Influence of MHC on genetic diversity and testicular expression of linked olfactory receptor genes

doi:10.21203/rs.3.rs-4905052/v1

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Influence of MHC on genetic diversity and testicular expression of linked olfactory receptor genes

https://doi.org/10.21203/rs.3.rs-4905052/v1

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Background

Olfactory receptor (OR) genes are highly polymorphic and form extensive families that recognize a wide range of vertebrate odorants. Although OR gene clusters are dispersed across many regions of vertebrate genomes, ORs expressed in the testes exhibit major histocompatibility complex (MHC)-linked structural conservation.

Results

In this study, we selected nine MHC-linked OR genes based on their expression levels in pig testes and developed a sequence-based typing method for these genes. We then performed high-resolution typing of these OR genes, along with three major classical MHC class I genes (SLA-1, -2, and − 3), in 48 pigs across six breeds. We observed significantly higher allelic diversity (P < 0.01) in ORs with strong linkage disequilibrium (LD) to SLA compared to those with weak or no LD, and we identified 48 SLA class I-OR haplotypes using the expectation-maximization algorithm. The genetic diversity of SLA-linked ORs was positively correlated with their expression levels in the testis. Specifically, SLA-linked ORs with higher testicular expression (FPKM ≥ 0.1) exhibited an increase in the number of codons under mutually diversifying selection with SLA compared to those with lower expression (FPKM < 0.1).

Conclusions

Our results suggest the presence of evolutionary interactions between the MHC and linked OR genes. These characteristics of SLA-linked ORs support the potential involvement of MHC-linked ORs in MHC-based mate selection.

Olfactory receptors (ORs) are G protein-coupled receptors (GPCRs) responsible for odorant recognition, primarily expressed in olfactory neurons [1]. OR genes are highly diverse, forming extensive gene families that recognize a wide range of odorants and are organized into clusters across multiple chromosomes in the vertebrate genome [2–5]. The number of OR genes and the extent of pseudogenization vary significantly among species, reflecting the importance of olfaction for their survival. For example, pigs (Sus scrofa), cattle (Bos taurus), mice (Mus musculus), and humans (Homo sapiens) possess 1,301 (14.5% pseudogenes), 1,071 (17.7% pseudogenes), 1,483 (23.1% pseudogenes), and 874 OR genes (55.5% pseudogenes), respectively [6–8]. Notably, marine mammals exhibit a reduced OR repertoire compared to terrestrial animals [9–12].

ORs are also implicated in functions beyond odor recognition and olfactory signal transduction, as they are expressed in a variety of tissues and cells, including the brain, tongue, heart, spleen, and pancreas [13–15]. Noncanonical OR expression has been associated with the modulation of cell-cell recognition, migration, proliferation, apoptosis, exocytosis, and pathfinding processes [16–20]. Furthermore, several ORs are expressed in the testes and sperm cells, where they detect specific odorant molecules that trigger Ca²⁺ signals, which are crucial for changes in sperm motility and chemotaxis [21–23]. Intriguingly, ORs expressed in the testes are genetically linked to highly variable major histocompatibility complexes (MHC) and GABBR1, which are involved in recognizing foreign antigens for the adaptive immune response and play a role in sperm acrosome reactions, respectively [15, 24–27]. The conserved architecture of OR-GABBR1-MHC genes across vertebrate species may contribute to MHC genotype-based mating preferences, favoring individuals with dissimilar MHC genotypes [28–32]. However, our understanding of this phenomenon remains limited.

Although ORs expressed in the testes appear to be genetically linked to the MHC, limited information is available on the diversity of MHC-linked ORs compared to ORs located in other chromosomal regions. Some studies have reported that MHC-OR haplotypes are highly variable and that specific OR-MHC allele combinations occur more frequently than expected by chance [33, 34].

To elucidate the genetic and functional relationships between MHC and MHC-linked ORs, we analyzed the evolutionary conservation and linkage disequilibrium (LD) between MHC and MHC-linked ORs by typing nine MHC-linked ORs and three classical MHC class I genes in pigs. We performed haplotype analysis and compared allelic diversity between MHC-linked and unlinked ORs to assess the influence of MHC genes on OR diversity. Additionally, we examined the testicular expression patterns of orthologous MHC-linked ORs in pigs and humans using transcriptome data.

Identification of MHC-linked orthologous OR genes across humans, mice, cattle, and pigs

The current genome assemblies for humans, mice, pigs, and cattle reveal that the numbers of OR genes mapped to MHC-residing chromosomes are 34, 67, 33, and 90, respectively (Supplementary Table 1). To determine the orthologous relationships of the ORs in the MHC-linked region among the four species, we identified 11 non-OR orthologous framework loci across these species using information from the NCBI Ortholog Database (www.ncbi.nlm.nih.gov/gene). These loci include ABT1, ZNF322, POM121L2, ZNF184, H1-5, ZSCAN26, ZSCAN12, GPX6, GABBR1, MOG, and ZFP57 (Fig. 1). Subsequently, we established the orthologous relationships of the ORs adjacent to the framework loci through interspecies pairwise BLAST analysis (Supplementary Table 2). OR gene pairs with e-values < 10^− 100 (E-100) and bit scores > 500 between species, along with their syntenic relationships from the comparative analysis of framework loci, were identified as putative OR orthologs.

