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Immunogenetics ISSN 0093-7711Volume 65Number 6 Immunogenetics (2013) 65:447-459DOI 10.1007/s00251-013-0697-6

Diversity and evolution of the highlypolymorphic tandem repeat LEI0258 in thechicken MHC-B region

Olympe Chazara, Chi-Sheng Chang,Nicolas Bruneau, Khalid Benabdeljelil,Jean-Claude Fotsa, Boniface B. Kayang,N’Goran E. Loukou, et al.

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ORIGINAL PAPER

Diversity and evolution of the highly polymorphic tandemrepeat LEI0258 in the chicken MHC-B region

Olympe Chazara & Chi-Sheng Chang & Nicolas Bruneau &

Khalid Benabdeljelil & Jean-Claude Fotsa & Boniface B. Kayang &

N’Goran E. Loukou & Richard Osei-Amponsah & Valentine Yapi-Gnaore &

Issaka A. K. Youssao & Chih-Feng Chen & Marie-Hélène Pinard-van der Laan &

Michèle Tixier-Boichard & Bertrand Bed’Hom

Received: 22 January 2013 /Accepted: 10 March 2013 /Published online: 26 March 2013# Springer-Verlag Berlin Heidelberg 2013

Abstract The chicken major histocompatibility complex(MHC) is located on the microchromosome 16 and is de-scribed as the most variable region in the genome. Thegenes of the MHC play a central role in the immune system.Particularly, genes encoding proteins involved in the antigenpresentation to T cells. Therefore, describing the geneticpolymorphism of this region is crucial in understandinghost–pathogen interactions. The tandem repeat LEI0258 islocated within the core area of the B region of the chickenMHC (MHC-B region) and its genotypes correlate withserology. This marker was used to provide a picture of theworldwide diversity of the chicken MHC-B region and tocategorize chicken MHC haplotypes. More than 1,600 ani-mals from 80 different populations or lines of chickens fromAfrica, Asia, and Europe, including wild fowl species, weregenotyped at the LEI0258 locus. Fifty novel alleles were

described after sequencing. The resulting 79 alleles wereclassified into 12 clusters, based on the SNPs and indels foundwithin the sequences flanking the repeats. Furthermore, hy-potheses were formulated on the evolutionary dynamics of theregion. This study constitutes the largest variability report forthe chicken MHC and establishes a framework for futurediversity or association studies.

Keywords Major histocompatibility complex .MHC .

LEI0258 . Chicken .Gallus gallus . Evolution

Introduction

Chicken genetic resources comprise a wide range of breedsand populations including wild fowl species, fancy breeds,

O. Chazara :N. Bruneau :M.-H. Pinard-van der Laan :M. Tixier-Boichard : B. Bed’HomGénétique Animale et Biologie Intégrative, INRA, AgroParisTech,UMR 1313, Jouy-en-Josas, France

O. Chazara (*)Department of Pathology, University of Cambridge and Centrefor Trophoblast Research, Tennis Court Road,CB2 1QP Cambridge, UKe-mail: olympechazara@gmail.com

C.-S. ChangInstitute of Statistical Science, Academia Sinica, Taipei, Taiwan

K. BenabdeljelilInstitut Agronomique et Vétérinaire Hassan II, DPBA,Rabat Institute, Rabat, Morocco

J.-C. FotsaInstitut de Recherche Agricole pour le Développement,Bamenda, Cameroon

B. B. Kayang :R. Osei-AmponsahDepartment of Animal Science, University of Ghana,Legon, Ghana

N. E. Loukou :V. Yapi-GnaoreCentre Nationale de Recherche Agronomique,Université de Cocody, Abidjan, Ivory Coast

I. A. K. YoussaoEcole Polytechnique d’Abomey-Calavi,Université d’Abomey-Calavi, Cotonou, Benin

C.-F. ChenDepartment of Animal Science, National Chung-Hsing University,Taichung, Taiwan

Immunogenetics (2013) 65:447–459DOI 10.1007/s00251-013-0697-6

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local breeds, commercial stocks, and experimental lines.Recent studies on chicken diversity have used neutralmarkers such as microsatellites (Rosenberg et al. 2001;Berthouly et al. 2008) or single nucleotide polymorphisms(SNPs) (Twito et al. 2007; Gärke et al. 2012) and mitochon-drial sequences (Liu et al. 2006). Only a few populationgenetic studies, however, have been conducted on genes orfunctionally important regions (Downing et al. 2009). Themajor histocompatibility complex (MHC) is a genomic re-gion comprising genes coding for proteins involved in pep-tide presentation to T cells. The chicken MHC is divided intwo genetically independent regions, B and Y, both locatedon the microchromosome 16 of the chicken (Fillon et al.1996; Miller et al. 1996; Delany et al. 2009; Solinhac et al.2010). The B region has been associated with resistance,susceptibility, and differential response to vaccine (Bacon etal. 1981; Pinard-van der Laan et al. 1998; Lee et al. 2004).Historically, the B region has been defined using serology.Numerous serotypes have been defined mainly in closedpopulations of White Leghorn chickens, and approximately20 are well known (Rogers and Kaufman 2008).Consequently, serological reagents are rarely available forother types of chicken, and comparative studies are difficult.For these animals, only indications of the MHC genotypescan be provided (O'Neill et al. 2009). Therefore, compari-sons between studies are difficult, and would be facilitatedby the generalization of the use of the reference marker forthe chicken MHC, identified in 1999 by McConnell et al.and described by Fulton et al. in 2006: the tandem repeatLEI0258. Allele sizes can be defined by gel electrophoresisand sequencing for this marker, and they correlate well withserology in the commercial or experimental lines tested(Fulton et al. 2006).

