Genome-wide association study
We had access to tissue samples of 43 German Landrace gilts, considered as affected based on their abnormally small vulvae. The 43 affected animals originated from 21 litters of 20 sows and 11 known boars and were from four breeding units; the sire of one litter was unknown (Additional file 1). Seventeen affected individuals were considered as cases in a genome-wide association study (GWAS) with 1,818 fertile sows from the Bavarian German Landrace population as controls. All sows included in the GWAS were genotyped on the Illumina Porcine SNP60 BeadChip. Association testing revealed a cluster of 18 significantly associated SNPs (P < 9.87 × 10-7) between 38.6 Mb and 108.7 Mb on the X-chromosome (Figure 1a). The most significant SNP (P = 8.81 × 10-43) on the X-chromosome (rs328794518, 88,499,366 bp, Sscrofa11.1 assembly) was an intron variant in the ENSSSCG00000012566 gene. We considered significantly associated, scattered SNPs on other chromosomes as artifacts due to the inflation of significant signals (λ = 1.26, Figure 1b).
Identification of the underlying mutation by analyzing whole-genome re-sequencing (WGS) data
Re-sequencing data of 42 pigs (22 German Landrace and 20 Piétrain boars and fertile sows) were available for identifying possible causal variants. The sire of eight affected gilts was among the 42 sequenced pigs. We located 828 coding variants within the associated region (38.6 Mb to 108.7 Mb), of which 366 were missense mutations, 30 were frameshift variants, and five were nonsense-mutations. The sire of the affected gilts was hemizygous for one of the five nonsense-mutations. This nonsense-mutation, with no previous reports, is a C to T substitution in the second exon of BMP15 (Sscrofa11.1_X:g.44618787C>T, NP_001005155.2:p.R212X). In addition to the sire of affected gilts, two German Landrace boars and one German Landrace sow were carriers of the nonsense-mutation (Additional file 2). None of the Piétrain pigs carried the mutation.
Validation of the nonsense mutation in BMP15
All 22 German Landrace pigs with whole-genome sequences were genotyped by Sanger sequencing to confirm the mutation technically (Additional file 3). Complete concordance with WGS derived genotypes corroborates the technical validity of the variant. Next, we genotyped 43 sows, assessed by the farmers to exhibit small vulvae, 29 full siblings with normal vulvae, and 4,869 fertile sows, using either a KASP™ genotyping assay or a customized version of the Illumina Porcine SNP60 BeadChip containing the p.R212X variant of BMP15. Of the 43 sows with abnormal vulvae, 36 were homozygous for the T allele, three were heterozygous (C/T), and four were homozygous for the wild type allele (C). Of the seventeen animals that we considered as cases in the GWAS, only eleven turned out to be homozygous for the mutant allele; three animals where heterozygous and three animals were homozygous for the wildtype allele. None of the 4,869 fertile sows and none of the 29 unaffected full siblings of affected gilts were homozygous for the T allele (Table 1). The frequency of the T allele in the 4,869 fertile sows was 0.062.
Table 1: Genotypes of the p.R212X variant of BMP15 for 4,941 German Landrace sows
Genotype
|
Sows with small vulvae
|
Full siblings with normal vulvae
|
Fertile German Landrace sows
|
C/C (ref/ref)
|
4
|
0
|
4,263
|
C/T (ref/alt)
|
3
|
29
|
606
|
T/T (alt/alt)
|
36
|
0
|
0
|
N sows
|
43
|
29
|
4,869
|
While fertile sows and gilts that were classified to have normal-sized vulvae were not homozygous for the nonsense mutation of BMP15, seven of the 43 presumably affected gilts carried one or two wild type alleles. This finding is not compatible with the hypothesis of the nonsense mutation causing small vulvae. However, assessing the size of the vulva is subjective, and the discrimination between affected and unaffected animals is prone to error. On the other hand, if an animal's status is assessed based on the objective criterion of fertility (i.e., an animal having one or more litters), the hypothesis that the nonsense mutation of BMP15 causes underdeveloped external sexual organs and consequent infertility remains valid.
Effect of the mutation on reproductive organs
The initial phenotyping consisted of on-farm observations of gilts with underdeveloped vulvae. A more thorough assessment of the phenotype in the field turned out to be impracticable. Therefore, we acquired two gilts that were heterozygous for BMP15:p.R212X and mated them on an experimental farm with a boar carrying the mutation. One of the resulting litters consisted of male piglets only, while the other litter consisted of thirteen piglets, ten of them female. Of the female piglets, five were homozygous for the T allele, and five were heterozygous (C/T). At the age of six months, eight of the ten female pigs (five homozygous and three heterozygous) were slaughtered, and their uteri and ovaries resected. The uterus horns of the homozygous animals were considerably smaller, specifically less voluminous (Figure 2a, b, c) or less coiled (Figure 2d and e) than those of their heterozygous full siblings (Figure 2f, g, h). The differences in the appearance of the ovaries were even more pronounced. As shown in Figure 3, the ovaries of homozygous gilts (Figure 3a to e) did not exhibit follicles, whereas multiple follicles were evident in the ovaries of their heterozygous full siblings (Figure 3f to h). Besides the morphologically assessed underdevelopment of the ovaries and uteri, there may also be functional deficits as indicated by cysts in two homozygously affected gilts (Figure 3d and e). It is noteworthy that the two gilts with cysts exhibit uterus horns that are more voluminous (Figure 2d and e) than those of their homozygous siblings (Figure 2a, b, c), but less coiled than those of their heterozygous siblings (Figure 2f to h).
