BACKGROUND
adaptive immunity in jawed vertebrates is mediated by b cells and t cells, two lymphocyte subsets characterised by the expression of highly diverse repertoires of heterodimeric antigen-specific receptors – immunoglobulins  and t cell receptors .
immunoglobulins are formed from heavy  and light  chains and tr from either α and β chains  or γ and δ tr chains .
the variable domains of individual ig and tr chains are formed in lymphocyte precursors by somatic recombination of single discontiguous variable , diversity  and joining  genes selected from multiple genomic copies of these genes.
this recombination is directed by the products of the rag1/ <dig> genes which introduce double-strand breaks at recombination signal  sequences flanking v, d and j genes  <cit>  that are subsequently repaired by non-homologous joining of the cleaved vj elements.
after recombination the vj product is spliced to a relatively invariant constant  gene.
the different permutations of recombined vj genes used to form individual ig and tr chains generate diversity  that is further amplified by the activities of exonuclease and terminal deoxynucleotide transferase which together modify the germline nucleotide sequence at the vj junction .
together these mechanisms enable the generation of hugely diverse ig and tr repertoires – for example, the potential repertoire of unique human αβ tr has been calculated to be  <dig> <cit> .
highly diverse ig and tr repertoires are crucial to facilitating recognition of the vast array of antigens to which hosts may be exposed and are therefore considered critical to an effective adaptive immune system  <cit> .
the constituent genes used to construct tr chains are located in  <dig> distinct chromosomal loci - trb for trb, trg for trg and tra/trd for both tra and trd chains.
sequencing of murine and human tr loci has enabled the full complement of the tr genes in these species to be described  and allowed the organisation and regulation of these immunologically significant loci to be analysed .
additionally, inter-species and inter-loci comparisons have provided insights into how these loci have evolved .
between mammalian species the orthologous tr loci generally demonstrate conservation of overall structure – for example the organisation of genes in the trb locus is similar in humans  <cit> , mice  <cit> , cattle  <cit>  and dog  <cit> .
despite this conservation there are marked inter-species differences in both the total number of v genes and the relative representation of individual v gene subgroups  located in orthologous loci, indicating that dynamic evolutionary development of v gene germline repertoires has occurred post-speciation.
tr v genes follow a ‘birth and death’ model of evolution whereby new genes are generated by repeated duplication , of which some are retained whilst others are deleted or become pseudogenes following acquisition of deleterious mutations .
dramatic evolutionary modifications of functional v gene germline repertoires are assumed to reflect selective pressures exerted on the adaptive immune system and in turn result in beneficial changes in the expressed tr repertoires.
the tra/trd locus is unique in containing the tr gene elements for  <dig> tr chains.
in humans the tra/trd locus spans ~1 mb and contains  <dig> trav genes,  <dig> dual usage trav/trdv genes,  <dig> trdv,  <dig> trdd,  <dig> traj,  <dig> trdj and single trac and trdc genes, whilst in mice the tra/trd locus occupies ~ <dig>  mb and contains  <dig> trav,  <dig> dual usage trav/trdv genes,  <dig> trdv,  <dig> trdd,  <dig> traj,  <dig> trdj and single trac and trdc genes  <cit> .
comparative genomics show that the human and murine tra/trd loci share a similar gene organisation with most of the v gene segments located at the 5′ end of the locus and the other tra/trd genes and a single trdv gene with an inverted orientation located at the 3′ end  <cit> .
two studies have analysed the genomic sequence of the bovine tra/trd locus  <cit> .
however, these studies were largely restricted to cataloguing the repertoires of either trd  <cit>  or trav/trdv  <cit>  genes, and an annotation of all of the tr genes within the locus has yet to be completed.
furthermore, although a main finding in both studies was the dramatic expansion of the v gene repertoires , analysis of the duplication events that generated this hugely expanded germline repertoire, which could inform our understanding of how the bovine trav/trdv repertoire has evolved, have not been conducted.
in this study, we use the most recent assembly of the bovine genome  to re-examine the genomic sequence of the tra/trd locus.
we sought to complete a comprehensive annotation of the tra/trd gene repertoire and use this as a basis to fulfil our primary aim of examining the evolutionary development of the massive expansion of bovine trav/trdv genes.
by combining analysis of the genome assembly and tra/trd transcripts we present data indicating that i) the majority of the trav/trdv gene expansion is attributable to a complex series of duplications of multiple homology units , ii) expansion of some trav subgroups may have occurred ‘collaterally’, as a consequence of selection for expanded functional repertoires of other subgroups , and iii) the expressed tra and trd repertoires preferentially use different trav/trdv subgroups, suggesting tra and trd selection have made distinct contributions to the evolutionary development of the genomic trav/trdv repertoire.
RESULTS
annotation of the tra/d gene repertoire
a prerequisite of our study was accurate, up-to-date information on the bovine genomic tra/trd gene repertoire.
annotation of the umd <dig>  bovine genome assembly identified a total of  <dig> trav/trdv,  <dig> trdj,  <dig> trdd,  <dig> traj,  <dig> trac and  <dig> trdc genes.
all of these tr genes, with the exception of  <dig> trav/trdv genes, were located within a  <dig>  mb region of chromosome  <dig> .
phylogenetic analysis indicated the existence of  <dig> distinct bovine trav/trdv gene subgroups .
of these,  <dig> were orthologous to human trav subgroups and  <dig> to human trdv subgroups and therefore were provisionally assigned the corresponding numbers .
of the remaining  <dig> subgroups, one contained a single gene with  <dig> % nucleotide identity with murine trdv <dig> and another contained  <dig> genes showing  <dig> - <dig> % nucleotide identity with murine trav <dig> – these were designated as btrdvy and btravy respectively.
the other  <dig> subgroups lacked directly orthologous human or murine subgroups.
genes from  <dig> of these subgroups had been previously described as btrdv <dig> genes  <cit>  – we have re-assigned this subgroup as btrdvb <dig> to distinguish it from the bovine trdv <dig> subgroup.
subsequent cdna analyses  demonstrated that members of the final subgroup recombined with traj genes – we consequently designated this subgroup as btravx.
all but  <dig> of the trav/trdv subgroups  are multi-membered and several subgroups  have undergone dramatic expansion, containing more than  <dig> members .table  <dig>
the human and bovine  trav/trdv repertoires
the numbers of total, functional and pseudogenes in each trav/trdv subgroup in the germline human and bovine  repertoires are shown.
the percentage of genes in each subgroup that are functional has been calculated .
for the bovine trav/trdv subgroups, the number of genes for which only incomplete genomic sequence was available is also shown.
details of the human trav/trdv repertoire were taken from the imgt database .
although our findings are broadly consistent with the findings from previous annotations  <cit>  there are several features that make this annotation distinct: i) the number of trav/trdv genes identified is lower than in previous annotations , ii) several novel subgroups have been identified, iii) the full length  of all trav/trdv genes has been annotated , and iv) all of the tra/trd genes have been included.
notably only about half of the trav/trdv genes identified here had 100% sequence identity to genes identified in the btau_ <dig>  assembly .
the structure of the 3′ end of the bovine tra/trd locus shows syntenic conservation with that of humans and mice
the umd <dig>  assembly of the 3′ end of the bovine tra/trd locus appears to be essentially complete.
all trac, traj, trdc, trdj, trdd genes and the trdv <dig> gene  are located on  <dig> large contigs  separated by a gap of approximately  <dig> kb.
the organisation of the genes demonstrates a marked syntenic conservation between mouse, human and bovine .
synteny is maintained within the traj repertoire with individual bovine traj genes sharing a conserved order with their murine and human orthologues .
additionally there is conservation of important regulatory elements at the 3′ end of the tra/trd locus, with bovine sequences showing identity with the human/murine tra enhancer , trd enhancer , csb and tea located in syntenic positions .
in addition to gene content and organisation, the size of the 3′ end of tra/trd locus is similar across the  <dig> species , with the exception of the trdd region which in bovine is ~ <dig> kb – approximately 8× the size of the equivalent regions in the mouse and human tra/trd loci.figure  <dig>
conserved synteny of the 3′ end of the bovine, human and murine tra/trd loci.
a schematic representation shows the conserved inter-species organisation of tr genes and regulatory elements at the 3′ end of the tra/trd locus.
the approximate positions of trdd , trdj , traj , trdc and trac , the single inverted trdv gene  and regulatory elements  are shown.
the regulatory elements represented are – tra enhancer , trd enhancer , t early alpha  and conserved sequence block .
the length of the region is also conserved across the species, with the exception of the area occupied by trdd genes, which is much greater in the bovine tra/trd locus  than in the mouse or human .
