# Unraveling IGK locus in dog breeds: IMGT® new insights into canine immunogenetics

Taismara Kustro Garnica, Ariadni Papadaki, Maria Georga, Guilhem Zeitoun, Joumana Jabado-Michaloud, Géraldine Folch, Véronique Giudicelli, Pablo Gonzalez, Jéssika Cristina Chagas Lesbon, Talita Gabriela Luna Alves, et al.

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## ► To cite this version:

Taismara Kustro Garnica, Ariadni Papadaki, Maria Georga, Guilhem Zeitoun, Joumana Jabado-Michaloud, et al. Unraveling IGK locus in dog breeds: IMGT® new insights into canine immunogenetics. NAR Genomics and Bioinformatics, 2026, 8 (1), pp.lqag020. 10.1093/nargab/lqag020. hal-05523590

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# Unraveling IGK locus in dog breeds: IMGT® new insights into canine immunogenetics

Taismara Kustro Garnica<sup>1,2</sup>, Ariadni Papadaki<sup>2</sup>, Maria Georga<sup>2</sup>, Guilhem Zeitoun<sup>2</sup>, Joumana Jabado-Michaloud<sup>2</sup>, Géraldine Folch<sup>2</sup>, Véronique Giudicelli<sup>2</sup>, Pablo Gonzalez<sup>2</sup>, Jéssika Cristina Chagas Lesbon<sup>1</sup>, Talita Gabriela Luna Alves<sup>1</sup>, Heidge Fukumasu<sup>1</sup>, Sofia Kossida<sup>2,3,*</sup>

## Abstract

Over thousands of years, selective breeding of domestic dogs (Canis lupus familiaris) has led to extensive genetic diversity, underscoring the need for refined genomic and immunogenetic investigations. This study presents a comprehensive analysis and biocuration of the immunoglobulin kappa light chain locus (IGK) across multiple dog breeds to identify breed-specific variations and assess their relevance to canine immunology and veterinary diagnostics. We examined nine canine genome assemblies to characterize structural variations, polymorphisms, and gene diversity, with the goal of enriching the IMGT® reference database and expanding its representativeness across breeds. Through in-depth annotation of seven breeds—Bernese Mountain Dog, Boxer, Cairn Terrier, Labrador Retriever, Great Dane, Basenji, and German Shepherd—we identified 40 genes and 97 alleles, highlighting both conserved regions and unique breed-specific variants. Variants were validated in *silico* against Sanger sequencing data. Importantly, discrepancies were observed in the CanFam3.1 Boxer reference genome, indicating possible sequencing or assembly artifacts, challenges in gene and allele nomenclature standardization, and a low-density genomic segment within the IGH locus. These findings refine current knowledge of IGH locus diversity and enhance IMGT® database accuracy, supporting future studies on immunogenetic variability, somatic hypermutation, and immune response mechanisms in canine health and disease.

## Introduction

Immunoglobulins (IGs) are composed of two identical heavy chains (IGH) and two identical light chains, either kappa (IGK) or lambda (IGL), connected by disulfide bonds. The heavy chains are encoded by variable (V), diversity (D), joining (J), and constant (C) genes, while the light chains are encoded by V, J, and C genes [1]. The IGH light chain exhibits less diversity than the IGH across species [2]. However, the IGH germline diversity in dog breeds is still largely uncharacterized, which can be attributed to the additional complexity introduced by high-frequency repeat regions, which makes the annotation of IG genes particularly challenging [3, 4].

Research on IG and T-cell receptor (TR) genes across dog breeds is crucial for building a comprehensive genetic database to advance veterinary medicine. Such research is essential for understanding breed-specific predispositions to immune-related diseases, including lymphoma—a hematological malignancy of lymphocytes—that is well documented in breeds like Doberman Pinschers, Rottweilers, Boxers, and Bernese Mountain Dogs [5, 6]. Moreover, identifying allelic variants and polymorphisms associated with conditions such as chronic lymphocytic leukemia and diffuse large B-cell lymphoma offers valuable insights into disease mechanisms [7, 8]. Despite these advances, the genetic diversity of IGs, which function as B-cell antigen receptors, remains underexplored

across different breeds, highlighting a critical gap in our understanding of canine immune genetics.

The International Immunogenetics Information System (IMGT®, https://www.imgt.org/), established in 1989, serves as the global standard for immunogenetics, providing databases and tools critical for investigating the adaptive immune response [9]. According to IMGT®, the canine IGH locus, the smallest of the IG loci, is located on chromosome 17, spanning 317–349 kb and comprising 31 genes (25 V, 5 J, 1 C) based on two reference assemblies: Basenji (Basenji_breed-1.0) and Boxer (CanFam3.1), corresponding to the IMGT sequence database IMGT/LIGM-DB [10] accession numbers IMGT000067 and IMGT000002, respectively [11]. However, discrepancies in the V-J-C-CLUSTER and locus orientation between these genomes require further investigation for accurate biocuration of novel canine assemblies.

To address these challenges, this study has four main objectives: (i) conduct a detailed comparative analysis to verify the orientation and gene positions in the dog IGH locus reference assemblies; (ii) perform extensive biocuration of the IGH locus across nine canine genome assemblies to uncover structural variations, polymorphisms, and gene and allele diversity; (iii) enhance the IMGT® databases, tools, reference directories, and web resources by integrating data from multiple dog breeds, thereby creating a more comprehensive and

<footnote n="1"><sup>1</sup>Laboratory of Comparative and Translational Oncology (LOCT), Department of Veterinary Medicine, Faculty of Animal Science and Food Engineering (FZEA), University of São Paulo (USP), 13635-900 Pirassununga, Brazil</footnote>

<footnote n="2"><sup>2</sup>IMGT®, IGH, Univ Montpellier, CNRS, 34000 Montpellier, France</footnote>

<footnote n="3"><sup>3</sup>Institut universitaire de France (IUF), 75005 Paris, France</footnote>

<footnote>*To whom correspondence should be addressed. Email: sofia.kossida@igh.cnrs.fr</footnote>
diverse genetic repository; and (iv) confirm the IGKV allelic variation through Sanger sequencing.

## Materials and methods

The annotation of the IGK locus was conducted according to the IMGT biocuration pipeline, as previously described [12]. The *Canis lupus familiaris* genomic sequences from new assemblies were analyzed and annotated according to the IMGT dog reference sequences from Boxer (CanFam3.1) and Basenji (Basenji_breed-1.1) (https://www.imgt.org/IMGTrepertoire/index.php?section=LocusGenes&repertoire=locusAssembly&species=dog&group=IGK).

## Criteria for the selection of novel dog assemblies

The canine assemblies were selected by searching for "*Canis lupus familiaris*" in the NCBI Datasets in December 2023 [13]. The selection criteria were defined by IMGT® on chromosomal-level organization, haploid assemblies, and complete genome representation (https://www.imgt.org/IMGTScientificChart/Assemblies/IMGTassemblyquality.php). Thus, nine assemblies from seven different breeds were chosen for the quality of their IGK locus, which fulfilled the standard IMGT criteria for assembly selection²: "Basenji" (UNSW_CanFamBas_1.2, GenBank Assembly ID: GCA_013276365.2), "Bernese Mountain Dog" (BD_1.0 and OD_1.0, GenBank Assembly ID: GCA_031010765.1 and GCA_031010635.1), "Boxer" (Dog10K.Boxer_Tasha, GenBank Assembly ID: GCA_000002285.4), "Cairn Terrier" (CA611_1.0, GenBank Assembly ID: GCA_031010295.1), "German Shepherd" (ASM864105v1, GenBank Assembly ID: GCA_008641055.1), "Great Dane" (UMICH_Zoey_3.1, GenBank Assembly ID: GCA_005444595.1), and "Labrador Retriever" (ASM1204501v1 and ROS_Cfam_1.0, GenBank Assembly ID: GCA_012045015.1 and GCA_014441545.1).

