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Fish & Shellfish Immunology (2002) 12, 287301doi:10.1006/fsim.2001.0371Available online at http://www.idealibrary.com on

Differences in MHC class I genes between strains ofrainbow trout (Oncorhynchus mykiss)

CHUNXIA1*, IKUNARIKIRYU1*, JOHANNESMARTINUSDIJKSTRA1, TERUO AZUMA2,TERUYUKINAKANISHI1 ANDMITSURUOTOTAKE1

1Inland Station/National Research Institute of Aquaculture, Tamaki,

Mie 519-0423, Japan and 2Nikko Branch, National Research Institute of

Aquaculture, Chugushi, Nikko, Tochigi 321-1661, Japan

(Received 28 February 2001, accepted after revision 4 July 2001, publishedelectronically 28 January 2002)

In rainbow trout there is only one dominant classical MHC class I locus,Onmy-UBA, for which four very di#erent allelic lineages have been described.The purpose of the present study was to determine if Onmy-UBA poly-morphism could be used for strain characterisation. This was performed bylineage-specific PCR investigation of 30 fish, each of the Nikko and Donaldsonstrains, and by sequence analysis of 25 of the amplified DNA fragments. Twonew MHC class I lineages were detected in addition to the four previouslydescribed lineages, thus six distinct lineages were observed within the fish

examined (Sal-MHCIa*AF). The distribution of lineages appeared to bestrain-specific. For example, the lineage Sal-MHCIa*A was very common inthe Nikko strain but could not be detected in the Donaldson strain. Analysisof MHC class I variation may help to elucidate relationships between strainsand the roles of MHC alleles in disease resistance. 2002 Elsevier Science Ltd.

Key words: MHC, polymorphism, lineages, strain, rainbow trout.

I. Introduction

Classical MHC class I molecules are involved in cell surface presentation

of peptides, derived from intracellular proteins, for recognition by T cellreceptors (TCR) of CD8 positive T lymphocytes [1,2]. When presented non-selfpeptides T cells can be stimulated, this results in killing the presenting cell.

This screening mechanism is the primary immune defence against many viraldiseases [3,4].

One characteristic of classical MHC class I is extensive polymorphism. The

polymorphism results in the presentation of di#erent sets of antigenic peptidesby each allomorph (protein expressed by an allele), and may result in

Present address: Department of Microbiology and Infection, College of Veterinary Medicine,China Agricultural University, Beijing 100094, P.R. China.

Present address: Department of Veterinary Medicine, College of Bioresource Sciences,Nihon University, Kameino, Fujisawa, Kanagawa 252-8510, Japan.

*Authors contributed equally to this work.Corresponding author. E-mail: ototake@a#rc.go.jp

28710504648/02/040287+15 $35.00/0 2002 Elsevier Science Ltd.

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di#erences in disease resistance. Well-characterised correlations between

MHC class I alleles and disease resistance are described for Mareks disease inchickens [5,6], and malaria[7]and HIV [8] in humans.

In aquaculture there is extensive interest in the characterisation of unique

strains within di#

erent fish species [9, 10]. Characterisation of strains withrespect to their histories and relationships is expected to guide strainimprovement, and knowledge regarding polymorphic genes could aid this

characterisation.Classical MHC class I is known to be one of the most polymorphic genes

[11], and its polymorphism has been successfully used to characterise popu-

lations of several species including humans [12], horses [13], and cattle[14].Natural populations of chinook and coho salmon have been distinguished bytheir MHC class I polymorphism [1518]. One advantage of MHC class I as a

genetic trait marker is that allelic di#erences may relate to di#erences indisease resistance.