The LEI0258 marker is an atypical variable number oftandem repeat (VNTR). It is highly polymorphic, with 26allele sizes reported previously (Fulton et al. 2006). It ischaracterized by the repetition of two tandem and conservedshort sequences of 12 and 13 bp plus several sequencepolymorphisms in the flanking regions (small insertionsand deletions (indels) and SNPs). It is the combination ofthe two repeats and the indels that determines the allele size.This VNTR is found in the central region of the B region,close to the gene BG1. This gene has been shown to beassociated with resistance to Marek's disease in the chicken(Goto et al. 2009). Preliminary evidence of a correlationbetween LEI0258 alleles and MHC-B SNPs or sequenceshas been observed in White Leghorn lines (Chazara et al.2008), White Leghorn, Rhode Island Red or Fayoumi ani-mals (Chazara et al. 2011), and Brazilian chickens (Lima-Rosa et al. 2005). Recently, the marker LEI0258 has beensuccessfully used in several diversity studies (Hoque et al.2011; Izadi et al. 2011; Chang et al. 2012) and inmany diseaseassociation studies (Owen et al. 2008; Petersen et al. 2008;

Schou et al. 2010; Bader et al. 2010; Banat et al. 2012; Suzukiet al. 2012).

This research reported herein has two aims. Firstly, toidentify the variability of alleles for the tandem repeatLEI0258 by examining an extensive and diverse collectionof 80 populations or lines of chicken from Africa, Asia, andEurope. Secondly, to compare the features of the allelescharacterized in order to infer the relationship betweensequences and therefore understand the dynamic evolutionof the marker.

Materials and methods

Genomic DNA origin

Animals were sampled from 80 different populations orlines of chickens (Gallus gallus). This included 51populations of various origins, and selection histories sam-pled in the frame of the European AvianDiv project(populations are numbered accordingly to the AvianDivdatabase, P04 to P34, P37 to P53, P101 and P102; http://aviandiv.tzv.fal.de, Hillel et al. 2003). Samples from otherpopulations include: 12 traditional French breeds (Berthoulyet al. 2008), nine populations of Asian chickens fromNational Chung-Hsing University (Taichung, Taiwan), fourimmune response lines and three lines fixed for different Bserotypes from INRA (Nouzilly, France; Minozzi et al.2008; Miller et al. 2004), a set of village chickens from fiveWest-African countries and from Morocco, and three wildGallus species kept in a zoological park (Clères, France).Many of these populations had previously been the subjectof diversity studies using neutral microsatellite markers(Table 1). DNA was made available from the AvianDivDNA bank kept at INRA, Jouy-en-Josas. Additionalextractions were performed for the African chickens atthe Department of Animal Science, University ofGhana, Legon, using the DNeasy Blood & Tissue Kit(QIAGEN), as well as for Asian and French breeds atINRA using the quick extraction procedure described inChang et al. (2006).

MHC serotypes were available for only the seven INRAexperimental lines reported as B12, B124 (Pinard 2002),B13, B15, B19, B21, B34, and for the AvianDiv B4 orB12 C lines (P32) and B15 sarcoma-resistant or susceptiblelines (P34).

Genotyping: PCR and electrophoresis

Typically, 10 to 50 animals were randomly chosen from thedifferent populations, except for the zoo animals which wereall included (Table 1). Amplifications were performed with theprimers LEI0258-F: CACGCAGCAGAACTTGGTAAGG

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Table 1 Description and diversity measurements for the different chicken populations

