doi: 10.1111/age.13003 Discovering lethal alleles across the turkey genome using a transmission ratio distortion approach E. A. Abdalla* , S. Id-Lahoucine* , A. Cánovas* , J. Casellas† , F. S. Schenkel* , B. J. Wood*,‡,§ and C. F. Baes*,¶ *Centre for Genetic Improvement of Livestock, Department of Animal Biosciences, University of Guelph, Guelph, ON N1G 2W1, Canada. † Departament de Ciència Animal i dels Aliments, Universitat Autònoma de Barcelona, Bellaterra 08193, Spain. ‡Hybrid Turkeys, C-650 Riverbend Drive, Suite C, Kitchener, ON N2K 3S2, Canada. §School of Veterinary Science, University of Queensland, Gatton, Qld 4343, Australia. ¶Institute of Genetics, Vetsuisse Faculty, University of Bern, Bern 3001, Switzerland. Summary Deviation from Mendelian inheritance expectations (transmission ratio distortion, TRD) has been observed in several species, including the mouse and humans. In this study, TRD was characterized in the turkey genome using both allelic (specific- and unspecific-parent TRD) and genotypic (additive- and dominance-TRD) parameterizations within a Bayesian framework. In this study, we evaluated TRD for 23 243 genotyped Turkeys across 56 393 autosomal SNPs. The analyses included 500 sires, 2013 dams and 11 047 offspring (trios). Three different haplotype sliding windows of 4, 10 and 20 SNPs were used across the autosomal chromosomes. Based on the genotypic parameterizations, 14 haplotypes showed additive and dominance TRD effects highlighting regions with a recessive TRD pattern. In contrast, the allelic model uncovered 12 haplotype alleles with the allelic TRD pattern which showed an underrepresentation of heterozygous offspring in addition to the absence of homozygous animals. For regions with the allelic pattern, only one particular region showed a parent-specific TRD where the penetrance was high via the dam, but low via the sire. The gene set analysis uncovered several gene ontology functional terms, Reactome pathways and several Medical Subject Headings that showed significant enrichment of genes associated with TRD. Many of these gene ontology functional terms (e.g. mitotic spindle assembly checkpoint, DRM complex and Aneuploidy), Reactome pathways (e.g. Mismatch repair) and Medical Subject Headings (e.g. Adenosine monophosphate) are known to be related to fertility, embryo development and lethality. The results of this study revealed potential novel candidate lethal haplotypes, functional terms and pathways that may enhance breeding programs in Turkeys through reducing mortality and improving reproduction rate. Keywords fertility, functional analysis, gene set enrichment, lethal haplotypes, trans- mission ratio distortion tend to increase inbreeding levels among individuals, and Introduction consequently the probability of mating parents carrying Owing to its considerable economic impact, reproduction lethal alleles may increase (Granleese et al. 2015). Turkeys has drawn the attention of turkey breeders and producers are not an exception, thus identifying genetic regions that (Saif & Nestor 2002; Huff et al. 2005; Emamgholi Begli et al. influence reproductive efficiency and mortality is relevant 2019). Lethal alleles may cause mortality before, during or and may enhance breeding programs in this species. after the embryonic stage, and hence reduce reproductive Many autosomal recessive lethal loci have been distin- performance. By their nature, livestock breeding programs guished in livestock species such as cattle (e.g. Dong et al. 2019; Guarini et al. 2019) and the correct mate allocation Address for correspondence is expected to reduce the economic losses (Cole et al. 2016). E. A. Abdalla, Department of Animal Biosciences, Centre for Genetic Several methods, such as screening for the absence of Improvement of Livestock, University of Guelph, 50 Stone Road East, homozygous haplotypes (e.g. VanRaden et al. 2011; Hoff Building 70, Room 21, Guelph, ON N1G 2W1, Canada. et al. 2017; Jenko et al. 2018) and transmission ratio E-mail: eabdalla@uoguelph.ca distortion (TRD; Casellas et al. 2012, 2014), can be used to Accepted for publication 28 August 2020 discover genomic regions with potentially lethal alleles. TRD 876 © 2020 The Authors. Animal Genetics published by John Wiley & Sons Ltd on behalf of Stichting International Foundation for Animal Genetics, 51, 876–889 This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Lethal alleles in turkey 877 is a process whereby the transmission of alleles from Allelic parametrization heterozygous parents to offspring deviates from Mendelian Following Casellas et al. (2014, 2017), the probability of allele ratios, regardless of the cause (Crow 1999; Pardo-Manuel transmission (p) from heterozygous parents to offspring can De Villena et al. 2000; Huang et al. 2013). Thus, TRD be parameterized, including TRD effects on allelic basis, as: reveals locus-specific signals that provide insight into genetics and evolutionary processes of individual fitness pðAÞ ¼ 1  pðBÞ ¼ 0:5 þ α j variation, population divergence and speciation (Fishman & McIntosh 2019). pðBÞ ¼ 1  pðAÞ ¼ 0:5  α j , The availability of genomic markers has facilitated the where A is the particular haplotype allele j being analysed, task of investigating lethal alleles. The decline in reproduc- B represents the remaining haplotype alleles and αj is the tive performance and ability of parents to contribute equally overall TRD for the allele j. To capture parent-specific TRD to next generations may alter the expected Mendelian