Previously, it has been shown that there is only one dominant expressed

classical MHC class I locus in rainbow trout, designated Onmy-UBA [19].Among 14 fish, of which five were distinct homozygous clones, 10 di#erentOnmy-UBA alleles were detected [19]. The alleles of the locus were shown tobe classical by conservation of key amino acids, high variability in the 1 and2 domains and ubiquitous expression. A remarkable variability in the 3

domain was observed, whereas for MHC class I loci in higher vertebrates andsharks [20] this region is quite well conserved. This may be due to the ancientcharacter of the Onmy-UBA locus or to di#erent stringencies with respect to

interactions with T lymphocytes. The 10 alleles in rainbow trout were placedinto four lineages (Sal-MHCIa*AD) based on similarity of 1, 2 and 3domains [19]. MHC class I sequences of other salmonid species could also be

placed into these lineages [19]. In the present study significant di#erences areshown in the Onmy-UBA alleles of the Nikko and Donaldson rainbow troutstrains present in Japan.

II. Materials and Methods

FISH

The fish used in this study were randomly selected 30100 g Nikko orDonaldson strain rainbow trout from the National Research Institute ofAquaculture (Nikko, Japan). Fish were held in 9C flowing water. Both

strains were maintained by moderate inbreeding using 510 males and 2030females for annual reproduction, which is a common practice in Japan. TheNikko strain was imported from the U.S.A. before 1951, but its exact origin is

unclear [21]. The Nikko strain fish have classic rainbow trout characteristics.The Donaldson strain [22] was imported from the U.S.A. in 1966 [21]. Largesize, rapid growth and high fecundity characterise these fish.

PRIMERS USED

The specificities of the primer combinations used for detection of theSal-MHCIa*AF lineages in Nikko and Donaldson strains are detailed below,

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amplification with the di#erent primer sets were: For the primer combinationspG-LPf with pG-3UTRr, pG-LPf with pI-3/TMr, and pIV-LPf withpG-3UTRr: First 60C for 98 min, then 94C for 2 min, then 35 cycles of 94Cfor 1 min followed by 55C for 5 min, and finally 55C for 7 min. For the primer

combinations pI-1f with p5-2r and pI-1f with pIII-3/TMr: First 60C for30 min, then 94C for 2 min, then 25 cycles of 94C for 1 min followed by 65Cfor 15 min, and finally 65C for 7 min. For the primer combinations pIII-1f

with pIII-3/TMr and pVI-1f with pII-3/TMr: First 60C for 98 min, then94C for 2 min, then 35 cycles of 94C for 1 min, 60C for 5 min, and finally60C for 7 min.

SEQUENCE ANALYSIS

Amplified RT-PCR fragments were cloned into the vector pGEM-T Easy(Promega Corporation, Wisconsin, U.S.A.). The nucleotide sequences weredetermined by the dideoxychain termination method using a CEQ Dye

terminator Cycle Sequencing Kit (Beckman Coulter, Inc., California, U.S.A.)and suitable primers. Sequence analysis was performed with an automated

sequencer (CEQ 2000 DNA analysis system, Beckman Coulter, Inc.). Compari-son of deduced amino acid sequences was performed using the programsSearch homology, Multiple alignment, and UPGMA of GENETYX version

10.1 (Software Development Co., Ltd, Tokyo, Japan) computer software.

GENBANK ACCESSION NUMBERS

New sequences described in this report have been deposited in GenBankunder the accession numbers AF318188AF318190.

III. Results

RT-PCR ANALYSIS

In a previous study four di#erent classical MHC class I lineages

Sal-MHCIa*-A, -B, -C and -D were detected in rainbow trout [19]. In thepresent study lineage-specific RT-PCR assays were established (see Materials

and Methods). These assays were based on primers that were expected to bindto all of the rainbow trout MHC class I sequences reported for each respectivelineage. Lineage definitions are discussed below.

