Diversity of the Major Histocompatibility Complex Region Among Seven Founders of the Crested Ibis Nipponia nippon in Japan

ABSTRACT

The Crested Ibis Nipponia nippon is a critically threatened avian species. The current Japanese population originates from only seven founders introduced from China. The major histocompatibility complex (MHC) is a highly polymorphic genomic region that plays a central role in the immune system. We previously reported that only three MHC class II haplotypes (HP1–3) with one to three copies of MHC‐IIA/IIB gene pairs (one copy of DAA/DAB locus and 0–2 copies of DBA/DBB loci) were detected among the five older founders, and four MHC‐IIB exon 2 alleles (Type I—IV) were identified. In this study, MHC class II haplotypes in two novel founders, and MHC class IA genotypes at UAA locus in the seven founders were investigated. Of the two novel founders, while one possessed known HP1 and HP2, another possessed known HP1 and a novel haplotype (HP4) with the combination of type II and type I in DAB and DBB exon 2 s, respectively. Analysis of the linkage between MHC class II haplotypes and UAA alleles revealed five distinct MHC haplotypes against seven founders. These results strongly suggested that the founders might be considerably closely related, and the novel HP4 was produced by recombination between HP1 and HP2.

1. Introduction

The Crested Ibis Nipponia nippon is a special natural treasure in Japan (Agency for Cultural Affairs ¹⁹⁵⁰) and specified as a national endangered species of Wild Fauna and Flora (Ministry of the Environment ¹⁹⁹²). Until the middle of the 18th century, this species had been distributed widely from Hokkaido to Kyushu, however, through historical circumstances and processes as previously reported (e.g., BirdLife International ²⁰⁰¹; Ministry of the Environment ^{2003, 2007, 2009}; Nagata and Yamagishi ²⁰¹³; Okahisa ²⁰²²; Ministry of the Environment ^{2024a, 2024b}; Kubota et al. ²⁰²⁵; Ministry of the Environment ²⁰²⁶), the last indigenous individual in Japan died in 2003 in captivity, and Japanese‐born ibis had completely disappeared.

Then, the new captive breeding population had been founded in Sado Island, originating from one pair (denoted as F1 and F2) gifted by China in 1999, and one female (F3) was introduced in 2000 from the Chinese captive population. Similarly, the additional 2 birds were introduced in 2007 (F4 and F5), resulting in a significant increase in the population size. In 2008, the reintroduction of the captive birds to the wild was launched and the project has been maintained to date (Nagata and Yamagishi 2013; Okahisa ²⁰²²). Furthermore, to further recover the genetic diversity of the Japanese population, two birds (F6 and F7) were introduced also from China in 2018. As a result, the current Japanese population, which consists of captive, released and wild‐born individuals, has originated from a total of seven founders.

A major concern in the conservation of endangered species such as the Crested Ibis is the maintenance and recovery of genetic diversity. Because the pedigree information among founders was not available, the application of molecular genetic tools is essential for the evaluation of genetic diversity.

The major histocompatibility complex (MHC) plays a central role in the vertebrate immune system. MHC class I and class II genes are responsible for presenting peptides derived from the processing of intracellular (class I) and extracellular (class II) pathogens, and for triggering adaptive immune reactions (Klein 1986). Polymorphisms in MHC class I and II genes facilitate the binding of diverse pathogens (Klein 1986), and such evolutionary selection pressures possibly contribute to high genetic variation in MHC loci (Zinkernagel and Doherty 1979). Therefore, it is thought that the diversity of the MHC region influences pathogen resistance, adaptation, and population survival (Sommer 2005; Minias et al. ^{2018, 2019}). However, to our knowledge, there are currently no documented cases where MHC haplotype diversity has been directly utilized to guide decisions in captive breeding or reintroduction programs. Moreover, the variability of MHC haplotypes might serve as one of the indicators representing population‐level genetic diversity (Minias et al. 2016, 2019; Cruz‐López et al. ²⁰²⁰; Murakami et al. ²⁰²⁵).

Because no Crested Ibis had naturally migrated into Japan from the Eurasian continent recently, the current Japanese population is a practically closed population. To monitor the future maintenance and recovery of genetic diversity in the conservation of the Crested Ibis in Japan, knowledge of the diversity of the MHC region among the founder population could be crucial.