the asterisk indicates the approximate location of the  <dig> kb gap between contigs daaa <dig>  and daaaa <dig> .
expansion of the bovine trav/trdv repertoire has predominantly occurred via duplication of homology units
the umd <dig>  assembly of the 5′ end of the locus is highly fragmented with the ~ <dig>  mb of sequence distributed across > <dig> separate contigs.
although this fragmentation precludes a description of the overall genomic structure of the 5′ end of the locus, regions of substantial contiguous sequence  enable examination of the organisation of sections of the trav/trdv region.
within these sections small areas demonstrating syntenic conservation with human trav/trdv genes were identified .
however, a more prominent feature of the trav/trdv gene organisation within these contigs was recurrent patterns of intercalation of genes from the multi-membered subgroups.
for example the ordered sequence of trav12-11-10- <dig> genes recurs  <dig> times in the umd <dig>  assembly , suggesting that replication of dna segments containing multiple trav/trdv genes has led to the formation of ‘homology units’.
detailed nucleotide identity analysis of these regions indicated the presence of  <dig> putative homology units incorporating between  <dig> and  <dig> trav/trdv genes and extending in size from ~ <dig> kb to > <dig> kb .table  <dig>
putative bovine trav/trdv gene homology units identified in the umd <dig>  assembly
for each homology unit the trav/trdv gene motif, the number of genes, the estimated minimal size and the number of replicons identified are shown.
due to the fragmentary nature of the umd <dig>  assembly many of the homology units presented here and many of the copies of the homology units are likely to be incomplete.
therefore the trav/trdv gene motif, number of trav/trdv genes and size of homology units should be considered a minimum.
similarly, we have rationalised the number of replicons for each homology unit to the minimum that could account for all of the partial replications observed in the umd <dig>  assembly and so this should also be considered a minimum.
for homology units  <dig>   <dig> and  <dig> no single copy covers the entirety of the homology unit and we have estimated its size by combining data from multiple copies; the number of genes in the most extensive copy of these homology units is indicated in parentheses in the ‘no.
of trav/trdv genes’ column.
in addition we have summarised where there is evidence in the umd <dig>  assembly of the trav/trdv gene content of copies of a homology unit differing due to either varying proportions of the homology unit being duplicated in different replicative iterations or post-replication insertion/deletion .
within the context of homology unit replication the expansion of some multi-membered subgroups appears evolutionarily simple.
for example, amplification of the trav <dig> and  <dig> subgroups is due to  <dig> tandem replications of homology unit  <dig>  resulting in a ~ <dig> kb region with alternating trav <dig> and trav <dig> genes at the 5′ end of the locus.
in contrast, expansion of other subgroups, especially those that have undergone prolific expansion, appears to be evolutionarily complex.
trdv <dig> subgroup members are found on  <dig> different homology units , intercalated with different permutations of genes including members of the trav <dig>   <dig>   <dig> and  <dig> subgroups.
the presence of multiple copies of genes from some subgroups  amplifies the expansive effect of homology unit replication on the size of these subgroups.
notably travx expansion is also largely a product of members being incorporated into multiple homology units  and the presence of multiple members of this subgroup being present in the homology units.
most of the homology units have undergone multiple replicative events  and in some cases variable proportions of the unit appear to have been duplicated  whilst others display evidence of post-replication deletion/insertion events modifying the gene content of some replicons.
for example, the  <dig> copies of homology unit  <dig> show disparity in the number of trav <dig> genes present .
the location of  <dig> out of the  <dig>  trav/trdv genes identified in the umd <dig>  assembly within homology units attests to replication of homology units being the principal mechanism employed in achieving the massively expanded bovine trav/trdv repertoire.
the multiplicity of identified homology units, the variation in replicon number and the modifications of homology unit content during and after replication suggest the evolutionary development of the bovine trav/trdv repertoire has been highly complex.
extensive duplication of the trdv <dig> subgroup has skewed the phylogenetic representation of the functional trav/trdv gene repertoire of cattle
the functional competency of v, d and j genes is dependent on the maintenance of a correct open reading frame , the presence of codons encoding certain critical amino acid residues and the presence of a rs.
in silico sequence analysis based on these parameters indicates that all trdd and trdj genes and  <dig> out of  <dig>  of traj genes are functional  and so the repertoire size of these genes is fairly comparable to that seen in mice and humans .table  <dig>
repertoire of functional tra/trd gene segments in the bovine , murine and human genomes
*for human and murine trav/trdv repertoires we represent the maximum number of potential functional genes .
the numbers in parentheses show the number of trav/trdv subgroups that include functional members.
of the  <dig> trav/trdv genes,  <dig>  were predicted to encode functional products and  <dig>  pseudogenes , whilst the functional competency of the remaining  <dig>  genes could not be assessed as the sequences were incomplete .
the predicted bovine functional trav/trdv repertoire incorporates genes from  <dig> subgroups and is approximately  <dig>  and  <dig> times larger than that of mice and humans respectively.
between multi-membered subgroups there is huge disparity in the proportion of genes that are functional - some having a high proportion of pseudogenes  whilst in others  every gene is predicted to be functional.
this extends to the massively expanded subgroups, with travx and trdv <dig> subgroups retaining a high proportion of functional genes  and trav <dig> having a low proportion .
notably members of subgroups with high proportions of pseudogenes are generally co-localised within homology units with genes from subgroups with low pseudogene proportions: for example many trav <dig>   <dig> and  <dig> pseudogenes are found with trdv <dig> genes on homology units 7– <dig> .
such juxtaposition suggests that homology unit replication due to selection for expansion of some subgroups  may have co-incidentally led to the expansion of other trav/trdv subgroups  for which there has been no evolutionary selection to generate/maintain an expanded functional genomic repertoire.
neighbour-joining analysis resolved functional murine, human and bovine trav/trdv genes into  <dig> monophylogenic groups .
in mice and humans there is marked inter-species similarity in the distribution between these monophylogenic groups, with groups  <dig>   <dig>   <dig> and  <dig> containing approximately 50%, 35%, 1% and 10% of the functional trav/trdv genes respectively in both species .
in contrast, the distribution of functional genes in bovine is different with a relative contraction of group  <dig>  and expansion of group  <dig> ; the latter due to the high number of trdv <dig> genes.
in contrast to mice and humans, in which the relative proportions of the  <dig> groups within the total gene repertoires  are similar to that observed in the functional repertoires, in bovine the representation of group  <dig> is higher  and that of group  <dig> is lower  in the total than in the functional repertoires.
thus, it is apparent that massive expansion of particular trav/trdv subgroups  combined with the preferential retention of functionality in some of these subgroups act synergistically to cause a profound phylogenetic shift in the repertoire of functional trav/trdv genes available for rearrangement of tra/trd chains.figure  <dig>
phylogenetic analysis of the repertoires of functional trav/trdv genes in mice, humans and cattle.
- neighbour-joining phylogenetic tree of all functional murine, human and bovine  trav/trdv genes.
analysis of the nucleotide sequence of the v-region  following pairwise deletion to remove gaps in the alignment.
the final dataset included  <dig> positions.
the sequence of btrbv3a was used to root the tree.
based on  <dig> boot strap replicates support for groups  <dig> ,  <dig>  and  <dig>  was high with pb >97%.
support for group  <dig>  was low  but examination of the data using upmga and minimum evolution models generated the same phylogenic groups, suggesting it was reliable .
h = human, b = bovine and m = murine.
percentage of the functional  and total  trav/trdv genes in humans, mice and cattle in the  <dig> phylogenetic groups defined from neighbour-joining analysis.
analysis of expressed tra and trd repertoires suggests preferential usage of particular trav/trdv subgroups
although the expressed bovine trd chain repertoire has been examined extensively in a number of studies , the published data on the expressed bovine tra chain repertoire is limited  <cit> .
a search of the ncbi est archive revealed a restricted number of entries for rearranged tra chains .
to facilitate a direct examination of the trav/trdv subgroups contributing to the expressed tra and trd chain repertoires, we designed a panel of trav/trdv subgroup-specific 5′ primers that could be used in conjunction with trac- and trdc-specific 3′ primers to amplify rearranged tra and trd chains from cdna.
sequencing of pcr amplicons generated from γδ t cells and αβ t cells demonstrated that; i) all of the trav subgroups predicted to include functional members  and also trdv <dig>  trdv <dig> and trdvb <dig> could rearrange to form functional tra chains and ii) genes from all of the trdv subgroups and trav <dig> can functionally rearrange with trdc.
