II genes are highly polymorphic and the highest level of polymorphism observed in the MHC genes is concentrated within the peptide-binding region (PBR). Specific MHC alleles of class Ia and class II genes have been discovered in some species and have been linked to some diseases [12e16].

T.-jun Xu et al. For instance, one MHC haplotype was found significantly linked with resistance to Marek’s disease in chicken [17]. In chicken, the MHC is also found to be associated with resistance towards other pathogens [18]. In salmonid species, association between major histocompatibility complex polymorphism and disease resistance was investigated by comparing individual alleles of MHC genes; specific alleles of MHC have been documented to correlate with some viral and bacterial diseases [19e23]. The association between disease susceptibility and resistance and MHC polymorphism has been established in some species. It is very important to detect resistant alleles of MHC in important marine aquaculture species for molecular marker-assisted selective breeding programs. Japanese flounder was chosen as our trial species due to established disease challenge trials, ease of generating numerous offspring and the availability of MHC class IIB sequence date [3]. Zhang et al. has identified 13 alleles and has made correlations with one bacterial disease by analyzing partial exon2 of MHC IIB gene from a Chinese population of flounder [3]. Family selective breeding provides an approach to increase resistance of fishes to diseases. Individuals from families are important trial materials for detecting molecular markers correlating with enhanced disease resistance due to its clear heredity information. Thus, it is more effective and objective to assess a link between MHC IIB gene polymorphism and the disease resistance to V. anguillarum using many full-sib families. The aim of the present study was to detect specific alleles which correlate with high resistance to V. anguillarum across 12 selected families of Japanese flounder, and to evaluate the heredity of MHC alleles.

Materials and methods Fish and rearing Japanese flounder for creating the base population of the test fish were collected from Japanese stock (JS), yellow sea wild stock (YS) and resistance stock (RS) to V. anguillarum which was selected according to the resistance against V. anguillarum as described by Zhang et al. [3]. Sixty fullsib families of Japanese flounder were established as described [24]. Fertilized eggs were incubated, hatched and reared at Aqua breeding station in Haiyang and were kept in separate tanks. The fry were fed a commercial diet according to a standard feeding scheme.

Challenge experiment Although there are some weaknesses in the intraperitoneal (ip) infection challenge model, it is a very effective and feasible experimental infection model in fish [11]. For the V. anguillarum challenge experiment in flounder, all the test fishes were inoculated by ip injection. In order to determine the median lethal concentration, different concentrations of V. anguillarum were tested in a pre-challenge experiment on fish of the same size as the test fish prior to the challenge trial. Each family was reared in a separate tank with a fresh water supply at 20  C. A total

MHC polymorphism and disease resistance to Vibrio anguillarum number of 6000 offspring from 60 full-sib families were inoculated ip with 9.8  105 colony forming units (CFU) of V. anguillarum, approximately 100 individuals from each family. The test lasted for approximately 6 days, mortality was recorded every 12 h and fins of all dead fish were stored in 100% ethanol.

Sampling and DNA isolation To examine whether MHC alleles are associated with resistance/susceptibility to V. anguillarum, samples of resistance and susceptible populations of each family were collected from the first 20 to die and the last survivors of the bacterial challenge. Meanwhile, the fin of parents was sampled and reserved in ethanol till use. We selected high-resistance families (HR, survivor rate (SR) > 50%) and low-resistance families (susceptible families, LR, SR < 20%) from the challenge tests. Both individuals that had died and those that had survived the infection were sampled from HR and LR families (Table 1). Genomic DNA was extracted from fin samples of 5 individuals per each HR and LR families with the method of phenolechloroform. The procedure is as follows: about 25 mg tissue was homogenized in 400 ml lysis buffer (10 mM TriseHCl, pH 8.0; 100 mM EDTA, pH 8.0; 100 mM NaCl; 0.5% SDS), and extracted with phenolechloroformeisoamyl alcohol (25:24:1). After centrifugation at 12,000 rpm for 10 min, RNase A was added to the supernatant (final concentration 100 mg/ml) for a 3 h incubation at 55  C. The mixture was extracted twice with phenolechloroformeisoamyl alcohol (25:24:1), and then precipitated with two volumes of 100% ethanol. The DNA was washed twice in 70% ethanol, dried and dissolved in 80e200 ml of TE buffer (10 mM Trise HCl, pH 8.0; 10 mM EDTA, pH 8.0). The quality and concentration of DNA were assessed by agarose gel electrophoresis and measured with a GeneQuant Pro (Pharmacia Biotech Ltd.) RNA/DNA spectrophotometer. Finally, DNA was adjusted to 100 ng/ml and was stored at 4  C for future use.