origin, a parent-specific model was also implemented on the inheritance patterns, resulting in an observable TRD. For basis of allelic parametrization, but including two different instance, it is possible to trace back the inheritance of each parameters: allele as well as the combination of two alleles inherited from parents to offspring using genotype trios with high ps ðAÞ ¼ 1  ps ðBÞ ¼ 0:5 þ αsj accuracy. Several Bayesian models have been developed to pd ðAÞ ¼ 1  pd ðBÞ ¼ 0:5 þ αdj , detect and analyze all types of TRD based on allelic and genotypic parameterizations (Casellas et al. 2012, 2014). where s and d represent sire and dam respectively, and αsj These two parametrizations have been already evaluated in and αdj are sire- and dam-specific TRD for allele j. For all a previous study (Casellas et al. In press) reporting the TRD parameters, flat priors were assumed within a para- relevance of implementing and comparing the different metric space ranging from −0.5 to 0.5. Under a Bayesian parametrizations to capture all types of TRD. The objective implementation, the conditional posterior probabilities of of this study was to assess TRD based on allelic and the TRD parameters are defined as: genotypic parameterizations and perform a functional gene pðα j jyÞ / pðyjα j Þpðα j Þ set enrichment analysis to uncover biological pathways associated with TRD in a purebred line of turkeys. pðαsj , αdj jyÞ / pðyjαsj , αdj Þpðαsj Þpðαdj Þ for overall and parent-specific TRD respectively, where y is a Materials and methods column vector of genotypes of the offspring generation. The likelihood of data consists of a straightforward multiplica- Data tion of the corresponding probabilities for each offspring (i.e. Q In this study, we used a turkey population with 23 243 n poff ðyi Þ), where n is the total number of offspring and poff (6867 males and 16 376 females) genotyped animals for and yi are the probability and the genotype of the ith 61 705 SNPs. The animals were hatched between late 2010 offspring respectively. The software TRDSCAN version 1.0 (Id- and early 2018, and genomic and pedigree information was Lahoucine et al. 2019) uses a multinomial process, hence provided by Hybrid Turkeys, Kitchener, Canada. The whole the likelihood of the data becomes: dataset combines 11 047 parent-offspring genotyped trios, Y 3 ni ! including 500 sires and 2013 dams. All birds were geno-  poff,i ðAAÞnAA,i  poff,i ðABÞnAB,i  poff,i ðBBÞnBB,i : i¼1 nAA,i !n AB,i !n BB,i ! typed with the same SNP genotyping platform array (65 000 SNP; Illumina, Inc.) and mapped to the Turkey In the above, ni is the sum of nAA,i, nAB,i and nBB,i offspring 5.0 Meleagris gallopavo assembly (Dalloul et al. 2010). genotypes, poff,i is the probability of an offspring genotype Quality control analyses were performed and resulted in from the ith mating and nAA,i, nAB,i and nBB,i are the number the removal of non-autosomal SNP markers and those with of AA, AB and BB offspring genotypes from the specific ith a call rate below 90%. Whereas all birds had a call rate mating respectively. For the parent-specific TRD model, five higher than 90% and passed the quality control criteria, the kinds of matings were differentiated in the multinomial Q number of SNPs retained for analysis was 56 393 out of the expression (i.e. 5i¼1 ). For the sampling process, uniform 61 705 markers. BEAGLE 5.0 (Browning et al. 2018) was used proposal distributions (flat priors) were used for both αg and to phase genotypes and impute the missing genotypes. δg within a deepened parametric space ranging from −1 to 1 (Casellas et al. 2012). Statistical analyses Genotypic parameterization To trace the haplotype allele inheritance from parents to offspring in this turkey population, two parametrizations This parameterization captures the interaction between were considered in this study. alleles of offspring genotypes. Additive (αg) and dominance © 2020 The Authors. Animal Genetics published by John Wiley & Sons Ltd on behalf of Stichting International Foundation for Animal Genetics, 51, 876–889 878 Abdalla et al. (δg) or over-dominance, both positive or negative, TRD parametrization, an arbitrary minimum magnitude of TRD parameters are considered regardless of the origin of the at least 0.20 and the number of underrepresented offspring allele. As described by Casellas et al. (2012, In press), the at least 1000 were considered to identify haplotype alleles probability of observing offspring (poff) from heterozygous- with strong allelic TRD patterns. The number of underrep- by-heterozygous mating can be estimated as follows: resented offspring is the total number of offspring expected, but not observed for a particular allele, which is also ð1 þ αgj  δgj Þ Poff ðAAÞ ¼ approximately equal to the number of informative offspring 4 multiplied by twice the magnitude of the TRD. These ð1 þ δgj Þ thresholds ensured the identification of target haplotypes Poff ðABÞ ¼ 2 with a moderate-to-high level of TRD (i.e. moderate-to-high penetrance) as well as a reasonable number of offspring that ð1  αgj  δgj Þ Poff ðBBÞ ¼ , are expected, but not observed (i.e. a minimum frequency 4 for the allele in the population). For the genotypic where αgj and δgj are additive and dominance TRD parametrization, an arbitrary minimum number of 10 parameters for the specific allele j respectively. non-observed homozygous offspring from heterozygous-by- Under a Bayesian implementation, the conditional poste- heterozygous matings with additive TRD effect less