thus, the expressed tra and trd chain repertoires can utilise genes from a minimum of  <dig> and  <dig> trav/trdv subgroups respectively.table  <dig>
summary of est and pcr data on bovine trav/trdv subgroup expression in re-arranged tra and trd chains
blast analysis of the ncbi bos taurus est archive  using the bovine trac and trdc sequences identified  <dig> subgroups participating in tra chain rearrangement  and  <dig> participating in trd rearrangement .
using a combination of bovine trav/trdv subgroup-specific and unbiased smart trac+ and trdc+ pcr   <dig> unique full or partial tra/trd chain transcripts were sequenced , providing a more comprehensive description of the breadth of trav/trdv subgroup usage in tra and trd transcripts.
amongst these sequences  <dig> unique trav/trdv gene sequences were identified , of which only  <dig>  matched any of the genomic sequences identified in the umd <dig>  assembly .
twenty-one of the trav/trdv gene sequences  were sufficiently dissimilar to genomic trav/trdv genes  to be considered as products of trav/trdv genes absent from the umd <dig>  assembly , whilst the other  <dig> sequences  showed >97% identity with genomic sequences and may either represent products of novel trav/trdv genes or allelic variants of the genes in the umd <dig>  assembly .
despite several attempts no amplification could be achieved with trav <dig> subgroup-specific primers and there was no est data supporting use of genes from this subgroup in rearranged tra or trd chains; consequently this is the only subgroup which was predicted to contain functional trav/trdv genes for which there is no evidence of expression in the tra/trd repertoires.
to determine at what frequencies the different trav/trdv subgroups were utilised in the expressed tra and trd repertoires, we used a smart race-pcr system that enables unbiased amplification of tr chain sequences  <cit>  to analyse tra and trd transcripts from αβ and γδ t cells of  <dig> mhc-disparate animals.
tra chain analysis showed utilisation of a diverse range of trav/trdv subgroups  in both animals .
although most subgroups were represented at low frequencies , in each animal there was a limited number of subgroups  that constituted a higher proportion of the repertoire.
although these highly represented subgroups generally differed between the  <dig> individuals, both animals showed a particularly high representation of trav <dig>  and travx .
comparison with the functional genomic trav/trdv gene repertoire revealed that these two trav/trdv subgroups, as well as trav <dig>   <dig> and  <dig>  were relatively over-represented in the expressed tra chain repertoire in both animals, whilst trav <dig> and trdv <dig> were under-represented .
with reference to the phylogenetic groups discussed above, groups  <dig> and  <dig> each accounted for approximately ~45% of the expressed tra chain sequences whilst the remaining ~10% used genes from group  <dig>  thus, in comparison to the genomic repertoire there is a preference for use of group  <dig> and  <dig> genes and bias against use of group  <dig> genes.figure  <dig>
usage of trav/trdv subgroups in the expressed tra and trd chain repertoires.
cdna from cd3+γδ-  and cd3+γδ+  populations isolated from the pbmc of  <dig> animals were subjected to unbiased smart trac+/ trdc+ pcr amplification and representative amplicons sequenced.
the subgroup of the trav/trdv gene expressed by each tra and trd chain sequence that was predicted from in silico analysis to be functional  was identified.
the percentage of the tra transcripts  and trd transcripts  that utilised genes from the different trav/trdv subgroups is shown  alongside the percentage of the genes in the genomic repertoire available for tra and trd rearrangement within each trav/trdv subgroup .
as anticipated from the trav/trdv subgroup-specific pcr, the trd chain repertoire in both animals showed a limited use of trav/trdv subgroups.
the trd chain sequences obtained from both animals were dominated by trdv <dig> and trdvb <dig>  which accounted for 90% and 10% of the sequences respectively in one animal and 60% and 32% in the other, with only single additional trdvy and trav <dig> sequences found in the second animal .
the dominance of trdv <dig> observed was consistent with results from previous analysis  <cit> .
with the exception of trdvy, all of the subgroups detected in the sequenced trd repertoire belong to phylogenetic group  <dig> and consequently the trd repertoire analysed here is almost completely restricted  to this group.
during this study,  <dig> and  <dig> unique functional tra and trd chains, utilizing a total of  <dig> distinct trav/trdv genes, were sequenced.
only  <dig>  of the expressed trav/trdv gene sequences were identical to a gene identified from the genome.
all of the remainder belonged to already identified subgroups;  <dig>  of them showed <97% identity to one or more genomic sequences  and thus most likely represent products of genes absent from the current assembly, whilst  <dig>  displayed nucleotide identities of between  <dig> - <dig> % to genomic sequences.
given the presence within the majority of multi-membered subgroups of genomic sequences sharing >97% identity , it is not possible to conclude whether these  <dig> trav/trdv sequences represent allelic variants of genes identified in the genome or products of trav/trdv genes absent from the current assembly.
in contrast, the expressed traj and trdj genes all corresponded to a genomic sequence – all  <dig> trdj genes and all functional traj genes with the exception of traj <dig>   <dig>  8– <dig>   <dig>   <dig>   <dig> were represented in the expressed tra/trd chains analysed.
discussion
gene duplication has been fundamental to the evolution of antigen-specific receptors, leading to the formation of the separate ig/tr loci  <cit>  and the expansion of the repertoire of d, j and most notably v genes within these loci.
inter-loci and inter-species comparisons demonstrate that the extent of duplication that has occurred in different ig/tr loci during their evolution varies dramatically.
the large number of bovine trav/trdv genes identified in previous genomic analyses  <cit>  demonstrates that duplication within the bovine tra/trd locus has been prolific.
in this study we have used genomic and cdna analyses to investigate the evolutionary basis for this dramatic expansion and its relationship with the expressed tra and trd chain repertoires.
the availability of an improved bovine genome assembly was critical to the study.
the umd <dig>  assembly is more accurate than the btau versions with a greater degree of completion, more contiguous sequence, corrections of snps and removal of erroneous segmental duplications  <cit> .
thus, although the catalogue of genes defined in our annotation is in general agreement with previous analyses  <cit>  there are substantive differences.
most significant of these are the identification of genes belonging to  <dig> novel bovine trav/trdv subgroups  and a reduction in the total number of trav/trdv genes identified .
comparative analyses indicated that btau_ <dig>  included many more trav/trdv genes showing 100% nucleotide identity, suggesting the discrepancy in trav/trdv gene number is largely attributable to the annotation of erroneous duplications in btau_ <dig> .
however, more pertinent to our study was the improved mapping of genes to the tra/trd locus on chromosome  <dig> and the larger fragments of contiguous tra/trd locus sequence.
despite these improvements, the umd <dig>  assembly, like the previous btau_ <dig>  and _ <dig>  assemblies, is i) derived from dna taken from a father-daughter pairing, which due to the highly polymorphic nature of trav/trdv genes, means that the potential for some proportion of the trav/trdv expansion to be artefactual cannot be excluded, and ii) highly fragmented, with cdna analysis suggesting that a substantial number of trav/trdv genes are still missing.
consequently, definition of the total bovine trav/trdv repertoire will be dependent on further work to obtain a completed assembly of the bovine tra/trd locus.
the high level of similarity in the organisation and gene content of the 3′ end of the bovine, murine and human tra/trd loci, which contains the traj, trdj, trac, trdc genes and the main regulatory elements, indicates that since the divergence of the primate, rodent and artiodactyl lineages  <dig> million years ago   <cit>  there has been strong conservative evolution of this region.
in direct contrast, the huge disparity in the numbers of trav/trdv genes located in the 5′ end of the human, murine and bovine tra/trd loci describes a divergent evolutionary development.
expansion of the human trav/trdv repertoire  has been limited with only  <dig> of the  <dig> trav/trdv subgroups having multiple-members and only trav <dig>  having > <dig> members.
in mice a higher proportion of subgroups are multi-membered  and these are in general larger  but the repertoire of  <dig> trav/trdv genes is still modest in comparison to the  <dig> genes in cattle.
duplication appears to be a general feature of bovine trav/trdv subgroups, with the majority  being multi-membered; however, the more remarkable feature is the extent of duplication within some of the subgroups – trdv <dig> has  <dig> members, and trav <dig>   <dig>   <dig>   <dig> and x all have > <dig> members.
in this respect there are clear parallels with the bovine trb locus where amplification of the trbv repertoire is characterised by the prodigious expansion of certain subgroups   <cit> .
the location of  <dig> % of bovine trav/trdv genes in homology units demonstrates that homology unit replication has been the principal mechanism for expansion of the repertoire.
in ig/tr loci expansion of the v gene repertoire by duplication of homology units incorporating genes from multiple subgroups is a common feature , although the extent to which this has occurred and its complexity differs markedly between loci.
