Deletion of an intronic polypyrimidine tract of porcine DNAH17 perturbs splicing and causes defective sperm flagella

Artificial insemination in pig (Sus scrofa domesticus) breeding involves the evaluation of the semen quality of breeding boars. Ejaculates that fulfill predefined quality requirements are processed, diluted and used for inseminations. Within short time, eight Swiss Large White boars producing immotile sperm that had multiple morphological abnormalities of the sperm flagella were noticed. The eight boars were inbred on a common ancestor suggesting that the novel sperm flagella defect is a recessive trait. Transmission electron microscopy cross-sections revealed that the immotile sperm had disorganized flagellar axonemes. Haplotype-based association testing involving microarray-derived genotypes at 41,094 SNPs of six affected and 100 fertile boars yielded strong association (P=4.22 x 10−15) at chromosome 12. Autozygosity mapping enabled us to pinpoint the causal mutation on a 1.11 Mb haplotype located between 3,473,632 and 4,587,759 bp. The haplotype carries an intronic 13-bp deletion (Chr12:3,556,401-3,556,414 bp) that is compatible with recessive inheritance. The 13-bp deletion excises the polypyrimidine tract upstream exon 56 of DNAH17 (XM_021066525.1:c.8510-17_8510-5del) encoding dynein axonemal heavy chain 17. Transcriptome analysis of the testis of two affected boars revealed that the loss of the polypyrimidine tract causes exon skipping which results in the in-frame loss of 89 amino acids from DNAH17. Disruption of DNAH17 impairs the assembly of the flagellar axoneme and manifests in multiple morphological abnormalities of the sperm flagella. Direct gene testing may now be implemented to monitor the defective allele in the Swiss Large White population and prevent the frequent manifestation of a sterilizing sperm tail disorder in breeding boars.

Artificial insemination is the most frequent method of breeding in pigs. The semen of 88 breeding boars is collected once or twice per week at semen collection centers. All 89 ejaculates are macro-and microscopically evaluated. Traits that are routinely 90 measured in all ejaculates include ejaculate volume, sperm concentration, motility, 91 and morphology. Only ejaculates that meet the quality requirements for artificial 92 insemination are processed, diluted and used for inseminations (Colenbrander et al., 93 1993), (Broekhuijse et al., 2011), (Holt et al., 1997). Semen quality and insemination 94 studies on male fertility carried out in livestock discovered novel phenotype-genotype 115 associations that improved our biological understanding of mammalian fertilization 116 (Pausch et al., 2014),(Lamas-Toranzo et al., 2020, p. 95), (Noda et al.,117 2020), (Hiltpold et al., 2020), (Ni et al., 2020, p. 19), (Noskova et al., 2020). 118 Ejaculates from hundreds of boars are evaluated every year at semen collection 119 centers as a service to the pig breeding industry. This rich resource of semen quality 120 records facilitates to investigate sporadically occurring sperm defects using case-121 control association testing (Sironen et al., 2006), (Sironen et al., 2011), (Noskova et 122 al., 2020). Once causative variants have been identified, direct gene tests and 123 genome-based mating strategies may be implemented to avoid the birth of infertile 124 males. Moreover, the discovery of genes that harbor pathogenic alleles that 125 compromise male fertility is important to enhance the diagnostic yield of genetic 126 testing also in species other than livestock (Xavier et al., 2020). 127 Here, we investigate an autosomal recessive sperm tail defect of Swiss Large White 128 boars. Using genome-wide association testing, we map the disorder to porcine 129 chromosome 12. The analysis of genome-wide DNA and RNA sequencing data of 130 affected boars revealed that the loss of an intronic polypyrimidine tract of porcine 131 DNAH17 is causal for the morphological abnormalities of the sperm flagella.