The number of OR genes within each OR cluster varied among species. OR genes with multiple matches in a given species from the BLAST analysis were considered to result from recent gene duplications. We observed three OR clusters on S. scrofa chromosome 7 (SSC7) and B. taurus chromosome 23 (BTA23), whereas H. sapiens chromosome 6 (HSA6) contained two clusters. An additional OR gene located between ABT1 and ZNF322 in Euungulata, but absent in other species, indicated an expansion of MHC-linked ORs within this lineage. In contrast, a single cluster of OR genes was present on M. musculus chromosome 17 (MMU17) due to the translocation of the OR cluster onto MMU13 (Fig. 1). Consequently, only a GABBR1-linked OR cluster was common to MHC-bearing chromosomes in all four species. The ORs between ZSCAN26 and H1-5, as well as between ZSCAN12 and GPX6, were conserved across cattle, humans, and pigs.

Development of a high-resolution typing method for nine MHC-linked OR genes in pigs

The high frequency of particular allele combinations at different loci may suggest the presence of LD or a functional association between them. To investigate the associations between MHC and linked ORs, we selected nine pig MHC-linked OR genes, including LOC100514111, LOC100516618, LOC100157348, LOC100515036, LOC100156552, LOC100522686, LOC100516811, OLF42-3, and OLF42-1, based on their cluster location, testicular expression level, and the presence of human orthologs (Supplementary Table 2). To develop a comprehensive typing method for these OR genes, we designed primers corresponding to the 500 bp upstream and downstream regions of each OR gene. Through iterative primer design and confirmation of amplification specificity, we identified a set of primers that enabled comprehensive amplification of the nine ORs (Table 1). We then developed sequencing primers for each amplicon, resulting in a sequence-based high-resolution typing method for ORs. For allelic determination, homozygous typing results were classified as novel alleles when observed for the first time. Heterozygous samples were separated by molecular cloning and sequenced to resolve the alleles. As a result, we successfully determined the allelic status of all typing results (n = 576) without any exceptions (Supplementary Tables 3 and 4). The sequence information for OR alleles was submitted to NCBI under accession numbers PP768337 to PP768442 (Supplementary Table 3). The number of alleles for ORs ranged from 5 to 22 (Supplementary Tables 3 and 5). Typing results for LOC100515036 revealed more than two alleles per individual across multiple samples, suggesting an association between gene duplications and copy number variations (CNVs).

Table 1

Primer information for typing nine SLA-linked OR and three SLA class I genes
Target loci	Primer usage	Sequence (5' to 3')	Amplicon size (bp)	Annealing (℃)
LOC100514111	gPCR^a	TTGACAGCAAGTAAATGTGTTTGT	1,302	58
	gPCR^a	GATGAGCTCTTCACCRAGTCCA	1,302	58
	Sequencing	GATAGGTTTGTAGCCA	-	50
	Sequencing	GCTTCATTTGCTGTTG	-	50
LOC100516618	gPCR	GCTAGGTAGAGGGGCAGGGGATACA	1,620	58
	gPCR	CTGGCGTCCACATTCATGGTTGGTT	1,620	58
	Sequencing	TCTGTAAACCTCTGCAC	-	50
	Sequencing	CCAATTARTGCTGGCATCT	-	50
LOC100157348	gPCR	GTTGAGTTACCTTTGCAGTTCATACA	2,323	58
	gPCR	TCCAAATTTTRTGTGAAAGGAACAAAAG	2,323	58
	Sequencing	ACTCCACTACATGCTG	-	50
	Sequencing	TCCACTTGGTTTTTGC	-	50
LOC100156552	gPCR	TGGTCACTATCCCTTGCTTTCT	1,834	58
	gPCR	CCTTACACTGTTTTAGTGACTTMAT	1,834	58
	Sequencing	ACTTGACCGCTATGC	-	50
	Sequencing	CTTGAGCAAAGGARGGAC
	Additional Sequencing^b	GCAAGCCCCTTCACTAC
LOC100515036	gPCR	CTCTCTCCCCTTGTCGAGC	1,803	58
	gPCR	AGAGAAAGTATATGTACAGCTGGG	1,803	58
	Sequencing	GCCCCTTCACTACACAG	-	50
	Sequencing	TTCATTTCTCCCACAGAGT	-	50
LOC100522686	gPCR	GGGTGTACTTCATTCCTCCTTCA	1,242	58
	gPCR	CCTTGCATTTCTTGAAGGTCTCC	1,242	58
	Sequencing	TGTGTGTAARCCTCTGA	-	50
	Sequencing	TCACAAAAGAAGTGGTCC	-	50
LOC100516811	gPCR	CAAGAGCTCACCTTTGCCCAT	1,720	58
	gPCR	GTGTGTGCTCACKTACTTACC	1,720	58
	Sequencing	GGCCTATGACCACTA	-	50
	Sequencing	AGGTRTCTCTGCATAC	-	50
OLF42-3	gPCR	TACCTTCAGTGAGTCTGAGGAAC	1,700	58
	gPCR	GCAGGTCCTAAMACGCCTCTC	1,700	58
	Sequencing	CCAACCCCTCCACTATG	-	50
	Sequencing	TGGATCTCATTGTAGGTG	-	50
OLF42-1	gPCR	AACCACTGACCTGCTGTAGTGCCTT	1,322	58
	gPCR	AAGCTTCGTCTGTTTTCCAGCGCAT	1,322	58
	Sequencing	CCAACCCCTCCACTATG	-	50
	Sequencing	ACAGGRGAGTCGAATTAGA	-	50
SLA-1 exon 2, 3	gPCR	MTAARCTCTCCRCCCASCCGGCTCTG	1,844	65
	gPCR	CGGGTCACATGTGTCYTTGGAGG	1,844	65
	Sequencing	TGCTATGCTGTGCGCCGARAGGAGGGT	-	50
	Sequencing	ATGCTGATTATCGCCCKCGTTGGWCGCG	-	50
SLA-3 exon 2, 3	gPCR	GGGGRCCCTGGCCCTGATT	1,655	65
	gPCR	CGGGTCACATGTGTCYTTGGAGG	1,655	65
	Sequencing	CTGTGAATGCTGCTGCGCCGAGAGGAGGGT	-	50
	Sequencing	TGTGAATGCTATGGTKGGTCGYGGC
	Additional Sequencing^b	GACCCCCTTTTCCTCT
SLA-2 exon 2, 3	gPCR	GCCTCGACACAGAATCTCCGATATATCCAAAGATG	1,679	65
	gPCR	CGGGTCACATGTGTCYTTGGAGG	1,679	65
	Sequencing	TGCTATGCTGTGCGCCGARAGGAGGGT	-	50
	Sequencing	GAGGGGAGATGGTGGAG
	Additional Sequencing^b	ATGCTGATTATCGCCCKCGTTGGWCGCG
	Additional Sequencing^b	TTCCTGGGGATGGGGATG
T7 universal	Colony PCR^c	TAATACGACTCACTATAGGG	n.a.	50
SP6 universal	Colony PCR^c	ATTTAGGTGACACTATAG	n.a.	50
^a Primer for genomic PCR
^b Additional sequencing primers for indel variants
^c PCR primers for target clone amplification