## IGK locus extraction

The IGK locus on chromosome 17 for each assembly was localized and extracted using BLAST (v. 2.15.0) by comparing sequences against IGK reference sequences from Basenji (Basenji_breed-1.1) and Boxer (CanFam3.1) in IMGT/GENE-DB (v. 3.1.37), corresponding to IMGT/LIGM-DB accession numbers IMGT000067 and IMGT000002, respectively [14]. The locus position and orientation were defined based on the flanking non-IG genes, known as "IMGT bornes," identified as paired box 8 (PAX8, NCBI Gene ID: 403927) and ribose 5-phosphate isomerase A (RPIA, NCBI Gene ID: 100856455). The IMGT bornes provide a standardized method for comparing IG and TR locus delimitation across species [1]. Distances between the IMGT bornes and the flanking IG genes (IGKV9-24 upstream and IGKC downstream) were measured. If the borne genes were absent or distances exceeded 10 kb, an extraction interval was set to extend 10 kb upstream of IGKV9-24 and 10 kb downstream of IGKC.

## Quality evaluation of the IGK locus and integration in IMGT

The extracted IGK locus sequence was evaluated by vector contamination to detect foreign DNAs such as vector, linker,

adapter, and primer regions using VecScreen [15]. The Comparative Genome Viewer (CGV) [16] from NCBI was employed to analyze the IGK locus between two assemblies, allowing for the comparison of locus orientation, and the investigation of potential deletions, insertions, translocations, and inversions that could affect IGK locus annotation. The default parameters included a minimum alignment size of 10 kb, with adjustments to the viewer to display non-best-placed alignments and show both forward and reverse alignments. The IMGT/LIGMotif internal tool was used to evaluate the orientation of genes in the locus, the total number of genes by group and subgroup, and to find and delimit the gaps when they were present [17]. After the quality evaluation of the locus, the corresponding nucleotide sequences were extracted from the NCBI chromosome sequences, and entries were created in IMGT/LIGM-DB with an IMGT accession number (IMGT' followed by 6 digits) [14].

## V, J, and C genes annotation

The V, J, and C genes were identified and delineated along the IMGT/LIGM-DB genomic sequences of the IGK locus using IMGT/LIGMotif [14, 17]. IGK genes were characterized and classified through BLAST alignments, Clustal Omega, and the application of IMGT's unique numbering and annotation rules according to the IMGT Scientific Chart [18–20]. The nomenclature for *Canis lupus familiaris* IGK genes and alleles, and the determination of the allele functionality, follow the IMGT-ONTOLOGY concepts [21] (https://www.imgt.org/IMGTScientificChart/Nomenclature/IMGTnomenclature.html). In agreement, the nomenclature for IGK genes of Labrador Retriever assembly (ROS_Cfam_1.0) was approved by the Vertebrate Gene Nomenclature Committee (VGNC) in 2024 [22]. The NCBI Third Party Annotation (TPA) accession numbers were assigned to two assemblies from the Bernese Mountain Dog breed: BD_1.0 and OD_1.0, corresponding to IMGT/LIGM-DB accession numbers BK066061 and BK066062, respectively (https://www.ncbi.nlm.nih.gov/genbank/tpa/). The whole data of IGK genes were integrated into IMGT/GENE-DB [14] and the biocuration data for genes, alleles, and proteins were entered into IMGT web resources (https://www.imgt.org/IMGTrepertoire/). The phylogenetic tree of IGKV alleles was constructed using NGPhylogeny.fr [23], employing MAFFT version 7 [24] for multiple sequence alignment, PhyML [25] for maximum likelihood phylogenetic inference, and iTOL v6 [26] for visualization.

## Primer design, PCR amplification, and sequence analysis

Seven forward primers and one consensus reverse primer were designed to validate alleles belonging to the *IGKV2* subgroup, considering the V-EXON region, which presented the highest number of alleles. The primer sequences are listed in Table 1. Additional information can be assessed in Supplementary Table S4.

Genomic DNA (gDNA) was extracted from non-lymphoid cardiac tissue samples of three dogs (two females and one male) obtained from the LOCT Laboratory Biobank (Ethics approval: CEUA 7602220224). Each polymerase chain reaction (PCR) contained 100 ng of gDNA, 200 nM of each
<table><caption>**Table 1.** Primers for validation of *Canis lupus familiaris* IGKV2 alleles</caption><thead><tr><th>Group</th><th>Primer</th><th>Sequence</th><th>Tm°C</th><th>CG%</th><th>nt</th><th>Matches in IMGT/GENE-DB</th></tr></thead><tbody><tr><td rowspan="10">IGKV2</td><td>FWD 3</td><td>TACTTTAGGATCCAGTGGG</td><td>57.1</td><td>47.4</td><td>19</td><td>4</td></tr><tr><td>FWD 5</td><td>TTACCTAGGATCCAGTGGG</td><td>59.5</td><td>52.6</td><td>19</td><td>1</td></tr><tr><td>FWD 6</td><td>TTACCTAGGATCCAGTGGG</td><td>62.6</td><td>52.6</td><td>19</td><td>3</td></tr><tr><td>FWD 7</td><td>AATTTTAGGATCCAGTGGG</td><td>58.4</td><td>42.1</td><td>19</td><td>3</td></tr><tr><td>FWD 8</td><td>AATTTTAGGATCCACTGGG</td><td>58.4</td><td>42.1</td><td>19</td><td>2</td></tr><tr><td>FWD 10</td><td>TTACCTAAGATCCAGTGGG</td><td>57.1</td><td>47.4</td><td>19</td><td>4</td></tr><tr><td>FWD 12</td><td>TATTTTAGGATCCGGTGGG</td><td>61.6</td><td>47.4</td><td>19</td><td>6</td></tr><tr><td>REV 1</td><td>GGTTTGTGTTCAGGGCTG</td><td>62.3</td><td>55.6</td><td>18</td><td>48</td></tr></tbody></table>

<table><caption>**Table 2.** Mainly data from IGK locus biocuration from nine dog assemblies</caption><thead><tr><th>IMGT/LIGM-DB</th><th>Assembly (breed)</th><th>V</th><th>J</th><th>C</th><th>Total</th><th>Length (kb)</th></tr></thead><tbody><tr><td>IMGT000184</td><td>ASM864105v1 (German Shepherd)</td><td>21</td><td>5</td><td>1</td><td>26</td><td>264</td></tr><tr><td>BK066061<sup>a</sup></td><td>BD_1.0 (Bernese Mountain Dog)</td><td>29</td><td>5</td><td>1</td><td>35</td><td>358</td></tr><tr><td>IMGT000179</td><td>CA611_1.0 (Cairn Terrier)</td><td>24</td><td>5</td><td>1</td><td>30</td><td>298</td></tr><tr><td>IMGT000185</td><td>Dog10K_Hoxer_Tasha (Boxer)</td><td>19</td><td>5</td><td>1</td><td>25</td><td>235</td></tr><tr><td>BK066062<sup>a</sup></td><td>OD_1.0 (Bernese Mountain Dog)</td><td>29</td><td>5</td><td>1</td><td>35</td><td>369</td></tr><tr><td>IMGT000181</td><td>ROS_Cfam_1.0 (Labrador Retriever)</td><td>21</td><td>5</td><td>1</td><td>27</td><td>274</td></tr><tr><td>IMGT000183</td><td>UMICH_Zoey_3.1 (Great Dane)</td><td>25</td><td>5</td><td>1</td><td>31</td><td>306</td></tr><tr><td>IMGT000180</td><td>UNSW_CanFamBas_1.2 (Basenji)</td><td>23</td><td>5</td><td>1</td><td>29</td><td>285</td></tr><tr><td>IMGT000182</td><td>Yella_v2 (Labrador Retriever)</td><td>22</td><td>5</td><td>1</td><td>28</td><td>279</td></tr></tbody></table>

primer, 1× Platinum™ PCR Master Mix (Invitrogen, USA), and molecular biology-grade water in a final volume of 50 µl.