Consent for publication 143
SUISAG, the Swiss pig breeding and competence center provided written consent to 144 the analyses performed and agreed to publish results and data. 145

Animals 146
Eight Swiss Large White boars with a sperm tail defect were considered in our study 147 (Table 1). Five of them were noticed at the semen collection center of SUISAG 148 because their ejaculates contained immotile spermatozoa that had multiple 149 morphological abnormalities of the flagella. Because the five boars were healthy and 150 pedigree analysis indicated that they were inbred on a common ancestor, recessive 151 inheritance of the sperm tail defect was suspected. A mating between two suspected 152 carrier animals was performed in the field to assess phenotypic manifestations in 153 their offspring. The pregnant sow was purchased and maintained at the research 154 barn of the Division of Swine Medicine, Vetsuisse Faculty, University of Zurich. The 155 sow gave birth to a litter with eleven piglets (eight females, three males). One of the 156 male piglets (Boar_1246) died at the age of 200 days due to hemorrhagic bowel 157 syndrome (Grahofer et al., 2017). The other two male boars (Boar_1249, 158 Boar_1254) were slaughtered at the age of 17 months at a regular slaughterhouse. 159 All three male piglets from the mating of suspected carrier animals expressed the 160 sperm tail defect (see below). Pedigree records were analyzed using the PYPEDAL 161 software package (Cole, 2007). 162 163 Sporadically missing genotypes were imputed and haplotypes were inferred using 12 219 iterations of the phasing algorithm implemented in the BEAGLE (version 5.0) software 220 (Browning et al., 2018), assuming an effective population size of 500 while all other 221 parameters were set to default values. 222

Genome-wide association testing 223
Six boars that produced sperm with multiple morphological abnormalities of the 224 flagella were considered as case group for a genome-wide association study. The 225 control group consisted of 100 randomly selected boars that produced normal sperm 226 and were fertile, i.e., each of them sired at least one litter in the Swiss Large White 227 breeding unit. 228 We tested the association between the affection status of the boars and 41,094 229 autosomal SNPs for which the frequency of the minor allele was greater than 5%. 230 Single marker-based case-control association testing was performed using Fisher's 231 exact tests of allelic association as implemented in the PLINK (version 1.9) software 232 (Chang et al., 2015). sliding windows considered, we tested the association of between 1 and 10 238 haplotypes that had frequency greater than 5% with the sperm tail defect using the autosomal SNPs using the PLINK software, a and b are effects of the principal 243 components and the haplotype (HT) tested, respectively, and e is a vector of 244 residuals that are assumed to be normally distributed. In total, we tested 47,685 245 haplotypes for association with the sperm tail defect. 246 SNPs and haplotypes that exceeded the Bonferroni-corrected significance threshold 247 (PSNP=1.22 x 10 -6 , PHAPLO=1.05 x 10 -6 ) were considered as significantly associated. 248 The inflation factors of the test statistics were calculated using the estlambda()-249 function of the GENABEL R package (Aulchenko et al., 2007). 250

Analysis of positional candidate genes 251
The top association signal revealed a 1.11 Mb segment of extended homozygosity at

Whole-genome sequencing and sequence variant genotyping 267
We sequenced five Swiss Large White boars that produced sperm that had multiple 268 morphological abnormalities of the sperm flagella using 2 ×150 bp paired-end reads. (https://github.com/Ensembl/VEP_plugins/blob/release/101/SpliceRegion.pm). We 293 considered 82 pigs with known pedigree as a control group for the present study. 294

Identification of candidate causal variants 295
We considered 14,806 SNPs and 3,856 Indels that were detected within the 1.11 Mb 296 segment (between 3,473,632 and 4,587,759 bp) of extended homozygosity at 297 chromosome 12 as positional candidate causal variants. To identify variants 298 compatible with recessive inheritance of the sperm tail disorder, we applied a filtering 299 strategy that takes into account flawed genotypes and the under-calling of 300 heterozygous genotypes due to relatively low sequencing coverage. Specifically, we 301 screened for alleles that had following frequency: 302 -³ 0.8 in five affected boars (at least 8 out of 10 alleles), 303 -£ 0.05 in 82 control pigs from different breeds (less than 8 alleles). 304 This filtration resulted in only one compatible variant which was a 13-bp intronic 305 deletion. 306

Whole transcriptome sequencing and read alignment 307
Testes were collected immediately after slaughter at an approved slaughterhouse 308 from two boars (Boar_80, Boar_81) that were homozygous for the 13-bp deletion 309 and produced sperm with multiple morphological abnormalities of the flagella. Tissue 310 samples were frozen in liquid nitrogen and stored at -80°C until RNA extraction.

