BACKGROUND
diverse αβtr repertoires are crucial to the maintenance of effective t cell-mediated immunity  <cit> .
estimates based on direct measurement indicate that in humans and mice individuals express a repertoire of approximately  <dig> ×  <dig>  <cit>  and  <dig> ×  <dig>  <cit>  unique αβtrs respectively.
as with the other antigen-specific receptors  diversity is generated in lymphocytic precursors by somatic recombination of discontiguous variable , diversity  and joining  genes to form the membrane-distal variable domains.
diversity is derived from both the different permutations of vj genes used to form the tra and trb chains expressed by individual thymocytes  and also by the activity of terminal deoxynucleotide transferase and exonuclease at the vj junction during recombination .
consequently, much of the diversity is focused in the third complementarity determining region , which is encoded by the vj junction and forms the most intimate association with the antigenic peptide component of the peptide-mhc  ligand of αβtrs, whereas the cdr <dig> and cdr <dig> of the tra and trb chains, that predominantly interact with the mhc, are encoded within the germline v genes  <cit> .
trb chain genes are located in the trb locus, which in humans is ~ <dig> kb long and situated on chromosome  <dig> and in mice is ~ <dig> kb and located on chromosome  <dig>  <cit> .
in both species, the organisation of trb genes is similar, with a library of trbv genes positioned at the 5' end and  <dig> djc clusters  followed by a single trbv gene with an inverted transcriptional orientation located at the 3'end  <cit> .
the germline repertoire of trbv genes in humans is composed of  <dig> genes belonging to  <dig> subgroups , whilst in mice the repertoire comprises  <dig> genes belonging to  <dig> subgroups  <cit>  the disparity between the number of trbv genes in the  <dig> species is the result of multiple duplication events within the human trb locus, most of which have involved tandem duplication of blocks of dna  containing genes from more than one subgroup  <cit> .
vj recombination is initiated by site-specific dna cleavage at recombination signal sequences  mediated by enzymes encoded by recombination activating genes   <dig> and  <dig>  <cit> .
rss comprise conserved heptamer and nonamer sequences separated by spacers of either  <dig> bp  or  <dig> bp .
correct vj assembly is achieved as recombination can only occur between genes flanked with rs of dissimilar length  and direct trbv/trbj recombination is prohibited by the 'beyond 12/23' phenomenon  <cit> .
as with other antigen-specific receptor loci, recombination in the trb locus is under strict lineage-, stage- and allele-specific regulation associated with control of rag accessibility to rss mediated through alterations in chromatin structure   <cit> .
numerous studies have shown that both the trb enhancer  and transcriptional promoters within the trb locus serve as rag accessibility control elements, playing a critical role in regulating chromatin structure and therefore recombination of trb genes  <cit> .
current knowledge of the trb gene repertoires of agriculturally important artiodactyl species  is limited.
published analyses of rearranged trb transcripts have demonstrated the expression of  <dig> trbv subgroups in pigs  <cit> ,  <dig> subgroups in sheep  <cit>  and  <dig> subgroups in cattle, some of which have undergone extensive duplication  <cit> .
information on the genomic organisation of the trb loci is predominantly restricted to the djc region, which in the pig was found to be composed of  <dig> tandemly arranged djc clusters  <cit>  but in sheep contained  <dig> tandemly arranged djc clusters  <cit> .
preliminary analysis of a bac clone corresponding to part of the djc region indicates that in cattle the djc region may also consist of  <dig> djc clusters  <cit> .
sequencing of the complete trb loci in human and mice allowed the repertoire of trb genes in these species to be fully characterised and also permitted analysis of the organisation, regulation and evolution of this immunologically important locus  <cit> .
in this study we have used the sequence of the third bovine genome assembly  to further study the bovine trb repertoire and trb locus.
although the sequence of the trb locus is incomplete, the results reveal that duplication within the locus has been prolific leading to a massive expansion of trbv gene numbers and the generation of a third djc cluster.
furthermore, the analysis shows that the genomic organisation of the trb locus and the non-coding elements that regulate trb expression are highly conserved in cattle when compared to that of humans and mice.
RESULTS
extensive duplication has generated a large germline repertoire of bovine trbv genes
a total of  <dig> trbv genes, distributed over  <dig> scaffolds was identified in btau_ <dig>  .
consistent with data from fluorescent in situ hybridisation studies  <cit> , the majority of the trbv genes were located on  <dig> scaffolds  mapped to chromosome  <dig>  whilst the remaining genes were located on  <dig> scaffolds  which have not been assigned a chromosomal location.
within the scaffolds are several regions of undetermined sequence, including large areas of ~ <dig> kb and ~ <dig> kb on chr <dig> .
<dig> and chr <dig> .
<dig> respectively.
each trbv gene is composed of i) a short leader  exon, generally of ~ <dig> bp, ii) a single intron of between ~ <dig> and ~ <dig> bp and iii) a variable  exon of ~ <dig> bp, immediately flanked at the 3'end with a 23-rs.
comparison of the nucleotide sequence of each of the bovine trbv genes with human trbv gene sequences, revealed maximum levels of similarity between the species ranging from  <dig> % to  <dig> % for all except one of the bovine trbv genes.
on the basis of these results, bovine trbv genes were considered orthologues of their most similar human counterpart and were assigned to subgroups named according to the orthologous human subgroup .
the single bovine trbv gene that lacked significant homology to any of the human trbv genes displayed  <dig> % identity with the murine trbv <dig> gene  and was placed in subgroup trbvx.
the subgroups thus established generally adhered to the definition of members within a subgroup exhibiting > 75% nucleotide sequence identity.
however, the single member of the trbv <dig> subgroup displayed > 75% identity to all of the trbv <dig> genes and the identity between members of the trbv <dig> and trbv <dig> subgroups was often > 75% .
conversely a single member of the trbv <dig> subgroup  showed only  <dig> – <dig> % nucleotide identity with the other members of this subgroup.
the total and functional repertoire of trbv genes in the human trb locus and in the third assembly of the bovine genome as well as the bovine trbv gene repertoire identified from cdna analysis.
details of the human repertoire are taken from the imgt database .
*denotes variation in germline repertoire due to insertion/deletion polymorphisms and † denotes trbv genes which have alternative functional and non-functional alleles .
of the  <dig> bovine subgroups present in the genome assembly,  <dig> have multiple members.
subgroups trbv <dig>   <dig> and  <dig> have all undergone substantial expansion, having  <dig>   <dig> and  <dig> members respectively – together representing 68% of the total btau_ <dig>  trbv gene repertoire.
southern blot analysis corroborates the presence of large numbers of trbv <dig> and  <dig> genes in the genome .
a prominent feature of the genomic organisation of trbv genes  is that members of expanded subgroups are generally intercalated with members of other expanded subgroups in a recurrent pattern.
thus, a  <dig> kb region of chr <dig> .
<dig> and virtually all of scaffold chrun.
<dig>  are composed of alternating trbv <dig> and  <dig> genes  obtained in southern blots of genomic dna when hybridised with trbv9- and trbv6-specific probes in figure 1), whilst the 3'end of chr <dig> .
<dig> and the 5' end of chr <dig> .