PCR amplification was performed on a C1000 Touch™ Thermal Cycler (Bio-Rad, USA) under the following conditions: initial denaturation at 94°C for 2 min; followed by 25 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s.

Amplicons were visualized on a 2% agarose gel and purified using the QIAquick PCR Purification Kit (Qiagen, Germany) according to the manufacturer's instructions. Sanger sequencing was performed on an ABI 3730 DNA Analyzer (Applied Biosystems, Thermo Fisher Scientific, USA) using the BigDye™ Terminator v3.1 Cycle Sequencing Kit.

The obtained sequences were analyzed with FastQC (v0.12.1) to assess the PHRED quality scores. Reads were trimmed using Sickle (v1.33; https://github.com/najoshi/sickle) with default Sanger parameters. Forward and reverse sequences were then analyzed using IMGT/V-QUEST (version 3.7.0; reference directory 202531-1) and compared with the IMGT reference database. Ambiguous nucleotides were resolved using overlapping information from the complementary sequence, when applicable.

# Results

## Comprehensive annotation of the IGK locus across dog breeds

### Quality evaluation of IGK locus assemblies

During the quality assessment of the loci, it was observed that three assemblies (33.3%) presented small gaps in the IGK locus. The Bernese Mountain Dog (OD_1.0), Labrador Retriever (Yella_v2), and Great Dane (UMICH_Zoey_3.1) with variable sizes from 99 to 200 bp (Supplementary Table S1). However, the genomic structure (the organization of V-J-C-CLUSTER) and orientation of the IGK locus

were not affected, and they passed the quality control of IMGT.

### Localization and description of the IGK locus

The biocuration of the dog IGK locus followed the IMGT standards and comprised seven distinct dog breeds: Basenji, Bernese Mountain Dog, Boxer, Cairn Terrier, German Shepherd, Great Dane, and Labrador Retriever. Accounting with five assemblies from female dogs and four from male dogs. The IGK locus in dogs had a variable length, ranging from 235 to 369 kb across the nine assemblies, with the longest locus belonging to the Bernese Mountain Dog assembly (OD_1.0) and the shortest to the Boxer (Dog10K_Hoxer_Tasha) (Table 2). The highest number of V genes was found in both Bernese Mountain Dog assemblies (BD_1.0 and OD_1.0) and the lowest was found in Boxer (Dog10K_Hoxer_Tasha). However, the number of J and C genes was conserved among the assemblies (Table 2).

A holistic IGK locus map was created to integrate all data obtained from the annotated assemblies (gene positions, allele functionality, V-J-C-CLUSTER organization, and locus orientation) (Fig. 1). The IGK locus was found forward-oriented (FWD) on chromosome 17, for all nine dog assemblies. The locus extends from 10 kb upstream, IGKV9-24, to 10 kb downstream, IGKC. The two non-IG flanking genes, referred to as “IMGT bornes,” were identified through BLAST alignment and annotated in all assemblies to define the boundaries of the IGK locus. According to the IGK holistic map for dogs, the 5' end IMGT flanking gene, “IMGT borne,” PAX8 (Gene ID: 403927) is 137 kb upstream of IGKV9-24, the most 5' gene in the locus, and the 3' end IMGT flanking gene, “IMGT borne,” RPIA (Gene ID: 100856455) is 35 kb downstream of IGKC, the most 3' gene in the locus. The IGK locus consists predominantly of a cluster of 34 V genes (V-CLUSTER) and a smaller cluster containing 5 J genes and 1 C gene (J-C-CLUSTER). Five genes were in the opposite orientation in the locus—IGKV4-15,

<footnote><sup>a</sup>Annotations available on TPA (NCBI) with the same accession number as IMGT/LIGM-DB.</footnote>
<figure><img src="image_3.png" /><figcaption>**Figure 1.** Holistic IMGT reference IGK map for dogs (*Canis lupus familiaris*). The dog IGK locus is on chromosome 17, and the orientation is forward (FWD). The colors are according to the IMGT® color menu for genes (https://www.imgt.org/IMGTScientificChart/RepresentationRules/colormenu.php#LOCUS).</figcaption></figure>

IGKV(I)-14, IGKV3-3, IGKV7-2, and IGKV4-1—across all assemblies.

The boxes representing the genes are not scaled, and the exons are not shown. IGK gene names follow the IMGT nomenclature [27]. The dog's IGKV genes belong to seven subgroups IGKV1, IGKV2, IGKV3, IGKV4, IGKV7, IGKV8, IGKV9 as well as IGKV(I) and IGKV(II), belonging to clans [a clan is a set of subgroups, eventually from different species, which appear related to phylogenetic trees (IGHV, IGKV, IGLV clans). The “clan” concept is part of the “CLASSIFICATION” concept of IMGT-ONTOLOGY] [28]. The dots represent the distance between the borne genes (PAX8 and RPIA), the upstream gene (IGKV9-24), and the downstream gene (IGKC) in the locus. Single arrows show genes whose orientation is opposite to that of the J-C-CLUSTER comprising IGKJ (J1–J5)-IGKC. The eight highlighted genes above the central axis represent genes not found in all assemblies. The central axis represents the functionality of the alleles found in the reference genomes (CanFam3.1 and Basenji_breed-1.1)—reproduction authorized by IMGT®, the international ImMunoGeneTics information system, available on the IMGT® website.

## Annotation of IGKV gene cluster

The biocuration of dog assemblies identified 34 IGKV genes (Fig. 1). In dogs, the IGKV genes are categorized into seven distinct subgroups (IGKV1, IGKV2, IGKV3, IGKV4, IGKV7, IGKV8, and IGKV9) and two clans, IGKV(I) and IGKV(II), according to the IMGT-ONTOLOGY and their sequence correspondence with the IGK human subgroups (Fig. 1). Among these, the IGKV2 subgroup was the most representative, encompassing 21 genes constituting 61.76% of the total V genes, followed by IGKV3 with 4 genes (11.76%).

Seven new genes were discovered during the annotation of nine dog assemblies (IGKV3-17-1, IGKV2-11D, IGKV2-9-1, IGKV2-7-2, IGKV2-7-1, IGKV2-6-1, and IGKV2-4-1), accounting for a discovery rate of 17.5%. However, these new genes were uncommon for all the assemblies (Table 3). The IGKV2-7-1 gene was found in two distinct breed assemblies from Bernese Mountain Dog (BD_1.0) and Cairn Terrier (CA611_1.0). The IGKV2-6-1 gene was found in four assemblies from Bernese Mountain Dog (BD_1.0 and OD_1.0), Cairn Terrier (CA611_1.0), and Basenji (UNSW_CanFamBas_1.2). Finally, the IGKV2-4-1
<table><caption>**Table 3.** New IGKV genes per assembly</caption><thead><tr><td>New IGKV genes</td><td>Breed (assembly)</td></tr></thead><tbody><tr><td>IGKV3-17-1</td><td>Great Dane (UMICH_Zoey_3.1)</td></tr><tr><td>IGKV2-11D</td><td>Great Dane (UMICH_Zoey_3.1)</td></tr><tr><td>IGKV2-9-1</td><td>Bernese Mountain Dog (OD_1.0)</td></tr><tr><td>IGKV2-7-2</td><td>Bernese Mountain Dog (BD_1.0)</td></tr><tr><td>IGKV2-7-1</td><td>Bernese Mountain Dog (BD_1.0)Cairn Terrier (CA611_1.0)</td></tr><tr><td>IGKV2-6-1</td><td>Bernese Mountain Dog (BD_1.0)Bernese Mountain Dog (OD_1.0)Cairn Terrier (CA611_1.0)Basenji (UNSW_CanFamBas_1.2).</td></tr><tr><td>IGKV2-4-1</td><td>Bernese Mountain Dog (BD_1.0)Bernese Mountain Dog (OD_1.0)</td></tr></tbody></table>

gene was found in two assemblies from the Bernese Mountain Dog (BD_1.0 and OD_1.0).