Total RNA from the blood of 60 rainbow trout was tested with Sal-MHCIa

lineage-specific primer combinations following reverse transcription. Anumber of the RT-PCR fragments (25) were sequenced (see below) and twoadditional lineages, Sal-MHCIa*-E and -F, were detected. For these new

lineages specific RT-PCRs were established as well. The RT-PCR results aresummarised inTable 1.The results indicated that in 41 of the 60 fish at leasttwo di#erent MHC class I sequences were present, and for one Donaldson fish

three di#erent fragments were amplified. The main di#erence between the tworainbow trout strains was that sequences belonging to lineage A were verycommon in the Nikko strain but were not detected in the Donaldson strain. In

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addition, the C lineage was absent in the Nikko strain fish and the E lineage

was absent in the Donaldson strain fish.In Table 1 the lineages Sal-MHCIa*-G, -H, and -K are indicated between

brackets. These lineages were discovered in other rainbow trout strainsduring the course of this study. Sequences belonging to these lineages would

probably be amplified by the RT-PCR systems used, but they were not detectedin the Nikko and Donaldson strains.

SEQUENCE ANALYSIS

The 25 amplified and sequenced RT-PCR fragments derived from 13 fish are

shown in Table 1. For each fragment the complete sequences from at least

three clones were determined to exclude PCR mistakes, the exact numberdepending on the quality of the sequence information obtained and whether

the fragment contained one or two Onmy-UBA sequences. The sequences

Table 1. RT-PCR specific for Sal-MHCIa lineages and sequence analysis. Total RNAsamples from 30 Nikko strain and 30 Donaldson strain rainbow trout were investigatedby seven RT-PCR systems: primer pG-LPfwith pG-3UTRr [A, C, D, F (G, H)], pG-LPfwithpI-3/TMr[A (H)], pIV-LPfwith pG-3UTRr [B (K)], pI-1fwith p5-2r (C), pI-1fwith pIII-3/TMr(D), pIII-1fwith pIII-3/TMr(E), and pVI-1fwith pII-3/TMr(F)

No. offish

RT-PCR for Sal-MHCIa lineages:

A, C, D, E, F (G, H) A (H) B (K) C D E F

Nikko10 1401(A)2a + 4901(F)1a

6 + + +4 1401(A)2b 1401(A)1b 401(B)1b

4 + +3 1401(A)1c + 701(D)1c

701(D)1c

1 1401(A) +1 +1 0501(E) + +

Donaldson8 +8 701(D)2d 401(B)1d 701(D)1d

4 + +3 + + +3 4901(F)1 + +2 501(C)2e 401(B)1e +1 + +1 701(D) 401(B) 701(D) 4901(F)

4901(F)

The + symbols or the names of sequenced fragments indicate positive RT-PCR results, whereasopen spaces indicate negative RT-PCR results. The number of fish showing the same PCR patternis indicated at the left. When sequencing analysis was performed, this is indicated by the name ofthe Onmy-UBA sequence, followed by the Sal-MHCIa lineage to which it belongs (indicatedbetween parentheses) and the number of fish analysed. The superscripts ae indicate thatsequenced PCR fragments were derived from the same fish; superscripts following a 2 refer toonly one of the two fish for which the amplified fragment was sequenced.

MHC CLASS I TYPING OF RAINBOW TROUT STRAINS 291

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detected were Onmy-UBA*-401, -501, -0501, -701, -1401, and -4901. The

sequences Onmy-UBA*-401, -701 and -4901 were present in both the Nikko andDonaldson strains. Onmy-UBA*-401, -501 and -701 had previously beendetected in other rainbow strains [19]. The sequences Onmy-UBA*-0501 and

-1401 were recently submitted to the GenBank by Shum et al. [23] (GenBankAF296362 and AF296371). The sequence Onmy-UBA*4901 has not beenpreviously published. The new sequences were numbered from 4901 down-

wards, since Shum et al. [23] recently submitted Onmy-UBA sequencesnumbered through Onmy-UBA*1601 to the GenBank database, and it wasnecessary to avoid giving the same designations. It is unfortunate that, more

or less simultaneously, sequences were designated as Onmy-UBA*-101 to -701[19] while others designated di#erent Onmy-UBA sequences as Onmy-UBA*-0101 to -0701 [23](Genbank AF091785, AF296359AF296364).