<dig> contain repeated units comprising trbv <dig>   <dig>   <dig> and  <dig> genes.
dot-plot analyses indicate that this organisation has arisen through a series of complex tandem duplication events within the regions in which trbv <dig> and  <dig> genes and trbv <dig>   <dig>   <dig> and  <dig> genes are located .
six homology units, ranging in size from ~ <dig> kb to ~ <dig> kb and encompassing from  <dig> to  <dig> trbv genes were identified.
three of these homology units  have undergone multiple  duplications: variation in the length of the different copies of these homology units , suggests that either i) distinct iterations of a duplication event have involved different components of the homology unit or ii) the different copies have been subject to different post-duplication deletions.
the levels of nucleotide identity between trbv genes in corresponding positions in homology units is frequently high:  <dig> pairs of trbv <dig> genes,  <dig> pairs of trbv <dig> and  <dig> pair each of trbv <dig> and trbv <dig> have identical coding sequences whilst  <dig> pair of trbv <dig> genes and  <dig> pairs of trbv <dig> as well as  <dig> triplets of trbv <dig> and  <dig> triplets of trbv <dig> genes have > 97% sequence identity in the coding region.
duplication has expanded the repertoire of trbd, trbj and trbc genes in the bovine genome
a total of  <dig> trbd,  <dig> trbj and  <dig> trbc genes were identified in the assembly .
these genes were all located within a ~ <dig> kb region of scaffold chr <dig> .
<dig> and organised into  <dig> tandemly arranged clusters, each of ~ <dig> kb length and composed of a single trbd gene, 5- <dig> trbj genes and a single trbc gene .
dot-plot analysis reveals the presence of a third djc cluster is attributable to duplication of a ~ <dig> kb region, one copy of which incorporates trbc <dig>  trbd <dig> and the trbj <dig> cluster whilst the other copy incorporates trbc <dig>  trbd <dig> and the trbj <dig> cluster .
numerous interruptions in the line representing the duplicated region indicate that there has been significant post-duplication deletion/insertion related modification of the duplicated region.
the nucleotide and deduced amino acid sequence of the  <dig> trbd and  <dig> trbj genes as well as the flanking rs are shown in figure 5a and 5b respectively.
the  <dig> bp  or  <dig> bp  trbd genes are g-rich and encode at least one glycine in all  <dig> potential reading frames with the exception of the 3rd reading frame of trbd <dig>  the trbj genes range in size from  <dig> bp to  <dig> bp in length and all encode the canonical fgxg amino acid motif that defines trbj genes.
as with all mammalian trbc genes so far characterised, bovine trbc <dig> and trbc <dig> genes are composed of  <dig> exons,  <dig> introns and a 3'utr region.
the structure of the trbc <dig> gene is anticipated to be the same but due to a region of undetermined sequence between exons  <dig> and  <dig> we were unable to identify exon  <dig>  the exon nucleotide sequences of trbc <dig> and  <dig> are very similar , resulting in the encoded  <dig> amino acid products differing by only  <dig> residues –  <dig> in the extra-cellular domain and  <dig> in the cytoplasmic domain .
the incomplete sequence for trbc <dig> is predicted to encode a product identical to that of trbc <dig>  in contrast to the high levels of pairwise identity between the exonic nucleotide sequences of all  <dig> trbc genes, the nucleotide sequences of the 3rd intron and the 3'utr regions of trbc <dig> show low identity with trbc <dig> and  <dig>  whereas the latter two genes show a high level of identity .
the similarity in the lengths of trbd <dig> and  <dig>  the phylogenetic clustering of trbj <dig> and trbj <dig> genes in corresponding genomic positions  and the similarity in the sequences of the 3rd introns and 3'utrs of trbc <dig> and  <dig> all reflect the duplication history of the djc region as described in figure  <dig>
the repertoire of functional trbv, trbd and trbj genes available for somatic recombination is large and phylogenetically diverse
computational analysis was used to predict the functional competency of the trbv, trbd and trbj genes present in the genome assembly.
fifty-five  of the trbv genes identified are predicted to encode pseudogenes , whilst trbj1- <dig>  and trbj1- <dig>  are also predicted to be non-functional .
thus, the functional repertoire comprises  <dig>  trbv genes  belonging to  <dig> different subgroups,  <dig> trbd genes and  <dig> trbj genes.
this provides a potential  <dig>  unique vdj permutations that can be used during somatic recombination of trb chains.
phylogenetic analysis demonstrates that the repertoire of functional trbv genes is diverse , with representatives in each of the  <dig> phylogenetic groups  described previously in humans and mice  <cit> .
the phylogenetic groupings were supported by high , bootstrap values , with the exception of group a .
maximum likelihood analysis using a variety of nucleotide models provides essentially similar phylogenetic clustering  indicating the reliability of the tree presented in figure  <dig>  the extensive intermingling of murine, human and bovine trbv subgroups is consistent with the establishment of distinct subgroups having occurred prior to mammalian radiation.
conversely, the formation of distinct clades of trbv genes of orthologous subgroups from different species  indicates that duplication within subgroups has predominantly occurred post-speciation.
despite this and the substantial disparity in the number of functional trbv genes present in the  <dig> species, the distribution amongst the different phylogenetic groups is similar .
phylogenetic groups c and f form a minor component of the functional trbv repertoire, whilst the contributions from groups b and d are also fairly modest.
in contrast, group e and to an even greater extent group a, are over-represented, together representing between  <dig> %  and  <dig> %  of the total functional repertoire.
phylogenetic analysis resolves the functional trbj genes in human, mice and btau_ <dig>  into  <dig> groups .
with the exception of group  <dig>  each group is supported by high pb values and is composed of orthologues that share a conserved order in the genome; consistent with the duplication history of the djc region, trbj genes from both the 2nd and 3rd bovine djc clusters group together with the respective genes from the 2nd murine and human djc clusters.
group  <dig>  which contains trbj2- <dig>  human and murine trbj2- <dig> and bovine trbj3- <dig> and 3– <dig> genes is only supported by a pb value of 57%.
the diversity of the functional trbj repertoire across the  <dig> species is comparable, with humans having functional genes in each of the  <dig> phylogenetic groups whilst in both mice and btau_ <dig>  only  <dig> groups lack functional members: groups  <dig>  and  <dig>  in btau_ <dig>  and groups  <dig>  and  <dig>  in mice.
comparison with cdna data identifies additional trbv gene sequences missing from the genome assembly
using a variety of rt-pcr based methods, our group has isolated and sequenced over  <dig> partial trb chain cdnas  <cit> .
with a few exceptions, these cdna sequences incorporated >  <dig> bp of the trbv gene  and in some cases the full length of the trbv gene.
based on the assumption that sequences sharing ≤ 97% nucleotide identity represent distinct genes, as applied in studies of human and murine trbv genes  <cit> , our analysis identified  <dig> putative unique trbv genes belonging to  <dig> subgroups .
analysis of the sequence data available for each cdna sequence indicated that only one of these genes is predicted to be non-functional , consistent with evidence that mrna expression of non-functional trb chains is down-regulated and therefore limited  <cit> .
all of the trbv subgroups identified in btau_ <dig>  were also identified in the cdna sequences.
in addition a single member of subgroup trbv <dig>  which is not represented in btau_ <dig> , was identified.
although the repertoire of cdna and functional genomic trbv genes is broadly similar in both size and distribution across the subgroups , detailed comparison shows that for the large subgroups there is substantial disparity between the genes present in the assembled genome and the cdna repertoire.
thus, only  <dig>  of the  <dig> trbv genes in subgroups trbv <dig>   <dig>   <dig>   <dig>   <dig> and  <dig> identified from cdna analysis had genomic sequences showing 100% sequence identity, whilst  <dig>  exhibited ≤ 97% identity to any genomic sequence.
the remaining  <dig>  sequences displayed nucleotide identities of 98–99% with genomic gene sequences.
given the presence in the genome of trbv genes exhibiting > 97% nucleotide identity, it is not possible to conclude whether these cdnas represent allelic variants of already identified genes or products of additional genes absent from the current assembly.
that at least some of the cdnas fall into the latter category, is supported by the identification of sequences exhibiting 100% identity with  <dig> of these cdna sequences, in the genome project's wgs trace archive .
conversely,  <dig>  of the  <dig> predicted functional genes identified in these subgroups within the genome did not have cdna sequences displaying 100% nucleotide identity.
twenty-two of these  showed 98–99% identity with cdna sequences, whilst the remaining  <dig>  exhibited < 97% identity to any of the cdna sequences.
in contrast to the findings with multi-member subgroups, cdnas corresponding to  <dig> subgroups with single members identified in the genome showed 100% identity with their respective genome sequence.
thus, comparison with cdna evidence suggests that substantial numbers of genes, predominantly from the large subgroups, are still absent from btau_ <dig> .
in contrast to the trbv situation, all trbd and trbc genes and the  <dig> functional trbj genes identified in btau_ <dig>  have been found expressed in cdna.
in addition, a functional allele of the trbj1- <dig> gene, which compared to the genomic sequence has a  <dig> bp insertion that restores the orf encoding the fgxg motif , has been identified.
no evidence for further trbd, trbj or trbc genes was found from cdna analysis, suggesting the repertoire of these genes in btau_ <dig>  is complete.
conserved synteny between the human trb locus and scaffolds chr <dig> .