Primer design and PCR Specific primers: fMPN (50 -CTCCCTCTTCTTCATCACGGT-30 ) and fMPC (50 -ACACACTCACCTGACTTCGT-30 ) from flounder MHC IIB gene were designed based on the published cDNA sequence [3]. The MHC IIB forward primer is located in exon1, and the reverse primer of MHC IIB is located in intron2. The primers were used to amplify partial exon1 Table 1 Numbers of high-resistance (HR, survivor rate (SR) > 50% when infected with the bacterium Vibrio anguillarum) and low-resistance (LR, SR < 20%) families of Japanese flounder from which dead, surviving individuals were sampled Family

Individuals per family Dead

HR LR Total

6 6

5

12

30

Total

Surviving 5

30 30

30

60

215

and intron2, complete intron1 and complete exon2 from flounder using PCR method. DNA was used as template in a 25 ml PCR reaction mixture containing 2.5 ml of 10  Taq polymerase buffer, 1.5 mM MgCl2, 0.2 mM dNTPs, 0.2 mM of the forward and reverse primers, and 1 unit of Taq polymerase (Promege). Cycling conditions were 94  C for 4 min followed by 30 cycles of 94  C for 40 s, 53  C for 40 s and 72  C for 50 s, followed by 1 cycle of 72  C for 10 min. PCR was performed on a PTC-200. The PCR product was electrophoresed on a 1% agarose gel to check for integrity and visualized by Molecular Imager Gel Doc XR system (Biorad).

Cloning and sequencing PCR product was purified on 1% agarose gel and extracted using QIAEX II Gel Extraction Kit (Qiagen). The purified fragments were ligated into PMD-18T vector (Takara) and cloned into TOP10 cells according to the standard protocol. Positive clones were screened via PCR with M13þ/primers. An average of 5 clones per individual was sequenced using the ABI 3730 automated sequencer with M13 primer.

Genotyping, sequence analysis and statistical tests Nucleotide sequences and the amino acid sequences were aligned using MEGA3.1 software. MEGA software was also used to calculate the rate of synonymous substitution (dS) and nonsynonymous substitution (dN) according to Nei and Gojobori [25]. Polymorphic value was analyzed using DnaSP4.0 analysis software [26] and DAMBE software. Statistic analysis was carried out with SPSS 13.0, differences in the frequencies of each allele were tested using Fisher’s exact test with and without sequential Bonferroni corrected significance levels [27] using each individual (n Z 60) and each family (n Z 12). The new alleles were identified and nominated on the basis of the rules reported by Davies et al. [28]. Names are based on the deduced amino acid sequences and consist of four digits, of which the first two digits indicate the major type; the next two digits indicate the subtype. Alleles that differ by less than five amino acid substitutions are considered as subtype within a single major type [29,30].

Results Disease resistance comparison of 12 Japanese flounder families After 20 h, the first specific mortality due to V. anguillarum was recorded. At the end of the challenge experiment a total cumulative survivability of 32.68% was reached 120 h post-challenge (Fig. 1). The within-family prevalence of survival ranged from 7.27% to 65.76%. Survival rate was collected on the basis of a family. Among the 60 families, we selected six high-resistance families and six low-resistance families to study whether MHC alleles are associated with resistance to V. anguillarum. The survival rates of six high-resistance families were higher than other families and the mean survival

Fraction survial

216

T.-jun Xu et al. 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0

24

36

48

60

72

84

96

120

Time (hours)

Figure 1 The fraction of Japanese flounder survivors in the Vibrio anguillarum challenge experiment from hours of inclusion until study termination.

rate was 56.95%; the survival rates of six low-resistance families were lower than other families and the mean survival rate was only 13.14%.