than rior probabilities of the TRD parameters are defined as: −0.50 and dominance TRD effect greater than 0.10 was considered to determine haplotype alleles with a substantial pðαgj , δgj jyÞ / pðyjαgj , δgj Þpðαg Þ pðδgj jαgj Þ, recessive TRD pattern. This threshold was considered to where y is a column vector of genotypes of the offspring maintain the most important regions. It should also be generation. For the sampling process, uniform proposal emphasized that a well-known lethal haplotype in cattle distributions (flat priors) were used for both αg and δg within industry (Holstein haplotype 3) was initially identified by an extended parametric space ranging from −1 to 1 VanRaden et al. (2011) with only seven non-observed (Casellas et al. 2012). Thus, as the parametric space for αg homozygous offspring from heterozygous sires and is initially [−1, 1], the parametric space for δg is restricted to heterozygous maternal grandsires matings. In addition, as [−1, |αg|]. Moreover, the parametric space of αg itself is different sliding windows were used, only the haplotype restricted to [−1 + δg to 1 − δg] if δg > 0 and this guaran- alleles with the largest BF within a region (with many tees that the sum of offspring’s genotypes probabilities for physically linked haplotypes) were selected as the best specific mating is equal to 1. candidates to explain the observed TRD in the region and Transmission ratio distortion was evaluated using three potentially harbor the causal mutations. Thus, it is impor- sliding windows: 4, 10, and 20 SNP. The analyses were tant to mention that the patterns of TRD observed in short carried out using TRDSCAN version 1.0 software (Id-Lahou- windows are displayed in larger haplotypes including the cine et al. 2019) based on a MCMC and the Metropolis–- same allele and also on other physically linked haplotypes, Hastings’ algorithm (Hastings 1970). A single MCMC of supporting the relevance of TRD in the corresponding 100 000 iterations was run for each analysis with the first particular locus. 10 000 iterations being discarded as burn-in. Bayes factor (BF; Kass & Raftery 1995), which is a ratio of probabilities Functional and gene set enrichment between full and null TRD models, was used to determine significant TRD (BF ≥ 100; decisive evidence according to Assignment of lethal haplotypes to genes Jeffreys’ (1984) scale). To obtain a reasonable statistical power and to minimize false TRD as a result of genotyping Genes associated with complex traits are expected to errors, only haplotype alleles with a minimum number of represent only a small fraction of the genetic variation 50 informative offspring (i.e. from heterozygous parents) and, hence, some genetic variants with small effects and and five heterozygous sires and/or dams were analyzed. disease risks may not ever be detected (Peng et al. 2010; The identified TRD regions were then filtered to minimize Abdalla et al. 2016). To further investigate the potential genotypic errors and to eliminate regions with random lethal haplotypes identified with TRD, the coordinates of TRD. First, an approximate empirical null distribution of these haplotype regions were used to mine for annotated TRD (Id-Lahoucine et al. 2019) at less than 0.001% margin genes using the Turkey 5.0 (release 102) assembly (Dalloul error was used to remove TRD generated by chance. Also, a et al. 2010). It has been reported that strong LD may extend minimal number of informative parents (≥5 heterozygous up to 10–30 kb in chickens (Rao et al. 2008; Megens et al. sires and/or heterozygous dams) were considered to mini- 2009; Qanbari et al. 2010). Thus, haplotypes were assigned mize possible false TRD from genotyping errors. Similarly, to genes if they were located within the genomic sequence regions with few heterozygous parents fully explaining the of an annotated gene or within 15 kb of the 50 or 30 ends of observed TRD in the corresponding region were discarded as the first and last exons respectively. The 15 kb distance was potential genotyping errors. Moreover, for the allelic used to capture proximal regulatory regions and other © 2020 The Authors. Animal Genetics published by John Wiley & Sons Ltd on behalf of Stichting International Foundation for Animal Genetics, 51, 876–889 Lethal alleles in turkey 879 functional sites that may lie outside (e.g. promoter regions) Gene network but close to each gene. If a haplotype was found to be The overlaps among significant genes associated with GO located within or close to more than one gene, all of these terms and Reactome pathways and their functional net- genes were included in the subsequent analyses. works were also examined. We retrieved close neighbor genes and then generated an aggregate interaction network Assignment of genes to functional categories based on physical protein interaction and co-expression of those genes using GENEMANIA software (Warde-Farley et al. Gene Ontology (GO; Ashburner et al. 2000), Pathway 2010). Knowledgebase (Reactome; Fabregat et al. 2018) and Medical Subject Headings (MeSH; Coletti & Bleich 2001; Nelson et al. 2004) databases were used to define functional Results and discussion sets of genes. Biological descriptors, known as GO terms, fall into three categories: biological process, molecular func- Prevalence of TRD across the turkey genome tions and cellular components. Reactome, on the other The prevalence of TRD was widely distributed across the hand, provides several biochemical networks including turkey genome as shown in Fig. 1. The initial numbers of metabolic and regulatory pathways. Finally, MeSH is a haplotype alleles detected with decisive significant evidence collection of descriptors or headings representing key topics (BF ≥ 100) of TRD, according to Jeffreys’ scale (Jeffreys discussed in the papers indexed in the MEDLINE database. 