<dig> and chr <dig> .108
the organisation of genes within chr <dig> .
<dig> and chr <dig> .
<dig> shows marked conservation in order with that of genes at the 5' and 3'ends of the human trb locus respectively .
genes belonging to orthologous trbv subgroups show a similar order although in some areas substantial tandem duplication has obscured synteny at the level of individual genes .
trbvx, the only bovine trbv gene that has no human orthologue, is located in a position  gene and trypsinogen genes) syntenic with its murine orthologue .
as mentioned previously, synteny is also shown in the organisation of trbj genes, with human and bovine orthologues occupying conserved positions in their relative clusters.
synteny also extends to non-trb genes located within and adjacent to the human trb locus.
the  <dig> trypsinogen genes located on chr <dig> .
<dig> and chr <dig> .
<dig> are syntenic to those located towards the 5'end and 3'end of the human trb locus respectively, and the dβh-like gene flanking the 5' end of the human trb locus and the ephrin type-b receptor  <dig> precursor , transient receptor potential cation channel subfamily v  member  <dig>  trpv <dig> and kell blood group glycoprotein  loci flanking the 3'end of the human trb locus all have bovine orthologues in syntenic positions on the  <dig> scaffolds.
although fluorescent in situ hybridisation studies have previously shown that the position of the trb locus with respect to the blue cone pigment  and chloride channel protein  <dig>  genes are conserved between ruminants and humans  <cit> , this analysis shows for the first time the high levels of synteny between human and bovine orthologues both within and adjacent to the trb locus.
extrapolation of this synteny predicts that chr <dig> .
<dig> and chr <dig> .
<dig>  should be juxtaposed on chromosome  <dig>  whilst chr <dig> .
<dig>  which contains bovine orthologues to numerous genes that in humans are telomeric to the trb locus  should be located 3' to chr <dig> .
<dig> and chr <dig> .
<dig>  which contains a bovine orthologue to the acylglycerol kinase  gene that in humans lies centromeric to the trb locus, should be positioned 5' to chr <dig> .
<dig>  this location of chr <dig> .
<dig> has also been predicted by clone paired-end analysis .
rs and regulatory elements sequences are conserved in the bovine trb locus
the rs sequences of the bovine trbv, trbj and trbd genes show a high degree of similarity to canonical rs sequences defined for the corresponding human and murine genes .
in the bovine trbv 23-rs sequences the cacag of the heptamer and a poly-a stretch in the centre of the nonamer show a high degree of intra- and inter-species conservation.
although conservation of the spacer is less marked, the ctgca sequence proximal to the heptamer is reasonably well conserved and similar to that of humans.
despite more limited conservation, the  <dig> bp proximal to the nonamer also displays a degree of cross-species similarity.
similarly, the bovine trbj rs exhibits intra- and inter-species conservation of the first  <dig> bp  of the heptamer sequence and a poly-a stretch in the nonamer.
conservation in the spacer is limited but overrepresentation of g at the position  <dig> bp from the heptamer and c  <dig> bp from the nonamer is seen in both the bovine and human.
we identified a  <dig> bp sequence ~ <dig>  kb 3' to the trbc <dig> gene that displays high nucleotide similarity with the sequences of the enhancers  identified in the murine  and human  trb loci  <cit> .
sequences of the protein binding sites described in the eβs of humans  and mice  are well conserved in the aligned bovine sequence ; several of the transcription binding sites shown to be functionally important in the regulation in eβ function  <cit> , such as the gata-binding site in βe1/tβ <dig> and the κe2-binding motif in βe <dig> are absolutely conserved, whilst others  show minimal sequence divergence.
in contrast, the sequence of the trbd <dig> promoter , which includes the ~ <dig> bp directly upstream of the trbd <dig> gene and has been well defined in the mouse  <cit> , displays a more limited nucleotide identity  with the bovine sequence.
as shown in figure 11b, some transcription factor binding sites demonstrated to be important for pdβ <dig> function  in mice and/or humans are absent from the bovine sequence, whereas others  have been well conserved  <cit> .
we were also able to identify a conserved camp responsive element  motif  in the ~80– <dig> bp upstream sequence of  <dig>  of the bovine trbv genes .
this motif is found within conserved decamer sequences in the promoter regions of some murine and human trbv genes  <cit>  and has been shown to specifically bind a splice variant of a cre binding protein preferentially expressed in the thymus  <cit> .
in general, the cre motif has been found in bovine genes that are members of subgroups that are orthologous to the human trbv subgroups in which the cre motif is also found  <cit> .
discussion
sequencing of the human and murine trb loci has defined the repertoire of trb genes in these species as well as provided insights into the organisation, evolution and regulation of this immunologically important locus  <cit> .
although the bovine trb locus sequence in the third bovine genome assembly is incomplete, the analysis conducted in the present study has provided insight into the nature of the bovine trb gene repertoire and its genomic organisation and evolution.
the most striking result from the study was the large number of trbv genes identified  which is over twice the number found in humans and four times that in mice  <cit> .
although  <dig> of the  <dig> bovine subgroups identified in the genome contain multiple genes, the majority of the trbv repertoire expansion is attributable to the extensive membership of just  <dig> subgroups, trbv <dig> ,  <dig>  and  <dig> .
by comparison, the largest subgroups in humans are trbv <dig> and trbv <dig>  with  <dig> members each, whilst in mice the only multi-membered subgroups are trbv <dig> and  <dig> with  <dig> members each.
as in humans the expansion of the trbv repertoire has predominantly occurred through the tandem duplication of dna blocks containing genes from more than  <dig> subgroup  <cit> .
dot-plot analyses shows that this duplication in the bovine is complex, leading to the generation of  <dig> homology units ranging in size from ~ <dig> kb to ~ <dig> kb and encompassing between  <dig> and  <dig> trbv genes.
unequal cross-over  between genome-wide repeats  has been proposed to act as the substrate for such duplication events in tr loci  <cit> .
although genome-wide repeats are found in the dna surrounding the bovine trbv genes , as in the human trb locus they are only rarely found at the boundaries of duplicated homology units , suggesting their contribution to mediating duplication is minimal  <cit> .
although gene conversion of trbv genes has been documented  <cit> , as with other multi-gene families in the immune system, trbv genes predominantly follow a 'birth-and-death' model of evolution  <cit> , by which new genes are created by repeated gene duplication, some of which are maintained in the genome whilst others are deleted or become non-functional due to mutation.
genes maintained following duplication are subject to progressive divergence, providing the opportunity for diversification of the gene repertoire.
gene duplication within the tr loci has occurred sporadically over hundreds of millions of years with ancient duplications accounting for the generation of different subgroups and more recent duplications giving rise to the different members within subgroups  <cit> .
the continuous nature of duplication and divergence of bovine trbv genes is evident in the multi-membered subgroups where nucleotide identity between members ranges between  <dig> % and 100%.
the complete identity observed between some trbv genes suggests that some of the duplication events have occurred very recently.