Effects of the mutation on fertility and other economically important breeding traits
Nonsense and missense mutations of BMP15 increase ovulation rate in heterozygous ewes but cause infertility in ewes that are homozygous for the mutant allele (first reported by Galloway et al. (2000) [3]). Therefore, we analyzed the effects of the BMP15 nonsense mutation (i. e., the T-allele) on fertility, measured as the number of live-born piglets, and several other economically important traits, most of them growth-related. Since genotypes were not available for all sows with breeding values, we derived the T-allele dosage based on the parents' genotypes and the T-allele frequencies in male and female animals. A total of 5,263 genotyped parents were available (4,263 C/C-sows, 606 C/T-sows, 367 C-boars, and 27 T-boars; T/T-females cannot be parents as they are infertile). The frequency of the T-allele amounts to qm = 0.069 in male, and qf = 0.062 in female parents. Table 2 lists the expected dosages and Table 3 the number of animals in each dosage group.
Table 2: Dosage of the T-allele (causing the nonsense mutation) based on the genotypes of the parents
|
C/C-sow
|
C/T-sow
|
C/N-sow
|
C-boar
|
0
|
0.5
|
qf = 0.062
|
T-boar
|
1
|
1
|
1
|
N-boar
|
qm = 0.069
|
qm+0.5 = 0.569
|
qm+qf = 0.131
|
qm : T allele frequency in boars; qf : T allele frequency in sows; C: wildtype allele; T: mutant allele; N: allele not known.
Table 3: Number of sows in T-allele dosage groups
|
T allele dosage
|
|
0.000
|
0.062
|
0.069
|
0.131
|
0.500
|
0.569
|
1.000
|
N sows
|
8,400 (4,156)
|
9,623 (9,623)
|
1,092 (1,092)
|
9,969 (9,969)
|
1,185 (1,185)
|
565 (565)
|
1,185 (579)
|
Numbers in parentheses indicate animals with allele dosage derived from the parents’ genotypes.
We applied a linear regression model to estimate the effect of the T-allele of the BMP15 nonsense mutation on breeding values (Table 4).
Table 4: Estimated effects of the p.R212X-mutation on economically important traits
Trait
|
N sows
|
Effect ± std. error
|
P-value
|
Signifi-cance
|
Live-born piglets (N)
|
20,549
|
+0.411 ± 0.021
|
4.50 × 10-83
|
****
|
Feed conversion ratio (kg/kg)
|
10,072
|
-0.028 ± 0.004
|
7.60 × 10-15
|
****
|
Daily gain (g/day)
|
10,072
|
-0.847 ± 1.684
|
6.15 × 10-01
|
|
Lean meat content (%)
|
10,072
|
-0.889 ± 0.059
|
2.50 × 10-51
|
****
|
Belly meat content (%)
|
10,072
|
-0.906 ± 0.057
|
2.21 × 10-56
|
****
|
Loin eye area (cm2)
|
10,072
|
-1.216 ± 0.097
|
1.12 × 10-35
|
****
|
Loin to fat ratio (cm2/ cm2)
|
10,072
|
-0.031 ± 0.002
|
1.06 × 10-51
|
****
|
Carcass length (cm)
|
10,072
|
-0.030 ± 0.045
|
5.13 × 10-01
|
|
Intramuscular fat content (%)
|
10,072
|
0.068 ± 0.007
|
6.20 × 10-25
|
****
|
pH1
|
10,072
|
0.002 ± 0.002
|
1.16 × 10-01
|
|
Drip loss (%)
|
10,072
|
-0.005 ± 0.004
|
2.16 × 10-01
|
|
N sows: number of sows with a breeding value reliability ≥40 %; Bonferroni corrected significance for eleven tests is P-value ≤ 4.5 × 10-03. **** significant at P ≤ 1 × 10-04
The T-allele has a significantly positive effect on the number of live-born piglets (+0.41 piglets). Thus, the BMP15 mutation increases the litter size, probably as a result of an increased ovulation rate, and causes infertility in a dosage-sensitive manner (one T-allele increases the litter size, two T-alleles cause infertility). The positive effect of the BMP15 mutation on litter size is contrasted by the significantly negative effect on traits of lean meat growth (lean meat content, belly meat content, loin eye area, loin to fat ratio) and the positively correlated feed conversion ratio. Intramuscular fat content is negatively correlated with lean meat growth and thus positively affected by the BMP15 mutation.