Sequence polymorphism analysis within exon2 of MHC IIB gene Sixty individuals from six high-resistance families and six low-resistance families were analyzed. Averages of five positive clones per individual were sequenced and 306 sequences were obtained. The size of the expected fragment was 408 bp. Based on sequence alignment with Japanese flounder MHC IIB cDNA complete sequence [3], 408 bp fragment spanned the complete exon2 of MHC IIB, partial exon1 (29 bp), complete intron1 (84/96 bp, comprising a 12 bp repeat loci). A 273 bp fragment, the complete exon2 of MHC IIB gene, was subsequently analyzed and revealed 76 different sequences, which presented 76 novel alleles (GenBank Accession No: EU380319eEU380394) belonging to 45 allele major types using allele nomenclature method. The full alignment of the 273 bp exon2 of MHC IIB gene did not show any gaps. The nucleotide sequence corresponded with a putative 91 amino acid peptide according to Zhang et al. [3]. There were 58 variable sites in exon2, of which 54 parsimony informative sites were observed. Analysis of all the 76 alleles showed 21.2% of the nucleotides were variable and 35.2% (32 out of 91) were variable. Table 2 shows that the nucleotide substitution happened at 58 sites involved in 44 mutation regions. The SNP variation sites include transition and transversion and are located tightly between 210 and 251 bp, the mutation hotspots of MHC exon2 are located from 14 to 33 bp and from 199 to 251 bp. The putative amino acid residues involved in antigen binding in Japanese flounder were based on the corresponding antigen-binding sites identified in humans [31]. Nineteen out of 23 positions of the PBR were variable and 15 (21.74%) nucleotides of 69 PBR sites were polymorphic. The rate of nonsynonymous substitution (dN) was 19.447 times higher than the rate of synonymous substitution (dS) in the peptide-binding regions. The rates of dN and dS of non-PBR were 0.0475 and 0.0314, respectively. These rates were calculated on the whole sequences. The rate of dN occurred at a significantly higher frequency than that of

dS in PBR, dN in PBR was higher than that in non-PBR, but dS in PBR was significantly lower than in non-PBR (Table 3). The average number of nucleotide diversity Pi (p) and Theta-W value of the whole sequences were 0.0732 and 0.0343, respectively. The haplotype diversity (H ) was 0.970; the average number of nucleotide differences (k) was 19.879. These polymorphic values were calculated using the computer application DnaSP4.0. Among the 12 families of flounder, the exon2 of MHC IIB revealed high nucleotide diversity. The spatial distribution of nucleotide diversity is shown in Fig. 2. There were two peaks in the upstream and downstream of the exon2 of MHC IIB gene, respectively, and there was lower Pi (p) value in the middle of the exon2 of MHC IIB gene.

Association between alleles and resistance to V. anguillarum The number of alleles per individual and its corresponding individual number are described in Table 4. Numbers of alleles per individual diversified from 1 to 5. Considering average five clones per individual were sequenced, we believed that there were at least three loci of MHC IIB gene per individual. Out of all the families, 93.3% examined individuals were heterozygous (all families were heterozygous) for exon2 of the MHC class IIB gene. Seventy-six different MHC II exon2 alleles were identified from the 60 individuals. The frequency of alleles was not distributed equally. Some alleles were low frequency or only existed in one family. Alleles with the low frequency have no value as molecular markers and these alleles were excluded from distribution analysis when discussing association between alleles and resistance to disease. Fifteen alleles were picked out for subsequent analysis. MHC class IIB allele frequencies differed significantly between high-resistance and low-resistance families (Fig. 3). For example, the Paol-DAB*4301 allele was significantly more frequency in individuals from high-resistance families than in individuals from low-resistance families (P Z 0.023, n Z 60 individuals), but the frequencies of some alleles did not differ significantly between HR and LR families, such as the Paol-DAB*0101 (P Z 1.000) allele and the Paol-DAB*0301 allele (P Z 0.448). Paol-DAB*4301 occurred more frequency in three high-resistance families than in only one low-resistance family. Some alleles only appeared in HR or LR families with high frequency, for example, the Paol-DAB*0601, Paol-DAB*2001, PaolDAB*3803, Paol-DAB*3805 and Paol-DAB*4001 were only discovered in HR families with comparatively high frequency, also some alleles such as Paol-DAB*1601, Paol-DAB*2201, Paol-DAB*2701 only appeared in LR families. Here, we believe that Paol-DAB*4301 was highly associated with resistance to V. anguillarum in Japanese flounder. The PaolDAB*0601, Paol-DAB*0801, Paol-DAB*2001, Paol-DAB*3803, which were discovered from two unrelated HR families, might associate with resistance to V. anguillarum. Alignment showed the conservation and difference in amino acid sequences of 15 alleles (Fig. 4). The amino acid substitution R5L, Y28H, Y51V and W59L might be responsible for the susceptibility or resistance, which revealed amino acid substitution between HR alleles and LR alleles.