similar features have been described for the murine tra and human igκ loci, within which recent duplications, <  <dig> million years ago , have created pairs of v genes exhibiting ~97% nucleotide identity  <cit> .
southern blot data showing differences in rflp banding patterns of trbv <dig> and  <dig> genes in dna from bos indicus and bos taurus animals , which only diverged between  <dig>  –  <dig> million years ago  <cit> , provides further evidence of recent evolutionary development of the trbv repertoire in cattle.
the distribution of trbv genes over  <dig> scaffolds and the presence of >  <dig> kb of undetermined sequence within two of the scaffolds indicate that characterisation of the genomic trbv repertoire remains incomplete.
comparison with cdna sequences data indicates that the number of undefined genes is substantial – only 36/ <dig>  of trbv genes identified from cdna analysis have corresponding identical sequences in btau_ <dig> .
most of the identified trbv genes missing from the assembly are members of the large subgroups trbv <dig>   <dig>   <dig>   <dig>   <dig> and  <dig>  further enhancing their numerical dominance.
although it is anticipated that completion of the trb locus sequence will incorporate significant numbers of additional trbv genes, the possible existence of insertion-deletion related polymorphisms , which can lead to intra-species variation in genomic trbv gene repertoires as described in human and murine trb loci  <cit> , may result in some of the genes identified in cdna being genuinely absent from the sequenced bovine genome
the proportion of trbv pseudogenes in btau_ <dig>  is 41%, comparable to that seen in both humans  and mice , suggesting that the 'death rate' in trbv gene evolution is generally high  <cit> .
pseudogene formation has occurred sporadically throughout the evolution of trbv genes, with genes that have lost function tending to subsequently accumulate further lesions  <cit> .
the majority of bovine trbv pseudogenes  contain a single lesion and thus appear to have arisen recently; the remaining 43% have multiple lesions of varying severity and complexity .
in addition to pseudogenes we also identified  <dig> sequences showing limited local similarity to trbv genes in btau_ <dig>  .
such severely mutated trbv 'relics',  <dig> of which have been identified in the human trb locus  <cit> ., are considered to represent the remnants of ancient pseudogene formation.
in contradiction to a previous report  <cit> , the repertoire of functional trbv genes in btau_ <dig>  exhibits a level of phylogenetic diversity similar to that of humans and mice.
phylogenetic groups a and e are over-represented in all  <dig> species, which in humans and cattle is largely attributable to expansion of subgroups trbv <dig>   <dig>   <dig> and  <dig> and trbv <dig>   <dig> and  <dig> respectively; in mice the expansion of subgroups trbv <dig> and  <dig> make a more modest contribution to this over-representation.
much of the expansion of human subgroups trbv <dig>   <dig> and  <dig> occurred 24– <dig> mya  <cit>  and similarly, as described above, in bovines much of the expansion of subgroups trbv <dig>   <dig> and  <dig> subgroups appears to be very recent.
as these expansions have occurred subsequent to primate/artiodactyl divergence   <cit> , over-representation of phylogenetic groups a and e must have occurred as parallel but independent events in these lineages, raising interesting questions about the evolutionary pressures that shape the functional trbv repertoire.
in contrast to the wide variation in the organisation of trbd, trbj and trbc genes in the trb locus seen in non-mammalian vertebrates  <cit> , in mammals the arrangement of tandemly located djc clusters is well conserved  <cit> .
although most placental species studied have  <dig>  variation in the number of djc clusters has been observed, with unequal cross-over events between trbc genes usually invoked as the most likely explanation for this variation  <cit> .
the results from this study provide the first description of the entire bovine djc region and confirm that like sheep, cattle have  <dig> complete djc clusters  <cit> .
dot-plot and sequence analyses indicate that unequal crossover between the ancestral trbc <dig> and trbc <dig> genes led to duplication of a region incorporating trbc <dig>  trbd <dig> and trbj <dig> genes, generating the djc <dig> cluster.
the similarity with the structure of the ovine djc region suggests that this duplication event occurred prior to ovine/bovine divergence  <dig>  mya  <cit> .
as with duplication of trbv genes, expansion of trbd and trbj gene numbers has increased the number of genes available to partake in somatic recombination – the  <dig> different vdj permutations possible from the functional genes present in btau_ <dig>  is considerably more than that for either humans  or mice .
interestingly, the sequence of bovine trbd <dig> gene is the first trbd gene described that doesn't encode a glycine residue  in all  <dig> reading frames  <cit> .
however, analysis of cdna reveals evidence of expression by functional trb chains of trbd1in the reading frame that doesn't encode a glycine but have generated a glycine codon by nucleotide editing at the vj junction .
in contrast to trbv, trbd and trbj genes which encode products that bind to a diverse array of peptide-mhc ligands, trbc gene products interact with components of the cd <dig> complex which are non-polymorphic.
consequently, due to structural restrictions trbc genes are subject to concerted evolutionary pressures with intra-species homogenization through gene conversion evident in both humans and mice  <cit> .
similarly, the bovine trbc genes were found to encode near identical products, most likely as a result of gene conversion, although in the case of trbc <dig> and trbc <dig> genes this more probably reflects minimal divergence following duplication.
comparison with the human and murine sequences shows that non-coding elements that regulate trb expression, such as the eβ, promoters and rss are highly conserved in the bovine.
this is consistent with work demonstrating that the critical role of rss has enforced a high level of evolutionary conservation  <cit>  and that eβ and pdβ <dig> sequences are well conserved in eutherian species  <cit> .
although transcriptional factor binding sites are less well conserved in the putative pdβ <dig> than the eβ sequence, the ikaros/lyf- <dig> and ap- <dig> binding sites of the pdβ <dig>  which are vital in enforcing stage-specific  are conserved  <cit> .
our analysis of putative trbv promoter elements was restricted to the well described cre motif  <cit> .
however, trbv promoters are complex and expression of trbv genes whose promoters lack the cre motif is maintained through the function of other transcriptional factor binding sites  <cit> .
a more detailed analysis of the bovine trbv promoters would be interesting given the potential influence this may have on shaping the expressed trbv repertoire  <cit> , but is beyond the scope of the current study.
the portion of the bovine trb locus described in btau_ <dig>  encompasses >  <dig> kb of sequence .
thus, although incomplete, the bovine trb locus is larger than that of either humans  or mice , mainly as a consequence of the duplications leading to the dramatic expansion of the v genes.
in contrast to v genes, duplication of trypsinogen genes within the trb locus is more limited in the bovine , where only  <dig> trypsinogen genes were identified, compared to the human and murine where more extensive duplication has lead to the presence of  <dig> and  <dig> trypsinogen genes respectively.
despite the differences in duplication events, the organisation of both tr and non-tr genes within and adjacent to the trb locus exhibits a striking conserved synteny between cattle, humans and mice  <cit> .
indeed, the organisation of genes within the trb locus and its position relative to adjacent loci is ancient, with marked conserved synteny also demonstrated between eutherian and marsupial mammalian species and, to a large extent, chickens  <cit> .
given the evidence for conserved synteny of trbv gene organisation despite dissimilar duplication/deletion events between mice, humans and cattle, the results of the analysis completed in this study suggest that several subgroups including trbv <dig>   <dig>   <dig>   <dig> and  <dig>  which were not identified in the genome assembly or from cdna sequences, may have been deleted from the bovine genome .
conservation of synteny would predict that the genomic location of the trbv <dig> gene identified from cdna analysis will be within the region of undetermined sequence in chr <dig> .
<dig> between the trbv <dig> and  <dig> genes .