Code Name N Type Geo LEI0258 Other microsatellite markers

Nall HnbLEI HobsLEI Hobs ref 1 Hobs ref 2 Hobs ref 3 Hobs ref 4 Hobs ref 5

P101 G. gallus gallus 25 1 Asia 8 0.86 0.92 0.60 0.63 0.66

P102 G. gallus spadiceus 24 1 Asia 14 0.84 0.79 0.64 0.58

RJF G. gallus 6 1 Asia 4 0.76 1.00

GJF G. sonneratii 5 1 Asia 5 0.82 0.80

SLJF G. lafayetii 4 1 Asia 2 0.54 0.75

BEN Benin 26 2 Africa 18 0.94 0.96 0.55

CAM Cameroon 23 2 Africa 15 0.92 0.91 0.63

CI Ivory Coast 24 2 Africa 9 0.86 0.71 0.58

GHA Ghana 25 2 Africa 14 0.90 0.96 0.57

MOR Morocco 21 2 Africa 10 0.86 0.90 0.58

P05 Bedouin 23 2 Africa 13 0.89 0.96 0.58 0.59

P16 Icelandic Landrace 18 2 Europe 9 0.83 0.67 0.48 0.51

P17 Finnish Landrace 24 2 Europe 6 0.82 0.79 0.59

BAZ Barbezieux 18 3 Europe 9 0.86 0.94 0.53

BNA Bourbonnaise 18 3 Europe 6 0.65 0.67 0.49

COU Coucou de Rennes 24 3 Europe 10 0.87 0.67 0.58

CRC Crèvecoeur 24 3 Europe 6 0.61 0.63 0.53

GAS Gasconne 24 3 Europe 9 0.83 0.88 0.54

GLD Gauloise dorée 11 3 Europe 4 0.40 0.18 0.41

GOU Gournay 21 3 Europe 6 0.73 0.81 0.54

NCH Noire de Challans 18 3 Europe 7 0.82 0.67 0.45

P06 Westfaeliche Totleger 10 3 Europe 6 0.80 0.60 0.44 0.32

P07 Sundheimer 14 3 Europe 3 0.46 0.50 0.49 0.48

P08 Light Brown Leghorn 12 3 Europe 4 0.71 0.83 0.41

P09 Owl-bearded 24 3 Europe 8 0.84 0.54 0.54

P10 Friesian fowl 12 3 Europe 6 0.79 0.58 0.39

P12 Houdan 11 3 Europe 3 0.65 0.55 0.22 0.43

P13 Marans 11 3 Europe 5 0.75 0.82 0.42 0.50 0.49

P14 Dorking 17 3 Europe 5 0.49 0.53 0.50

P15 Cochin 20 3 Europe 5 0.63 0.65 0.54

P19 Jaerhoens 12 3 Europe 2 0.65 0.75 0.34 0.45

P20 Sicilienne Buttercup 18 3 Europe 4 0.65 0.67 0.50

P21 Padovana 12 3 Europe 3 0.54 0.58 0.17 0.36

P22 Black Castellana 12 3 Europe 5 0.70 0.75 0.44

P23 Red Villafranquina 18 3 Europe 4 0.57 0.72 0.48

P24 Czech Golden Pencilled 19 3 Europe 7 0.67 0.63 0.49

P25 Oravka hen 24 3 Europe 6 0.79 0.88 0.52

P26 Transsylvania N. Neck 24 3 Europe 5 0.60 0.67 0.53 0.44

P27 Green-legged Partridge 24 3 Europe 5 0.71 0.67 0.53 0.44

P28 Orlov 24 3 Europe 3 0.51 0.63 0.60 0.59

P29-1 Yurlov crower (Russia) 13 3 Europe 4 0.62 0.62 0.62

P29-2 Yurlov crower (Ukraine) 12 3 Europe 6 0.78 0.92 0.58

P30 Ukrainian bearded 23 3 Europe 9 0.83 1.00 0.57

P31 Poltava clay 18 3 Europe 4 0.75 0.83 0.51

SLK Silkie 25 3 Asia 4 0.72 0.48

T Hsin-Yi 47 3 Asia 4 0.61 0.51 0.53

J Ju-Chi 48 3 Asia 4 0.38 0.40 0.46

F Hua-Tung 47 3 Asia 7 0.84 0.85 0.58

KM Quemoy 47 3 Asia 4 0.61 0.64 0.49

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forward and LEI0258-R: AGCTGTGCTCAGTCCTCAGTGC reverse (McConnell et al. 1999) in 10 μL with20 ng of DNA, 1× of HotStarTaq Master Mix Kit(QIAGEN), and primer concentrations of 0.1 μM. ThePCR cycle setup was 94 °C for 15 min, then 35 cyclesof 94 °C for 45 s, 60 °C for 1 min 30 s, and 72 °C for

1 min with a final extension of 72 °C for 15 min. Thetotal volume of PCR product was then loaded on a 4 %agarose gel containing ethidium bromide. The gel wasexposed to UV light to reveal the amplified fragmentsand their size, by comparison with a 100 bp DNA ladder(New England BioLabs).

Table 1 (continued)