Higher genetic diversity of SLA-linked ORs compared to SLA-unlinked ORs

Extreme genetic polymorphisms of the MHC can influence the diversity of linked genes. To assess the impact of LD with the MHC on the diversity of OR genes, we performed sequence-based typing of SLA-1, SLA-2, and SLA-3 in animals with existing OR typing data, using previously established methods [35–37]. High-resolution typing results were obtained for all three SLA class I genes, identifying 34, 29, and 17 alleles for SLA-1, SLA-2, and SLA-3, respectively. CNV was observed in SLA-1, consistent with previous findings [35, 38] (Supplementary Table 4). No novel alleles were detected for SLA-1 and SLA-2; however, a new non-functional allele was identified for SLA-3 (PQ037598), which contains a premature stop codon. Regarding genetic diversity, the observed heterozygosity (Ho) for SLA-1, SLA-2, and SLA-3 ranged from 0.646 to 0.813, with a mean of 0.750, while the expected heterozygosity (He) ranged from 0.942 to 0.961, with a mean of 0.954, consistent with previously reported diversity of SLA class I genes [35–37] (Supplementary Table 5).

When comparing the allelic diversity between nine SLA-linked ORs typed in this study and 20 SLA-unlinked ORs reported in a previous study, which examined mostly the same populations as in this study [39], the mean allele numbers for SLA-linked and SLA-unlinked ORs were 11.78 ± 5.38 and 6.65 ± 3.01, respectively (Fig. 2, Supplementary Table 5), indicating a significant difference between the two groups (P-value < 0.05). The mean Ho and He of SLA-linked OR genes were also significantly higher than those of SLA-unlinked ORs, with Ho = 0.68 ± 0.08 vs. 0.33 ± 0.18 (P-value < 0.01) and He = 0.81 ± 0.10 vs. 0.57 ± 0.24 (P-value < 0.01) (Fig. 2, Supplementary Table 5). Notably, three SLA-linked ORs—OLF42-1, OLF42-3, and LOC100515036—exhibited particularly high allele numbers. CNVs were observed in LOC100515036, suggesting gene duplication at this locus. However, the number of alleles was also high for the SLA-unlinked OR sOR6T3 (15 alleles), indicating that the specific functions of certain ORs may also contribute to genetic diversity, although we were unable to determine functional differences among these ORs.

Determination of 48 haplotypes for SLA-1, SLA-2, SLA-3, and nine SLA-linked ORs

We identified a combined total of 106 alleles for nine ORs and 80 alleles for three classical SLA class I genes by typing 48 individuals from six pig breeds (Supplementary Tables 3 and 4). The allele numbers for SLA-1 and LOC100515036, which exhibit CNVs, ranged from 0 (null) to 2 and 1 to 3 per haplotype, respectively, while the remaining genes showed one allele per haplotype, as expected. To determine haplotypes, preliminary haplotype phasing was performed without CNV-related genes to reduce the complexity of haplotype determination, using the expectation-maximization (EM) algorithm. Subsequently, final haplotypes were manually refined by incorporating the genotypes of the CNV-related genes, following the strategy described in Supplementary Fig. 1. As a result, 48 combined haplotypes were established for SLA-1, SLA-2, SLA-3, and nine SLA-linked ORs (Supplementary Table 6). When the haplotype sequences were translated into amino acid sequences, the number of haplotypes was reduced by only one, indicating a high rate of nonsynonymous substitutions in this region (Supplementary Table 6).

Identification of five putative breakpoint hotspots at locus junctions within the SLA-OR region

We identified candidate recombination breakpoint sites based on codon alignment analysis of 48 haplotype sequences within the SLA-OR region using RDP software (Fig. 3). Five sites emerged as prominent breakpoint hotspots with > 99% confidence, as determined by the observed haplotype patterns in the region (Fig. 3, Supplementary Fig. 2). The deduced breakpoint hotspots within the SLA and linked OR gene regions were primarily located at the locus boundaries between LOC100516618 and LOC100157348, LOC100156552 and LOC100522686, and OLF42-1 and SLA genes. Consistent with these findings, when pairwise LD among three SLA class I and nine linked OR genes was estimated by calculating normalized entropy differences between two loci (ε) using eLD software, LOC100157348, LOC100515036, LOC100156552, OLF42-3, and OLF42-1 showed higher LD with SLA-1, -2, and − 3 (mean ε ≥ 0.3) compared to other analyzed ORs (Fig. 4).