We previously reported that the genomic structure of the Crested Ibis MHC class II region and their genetic diversity among five founders introduced from 1999 to 2007 (Taniguchi et al. 2014). MHC class II genes (MHC‐IIA/IIB gene pairs) were located between COL11A2 and BRD2 genes, and copy number variations of MHC‐IIA/IIB gene pair were observed. Among five founders, only three MHC class II haplotypes (HP1–3) with one to three copies of MHC‐IIA/IIB gene pairs (one copy of DAA/DAB locus and 0–2 copies of DBA/DBB loci) were detected, and the number of MHC‐IIB exon 2 alleles was four (Type I—IV) (see Figure S1). However, MHC class II haplotypes in two novel founders introduced in 2018 have not yet been investigated.

The Crested Ibis MHC region contains five MHC‐IA genes (UAA, UBA, UCA1, UCA2, and UDA) (Chen et al. 2015; Chang et al. ²⁰¹⁶). One major UAA locus is located in the Core Region, including MHC‐IIA/IIB, DM, and TAP genes (see Figure S1), whereas the other four class I genes are in the Class I region, apart from the Core Region. Regarding polymorphisms of MHC‐IA genes in the Chinese population, two variants were detected in UAA, UBA, and UCA2 loci, but nucleotide substitutions in UBA and UCA2 exon 3 s were silent, and only UAA exon 3 was dimorphic at the protein level (Lan et al. 2019). However, there is no information on polymorphisms in the MHC‐IA gene among seven founders in Japan.

The purpose in this study was to identify all variants of the MHC region covering class I and class II genes within seven founders. Therefore, we performed haplotyping of the MHC class II region for two novel founders by PCR direct Sanger sequencing. In addition, genotypes of UAA exon 3 allele (UAA*01 and *02) were determined by locus‐ and allele‐specific PCR method. The diversity of the MHC region in seven founders was evaluated by analysis of the linkage between MHC class II haplotypes and UAA alleles.

2. Materials and Methods

2.1. Samples

Blood samples from seven founders of the Crested Ibis (F1–F7) were provided by Sado Japanese Crested Ibis Conservation Center (Niigata, Japan). Sample‐collection protocols were based on a conservation project of the Ministry of the Environment of Japan and approved by the Animal Research Committee of Niigata University. The genomic DNAs were prepared from the whole blood using the Wizard SV Genomic DNA Purification System (Promega, Madison, USA) according to the manufacturer's instructions.

2.2. Haplotyping of the MHC Class II Region Covering DAB and DBB Loci

Firstly, to investigate copy numbers of MHC‐IIA/IIB gene pairs, the PCR with the primer‐pair DAA‐F1/DXB‐R1 (Figure S1 and Table S1) was applied against two novel founders F6 and F7. The PCR was performed by a three‐step method (initial denaturation at 95°C for 5 min, and 30 cycles of denaturation at 95°C for 30 s, annealing at 64°C for 30 s and extension at 72°C for 4 min) using KOD FX Neo DNA polymerase (TOYOBO, Osaka, Japan).

For haplotyping of the individuals with both one‐copy and two‐copy MHC‐IIA/IIB configurations, three MHC‐IIB loci (DAB of 3.0 kb in one‐copy, DAB of 3.1 kb and DBB of 3.3 kb in two‐copies) were independently amplified with the primer‐pairs DAA‐F1/DXB‐R1, DAA‐F1/DAB‐R2 and DBA‐F2/DXB‐R1, respectively (Figure S1 and Table S1). The PCR was performed as described above except for extension at 72°C for 2 min. PCR products were gel‐purified and then directly Sanger sequenced using the primer DXBint2‐seqR for DAB and DBB exon 2 loci. Sanger sequencing was performed on an Applied Biosystems 3730xl DNA Analyzer at Eurofins Genomics K. K. (Tokyo, Japan).