MHC polymorphism and disease resistance to Vibrio anguillarum

Table 2 Serial number

Distribution of SNP sites within exon2 of MHC IIB allelic sequences Position

1

14

2

16

3

17e18

4

22

5

23

6

28

7

29e31

8

32

9

33

10

55

11

66

12

71

13

217

74e76

14

77

15

78

16

82

17

100

18

102e104

19

106

20

134

21

142

22

151

23

152

24

173e174

Base type

Allele no. (n Z 76)

Frequency

Serial number

Position

Base type

Allele no. (n Z 76)

Frequency

T G T C TC AT G A C T G A CAG ACA GCA G A G C T C A C T A CAG CAC TCT G T C T G C T A T TA TT AT G A T A A T C T G A T GG GT AG

29 47 31 45 2 74 36 40 15 61 33 43 35 24 7 27 49 7 69 1 75 8 68 16 60 22 36 18 61 15 3 18 55 15 61 5 71 49 7 20 28 48 37 39 1 75 26 25 25 37 39 50 14 12

0.382 0.618 0.408 0.592 0.026 0.974 0.474 0.526 0.197 0.803 0.434 0.566 0.461 0.316 0.092 0.355 0.645 0.092 0.908 0.013 0.987 0.105 0.895 0.211 0.789 0.289 0.474 0.237 0.803 0.197 0.039 0.237 0.724 0.197 0.803 0.066 0.934 0.645 0.092 0.263 0.368 0.632 0.487 0.513 0.013 0.987 0.342 0.329 0.329 0.487 0.513 0.658 0.184 0.158

25

176e177

26

188

27

199

28

204

29

209

30

210e211

31

212

32

213

33

222

34

223e224

35

231e232

36

233

37

244

38

245

39

247e248

40

249

41

250e251

42

253

43

256

44

260

TT TG GG A C G A T A T C G C TT CC TG G C T A T G C G GT TA GA TT TC A C G G A C A T GA TT CT A C GT AT AC A G G T C A

1 15 60 17 59 28 22 26 64 11 1 32 44 5 26 45 1 3 5 67 5 71 24 52 3 73 18 5 53 49 6 21 35 41 4 12 60 32 43 1 4 72 31 1 44 1 75 25 51 72 4

0.013 0.197 0.789 0.224 0.776 0.368 0.289 0.342 0.842 0.145 0.013 0.421 0.579 0.066 0.342 0.592 0.013 0.039 0.066 0.882 0.066 0.934 0.316 0.684 0.039 0.961 0.237 0.066 0.697 0.645 0.079 0.276 0.461 0.539 0.053 0.158 0.789 0.553 0.566 0.013 0.053 0.947 0.408 0.013 0.579 0.013 0.987 0.329 0.671 0.947 0.053

218

T.-jun Xu et al.

Table 3 Synonymous (dS) and nonsynonymous (dN) substitution ratio in the putative peptides binding region (PBR) and non-peptides binding region (non-PBR) among flounder alleles Region

No. of dN (SE) codons

PBR 23 Non-PBR 68 Total 91

dS (SE)

Table 4 The number of allele per individual and its corresponding individual number Allele no. Individual no.