1984), were 48 951, 52 896 and 54 287 for 4, 10 and 20 Whereas MeSH terms are classified into 19 categories, in SNP haplotype windows respectively. Despite this high this study, we were interested in only four: anatomy, number of regions, some of them had a large BF (10100) disease, phenomena and processes, and lastly chemicals and suggesting virtually 0 probability of the null TRD model. In drugs. addition, most of the detected TRD regions had low frequencies (i.e. rare variants); however, these haplotype Pathway-based association analysis alleles were supported by the large dataset. It is important to mention that it has been suggested that rare variants are The Fisher’s exact test was used to declare the association of more likely to be functional than common variants (Gorlov a given GO term, Reactome pathway and MeSH heading et al. 2008; Karaca et al. 2015), emphasizing the impor- with TRD. This test was performed to search for an tance of TRD regions despite their low frequencies. It is overrepresentation of significant genes in a given functional noteworthy to mention that the majority of the regions category among all genes. The P-value of observing g were detected with more than one of the models applied, but significant genes in the term was calculated as follows: with different fits and statistical significance. Thus, after the    S N S characterization of the TRD across the whole genome with g1 i ki the filtration criteria provided in the ‘Materials and meth- P  value ¼ 1  ∑   i¼0 N ods’ section, the list of the most relevant haplotypes is k provided in Tables 1 and 2 for regions with allelic and recessive TRD patterns respectively. These haplotypes indi- where N is the total number of genes analyzed in the study, cate candidate regions potentially carrying deleterious S is the total number of genes that were deemed signifi- alleles or genes affecting reproduction. However, these cantly associated with TRD and k is the total number of TRD findings were obtained under the assumption of no genes in the functional category in the database under selection of offspring within a family to be genotyped. The consideration. violation of this must be taken into consideration by further Owing to a lack of biological information related to investigating the source of the observed TRD signals. This is turkeys, both the Turkey 5.0 assembly (Dalloul et al. because the pre-selection of offspring to be genotyped within 2010) and the GRCg6a chicken assembly (International families (Id-Lahoucine & Casellas 2017) could be a source of Chicken Genome Sequencing Consortium 2004) were used bias on TRD analyses as discussed by Id-Lahoucine et al. to perform the pathway analysis. It is important to (2019). It is well known that selection of data has been a emphasize that all potential genes defined in this study concern and a limitation for many types of analyses. In are conserved across many organisms, including humans particular, for TRD analyses, major genes that present and chickens. The GO and Reactome enrichment analyses crucial and large impact in reproductive performance are were carried out using GO (Ashburner et al. 2000) and targeted. The genetic selection performed in turkey popu- PANTHER (Mi et al. 2016) respectively, whereas the MeSH lations is based on selection indexes targeting a multiple- enrichment analysis was performed using the MESHR trait breeding objective (mainly highly polygenic production package (Tsuyuzaki et al. 2015) available in the R traits). Thus, the chance to observe TRD signals with environment (R Core Team 2019). absence of homozygous offspring as a result of selection is © 2020 The Authors. Animal Genetics published by John Wiley & Sons Ltd on behalf of Stichting International Foundation for Animal Genetics, 51, 876–889 880 Abdalla et al. Figure 1 Bayes factor for haplotypes with transmission ratio distortion across the turkey genome. Significant haplotype alleles were determined based on log10 Bayes factor ≥2 according to Jeffreys’ scale (Jeffreys 1984) less likely. Here, we are using the TRD method as an number is still substantially lower than expected, indicating alternative strategy to first scan possible relevant regions lower viability. This could be due to the variation in specific harboring lethal alleles that will require further research to TRD between males and females. As the probability of exclude both genotyping errors and biases from pre-selec- transmitting this lethal allele from heterozygous sires is tion in the offspring generation. close to the Mendelian expectation (0.5–0.03 = 0.47), the probability of observing live homozygous parents/offspring increases (4 instead of 0). Matings with either parent Haplotypes with allelic TRD pattern using the allelic carrying the lethal allele in the homozygous state for the parametrization model remaining 11 regions were not observed, supporting the Across the turkey genome, 12 potentially lethal haplotype lethal effect of this haplotype allele. On the other hand, it candidates were detected with allelic TRD, as shown in must be emphasized that these regions were also detected by Table 1. The number of informative offspring detected in the