CONCLUSIONS
the primary purpose of this study was to analyse the sequence data made available from the third bovine genome assembly to gain a better understanding of the bovine trb gene repertoire and the organization and evolution of the bovine trb locus.
the results of this analysis have shown that:  the bovine trbv genomic repertoire has been dramatically expanded through a complex series of duplication events and although incomplete, is the largest described to date.
these duplication events have led to massive expansion in the membership of certain trbv subgroups, particularly trbv <dig>   <dig> and 21;  duplication has generated  <dig> djc clusters compared to  <dig> in humans and mice;  the elements that regulate trb expression and the organisation of genes within and adjacent to the trb loci exhibit high levels of conservation between humans, mice and cattle.
cdna evidence indicates that a substantial number of trbv genes, predominantly from the larger subgroups are absent from the current assembly.
notwithstanding the incomplete assembly of the trb locus, the results of these analyses clearly demonstrate that cattle possess a phylogenetically diverse repertoire of functional trb genes that is substantially larger than that described for other species.
these findings, together with emerging evidence of similar expansions of gene repertoires for other tr chains in ruminants  <cit>  suggest that strong evolutionary pressures have driven a generic enlargement of tr gene numbers, and thus greater potential tr diversity, in the ruminant lineage.
further studies are required to define the full extent of these expansions and to understand their evolutionary basis.

 diverse αβtr repertoires are crucial to the maintenance of effective t cell-mediated immunity. estimates based on direct measurement indicate that in humans and mice individuals express a repertoire of approximately × and × unique αβtrs respectively. as with the other antigen-specific receptors diversity is generated in lymphocytic precursors by somatic recombination of discontiguous variable, diversity and joining genes to form the membrane-distal variable domains. diversity is derived from both the different permutations of vj genes used to form the tra and trb chains expressed by individual thymocytes and also by the activity of terminal deoxynucleotide transferase and exonuclease at the vj junction during recombination. consequently, much of the diversity is focused in the third complementarity determining region, which is encoded by the vj junction and forms the most intimate association with the antigenic peptide component of the peptide-mhc ligand of αβtrs, whereas the cdr and cdr of the tra and trb chains, that predominantly interact with the mhc, are encoded within the germline v genes. trb chain genes are located in the trb locus, which in humans is ~ kb long and situated on chromosome and in mice is ~ kb and located on chromosome. in both species, the organisation of trb genes is similar, with a library of trbv genes positioned at the 5' end and djc clusters followed by a single trbv gene with an inverted transcriptional orientation located at the 3'end. the germline repertoire of trbv genes in humans is composed of genes belonging to subgroups, whilst in mice the repertoire comprises genes belonging to subgroups the disparity between the number of trbv genes in the species is the result of multiple duplication events within the human trb locus, most of which have involved tandem duplication of blocks of dna containing genes from more than one subgroup. vj recombination is initiated by site-specific dna cleavage at recombination signal sequences mediated by enzymes encoded by recombination activating genes and. rss comprise conserved heptamer and nonamer sequences separated by spacers of either bp or bp. correct vj assembly is achieved as recombination can only occur between genes flanked with rs of dissimilar length and direct trbv/trbj recombination is prohibited by the'beyond 12/23' phenomenon. as with other antigen-specific receptor loci, recombination in the trb locus is under strict lineage-, stage- and allele-specific regulation associated with control of rag accessibility to rss mediated through alterations in chromatin structure. numerous studies have shown that both the trb enhancer and transcriptional promoters within the trb locus serve as rag accessibility control elements, playing a critical role in regulating chromatin structure and therefore recombination of trb genes. current knowledge of the trb gene repertoires of agriculturally important artiodactyl species is limited. published analyses of rearranged trb transcripts have demonstrated the expression of trbv subgroups in pigs, subgroups in sheep and subgroups in cattle, some of which have undergone extensive duplication. information on the genomic organisation of the trb loci is predominantly restricted to the djc region, which in the pig was found to be composed of tandemly arranged djc clusters but in sheep contained tandemly arranged djc clusters. preliminary analysis of a bac clone corresponding to part of the djc region indicates that in cattle the djc region may also consist of djc clusters. sequencing of the complete trb loci in human and mice allowed the repertoire of trb genes in these species to be fully characterised and also permitted analysis of the organisation, regulation and evolution of this immunologically important locus. in this study we have used the sequence of the third bovine genome assembly to further study the bovine trb repertoire and trb locus. although the sequence of the trb locus is incomplete, the results reveal that duplication within the locus has been prolific leading to a massive expansion of trbv gene numbers and the generation of a third djc cluster. furthermore, the analysis shows that the genomic organisation of the trb locus and the non-coding elements that regulate trb expression are highly conserved in cattle when compared to that of humans and mice.  extensive duplication has generated a large germline repertoire of bovine trbv genes a total of trbv genes, distributed over scaffolds was identified in btau_. consistent with data from fluorescent in situ hybridisation studies, the majority of the trbv genes were located on scaffolds mapped to chromosome whilst the remaining genes were located on scaffolds which have not been assigned a chromosomal location. within the scaffolds are several regions of undetermined sequence, including large areas of ~ kb and ~ kb on chr. and chr. respectively. each trbv gene is composed of i) a short leader exon, generally of ~ bp, ii) a single intron of between ~ and ~ bp and iii) a variable exon of ~ bp, immediately flanked at the 3'end with a 23-rs. comparison of the nucleotide sequence of each of the bovine trbv genes with human trbv gene sequences, revealed maximum levels of similarity between the species ranging from % to % for all except one of the bovine trbv genes. on the basis of these results, bovine trbv genes were considered orthologues of their most similar human counterpart and were assigned to subgroups named according to the orthologous human subgroup. the single bovine trbv gene that lacked significant homology to any of the human trbv genes displayed % identity with the murine trbv gene and was placed in subgroup trbvx. the subgroups thus established generally adhered to the definition of members within a subgroup exhibiting > 75% nucleotide sequence identity. however, the single member of the trbv subgroup displayed > 75% identity to all of the trbv genes and the identity between members of the trbv and trbv subgroups was often > 75%. conversely a single member of the trbv subgroup showed only – % nucleotide identity with the other members of this subgroup. the total and functional repertoire of trbv genes in the human trb locus and in the third assembly of the bovine genome as well as the bovine trbv gene repertoire identified from cdna analysis. details of the human repertoire are taken from the imgt database. *denotes variation in germline repertoire due to insertion/deletion polymorphisms and † denotes trbv genes which have alternative functional and non-functional alleles. of the bovine subgroups present in the genome assembly, have multiple members. subgroups trbv and have all undergone substantial expansion, having and members respectively – together representing 68% of the total btau_ trbv gene repertoire. southern blot analysis corroborates the presence of large numbers of trbv and genes in the genome. a prominent feature of the genomic organisation of trbv genes is that members of expanded subgroups are generally intercalated with members of other expanded subgroups in a recurrent pattern. thus, a kb region of chr. and virtually all of scaffold chrun. are composed of alternating trbv and genes obtained in southern blots of genomic dna when hybridised with trbv9- and trbv6-specific probes in figure 1), whilst the 3'end of chr. and the 5' end of chr. contain repeated units comprising trbv and genes. dot-plot analyses indicate that this organisation has arisen through a series of complex tandem duplication events within the regions in which trbv and genes and trbv and genes are located. six homology units, ranging in size from ~ kb to ~ kb and encompassing from to trbv genes were identified. three of these homology units have undergone multiple duplications: variation in the length of the different copies of these homology units, suggests that either i) distinct iterations of a duplication event have involved different components of the homology unit or ii) the different copies have been subject to different post-duplication deletions. the levels of nucleotide identity between trbv genes in corresponding positions in homology units is frequently high: pairs of trbv genes, pairs of trbv and pair each of trbv and trbv have identical coding sequences whilst pair of trbv genes and pairs of trbv as well as triplets of trbv and triplets of trbv genes have > 97% sequence identity