Code Name N Type Geo LEI0258 Other microsatellite markers

Nall HnbLEI HobsLEI Hobs ref 1 Hobs ref 2 Hobs ref 3 Hobs ref 4 Hobs ref 5

KT Shek-Ki 47 3 Asia 4 0.73 0.70 0.42

Ng Nagoya 48 3 Asia 3 0.53 0.58 0.44

P04 Fayoumi 12 3 Africa 5 0.71 0.75 0.35 0.44

P18 Old Scand. Ref. Pop. 18 4 9 0.85 0.89 0.45 0.46

BBL Bresse blanche 24 4 5 0.82 0.88 0.56

GLG Gauloise grise 18 4 6 0.71 0.83 0.48

GLN Gauloise noire 17 4 5 0.76 0.82 0.47

GLT Geline de Touraine 24 4 6 0.71 0.67 0.58

LIM Limousin 23 4 8 0.77 0.91

P11 Bresse noire 18 4 8 0.80 0.72 0.50

P33 Godollo Nhx 23 4 8 0.85 0.87 0.60 0.61

P34 Line Sarcoma R or S 12 4 1 0.00 0.00 0.37 0.34

P37 White egg layer A 12 4 1 0.00 0.00 0.33 0.35 0.30

P38 Brown egg layer A 18 4 3 0.30 0.22 0.46 0.44

P39 Broiler dam line A 18 4 3 0.45 0.50 0.57 0.58

P40 Broiler sire line A 23 4 6 0.80 0.78 0.55 0.53

P41 Broiler dam line B 18 4 5 0.50 0.50 0.49 0.48

P42 Broiler sire line B 18 4 6 0.82 0.83 0.47 0.51

P43 Brown egg layer B 18 4 4 0.75 0.89 0.45 0.36

P44 Brown egg layer C 12 4 2 0.23 0.25 0.44 0.43

P45 Brown egg layer D 18 4 3 0.40 0.39 0.47 0.49

P46 Brown egg layer E 12 4 5 0.78 0.75 0.39

P47 Broiler sire line C 12 4 6 0.76 0.83 0.44 0.51

P48 Broiler dam line C 11 4 2 0.25 0.27 0.42

P49 Broiler sire line D 12 4 6 0.79 0.83 0.44 0.48

P50 Broiler dam line D 22 4 10 0.86 0.68 0.62 0.61

P51 Ab line, high 20 4 3 0.70 0.60 0.51 0.49

P52 Ab line, low 20 4 3 0.54 0.65 0.46

P53 Ab line, control 17 4 4 0.55 0.59 0.51

L2 Taiwan Layer Native 50 4 6 0.45 0.42

B Taiwan Broiler Native 49 4 7 0.74 0.69

I.R.1 INRA I.R. line 1 49 4 4 0.70 0.71 0.37

I.R.2 INRA I.R. line 2 50 4 5 0.65 0.66 0.46

I.R.3 INRA I.R. line 3 49 4 4 0.60 0.60 0.39

I.R.4 INRA I.R. line 4 49 4 4 0.67 0.68 0.39

P32 C line 12 5 2 0.52 0.00 0.05 0.05

N number of animals

Type; population history, 1 wild species, 2 domesticated unselected, 3 standardized breed, 4 selected line, 5 inbred line (as defined for the AvianDivproject, Hillel et al. 2003)

Nall number of alleles for the LEI0258 marker, Hn.b.LEI unbiased expected heterozygosity for the LEI0258 marker (Nei 1978), HobsLEI observedheterozygosity for the LEI0258marker, Hobs average value of observed heterozygosity for microsatellite markers, obtained in previous studies; ref 1Hillel et al. 2003, ref 2 Granevitze et al. 2007, ref 3 Berthouly et al. 2008; Berthouly et al. 2009, ref 4 Leroy et al. 2012, ref 5 Loywyck et al. 2008

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Genotyping: sequencing

For each population, the different alleles identified by sizewere amplified again for sequencing from at least two in-dividuals. Four hundred thirty-eight animals were used;homozygous individuals were selected when possible, butheterozygous animals were used for the rare alleles. Theamplifications were performed in 50 μL with 40 ng of DNAusing the same primers, in the conditions described above.PCR products were sent directly to sequencing for thehomozygotes or after separation of the two alleles on 4 %agarose gels and purification using the QIAquick PCRPurification Kit from QIAGEN, for the heterozygotes.Sequencing was carried out by Eurofins MWG Operon,using their standard protocol for purified PCR products.

Sequence and data analysis

Sequences were analyzed with BioEdit (Hall 1999).Polymorphisms in the flanking sequences, (i.e., SNPs andindels) only and not the variation of the VNTR itself, wereused for this analysis. They were encoded as binary se-quences, and a median-joining network was constructedusing SplitsTree version 4.10 (Huson and Bryant 2006;Bandelt et al. 1999).

Population analysis

All animals were genotyped on gels, and the exact size ofthe nonsequenced alleles was extrapolated from the closestsequenced allele from the same population. For each popu-lation, the number of alleles and expected and observedheterozygosity values were calculated using GENETIX 4.4(Belkhir et al. 2000).

Results

Polymorphisms of the tandem repeat LEI0258

As described by Fulton et al. (2006), the two levels ofpolymorphism for the marker LEI0258 are the number ofrepetitions of the two motifs: R13 and R12 (R13:ATGTCTTCTTTCT, R12: TTCCTTCTTTCT) and the se-quence of the 62 bp upstream and 51 bp downstream re-gions. The polymorphisms previously reported were twodeletions and two SNPs upstream and two deletions andthree SNPs downstream. In comparison, we could onlyobserved one of the upstream deletions (the TT indel atposition −31–32 bp, i.e., 31 bp before the R13 repeats)and found two additional SNPs, one upstream at position−2 bp and one downstream at position +13 bp (i.e., 13 bpafter the R12 repeats). We redefined the largest deletion,

found in the downstream sequence, as a deletion involving8 bp instead of 7 bp: ATTTTGAG. This was discussed withthe authors of the 2006 publication (Fulton, personalcommunication), and this deletion will now be definedas position +21 to +28.

Global allelic variability

One of the aims of the study was to use the marker LEI0258in order to evaluate the variability of the MHC in diversepopulations of chickens. To achieve this, 534 alleles weresequenced, originating from 80 different populations ofchickens. In total, 79 different alleles are now identifiedand described by their number of repetitions, total size,combination of the indels, and the SNPs in the flankingregions (Table 2). Fifty are novel alleles, not reported byFulton et al. (2006). The number of alleles may be higher,since four additional allele sizes have been reported inChinese breeds (216, 324, 437, or 456 bp; Izadi et al.2011). However, in the absence of sequence information,they were excluded from this analysis.