Nevertheless, four genes were found exclusively in one assembly. The IGKV3-17-1 and IGKV2-11D genes were identified only in the Great Dane assembly (UMICH_Zoey_3.1), and the duplicated gene was found in this assembly at the contig level. The IGKV2-9-1 gene was unique to the Bernese Mountain Dog assembly (OD_1.0), while the IGKV2-7-2 gene was exclusively in the Bernese Mountain Dog assembly (BD_1.0). According to our results, most new IGKV genes (5 out of 7, 71.42%) were identified in the Bernese Mountain Dog breed assemblies (Table 3).

## Annotation of IGKJ and IGKC gene clusters

All nine canine assemblies conserved five IGKJ genes (J-CLUSTER), each belonging to a different set (J1, J2, J3, J4, and J5) and one IGKC gene (C-CLUSTER).

## Insights into canine immunogenetic diversity

### Patterns of gene conservation across dog breeds

The overall conservation of IGK genes across dog assemblies was 50%, with 20 out of 40 IGKV, IGKJ, and IGKC genes being shared. This analysis included genes identified in all annotated dog assemblies available on the IMGT website, comprising two IMGT reference assemblies and nine newly annotated assemblies (Fig. 2). Conversely, the remaining 50% of IGK genes, all from the IGKV group, exhibited variability across the dog assemblies. Notably, a unique gene, IGKV2S13, identified in the IMGT reference Boxer assembly (CanFam3.1), was absent from all newly annotated dog assemblies in this study.

### Discovery of new alleles

During this study, it was possible to characterize 97 IGK alleles, most of which belonged to the variable genes (IGKV). No new alleles were identified for the IGKJ and IGKC genes beyond those previously characterized in the IMGT reference assemblies (CanFam3.1 and Basenji_breed-1.1). A single allele was found for each IGKJ gene (IGKJ1*01, IGKJ2*01, IGKJ3*01, IGKJ4*01, and IGKJ5*01), and two alleles were identified for the IGKC constant gene (IGKC*01 and IGKC*02).

Ninety alleles were identified across 34 IGKV genes (Fig. 3). Of these, 29 genes were polymorphic, while only 5 genes exhibited the same allele across all dog assemblies: IGKV1-23, IGKV2-19, IGKV4-15, IGKV(I)-14, and IGKV(II)-1-1 (Fig. 4). The new alleles were defined based on the percentage of iden-

tity in the V-REGION ranging from 96% to 99%. The newly identified alleles for each assembly are highlighted in yellow, along with their respective identity percentages, and can be found in the Supplementary material (Supplementary Table S2). The majority of dog IGKV genes belong to the IGKV2 subgroup (Fig. 3 and Table 4). The varying population sizes within a given immunoglobulin V subgroup can be explained by factors such as genetic evolution, selective pressures, and recombination preferences observed in other species [29].

Our analysis found a common "low coverage region" in the V-CLUSTER of all the assemblies. The region between IGKV2-9 and IGKV2-4 had fewer genes than the locus's upstream and downstream regions. Interestingly, the genes at low frequency in this region had a high diversity of alleles, as we can see from IGKV2-5 and IGKV2-4 (Fig. 4).

Considering the functionality of the IGKV alleles, most of them are functional. The IGKV2 subgroup exhibited the highest allele count, with 60 alleles (66.6%), predominantly functional, including 43 F and 17 P alleles. The IGKV3 subgroup contained the second largest number of alleles, comprising six functional alleles (F) and four pseudogenes (P). Subgroups IGKV4 and IGKV9 contained two open reading frames (ORFs). Subgroup IGKV1 and clan IGKV(I) presented a unique allele, both P. In total, the analysis identified 48 F (53.3%), 38 P (42.2%), and 4 ORF (4.5%) IGKV alleles (Table 5).

## Validation of new alleles by Sanger sequencing

Most of the functional genes are IGKV2; the objective is to confirm by a second method the existence of allelic polymorphism and eliminate potential sequencing errors. For this purpose, nine PCR products were successfully sequenced using the Sanger method with the seven designed primers. Among them, two out of seven primers yielded sequences showing 100% alignment identity with IGKV2 alleles deposited in the IMGT/GENE-DB database. Specifically, Primer 6 (FWD) amplified and confirmed the allele IGKV2-12*03 (F), while Primer 12 (FWD) identified the allele IGKV2-40*4 (F), confirming their existence and homozygosity in the analyzed samples (Fig. 5).

The remaining five primers produced sequences with alignment identities ranging from 92.08% to 97.69%, suggesting the presence of heterozygous loci or allelic variants not yet represented in the reference database (Supplementary Table S4).

## Challenges and limitations

### Locus annotation discrepancies in IMGT references

Before initiating annotations for the new dog assemblies, a thorough analysis of the IMGT reference sequences for the dog's IGK locus was conducted. This step was necessary due to discrepancies in locus orientation, which could affect the accurate identification and extraction of this locus in subsequent assemblies. For the *Canis lupus familiaris* IGK locus, two reference assemblies from distinct breeds are available in the IMGT® database: the Boxer (CanFam3.1; accession number IMGT000002) and the Basenji (Basenji_breed-1.1; accession number IMGT000067), both listed in the IMGT/LIGM-DB [10].

The IGK locus in dogs is located on chromosome 17, with a reverse (REV) orientation in the Boxer (CanFam3.1), and a forward (FWD) orientation in the Basenji (Fig. 5A and
IGK Genes: 40
Dog Assemblies: 11
Dog Breeds: 7
(IMGT/GENE-DB)

Non-common Genes: 20
(50%)

IGKV9-24
IGKV1-23
IGKV8-22
IGKV3-17-1
IGKV3-14-1
IGKV2S13
IGKV2-11D
IGKV2-11
IGKV2-10
IGKV2-9-1
IGKV2-9
IGKV2-8
IGKV2-7-2
IGKV2-7-1
IGKV2-7
IGKV2-6-1
IGKV2-6
IGKV2-5
IGKV2-4-1
IGKV2-4

GENEDB

<figure><img src="image_4.png" /></figure>

Common Genes: 20
(50%)

IGKV2-21
IGKV2-20
IGKV2-19
IGKV3-18
IGKV(II)-17
IGKV2-16
IGKV4-15
IGKV(I)-14
IGKV2-13
IGKV2-12
IGKV3-3
IGKV7-2
IGKV(II)-1
IGKV4-1
IGKJ1
IGKJ2
IGKJ3
IGKJ4
IGKJ5
IGKC

<figure><img src="image_5.png" /><figcaption>Bernese Mountain Dog
(BD_1.0, OD_1.0)</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>Cairn Terrier
(CA611_1.0)</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>Basenji
(UNSW_CanFamBas_1.2,
Basenji_breed-1.1*)</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>Labrador
(ROS_Cfam_1.0, Yella_v2)</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>Great Dane
(UMICH_Zoey_3.1)</figcaption></figure>

<figure><img src="image_10.png" /><figcaption>German Shepherd
(ASM864105v1)</figcaption></figure>

<figure><img src="image_11.png" /><figcaption>Boxer
(CanFam3.1*, Dog10K_Hoxer_Tasha)</figcaption></figure>