in the coding region. duplication has expanded the repertoire of trbd, trbj and trbc genes in the bovine genome a total of trbd, trbj and trbc genes were identified in the assembly. these genes were all located within a ~ kb region of scaffold chr. and organised into tandemly arranged clusters, each of ~ kb length and composed of a single trbd gene, 5- trbj genes and a single trbc gene. dot-plot analysis reveals the presence of a third djc cluster is attributable to duplication of a ~ kb region, one copy of which incorporates trbc trbd and the trbj cluster whilst the other copy incorporates trbc trbd and the trbj cluster. numerous interruptions in the line representing the duplicated region indicate that there has been significant post-duplication deletion/insertion related modification of the duplicated region. the nucleotide and deduced amino acid sequence of the trbd and trbj genes as well as the flanking rs are shown in figure 5a and 5b respectively. the bp or bp trbd genes are g-rich and encode at least one glycine in all potential reading frames with the exception of the 3rd reading frame of trbd the trbj genes range in size from bp to bp in length and all encode the canonical fgxg amino acid motif that defines trbj genes. as with all mammalian trbc genes so far characterised, bovine trbc and trbc genes are composed of exons, introns and a 3'utr region. the structure of the trbc gene is anticipated to be the same but due to a region of undetermined sequence between exons and we were unable to identify exon the exon nucleotide sequences of trbc and are very similar, resulting in the encoded amino acid products differing by only residues – in the extra-cellular domain and in the cytoplasmic domain. the incomplete sequence for trbc is predicted to encode a product identical to that of trbc in contrast to the high levels of pairwise identity between the exonic nucleotide sequences of all trbc genes, the nucleotide sequences of the 3rd intron and the 3'utr regions of trbc show low identity with trbc and whereas the latter two genes show a high level of identity. the similarity in the lengths of trbd and the phylogenetic clustering of trbj and trbj genes in corresponding genomic positions and the similarity in the sequences of the 3rd introns and 3'utrs of trbc and all reflect the duplication history of the djc region as described in figure the repertoire of functional trbv, trbd and trbj genes available for somatic recombination is large and phylogenetically diverse computational analysis was used to predict the functional competency of the trbv, trbd and trbj genes present in the genome assembly. fifty-five of the trbv genes identified are predicted to encode pseudogenes, whilst trbj1- and trbj1- are also predicted to be non-functional. thus, the functional repertoire comprises trbv genes belonging to different subgroups, trbd genes and trbj genes. this provides a potential unique vdj permutations that can be used during somatic recombination of trb chains. phylogenetic analysis demonstrates that the repertoire of functional trbv genes is diverse, with representatives in each of the phylogenetic groups described previously in humans and mice. the phylogenetic groupings were supported by high, bootstrap values, with the exception of group a. maximum likelihood analysis using a variety of nucleotide models provides essentially similar phylogenetic clustering indicating the reliability of the tree presented in figure the extensive intermingling of murine, human and bovine trbv subgroups is consistent with the establishment of distinct subgroups having occurred prior to mammalian radiation. conversely, the formation of distinct clades of trbv genes of orthologous subgroups from different species indicates that duplication within subgroups has predominantly occurred post-speciation. despite this and the substantial disparity in the number of functional trbv genes present in the species, the distribution amongst the different phylogenetic groups is similar. phylogenetic groups c and f form a minor component of the functional trbv repertoire, whilst the contributions from groups b and d are also fairly modest. in contrast, group e and to an even greater extent group a, are over-represented, together representing between % and % of the total functional repertoire. phylogenetic analysis resolves the functional trbj genes in human, mice and btau_ into groups. with the exception of group each group is supported by high pb values and is composed of orthologues that share a conserved order in the genome; consistent with the duplication history of the djc region, trbj genes from both the 2nd and 3rd bovine djc clusters group together with the respective genes from the 2nd murine and human djc clusters. group which contains trbj2- human and murine trbj2- and bovine trbj3- and 3– genes is only supported by a pb value of 57%. the diversity of the functional trbj repertoire across the species is comparable, with humans having functional genes in each of the phylogenetic groups whilst in both mice and btau_ only groups lack functional members: groups and in btau_ and groups and in mice. comparison with cdna data identifies additional trbv gene sequences missing from the genome assembly using a variety of rt-pcr based methods, our group has isolated and sequenced over partial trb chain cdnas. with a few exceptions, these cdna sequences incorporated > bp of the trbv gene and in some cases the full length of the trbv gene. based on the assumption that sequences sharing ≤ 97% nucleotide identity represent distinct genes, as applied in studies of human and murine trbv genes, our analysis identified putative unique trbv genes belonging to subgroups. analysis of the sequence data available for each cdna sequence indicated that only one of these genes is predicted to be non-functional, consistent with evidence that mrna expression of non-functional trb chains is down-regulated and therefore limited. all of the trbv subgroups identified in btau_ were also identified in the cdna sequences. in addition a single member of subgroup trbv which is not represented in btau_, was identified. although the repertoire of cdna and functional genomic trbv genes is broadly similar in both size and distribution across the subgroups, detailed comparison shows that for the large subgroups there is substantial disparity between the genes present in the assembled genome and the cdna repertoire. thus, only of the trbv genes in subgroups trbv and identified from cdna analysis had genomic sequences showing 100% sequence identity, whilst exhibited ≤ 97% identity to any genomic sequence. the remaining sequences displayed nucleotide identities of 98–99% with genomic gene sequences. given the presence in the genome of trbv genes exhibiting > 97% nucleotide identity, it is not possible to conclude whether these cdnas represent allelic variants of already identified genes or products of additional genes absent from the current assembly. that at least some of the cdnas fall into the latter category, is supported by the identification of sequences exhibiting 100% identity with of these cdna sequences, in the genome project's wgs trace archive. conversely, of the predicted functional genes identified in these subgroups within the genome did not have cdna sequences displaying 100% nucleotide identity. twenty-two of these showed 98–99% identity with cdna sequences, whilst the remaining exhibited < 97% identity to any of the cdna sequences. in contrast to the findings with multi-member subgroups, cdnas corresponding to subgroups with single members identified in the genome showed 100% identity with their respective genome sequence. thus, comparison with cdna evidence suggests that substantial numbers of genes, predominantly from the large subgroups, are still absent from btau_. in contrast to the trbv situation, all trbd and trbc genes and the functional trbj genes identified in btau_ have been found expressed in cdna. in addition, a functional allele of the trbj1- gene, which compared to the genomic sequence has a bp insertion that restores the orf encoding the fgxg motif, has been identified. no evidence for further trbd, trbj or trbc genes was found from cdna analysis, suggesting the repertoire of these genes in btau_ is complete. conserved synteny between the human trb locus and scaffolds chr. and chr.108 the organisation of genes within chr. and chr. shows marked conservation in order with that of genes at the 5' and 3'ends of the human trb locus respectively. genes belonging to orthologous trbv subgroups show a similar order although in some areas substantial tandem duplication has obscured synteny at the level of individual genes. trbvx, the only bovine trbv gene that has no human orthologue, is located in a position gene and trypsinogen genes) syntenic with its murine orthologue. as mentioned previously, synteny is also shown in the organisation of trbj genes, with human and bovine orthologues occupying conserved positions in their relative clusters. synteny also extends to non-trb genes located within and adjacent to the human trb locus. the trypsinogen genes located on chr. and chr. are syntenic to those located towards the 5'end and 3'end of the human trb locus respectively, and the dβh-like gene flanking the 5' end of the human trb locus and the ephrin type-b receptor precursor, transient receptor potential cation channel subfamily v member trpv and kell blood group glycoprotein loci flanking the 3'end of the human trb locus all have bovine orthologues in syntenic positions on the scaffolds. although fluorescent in situ hybridisation studies have previously shown that the position of the trb locus with respect to the blue cone pigment and chloride channel protein genes are conserved between ruminants and humans, this analysis shows for the first time the high levels of synteny between human and bovine orthologues both within and adjacent to the trb locus. extrapolation of this synteny predicts that chr. and chr. should be juxtaposed on chromosome whilst chr. which