2.3. Genotyping of UAA Exon 3 by Cloning and Locus‐/Allele‐Specific PCR

Because five MHC‐IA loci (UAA, UBA, UCA1, UCA2, and UDA) were highly conserved, designing of primers for UAA locus‐specific PCR was very difficult. In this study, the primer‐pair amplifying three MHC‐IA exon 3 loci (UAA, UBA, and UDA) were used (Figure S2). In addition, because the UAA*02 allele‐specific primer‐pair used here also amplified UBA and UDA loci (Figure S4), PCR products from UAA locus must be distinguished from those from UBA and UDA loci before UAA allele‐specific PCRs. For these reasons, genotyping of UAA exon 3 alleles was performed by a method combining cloning with locus‐/allele‐specific PCRs. The scheme was shown in Figure 1. Briefly, in the 1st PCR, MHC‐IA exon 3 fragments from three loci—UAA, UBA, and UDA—were amplified from seven founders with the primer‐pair MHC‐IAnt2‐F1/MHC‐IAint3‐R1 (Figure S2 and Table S1). The PCR was performed by a three‐step method (initial denaturation at 95°C for 5 min, and 35 cycles of denaturation at 95°C for 30 s, annealing at 68°C for 30 s and extension at 72°C for 30 s) using KOD FX Neo DNA polymerase. The 1st PCR products were diluted 200‐fold with DDW and then used as templates in the 2nd PCR. In the 2nd PCR with the primer‐pair MHC‐IAint2‐F2Eco/MHC‐IAexo3‐R2Eco (Figure S2 and Table S1), EcoRI sites were added to both ends of the PCR products. The PCR was performed by a three‐step method (initial denaturation at 95°C for 5 min, 5 cycles of denaturation at 95°C for 30 s, annealing at 52°C for 30 s and extension at 72°C for 30 s, and then 23 cycles of denaturation at 95°C for 30 s, annealing at 62°C for 30 s and extension at 72°C for 30 s) using KOD FX Neo DNA polymerase. The 2nd PCR products were gel‐purified, digested with EcoRI, and then cloned into pBluescriptII (Stratagene, La Jolla, USA) at EcoRI site. For transformation, E. coli HST16CR Competent Cells (TAKARA BIO, Otsu, Japan) were used. Forty‐eight clones were picked up, and replica plate was prepared. Firstly, to investigate insert lengths, colony‐PCRs were performed with T3 and T7 primers on pBluescriptII. The colony‐PCR was performed by a three‐step method (initial denaturation at 95°C for 5 min, and 28 cycles of denaturation at 95°C for 30 s, annealing at 50°C for 30 s and extension at 72°C for 30 s) using KOD FX Neo DNA polymerase. Next, clones with a proper insert length, were subjected to UDA locus‐specific colony‐PCR with the primer‐pair UNAexo3‐S1/UDAexo3‐A1 (Figure S3 and Table S1). UDA negative clones were selected and subjected to UAA locus‐specific colony‐PCR with the primer‐pair UNAexo3‐S1/UAAexo3‐A1 (Figure S3 and Table S1). Finaly, UAA positive clones were subjected to UAA allele‐specific colony‐PCR with the primer‐pairs UAA*01‐spF1/‐spR1 and UAA*02‐spF2/‐spR2 (Figure S4 and Table S1). UDA and UAA double‐negative clones were tested by UBA locus‐specific colony‐PCR. All locus‐ and allele‐specific colony‐PCRs were performed by a three‐step method (initial denaturation at 94°C for 5 min, and 28 cycles of denaturation at 94°C for 30 s, annealing at 56°C–60°C for 30 s and extension at 72°C for 30 s) using Go Taq DNA Polymerase (Promega, Madison, USA).

FIGURE 1.

Scheme of genotyping for UAA exon 3 allele by cloning and locus‐/allele‐specific PCRs.

2.4. Genotyping of BRD2 Marker

We found a polymorphism (termed BRD2 marker) near the XhoI site between DBB and BRD2 loci in the analysis of the genome structure of the Crested Ibis MHC class II region (Figure S7) (Taniguchi et al. 2014). Genotyping of BRD2 marker was performed by locus‐ and allele‐specific PCRs as described follows (Figure S7). In founders heterozygous for MHC class II haplotype HP1/HP2, HP1/HP3 and HP1/HP4, two genomic fragments spanning from DAB or DBB to a part of BRD2 were independently amplified with the primer‐pairs DAA‐F1/BRD2‐R1 and DBA‐F2/BRD2‐R1, respectively (Figures S1 and S7 and Table S1). In contrast, in founders with HP2/HP3, a mixture from both haplotypes were obtained in PCR with the primer‐pair DBA‐F2/BRD2‐R1 (Figures S1 and S7 and Table S1). The PCR was performed by a two‐step method (initial denaturation at 94°C for 5 min, and 30 cycles of denaturation at 94°C for 30 s, annealing and extension at 68°C for 7 min) using KOD FX Neo DNA polymerase (TOYOBO). These PCR products were gel‐purified and subjected to BRD2 allele‐specific PCRs with the primer‐pairs BRD2‐F2/BRD2*01‐R2 and BRD2‐F2/BRD2*02‐R2 (Figure S7 and Table S1). The PCR was performed by a three‐step method (initial denaturation at 95°C for 5 min, 25 cycles of denaturation at 95°C for 30 s, annealing at 66°C for 30 s, and extension at 72°C for 60 s) using KOD FX Neo DNA polymerase.

