Significant departures from expected Mendelian inheritance ratios (transmission ratio distortion, TRD) are frequently observed in both experimental crosses and natural populations. TRD on mouse Chromosome (Chr) 2 has been reported in multiple experimental crosses, including the Collaborative Cross (CC). Among the eight CC founder inbred strains, we found that Chr 2 TRD was exclusive to females that were heterozygous for the WSB/EiJ allele within a 9.3 Mb region (Chr 2 76.9 – 86.2 Mb). A copy number gain of a 127 kb-long DNA segment (designated as responder to drive, R2d) emerged as the strongest candidate for the causative allele. We mapped R2d sequences to two loci within the candidate interval. R2d1 is located near the proximal boundary, and contains a single copy of R2d in all strains tested. R2d2 maps to a 900 kb interval, and the number of R2d copies varies from zero in classical strains (including the mouse reference genome) to more than 30 in wild-derived strains. Using real-time PCR assays for the copy number, we identified a mutation (R2d2 WSBdel1 ) that eliminates the majority of the R2d2 WSB copies without apparent alterations of the surrounding WSB/EiJ haplotype. In a three-generation pedigree segregating for R2d2 WSBdel1 , the mutation is transmitted to the progeny and Mendelian segregation is restored in females heterozygous for R2d2 WSBdel1 , thus providing direct evidence that the copy number gain is causal for maternal TRD. We found that transmission ratios in R2d2 WSB heterozygous females vary between Mendelian segregation and complete distortion depending on the genetic background, and that TRD is under genetic control of unlinked distorter loci. Although the R2d2 WSB transmission ratio was inversely correlated with average litter size, several independent lines of evidence support the contention that female meiotic drive is the cause of the distortion. We discuss the implications and potential applications of this novel meiotic drive system.

One of the strongest expectations in genetics is that chromosomes segregate randomly during meiosis. However, genetic loci that exhibit transmission ratio distortion (TRD) are sometimes observed in offspring of F1 hybrids. Meiotic drive is a type of non-Mendelian inheritance in which a “selfish” genetic element exploits asymmetric female meiotic cell division to promote its preferential inclusion in ova. We previously reported TRD on Chr 2 in the CC, a mouse recombinant inbred panel with contributions from three Mus musculus subspecies. Here we show that maternal TRD consistent with a novel meiotic drive system is caused by a copy number gain. This mutation is similar in size and structure to other known meiotic drive responders, such as the knobs of maize. A deletion of most of the copies is sufficient to restore Mendelian segregation, proving that the copy number variant is causative of the observed TRD. In the CC, and also the related DO population, the transmission frequency of the favored allele varies dependent on genetic background, demonstrating that this system is under genetic control. In conclusion, we describe a novel wild-derived meiotic drive locus on mouse Chr 2 that exploits female meiosis asymmetry to violate the Laws of Mendelian inheritance.

Funding: We acknowledge grants National Institute of General Medical Sciences/National Institutes of Health GM076468 and GM078452 (PMP); National Institute of General Medical Sciences/National Institutes of Health 2P50GM076468-06 (EJC, GAC); National Institute of Mental Health/National Institutes of Health K01MH094406 (JJC); The National Institute of Diabetes and Digestive and Kidney Diseases/National Institutes of Health RC1DK087510 (DWT, AHH); National Cancer Institute/National Institutes of Health ZIA BC011255 (KHun); National Institute of Child Health and Human Development/National Institutes of Health R01HD065024 (DAO, FPMdV); NHGRI/National Institutes of Health R01 HG00941 (LBR); National Cancer Institute/National Institutes of Health U01CA105417 (DWT, DP); National Cancer Institute/National Institutes of Health R01CA079869 (DWT); National Institute of Diabetes and Digestive and Kidney Diseases/National Institutes of Health DK056350 (DP); National Cancer Institute/National Institutes of Health RC1 CA145504-02 (GAC); National Institute of Mental Health/National Institutes of Health 1R21 MH096261-01 (FPMdV); National Institute of Mental Health/National Institutes of Health 1F30 MH103925 (APM); National Human Genome Research Institute/National Institutes of Health 1U54 HG004968 (GMW); National Human Genome Research Institute/National Institutes of Health (funded the JAX interspecific backcross panels); the National Science Foundation IOS-1121273 (TG). This research was supported also by Vaadia-BARD Postdoctoral Fellowship Award No. FI-478-13 from BARD, The United States - Israel Binational Agricultural Research and Development Fund (LY). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Here we report our extensive genetic characterization of Chr 2 TRD in the CC and in the Diversity Outbred (DO), an outbred population initiated from 144 incompletely inbred CC lines and specifically tailored for high resolution mapping of complex traits [ 33 – 36 ]. Using a combination of classical genetics, whole genome sequence analysis and bioinformatics, we demonstrate conclusively that maternal transmission distortion is caused by a large copy number gain of a 127 kb DNA segment containing a single gene, Cwc22. We also provide compelling evidence that meiotic drive is required to explain the TRD in the progeny of heterozygous dams. Finally, we show that there exist several genetically determined levels of TRD controlled by unlinked genetic variation, which, to our knowledge, is unique among meiotic drive systems.