the identification in the umd <dig>  assembly of  <dig> putative homology units, ranging in size from  <dig> to  <dig> kb and containing 2– <dig> trav/trdv genes, many of which had undergone repeated replication, shows that the expansion of the bovine trav/trdv repertoire has involved a particularly complex series of duplication events.
in direct contrast, the majority of the murine trav/trdv expansion is accounted for by a recent single duplication event in which a ~ <dig> kb segment containing  <dig> trav/trdv genes was replicated, whilst in the human tra/trd locus homology unit duplication has not been documented.
similarly, homology unit replication in other mammalian tr loci is much simpler than that described here.
due to the fragmented nature of the tra/trd locus in the umd <dig>  assembly, many of the homology unit copies analysed were incomplete and, with the exception of homology unit  <dig> , it was not possible to define the full extent of the homology units.
therefore, although our data clearly demonstrate the key role homology unit duplication has had in evolution of the bovine trav/trdv repertoire, analysis of improved, more contiguous assemblies of the bovine tra/trd locus will be required to fully appreciate the extent and complexity of homology unit duplication.
in addition to a prolific ‘birth’ rate, bovine trav/trdv genes also exhibit a high ‘death’ rate  compared to humans  and mice .
there was marked variation in the percentages of non-functional genes between the multi-membered subgroups; for example amongst the  <dig> largest subgroups it ranged from  <dig> %  to 20% .
although observed in ig v subgroups  <cit> , the high proportion of pseudogenes seen in several of the bovine trav/trdv subgroups, is rare in tr v subgroups.
based on the assumption that within the context of ‘birth-and-death’ evolution, homology unit replication is utilised to expand the functional trav/trdv gene repertoire, the frequent co-localisation within homology units of genes from low  and high  ‘death’ rate subgroups, suggests that expansion of some high ‘death’ rate trav/trdv subgroups may have occurred as a ‘collateral’ consequence of selection for juxtaposed low ‘death’ rate subgroups.
such ‘collateral’ expansion of v gene repertoires appears to be a feature of the evolution of other bovine ig/tr loci.
genomic analysis of the bovine iglh locus suggests that the non-functional ighv <dig> subgroup may have been expanded due to co-localisation of ighv <dig> and functional ighv <dig> genes in a duplicated homology unit  <cit>  and similarly in the igl locus many members of the expanded but predominantly non-functional iglv <dig> subgroup are found juxtaposed to functional iglv <dig> subgroup  <dig> genes  <cit> .
many bovine trav/trdv pseudogenes in equivalent/analogous locations within different replicates of a homology unit share a common mutational lesion  whilst conversely there are examples of analogous genes in homology unit replicates being a mixture of pseudogenes with non-identical lesions and functional genes .
this indicates that ‘collaterally’ expanded genes may either be pseudogenes prior to duplication or may, due to absence of selection, lose functionality following replication.
notably, trdv <dig> genes are co-located in  <dig> different homology units with different permutations of genes from  <dig> of the other massively expanded subgroups - trav <dig>   <dig>   <dig> and  <dig>  whilst the percentage of trdv <dig> pseudogenes  is much lower than the bovine trav/trdv average  that of the other subgroups is higher, ranging from  <dig> - <dig> %.
thus, under the model of ‘collateral’ expansion, replication of homology units 7– <dig> and the consequent expansion of subgroups trav <dig>   <dig>   <dig> and  <dig> may have largely been due to evolutionary selection for a large functional trdv <dig> repertoire.
as these  <dig> subgroups together account for nearly half of the annotated bovine trav/trdv genes, trdv <dig> selection may have been a critical force in the evolution of the bovine trav/trdv repertoire.
a unique feature of the trav/trdv locus is the co-localisation and sharing of v genes available for rearrangement of  <dig> distinct antigen-specific receptor chains expressed by  <dig> distinct lineages of lymphocytes.
consequently, to understand the evolution of the bovine trav/trdv gene repertoire in the context of the tr expressed by αβ and γδ t cells we undertook an analysis of the tra and trd transcripts expressed by these t cell subsets.
phylogenically defined trav/trdv gene orthologues don’t necessarily have the same capacity to partake in tra and trd rearrangements – for example mtrdv <dig> and htrav <dig> are phylogenetic orthologues but rearrange exclusively with trdc and trac respectively.
it was therefore necessary to first empirically evaluate trav/trdv subgroup usage in rearranged tra and trd transcripts using trav/trdv subgroup-specific and trac/trdc specific primers.
this analysis confirmed that each of the subgroups assigned as trav based on orthology to human trav subgroups were represented in rearranged tra chain transcripts  and that genes from all of the subgroups assigned as trdv were demonstrated to be represented in trd chain transcripts.
only genes from subgroups trav <dig>  trdv <dig>  trdv <dig> and trdvb <dig> were expressed in both rearranged tra and trd chains, extending the previous observations of dual usage of these subgroups  <cit> .
thus, in contrast to humans and mice, dual usage of trav/trdv genes in cattle makes a minimal contribution to diversifying the available trdv germline repertoire but instead has been employed to further diversify the germline repertoire of v genes available for tra rearrangement.
commensurate with the number of trav/trdv subgroups available for rearrangement, ‘unbiased’ transcript analysis demonstrated that the tra repertoire uses genes from a diverse range of trav/trdv subgroups, most of which are represented at <5%, whereas the trd repertoire predominantly expressed trdv <dig> genes, with minor contributions from a limited number of additional trav/trdv subgroups.
the dominance of trdv <dig> in the expressed trd repertoire suggests a functional advantage conferred by trd chains utilising trdv <dig> genes may have selected for the expansion of the trdv <dig> genomic repertoire and consequently been a major determinant in the replication of homology units 7– <dig>  conversely the under-representation of trav <dig>  genes in the expressed tra repertoire suggests functional selection through tra chain expression has not been a primary factor in replication of these homology units, but rather supports the concept that some trav subgroups have been expanded ‘collaterally’ as a consequence of being within the same homology units as trdv <dig>  however, over-representation of trav <dig> and travx gene usage in the expressed tra repertoire indicates that tra selection has driven replication of other homology units .
as such the transcript data indicate that tra and trd selection may have driven expansion of different components of the genomic bovine trav/trdv gene repertoire, with trd selection playing an apparently disproportionately large role.
the significance of trd selection in shaping evolutionary development of the functional bovine trav/trdv genomic repertoire is suggested by the marked increase in the proportion of group  <dig> genes  and concomitant decrease in the proportion of group  <dig> genes  seen in cattle compared to mice and humans.
although our studies have provided an insight into the potential mechanisms of the evolution of the bovine tra/trd locus, the biological factors that have led to the massive expansion of the trav/trdv repertoire remain a matter of conjecture.
cattle, along with other artiodactyl species have proportionally higher numbers of circulating γδ t cells than humans and mice; this is assumed to reflect a greater immunological role for γδ t cells in these species  <cit> .
a parallel but apparently evolutionarily independent expansion of the trdv <dig> subgroup in pigs  may signify that in artiodactyls common selective forces have led to high frequencies of γδ t cells and trdv <dig> gene duplication.
incomplete knowledge on the functional roles of γδ t cells and the ligands recognised by γδ tr limit our ability to construct a hypothesis of the evolutionary advantage conferred to artiodactyls by the expansion of trdv <dig> genes.
however, it is well recognised in mice and humans that subsets of γδ t cells defined by specific trdv  gene expression have distinct functional phenotypes .
in addition to roles such as cytotoxicity and ifnγ production, human trdv1+ γδ t cells have been shown to express an immune-regulatory phenotype following stimulation with a tr-independent agonist  <cit> .
recent evidence demonstrating that γδ t cells rather than foxp3+cd4+t cells contain the dominant regulatory t cell subset in cattle  <cit>  raises the intriguing possibility that the requirement for a substantial immune-regulatory t cell population may have been a factor leading to the expansion of trdv1+ γδ t cells in cattle and other artiodactyls.
CONCLUSIONS
a comprehensive understanding of the evolutionary development of complex multi-gene families, such as the v genes of ig/tr loci requires integration of a variety of data.
previous studies cataloguing the bovine trav/trdv genes have demonstrated the unprecedented size of this repertoire but have not addressed the underlying evolutionary processes leading to its expansion.
by combining data on gene characterisation, function, organisation and expression we have shown that the expansion of bovine trav/trdv genes has been achieved primarily through a highly complex series of homology unit replications.
we provide evidence indicating that as a consequence of this mode of replication a substantial proportion of the expanded trav gene repertoire may have occurred ‘collaterally’ as a consequence of positive selection for duplication of adjacent trav/trdv segments, most notably trdv <dig> genes.
as trd selection appears to be the main driver of trdv <dig> expansion, this suggests that in bovine  trd selection may have been a dominant factor in the genomic expansion of a number of trav subgroups.
such data provide an important novel perspective of the evolutionary dynamics that have led to the development of the dramatically expanded bovine trav/trdv repertoire and may have relevance to other ig/tr loci with enlarged v gene repertoires.