Overall, 50 different allele sizes were observed, rangingfrom 181 to 552 bp. This is consistent with the size rangepreviously described (182 to 552 bp) with the 27 novelallele sizes found being mostly intermediate. The allele sizesprimarily reflect the combination of the repeats. The R13and R12 VNTR were found repeated 1 to 28 and 2 to 24times, respectively, but with only a limited number of com-binations (Fig. 1). The number of mathematically possiblecombinations is 644, but only 37 were observed, with themajority of alleles containing only one R13 motif (23 out of37 combinations). This is presented visually in Fig. 1.

Population analysis

The 80 populations, breeds or lines studied, were classifiedinto five types according to their selection history (Table 1):from the weakly selected (wild species and unselected do-mestic birds, standardized breeds) to the strongly selected(commercial lines and inbred lines). The observed hetero-zygosity for the LEI0258 marker was calculated for allpopulations (Table 1). The average observed heterozygositywas the highest for the unselected populations (Type 2,average HobsLEI=0.86 over eight populations) and for thewild species (Type 1, average HobsLEI=0.85 over five taxa),with a value of HobsLEI=1.00 for a Red Junglefowl (RJF)population. These two categories generally had many allelesfor the marker. The observed heterozygosity was more var-iable for standardized breeds (Type 3, 39 breeds) with anaverage HobsLEI of 0.67. The maximal value for all domesticchickens was obtained for the Ukrainian bearded breed, atraditional breed from Eastern Europe (HobsLEI=1.00). Theminimal value was observed for a traditional French breed,

Immunogenetics (2013) 65:447–459 451

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Tab

le2

Polym

orph

ismsandorigin

ofthe79

LEI025

8alleles

Allele

name

Size(bp)

Cluster

Upstream

R13

R12

Dow

nstream

NSerolog

yPop

ulation

Position

−31–32

−30

−13

−2

313

21–28

3136

43

Character

nb1

23

45

67

89

10

TT/Δ

G/A

G/A

Cn

nC/T

T/C

ATTTTGAG/Δ

Δ/A

A/T

T/A

181C

181

C1

2Δ

1Ng

182A

182

AA

12

ΔA

9B4

BEN,CI,GHA,KT,

P32

,WL*

193B

193

B1

3T

Δ35

B15.1,B11,B61

,B27

ANC*,

B,BBL,BEN,BNA,CAM,

CI,COU,F,

GHA,GJF,GLN,J,

L2,

MOR,P04

,P08

,P09,P10,

P102,

P11,P24,P29-2,P30,P31,

P33,P50

,T,

WL*

194A

194

AA

13

ΔA

10BW3

BAZ,GLD,LIM

,P09

,P22,P42,

P47,RJF*

205B

205

B1

4T

Δ16

B13

CI,GLT

,J,KM,P07

,P10,P17

,P18

,P40,P42

,p4

9,P50,WL*

205C

205

C1

4Δ

15B13.2,B17,BW11

BBL,BEN,BNA,CAM,COU,P05

,P101,

P102,

P17

,P24

,P27

,P28,

P29-2,RJF*,

WL*

217B

217

B1

5T

Δ2

P16

,P18

217C

217

C1

5Δ

23B,C

AM,G

AS,G

OU,P

05,P

13,P

15,

P16,P

23,P

25,P

27,P

40,P

46,p

49,

T

235G

235

GΔ

16

A2

CAM,CRC

237D

237

D1

6T

1F

237F

237

F1

6A

2BEN,GHA

239L

239

LΔ

17

Δ1

GJF

241C

241

C1

7Δ

18BAZ,COU,GAS,GLN,GOU,KT,

NCH,P

07,P

11,P

14,P

22,P

27,P

41

247G

247

GΔ

17

A1

B18

WL*

249D

249

D1

7T

32B15.2,B22,B73

B,BEN,BNA,CAM,CI,F,

FAY*,

GAS,GLG,J,KM,MOR,Ng,

NH*,

P05

,P07

,P17

,P23,P24,

P25,P29

-1,P38,P50

,P52

,P53,

WPR*

249E

249

E1

73

P05

,P52

249F

249

F1

7A

13BEN,NCH,P10

,P10

2,P23

,P26

,P27,P39

,P50

259G

259

GΔ

18

A3

P05

,P29

-2

259H

259

HΔ

18

CA

3BEN,CAM,GHA

261D

261

D1

8T

2GHA,GLG

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Tab

le2

(con

tinued)

Allele

name

Size(bp)