Similarity in the testicular expression of MHC-linked ORs between humans and pigs

To investigate the expression patterns of MHC-linked ORs, we analyzed the expression levels of nine SLA-linked ORs using publicly available transcriptome data from pig testes (from three 120-day-old pigs, GEO accession: GSE171756) and olfactory epithelium (OE) (from four newborn pigs, GEO accession: GSE197184) (Table 2, Supplementary Table 7). The expression levels of OLF42-1 and OLF42-3 in the proximal OR cluster of SLA class I and LOC100515036 and LOC1001156552 in the distal OR cluster were higher (FPKM 0.102–0.742) than those of other ORs (FPKM 0–0.055; Table 2), indicating differential expression levels in the testis. However, this pattern differed from that of ORs expressed in OE, suggesting tissue-specific regulation of gene expression in these ORs (Table 2, Supplementary Table 7). Additionally, we analyzed the expression patterns of putative human OR orthologs of the nine pig ORs using publicly available transcriptome data from human tissues (GEO accession: GSE30611 for human testis and GSE80249 for human OE) and compared them with their expression in pigs. The expression of OR2H1 and OR2H2, human orthologs of pig OLF42-1 and OLF42-3, respectively, was also higher in the human testis (mean FPKM = 0.603) compared to other ORs (mean FPKM = 0.007), reflecting a similar expression pattern to that observed in pigs. Notably, OR2B8P, the putative human ortholog corresponding to pig LOC100515036 and LOC1001156552 (likely duplicated in pigs), has been pseudogenized in humans. These results indicate that the testicular expression pattern of the analyzed functional HLA-linked ORs is similar between pigs and humans (Table 2, Supplementary Table 7, Supplementary Fig. 3).

Table 2

Comparison of SLA-linked OR expression in testis and olfactory epithelium between pigs and humans
Group^a	Gene symbol	Human ortholog	Expression (FPKM)
			Pig		Human
			Testis^e	OE^f	Testis^g	OE^h
Distal OR	LOC100514111	OR2B2	0.055	5.717	0.003	0.003
	LOC100516618	OR2W6P^b	0.005	0.753	0.018	0.003
	LOC100157348	OR2B7P^b	0.003	0.465	0.019	0.039
	LOC100515036	OR2B8P^b	0.127	0.182	0.003	0.104
	LOC100156552	OR2B8P^b	0.102	0.107	0.003	0.104
	LOC100522686	OR2E1P^b	0.000	n.a.	0.003	0.188
Proximal OR	LOC100516811^b	OR11A1	0.000	n.a.	0.003	2.479
	OLF42-3	OR2H2	0.300	0.406	0.799	0.136
	OLF42-1	OR2H1	0.742	0.710	0.406	0.017
Control	ACTG1^c		1106.680	1315.007	664.920	911.566
Control	NPY^d		0.012	10.273	0.870	3.028
^a OR groups were categorized based on the physical distance to SLA.
^b Putative pseudogene
^{c, d} Housekeeping and olfactory epithelium (OE)-specific genes, respectively.
^{e, f, g, h} GEO accession numbers for data sets: GSE171756, GSE197184, GSE30611, and GSE80249, respectively.
^f FPKM values of pig OE were not available and indirectly calculated from read counts.

Positive correlation between heterozygosity levels and testicular expression of SLA-linked ORs

From the expression data of nine SLA-linked pig OR genes in regions conserved between pigs and humans, we categorized the genes into two groups based on testicular expression levels exceeding 0.1 FPKM, using the round-off mean value (mean FPKM = 0.098) for expressed ORs in pig testes (Supplementary Table 7, Supplementary Fig. 3). LOC100515036, LOC100156552, OLF42-3, and OLF42-1 showed mean expression levels of ≥ 0.1 FPKM (n = 3), while LOC100514111, LOC100516618, LOC100157348, LOC100522686, and LOC100516811 had mean expression levels of < 0.1 FPKM (n = 3). When comparing these expression levels to the genetic diversity of each locus, Ho differed between the two groups, with mean Ho = 0.750 for FPKM ≥ 0.1 versus Ho = 0.617 for FPKM < 0.1 (P-value < 0.01; Fig. 2 and Supplementary Table 5). The mean Ho of ORs with FPKM ≥ 0.1 (Ho = 0.750) was similar to that of SLA class I genes (Ho = 0.750; P-value = 0.5) but differed from ORs with FPKM < 0.1 (Ho = 0.617; P-value < 0.05). Additionally, allele numbers were greater for ORs with FPKM ≥ 0.1 (mean n = 16.25) compared to those with FPKM < 0.1 (mean n = 8.20), suggesting a positive correlation between allele diversity and testicular expression levels for SLA-linked ORs.

Positive correlation between testicular expression and the number of codons under diversifying selection for SLA and linked ORs

We estimated selection pressure for 3,032 codons constituting nine OR and three SLA genes using a random-site model in PAML. We also calculated LD between all possible SNP pairs from 550 SNPs identified in 48 OR-SLA haplotype sequences (Supplementary Table 8). Our analysis revealed that 41 codons from nine ORs had dN/dS (ω) values > 1.00 according to both M2 and M8 models, indicating diversifying selection. Among these 41 diversifying codons, 25 were in significant LD (D’ > 0.5, LOD > 2) with diversifying codons of SLA genes (Table 3, Supplementary Table 9). Further analysis showed that 15 out of 16 (94%) functional OR genes with FPKM ≥ 0.1 (LOC100515036, LOC100156552, OLF42-3, OLF42-1) were in significant LD with SLA, compared to only 10 out of 25 (40%) for ORs with FPKM < 0.1 (LOC100514111, LOC100516618, LOC100157348, LOC100522686), except for LOC100522686 (89%). This finding suggests a correlation between OR expression in the testis and evolutionary selection between SLA and SLA-linked ORs.