 adaptive immunity in jawed vertebrates is mediated by b cells and t cells, two lymphocyte subsets characterised by the expression of highly diverse repertoires of heterodimeric antigen-specific receptors – immunoglobulins and t cell receptors. immunoglobulins are formed from heavy and light chains and tr from either α and β chains or γ and δ tr chains. the variable domains of individual ig and tr chains are formed in lymphocyte precursors by somatic recombination of single discontiguous variable, diversity and joining genes selected from multiple genomic copies of these genes. this recombination is directed by the products of the rag1/ genes which introduce double-strand breaks at recombination signal sequences flanking v, d and j genes that are subsequently repaired by non-homologous joining of the cleaved vj elements. after recombination the vj product is spliced to a relatively invariant constant gene. the different permutations of recombined vj genes used to form individual ig and tr chains generate diversity that is further amplified by the activities of exonuclease and terminal deoxynucleotide transferase which together modify the germline nucleotide sequence at the vj junction. together these mechanisms enable the generation of hugely diverse ig and tr repertoires – for example, the potential repertoire of unique human αβ tr has been calculated to be. highly diverse ig and tr repertoires are crucial to facilitating recognition of the vast array of antigens to which hosts may be exposed and are therefore considered critical to an effective adaptive immune system. the constituent genes used to construct tr chains are located in distinct chromosomal loci - trb for trb, trg for trg and tra/trd for both tra and trd chains. sequencing of murine and human tr loci has enabled the full complement of the tr genes in these species to be described and allowed the organisation and regulation of these immunologically significant loci to be analysed. additionally, inter-species and inter-loci comparisons have provided insights into how these loci have evolved. between mammalian species the orthologous tr loci generally demonstrate conservation of overall structure – for example the organisation of genes in the trb locus is similar in humans, mice, cattle and dog. despite this conservation there are marked inter-species differences in both the total number of v genes and the relative representation of individual v gene subgroups located in orthologous loci, indicating that dynamic evolutionary development of v gene germline repertoires has occurred post-speciation. tr v genes follow a ‘birth and death’ model of evolution whereby new genes are generated by repeated duplication, of which some are retained whilst others are deleted or become pseudogenes following acquisition of deleterious mutations. dramatic evolutionary modifications of functional v gene germline repertoires are assumed to reflect selective pressures exerted on the adaptive immune system and in turn result in beneficial changes in the expressed tr repertoires. the tra/trd locus is unique in containing the tr gene elements for tr chains. in humans the tra/trd locus spans ~1 mb and contains trav genes, dual usage trav/trdv genes, trdv, trdd, traj, trdj and single trac and trdc genes, whilst in mice the tra/trd locus occupies ~  mb and contains trav, dual usage trav/trdv genes, trdv, trdd, traj, trdj and single trac and trdc genes. comparative genomics show that the human and murine tra/trd loci share a similar gene organisation with most of the v gene segments located at the 5′ end of the locus and the other tra/trd genes and a single trdv gene with an inverted orientation located at the 3′ end. two studies have analysed the genomic sequence of the bovine tra/trd locus. however, these studies were largely restricted to cataloguing the repertoires of either trd or trav/trdv genes, and an annotation of all of the tr genes within the locus has yet to be completed. furthermore, although a main finding in both studies was the dramatic expansion of the v gene repertoires, analysis of the duplication events that generated this hugely expanded germline repertoire, which could inform our understanding of how the bovine trav/trdv repertoire has evolved, have not been conducted. in this study, we use the most recent assembly of the bovine genome to re-examine the genomic sequence of the tra/trd locus. we sought to complete a comprehensive annotation of the tra/trd gene repertoire and use this as a basis to fulfil our primary aim of examining the evolutionary development of the massive expansion of bovine trav/trdv genes. by combining analysis of the genome assembly and tra/trd transcripts we present data indicating that i) the majority of the trav/trdv gene expansion is attributable to a complex series of duplications of multiple homology units, ii) expansion of some trav subgroups may have occurred ‘collaterally’, as a consequence of selection for expanded functional repertoires of other subgroups, and iii) the expressed tra and trd repertoires preferentially use different trav/trdv subgroups, suggesting tra and trd selection have made distinct contributions to the evolutionary development of the genomic trav/trdv repertoire.  annotation of the tra/d gene repertoire a prerequisite of our study was accurate, up-to-date information on the bovine genomic tra/trd gene repertoire. annotation of the umd bovine genome assembly identified a total of trav/trdv, trdj, trdd, traj, trac and trdc genes. all of these tr genes, with the exception of trav/trdv genes, were located within a  mb region of chromosome. phylogenetic analysis indicated the existence of distinct bovine trav/trdv gene subgroups. of these, were orthologous to human trav subgroups and to human trdv subgroups and therefore were provisionally assigned the corresponding numbers. of the remaining subgroups, one contained a single gene with % nucleotide identity with murine trdv and another contained genes showing - % nucleotide identity with murine trav – these were designated as btrdvy and btravy respectively. the other subgroups lacked directly orthologous human or murine subgroups. genes from of these subgroups had been previously described as btrdv genes – we have re-assigned this subgroup as btrdvb to distinguish it from the bovine trdv subgroup. subsequent cdna analyses demonstrated that members of the final subgroup recombined with traj genes – we consequently designated this subgroup as btravx. all but of the trav/trdv subgroups are multi-membered and several subgroups have undergone dramatic expansion, containing more than members.table the human and bovine trav/trdv repertoires the numbers of total, functional and pseudogenes in each trav/trdv subgroup in the germline human and bovine repertoires are shown. the percentage of genes in each subgroup that are functional has been calculated. for the bovine trav/trdv subgroups, the number of genes for which only incomplete genomic sequence was available is also shown. details of the human trav/trdv repertoire were taken from the imgt database. although our findings are broadly consistent with the findings from previous annotations there are several features that make this annotation distinct: i) the number of trav/trdv genes identified is lower than in previous annotations, ii) several novel subgroups have been identified, iii) the full length of all trav/trdv genes has been annotated, and iv) all of the tra/trd genes have been included. notably only about half of the trav/trdv genes identified here had 100% sequence identity to genes identified in the btau_ assembly. the structure of the 3′ end of the bovine tra/trd locus shows syntenic conservation with that of humans and mice the umd assembly of the 3′ end of the bovine tra/trd locus appears to be essentially complete. all trac, traj, trdc, trdj, trdd genes and the trdv gene are located on large contigs separated by a gap of approximately kb. the organisation of the genes demonstrates a marked syntenic conservation between mouse, human and bovine. synteny is maintained within the traj repertoire with individual bovine traj genes sharing a conserved order with their murine and human orthologues. additionally there is conservation of important regulatory elements at the 3′ end of the tra/trd locus, with bovine sequences showing identity with the human/murine tra enhancer, trd enhancer, csb and tea located in syntenic positions. in addition to gene content and organisation, the size of the 3′ end of tra/trd locus is similar across the species, with the exception of the trdd region which in bovine is ~ kb – approximately 8× the size of the equivalent regions in the mouse and human tra/trd loci.figure conserved synteny of the 3′ end of the bovine, human and murine tra/trd loci. a schematic representation shows the conserved inter-species organisation of tr genes and regulatory elements at the 3′ end of the tra/trd locus. the approximate positions of trdd, trdj, traj, trdc and trac, the single inverted trdv gene and regulatory elements are shown. the regulatory elements represented are – tra enhancer, trd enhancer, t early alpha and conserved sequence block. the length of the region is also conserved across the species, with the exception of the area occupied by trdd genes, which is much greater in the bovine tra/trd locus than in the mouse or human. the asterisk indicates the approximate location of the kb gap between contigs daaa and daaaa. expansion of the bovine trav/trdv repertoire has predominantly occurred via duplication of homology units the umd assembly of the 5′ end of the locus is highly fragmented with the ~  mb of sequence distributed across > separate contigs. although this fragmentation precludes a description of the overall genomic structure of the 5′ end of the locus, regions of substantial contiguous sequence enable examination of the organisation of sections of the trav/trdv region. within these sections small areas demonstrating syntenic conservation with human trav/trdv genes were identified. however, a more prominent feature of the trav/trdv gene organisation within these contigs was recurrent patterns of intercalation of genes from the multi-membered subgroups. for example the ordered sequence of trav12-11-10- genes recurs times in the umd assembly, suggesting that replication of dna segments containing multiple trav/trdv genes has led to the formation of ‘homology units’. detailed nucleotide identity analysis of these regions indicated the presence of putative homology units incorporating between and trav/trdv genes and extending in size from ~ kb to > kb.table putative bovine trav/trdv gene homology units identified in the umd assembly for each homology