contains bovine orthologues to numerous genes that in humans are telomeric to the trb locus should be located 3' to chr. and chr. which contains a bovine orthologue to the acylglycerol kinase gene that in humans lies centromeric to the trb locus, should be positioned 5' to chr. this location of chr. has also been predicted by clone paired-end analysis. rs and regulatory elements sequences are conserved in the bovine trb locus the rs sequences of the bovine trbv, trbj and trbd genes show a high degree of similarity to canonical rs sequences defined for the corresponding human and murine genes. in the bovine trbv 23-rs sequences the cacag of the heptamer and a poly-a stretch in the centre of the nonamer show a high degree of intra- and inter-species conservation. although conservation of the spacer is less marked, the ctgca sequence proximal to the heptamer is reasonably well conserved and similar to that of humans. despite more limited conservation, the bp proximal to the nonamer also displays a degree of cross-species similarity. similarly, the bovine trbj rs exhibits intra- and inter-species conservation of the first bp of the heptamer sequence and a poly-a stretch in the nonamer. conservation in the spacer is limited but overrepresentation of g at the position bp from the heptamer and c bp from the nonamer is seen in both the bovine and human. we identified a bp sequence ~ kb 3' to the trbc gene that displays high nucleotide similarity with the sequences of the enhancers identified in the murine and human trb loci. sequences of the protein binding sites described in the eβs of humans and mice are well conserved in the aligned bovine sequence; several of the transcription binding sites shown to be functionally important in the regulation in eβ function, such as the gata-binding site in βe1/tβ and the κe2-binding motif in βe are absolutely conserved, whilst others show minimal sequence divergence. in contrast, the sequence of the trbd promoter, which includes the ~ bp directly upstream of the trbd gene and has been well defined in the mouse, displays a more limited nucleotide identity with the bovine sequence. as shown in figure 11b, some transcription factor binding sites demonstrated to be important for pdβ function in mice and/or humans are absent from the bovine sequence, whereas others have been well conserved. we were also able to identify a conserved camp responsive element motif in the ~80– bp upstream sequence of of the bovine trbv genes. this motif is found within conserved decamer sequences in the promoter regions of some murine and human trbv genes and has been shown to specifically bind a splice variant of a cre binding protein preferentially expressed in the thymus. in general, the cre motif has been found in bovine genes that are members of subgroups that are orthologous to the human trbv subgroups in which the cre motif is also found. discussion sequencing of the human and murine trb loci has defined the repertoire of trb genes in these species as well as provided insights into the organisation, evolution and regulation of this immunologically important locus. although the bovine trb locus sequence in the third bovine genome assembly is incomplete, the analysis conducted in the present study has provided insight into the nature of the bovine trb gene repertoire and its genomic organisation and evolution. the most striking result from the study was the large number of trbv genes identified which is over twice the number found in humans and four times that in mice. although of the bovine subgroups identified in the genome contain multiple genes, the majority of the trbv repertoire expansion is attributable to the extensive membership of just subgroups, trbv, and. by comparison, the largest subgroups in humans are trbv and trbv with members each, whilst in mice the only multi-membered subgroups are trbv and with members each. as in humans the expansion of the trbv repertoire has predominantly occurred through the tandem duplication of dna blocks containing genes from more than subgroup. dot-plot analyses shows that this duplication in the bovine is complex, leading to the generation of homology units ranging in size from ~ kb to ~ kb and encompassing between and trbv genes. unequal cross-over between genome-wide repeats has been proposed to act as the substrate for such duplication events in tr loci. although genome-wide repeats are found in the dna surrounding the bovine trbv genes, as in the human trb locus they are only rarely found at the boundaries of duplicated homology units, suggesting their contribution to mediating duplication is minimal. although gene conversion of trbv genes has been documented, as with other multi-gene families in the immune system, trbv genes predominantly follow a'birth-and-death' model of evolution, by which new genes are created by repeated gene duplication, some of which are maintained in the genome whilst others are deleted or become non-functional due to mutation. genes maintained following duplication are subject to progressive divergence, providing the opportunity for diversification of the gene repertoire. gene duplication within the tr loci has occurred sporadically over hundreds of millions of years with ancient duplications accounting for the generation of different subgroups and more recent duplications giving rise to the different members within subgroups. the continuous nature of duplication and divergence of bovine trbv genes is evident in the multi-membered subgroups where nucleotide identity between members ranges between % and 100%. the complete identity observed between some trbv genes suggests that some of the duplication events have occurred very recently. similar features have been described for the murine tra and human igκ loci, within which recent duplications, < million years ago, have created pairs of v genes exhibiting ~97% nucleotide identity. southern blot data showing differences in rflp banding patterns of trbv and genes in dna from bos indicus and bos taurus animals, which only diverged between – million years ago, provides further evidence of recent evolutionary development of the trbv repertoire in cattle. the distribution of trbv genes over scaffolds and the presence of > kb of undetermined sequence within two of the scaffolds indicate that characterisation of the genomic trbv repertoire remains incomplete. comparison with cdna sequences data indicates that the number of undefined genes is substantial – only 36/ of trbv genes identified from cdna analysis have corresponding identical sequences in btau_. most of the identified trbv genes missing from the assembly are members of the large subgroups trbv and further enhancing their numerical dominance. although it is anticipated that completion of the trb locus sequence will incorporate significant numbers of additional trbv genes, the possible existence of insertion-deletion related polymorphisms, which can lead to intra-species variation in genomic trbv gene repertoires as described in human and murine trb loci, may result in some of the genes identified in cdna being genuinely absent from the sequenced bovine genome the proportion of trbv pseudogenes in btau_ is 41%, comparable to that seen in both humans and mice, suggesting that the'death rate' in trbv gene evolution is generally high. pseudogene formation has occurred sporadically throughout the evolution of trbv genes, with genes that have lost function tending to subsequently accumulate further lesions. the majority of bovine trbv pseudogenes contain a single lesion and thus appear to have arisen recently; the remaining 43% have multiple lesions of varying severity and complexity. in addition to pseudogenes we also identified sequences showing limited local similarity to trbv genes in btau_. such severely mutated trbv'relics', of which have been identified in the human trb locus., are considered to represent the remnants of ancient pseudogene formation. in contradiction to a previous report, the repertoire of functional trbv genes in btau_ exhibits a level of phylogenetic diversity similar to that of humans and mice. phylogenetic groups a and e are over-represented in all species, which in humans and cattle is largely attributable to expansion of subgroups trbv and and trbv and respectively; in mice the expansion of subgroups trbv and make a more modest contribution to this over-representation. much of the expansion of human subgroups trbv and occurred 24– mya and similarly, as described above, in bovines much of the expansion of subgroups trbv and subgroups appears to be very recent. as these expansions have occurred subsequent to primate/artiodactyl divergence, over-representation of phylogenetic groups a and e must have occurred as parallel but independent events in these lineages, raising interesting questions about the evolutionary pressures that shape the functional trbv repertoire. in contrast to the wide variation in the organisation of trbd, trbj and trbc genes in the trb locus seen in non-mammalian vertebrates, in mammals the arrangement of tandemly located djc clusters is well conserved. although most placental species studied have variation in the number of djc clusters has been observed, with unequal cross-over events between trbc genes usually invoked as the most likely explanation for this variation. the results from this study provide the first description of the entire bovine djc region and confirm that like sheep, cattle have complete djc clusters. dot-plot and sequence analyses indicate that unequal crossover between the ancestral trbc and trbc genes led to duplication of a region incorporating trbc trbd and trbj genes, generating the djc cluster. the similarity with the structure of the ovine djc region suggests that this duplication event occurred prior to ovine/bovine divergence mya. as with duplication of trbv genes, expansion of trbd and trbj gene numbers has increased the number of genes available to partake in somatic recombination – the different vdj permutations possible from the functional genes