1 4

2 25

3 21

4 8

5 2

dN/dS

0.0739  0.0312 0.0038  0.0034 19.447 0.0475  0.0161 0.0314  0.0162 1.513 0.0900  0.0183 0.0284  0.0118 3.169

The heredity of alleles associating with disease resistance Sequences analysis of their sires and dams in 12 selective families showed, as expected, alleles (Paol-DAB*0601, PaolDAB*0801, Paol-DAB*2001, Paol-DAB*3803, Paol-DAB*4001 and DAB*4301) associating with disease resistance in their sires or dams. This revealed that the MHC IIB alleles were transmitted to the progeny. For example, Paol-DAB*4301 and Paol-DAB*0801 were discovered in the offspring of family 68. Paol-DAB*0801 was found in their dam (107#) and Paol-DAB*4301 was found in their sire (218#) (Fig. 5). To confirm the hereditary stability of the Paol-DAB*4301, we sequenced exon2 of MHC class IIB gene of another three families (21, 36, 70), in which their parent had the PaolDAB*4301 allele. This allele has also been found in their family’s offspring (Fig. 5). Noticeably, the mean survival rate of the six families having Paol-DAB*4301 allele was 49.46%, which was higher than the mean survival rate of the other 60 families. This result demonstrated that the Paol-DAB*4301 allele was associated with disease resistance.

Discussion Because MHC genes are significant elements of both innate and adaptive immunity in vertebrates, they have been attracting attention of many scientists. In the present

Figure 2 The nucleotide diversity within exon2 sequences of MHC IIB genes at the 76 alleles denoted by Pi (p). Sliding window length: 100; step size: 10.

study, MHC class IIB gene polymorphism in different families and associations between alleles and resistance or susceptibility to V. anguillarum were presented. To our knowledge, this is the first study on MHC IIB variation and disease resistance on the basis of different families in Japanese founder. Among 44 mutation regions, 11 regions were multinucleotide co-mutation, and this indicated that interalleles recombination occurred at these regions. Frequencies of substituted nucleotides per mutation region were not equally distributed. The different frequency ratio of SNP sites indicated that different regions might have evolved from the different stage of mutation life. Moreover, no stop codon has been discovered, which revealed that all these alleles were functional genes. The ratio of nonsynonymous substitutions to synonymous substitutions in PBR and non-PBR in exon2 of MHC IIB in flounder was analyzed. It was significantly different as expected, dN/dS was significantly higher in PBR than nonPBR, which corresponds with results of some other species [32]. The location of the PBR sites in fish MHC genes is uncertain, the PBR in fish was identified based on the similarity to human HLA II molecule, but it is possible that the PBR sites in fish do not exactly correspond to human [33]. In mammals, MHC polymorphism is maintained over long periods of time by balancing selection at the nonsynonymous sites specifying the PBR of the MHC molecule [8]. The rate of nonsynonymous substitutions significantly exceeds that of synonymous substitutions in MHC genes, the dN/dS ratio is greater than 1, as would be expected if the locus were evolving under balancing selection [34]. Sequence analysis revealed the high polymorphism of MHC IIB gene in flounder. Numbers of alleles per individual diversified from 1 to 5, there were at least three loci of MHC IIB gene per individual, this result was similar to that described by Chen et al. [35] and Zhang and Chen [36]. The numbers of alleles in the 60 individuals were 76 and polymorphism of MHC IIB was higher in flounder than in cyprinid fish [33] and Atlantic salmon [37]. Some hypotheses were reported to explain the vast polymorphism of the MHC genes, such as heterozygous advantage, over-dominant selection, and frequency-dependent or balancing selection [38]. The pathogen-driven selection favors genetic diversity of the MHC genes through both heterozygote advantage (over-dominance) and frequency-dependent selection [11]. In this study, the high ratio of dN/dS value and high allelic diversity observed in flounder suggested a balancing selection in the exon2 of the MHC class IIB gene, which explained the high polymorphism of MHC IIB gene in Japanese flounder. In general, high polymorphism of MHC genes in animals is believed to confer high disease resistance. In this study, the different distribution of alleles indicated the association between MHC class IIB alleles and resistance to V. anguillarum in Japanese flounder. The Paol-DAB*4301 allele which