unit the trav/trdv gene motif, the number of genes, the estimated minimal size and the number of replicons identified are shown. due to the fragmentary nature of the umd assembly many of the homology units presented here and many of the copies of the homology units are likely to be incomplete. therefore the trav/trdv gene motif, number of trav/trdv genes and size of homology units should be considered a minimum. similarly, we have rationalised the number of replicons for each homology unit to the minimum that could account for all of the partial replications observed in the umd assembly and so this should also be considered a minimum. for homology units and no single copy covers the entirety of the homology unit and we have estimated its size by combining data from multiple copies; the number of genes in the most extensive copy of these homology units is indicated in parentheses in the ‘no. of trav/trdv genes’ column. in addition we have summarised where there is evidence in the umd assembly of the trav/trdv gene content of copies of a homology unit differing due to either varying proportions of the homology unit being duplicated in different replicative iterations or post-replication insertion/deletion. within the context of homology unit replication the expansion of some multi-membered subgroups appears evolutionarily simple. for example, amplification of the trav and subgroups is due to tandem replications of homology unit resulting in a ~ kb region with alternating trav and trav genes at the 5′ end of the locus. in contrast, expansion of other subgroups, especially those that have undergone prolific expansion, appears to be evolutionarily complex. trdv subgroup members are found on different homology units, intercalated with different permutations of genes including members of the trav and subgroups. the presence of multiple copies of genes from some subgroups amplifies the expansive effect of homology unit replication on the size of these subgroups. notably travx expansion is also largely a product of members being incorporated into multiple homology units and the presence of multiple members of this subgroup being present in the homology units. most of the homology units have undergone multiple replicative events and in some cases variable proportions of the unit appear to have been duplicated whilst others display evidence of post-replication deletion/insertion events modifying the gene content of some replicons. for example, the copies of homology unit show disparity in the number of trav genes present. the location of out of the trav/trdv genes identified in the umd assembly within homology units attests to replication of homology units being the principal mechanism employed in achieving the massively expanded bovine trav/trdv repertoire. the multiplicity of identified homology units, the variation in replicon number and the modifications of homology unit content during and after replication suggest the evolutionary development of the bovine trav/trdv repertoire has been highly complex. extensive duplication of the trdv subgroup has skewed the phylogenetic representation of the functional trav/trdv gene repertoire of cattle the functional competency of v, d and j genes is dependent on the maintenance of a correct open reading frame, the presence of codons encoding certain critical amino acid residues and the presence of a rs. in silico sequence analysis based on these parameters indicates that all trdd and trdj genes and out of of traj genes are functional and so the repertoire size of these genes is fairly comparable to that seen in mice and humans.table repertoire of functional tra/trd gene segments in the bovine, murine and human genomes *for human and murine trav/trdv repertoires we represent the maximum number of potential functional genes. the numbers in parentheses show the number of trav/trdv subgroups that include functional members. of the trav/trdv genes, were predicted to encode functional products and pseudogenes, whilst the functional competency of the remaining genes could not be assessed as the sequences were incomplete. the predicted bovine functional trav/trdv repertoire incorporates genes from subgroups and is approximately and times larger than that of mice and humans respectively. between multi-membered subgroups there is huge disparity in the proportion of genes that are functional - some having a high proportion of pseudogenes whilst in others every gene is predicted to be functional. this extends to the massively expanded subgroups, with travx and trdv subgroups retaining a high proportion of functional genes and trav having a low proportion. notably members of subgroups with high proportions of pseudogenes are generally co-localised within homology units with genes from subgroups with low pseudogene proportions: for example many trav and pseudogenes are found with trdv genes on homology units 7–. such juxtaposition suggests that homology unit replication due to selection for expansion of some subgroups may have co-incidentally led to the expansion of other trav/trdv subgroups for which there has been no evolutionary selection to generate/maintain an expanded functional genomic repertoire. neighbour-joining analysis resolved functional murine, human and bovine trav/trdv genes into monophylogenic groups. in mice and humans there is marked inter-species similarity in the distribution between these monophylogenic groups, with groups and containing approximately 50%, 35%, 1% and 10% of the functional trav/trdv genes respectively in both species. in contrast, the distribution of functional genes in bovine is different with a relative contraction of group and expansion of group; the latter due to the high number of trdv genes. in contrast to mice and humans, in which the relative proportions of the groups within the total gene repertoires are similar to that observed in the functional repertoires, in bovine the representation of group is higher and that of group is lower in the total than in the functional repertoires. thus, it is apparent that massive expansion of particular trav/trdv subgroups combined with the preferential retention of functionality in some of these subgroups act synergistically to cause a profound phylogenetic shift in the repertoire of functional trav/trdv genes available for rearrangement of tra/trd chains.figure phylogenetic analysis of the repertoires of functional trav/trdv genes in mice, humans and cattle. - neighbour-joining phylogenetic tree of all functional murine, human and bovine trav/trdv genes. analysis of the nucleotide sequence of the v-region following pairwise deletion to remove gaps in the alignment. the final dataset included positions. the sequence of btrbv3a was used to root the tree. based on boot strap replicates support for groups, and was high with pb >97%. support for group was low but examination of the data using upmga and minimum evolution models generated the same phylogenic groups, suggesting it was reliable. h = human, b = bovine and m = murine. percentage of the functional and total trav/trdv genes in humans, mice and cattle in the phylogenetic groups defined from neighbour-joining analysis. analysis of expressed tra and trd repertoires suggests preferential usage of particular trav/trdv subgroups although the expressed bovine trd chain repertoire has been examined extensively in a number of studies, the published data on the expressed bovine tra chain repertoire is limited. a search of the ncbi est archive revealed a restricted number of entries for rearranged tra chains. to facilitate a direct examination of the trav/trdv subgroups contributing to the expressed tra and trd chain repertoires, we designed a panel of trav/trdv subgroup-specific 5′ primers that could be used in conjunction with trac- and trdc-specific 3′ primers to amplify rearranged tra and trd chains from cdna. sequencing of pcr amplicons generated from γδ t cells and αβ t cells demonstrated that; i) all of the trav subgroups predicted to include functional members and also trdv trdv and trdvb could rearrange to form functional tra chains and ii) genes from all of the trdv subgroups and trav can functionally rearrange with trdc. thus, the expressed tra and trd chain repertoires can utilise genes from a minimum of and trav/trdv subgroups respectively.table summary of est and pcr data on bovine trav/trdv subgroup expression in re-arranged tra and trd chains blast analysis of the ncbi bos taurus est archive using the bovine trac and trdc sequences identified subgroups participating in tra chain rearrangement and participating in trd rearrangement. using a combination of bovine trav/trdv subgroup-specific and unbiased smart trac+ and trdc+ pcr unique full or partial tra/trd chain transcripts were sequenced, providing a more comprehensive description of the breadth of trav/trdv subgroup usage in tra and trd transcripts. amongst these sequences unique trav/trdv gene sequences were identified, of which only matched any of the genomic sequences identified in the umd assembly. twenty-one of the trav/trdv gene sequences were sufficiently dissimilar to genomic trav/trdv genes to be considered as products of trav/trdv genes absent from the umd assembly, whilst the other sequences showed >97% identity with genomic sequences and may either represent products of novel trav/trdv genes or allelic variants of the genes in the umd assembly. despite several attempts no amplification could be achieved with trav subgroup-specific primers and there was no est data supporting use of genes from this subgroup in rearranged tra or trd chains; consequently this is the only subgroup which was predicted to contain functional trav/trdv genes for which there is no evidence of expression in the tra/trd repertoires. to determine at what frequencies the different trav/trdv subgroups were utilised in the expressed tra and trd repertoires, we used a smart race-pcr system that enables unbiased amplification of tr chain sequences to analyse tra and trd transcripts from αβ and γδ t cells of mhc-disparate animals. tra chain analysis showed utilisation of a diverse range of trav/trdv subgroups in both animals. although most subgroups were represented at low frequencies, in each animal there was a limited number of subgroups that constituted a higher proportion of the repertoire. although these highly represented subgroups generally differed between the individuals, both animals showed a particularly high representation of trav and travx. comparison with the functional genomic trav/trdv gene repertoire revealed that these two trav/trdv subgroups, as well as trav and were relatively over-represented in the expressed tra chain repertoire in both animals, whilst trav and trdv