InFig. 1the deduced amino acid sequences of the extracellular domains ofthe di#erent alleles detected in the present study are compared, with adescription of features typical of classical MHC class I molecules.

HOMOLOGY COMPARISON

The new sequences obtained were placed into lineages based on similaritiesin 1, 2 and 3 domains as done by Aoyagi et al.[19]. Sequences were placed

into the same Sal-MHCIa* lineage when (i) the 1 and 2 domains each hadover 67% amino acid identity with the bulk of the other lineage members andwere grouped together by UPGMA tree analysis, and (ii) the 3 domains had

a similar length.A simplified form of the UPGMA tree analysis for the 1 and 2 domains, in

which homologous sequences are indicated as groups instead of individual

sequences, is shown in Fig. 2. In addition to salmonid sequences severalcyprinid MHC class I sequences were compared, in order to show conservationof lineages between evolutionarily distant fish. The figure legend describes

which sequences were compared. Sequences within the 1 and 2 homologygroups, indicated with Roman numerals, share greater than 67% amino acididentities with the bulk of the other members in the group. With this definition

six di#erent 1 groups and two di#erent 2 groups were established forrainbow trout. The 1 homology group V was divided into Va and Vb, as the

UPGMA analysis clustered them close together, but the cyprinid sequenceBrre-UAA*01 showed greater than 67% amino acid identity with only a few ofthe members of group Va. The 3 region was divided into three di#erentgroups, each of them with a di#erent length of the C-terminus: I has no

extension, II has a short extension, and III a long extension (compareFig. 1and Table 2). The C-termini of the 3 domains were based on the expectedintron-exon borders, in comparison with the sequences Onmy-UBA*-401, -501,

and -701 for which these borders had been determined (unpublished data).The sequences compared inFig. 2that encompass the 1, 2 and 3 domains

are schematically compared inTable 2.The Roman numbers in Table 2refer

to those in Fig. 2. By lineage definition the sequence Onmy-UBA*0501 canbe placed into a new lineage Sal-MHCIa*E, and Onmy-UBA*4901 can beplaced into a new lineage Sal-MHCIa*F. Table 2a shows the Sal-MHCIa

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Fig.

1.

PredictedaminoacidsequencesoftheextracellulardomainsoftheOnmy-U

BA*-1

401,-4

01,-5

01,-7

01,-05

01,

and-4

901moleculesdet

ectedinNikkoandDonaldso

nstrainrainbowtrout.ThesequencesOnmy-U

BA*-4

01,-5

01

and-7

01havebeenalreadydescribedbyAoyagiet

al.[19](GenBankAF287487

,AF287488,

AF287492)andt

he

sequenceOnmy-U

BA*-0501and-1

401byShum

eta

l.[23](GenBankAF296362andAF296371).Alignmentw

as

performedbycomputersoftwareandmodifiedby

hand.

Numberingofamino

acidswasperformedfort

he

Onmy-U

BA*1401sequence,

startingfromthe1domain.

Dashesindicateidenticalaminoacids;asterisksindicate

gapsinthesequence;fi

lledcirclesindicatetheconservedpositionsbelievedtointeractwithantigenicpeptide

terminiinmammals(M

adden[32]);Pindicatespositionsbelievedtointeractwiththeantigenicpeptidea

nd

determineitsspecificity

inmammals(Matsumuraet

al.[33]);Cindicatesconservedcysteineresidues.Thebox

at

position86indicatesaconservedN-g

lycosylationsite.