Cluster

Upstream

R13

R12

Dow

nstream

NSerolog

yPop

ulation

Position

−31–32

−30

−13

−2

313

21–28

3136

43

Character

nb1

23

45

67

89

10

TT/Δ

G/A

G/A

Cn

nC/T

T/C

ATTTTGAG/Δ

Δ/A

A/T

T/A

261F

261

F1

8A

50B15,B2,

B29

CAM,COU,GAS,GLD,GLN,

GOU,IR,L2,

LIM

,MOR,P09,

P10,P

11,P

12,P

16,P

17,P

18,P

19,

P21,P

22,P

23,P

28,P

30,P

34,P

37,

P47,p4

9,P50,WL*

271H

271

HΔ

19

CA

1CI

273F

273

F1

9A

13BEN,CAM,CRC,GLN,KT,

P10

1,P102,

P11,P12,P47,T

283I

283

IΔ

G1

10A

1P05

283J

283

JΔ

110

1P10

1

285F

285

F1

10A

4GAS,NCH,P10

2,P25

295G

295

GΔ

111

A2

CAM,P13

295H

295

HΔ

111

CA

1GHA

295I

295

IΔ

G1

11A

1GLT

295J

295

JΔ

111

22B5

BAZ,BEN,F,

MOR,P05

,P08

,P09

,P11,P13

,P18,P20,P29

-2,P31,

P33,P47

,p4

9,P50,WL*

295K

295

KΔ

A1

113

B11.1

ANC*,

COU,P101

297F

297

F1

11A

3P10

2

298F

298

F2

10A

2SLJF

307G

307

GΔ

112

A4

BBL,BNA,P15

,P23

307I

307

IΔ

G1

12A

8COU,GLG,GLT

,GOU,P15,P26

307J

307

JΔ

112

2P05

,P21

307K

307

KΔ

A1

1212

B72,B78

P04

,P20

,P25,P43,P45

,P51

,P53,

RIR*,

WPR*

309D

309

D1

12T

36B10,B24,B26

,B76

B,CAM,CRC,F,

GAS,J,KM,L2,

MOR,NCH,Ng,

NH*,

P08

,P16

,P17,P24

,P25

,P29

-1,P29

-2,P30

,P33,P38

,P44

,P45

,P46,P52,

RIR*,

T,WL*,

WPR*

309E

309

E1

121

CI

309F

309

F1

12A

2CI,P10

1

318E

318

E10

31

P10

319G

319

GΔ

113

A1

NCH

319H

319

HΔ

113

CA

3P06

,P10

,P26

319K

319

KΔ

A1

1310

F,GHA,P04

,P102,

SLK

Immunogenetics (2013) 65:447–459 453

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Tab

le2

(con

tinued)

Allele

name

Size(bp)

Cluster

Upstream

R13

R12

Dow

nstream

NSerolog

yPop

ulation

Position

−31–32

−30

−13

−2

313

21–28

3136

43

Character

nb1

23

45

67

89

10

TT/Δ

G/A

G/A

Cn

nC/T

T/C

ATTTTGAG/Δ

Δ/A

A/T

T/A

321D

321

D1

13T

1B74

WPR*

321F

321

F1

13A

1CAM

331K

331

KΔ

A1

141

P06

333D

333

D1

14T

6BEN,P10

1,P10

2

333F

333

F1

14A

8BW4

BEN,GJF,P05

,P11,RJF*

345D

345

D1

15T

8B14

GHA,KT,

P08

,P11,P18

,T,

WL*

345F

345

F1

15A

3P07

,P10

1

357D

357

D1

16T

49B130,

B131,

B201,

B5.1,

B6.1,

B21

,B75

B,BAZ,BBL,BEN,BNA,CAM,

COU,GAS,GLD,GLT

,L2,

MOR,

NH*,

P09

,P13

,P15

,P17,P18,

P22,P24

,P26

,P27

,P28,P29-2,

P31,P

38,P

46,P

47,P

48,P

51,P

53,

RIR*,

T,WC*,

WL*,

WPR*

357E

357

E1

163

BBL,BEN,GLG

357F

357

F1

16A

6B23

F,GOU,NH*,

P102,

P51

367G

367

GΔ

117

A1

BC

CRJF*

369D

369

D1

17T

3B21.1,BQ,BW1

NCH,RJF,RJF*,

SP*

369F

369

F1

17A

7BAZ,CAM,LIM

,P10

2,P50

381D

381

D1

18T

20B13.1

CI,GHA,J,KM,MOR,P04,P05

,P102,

P47,P48

,p4

9,P50,WL*

381F

381

F1

18A

8CRC,GJF,P09

,P21,P30

391K

391

KΔ

A1

191

BAZ

392K

392

KΔ

A2

181

P06

393D

393

D1

19T

4B1

BEN,CI,P27

,WL*

405D

405

D1

20T

4B8

ANC*,

GHA,L2,

P05

417D

417

D1

21T

4P05

,P10

1,P102

419E

419

E15

61

F

420E

420

E16

58

B34,B62

BAZ,GAS,IR,P18

,P25

,WL*

427K

427

KΔ

A1

221

GLT

429D

429

D1

22T

1GHA

441F

441

F1

23A

1GJF

443E

443

E15

88

B6

B,COU,GAS,MOR,P05,P30,T,

WL*

445E

445

E17

62

P20

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the Gauloise dorée (HobsLEI=0.18), consistent with overalllow heterozygosity for this breed (Berthouly et al. 2008,2009). The observed heterozygosity in selected lines (Type4, 32 lines) was generally lower with an average of 0.62; thelowest values were obtained for an experimental line (P34),which, as the inbred line (Type 5, P32) has been fixed forthe MHC-B region (HobsLEI=0, as expected) and four com-mercial lines potentially under selection for certain sero-types (P37, P38, P44, P48, HobsLEI=0 to HobsLEI=0.27).Since many of these populations had been previously ex-amined for heterozygosity at neutral microsatellite sites, thelevel of heterozygosity could be compared between neutralloci and the LEI0258 locus, which is presumably underselection due to its location within the MHC. However, themarker LEI0258 is a VNTR, its evolution might differcompared to the nearby sequences, which could lead to anoverestimation of the variability of the region.