were under-represented. with reference to the phylogenetic groups discussed above, groups and each accounted for approximately ~45% of the expressed tra chain sequences whilst the remaining ~10% used genes from group thus, in comparison to the genomic repertoire there is a preference for use of group and genes and bias against use of group genes.figure usage of trav/trdv subgroups in the expressed tra and trd chain repertoires. cdna from cd3+γδ- and cd3+γδ+ populations isolated from the pbmc of animals were subjected to unbiased smart trac+/ trdc+ pcr amplification and representative amplicons sequenced. the subgroup of the trav/trdv gene expressed by each tra and trd chain sequence that was predicted from in silico analysis to be functional was identified. the percentage of the tra transcripts and trd transcripts that utilised genes from the different trav/trdv subgroups is shown alongside the percentage of the genes in the genomic repertoire available for tra and trd rearrangement within each trav/trdv subgroup. as anticipated from the trav/trdv subgroup-specific pcr, the trd chain repertoire in both animals showed a limited use of trav/trdv subgroups. the trd chain sequences obtained from both animals were dominated by trdv and trdvb which accounted for 90% and 10% of the sequences respectively in one animal and 60% and 32% in the other, with only single additional trdvy and trav sequences found in the second animal. the dominance of trdv observed was consistent with results from previous analysis. with the exception of trdvy, all of the subgroups detected in the sequenced trd repertoire belong to phylogenetic group and consequently the trd repertoire analysed here is almost completely restricted to this group. during this study, and unique functional tra and trd chains, utilizing a total of distinct trav/trdv genes, were sequenced. only of the expressed trav/trdv gene sequences were identical to a gene identified from the genome. all of the remainder belonged to already identified subgroups; of them showed <97% identity to one or more genomic sequences and thus most likely represent products of genes absent from the current assembly, whilst displayed nucleotide identities of between - % to genomic sequences. given the presence within the majority of multi-membered subgroups of genomic sequences sharing >97% identity, it is not possible to conclude whether these trav/trdv sequences represent allelic variants of genes identified in the genome or products of trav/trdv genes absent from the current assembly. in contrast, the expressed traj and trdj genes all corresponded to a genomic sequence – all trdj genes and all functional traj genes with the exception of traj 8– were represented in the expressed tra/trd chains analysed. discussion gene duplication has been fundamental to the evolution of antigen-specific receptors, leading to the formation of the separate ig/tr loci and the expansion of the repertoire of d, j and most notably v genes within these loci. inter-loci and inter-species comparisons demonstrate that the extent of duplication that has occurred in different ig/tr loci during their evolution varies dramatically. the large number of bovine trav/trdv genes identified in previous genomic analyses demonstrates that duplication within the bovine tra/trd locus has been prolific. in this study we have used genomic and cdna analyses to investigate the evolutionary basis for this dramatic expansion and its relationship with the expressed tra and trd chain repertoires. the availability of an improved bovine genome assembly was critical to the study. the umd assembly is more accurate than the btau versions with a greater degree of completion, more contiguous sequence, corrections of snps and removal of erroneous segmental duplications. thus, although the catalogue of genes defined in our annotation is in general agreement with previous analyses there are substantive differences. most significant of these are the identification of genes belonging to novel bovine trav/trdv subgroups and a reduction in the total number of trav/trdv genes identified. comparative analyses indicated that btau_ included many more trav/trdv genes showing 100% nucleotide identity, suggesting the discrepancy in trav/trdv gene number is largely attributable to the annotation of erroneous duplications in btau_. however, more pertinent to our study was the improved mapping of genes to the tra/trd locus on chromosome and the larger fragments of contiguous tra/trd locus sequence. despite these improvements, the umd assembly, like the previous btau_ and _ assemblies, is i) derived from dna taken from a father-daughter pairing, which due to the highly polymorphic nature of trav/trdv genes, means that the potential for some proportion of the trav/trdv expansion to be artefactual cannot be excluded, and ii) highly fragmented, with cdna analysis suggesting that a substantial number of trav/trdv genes are still missing. consequently, definition of the total bovine trav/trdv repertoire will be dependent on further work to obtain a completed assembly of the bovine tra/trd locus. the high level of similarity in the organisation and gene content of the 3′ end of the bovine, murine and human tra/trd loci, which contains the traj, trdj, trac, trdc genes and the main regulatory elements, indicates that since the divergence of the primate, rodent and artiodactyl lineages million years ago there has been strong conservative evolution of this region. in direct contrast, the huge disparity in the numbers of trav/trdv genes located in the 5′ end of the human, murine and bovine tra/trd loci describes a divergent evolutionary development. expansion of the human trav/trdv repertoire has been limited with only of the trav/trdv subgroups having multiple-members and only trav having > members. in mice a higher proportion of subgroups are multi-membered and these are in general larger but the repertoire of trav/trdv genes is still modest in comparison to the genes in cattle. duplication appears to be a general feature of bovine trav/trdv subgroups, with the majority being multi-membered; however, the more remarkable feature is the extent of duplication within some of the subgroups – trdv has members, and trav and x all have > members. in this respect there are clear parallels with the bovine trb locus where amplification of the trbv repertoire is characterised by the prodigious expansion of certain subgroups. the location of % of bovine trav/trdv genes in homology units demonstrates that homology unit replication has been the principal mechanism for expansion of the repertoire. in ig/tr loci expansion of the v gene repertoire by duplication of homology units incorporating genes from multiple subgroups is a common feature, although the extent to which this has occurred and its complexity differs markedly between loci. the identification in the umd assembly of putative homology units, ranging in size from to kb and containing 2– trav/trdv genes, many of which had undergone repeated replication, shows that the expansion of the bovine trav/trdv repertoire has involved a particularly complex series of duplication events. in direct contrast, the majority of the murine trav/trdv expansion is accounted for by a recent single duplication event in which a ~ kb segment containing trav/trdv genes was replicated, whilst in the human tra/trd locus homology unit duplication has not been documented. similarly, homology unit replication in other mammalian tr loci is much simpler than that described here. due to the fragmented nature of the tra/trd locus in the umd assembly, many of the homology unit copies analysed were incomplete and, with the exception of homology unit, it was not possible to define the full extent of the homology units. therefore, although our data clearly demonstrate the key role homology unit duplication has had in evolution of the bovine trav/trdv repertoire, analysis of improved, more contiguous assemblies of the bovine tra/trd locus will be required to fully appreciate the extent and complexity of homology unit duplication. in addition to a prolific ‘birth’ rate, bovine trav/trdv genes also exhibit a high ‘death’ rate compared to humans and mice. there was marked variation in the percentages of non-functional genes between the multi-membered subgroups; for example amongst the largest subgroups it ranged from % to 20%. although observed in ig v subgroups, the high proportion of pseudogenes seen in several of the bovine trav/trdv subgroups, is rare in tr v subgroups. based on the assumption that within the context of ‘birth-and-death’ evolution, homology unit replication is utilised to expand the functional trav/trdv gene repertoire, the frequent co-localisation within homology units of genes from low and high ‘death’ rate subgroups, suggests that expansion of some high ‘death’ rate trav/trdv subgroups may have occurred as a ‘collateral’ consequence of selection for juxtaposed low ‘death’ rate subgroups. such ‘collateral’ expansion of v gene repertoires appears to be a feature of the evolution of other bovine ig/tr loci. genomic analysis of the bovine iglh locus suggests that the non-functional ighv subgroup may have been expanded due to co-localisation of ighv and functional ighv genes in a duplicated homology unit and similarly in the igl locus many members of the expanded but predominantly non-functional iglv subgroup are found juxtaposed to functional iglv subgroup genes. many bovine trav/trdv pseudogenes in equivalent/analogous locations within different replicates of a homology unit share a common mutational lesion whilst conversely there are examples of analogous genes in homology unit replicates being a mixture of pseudogenes with non-identical lesions and functional genes. this indicates that ‘collaterally’ expanded genes may either be pseudogenes prior to duplication or may, due to absence of selection, lose functionality following replication. notably, trdv genes are co-located in different homology units with different permutations of genes from of the other massively expanded subgroups - trav and whilst the percentage of trdv pseudogenes is much lower than the bovine trav/trdv average that of the other subgroups is higher, ranging from - %. thus, under the model of ‘collateral’ expansion, replication of homology units 7– and the consequent expansion of subgroups trav and may have largely been due to evolutionary selection for a large functional trdv repertoire. as these subgroups together account for nearly half of the annotated bovine trav/trdv genes, trdv selection may have been a critical force in the evolution of the bovine trav/trdv repertoire. a unique feature of the trav/trdv locus is the co-localisation and