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III

II0.248

I0.372

VI0.325

Va0.224

Vb

0.123

IV0.391

VII0.473

1

I0.3652

II

(3rt; 0.006)

(4rt; 0.012)

(36rt, 6bt 1as, 1ps; 0.145)

(2rt, 2bt, 1ca; 0.145)

(5rt, 1bt; 0.166)

(1zb)

(8rt, 1bt; 0.052)

(1zb)

(38rt, 9bt, 1as, 1ps; 0.217)

(17rt, 1bt, 1ca, 2zb; 0.181)

Fig. 2. UPGMA analysis. The amino acid sequences of the 1 and 2 regions ofpublished rainbow trout (rt) MHC class Ia molecules and those of several other fish[brown trout (bt), Atlantic salmon (as), pink salmon (ps), common carp (ca) andzebrafish (zb)] are compared. Amino acid substitutions per site is indicated on thehorizontal bars and is also represented by bar length. Division into 1 and 2 regionsis in agreement withFig. 1. The clustering of the individual sequences is shown ina simplified manner by depicting only the homology groups, indicated with Romannumerals. The number of sequences included in each group, as well as the amino

acid substitutions per site between group members, is indicated between parenth-eses. Compared are: the rainbow trout sequences Onmy-UBA*101701 by Aoyagiet al. [19](GenBank AF287483AF287492), Onmy-UBA*48014901 (this report; Gen-Bank AF318188AF318190) and Onmy-UBA*01011601 by Shumet al.[23] (GenBankAF091785, AF296359AF296373, AJ007847). Also the partial rainbow troutsequences Onmy-A*- and Onmy-UA*1 reported by Miller et al. [17] (GenBankAF1045234529 and AF1045794582), Onmy-UA*-, Onmy-UAA*-, Onmy-UCA*-, andOnmy-UBA*- reported by Hansen et al . [30] (GenBank AF0021712179 andAF1155185528), the brown trout sequences Satr-UBA*01011001 by Shum et al.[23](GenBank AF296374AF296383), the Atlantic salmon sequence Sasa-p30 byGrimholt et al. [34] (GenBank L07606), the pink salmon sequence Ongo-UA-(92H)by Katagiri et al.[35] (GenBank D58386), the common carp sequence Cyca-UA1*01

by van Erp et al.[31] (GenBank X91015), and the zebrafish sequences Brre-UAA*01and Brre-UBA*01 by Takeuchi et al. [36] (GenBank Z46776 and Z46777).

The homology groups to which the individual sequences belong can be found inTable 2, except for the single domain sequences of Miller et al. [17] and thetwo-domain sequences of Hansen et al.[30]. For the sequences not depicted in Table2the following groupings were made: the 1 domain sequences Onmy-A*-1, -2, -3, -4,-5, -6 and -7 to 1 group I, the 2 domain sequences Onmy-A*-1, -2, and -3 to 2 groupI and Onmy-UA*1 to 2 group II. The 1 domains of Onmy-UA*-b13, -K19, -b4.10 andOnmy-UCA*KD2.11 belong to 1 group I, the 1 domains of Onmy-UA*A4.10,Onmy-UAA*KD4.5 and -SP1.3 belong to 1 group II, the 1 domains of Onmy-UA*A4.3 and Onmy-UCA*KD2.9 belong to 1 group III, the 1 domains of Onmy-UA*-B3, -K18, -A5.1 and Onmy-UBA*Spu3.1 belong to 1 group IV and the 1

domains of Onmy-UA*A4.11 and Onmy-UAA*KD1.5 belong to 1 group V; whereasthe a2 domains of Onmy-UA*-b13, -K19, -A4.10, -B3, -K18, -A4.11, Onmy-UAA*KD4.5,-SP1.3, -KD1.5 and Onmy-UBA*Spu3.1 belong to 2 group I, and the 2 domains ofOnmy-UA*b4.10, -A4.3, -A5.1, Onmy-UCA*KD2.9 and -KD2.11 belong to 2 group II.