MHC polymorphism and disease resistance to Vibrio anguillarum

219

0.35

Precentage

0.3 0.25 0.2

HR

*

LR *

*

*

0.15

*

*

* *

*

0.1 0.05

Pa ol -D Pa AB* ol -D 01 0 1 Pa AB* ol -D 03 0 1 Pa AB* ol -D 06 0 1 Pa AB* ol -D 08 0 1 Pa AB* ol -D 16 0 1 Pa AB* ol -D 17 0 1 Pa AB* ol -D 20 0 1 Pa AB* ol -D 20 0 2 Pa AB* ol -D 22 0 1 Pa AB* ol -D 27 0 1 Pa AB* ol -D 32 0 1 Pa AB* ol -D 38 0 3 Pa AB* ol -D 38 0 5 Pa AB* ol -D 40 0 1 AB *4 30 1

0

Allele

Figure 3 Distribution of MHC class IIB alleles in high-resistance families individuals (open bars) and low-resistance families individuals (filled bars) of Japanese flounder. Asterisks indicate P < 0.05.

existed in three HR families and was significantly more frequency in individuals from HR families than in individuals from LR families might demonstrate the association of the allele with resistance to V. anguillarum in Japanese flounder. The Paol-DAB*1601 was discovered from three LR families, and the frequency was 20% in individuals, this allele might associate with susceptibility to V. anguillarum in Japanese flounder. In this study, the associations between alleles and resistance or susceptibility to V. anguillarum in flounder were revealed using statistical analysis. However, it is difficult to identify that a single allele was discovered from all HR families or all LR families. It might be at least partially explained by the vast polymorphism. Association between MHC polymorphism and resistance or susceptibility to some diseases in Atlantic salmon has been successfully documented, Kjøglum et al. [6] reported that UBA*0201 and UBA*0301 were significantly the most resistant alleles against infectious salmon anaemia in Atlantic salmon, while UBA*0601 and DAA*0301 were significantly the most susceptible alleles; Wynne et al. [22] showed that a significant association was found between AGD severity and the presence of two Sasa-DAA-3UTR genotypes in Atlantic salmon; Glover et al. [23] revealed that the SasaDAA-3UTR 248/278 genotype displayed a significantly higher abundance of lice compared with the Sasa-DAA-

3UTR 208/258 genotype within one family of Atlantic salmon; these results indicated a link between MHC class II and susceptibility to lice. Miller et al. [39] found one allele (Sasa-B-04) from a potentially non-classical class I locus was highly associated with resistance to infectious hematopoietic necrosis. In the present study, high polymorphism of MHC IIB gene and at least three loci were discovered in Japanese flounder. Associations between certain MHC class IIB alleles and the resistance to bacterial infection have been found. The alleles Paol-DAB*4301 and Paol-DAB*1601 were associated with resistance and susceptibility to V. anguillarum in flounder, and the steady heredity of the alleles were observed. Association between a gene and a disease can be due to effects of the gene itself or can arise if the studied gene is in linkage disequilibrium with another gene that causes the resistance [40,41]. We cannot exclude that another linked gene has possibly caused the observed association, but we found that the family having Paol-DAB*4301 allele had higher resistance to V. anguillarum. Thus, we believe our research might be potentially applied as disease resistance-related MHC markers for molecular marker-assisted selective breeding in the Japanese flounder with enhanced resistance to disease caused by bacterial infection. In our future research, we will use this marker to select

Figure 4 Putative amino acid sequences for MHC IIB exon2 alleles of Japanese flounder. Dots indicate identity with the top sequences; Asterisks indicate the codons involved in antigen-binding region.

220

T.-jun Xu et al. 107# DAB*0801

218# DAB*4301

Family 68 DAB*0801/DAB*4301 SR=62.08

128#

165#

[7]

Family 70 DAB*4301 SR=49.90

[8]

[9]

Family 21(SR=43.85 ) DAB*4301

[10] 218# DAB*4301

205#

Family 36(SR=37.38 ) DAB*4301

[11] 131#

Family 51(SR=54.13 ) DAB*4301

182# Family 66(SR=49.39 ) DAB*4301

Figure 5 The heredity of allele DAB*4301, showing the allele was transmitted to the progeny.

individuals for mating and it will provide help for selective breeding of Japanese flounder with enhanced disease resistance.

[12]

[13]

[14]

[15]

Acknowledgements This work was supported by the National Major Basic Research Program of China (2004CB117403), Nation Nature Science Foundation of China (30671623), State 863 Hightechnology R&D project of China (2006AA10A402, 2006AA10A404), and Taishan scholar project of Shan-dong province, China.

[16]

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