It is rare for TRD at any single locus to be observed in multiple independent genetic backgrounds. An exception is TRD on mouse Chromosome (Chr) 2, which was reported first in interspecific backcrosses between C57BL/6J (a classical inbred strain, primarily of Mus musculus domesticus origin [ 24 – 28 ]) and SPRET/EiJ (a Mus spretus wild-derived inbred strain) [ 25 – 28 ]. In offspring from two different (C57BL/6JxSPRET/EiJ)xC57BL/6J backcrosses, the SPRET/EiJ allele was overrepresented across a 40 cM region on Chr 2 [ 25 , 28 ] and a ~140 Mb region on Chr 2 with a maximum transmission frequency of 0.66 [ 25 , 29 ]. TRD in Chr 2 was also reported in an F2 cross between two body weight selection lines, one of which (high body weight; M16i) was derived from the Hsd:ICR outbred stock (also known as CD-1) [ 29 , 30 ]. Additionally, in an advanced intercross between the Hsd:ICR-derived high-running selection line HR8 [ 30 – 32 ] and C57BL/6J, TRD in Chr 2 was present in the primary data but not reported in the corresponding manuscripts [ 31 – 35 ]. And recently, we reported TRD in Chr 2 in the Collaborative Cross (CC) [ 33 – 35 ]. The CC is a mouse recombinant inbred panel derived from eight genetically diverse inbred strains: the classical strains A/J, C57BL/6J, 129S1/SvImJ, NOD/ShiLtJ and NZO/HlLtJ and the wild-derived strains PWK/PhJ (M. m. musculus origin), CAST/EiJ (M. m. castaneus) and WSB/EiJ (M. m. domesticus) [ 35 ]. We reported TRD in favor of the WSB/EiJ allele across a ~50 Mb region in the middle of Chr 2 in three largely independent sets of CC lines. In the largest sample, involving 350 genetically independent CC lines, the WSB/EiJ allele was present on 22% of Chr 2 [ 35 , 36 ], a significant over-representation compared to the expected frequency of 12.5% (1/8).

In most plant and animal species meiotic drive is restricted to females, which undergo asymmetric meiosis. At the locus where TRD is observed, an allele that is subject to preferential segregation is termed a responder [ 19 – 21 ]. There are examples in many species of meiotic drive responder alleles that, when in heterozygosity, succeed in being transmitted to the functional product of the asymmetric meiosis more than half of the time ( S1 Fig. ). Responders in known meiotic drive systems typically involve multi-megabase, highly repetitive and heterochromatic sequences, such as the D locus in monkeyflower [ 11 , 21 ], knobs in maize [ 11 , 22 ], homogenously staining regions (HSRs) in wild mice [ 12 , 17 , 22 , 23 ] and centromeres and B chromosomes in multiple species [ 12 , 17 , 23 , 24 ]. Those systems have mostly proven intractable to molecular characterization, and thus the mechanism(s) by which they gain a segregation advantage are largely unknown. Meiotic drive may be promoted or suppressed by distorter loci (alternately referred to in some publications as effectors, modifiers or drivers).

Most observations of TRD are due to selection acting upon the products of meiosis (gamete selection) or fertilization (differential pre- or post-natal survival) [ 5 – 8 ]. The latter is a relatively common occurrence in experimental crosses in many types of organisms including plants and animals [ 8 , 9 ], and is routinely used to classify the essentiality of genes and alleles [ 9 – 14 ]. However, a small but increasing number of observations of TRD can be ascribed to the differential segregation of alleles during meiosis, a process called meiotic drive [ 1 , 10 – 14 ]. To qualify as such, meiotic drive systems must exhibit three characteristics: 1) asymmetry in the meiotic division(s) with respect to cell fate; 2) functional asymmetry of the meiotic spindle poles; and 3) functional heterozygosity at a locus that mediates attachment of a chromosome or a chromatid to the meiotic spindle [ 1 , 15 , 16 ]. Meiotic drive is an evolutionary force thought to contribute to karyotypic evolution [ 15 – 17 ] and maintenance of non-essential “B chromosomes” in multiple clades [ 17 , 18 ]. The incidence of meiotic drive is unknown, but given that it is a relatively strong evolutionary force that can lead to the rapid fixation of a selfish allele, it should be rare to observe in action [ 18 – 20 ].