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lineages detected in the Nikko and Donaldson strains in Japan, 2b shows

lineages detected only in other rainbow trout strains, 2c shows browntrout lineages for which no rainbow trout sequences were found and 2dshowsseveral cyprinid sequences. The lineages Sal-MHCIa*AK can be distin-

guished when comparing all rainbow trout sequences, and the lineages

Sal-MHCIa*AO when comparing all salmonid sequences.The 3 domains of the cyprinid sequences are not indicated in Table 2

(despite being known for Cyca-UA*01 and Brre-UBA*01) since they are very

Table 2. Organisation of the full length classical MHC class I sequences detected insalmonids into lineages

1 2 3 LineageSal-MHCIa*-

(a)Rainbow trout: Onmy-UAA*KD6, -HC-01,-OSU-01, Onmy-UBA*101, -102, -201, -301, -0101,Onmy-UBA*1001, -1101, -1201, -1301, -1401.Brown trout: Satr-UBA*-0101, -0201, -0301.Pink salmon: Ongo-UA-(92H)

I I I A

Rainbow trout: Onmy-UBA*-SP3, -401 IV I I BRainbow trout: Onmy-UBA*-501, -502, -503, 1501,-1601. Atlantic salmon: Sasa-p30. Brown trout:Satr-UBA*0401

I I II C

Rainbow trout: Onmy-UBA*-601, -701, -0301, -0401,

-0601, Onmy-UCA*C32. Brown trout: Satr-UBA*0901

I II III D

Rainbow trout: Onmy-UBA*0501 III II III ERainbow trout: Onmy-UBA*4901 VI I II F

(b)Rainbow trout: Onmy-UBA*-4801, -4802 V II III GRainbow trout: Onmy-UBA*0901 II I I HRainbow trout: Onmy-UBA*0801 V I I IRainbow trout: Onmy-UBA*0701. Brown trout:Satr-UBA*0701

VI I I J

Rainbow trout: Onmy-UBA*-0201, -0202 IV II III K

(c)

Brown trout: Satr-UBA*0601 I I III LBrown trout: Satr-UBA*0801 V I II MBrown trout: Satr-UBA*0501 VI I III NBrown trout: Satr-UBA*1001 IV I II O

(d)Zebrafish: Brre-UAA*01 V IICarp: Cyca-UA*01 VI IIZebrafish: Brre-UBA*01 VII II

The Roman numerals for the 1 and 2 domains refer to the homology groups indicated inFig. 2.The sequences of the 3 groups I, II and III have no extension at the C-terminus, a shortextension and a long extension respectively. (a) Sal-MHCIa lineages detected in the present study,(b) Sal-MHCIa lineages detected in other rainbow trout, (c) Sal-MHCIa lineages detected only inbrown trout and (d) cyprinid sequences. The salmonid sequences are organised into lineagesSal-MHCIa*AO based on having their extracellular domains placed into the same groups. Thesequences detected in this study are underlined.


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di#erent from the salmonid sequences [19]. Table 2 clearly shows a mosaic

structure of domains, some which are shared between cyprinid and salmonidMHC class I sequences.

IV. Discussion

The present study investigated MHC class I variability between two

rainbow trout strains. The purpose was to investigate the distribution ofOnmy-UBAalleles between rainbow trout strains maintained in captivity, andto determine whether Onmy-UBA polymorphism can be used as a genetic

marker.Characterisation of fish strains in order to understand their historical

relationships and di#erences in properties such as disease resistance has long

been of interest in aquaculture, resulting in the discovery of a variety ofmarkers including allozymes, DNA-fingerprints and microsatellites [2427].For rainbow trout specifically, DNA finger printing has been established [28]

and a number of microsatellites and allozyme markers have been described[29].

MHC class I polymorphism has been used previously for the characteris-ation of salmonid populations by Miller et al. [1518]. They found that forBritish Columbia coho and chinook populations, the MHC class I variability

was similar to the variability found in microsatellites [17]. However, theiranalysis was based on genomic PCR analysis of single 1 and 2 domains andno distinction between expressed and non-expressed genes could be made.