<caption>**Figure 2.** Representation of the IGK locus genes in dogs (*Canis lupus familiaris*) that are either common (100% frequency) or noncommon (<100% frequency) across 11 annotated dog breed assemblies (2 IMGT reference and 9 new assemblies) available on IMGT/GENE-DB (https://www.imgt.org/genedb/) [14]. Notably, the conservation rate of IGK genes within this locus is 50% across all breeds. (https://imgt.org/IMGTrepertoire/LocusGenes/locusdesc/assembly_compare.php?species=Canis&lupus&familiaris&locus=IGK). (*) represents the reference assemblies in IMGT®.</caption>

B). In the Boxer (CanFam3.1) assembly, the position of the V-J-CLUSTER reveals unusual organization, with the J-C-CLUSTER positioned centrally within the IGK locus rather than at the expected 3' end. In contrast, the Basenji assembly displays an IGK locus organization more consistent with other veterinary species, such as the cat, goat, sheep, horse, bovine, and pig, as represented in IMGT® > Repertoire > Locus representations (https://www.imgt.org/IMGTrepertoire/LocusGenes/#B).

The length of the IGK locus was 349 kb in the Boxer (Can-Fam3.1), assembly and 317 kb in the Basenji (Basenji_breed-1.1) assembly, showing two gaps in the Boxer, in the region of the IGK locus on chromosome 17. The smallest gap was 226 bp, between the positions 227 525 and 227 750 bp, while the largest gap was 950 bp, between the positions 337 118 and

338 067 bp. The 950 bp size gap was positioned between the IGKC*02 and IGKV2-12*02 genes, and the 226 bp gap occurred within the IGKV3-18 gene. Although for the IGK locus in Basenji (Basenji_breed-1.1), no gap was found (Fig. 6A and B). The flanking genes, referred to as "IMGT bornes," for the IGK locus were present in both reference assemblies, though the position of RPIA differed notably. In the Basenji assembly (Basenji_breed-1.1), the 5' IMGT flanking gene, PAX8 (PAX8, Gene ID: 403927), is located 137 kb upstream of IGKV9-24, the most 5' gene in the locus, while the 3' IMGT flanking gene, RPIA (RPIA, Gene ID: 100856455), is positioned 35 kb downstream of IGKC. In the Boxer reference (CanFam3.1), PAX8 is 161 kb upstream of IGKV2-11, the most 5' gene in the locus, and RPIA is situated between the IGKC and IGKV2-12 genes (Fig. 6C).
<figure><img src="image_12.png" /><figcaption>**Figure 3.** A comprehensive phylogenetic tree displaying 90 IGKV alleles of the dog (*Canis lupus familiaris*) categorized by their respective IGKV subgroups, based on V-REGION sequence comparison. The genes belong to the same subgroup if they share at least 75% nucleotide sequence identity in their V-REGION (V gene) and J-REGION (J gene) according to IMGT® criteria. The tree was constructed using NGPhylogeny.fr [23], employing MAFFT version 7 [24] for multiple sequence alignment, PhyML [25] for maximum likelihood phylogenetic inference, and iTOL v6 [26] for visualization.</figcaption></figure>

The IGK locus in the Boxer (CanFam3.1) contains 30 genes, comprising 24 V-GENES, 5 J-GENES, and 1 C-GENE, whereas the Basenji (Basenji_breed-1.1) assembly includes 31 genes, totaling 25 V-GENES, with the same number of J-GENES [5] and the C-GENE [1] as in the Boxer (CanFam3.1). The two reference assemblies share the most IGK genes, with 28 out of 33 genes (84.4%) being common, including 22 IGKV, 5 IGKJ, and 1 IGKC gene. However, five genes (15.6%) differ between the assemblies: IGKV3-14-1 and IGKV2S13 were found only in the Boxer (CanFam3.1) assembly, while IGKV9-24, IGKV1-23, and IGKV8-22 were present only in the Basenji (Basenji_breed-1.1) assembly, and were located at the (5' FWD) end of the locus (Fig. 6D and E). Most of the IGKV genes in the Basenji reference assembly (Basenji_breed-1.1) were pseudogenes, whereas in the Boxer reference assembly (CanFam3.1), most IGKV genes were functional (Fig. 6D and E).

## Intra-individual IGK locus discrepancies: boxer case study

Given the discrepancies in IGK locus orientation, V-J-C-CLUSTER position, and gaps in the IMGT reference Boxer (CanFam3.1), the release of a new Boxer assembly (Dog10K_Hoxer_Tasha), which is based on sequencing from

the same individual as the reference (CanFam3.1), has facilitated improvements and clarify the discrepancies in the annotations of the IGK locus for the first Boxer assembly (CanFam3.1). The locus orientation differs; the Boxer (Dog10k_Hoxer_Tasha) has a forward orientation (FWD), contrary to the old reference Boxer (CanFam3.1) (Fig. 7A and B).

The CGV tool also confirmed that the Boxer assembly (Dog10k_Hoxer_Tasha) has the 5'end IMGT flanking gene, "IMGT borne," PAX8 (PAX8, Gene ID: 403927) at 168 kb upstream of IGKV9-24, the most 5'gene in the locus, and the 3' end IMGT flanking gene, "IMGT borne," RPIA (RPIA, Gene ID: 100856455) at 36 kb downstream of IGKC (Fig. 7C). The IGK locus length for the Boxer assembly (Dog10k_Hoxer_Tasha) spans 235 kb, with no gaps. Interestingly, the IGK locus in the Boxer assembly (Dog10k_Hoxer_Tasha) was smaller than the IMGT Boxer reference (CanFam3.1). Both the orientation of the IGK locus and the position of the V-J-C-CLUSTER in the Boxer (Dog10k_Hoxer_Tasha) were like the organization observed in the IMGT Basenji reference (Basenji_breed-1.1) (Fig. 6B). This suggests the possibility of annotation errors in the IGK locus of reference Boxer assembly (CanFam3.1), which may
<figure><img src="image_13.png" /><figcaption>**Figure 4.** The heatmap displays the allelic polymorphism at the gDNA level of the dog (*Canis lupus familiaris*) IGK locus, based on 11 genome assemblies from distinct dog breeds. Each row represents a locus from a specific assembly/breed, and the columns represent each IGK gene in their respective order within the locus (V-J-C-CLUSTER). Each color corresponds to different alleles (from *01 to *06). Blank squares indicate missing genes in each assembly (unassigned). Squares with a diamond shape represent a specific allele, IGKV2S13*01 found only in one assembly (CanFam3.1) with provisional nomenclature. Gene frequencies are presented below. (*) represents the reference assemblies in IMGT®.</figcaption></figure>

have been due to incorrect assembly reconstruction and the gaps in the region of this locus.