Mendel’s Laws provide the theoretical foundation of transmission genetics and explain many of the inheritance patterns of biological traits in sexually reproducing organisms. The Laws state that each gamete receives a random collection of alleles—exactly one per pair of homologous loci—and that gametes unite at random. However, reports of exceptions to Mendelian inheritance date back almost to the rediscovery of Mendel’s Laws, and have been instrumental in elucidating the mechanisms of genetic inheritance [ 1 – 4 ]. Transmission ratio distortion (TRD) is defined as a significant and reproducible violation of the inheritance ratios expected under Mendel’s Laws [ 1 , 5 – 7 ].

Further support for meiotic drive was provided by the analysis of the DO-G13–44 pedigree ( Fig. 5 C ) and crosses between (NZO/HILtJxWSB/EiJ)F1 dams and FVB/NJ sires (cross 15 in Table 1 ; Fig. 5 D ). The average litter size of DO-G13–44 G3 dams inheriting the mutant R2d2 WSB allele (R2d2 WSBdel1 ) was larger than in dams inheriting the standard R2d2 WSB allele (9.4 ± 2.9 and 6.8 ± 1.6, respectively), but the observed average litter size in dams with TRD is significantly greater than predicted based on TR (p = 0.02; S7 Fig. ). Similarly, in the (NZO/HILtJxWSB/EiJ)F1 crosses the average litter size (7.7 ± 2.4; Fig. 5 D ) was comparable to DO-G13 and DO-G16 dams without TRD, and was greater than predicted based on TR (p = 0.09; Fig. 5 ). There was little direct evidence of embryonic lethality at mid-gestation (1.8 ± 1.6 and 0.4 ± 0.5 resorbed embryos, respectively; S8 Fig. ). Furthermore, DO-G13–44 G3 dams with different R2d2 alleles differed significantly in the average absolute number of offspring per litter inheriting the R2d2 WSB allele (in dams with TRD) compared to the R2d2 WSBdel1 allele (in dams with Mendelian segregation; 5.3 ± 2.0 and 4.64 ± 2.4, respectively, p = 0.07; Fig. 5 C ). Similar results are observed when comparing the absolute number of offspring per litter that inherited the R2d2 WSB allele in the (NZO/HILtJxWSB/EiJ)F1 crosses to the DO dams without TRD (5.1 ± 1.0 and 4.1 ± 1.1, respectively, p = 0.03; Fig. 5 D ). In summary, all data from four independent experimental populations were consistent with an explanation of Chr 2 TRD that requires the joint presence meiotic drive and low-level embryonic lethality.

Although embryonic lethality can change the proportion of progeny inheriting alternative alleles at R2d2, only meiotic drive can lead to an increase in the absolute number of progeny inheriting the R2d2 WSB allele per litter in dams with TRD compared to dams with Mendelian segregation. To test whether meiotic drive was responsible for TRD, we determined the average absolute number of offspring per litter that inherited the R2d2 WSB and R2d2 NotWSB alleles in the progenies of the DO-G13 and DO-G16 DO dams with either TRD or Mendelian segregation. In dams with Mendelian segregation, the average numbers of offspring per litter that inherited either allele were not different (3.80 R2d2 WSB versus 3.96 R2d2 NotWSB , p = 0.73 in DO-G13 dams; 4.13 R2d2 WSB versus 4.03 R2d2 NotWS , p = 0.29 in DO-G16 dams; Fig. 5 A, B ). In contrast, in the progenies of dams with TRD the average number of offspring per litter that inherited the R2d2 WSB allele (4.51 and 4.89 in the DO-G13 and DO-G16 dams, respectively) was significantly greater than the absolute number of either allele in the offspring of dams without distortion (p = 0.006 and 0.049 for the R2d2 WSB and R2d2 NotWSB alleles in DO-G13; p = 0.005 and 4x10 –4 for the R2d2 WSB and R2d2 NotWSB alleles in DO-G16; Fig. 5 A, B ). The same result holds true for live embryos at mid-gestation: the average numbers of offspring that inherited R2d2 WSB and R2d2 NotWSB alleles were 5.0 ± 2.2 and 1.6 ± 1.8 for dams with TRD versus 4.3 ± 1.6 and 3.4 ± 1.8 for dams without TRD. Based on the consistent and significant excess average absolute number of R2d2 WSB alleles in the litters of dams with TRD, we conclude again that meiotic drive is required to explain TRD at R2d2.