sharing of v genes available for rearrangement of distinct antigen-specific receptor chains expressed by distinct lineages of lymphocytes. consequently, to understand the evolution of the bovine trav/trdv gene repertoire in the context of the tr expressed by αβ and γδ t cells we undertook an analysis of the tra and trd transcripts expressed by these t cell subsets. phylogenically defined trav/trdv gene orthologues don’t necessarily have the same capacity to partake in tra and trd rearrangements – for example mtrdv and htrav are phylogenetic orthologues but rearrange exclusively with trdc and trac respectively. it was therefore necessary to first empirically evaluate trav/trdv subgroup usage in rearranged tra and trd transcripts using trav/trdv subgroup-specific and trac/trdc specific primers. this analysis confirmed that each of the subgroups assigned as trav based on orthology to human trav subgroups were represented in rearranged tra chain transcripts and that genes from all of the subgroups assigned as trdv were demonstrated to be represented in trd chain transcripts. only genes from subgroups trav trdv trdv and trdvb were expressed in both rearranged tra and trd chains, extending the previous observations of dual usage of these subgroups. thus, in contrast to humans and mice, dual usage of trav/trdv genes in cattle makes a minimal contribution to diversifying the available trdv germline repertoire but instead has been employed to further diversify the germline repertoire of v genes available for tra rearrangement. commensurate with the number of trav/trdv subgroups available for rearrangement, ‘unbiased’ transcript analysis demonstrated that the tra repertoire uses genes from a diverse range of trav/trdv subgroups, most of which are represented at <5%, whereas the trd repertoire predominantly expressed trdv genes, with minor contributions from a limited number of additional trav/trdv subgroups. the dominance of trdv in the expressed trd repertoire suggests a functional advantage conferred by trd chains utilising trdv genes may have selected for the expansion of the trdv genomic repertoire and consequently been a major determinant in the replication of homology units 7– conversely the under-representation of trav genes in the expressed tra repertoire suggests functional selection through tra chain expression has not been a primary factor in replication of these homology units, but rather supports the concept that some trav subgroups have been expanded ‘collaterally’ as a consequence of being within the same homology units as trdv however, over-representation of trav and travx gene usage in the expressed tra repertoire indicates that tra selection has driven replication of other homology units. as such the transcript data indicate that tra and trd selection may have driven expansion of different components of the genomic bovine trav/trdv gene repertoire, with trd selection playing an apparently disproportionately large role. the significance of trd selection in shaping evolutionary development of the functional bovine trav/trdv genomic repertoire is suggested by the marked increase in the proportion of group genes and concomitant decrease in the proportion of group genes seen in cattle compared to mice and humans. although our studies have provided an insight into the potential mechanisms of the evolution of the bovine tra/trd locus, the biological factors that have led to the massive expansion of the trav/trdv repertoire remain a matter of conjecture. cattle, along with other artiodactyl species have proportionally higher numbers of circulating γδ t cells than humans and mice; this is assumed to reflect a greater immunological role for γδ t cells in these species. a parallel but apparently evolutionarily independent expansion of the trdv subgroup in pigs may signify that in artiodactyls common selective forces have led to high frequencies of γδ t cells and trdv gene duplication. incomplete knowledge on the functional roles of γδ t cells and the ligands recognised by γδ tr limit our ability to construct a hypothesis of the evolutionary advantage conferred to artiodactyls by the expansion of trdv genes. however, it is well recognised in mice and humans that subsets of γδ t cells defined by specific trdv gene expression have distinct functional phenotypes. in addition to roles such as cytotoxicity and ifnγ production, human trdv1+ γδ t cells have been shown to express an immune-regulatory phenotype following stimulation with a tr-independent agonist. recent evidence demonstrating that γδ t cells rather than foxp3+cd4+t cells contain the dominant regulatory t cell subset in cattle raises the intriguing possibility that the requirement for a substantial immune-regulatory t cell population may have been a factor leading to the expansion of trdv1+ γδ t cells in cattle and other artiodactyls.  a comprehensive understanding of the evolutionary development of complex multi-gene families, such as the v genes of ig/tr loci requires integration of a variety of data. previous studies cataloguing the bovine trav/trdv genes have demonstrated the unprecedented size of this repertoire but have not addressed the underlying evolutionary processes leading to its expansion. by combining data on gene characterisation, function, organisation and expression we have shown that the expansion of bovine trav/trdv genes has been achieved primarily through a highly complex series of homology unit replications. we provide evidence indicating that as a consequence of this mode of replication a substantial proportion of the expanded trav gene repertoire may have occurred ‘collaterally’ as a consequence of positive selection for duplication of adjacent trav/trdv segments, most notably trdv genes. as trd selection appears to be the main driver of trdv expansion, this suggests that in bovine trd selection may have been a dominant factor in the genomic expansion of a number of trav subgroups. such data provide an important novel perspective of the evolutionary dynamics that have led to the development of the dramatically expanded bovine trav/trdv repertoire and may have relevance to other ig/tr loci with enlarged v gene repertoires. 
annotation of the umd assembly identified trav/trdv genes, trdj, trdd, traj and single trac and trdc genes, most of which were located within a  mb region of chromosome most of the trav/trdv subgroups have multiple members and several have undergone dramatic expansion, most notably trdv. previous studies have demonstrated that the bovine tra/trd locus contains an exceptionally large number of trav/trdv genes. such data demonstrate how integration of genomic and transcript data can provide a more nuanced appreciation of the evolutionary dynamics that have led to the dramatically expanded bovine trav/trdv repertoire. wide variation in the proportion of pseudogenes within individual subgroups, suggest that differential ‘birth’ and ‘death’ rates have been used to form a functional bovine trav/trdv repertoire which is phylogenetically distinct from that of humans and mice. transcript analysis was used to define the trav/trdv subgroups available for recombination of tra and trd chains and demonstrated preferential usage of different subgroups by the expressed tra and trd repertoires, indicating that tra and trd selection have had distinct impacts on the evolution of the trav/trdv repertoire. both tra and trd selection have contributed to the evolution of the bovine trav/trdv repertoire. frequent co-localisation within homology units of genes from subgroups with low and high pseudogene proportions suggest that replication of homology units driven by evolutionary selection for the former may have led to a ‘collateral’ expansion of the latter. however, our data suggest that due to homology unit duplication trd selection for trdv subgroup expansion may have substantially contributed to the genomic expansion of several trav subgroups. the tra/trd locus contains the genes for vj somatic rearrangement of tra and trd chains expressed by αβ and γδ t cells respectively. in this study we combine genomic and transcript analysis to provide insights into the evolutionary development of the bovine tra/trd locus and the remarkable trav/trdv gene repertoire. the expansion of the bovine trav/trdv gene repertoire has predominantly been achieved through a complex series of homology unit replications.
 the tra/trd locus contains the genes for vj somatic rearrangement of tra and trd chains expressed by αβ and γδ t cells respectively. previous studies have demonstrated that the bovine tra/trd locus contains an exceptionally large number of trav/trdv genes. in this study we combine genomic and transcript analysis to provide insights into the evolutionary development of the bovine tra/trd locus and the remarkable trav/trdv gene repertoire.  annotation of the umd assembly identified trav/trdv genes, trdj, trdd, traj and single trac and trdc genes, most of which were located within a  mb region of chromosome most of the trav/trdv subgroups have multiple members and several have undergone dramatic expansion, most notably trdv. wide variation in the proportion of pseudogenes within individual subgroups, suggest that differential ‘birth’ and ‘death’ rates have been used to form a functional bovine trav/trdv repertoire which is phylogenetically distinct from that of humans and mice. the expansion of the bovine trav/trdv gene repertoire has predominantly been achieved through a complex series of homology unit replications. frequent co-localisation within homology units of genes from subgroups with low and high pseudogene proportions suggest that replication of homology units driven by evolutionary selection for the former may have led to a ‘collateral’ expansion of the latter. transcript analysis was used to define the trav/trdv subgroups available for recombination of tra and trd chains and demonstrated preferential usage of different subgroups by the expressed tra and trd repertoires, indicating that tra and trd selection have had distinct impacts on the evolution of the trav/trdv repertoire.  both tra and trd selection have contributed to the evolution of the bovine trav/trdv repertoire. however, our data suggest that due to homology unit duplication trd selection for trdv subgroup expansion may have substantially contributed to the genomic expansion of several trav subgroups. such data demonstrate how integration of genomic and transcript data can provide a more nuanced appreciation of the evolutionary dynamics that have led to the dramatically expanded bovine trav/trdv repertoire. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords bovinetra/trd locustrduplicationevolutionissue-copyright-statement© the author 2014 