3. Results

3.1. Haplotyping of the MHC Class II Region in Two Novel Founders

We previously reported that three haplotypes of the MHC class II region (HP1–3) with one to three copies of MHC‐IIA/IIB gene pairs were detected among five founders (F1–F5) introduced from 1999 to 2007 (Taniguchi et al. 2014). To investigate copy numbers of MHC‐IIA/IIB gene pairs in two novel founders (F6 and F7) introduced in 2018, the PCR with primer‐pair DAA‐F1/DXB‐R1 (Figure S1 and Table S1) was performed (Figure 2). In both F6 and F7, major 3 kb and minor 8 kb products were detected, suggesting that they possessed both one‐ and two‐copy MHC‐IIA/IIB configurations.

FIGURE 2.

Locus‐specific PCR of MHC class II genes in founder F6 and F7. Three MHC‐IIB loci (DAB of 3.0 kb in one‐copy configuration, DAB of 3.1 kb and DBB of 3.3 kb in two‐copy configuration) were independently amplified with the primer‐pairs DAA‐F1/DXB‐R1, DAA‐F1/DAB‐R2 and DBA‐F2/DXB‐R1, respectively. In the PCR with the primer‐pair DAA‐F1/DXB‐R1, a minor 8 kb product representing a two‐copy configuration was detected besides a major 3 kb product.

In such cases, three MHC‐IIB loci (DAB in one‐copy configuration, DAB and DBB in two‐copy configuration) could be independently amplified (Figure S1). In addition to major 3 kb products of DAB in one‐copy configuration as described above, two PCR products (3.1 kb of DAB and 3.3 kb of DBB in two‐copy configuration) were amplified in each founder with the primer‐pairs DAA‐F1/DAB‐R2 and DBA‐F2/DXB‐R1, respectively (Figure 2). Three PCR products were gel‐purified and then exon 2 s in each locus were directly Sanger sequenced (Table 1). While founder F6 possessed known HP1 and HP2, F7 possessed known HP1 and a novel haplotype (HP4) with the combination of type II and type I in DAB and DBB exon 2 s respectively. In total, four MHC class II haplotypes (HP1–4) were identified among seven founders.

TABLE 1.

MHC class II haplotypes and DAB/DBB exon 2 alleles in founder F6 and F7.

Founder	One copy configuration		Two copy configuration
	DAB exon 2	MHC class II haplotype	DAB exon 2	DBB exon 2	MHC class II haplotype
F6	Type II	HP1	Type IV	Type I	HP2
F7	Type II	HP1	Type II	Type I	HP4

3.2. Variation of the MHC Region Covering the MHC Class II Region and the UAA Locus

Genotyping of the UAA exon 3 for seven founders was performed by a method combining cloning with locus‐ and allele‐specific PCRs. Results of locus‐ and allele‐specific PCRs for 48 clones in each founder were summarized in Table S2. One example of typing process and results of UAA exon 3 allele‐specific PCRs in seven founders were shown in Figures S5 and S6, respectively. MHC class II haplotypes and UAA exon 3 genotypes were summarized in Table 2. The founder F4, whose MHC class II haplotype was homozygous for HP1, possessed both UAA*01 and UAA*02 alleles, indicating that HP1 haplotype linked to both UAA*01 and UAA*02 alleles (termed HP1a and HP1b for MHC haplotypes covering the MHC class II region and the UAA locus, respectively) (Figure 3). Moreover, genotypes of founders F1–F5 suggested that HP2 and HP3 linked to UAA*02 and UAA*01, respectively (Table 2 and Figure 3). However, at this time, because HP1 in founder F7 could not be identified as either HP1a or HP1b, it remained unknown which allele of UAA*01 or UAA*02 linked to HP4. Therefore, genotypes of BRD2 marker were used (Figure S7 and Table 2). Only HP3 linked to BRD2*02 allele, and all other MHC class II haplotypes linked to BRD2*01 allele. These linkages were confirmed by sequencing of lambda phage clones corresponding HP1–3 (Taniguchi et al. 2014) These results suggested that DBB locus (Type I) in HP4 originated from HP2, not HP3, i.e., HP4 linked to UAA*02 allele (Figure 3). Therefore, the MHC haplotype of F7 was HP1a/HP4 (Table 2).