Their PCR system led them to the possibly false assumption that genes with2 group I and II sequences can be expressed from di#erent loci. A problemwhen only 1 or 2 domains are analysed is that non-classical MHC class I loci

in rainbow trout have domains very similar to those in some Onmy-UBA

alleles. Besides theOnmy-UBAlocus at least two closely related non-classicalMHC class I loci (other than Onmy-UAA[23] which shows only low homology

with Onmy-UBA) can be detected, and the 1 domains of these loci may beindistinguishable from some Onmy-UBA sequences (unpublished observa-tions). Therefore, for MHC class I analysis of relatively small populations as

maintained in hatcheries, full length gene analysis is preferable, in order toavoid unnecessary complexity due to the existence of closely related non-

classical MHC class I sequences. For the screening of large populations, themethod of Miller et al., who rapidly analysed short PCR fragments bydenaturing gradient gel electrophoresis (DGGE), is certainly the bettermethod of choice; they succeeded in analysing samples from 20 000 coho and

chinook salmon [18].In the present study the Nikko and Donaldson rainbow trout strains

maintained in Japan were shown to have distinguishing di#erences in MHC

class I sequence lineages. Specifically, MHC class I sequences belonging tolineages Sal-MHCIa*-A and -E were common in the Nikko strain but not in theDonaldson strain. Although more than 30 classical MHC class I sequences

have been described for rainbow trout, probably representing alleles (seebelow), in the Nikko and Donaldson strains only five and four di#erentsequences were detected respectively. It must be recognised that these results

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pertain only to these strains as maintained in one hatchery, and that due to

inbreeding practices the Onmy-UBAalleles in fish from other hatcheries maybe di#erent. The variability in MHC class I genes is probably representativefor rainbow trout strains maintained in Japan, since the breeding practice

applied to maintain the strains used in this study is in general use throughoutthe country.The definitions used in this study (also in Aoyagi et al. [19]) for classical

MHC class I lineages in salmonids were based on similarities of1, 2 and 3domains. These distinctions seem to represent natural divisions for the 1 and2 domains, since all reported classical MHC class I sequences of salmonids fit

in easily. The 3 region is more problematic, since we could not find a divisionfor all salmonid 3 regions in which length, homology and UPGMA analysisagree, although these parameters match rather well when 3 domains of only

the rainbow trout sequences are compared (not shown). The transmembraneand cytoplasmic regions are not used in this definition, since natural-appearing lineages could not be detected and the functional importance of the

variability in this region is questionable. The lineage definition was based onindividual domains, rather than on overall homology, since recombination

events creating di#erent combinations of1 and 2 groups seem to have beena major cause of polymorphism. For example, the sequences Onmy-UBA*-101and -701 have a completely identical 1 region, but a very di#erent 2 region,

with di#erences beginning at the domain border [19]. And for exampleOnmy-UBA*601 [19]and Onmy-UA*A51 [30] have an identical 2 domain, buta very di#erent 1 domain. A probable reason for this pattern is that the intron

between the 1 and 2 domains is very large, which would stimulate recombi-nation events. This large intron size has not been proven, but is expectedbased on the genome analysis of the carp MHC class I sequence Cyca-12 [31]

and the fact that several groups, including ours, have been unsuccessful toamplify the complete intron at this location in Onmy-UBA.

The lineage definition was kept similar to that in Aoyagi et al.[19] in order

to be consistent, but in the future lineage definitions may need improve-ment with respect to the 3 domain. It is fascinating, however, that only threediscrete lengths of 3 domains are found for the growing number of salmonid

classical MHC class I sequences. The sequences of Atlantic salmon,brown trout, pink salmon and rainbow trout compared in Table 2 have 3

domains with C-termini lengths similar to Onmy-UBA*401 (no extension),Onmy-UBA*501 (6 aa extensions) or Onmy-UBA*701 (11 aa extensions) (Fig. 1)instead of continuous variation between absent and 11 aa extensions. Futureresearch is necessary to determine if there is a functional reason for these

discrete length di#erences, since there are presently no studies on otherspecies that show the same phenomenon.