The IGK locus of Boxer (Dog10k.Boxer_Tasha) contains 25 genes, comprising 19 V-GENES, 5 J-GENES, and 1 C-GENE. Compared to the Boxer reference (CanFam3.1), the Boxer assembly (Dog10k.Boxer_Tasha) was missing five V-GENES (Fig. 7). To determine whether the high gene count in the Boxer reference (CanFam3.1) was due to gene duplications, we employed the Clustal Omega tool, which confirmed the absence of duplicated genes (data not shown). The Boxer assemblies shared 22 out of 33 IGK

genes (66.6%), with a discrepancy of 11 genes (33.3%). The eight genes (IGKV2S13, IGKV2-11, IGKV2-10, IGKV2-9, IGKV2-8, IGKV2-7, IGKV2-6, IGKV2-4) were present only in the Boxer reference assembly (CanFam3.1), while three genes (IGKV9-24, IGKV1-23, and IGKV8-22) were present only in the Boxer assembly (Dog10k.Boxer_Tasha) (Fig. 7D and E). Most of the IGKV genes in the new Boxer assembly (Dog10k.Boxer_Tasha) were pseudogenes, whereas in the Boxer reference assembly (CanFam3.1), most IGKV genes were functional. Notably, 4 out of 22 genes (18.2%) shared between the Boxer assemblies [IGKV(II)-17,
<figure><img src="image_14.png" /><figcaption>**Figure 6.** Comparative analysis of the IGK locus in the dog (*Canis lupus familiaris*) IMGT reference genomes: Boxer (CanFam3.1) and Basenji (Basenji_breed-1.1). (A) IMGT reference map of the IGK locus for the Boxer (CanFam3.1) genome, displayed in reverse (REV) orientation, with the presence of two gaps indicated by double slashes. (B) IMGT reference map of the IGK locus for the Basenji (Basenji_breed-1.1) genome in forward (FWD) orientation. Notably, the positions of the V-J-CLUSTER and IMGT 3' Borne RPIA (Gene ID: 100856455) differ between the two references, with an uncommon pattern observed in the Boxer reference. Reproduction is authorized by IMGT®, the international ImMunoGeneTics information system, and is available on the IMGT® website. (C) Comparative analysis of chromosome 17 between the two IMGT reference assemblies using the CGV. Rectangles delineate the positions of the IGK locus in each assembly on the chromosome. The blue region (hourglass shape) represents alignment sequences in reverse orientation, while white represents nonmatched sequences and green denotes matched sequences. Notably, the IMGT 3' Borne RPIA (Gene ID: 100856455) is mispositioned within the middle of the IGK locus in the Boxer (CanFam3.1) assembly. (D) The Venn diagram illustrates the number of shared IGK genes between the Boxer and Basenji assemblies. (E) Venn diagram illustrating the number of shared IGK alleles between the Boxer and Basenji assemblies. Gray boxes highlight distinct genes (D) and alleles (E) in each assembly, with their respective functional classifications: functional (F) or pseudogene (P). The diagram was generated using Eulerr (https://eulerr.co).</figcaption></figure>
<figure><img src="image_15.png" /><figcaption>**Figure 7.** Comparative analysis of the IGK locus in the dog (*Canis lupus familiaris*) IMGT reference genome for the Boxer (CanFam3.1) and the latest version of the Boxer genome (Dog10K.Boxer_Tasha), derived from the same individual but two distinct genome assemblies. (A) IMGT reference map of the IGK locus for the Boxer (CanFam3.1) genome, displayed in reverse (REV) orientation, with the presence of two gaps indicated by double slashes. (B) The IMGT map of the IGK locus for the latest version of the Boxer genome (Dog10K.Boxer_Tasha) in forward (FWD) orientation. Notably, the positions of the V-J-CLUSTER and IMGT 3' Borne RPIA (Gene ID: 100856455) differ between the two references, with an uncommon pattern observed in the IMGT Boxer reference (CanFam3.1). Reproduction is authorized by IMGT®, the international ImMunoGeneTics information system, and is available on the IMGT® website. (C) Comparative analysis of chromosome 17 between two IMGT Boxer assemblies using the CGV. Rectangles indicate the IGK locus positions in each assembly. The blue hourglass-shaped region represents aligned sequences in reverse orientation, while white areas denote nonmatched sequences, and green indicates matched sequences. In the Boxer reference (CanFam3.1), the RPIA gene is located within the IGK locus, whereas in the new Boxer assembly (Dog10k.Boxer_Tasha), RPIA is positioned 35 kb downstream, just after the IGK locus. Additionally, the older assembly (+114 kb) encompasses a larger locus, which may explain the higher number of IGK genes observed in CanFam3.1 compared to Dog10k.Boxer_Tasha. (D) Venn diagram illustrating the number of shared IGK genes between the Boxer assemblies. (E) Venn diagram illustrating the number of shared IGK alleles between the Boxer assemblies. Grey boxes highlight distinct genes (D) and alleles (E) in each assembly, with their respective functional classifications: functional (F) or pseudogene (P). Notably, four genes exhibited allelic polymorphisms between the assemblies. The diagram was generated using Eulerr (https://eulerr.co).</figcaption></figure>
<figure><img src="image_16.png" /><figcaption>**Figure 8.** Comparative analysis of the IGK locus between the Bernese Mountain Dog assemblies BD_1.0 and OD_1.0. IMGT maps of the IGK locus for the Bernese Mountain Dog assemblies BD_1.0 (A) and OD_1.0 (B) are presented in forward (FWD) orientation on chromosome 17. Red arrows highlight alleles exhibiting 100% identity in the V-region, suggesting a possible duplication. The blue arrow points to the allele with 100% identity in the V-region shared between the BD_1.0 (A) and OD_1.0 (B) assemblies, indicating a potential translocation. Notably, the OD_1.0 (B) assembly shows a gap (double slash) near the IGKV2-9 allele. Reproduction authorized by IMGT®, the international ImMunoGeneTics information system, available on the IMGT® website. (C) Comparative analysis of chromosome 17 between the two IMGT Bernese Mountain Dog assemblies using the CGV. Rectangles represent the IGK locus positions in each assembly. Red arrows indicate sequence matches of different sizes between the assemblies, suggesting a possible duplication. Blue arrows indicate sequence matches in different locations, implying a possible translocation.</figcaption></figure>
complicating confirmation of the translocation and validation
of the positioning of IGKV2-4-1\*02 (OD_1.0) and IGKV2-
9\*02 (BD_1.0). Notably, the newly identified genes IGKV2-
4-1\*01 and IGKV2-4-1\*02 were exclusively observed in the
Bernese Mountain Dog assemblies. In contrast, the new al-
leles IGKV2-6\*02 and IGKV2-9\*02 were shared with the
Labrador Retriever assemblies (ROSCfam_1.0 and Yella_v2)
and were in their expected genomic positions without evi-
dence of translocations or duplications. Based on these find-
ings, we opted not to alter the existing gene names. The ob-
served assembly inconsistencies highlight the challenges of as-
igning gene nomenclature (based on position) when assem-
bly errors, such as duplications, translocations, and gaps, are
present.

Discussion

To the best of our knowledge, our study is the first to inves-
tigate the IGK gene at the germline level across various dog
breeds, offering novel insights into canine immunogenetics.
This achievement was made possible by the growing avail-
ability of high-quality dog genome assemblies published since
2019, driven by advancements in long-read sequencing tech-
nologies such as Oxford Nanopore and PacBio [30–32]. The
comprehensive annotation of multiple assemblies provides
several key advantages, including identifying conserved genes
and functions, enhanced inter- and intrabreed comparisons,
improved annotation accuracy, deeper insight into genetic
variability, and the development of a more comprehensive and
reliable genomic database.

The comprehensive annotation of the IGK locus
across dog breeds

The comprehensive annotation of the IGK locus across seven
dog breeds identified 40 IGK genes, comprising 34 IGKV
genes, 5 IGKJ genes, and 1 IGKC gene. In contrast, a previous
study annotated 19 IGKV genes, 5 IGKJ genes, and 1 IGKC
gene, identifying fewer V genes [11]. The discrepancy may be
attributed to differences in methodology, as the authors used
RNA-Seq data and based their annotations on rearranged IGK
genes rather than genomic sequences.