present in btau_ is considerably more than that for either humans or mice. interestingly, the sequence of bovine trbd gene is the first trbd gene described that doesn't encode a glycine residue in all reading frames. however, analysis of cdna reveals evidence of expression by functional trb chains of trbd1in the reading frame that doesn't encode a glycine but have generated a glycine codon by nucleotide editing at the vj junction. in contrast to trbv, trbd and trbj genes which encode products that bind to a diverse array of peptide-mhc ligands, trbc gene products interact with components of the cd complex which are non-polymorphic. consequently, due to structural restrictions trbc genes are subject to concerted evolutionary pressures with intra-species homogenization through gene conversion evident in both humans and mice. similarly, the bovine trbc genes were found to encode near identical products, most likely as a result of gene conversion, although in the case of trbc and trbc genes this more probably reflects minimal divergence following duplication. comparison with the human and murine sequences shows that non-coding elements that regulate trb expression, such as the eβ, promoters and rss are highly conserved in the bovine. this is consistent with work demonstrating that the critical role of rss has enforced a high level of evolutionary conservation and that eβ and pdβ sequences are well conserved in eutherian species. although transcriptional factor binding sites are less well conserved in the putative pdβ than the eβ sequence, the ikaros/lyf- and ap- binding sites of the pdβ which are vital in enforcing stage-specific are conserved. our analysis of putative trbv promoter elements was restricted to the well described cre motif. however, trbv promoters are complex and expression of trbv genes whose promoters lack the cre motif is maintained through the function of other transcriptional factor binding sites. a more detailed analysis of the bovine trbv promoters would be interesting given the potential influence this may have on shaping the expressed trbv repertoire, but is beyond the scope of the current study. the portion of the bovine trb locus described in btau_ encompasses > kb of sequence. thus, although incomplete, the bovine trb locus is larger than that of either humans or mice, mainly as a consequence of the duplications leading to the dramatic expansion of the v genes. in contrast to v genes, duplication of trypsinogen genes within the trb locus is more limited in the bovine, where only trypsinogen genes were identified, compared to the human and murine where more extensive duplication has lead to the presence of and trypsinogen genes respectively. despite the differences in duplication events, the organisation of both tr and non-tr genes within and adjacent to the trb locus exhibits a striking conserved synteny between cattle, humans and mice. indeed, the organisation of genes within the trb locus and its position relative to adjacent loci is ancient, with marked conserved synteny also demonstrated between eutherian and marsupial mammalian species and, to a large extent, chickens. given the evidence for conserved synteny of trbv gene organisation despite dissimilar duplication/deletion events between mice, humans and cattle, the results of the analysis completed in this study suggest that several subgroups including trbv and which were not identified in the genome assembly or from cdna sequences, may have been deleted from the bovine genome. conservation of synteny would predict that the genomic location of the trbv gene identified from cdna analysis will be within the region of undetermined sequence in chr. between the trbv and genes.  the primary purpose of this study was to analyse the sequence data made available from the third bovine genome assembly to gain a better understanding of the bovine trb gene repertoire and the organization and evolution of the bovine trb locus. the results of this analysis have shown that: the bovine trbv genomic repertoire has been dramatically expanded through a complex series of duplication events and although incomplete, is the largest described to date. these duplication events have led to massive expansion in the membership of certain trbv subgroups, particularly trbv and 21; duplication has generated djc clusters compared to in humans and mice; the elements that regulate trb expression and the organisation of genes within and adjacent to the trb loci exhibit high levels of conservation between humans, mice and cattle. cdna evidence indicates that a substantial number of trbv genes, predominantly from the larger subgroups are absent from the current assembly. notwithstanding the incomplete assembly of the trb locus, the results of these analyses clearly demonstrate that cattle possess a phylogenetically diverse repertoire of functional trb genes that is substantially larger than that described for other species. these findings, together with emerging evidence of similar expansions of gene repertoires for other tr chains in ruminants suggest that strong evolutionary pressures have driven a generic enlargement of tr gene numbers, and thus greater potential tr diversity, in the ruminant lineage. further studies are required to define the full extent of these expansions and to understand their evolutionary basis. 
in this study we analyze the sequence of the third bovine genome assembly to characterize the germline repertoire of bovine trb genes and compare the organization, evolution and regulatory structure of the bovine trb locus with that of humans and mice. the trb locus in the third bovine genome assembly is distributed over scaffolds, extending to ~ kb. analyses of cdna data confirms the diversity of the trbv genes and, in addition, identifies a substantial number of trbv genes, predominantly from the larger subgroups, which are still absent from the genome assembly. similarly, duplication has lead to the generation of a third djc cluster. dot-plot analyses demonstrate that expansion of the genomic trbv repertoire has occurred via a complex and extensive series of duplications, predominantly involving dna blocks containing multiple genes. similar expansions in other ruminant tr loci suggest strong evolutionary pressures in this lineage have selected for the development of enlarged sets of tr genes that can contribute to diverse tr repertoires. genomic studies have been pivotal in cataloguing the v, d, j and c genes present in the various tr/ig loci and describing how duplication events have expanded the number of these genes. these duplication events have resulted in massive expansion of several trbv subgroups, most notably trbv and which contain and members respectively. the analyses completed in this study reveal that, although the gene content and organization of the bovine trb locus are broadly similar to that of humans and mice, multiple duplication events have led to a marked expansion in the number of trb genes. the observed gene duplication within the bovine trb locus has created a repertoire of phylogenetically diverse functional trbv genes, which is substantially larger than that described for humans and mice. the available sequence contains trbv genes, assigned to subgroups, and clusters of djc genes, each comprising a single trbd gene, 5– trbj genes and a single trbc gene. seventy-nine of the trbv genes are predicted to be functional. comparison with the human and murine trb loci shows that the gene order, as well as the sequences of non-coding elements that regulate trb expression, are highly conserved in the bovine.
 diverse tr and ig repertoires are generated by vj somatic recombination. genomic studies have been pivotal in cataloguing the v, d, j and c genes present in the various tr/ig loci and describing how duplication events have expanded the number of these genes. such studies have also provided insights into the evolution of these loci and the complex mechanisms that regulate tr/ig expression. in this study we analyze the sequence of the third bovine genome assembly to characterize the germline repertoire of bovine trb genes and compare the organization, evolution and regulatory structure of the bovine trb locus with that of humans and mice.  the trb locus in the third bovine genome assembly is distributed over scaffolds, extending to ~ kb. the available sequence contains trbv genes, assigned to subgroups, and clusters of djc genes, each comprising a single trbd gene, 5– trbj genes and a single trbc gene. seventy-nine of the trbv genes are predicted to be functional. comparison with the human and murine trb loci shows that the gene order, as well as the sequences of non-coding elements that regulate trb expression, are highly conserved in the bovine. dot-plot analyses demonstrate that expansion of the genomic trbv repertoire has occurred via a complex and extensive series of duplications, predominantly involving dna blocks containing multiple genes. these duplication events have resulted in massive expansion of several trbv subgroups, most notably trbv and which contain and members respectively. similarly, duplication has lead to the generation of a third djc cluster. analyses of cdna data confirms the diversity of the trbv genes and, in addition, identifies a substantial number of trbv genes, predominantly from the larger subgroups, which are still absent from the genome assembly. the observed gene duplication within the bovine trb locus has created a repertoire of phylogenetically diverse functional trbv genes, which is substantially larger than that described for humans and mice.  the analyses completed in this study reveal that, although the gene content and organization of the bovine trb locus are broadly similar to that of humans and mice, multiple duplication events have led to a marked expansion in the number of trb genes. similar expansions in other ruminant tr loci suggest strong evolutionary pressures in this lineage have selected for the development of enlarged sets of tr genes that can contribute to diverse tr repertoires. 