TABLE 2.

Summary of class II haplotype, genotypes of BRD2 marker and UAA exon 3 alleles, and MHC haplotype.

Founder	Class II haplotype	BRD2 marker	UAA exon 3 allele	MHC haplotype
F1	HP1/HP3	*01/*02	*01/*02	HP1‐BRD2*01‐UAA*02/HP3‐BRD2*02‐UAA*01 (HP1b/HP3)
F2	HP1/HP2	*01/*01	*02/*02	HP1‐BRD2*01‐UAA*02/HP2‐BRD2*01‐UAA*02 (HP1b/HP2)
F3	HP1/HP3	*01/*02	*01/*01	HP1‐BRD2*01‐UAA*01/HP3‐BRD2*02‐UAA*01 (HP1a/HP3)
F4	HP1/HP1	*01/*01	*01/*02	HP1‐BRD2*01‐UAA*01/HP1‐BRD2*01‐UAA*02 (HP1a/HP1b)
F5	HP2/HP3	*01/*02	*01/*02	HP2‐BRD2*01‐UAA*02/HP3‐BRD2*02‐UAA*01 (HP2/HP3)
F6	HP1/HP2	*01/*01	*01/*02	HP1‐BRD2*01‐UAA*01/HP2‐BRD2*01‐UAA*02 (HP1a/HP2)
F7	HP1/HP4	*01/*01	*01/*02	HP1‐BRD2*01‐UAA*01/HP4‐BRD2*01‐UAA*02 (HP1a/HP4)

FIGURE 3.

Five MHC haplotypes detected in seven founders of the Crested Ibis in Japan. I–IV in MHC‐IIB genes indicate exon 2 allele. 01 or 02 indicates allele of BRD2 marker and UAA exon 3.

4. Discussion

The current Japanese population of the Crested Ibis originates from only seven founders introduced from China. To investigate variation in the MHC region covering the MHC class II region and the UAA locus among them, the linkages between two loci were analyzed, and five distinct MHC haplotypes were detected (Table 2 and Figure 3). HP4 detected in founder F7 introduced in 2018 was a novel haplotype with the combination of Type II and Type I in DAB and DBB exon 2 s, respectively (Table 1). Moreover, HP4 linked to BRD2*01 and UAA*02 alleles. Consequently, these results strongly suggested that type I allele of DBB locus in HP4 originated from HP2 (Figure 3). HP4 had not been reported in the Chinese population (n = 307 collected from 1990 to 2012) (Lan et al. 2019). Therefore, it was inferred that HP4 was recently produced by recombination between HP1 and HP2 in the Chinese population (Figure 4). Crossing over and/or gene conversion are now recognized to have crucial roles in generating and maintaining MHC diversity (Minias et al. 2016). The current result in the Created Ibis would support this observation.

FIGURE 4.

Model of production of HP4 by recombination between HP1 and HP2 in the Crested Ibis MHC.

Although a novel MHC haplotype was detected in the founder F7, the number of MHC‐IIB exon 2 alleles remained at four, and the diversity of MHC class II molecules at the protein level did not increase (Table 2 and Figure 3). In addition, the other three MHC haplotypes in two novel founders F6 and F7 were the same as those in five founders introduced from 1999 to 2008. The genetic diversity of the MHC region would be low even considering the situation of seven founders, strongly suggesting that the founders were considerably closely related. Therefore, the degree of inbreeding in the Japanese population might be higher than that estimated from the pedigree analysis by Kubota et al. (2025).

In this study, five MHC haplotypes covering the MHC class II region and the UAA locus were detected among seven founders. Previous studies using genome‐wide markers in the Japanese population of the Crested Ibis have shown that allele numbers of microsatellite markers and haplotype numbers of 202‐bp sequences containing multiple SNP sites were two or three in many loci (Taniguchi et al. 2013; Urano et al. ²⁰¹³; Tsubono et al. ²⁰¹⁴). These results might indicate that the MHC diversity is consistent with the genetic diversity measured using genome‐wide markers in a population that has recently experienced a severe bottleneck.as well as in the Oriental stork (Murakami et al. 2025).