Network analysis and allele relationships

In VNTR, tandem expansion and contraction are consideredto be very frequent compared to point mutations. Therefore,we only use the latter (SNPs and indels) for the networkconstruction. Indeed, for the marker LEI0258, the differenceof length between alleles is a poor estimate of allele diver-gence, as two alleles different in sequence can have identicallengths. Nevertheless, allele sizes and number of repeats arereported on the network (Fig. 2).

The network constructed from the 79 alleles discriminated12 clusters identified as A to L, each containing one to 15alleles (Fig. 2). The unique allele in cluster L was found in aGrey Junglefowl (GJF, Gallus sonneratii). The network wasbuilt independently of the number of repeat and allele sizeinformation, utilizing only SNP and indel information.T

able

2(con

tinued)

Allele

name

Size(bp)

Cluster

Upstream

R13

R12

Dow

nstream

NSerolog

yPop

ulation

Position

−31–32

−30

−13

−2

313

21–28

3136

43

Character

nb1

23

45

67

89

10

TT/Δ

G/A

G/A

Cn

nC/T

T/C

ATTTTGAG/Δ

Δ/A

A/T

T/A

453D

453

D1

24T

1P10

2

474E

474

E22

32

B12.2

BEN,WL*

484E

484

E20

64

BAZ,GLN,P11,SLK

487E

487

E23

39

B12,B124

B,IR,P18,P32,WL*

513E

513

E25

31

B12.3

WL*

526E

526

E26

32

P19

539E

539

E27

36

B19

L2,

IR,P16

,P20,P50,WL*

552E

552

E28

34

B19.1

IR,P18

,P30,WL*

Nnu

mberof

anim

als

Pop

ulationnames

areaccordingto

Table1except

whenastar

indicatesasequ

ence

andabreeddescribedin

Fultonet

al.20

06

2-10

10-3

2-18

15-616-5

15-8

17-6

22-3

20-6

23-3

25-326-327-328-3

1

1 10 20

Number of R12 repeats

1-2 1-24

Num

ber

of R

13 r

epea

ts

10

20

Fig. 1 The 37 combinations of repeat numbers observed in the 79alleles at the LEI0258 locus

Immunogenetics (2013) 65:447–459 455

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However, a pattern emerged from the clusters: the shortalleles (R13=1, R12=2 to 7; allele size ≤241 bp) weregrouped on one side in the A, B, C clusters, and most ofthe long alleles, (R13=15 to 28; allele size ≥419 bp), weregrouped into a large cluster, cluster E (Figs. 2 and 3). Inaddition, geographical information (described in Table 1),was used to create a pie chart for each allele cluster. Onlybreeds classified as types 1, 2, or 3 had relevant geographicalinformation and thus could be included within these piecharts. Each pie presents the percentages of each geographicalorigin for the breeds included in the cluster. Interestingly, noAsian breeds are found in clusters G, H, I.

Discussion

Population analysis

For 74 out of 80 populations of chicken, the measuredheterozygosity for the MHC region was compared with theheterozygosity at neutral microsatellite markers published inseveral studies (Berthouly et al. 2008, 2009; Granevitze etal. 2007; Hillel et al. 2003; Leroy et al. 2012; Loywyck et al.2008). Firstly, for the wild species and the unselectedpopulations, the observed heterozygosity was always higherwith the LEI0258 marker compared to the neutral markers.

E

F

G

H

I

J

L K

A

B

C

D

10

10

4

2

1

1

7

7

5

3, 8

6

Fig. 2 Median-joining network of the 79 alleles defined for the VNTRLEI0258. The network is based only on SNP and indels within theflanking regions. Clusters are identified as A to L. Alleles highlightedin gray are found in wild birds. Each pie represents the percentages of

each geographical origin for the type 1, 2, or 3 breeds. The characterchanges are indicated on each branch and correspond to the characternumber reported in Table 2

456 Immunogenetics (2013) 65:447–459

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This result indicates high genetic diversity of MHC regionand suggests balancing selection which maintains polymor-phisms and increases within-population diversity (Nielsen2005). It was also observed for 33 out of the 39 traditionalbreeds. Secondly, in commercial white-egg layers, the het-erozygosity was low overall, indicating a loss of geneticdiversity. This could simply be due to commercial selectionas reported by Muir et al. (2008), or because all commercialwhite egg layers are derived from a single breed, the WhiteLeghorn breed. Finally, three lines of commercial brown-egg layers out of five showed heterozygosities lower for theLEI0258 compared to the neutral markers. This also clearlyindicates selection targeted at the MHC, since any conse-quences of population size, bottleneck, or founder effectwould have affected all the markers.

In summary, the MHC region which is associated withdisease resistance in chicken is characterized by naturallybalanced selection. Therefore, the short-term benefits ofselecting for this region in commercial chickens should beweighed carefully. However, even if the nucleus populationsof the different selection schemes have lost genetic variabil-ity for the MHC, the terminal crosses can still combineseveral MHC haplotypes if the different grand-parental linesused in the cross are selected on different criterions.

LEI0258 and serology

Limited serological information was available for the animalsused and will very likely remain unavailable due to the lack ofreagents and complexity of comparison between breeds.