The primer sets used in this study were developed to distinguish among

sequences of the lineages Sal-MHCIa*AF, but rainbow trout sequencesbelonging to the Sal-MHCIa*-G, -H and -K lineages would also have beenamplified (as indicated in the Materials and Methods section); therefore those

lineages are likely to be absent in the Nikko and Donaldson strain rainbowtrout investigated. A few of the primers used in the present study bindupstream or downstream from the reported rainbow trout sequences of


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lineages Sal-MHCIa*-I and -J (Table 2), and therefore it cannot be predicted if

they would have been amplified.Since the lineages definitions do not include precise sequences, and the

classical MHC class I sequences in salmonid species are highly variable,

lineage determination can never be performed by PCR alone. Nevertheless,the lineage-specific PCR system indicated inTable 1would amplify most of therainbow trout sequences published and give substantive clues regarding their

identity. Exceptions do occur, however, such as the Nikko strain fish forwhich the partialOnmy-UBA*1401 sequence could be amplified by the primerset pG-LPf/pI-3/TMr but not by the primer set pG-LPf/pG-3UTRr (Table 1).

As the forward primer binding to the leader peptide region is identical in bothprimer sets, the lack of amplification by the latter set is probably due to amutation in the 3UTR (where the reverse primer should bind). Di#erences

in the 3UTR have been previously observed: Onmy-UBA*401 [19] andOnmy-UBA*SP3 [30]are identical, except that Onmy-UBA*SP3 has a 274 bpinsertion in its 3UTR.

Expression from the rainbow trout locus Onmy-UBA had previously beenshown only for sequences of the lineages Sal-MHCIa*-A, -B, -C, and -D [19].

Since Onmy-UBA is most likely the only classical MHC class I locus inrainbow trout, we gave the new sequence belonging to the Sal-MHCIa*Flineage a name starting with Onmy-UBA, and followed the Onmy-UBA

designation of Shum et al. [23] (Genbank) for the detected sequence belong-ing to the lineage Sal-MHCIa*E. The fact that in this study only one or twosequences were amplified per individual from 59 fish, despite an intensive

search for presence of di#erent sequences, strongly supports the idea thatall classical MHC class I sequences are derived from the same locus.However, for one Donaldson strain fish, three sequences belonging to the

lineages Sal-MHCIa*-B, -D, and -F were obtained. This might be due totriploidy in this individual since it is not consistent with the other results,but further studies on locus identification of the Sal-MHCIa*F lineage are

necessary. Although the allomorph Onmy-UBA*401 has a somewhat di#erent1 domain, lacking tyrosine at position 59 (Fig. 1; [30]), locus identificationand expression analysis suggested that it functions as a classical MHC class

I molecule [19].From the nomenclature for their sequences released to GenBank (Table 2),

it appears that Shum et al. [23] share the view that there is most likely onlyone classical MHC class I locus. However, Miller et al. [17] and Hansenet al . [30] suggested that the di#erent classical MHC class I lineagesmight be derived from di#erent loci, and designated sequences accordingly

[17] or state that the designation is based on lineage rather than on locus[30]. This discussion on loci and lineages is disturbing clear nomenclatureshared by di#erent groups (as seen in Table 2), and should change in the

future.Despite the confusing nomenclature, all investigators seem to agree on

several important aspects of MHC class I molecules in rainbow trout: that

they are highly variable and that both their structure and the detection ofother genes involved in the MHC class I pathway [30] indicates that the MHCclass I molecules play an important role similar to their counterparts in higher

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vertebrates. This makes research on trout MHC class I genes an important

and promising field.In conclusion this study employed a convenient system to analyse

Onmy-UBA polymorphism in rainbow trout. The Nikko and Donaldson

rainbow trout strains appear to have di#

erent Onmy-UBA alleles. Futurestudies will address relationships between Onmy-UBA alleles and di#erencesin disease resistance.

This study was supported by the promotion of basic research activities forinnovative biosciences funded by Bio-oriented Technology Research AdvancementInstitution (BRAIN), Japan, and by postdoctoral fellowships for C. Xia and J. M.Dijkstra from the Science and Technology Agency of Japan. We thank Dr James D.Moore, Bodega Marine Laboratory, U.S.A. for his help to edit this manuscript.

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