Table 3

Association of the number of codons under diversifying selection with testicular expression level in SLA-linked olfactory receptor genes
Gene group	Loci	No. of SNP (A)	No. of SNP in significant LD with SLA (B)	B/A ratio	No. of diversifying codons (C)	No. of diversifying codons in significant LD with SLA (D)	D/C ratio
OR with FPKM ≥ 0.1	LOC100514111	4	1	0.25	1	1	1.00
	LOC100516618	25	2	0.08	9	0	0.00
	LOC100157348	21	8	0.38	6	1	0.17
	LOC100522686	30	23	0.77	9	8	0.89
OR with FPKM < 0.1	LOC100515036	54	36	0.67	6	5	0.83
	LOC100156552	48	14	0.29	3	3	1.00
	OLF42-3	59	50	0.85	6	6	1.00
	OLF42-1	24	21	0.88	1	1	1.00
Total		265	155	-	41	25	-
Note. Selection pressure was calculated from the codon alignment using 48 haplotypes for the SLA class I-OR region. Significant LD indicates LD with D’ > 0.5 and log-likelihood odds ratio (LOD) > 2 between SNPs.

Breed differences in SLA-OR haplotypes

We analyzed population differences in MHC-OR haplotype repertoires for SLA-1, -2, -3, and nine linked OR regions among six pig breeds: Berkshire (BER), Duroc (DUR), Landrace (LAN), Yorkshire (YOR), Korean native pigs (KNP), and SNU minipigs (SNU). Principal component analysis (PCA) of 48 haplotypes showed a high degree of genetic relatedness among European breeds (BER, DUR, LAN, and YOR), whereas KNP and SNU were more distantly related to the European pig cluster (Fig. 5). The mean FST value was highest for SNU (mean FST = 0.265), followed by KNP (mean FST = 0.222), and BER (mean FST = 0.212), with DUR (mean FST = 0.181), YOR (mean FST = 0.172), and LAN (mean FST = 0.156) showing lower values (Supplementary Fig. 4). This indicates that BER has relatively distinct MHC-OR haplotypes compared to other European breeds. In the minimum spanning network (MSN) plot, LAN occupied a central position with the largest number of haplotypes (n = 12) among the six breeds (Supplementary Fig. 5). LAN haplotypes h1, h15, and h28 were shared with YOR and DUR. BER was closely positioned to other European breeds in its haplotype composition but did not directly share haplotypes with them. KNP shared haplotype h7 with DUR. SNU pigs had the lowest number of haplotypes in the inbred population.

About 1,300 OR genes are present in the pig genome, and the increased OR gene repertoire and allelic variation may have contributed to enhancing the survival of the species [5, 6, 40, 41]. In particular, 26 functional and 7 pseudogenes were annotated on pig chromosome 7 harboring SLA genes (Supplementary table 7). MHC molecules have evolved to be highly polymorphic and initiate immune responses against an almost unlimited number of foreign molecules [42–45]. Such evolutionary forces of MHC could affect the diversity of neighboring genes through mechanisms such as hitchhiking or functional association. Because many OR genes are present in SLA-linked regions, we investigated the genetic and evolutionary influence of MHC on the diversity of neighboring OR genes by comparing genetic variations between SLA-linked and unlinked ORs.

The typing results of SLA-linked ORs in this study showed a significant increase in the genetic diversity of SLA-linked ORs compared to unlinked ORs (Fig. 2, Supplementary table 5), suggesting MHC as a possible driving force for increasing the genetic diversity of neighboring ORs. The influence of MHC on the genetic diversity of linked ORs was also evidenced by the increased allelic diversity and observed heterozygosity of ORs with strong LD to SLA compared to those with weak or no LD to SLA (Fig. 2, Supplementary table 5).

The evolutionary conservation of OR-MHC architecture in vertebrates has been well described in previous versions of genome assemblies [46]. In this study, we conducted a comparative analysis of MHC-linked ORs using current genome assemblies and annotations, and provided an updated and more detailed picture of the comparative OR-MHC architecture among cattle, humans, mice, and pigs. For example, several OR genes within the extended MHC (xMHC) region were mapped differently in the previous genome assembly (Sscrofa10.2) than in the current genome assembly, Sscrofa11.1 (Supplementary table 10). In addition, the number of GABBR1-linked pig ORs mapped to this region appeared to be smaller than those of other species (Fig. 1). However, we confirmed the structural conservation of the major OR cluster linked to MHC among the four species, despite the presence of species specificity.

The conservation of the major MHC-linked OR cluster linked to GABBR1 in the xMHC region of vertebrates may indicate possible functional relationships between these ORs and MHC [25, 46]. Pig ORs, OLF42-3 and OLF42-1, which showed higher genetic diversity and testicular expression than the other ORs, were also present in the species-conserved region. A previous study suggested that the strong genetic linkage between GABBR1-linked ORs and HLA could be related to the functional interactions of these molecules [25, 46]. The orthologs of pig OLF42-1 and OLF42-3 which are highly expressed in pig testes, are also expressed in human sperm [22]. HLA class I expression is observed in granulosa cells surrounding oocytes in humans [26, 47]. These results suggested that MHC-linked ORs play a role in mate selection, showing a preference for partners with different MHC class I genotypes, as previously suggested [28, 29, 48].

