Genomic and comparative analysis of the T cell receptor gamma locus in two Equus species

Abstract

The genus Equus is the only extant genus of the Equidae family, which belongs to Perissodactyla, an order of mammals characterized by an odd number of toes (odd-toes ungulates). Taking advantage of the latest release of the genome assembly, we studied, for the first time in two organisms belonging to the Equus genus, the horse (Equus caballus) and the donkey (Equus asinus), the T cell receptor gamma (TRG) locus encoding the gamma chain of the γδ T cell receptor. Forty-five Variable (TRGV) genes belonging to the seven IMGT-NC validated mammalian TRGV subgroups, 25 Joining (TRGJ) and 17 Constant (TRGC) genes organized in 17 V-J-(J)-C cassettes, in tandem on about 1100 Kb, characterize the horse TRG locus, making the horse TRG locus the one with the greatest extension and with a significantly higher number of genes than the orthologous loci of the other mammalian species. A clonotype analysis of an RNA-seq transcriptomic dataset derived from spleen of an adult healthy horse, using the complete set of the horse TRGJ germline gene sequences as a probe, revealed that, in addition to the most prominent V-J rearrangements within each cassette, there is a relevant proportion of trans-cassette V-J recombination, whereby the same TRGV genes can recombine with different TRGJ genes spliced to the corresponding TRGC genes. This recombinant event strongly contributes to the diversity of the γ chain repertoire. In the donkey TRG locus, 34 TRGV, 21 TRGJ and 14 TRGC genes distributed in 14 V-J-(J)-C cassettes were found in a region of approximately 860 kb. Although the donkey's TRG is smaller than that of the horse, in Equus genus, this is still the second largest locus so far found in any mammalian species. Finally, the comparative analysis highlighted differences in size and gene content between the horse and donkey TRG loci, despite belonging to the same genus, indicating a good level of diversification within Equus. These data is in agreement with the evolutionary idea of the existence of a Equus recent common ancestor in rapid evolution, for which a mutation rate between horses and donkeys is more comparable to that between species belonging to different genera rather than to species of the same genus.

Keywords: Equus; Perissodactyla; TRG genes; TRG locus; equid genome; evolution; gamma-delta T-cell; immunogenomics.

Figure 1

Schematic representation of the genomic organization of the horse TRG locus deduced from the EquCab3.0 genomic assembly. The name and the orientation of each TRGC cassette is indicated by an arrow. The TRGV1-2 and TRGV1-3 genes are indicated in the map with a dashed line as they are incomplete due to a gap in the genomic assembly. They are showed as functional since they were found within transcripts (see text). The diagram shows the position of all related and unrelated TRG genes according to nomenclature. The boxes representing the genes are not to scale. The vestigial TRGC6 gene is indicated by a gray box. The exons are not shown. The arrow indicates the transcriptional orientation of the STARD3NL gene. All gaps in the genomic sequence are indicated by the symbols (–//–).

Figure 2

The neighbor-joining (NJ) tree inferred from the horse, dolphin, dromedary, pig, dog, rabbit, mouse and human TRGV gene sequences. The evolutionary analyses were conducted in MEGA X (33, 34). The optimal tree is shown. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the p-distance method (35) and are in the units of the number of base differences per site. This analysis involved 103 nucleotide sequences. Codon positions included were 1st + 2nd + 3rd + Noncoding. All ambiguous positions were removed for each sequence pair (pairwise deletion option). There was a total of 410 positions in the final dataset. The horse TRGV gene are marked with coloured circles. The different colours highlight the distribution of the phylogenetic groups. The branches highlighted by the letters group mammalian genes described in the text. The gene functionality according to IMGT rules (F: functional, ORF: open reading frame, P: pseudogene) is indicated. The IMGT six-letter standardized abbreviations for species (Equus caballus (horse), Homo sapiens (human), Mus musculus (mouse), Sus scrofa (pig), Camelus dromedarius (dromedary), Tursiops truncatus (dolphin), Oryctolagus cuniculus (rabbit)) and nine-letter standardized abbreviation for subspecies (Canis lupus familiaris, dog) taxa are used.