Serology was only available for eight B types: B4, B12from two origins, B124, B13, B15 from two origins, B19,B21, and B34. All originated from White Leghorns. For B4,B12, B13, B15, B19, and B21, a corresponding LEI0258allele had already been reported by Fulton et al. (2006), andour findings matched their observations. B124 has been de-fined in the INRA I.R. lines as serologically identical to theB124 described in ISA Warren experimental lines (high and

low antibody response to sheep red blood cells) and wasshown to be distinct from B12 on the basis of protein analysis(Pinard and Hepkema 1993). The LEI0258 allele for B124,however, is similar to B12 (487E, Table 2). B34 has beendescribed in the same INRA lines and is associated with thesame LEI0258 allele than B62 (420E, Table 2), which is foundin a commercial line of White Leghorns (Fulton et al. 2006).

In addition, out of the eight most common LEI0258alleles highlighted by Fig. 3, one has no correspondingserology. This is the 217C allele, which is found in clusterC. Similarly, serological information is absent for all thealleles of the H and I clusters. Therefore, as expected,serological studies are restricted to the subset of MHChaplotypes found in experimental lines which is a biasedrepresentation of the diversity of chicken breeds.

LEI0258 evolution

The evolution of the number of repeats of the LEI0258marker is probably based on a step-wise model, one stepbeing the duplication or the loss of a repeat. Indeed an allele(552E), absent in the founder animals, has been observed inthe I.R. lines at generation 11. This observation can beexplained by the duplication of a R13 repeat in a 539Eallele, common in these lines. As noted above, however,the combinations of the R13 and R12 motifs follow a patternwith always one motif having a low and one motif having ahigh number of repeats. A hypothesis explaining the limitednumber of possibilities observed could be a limitation of thetotal allele size. This is supported by the compact organiza-tion of the MHC in the chicken, characterized by smallintergenic sequences (Kaufman et al. 1999).

In comparison, the flanking regions are highly polymor-phic. Point mutations such as SNPs and indels can be usedto categorize alleles because they are likely to be underslower rates of evolution compared to repeats. Indeed, anumber of LEI0258 alleles have the same size and combina-tion of repeats but different flanking sequences illustrating

193B (6%)

217C (4%)

249D (6%)

309D (6%)

357D (9%)

381D (4%)

261F (9%)

295J (4%)

A

B

C

D

E

F

G

H

I

J

K

L

150 200 250 290 350 400 450 500 550 600

Clu

ster

Allele size (bp)

Fig. 3 Frequencies of the 79LEI0258 alleles. The area ofeach bubble represents thenumber of observations foreach allele. The name of theallele and its frequency areindicated for the eight mostfrequent alleles

Immunogenetics (2013) 65:447–459 457

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that the issue of homoplasy (when two identical states are notidentical by descent) is more relevant for repeats than forpoint mutations. In the network analysis, however, it appearsthat some mutations happened twice or that recombinationoccurred to explain the observed alleles. Therefore, the net-work is only a representation of the relationships betweenalleles, not a phylogeny.

Different combinations of the R13 and R12 motifs areassociated with different parts of the network. By utilizingonly the 8 bp and the 2 bp information, the network can bedivided into three parts: three clusters without deletion (D, E,F), five clusters with the 2 bp deletion (G, H, I, J, K), and fourclusters with the 8 bp deletion (A, B, C, L). Subsequently, thealleles found in wild species of Junglefowls, namely GreyJunglefowl (GJF), Red Junglefowl (RJF), and Sri-LankaJunglefowl (SLJF) can be found in eight of the 12 clustersdefined by the point mutations (A, B, C, D, F, J, K, L), and inall the three clusters defined above (Fig. 2). It has beensuggested that for chickens, several domestication events tookplace in Asia, mainly from the Red Junglefowl (Liu et al.2006) but with contribution from the Grey Junglefowl(Eriksson et al. 2008). In the case of the MHC, our observa-tions indicate that most of the diversity in domestic chickensoriginated in the ancestral species and has been maintained.There is, however, one major cluster containing only allelesfrom domesticated chickens, cluster E, and three clustersrestricted to chicken from Europe and Africa (clusters G, H,I). This can probably be attributed to genetic drift and thesmall sample sizes used for the wild species.

Conclusions

The marker LEI0258 was used to describe the MHC-Bregion variability in various chicken populations includingwild species of fowls. Traditional breeds and commerciallines were compared with experimental chickens defined byserology. A comprehensive reference set of alleles is nowavailable to identify and classify MHC genotypes andshould be used to further study local populations. In thesepopulations, novel LEI0258 alleles should be sequenced anddefined by both the combination of repeats and the poly-morphisms in the flanking sequences. Therefore, this studyprovides a general framework to study variability within theMHC and disease resistance or susceptibility in chickens.

Acknowledgments The authors thank Anne-Marie Chaussé (INRA,Nouzilly, France), Xavier Rognon (INRA, Jouy en Josas, France),Yannick Roman (Zoo de Clères, Clères, France), and Cheng Yu-Shin(Livestock Research Institute, Sinhua, Taiwan) for their help collectingDNA samples. They also thank Janet Fulton (Hy-Line International,Dallas Center, USA) for the helpful comments and discussion. Thiswork was supported by a studentship awarded by the French ministryof Higher Education and Research.

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