Our analysis identified seven novel IGK genes, with the ma-
jority (five out of seven) located within the Bernese Mountain
Dog assemblies (BD_1.0 and OD_1.0) [31]. These assemblies
displayed the longest IGK locus lengths, potentially enabling
the discovery of additional genes, consistent with findings that
larger locus coverage can uncover greater genomic diversity
[33]. However, despite including seven dog breeds, the limited
sample size per breed restricted our ability to detect breed-
specific genes or deletions, which may reflect the effects of in-
breeding or selective pressures. Inbreeding is known to reduce
genetic diversity, increase susceptibility to inherited disorders,
and elevate genetic load [34, 35]. Addressing this limitation,
future studies aligned with the Dog10K consortium's objec-
tives could incorporate larger sample sizes across breeds, en-
abling more robust detection of breed-specific genomic vari-
ations and enhancing our understanding of haplotype struc-
tures within canine populations [36].

In dogs, the majority of IGKV genes belong to the IGKV2
subgroup, highlighting its predominance within the canine
immune repertoire. Consistent with our findings, Cullen *et al.*
demonstrated that IGKV2 genes constituted 99.7% of all

functional V genes in healthy dogs using 5' RACE sequencing
[3]. Similarly, Martin *et al.* reported that 14 out of 19 IGKV
genes (73.7%) were classified within the IGKV2 subgroup, re-
inforcing its dominance [11]. Both studies utilized RNA-Seq to
analyze expressed immune repertoires, underscoring the criti-
cal role of IGKV2 genes. This subgroup's predominance may
be shaped by evolutionary pressures or specialized immune
functions that confer selective advantages. Understanding the
dominance of IGKV2 genes could provide valuable insights
into breed-specific immune responses, genetic susceptibilities,
and opportunities for developing targeted immunotherapies
[37].

Patterns of gene conservation

Among the 40 identified IGK genes, 20 (14 IGKV, 5 IGKJ,
and 1 IGKC) were consistently found across all assemblies,
reflecting 50% conservation (Fig. 2). Notably, 10 of these con-
served IGK genes are monomorphic, including IGKV1-23\*01,
IGKV2-19\*01, IGKV4-15\*01, IGKV(I)-14\*01, IGKV(II)-1-
1\*01, IGKVJ1\*01, IGKVJ2\*01, IGKVJ3\*01, IGKVJ4\*01,
and IGKVJ5\*01. The monomorphic nature of these genes
may indicate fixation within populations, potentially driven
by strong selective pressures that favor their retention [38].
Such pressures might arise from their critical roles in immune
function or adaptations to specific environmental challenges.
Further exploration of these monomorphic genes could pro-
vide insights into the evolutionary dynamics shaping the ca-
nine immune repertoire and their implications for disease re-
sistance or susceptibility. Additionally, the growing number of
annotated assemblies from different breeds—or even multiple
individuals within the same breed—may reveal previously un-
detected polymorphisms in these genes.

Recent studies of immune repertoire analysis have under-
scored the predominant usage of specific IGKV-J genes, with
five genes accounting for more than 5% of rearrangements:
IGKV2-16 (38.7%), IGKV2-5 (20.3%), IGKV2-11 (13.2%),
IGKV2-4 (7.6%), and IGKV2-12 (5.7%). Among kappa chain
rearrangements, IGKJ1 (33.8%) and IGKJ4 (50.3%) were
identified as the most prevalent J gene sets [3], highlighting a
highly skewed expressed repertoire. Interestingly, these IGKV
genes were not conserved across all annotated assemblies in
our study, suggesting potential differences in methodology or
underlying genetic diversity. Notably, Cullen *et al.* relied ex-
clusively on IMGT references for sequence alignment, which
may have resulted in a more limited identification of genes [3].
Despite these differences, such findings are pivotal for advanc-
ing immune-based therapies and diagnostic tools in veterinary
medicine, offering insights into the canine immune system's
structure and function.

Discovery of new alleles

Through the analysis of nine canine genome assemblies and
IMGT references, a total of 97 IGK alleles were identified,
comprising 90 IGKV, 5 IGKJ, and 2 IGKC alleles. Notably,
85.3% of the 34 IGKV genes exhibited polymorphisms, high-
lighting substantial allelic diversity. In contrast, a study by
Martin *et al.*, which analyzed a larger cohort of 107 dogs
across 19 breeds, identified a greater number of nonannotated
IGK alleles [11]. Their analysis revealed 108 IGK alleles (107
IGKV and 1 IGKC), with the majority (72.8%) maintaining
functionality comparable to the reference allele.
Our findings are consistent with those of Martin *et al.* and Cullen *et al.*, both of which reported a predominance of func- tional IGK genes [11, 3]. In our study, 59 out of 97 alleles were functional. Among the identified IGK genes, 28 (82.35%) ex- hibited alleles with identical functionality to their reference alleles, while six IGKV genes presented functional variations from their reference alleles. Notably, the genes IGKV2-5 (5 al- leles), IGKV2-6-1 (4 alleles), IGKV2-8 (3 alleles), IGKV2-9 (3 alleles), and IGKV4-1 (4 alleles) demonstrated functional di- versity. However, the gene IGKV2-4 exhibited the highest de- gree of polymorphism, with six alleles, all of which were func- tional. Interestingly, genes exhibiting notable polymorphism were in regions of the locus characterized by “low density,” suggesting potential challenges in assembly or annotation in these areas.

Through Sanger sequencing, we confirmed the *in silico* an-
notation of two functional *IGKV2* alleles, *IGKV2-12*\*03 and
*IGKV2-4*\*04, which had been previously identified in the
IMGT/GENE-DB database. The allele *IGKV2-12*\*03 was de-
tected in ten independent canine genome assemblies and was
assigned an IMGT confidence score of 3, indicating a high
level of reliability in the annotation. The allele *IGKV2-4*\*04,
identified in the Cairn Terrier assembly with an IMGT score
of 1, also showed full sequence identity with our experimental
data. Both alleles were amplified and validated using specific
forward primers, confirming their presence and homozygosity
in the tested samples.

These results provide experimental evidence supporting the
accuracy of the *in silico* annotation and reinforce the exist-
ence of functional diversity within the *IGKV2* subgroup. The
concordance between the predicted and experimentally vali-
dated alleles highlights the reliability of IMGT-based anno-
tations and demonstrates the utility of combining computa-
tional and experimental approaches for IG gene validation.
Moreover, the successful amplification of these alleles from
non-lymphoid tissues indicates that such variants are part of
the germline repertoire rather than products of somatic rear-
rangements.

Altogether, these findings expand the current understanding
of canine IG light chain variability and contribute to improv-
ing the genomic characterization of the *IGKV* locus across
breeds.

Challenges and limitations

IMGT annotated the dog IGK locus in 2017 using the Boxer
genome assembly (CanFam3.1), when no alternative assem-
blies were available. This annotation, based on orthology with
the human IGK locus, consists of two large, inverted segmen-
tal duplications and was complicated by gaps in the Boxer
genome in the IGK region [39, 40]. Consequently, the anno-
tation placed half of the IGKV genes upstream and the other
half downstream of the IGKJ and IGKC genes, deviating from
the typical organization seen in other species [2]. In dogs, a
cluster of IGKV genes is found 5' of a few IGKJ genes, with
the IGKJ cluster positioned 5' of a single IGKC gene. Initially,
this atypical structure was interpreted as an evolutionary vari-
ation specific to dogs rather than a potential assembly or an-
notation error, as noted by Collins *et al.* and Martin *et al.*
[2, 11].

Given that the Boxer genome (CanFam3.1) has served
as the reference for dogs for 13 years and has been cited

in over 130 studies, the identification and dissemination of
errors in reference genomes and annotations are essential
for the scientific community. The publication of the Basenji
genome in 2019 [41] and the *de novo* assembly of the Boxer
Tasha genome in 2021 [30] provided the opportunity to study
and validate the IGK locus orientation in dogs, as discussed
in the “Challenges and limitations” section of this article
(Figs 5 and 6).