genotypic model. Nevertheless, the allelic model had those 12 potentially lethal haplotype regions ranged better goodness-of-fit than the genotypic model in terms of between 1400 and 3548, whereas the number of under- deviance information criterion units and accurate TRD represented offspring reached 1564. Most of the haplotypes estimates with short credible intervals. showed relatively small differences between male- and female-specific TRD, supporting their unspecific-parent TRD Haplotypes with recessive TRD pattern using the pattern. In contrast, one haplotype allele located on genotypic parametrization model chromosome 16 showed parent-specific TRD, where it was high for the dam (−0.22) and low via the sire (−0.03). Based on the genotypic parametrization model, 14 poten- Although this region had some offspring (4 and 15) from tially lethal haplotypes were identified with additive- and homozygous-by-heterozygous (AB × AA) matings (i.e. the dominance-TRD resulting in lethal homozygous offspring homozygous parent carries two copies of the lethal allele), from heterozygous-by-heterozygous matings as shown in the numbers of offspring were extremely small compared Table 2. The additive TRD component of these regions with the expectations. Similarly, the matings of heterozy- ranged from −0.54 to −0.94, and interestingly, the gous-by-heterozygous (AB × AB) in this region produced negative effects of these haplotypes are counteracted by some (34) homozygous (AA) individuals. However, this the dominance TRD effects, which ranged between 0.11 © 2020 The Authors. Animal Genetics published by John Wiley & Sons Ltd on behalf of Stichting International Foundation for Animal Genetics, 51, 876–889 Table 1 Potential candidate lethal or semi-lethal alleles identified with allelic transmission ratio distortion patterns by the allelic model AB4 × AA AB × BB AB × AB TRD effects6 Chromosome Region (kbp) SNP1 Hetero sires2 Hetero dams3 Frequency (%) AA5 AB AB BB AA AB BB α αs αd © 2020 The Authors. Animal Genetics published by 1 173 127–173 367 10 17 360 4.54 0 0 641 1688 1 5 15 −0.23 −0.33 −0.23 2 71 543–71 622 4 21 305 2.93 0 0 397 1664 0 7 2 −0.31 −0.21 −0.32 2 99 815–99 884 4 67 384 4.44 0 0 654 1915 0 27 39 −0.25 −0.25 −0.25 5 36 746–36 854 4 15 262 2.77 0 0 403 1505 0 2 3 −0.29 −0.31 −0.29 6 50 141–50 184 4 22 182 1.37 0 0 146 1242 0 1 11 −0.40 −0.44 −0.39 7 10 128–10 171 4 61 277 2.66 0 0 342 1344 0 14 42 −0.31 −0.26 −0.32 10 10 424–10 465 4 15 235 2.34 0 0 327 1338 0 5 6 −0.31 −0.38 −0.30 11 15 529–15 551 4 25 410 4.41 0 0 639 2157 0 10 22 −0.28 −0.16 −0.29 14 16 726–16 743 4 32 287 2.96 0 0 383 1557 0 9 9 −0.31 −0.31 −0.31 16 10 429–10 450 4 95 457 8.21 4 15 1136 2025 34 189 145 −0.15 −0.03 −0.22 23 69–81 4 27 92 1.31 0 0 189 1271 0 8 30 −0.38 −0.42 −0.30 28 4507–4549 10 29 264 2.8 0 0 372 1597 0 8 21 −0.32 −0.27 −0.32 1 Number of SNP in a haplotype window. 2 Number of heterozygous sires. 3 Number of heterozygous dams. 4 Genotypes of parents. John Wiley & Sons Ltd on behalf of Stichting International Foundation for Animal Genetics, 51, 876–889 5 Genotypes of offspring. 6 α, αs and αd are overall, sire and dam allelic transmission ratio distortion respectively. Lethal alleles in turkey 881 882 Abdalla et al. Table 2 Potential candidate lethal alleles identified with recessive transmission ratio distortion patterns by the genotypic model AB5 × AA AB × BB AB × AB TRD effects7 Chr1 Region (Kbp) SNP2 Hetero sires3 Hetero dams4 Frequency (%) AA6 AB AB BB AA AB BB αg δg 1 27 680–27 924 10 24 101 2.51 0 0 450 447 0 17 10 −0.58 0.29 1 29 253–29 484 10 30 108 4.33 0 0 305 265 0 20 11 −0.54 0.35 2 40 083–40 199 4 13 86 1.83 0 0 336 481 0 23 15 −0.77 0.14 3 29 955–30 188 10 5 39 0.67 0 0 106 142 0 15 10 −0.71 0.16 4 60 400–60 941 20 9 64 1.93 0 0 252 321 0 32 16 −0.72 0.21 4 65 823–65 908 4 10 48 0.91 0 0 153 232 0 20 11 −0.77 0.11 5 3900–3975 4 50 170 4.92 0 0 950 967 0 26 17 −0.62 0.30 6 8973–9097 10 19 81 2.48 0 0 431 458 0 21 11 −0.63 0.27 11 23 084–23 137 4 31 107 3.04 0 0 573 673 0 22 12 −0.67 0.23 12 15 689–15 829 20 9 49 1.84 0 0 285 304 0 15 10 −0.61 0.26 19 4054–4196 20 43 156 4.39 0 0 763 756 0 62 18 −0.63 0.33 21 9664–9680 4 38 129 3.03 0 0 525 573 0 52 22 −0.67 0.29 24 672–742 10 13 54 1.86 0 0 323 384 0 17 10 −0.66 0.22 28 1640–1794 20 20 107 2.96 0 0 484 476 0 15 10 −0.57 0.29 1 Chromosome. 2 Number of SNP on a haplotype window. 3 Number of heterozygous sires. 4 Number of heterozygous dams. 5 Genotypes of parents. 6 Genotypes of offspring. 7 αg and δg are additive and dominance transmission ratio distortion respectively. Table 3 Biological process function terms significantly overrepresented with genes statistically associated with transmission ratio distortion Number of Number of genes significant GO ID Term (GO hierarchy level) in the GO term genes P-value1 0051315 Attachment of mitotic spindle microtubules to kinetochore (10) 10 1 0.023 0033567 DNA replication, Okazaki fragment processing (11) 3 1 0.008 1902969 Mitotic DNA replication (11) 8 1 0.019 0007094 Mitotic spindle assembly checkpoint (14) 18 1 0.039 0071174 Mitotic spindle checkpoint (9) 18 1 0.039 2000697 Negative regulation of epithelial cell differentiation involved in kidney development (12) 2 1 0.006 2000094 Negative regulation of mesonephric nephron tubule epithelial cell differentiation (15) 1 1 0.004 0061218 Negative regulation of mesonephros development (11) 4 1 0.001 0045841 Negative regulation of mitotic metaphase/anaphase transition (12) 20 1 0.044 2000816 Negative regulation of mitotic sister chromatid separation (11) 21 1 0.046 0072183 Negative regulation of nephron tubule epithelial cell differentiation (14) 1 1 0.004 1903461 Okazaki fragment processing involved in mitotic DNA replication (12) 1 1 0.004 2000093 Regulation of mesonephric nephron tubule epithelial cell differentiation (12) 1 1 0.004 Significance declared at P < 0.05. 