Figure 3

The neighbor-joining (NJ) tree inferred from the horse, dolphin, dromedary, pig, dog, rabbit, mouse and human TRGJ gene sequences. The evolutionary analyses were conducted in MEGA X (33, 34). The optimal tree is shown. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the p-distance method (35) and are in the units of the number of base differences per site. This analysis involved 68 nucleotide sequences. Codon positions included were 1st + 2nd + 3rd + Noncoding. All ambiguous positions were removed for each sequence pair (pairwise deletion option). There was a total of 129 positions in the final dataset. The branches highlighted by the letters group mammalian genes described in the text. The gene functionality according to IMGT rules (F: functional, ORF: open reading frame, P: pseudogene) is indicated. The IMGT six-letter standardized abbreviations for species Equcab (Equus caballus, horse), Homsap (Homo sapiens, human), Musmus (Mus musculus, mouse), Susscr (Sus scrofa, pig), Camdro (Camelus dromedarius, dromedary), Turtur (Tursiops truncatus, dolphin), Orycun (Oryctolagus cuniculus, rabbit), Galgal (Gallus gallus, chicken) and nine-letter standardized abbreviation for subspecies Canlupfam (Canis lupus familiaris, dog) taxa are used.

Figure 4

The neighbor-joining (NJ) tree inferred from the horse, dolphin, dromedary, pig, dog, rabbit, mouse and human TRGC gene sequences. The evolutionary analyses were conducted in MEGA X (33, 34). The optimal tree is shown. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the p-distance method (35) and are in the units of the number of base differences per site. This analysis involved 36 nucleotide sequences. Codon positions included were 1st + 2nd + 3rd + Noncoding. All ambiguous positions were removed for each sequence pair (pairwise deletion option). There was a total of 707 positions in the final dataset. The IMGT six-letter standardized abbreviations for species Equcab (Equus caballus, horse), Homsap (Homo sapiens, human), Musmus (Mus musculus, mouse), Susscr (Sus scrofa, pig), Camdro(Camelus dromedarius, dromedary), Turtur, (Tursiops truncatus, dolphin), Orycun (Oryctolagus cuniculus, rabbit), Galgal (Gallus gallus, chicken) and nine-letter standardized abbreviation for subspecies Canlupfam (Canis lupus familiaris, dog) taxa are used.

Figure 5

Schematic representation of the genomic organization of the donkey TRG locus deduced from the ASM1607732V2 genomic assembly. The name and the orientation of each TRGC cassette is indicated by an arrow. The diagram shows the position of all related and unrelated TRG genes according to nomenclature. The boxes representing the genes are not to scale. The vestigial TRGC6 gene is indicated by a gray box. The exons are not shown. The arrow indicates the transcriptional orientation of the STARD3NL gene.

Figure 6

The neighbor-joining (NJ) tree inferred from the donkey, horse and human TRGV (A) and TRGC (B) gene sequences. The evolutionary analyses were conducted in MEGA X (33, 34). The optimal tree is shown. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the p-distance method (35) and are in the units of the number of base differences per site. Codon positions included were 1st + 2nd + 3rd + Noncoding. All ambiguous positions were removed for each sequence pair (pairwise deletion option). In (A), the analysis involved 83 nucleotide sequences. There was a total of 403 positions in the final dataset. The donkey and horse TRGV genes are marked with two distinct coloured circles. The phylogenetic distribution highlights the grouping of the orthologous genes of the two equid species. The gene functionality according to IMGT rules (F: functional, ORF: open reading frame, P: pseudogene) is indicated. In (B), the analysis involved 24 nucleotide sequences. There was a total of 630 positions in the final dataset. The donkey and horse TRGV genes are marked with two distinct coloured circles. The IMGT six-letter standardized abbreviations for species Equasi (Equus asinus, donkey), Equcab (Equus caballus, horse) and Homsap (Homo sapiens, human) are used.