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The pedigree contains only obligate mutation carriers. Red color indicates the eight affected boars.

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The numbers within the boxes correspond to the IDs in Table 1

398
The boars that produced defective sperm were closely related ( Figure 1E). Two 399 affected boars were fullsibs. The average relationship coefficient between the boars 400 was 0.28 and it ranged from 0.14 to 0.63. Their average coefficient of inbreeding was 401 0.087, which is slightly higher than in the fertile boars (F=0.06). All affected boars  The haplotype-based association study peaked at a 1.11 Mb segment of extended 463 homozygosity (between 3,473,632 and 4,587,759 bp) at porcine chromosome 12 464 that was identical by descent in all six affected boars, corroborating recessive 465 inheritance ( Figure 2C). According to the Refseq annotation of the porcine genome, 466 the segment of extended autozygosity encompasses 15 genes ( Figure 2D). 467 We suspected that the mutation causing the sperm tail defect affects a transcript that 468 is abundant in the testes. We found expression levels greater than 10 transcripts per 469

An intronic 13-bp deletion is associated with the sperm tail defect 480
We sequenced five affected boars to an average coverage of 10.9-fold using short 481 paired-end reads. We also considered 82 sequenced pigs from our in-house variant 482 database that were not affected by the sperm tail disorder. The analysis of 483   SNPs on chromosomes 11, 12 and 13 were significantly associated with the sperm 586 disorder using Fisher's exact test of allelic association. This result was puzzling 587 because an oligogenic inheritance of the sperm flagella defect was unlikely. 588 Phenotypic misclassification and genetic heterogeneity could lead to an inconclusive 589 association study (Manchia et al., 2013). However, the morphological abnormalities 590 of the sperm flagella were strikingly similar in the ejaculates of eight boars. 591 Moreover, pedigree analysis suggested monogenic recessive inheritance. Principal 592 components analysis and a genomic inflation factor of 1.55 indicated that population 593 stratification confounded our SNP-based association study (Price et al., 2010). 594 Although Fisher's exact tests have been widely applied to map binary traits (Balding, 595 2006), these tests are prone to type I errors when cases and controls differ in their  (Abramowicz and Gos, 2018). To the best of our knowledge, our study reports 639 the first phenotype-genotype association for a mutation affecting an intronic 640 polypyrimidine tract in pigs. We provide evidence that the loss of the polypyrimidine 641 tract in porcine DNAH17 intron 55 prevents recognition of the 3' splice site which 642 leads to the skipping of exon 56, thus causing defective sperm flagella. Sequence 643 motifs that govern the assembly of the spliceosome may be more distant to 3' splice 644 sites than the polypyrimidine tract in porcine DNAH17 intron 55 (Zhang et al., 2017). 645 However, standard sequence variant annotation tools are largely blind to putative 646 consequences arising from mutations within intronic motifs. Thus, the systematic 647 characterization of branch points and polypyrimidine tracts seems warranted to 648 refine the functional classification of intronic variants. 649 Our findings enable the monitoring of the 13-bp deletion and unambiguous 650 identification of carrier and homozygous animals using direct gene testing. While 651 homozygosity for the 13-bp deletion is easily recognized in artificial insemination 652 boars using microscopic semen analysis, it may remain undetected in sows and 653 natural service boars. However, affected natural service boars will be noticed after 654 few matings due to low fertility. In agreement with previous findings on loss-of-