1 and 0.35. The interaction of additive and dominance TRD pattern, it is expected that there will be similar numbers of effects provides an equal chance for heterozygous (carriers) heterozygous (AB) and homozygous (BB) offspring from offspring to survive as non-carrier birds. From the heterozy- heterozygous-by-homozygous matings (AB × BB). Never- gous-by-heterozygous (AB × AB) matings, no homozygous theless, slightly more heterozygous offspring (AB) in (AA) individuals were observed, indicating the lethality of heterozygous-by-homozygous matings were observed, the haplotypes in the homozygous state. For these regions, which could be partly explained as a result of a random the numbers of carrier parents were between five and 50 for TRD (i.e. TRD generated by chance; Id-Lahoucine et al. sires and between 39 and 170 for dams. The number of 2019). informative offspring ranged from 273 to 1960, whereas It is worth noting that most of these regions were only the number of expected homozygotes that were not detected with the genotypic model whereas no statistical observed ranged from 10 to 22. For a fully recessive TRD evidence was found using the allelic model. This is due to © 2020 The Authors. Animal Genetics published by John Wiley & Sons Ltd on behalf of Stichting International Foundation for Animal Genetics, 51, 876–889 Lethal alleles in turkey 883 Table 4 Cellular component function terms significantly overrepresented in genes statistically associated with transmission ratio distortion Number of genes in Number of GO ID Term (GO hierarchy level) the GO term significant genes P-value1 0044444 Cytoplasmic part (7) 5503 18 0.038 0070176 DRM complex (15) 1 1 0.004 0034709 Methylosome (8) 6 1 0.014 0097431 Mitotic spindle pole (12) 18 1 0.039 0032021 NELF complex (14) 2 1 0.006 0090568 Nuclear transcriptional repressor complex (13) 19 2 0.000 0090571 RNA polymerase II transcription repressor complex (14) 4 2 0.000 Significance declared at P < 0.05. 1 Table 5 Molecular function terms significantly overrepresented in genes statistically associated with transmission ratio distortion Number of genes in Number of GO ID Term (GO hierarchy level) the GO term significant genes P-value1 0005488 Binding (2) 9725 28 0.017 0015187 Glycine transmembrane transporter activity (13) 5 1 0.012 0015375 Glycine: sodium symporter activity (16) 1 1 0.004 0060090 Molecular adaptor activity (3) 124 2 0.028 0015175 Neutral amino acid transmembrane transporter activity (12) 23 1 0.048 0030674 Protein binding, bridging (4) 112 2 0.023 1990756 Protein binding, bridging involved in substrate 3 1 0.008 recognition for Ubiquitination (11) Significance declared at P < 0.05. 1 their different parameterizations, where the genotypic metaphase/anaphase transition (GO:0045841), which is, in model includes the interaction between alleles, allowing turn, a negative regulation of mitotic sister chromatid separa- detection of recessive TRD patterns. Specifically, the nega- tion (GO:2000816). Mitosis, which is associated with TRD, tive effect of the additive TRD component can be contrasted is a specialized division of chromosomes that occur during with the positive effect of the dominance TRD component in the formation of reproductive cells (Nicklas 1971). A similar heterozygous offspring, allowing for capture of recessive significant (P-value < 0.05) cellular component GO term, TRD patterns. In contrast, the fact that the allelic Mitotic spindle pole (GO:0097431), was also associated with parametrization is based on targeting the transmission of TRD (Table 4). an allele from a specific parent to offspring, which is Three GO terms detected in the analysis classified into the separate from possible interaction in offspring generation, biological process domain showed significant association prevents its ability to capture recessive TRD patterns. with TRD (Table 3). In addition to their similar functions in mitosis, these three terms are close in the GO hierarchy. Okazaki fragment processing involved in mitotic DNA replication Functional analysis and gene set enrichment (GO:1903461) is a DNA replication, Okazaki fragment Sixty-seven biological process, 17 cellular components, 35 processing (GO:0033567) and part of mitotic DNA replication molecular function GO terms, in addition to 19 Reactome (GO:1902969). In cell biology, Okazaki fragments (Sakabe pathways and nine MeSH terms showed a significant & Okazaki 1966) comprise the processes involved in any overrepresentation (P-value < 0.05) of genes associated mitotic cell cycle DNA replication, a necessary step in the with TRD in turkeys. All of these significant terms and cell cycle. pathways are listed in Tables S1–S5. It is important to The Negative regulation of mesonephric nephron tubule mention that these terms, pathways and genes should be epithelial cell differentiation (GO:2000094) term was detected further investigated and validated to avoid false positives. as significantly enriched (P-value < 0.01) with genes asso- Four biological process GO terms, in a close GO hierarchy ciated with TRD and related to cell differentiation. This GO relationship and related to mitosis, showed