In studies on human testicular expression of ORs, the majority of OR genes expressed in the testis were genetically linked to HLA [49–51]. These testicular HLA-linked OR genes show significant variations in expression levels and exhibit a non-canonical expression pattern through long-distance splicing and exon sharing among OR genes [24, 52]. Interestingly, this promiscuous expression plays a critical role in the establishment of self-tolerance in T cells [53]. Therefore, MHC-linked ORs that are highly expressed in the testes or germ cells may play a role in mediating MHC genotypes through strong LD between MHC and OR genes. This is consistent with our results in which the frequency of diversifying codons in significant LD with SLA was much higher for functional OR genes of FPKM ≥ 0.1 than those of FPKM < 0.1 (Table 3). Furthermore, several testicular MHC-linked ORs are expressed in sperm [22, 51], and GABBR1, which is involved in OR signaling, is also expressed in the testis [27, 54]. These findings suggest that MHC diversity plays a role in OR function in testes and sperm.

A study of the human OR2E1P pseudogene and OR2H1, the orthologs of pig LOC100522686 and OLF42-1, respectively, revealed that OR2E1P can form spliced variant transcripts with OR2H1 [52]. Interestingly, the alternative splice sites involved in the exon splicing of OR2H1 were also conserved in porcine OLF42-1 (Supplementary Fig. 6), demonstrating the interspecies conservation of evolutionary and functional characteristics among MHC-linked ORs.

Vomeronasal receptors (VRs), a class of ORs that function as pheromone receptors, recognize the diversity of MHC peptide ligands and trigger responses from vomeronasal sensory neurons expressing these receptors [55, 56]. It has been suggested that MHC peptide ligands can serve as olfactory cues [57]. However, it is unclear whether MHC-linked OR genes expressed in the testes and sperm recognize MHC particles and elicit responses. Human OR2H1 recognizes the odor molecule methional, and spermatozoa elicit a crucial Ca²⁺ signal necessary for sperm chemotaxis via this molecule [22]. Recognition of cryptic MHC by ORs may involve distinct odorant molecules, resulting in mate selection. A potential explanation regarding MHC-OR interactions could be the involvement of MHC-linked ORs in MHC-based sexual selection [26, 58].

We identified 48 haplotypes for the nine SLA-linked ORs and three major SLA class I genes using sequence-based genotyping of 48 animals from six pig breeds (Supplementary table 3 and 4). Sampling bias can be a major cause of error in haplotype estimation using the EM algorithm [59]. To overcome the concerns of a small sample size and large number of loci for haplotype estimation in our dataset, we conducted indirect validation of our haplotype analysis results by comparing the population distribution of deduced haplotypes in this study with the outcomes of previous studies independently conducted for 20 MHC-unlinked ORs and the same set of SLA class I genes from populations of mostly overlapping breeds of pigs [39, 60, 61]. The allelic and haplotype distributions of SLA-linked ORs for each breed in this study were highly consistent with those of SLA-unlinked ORs reported in a previous study, supporting the reliability of our OR-SLA haplotype information.

Genetic analysis of ORs in the vertebrate genome is challenging because of the presence of high sequence homology, CNV, and pseudogenes, as well as the large number of loci in the gene family. In the present study, we developed sequence-based high-resolution typing methods for nine SLA-linked OR genes and analyzed their genetic and evolutionary characteristics in relation to MHC class I genes in pigs. Our results indicated a strong influence of SLA diversity on that of the linked OR genes, in which the allelic diversity of ORs was significantly higher than that of other chromosomal regions, and the testicular expression of more polymorphic ORs was higher than that of the less polymorphic SLA-linked ORs. We also showed the conservation of the testicular expression patterns of MHC-linked orthologous ORs between humans and pigs. Our results suggest the possibility of functional and evolutionary interactions between MHC molecules and their linked ORs during animal reproduction. The OR-MHC haplotypes defined in this study contribute to elucidating the relationship between MHC and MHC-like ORs and could be applicable to pig breeding and reproduction.

Animals and tissues

Tissue samples of six pig breeds, BER, DUR, LAN, YOR, KNP, and SNU were collected and stored at -80°C in previous studies [35, 62]. Eight individuals from each breed were randomly selected. All experiments were approved and performed in accordance with the guidelines and regulations set by the Institute of Animal Care and Use Committee and the Center for Research Ethics of Konkuk University.

Preparation of genomic DNA

Genomic DNA was extracted from tissues (0.5 g) using a standard protocol. Briefly, tissues were incubated with 700 µL lysis buffer (50 mM Tris pH 8.0, 0.1 M EDTA pH 8.0, 0.5% (w/v) sodium dodecyl sulfate, 20 µg/mL DNase-free pancreatic RNase) and 20 µL of proteinase K (20 mg/mL) at 50°C overnight. Subsequently, gDNA was purified by phenol: chloroform: isoamyl alcohol (pH 8.0) extraction, precipitated using ethanol, and dissolved in 50 mM Tris-EDTA buffer (pH 8.0).

Determination of syntenic relationship

The genome assemblies and annotations used in this study were GRCh38.p14, Sscrofa11.1, ARS-UCD2.0, and GRCm39 for humans, pigs, cattle, and mice, respectively. The construction of a synteny plot was carried out using the Python package “pyGenomeViz” (github.com/moshi4/pyGenomeViz) on Python version 3.8.15 (www.python.org/).