Interestingly, the *de novo* Boxer assembly (Dog10K Boxer Tasha) could not replace the previous Boxer assembly (CanFam3.1) in the IMGT reference annotations, as it contained five fewer genes than its predecessor. Although the newer Boxer assembly, generated using long-read sequencing technology, demonstrated superior quality, the earlier assembly was based on Sanger sequencing. This discrepancy underscores the importance of annotating multiple genome assemblies to ensure accuracy and reduce errors arising from limitations inherent in single-assembly references [42, 43].

Finally, nomenclature issues arose from the IGKV2-4-
1*01 and IGKV2-4-1*02 genes, which shared the same V-
REGION sequences as the newly identified alleles IGKV2-
6*02 and IGKV2-9*02 in the Bernese Mountain Dog assem-
blies BD_1.0 and OD_1.0. These new genes and alleles will be
retained in the IMGT databases and tools; however, we em-
phasize that if future studies demonstrate the nonexistence of
any of these variants, they will be removed. In such cases, the
nomenclature associated with these genes will not be reused
for this locus or species (i.e. nonrecyclable names).

Future perspectives

Enriching the IMGT database with canine sequences from var-
ious breeds enables future studies to explore inter and intra-
breed differences in the immune repertoire. This study repre-
sents the first annotation of the germline sequences of IGK
genes in dogs, which provides the advantage of identifying
functional and nonexpressed genes, such as ORFs and pseu-
dogenes. These findings are crucial for understanding V-J gene
usage and for validating newly identified alleles and genes.
Furthermore, the inclusion of alleles in the database will sup-
port future research on the characterization of mutations in
the IG genes of the IGK locus. However, this area has not been
sufficiently investigated in dogs due to the absence of IG alleles
reference sets from various breeds [7, 8].

Additionally, our research identified specific challenges in
canine genome sequencing, particularly in regions with low
gene frequency (low density). It could be attributed to com-
plex repetitive regions and high heterozygosity of IG loci,
as observed in humans, presenting significant challenges for
current sequencing methods [44]. To our knowledge, 2 dedi-
cated tools were designed to evaluate the accuracy and com-
pleteness of genome assemblies in complex regions, provid-
ing a valuable framework for overcoming these challenges:
*CloseRead* [45], and more recently IMGT/StatAssembly
[46].

In conclusion, our findings reveal substantial allelic vari-
ability within the canine IGK locus. Through Sanger sequenc-
ing, we experimentally validated two newly annotated IGKV2
alleles, both previously identified *in silico* and classified as
functional. This allelic diversity represents an important fac-
tor to consider in the development of immunologically based
therapeutic strategies, including vaccines, immunotherapies, and monoclonal antibody treatments for cancer and other immune-related diseases.

## Acknowledgements

We would like to express our heartfelt gratitude to the whole IMGT® team for their expertise and incredible collaboration in carrying out this study. We are also grateful to Prof. Marie-Paule Lefranc for her invaluable contribution to building the dog's IGK reference annotations on IMGT. We also extend special thanks to Dr Marina Omelchenko, responsible for the CGV tool at NCBI, for her support in assisting us and providing comparisons between the canine genomes. We extend our thanks to Dr Ruth Seal and her team from the VGNC for recognizing the nomenclature of the annotated genes and making it accessible in their database. Their efforts have significantly supported the standardization and dissemination of genetic information. We thank the São Paulo Research Foundation (FAPESP), grants number 23/05951-5 and 21/09982-7, and the Brazilian Coordination for the Improvement of Higher Education Personnel (CAPES) for the financial support of the TKG Ph.D. thesis and internship at IMGT®. IMGT® is a member of the French Infrastructure Institut Français de Bioinformatique (IFB) as well as a member of BioCampus, MAblmprove, and IBiSA. IMGT/HighV-QUEST was provided with computing HPC and storage resources by Grand Equipement National de Calcul Intensif (GENCI) at IDRIS and TGCC thanks to the grant 2010-2026 (036029).

**Author Contributions:** Taismara Kustro Garnica (Conceptualization [lead], Data curation [lead], Formal analysis [lead], Investigation [lead], Methodology [lead], Validation [lead], Writing—original draft [lead], Writing—review & editing [lead]), Ariadni Papadaki (Conceptualization [equal], Data curation [equal], Formal analysis [equal], Investigation [equal], Methodology [equal], Validation [equal], Writing—review & editing [equal]), Maria Georga (Conceptualization [equal], Data curation [equal], Formal analysis [equal], Investigation [equal], Methodology [equal], Validation [equal], Writing—review & editing [equal]), Guilhem Zeitoun (Data curation [equal], Formal analysis [equal], Methodology [equal], Resources [equal], Software [equal], Validation [equal], Visualization [equal], Writing—original draft [equal], Writing—review & editing [equal]), Joumana Michaloud (Resources [equal], Software [equal], Validation [equal], Visualization [equal], Writing—review & editing [equal]), Géraldine Folch (Resources [equal], Software [equal], Validation [equal], Visualization [equal], Writing—review & editing [equal]), Véronique Giudicelli (Resources [equal], Software [equal], Validation [equal], Visualization [equal], Writing—review & editing [equal]), Pablo Gonzalez (Data curation [equal], Investigation [equal], Writing—review & editing [equal]), Jéssika Cristina Chagas Lesbon (Investigation [equal], Validation [equal], Writing—review & editing [equal]), Talita Gabriela Luna Alves (Investigation [equal], Validation [equal], Writing—review & editing [equal]), Heidge Fukumasu (Conceptualization [equal], Funding acquisition [lead], Investigation [equal], Methodology [equal], Project administration [lead], Resources [lead], Supervision [lead], Writing—review & editing [equal]), and Sofia Kossida (Conceptualization [lead], Funding acquisition [lead], Project administration [lead], Resources [lead], Software [lead], Supervision [lead], Writing—review & editing [equal]).

## Supplementary data

Supplementary data is available at NAR Genomics & Bioinformatics online.

## Conflict of interest

None declared.

## Funding

IMGT® is granted access to the High-Performance Computing (HPC) resources of Meso@LR and of the Centre Informatique National de l'Enseignement Superieur (CINES), to Tres Grand Centre de Calcul (TGCC) of the Commissariat a l'Energie Atomique et aux Energies Alternatives (CEA) and Institut du developpement et des ressources en informatique scientifique (IDRIS) [036029 (2010-2024)] made by GENCI (Grand Equipement National de Calcul Intensif). IMGT® is currently supported by the Centre National de la Recherche Scientifique (CNRS) and the University of Montpellier. TGK's PhD thesis and internship project at IMGT® were funded by the São Paulo Research Foundation (FAPESP) in Brazil (Grants number 2024/18875-8, 2023/05951-5, 2022/09378-5, and 2021/09982-7) and the Coordination for the Improvement of Higher Education Personnel (CAPES), Brazil (Finance Code 001). We acknowledge the support of Immun4Cure IHU "Institute for innovative immunotherapies in autoimmune diseases" (France 2030/ANR-23-IHUA-0009) as well as the support of the Institut Universitaire de France.

## Data availability

IGK locus sequences and all related annotations from nine dog assemblies are available in the IMGT/LIGM database (ref) with the accession numbers: BK066061, BK066062, IMGT000179, IMGT000180, IMGT000181, IMGT000182, IMGT000183, IMGT000184, and IMGT000185. The annotations are also available in the TPA database on the NCBI for BK066061 and BK066062. IGK genes are managed and distributed through IMGT/GENE-DB [14] and the IMGT reference directory for the analysis of expressed repertoire with IMGT/V-QUEST, and IMGT/HighV-QUEST [47, 48] is available at https://www.imgt.org/IMGTindex/directory.php [(additionally, the Supplementary Table S3 provides all links and datasets generated in this study that are publicly available through the IMGT database and associated IMGT tools (web resources)].

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