a significant term is close in the GO hierarchy and in function to four overrepresentation (P-value < 0.05) of genes statistically other GO terms, which all can be linked to TRD: associated with TRD (Table 3). Mitotic spindle assembly GO:2000093, GO:0061218, GO:0072183 and checkpoint (GO:0007094) is a mitotic spindle checkpoint GO:2000697. Cellular differentiation is the process in (GO:0071174) and a negative regulation of mitotic which a simple cell changes from one cell type to a more © 2020 The Authors. Animal Genetics published by John Wiley & Sons Ltd on behalf of Stichting International Foundation for Animal Genetics, 51, 876–889 884 Abdalla et al. Table 6 Reactome terms significantly overrepresented in genes statistically associated with transmission ratio distortion. Number of genes Number of Reactome ID Reactome term name in the pathway significant genes P-value1 R-GGA-5358508 Mismatch repair 11 1 0.025 R-GGA-5358606 Mismatch repair (MMR) 3 1 0.008 directed by MSH2:MSH3 (MutSbeta) Significance declared at P < 0.05. 1 Table 7 Disease and chemicals and drugs MeSH terms significantly is an RNA polymerase II transcription repressor complex overrepresented in genes statistically associated with transmission ratio (GO:0090571), which is, in turn, a nuclear transcriptional distortion repressor complex (GO:0090568). Interestingly, a connec- Category MeSH term ID MeSH term name P-value1 tion between RNA polymerase II and TRD has been previously reported (Paterson et al. 2009). Moreover, Chemicals and drugs D000249 Adenosine 0.012 monophosphate Harrison et al. (2006) reported that DRM complex is a Disease D000708 Anaplasia 0.048 transcriptional repressor complex and involved in cell fate specification. Significance declared at P < 0.05. 1 Three GO terms, which are close in the GO hierarchy (Table 5), were classified into the molecular function category and showed a significant overrepresentation of specialized type, which may occur in numerous steps (Jones genes statistically associated with TRD (Table 4). Glycine:- & Taylor 1980; Slack 2007). In a study on the development sodium symporter activity (GO:0015375) is a glycine trans- of the kidney in mice, McCright et al (2001) reported that membrane transporter activity (GO:0015187), which is in animals died perinatally owing to the lack of normal turn a neutral amino acid transmembrane transporter activity capillary tufts, which is a result of defects in kidney (GO:0015175). Genetic factors that stop coding glycine development. cause late-stage embryo lethality and hence TRD (Seidel The terms NELF complex (GO:0032021) and two similar et al. 2011). Protein binding, bridging involved in substrate GO terms, methylosome (GO:0034709) and cytoplasmic part recognition for ubiquitination (GO:1990756) is a protein (GO:0044444), are classified under the cellular component binding, bridging (GO:0030674). The molecular function of domain and show a significant overrepresentation of genes binding and sperm motility was reported by Bauer et al. statistically associated with TRD (Table 4). The NELF (2007). As the authors mentioned, the binding partner for complex is a key regulatory step of the transcription cycle Ropporin and Ras proteins to the outer surface of the (Tamborrini & Piatti 2019). The GO term DRM complex dense fiber proteins plays a crucial function in sperm (GO:0070176), located in the cellular component category, motility. Cellular component Molecular function Biological process Reactome Figure 2 Venn diagram showing overlaps between significant genes associated with the terms of three GO domains (presented in Tables 3–5) and the Reactome pathways (presented in Table 6) © 2020 The Authors. Animal Genetics published by John Wiley & Sons Ltd on behalf of Stichting International Foundation for Animal Genetics, 51, 876–889 Lethal alleles in turkey 885 Physical interaction Co-expression Function: Figure 3 Network integration of gene MAD1L1 based on physical protein interaction and co-expression. The gene APITD1 is associated with MAD1L1 through pathways (links are not shown) © 2020 The Authors. Animal Genetics published by John Wiley & Sons Ltd on behalf of Stichting International Foundation for Animal Genetics, 51, 876–889 886 Abdalla et al. Physical interaction Co-expression Function: Figure 4 Network integration of gene LIG1 based on physical protein interaction and co-expression. The gene UBE2R2 is associated with LIG1 through prediction (links are not shown) © 2020 The Authors. Animal Genetics published by John Wiley & Sons Ltd on behalf of Stichting International Foundation for Animal Genetics, 51, 876–889 Lethal alleles in turkey 887 Two Reactome pathways were detected as significantly through prediction. The functions for these genes include (P-value < 0.01) enriched with genes related to TRD DNA replication and cell cycle DNA replication, which are (Table 6). Mismatch repair (MMR) directed by MSH2:MSH3 among the most important cell activaties concerning (MutSbeta; R-GGA-5358606) and Mismatch Repair (R-GGA- reproduction and cell deviation. 