Primer design

Primers that specifically amplify the entire coding sequence (CDS) of each of the nine MHC-linked OR genes were designed using NCBI Primer-BLAST (www.ncbi.nlm.nih.gov/tools/primer-blast/). The 500-bp upstream and downstream regions of the selected OR genes, based on the pig genome assembly (NC_010449.5, Sscrofa11.1), were used as queries. The maximum product size was set to 2500 bp. Primer sites containing nucleotide variations reported for dbSNPs (www.ncbi.nlm.nih.gov/snp/) were excluded. Off-target or multiple-target amplification of the primers was performed using BLAST analysis. Primer design was repeated until the optimal primers for the specific amplification of the target ORs were obtained. Primer information for the analysis of SLA-1, -2, and − 3 was based on previous studies [35–37] and is described in Table 1.

Polymerase chain reaction and sequencing

For a 10 µL reaction for nine OR genes including LOC100514111, LOC100516618, LOC100157348, LOC100515036, LOC100156552, LOC100522686, LOC100516811, OLF42-3, OLF42-1, the reaction mixture consisted of 1 µM of locus-specific primers (Table 1), 250 µM dNTPs, 1 unit of Supertherm™ Taq DNA polymerase (JMR Holdings, Kent, UK), 10× reaction buffer containing 15 mM MgCl₂, and 25 ng of DNA. The amplification reaction was conducted on an ABI9700 thermocycler (Applied Biosystems, Foster City, CA) with an initial pre-denaturation step at 94°C for 3 minutes, followed by 30 cycles of denaturation at 94°C for 30 seconds, annealing at the specific primer annealing temperature (Table 1) for 45 seconds, and elongation at 72°C for 90 seconds. Subsequently, the final elongation was conducted at 72°C for 10 min. The amplicons were electrophoresed on 1% agarose gel to confirm target amplification. To sequence OR amplicons, sequencing primers with lengths of 15–20 bp were designed at conserved regions in both the forward and reverse directions with an overlap to cover the entire CDS of each OR gene using the Primer Designer of CLC Main Workbench version 7.8.1 (CLC bio, Aarhus, Denmark) (Table 1). Before sequencing, 5 µL of PCR products were mixed with 0.25 U of shrimp alkaline phosphatase (USB Corporation, Cleveland, OH), 15 U of exonuclease I (Fermentas, Massachusetts, USA), and incubated at 37 ℃ for 30 min. The sequencing reaction was performed using the Applied Biosystems BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Massachusetts, USA) with 2 pmol of a sequencing primer under the conditions of pre-denaturation at 96°C for 1 min, 25 cycles of 96°C for 10 s, 50°C for 5 s, and 60°C for 4 min. The reaction products were purified using ethanol precipitation, resuspended in 10 µL of Hi-Di™ Formamide (Applied Biosystems, Massachusetts, USA), and analyzed on an ABI3730 DNA Analyzer (Applied Biosystems). Specific amplification and sequencing of SLA-1, -2, and − 3 were conducted as previously described (Table 1) [35–37].

Allelic differentiation and haplotype determination

In principle, alleles that exist as homozygotes in sequence-based typing are considered novel alleles. Alleles that existed as heterozygotes in the typing results were separated into individual alleles through TA cloning using pGEM®-T Easy Vector Systems (Promega, Wisconsin, USA). For cloning, the ligation products were transformed into DH10B competent cells (Thermo Fisher Scientific, Waltham, MA, USA) using electrotransformation. Target inserts were amplified in 10 µL of colony PCR mixture containing a piece of a single bacterial colony as a DNA template, 1 µM of T7 and SP6 universal primers (Table 1), 250 µM dNTP, 1U of Supertherm™ Taq DNA polymerase (JMR Holdings, Kent, UK), and 10× reaction buffer with 15 mM MgCl₂ (JMR Holdings, Kent, UK). The thermal profile for the colony PCR consisted of 5 min of pre-denaturation at 94°C, 30 cycles of denaturation at 94°C for 30 s, primer annealing at 50°C for 30 s, and elongation at 72°C for 60 s, followed by a final elongation at 72°C for 7 min. The PCR amplicons were sequenced using primers specific to each OR gene (Table 1). At least eight independent clones were sequenced for allelic determination using the T7 and SP6 universal primers. Allele differentiation for SLA-1, -2, and − 3 was conducted as described in previous studies [35–37]. Haplotype determination of SLA- and SLA-linked OR genes was conducted using the haplotype inference function, employing the EM algorithm provided in Arlequin version 3.5.2.2 [63]. The detailed procedure is shown in Supplementary Fig. 1.

Calculation of genetic diversity

The observed (Ho) and expected heterozygosities (He) of the loci were calculated using Arlequin version 3.5.2.2 [63]. Data normality and equality in variance distribution for the observed indices were tested using the Shapiro-Wilk normality test and an equal-variance test [64, 65]. Differences in the average values of the observed indices were tested using the Student’s t-test [66].

Population genetic analyses

LD index (ε) between loci was calculated after allele differentiation of genotyping results using eLD R script [67] on R version 4.1.2 (www.r-project.org). To generate an MSN plot, haplotype data was prepared for R library “adegenet” version 2.1.6 [68], and generation of a distance matrix and visualization was conducted using R library “poppr” version 2.9.4 [69]. A dataset for PCA was prepared using the genotype data of nine OR and three SLA class I genes from 48 individuals of six pig breeds, using the adegenet R package. PCA was conducted using the dudi.pca function of the R library “ade4” version 1.7.19 [70].