The frequency of MHC class II haplotype HP1 (50%) among the founder population was higher than those of the other three haplotypes (7%–21%) (Table 2). To recover at least partially the effective population size of the Crested Ibis in Japan, it would be required to increase frequencies of HP2–4 when novel founders will not be introduced from the Chinese population. In the Chinese population, seven MHC haplotypes (HT01–07) were detected (Lan et al. 2019). Out of them, three haplotypes (HT01, 06, and 07) were not found in the Japanese founder population. Because all founders of the Japanese population were introduced from China, the finding of lower genetic diversity among them compared with the Chinese population would be reasonable. It has been reported that MHC genotypes influenced mate choice; therefore, they are involved in reproductive rate in the Crested Ibis (Sun et al. 2019), suggesting that maintaining and recovering the genetic diversity in the MHC region are critical issues. The MHC haplotype HT01 has four copies of MHC‐IIA/IIB pairs and contains different MHC‐IIB exon 2 alleles from those of other haplotypes (Chen et al. 2015; Chang et al. ²⁰¹⁶). Introduction of such individuals would be very effective for recovering the genetic diversity of the Japanese population.

The MHC diversity (4 MHC‐IIB exon 2 alleles or 5 MHC haplotypes) within the Crested Ibis founders in Japan was lower than those in other endangered avian species or domestic breeds. For example, 14 and 17 alleles of exon 2 from multiple MHC‐IIB loci were detected in the Japanese golden eagle Aquila chrysaetos japonica (n = 41) (Naito‐Liederbach et al. ²⁰²¹) and the white‐tailed eagle Haliaeetus albicilla (n = 67) (Minias et al. 2019), respectively. Also, 36 MHC‐B haplotypes were found in the Finnish Landrace breed of chicken (n = 195) (Fulton et al. 2017).

Both the Crested Ibis and the Oriental stork are endangered avian species, and similar projects for their conservation are being implemented in Japan. The current Oriental stork population in Japan originates from 26 founders, and 28 MHC haplotypes were detected (Murakami et al. 2025), indicating that the genetic diversity of the MHC region is higher in the Oriental stork than in the Crested Ibis. It would be of further interest to investigate how the genetic diversity among founder populations influences the conservation processes.

In the conservation of endangered species, maintenance and recovery of genetic diversity are a critical concern. To monitor the future status and transition of genetic diversity, knowledge of the genetic diversity in founder population is essential. Because matings between wild individuals are out of human‐induced management, it is very important to maintain the genetic diversity detected among founders in captive populations as genetic reservoirs. Genotypes of MHC region could be available to design mating pairs in captive breeding. We believe that the diversity of the Crested Ibis MHC covering class I and II regions among seven founders will provide valuable information for genetic management in conservation programs of the Crested Ibis.

Funding

This work was supported by JSPS KAKENHI (JP20K06377).

Ethics Statement

Sample‐collection protocols were based on a conservation project of the Ministry of the Environment of Japan and approved by the Animal Research Committee of Niigata University.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Table S1: List of primers used in this study.

Table S2: Summary of genotyping of MHC class IA genes.

Figure S1: Genomic structure of the Crested Ibis MHC Core region and primers.

Figure S2: Alignment of MHC‐IA exon 3 sequences and primers amplifying UAA, UBA and UDA loci.

Figure S3: Alignment of MHC‐IA exon 3 sequences and positions of locus‐specific primers for UAA, UBA and UDA loci.

Figure S4: Alignment of MHC‐IA exon 3 sequences and positions of UAA allele‐specific primers.

Figure S5: Colony‐PCR for identification of locus in MHC‐IA genes against 48 clones in founder F4. The Scheme of locus‐specific PCRs was shown in Figure 1.

Figure S6: asj70220‐sup‐0008‐Supplementary_FigureS6.pdf. UAA*01 and *02 allele‐specific colony‐PCR against UAA positive clones in 7 founders. Locus B and D were used as negative (for UAA*01 allele) and positive (for UAA*02 allele) control, since the UAA*02 allele‐specific primer pair amplified UBA and UDA loci.

Figure S7: Genotyping of BRD2 marker by allele‐specific colony‐PCR in 7 founders.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.