5358508) are similar in their activities to the GO term Mitotic spindle assembly checkpoint (GO:0007094) and rep- Conclusions resent the GO biological process mismatch repair (GO:0006298). Analogous to the GO terms related to In this study, we applied allelic and genotypic parameter- mitosis and cell cycle, these two pathways are associated izations of TRD to detect potential lethal haplotypes in with DNA mismatch repair. It has been demonstrated that turkeys. The two methods revealed relevant regions across their activity increases and reaches the highest levels during the turkey genome with either a classical recessive inher- the S phase of the cell cycle (Edelbrock et al. 2009). itance pattern (i.e. lethal only in the homozygous state) or Mismatch Repair corrects single base mismatches and small allelic patterns (i.e. reduced viability of the carrier offspring). insertion and deletion loops of unpaired bases. Mismatch In addition to 19 Reactome pathways and nine MeSH repair directed by MSH2:MSH3 (MutSbeta) binds unpaired terms, 67 biological process, 17 cellular components and 35 loops of two or more nucleotides (Palombo et al. 1996). molecular function GO terms showed a significant (P- A MeSH term, Adenosine Monophosphate (D000249), value < 0.05) overrepresentation of genes statistically asso- classified into the chemicals and drugs category, showed a ciated with TRD. Functional networks among several significant overrepresentation of genes statistically associ- significant genes also showed links to mitosis and cell ated with TRD (Table 7). Interestingly, various reproductive replication. These highlighted pathways and gene ontolo- functions, such as those requiring hormone synthesis and gies, along with the overall findings of this study, will maintenance of fluid composition, are modulated by contribute in developing novel turkey breeding and man- adenosine (Zhou et al. 2006; Aliagas et al. 2010). Another agement strategies, as well as to specific mating programs significant (P-value < 0.05) MeSH term, Anaplasia for turkeys. (D000708), is classified into the disease domain (Table 7). Anaplasia is related to neoplastic cells and refers to the loss of Acknowledgements mature or specialized features of differentiated neoplastic cells (Rodriguez et al. 2010; Pujadas et al. 2019). Previous The authors extend their gratitude to Jeff Mohr and studies have shown that genetic variation among inbred personnel of the Hybrid Turkeys pedigree farm (Kitchener, mice as a result of continuous uploading and removal of Canada) for collecting and providing data used in this study. rare variation (new mutations) may generate TRD (Casellas & Medrano 2008; Niu & Liang 2009). Conflict of interests All authors declare no conflict of interest. Gene network The Venn diagram (Fig. 2) depicted the intersections Funding between the significant genes associated with the terms of three GO domains (Tables 3–5) and the Reactome pathways This study was conducted as part of the project entitled (Table 6). Notably, the gene MAD1L1 is significant across ‘Application of genomic selection in turkeys for health, the three GO domains and two of these GO domains welfare, efficiency and production traits’ funded by the (biological process and molecular function) overlap with the Government of Canada through the Genome Canada Reactome pathways in the gene LIG1. The aggregate Genomic Application Partnership Program and adminis- interaction networks for MAD1L1 and LIG1 genes, based tered by Ontario Genomics (recipients: C.F. Baes (Aca- on physical protein interaction and co-expression, are demic), B.J. Wood (Industry)). This study was also shown in Figs 3 and 4 respectively. The functional net- financially supported by Hybrid Turkeys (Kitchener, works of gene MAD1L1 with 19 other genes indicate that Canada). The authors declare that this study received this group of genes is involved in several activates related to funding from Hybrid Turkeys. The funder was involved in mitosis such as regulation of mitosis and negative regula- providing the datasets used in this study. tion of mitosis cell cycle phase transition. The gene APITD1 was not linked to MAD1L1 via physical protein interaction Data availability statement or co-expression networks, but through the pathways network (this connection is not shown in Fig. 3). Similarly, Data that support the findings of this study are available the functional network for gene LIG1 revealed 19 genes from Hybrid Turkeys upon reasonable request and were associated with it through physical protein interaction and used under license for the current study, and thus are not co-expression networks (Fig. 4) and one gene (i.e. UBE2R2) publicly available. © 2020 The Authors. Animal Genetics published by John Wiley & Sons Ltd on behalf of Stichting International Foundation for Animal Genetics, 51, 876–889 888 Abdalla et al. 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Euro- Table S1 Biological process terms significantly overrepre- pean Journal of Human Genetics 18, 111–7. sented with genes statistically associated with TRD Pujadas E., Chen L. & Rodriguez F.J. (2019) Pathologic and Table S2 Cellular component function terms significantly molecular aspects of anaplasia in circumscribed gliomas and overrepresented with genes statistically associated with TRD glioneuronal tumors. Brain Tumor Pathology 36, 40–51. Table S3 Molecular function terms significantly overrep- Qanbari S., Hansen M., Weigend S., Preisinger R. & Simianer H. resented with genes statistically associated with TRD (2010) Linkage disequilibrium reveals different demographic Table S4 Reactome pathways significantly overrepre- history in egg laying chickens. BMC Genetics 11, 103. sented with genes statistically associated with TRD Rao Y.S., Liang Y., Na Xia M., Shen X., Du Jun Y., Glong Luo C., Table S5 MeSH terms significantly overrepresented with Hua Nie Q., Zeng H. & Quan Zhang X. 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