Estimation of haplotype breakpoints

To generate input nucleotide sequences for haplotype codon alignment, stop codons at the end of the full-length coding sequences (CDS) for 11 genes were removed, and the sequences were sequentially connected to produce a single sequence contig relative to the chromosomal order, except LOC100516811, a pseudogene. The input sequences were aligned using the codon alignment function of MUSCLE in MEGA version 11.0.10 [71]. The generated codon alignment was used as an input file for RDP v4.101 [72]. Haplotype breakpoints were deduced using RDP, GENECONV, MaxChi, BootScan, and SiScan in RDP software. Statistical significance of the breakpoints was tested using a breakpoint P distribution plot (1000 permutations and window size = 200bp). Breakpoints with an estimated P-value < 0.01 were suggested as breakpoint hotspots.

Codon selection test and LD calculation

The haplotype phylogenetic tree was constructed from the haplotype codon alignment using the "Create Tree" feature of CLC Main Workbench version 7.8.1 (CLC bio, Aarhus, Denmark). The neighbor-joining method and Kimura 80 nucleotide substitution model were applied with 5000 bootstrap replications. The created haplotype phylogenetic tree was unrooted using the R library “ape” version 5.6.2. [73] and was used as the input file for CodeML of pamlX version 1.3.1 [74]. All analysis options were set to default except for codon frequency, which was configured using the 2:F3ⅹ4 model. To test selection signatures of codons under Random-sites models, we used site models to allow the dN/dS (ω) ratio to vary among sites using Models 0, 1, 2, 7, and 8 under different assumptions on the distribution of ω ratio [74]. Empirical Bayes analysis using Models 2 and 8 was used to deduce the positively selected codons [75]. Codons with a confidence level > 95% were identified as positively selected codons. LD values between haplotype SNPs were estimated using Haploview version 4.2 from the haplotype codon alignment [76].

Comparison of OR expression

Expression data for SLA-linked OR genes were obtained from the NCBI Gene Expression Omnibus database (www.ncbi.nlm.nih.gov/geo/; accession numbers GSE171756 (pig testis) and GSE197184 (pig olfactory epithelium (OE)). Expression data for human ORs were retrieved from the same database with accession numbers GSE30611 (human testis) and GSE80249 (human OE). Because the OR expression data for porcine OE were available only as read counts, the values were converted to relative FPKM values for expression comparison using the following formula: $$\:\text{O}\text{R}\:\text{g}\text{e}\text{n}\text{e}\:\text{F}\text{P}\text{K}\text{M}\:\text{i}\text{n}\:\text{O}\text{E}=\:\text{O}\text{R}\:\text{g}\text{e}\text{n}\text{e}\:\text{r}\text{e}\text{a}\text{d}\:\text{c}\text{o}\text{u}\text{n}\text{t}\:\text{i}\text{n}\:\text{O}\text{E}\:\times\:\left(\frac{\text{O}\text{R}\:\text{g}\text{e}\text{n}\text{e}\:\text{F}\text{P}\text{K}\text{M}\:\text{i}\text{n}\:\text{t}\text{e}\text{s}\text{t}\text{i}\text{s}}{\text{O}\text{R}\:\text{g}\text{e}\text{n}\text{e}\:\text{r}\text{e}\text{a}\text{d}\:\text{c}\text{o}\text{u}\text{n}\text{t}\:\text{i}\text{n}\:\text{t}\text{e}\text{s}\text{t}\text{i}\text{s}\text{}}\right)\:$$

Availability of data and materials

A total of 106 SLA-linked OR allele sequences analyzed in this study were submitted to the NCBI GenBank (www.ncbi.nlm.nih.gov/genbank/) under the accession numbers listed in Supplementary table 3 (PP768351-PP768442). All other information can be found in the text and supporting information.

Ethics approval

All experiments were approved and performed in accordance with the guidelines and regulations of the Institute of Animal Care and Use Committee and the Center for Research Ethics of Konkuk University.

Consent for publication

Not applicable

Competing interests

The authors declare no competing interests.

Funding

This study was supported by the Cooperative Research Program for Agriculture, Science, and Technology Development (Project No. PJ016221), Rural Development Administration, Republic of Korea.

Authors' contributions

Conceptualization and sample collection: M.K., B.A., and J.L. Data curation: M.K., H.C., and C.P. Bioinformatic analysis: M.K., B.A., and J.S. Methodology: M.K., and C.P. Manuscript writing: M.K. and C.P. Comments and discussion: C.P.

Acknowledgements

The authors have no specific acknowledgements to declare.

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Influence of MHC on genetic diversity and testicular expression of linked olfactory receptor genes

Status:

Version 1

Abstract

Background

Results

Conclusions

Figures

Background

Results

Identification of MHC-linked orthologous OR genes across humans, mice, cattle, and pigs

Development of a high-resolution typing method for nine MHC-linked OR genes in pigs

Higher genetic diversity of SLA-linked ORs compared to SLA-unlinked ORs

Identification of five putative breakpoint hotspots at locus junctions within the SLA-OR region

Similarity in the testicular expression of MHC-linked ORs between humans and pigs

Positive correlation between heterozygosity levels and testicular expression of SLA-linked ORs

Breed differences in SLA-OR haplotypes

Discussion

Conclusions

Materials and Methods

Animals and tissues

Preparation of genomic DNA

Determination of syntenic relationship

Primer design

Polymerase chain reaction and sequencing

Allelic differentiation and haplotype determination

Calculation of genetic diversity

Population genetic analyses

Estimation of haplotype breakpoints

Codon selection test and LD calculation

Comparison of OR expression

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1