Relationship between maternal TR and average litter size (top panels) and average number of offspring inheriting alternative alleles at R2d2 (bottom panels) for A) DO G13 dams, B) DO G16 dams, C) G3 dams in the D0-G13–44 pedigree and D) (NZO/HILtJxWSB/EiJ)F1 dams. Top panels: gray circles are dams without TRD (A, B, D) or having the low-copy allele (C); blue circles are dams with TRD (A, B, D) or having the high-copy allele (C). For each point, bars show standard error for TR (horizontal) and average litter size (vertical). Dotted lines show mean litter sizes for each type of female. Red line shows a linear fit to TR and average litter size. Bottom panels: left and right pairs of boxplots show average number of offspring per litter in females without and with TRD (A, B, D) or having the low- and high-copy allele (C) that inherit a WSB/EiJ (purple) or non-WSB/EiJ (gray) allele. Females with a mutant R2d2 WSB allele are excluded. Note that there are significantly more WSB/EiJ offspring of dams with TRD in F1 hybrid dams than in DO without TRD.

The results presented above demonstrate that TRD at R2d2 is only observed in the progeny of heterozygous dams. This restricts the plausible causes of TRD to meiotic drive, genotype-dependent embryonic lethality (including genotype-dependent competition between embryos) or a combination of both. To identify the cause of TRD, we first determined whether TR levels ( S6 Fig. ; S1 Table ) were correlated with litter size in 127 DO dams (these 56 DO-G13 and 71 DO-G16 females are a random sample from an outbred population). We observed a strong inverse correlation between average litter size and TR at R2d2 (r = -0.65, p = 7.2x10 –8 and r = -0.40, p = 5x10 –4 in the DO-G13 and DO-G16 dams, respectively; Fig. 5 A, B ). We conclude that the presence and the strength of TRD are significantly associated with reduced litter sizes and thus with some type of embryonic lethality. We determined the relationship between TRD and litter size under the assumption of TRD caused exclusively by embryonic lethality [ 40 , 41 ] ( S7 Fig. ). Under this scenario, in both the DO-G13 and DO-G16 samples the observed average litter size is significantly greater than predicted based on TR (p = 0.021 and 6.0x10 –5 for DO-G13 and DO-G16 dams, respectively; S7 Fig. ). We conclude that embryonic death alone could only account for a fraction of the “missing” progeny inheriting a non-WSB/EiJ (R2d2 NotWSB ) allele. We determined directly the levels of embryonic lethality in DO-G13 dams at mid-gestation (see Materials and Methods ). We observed that dams with TRD had slightly, but not significantly, higher numbers of resorbed embryos present in utero than did dams with Mendelian segregation (1.3 ± 1.5 and 1.1 ± 1.2 resorbed embryos, respectively, p = 0.66; N = 29 and 19 dams, respectively; S8 Fig. ). We conclude that embryonic lethality alone is insufficient to explain TRD at R2d2.

We were also able to estimate that G3 dams with the low-copy allele had a copy number of ~11. We conclude that the loss of ~22 copies of R2d was sufficient to rescue Mendelian transmission, thus demonstrating that the copy number gain is causative of TRD.

A) Pedigree of DO-G13–44xCC cross. Female DO-G13–44, mother of the G3 dams phenotyped for TR, is segregating for a copy-number variant at R2d2. G3 dams inheriting the maternal WSB/EiJ haplotype associated with the high-copy allele (R2d2 WSB ) are colored black; those inheriting the WSB/EiJ haplotype associated with the low-copy allele (R2d2 WSBdel1 ) are colored red. Genotypes at marker chr2:85.65Mbp is denoted -/- (homozygous non-WSB), +/- (heterozygous WSB/EiJ) or +/+ (homozygous WSB/EiJ). ΔC t , normalized cycle threshold by TaqMan qPCR assay; TR, transmission ratio, denoted as count of progeny inheriting a WSB/EiJ allele: count of progeny not inheriting a WSB allele; the paternal haplotype at chr2:83.6 Mb as determined by genotypes from the MegaMUGA array using the standard CC abbreviations is shown, A = A/J, E = NZO/HILtJ, ? = haplotype unknown. B) Distribution of ΔC t values among 27 G3 dams. Points are colored as in panel A. C) TR among 27 G3 dams partitioned according to copy-number (CN) haplotype at R2d2. Points are colored as in panel A. D) QTL scan for TRD, treated as a binary phenotype, in 25 G3 dams genotyped with MegaMUGA. Only the maternal signal from Chr 2 is shown. Grey dashed line indicates threshold for significance at α = 0.01 obtained by unrestricted permutation. Candidate interval for R2d is shaded yellow. E) Empirical cumulative distribution of both maternal and paternal LOD scores genome-wide, with α = 0.01 significance threshold indicated by grey dashed line.

We used the TaqMan assay to confirm R2d copy number in all heterozygous females tested for TRD ( S1 Table ; S5 Fig. ). We identified a dam (DO-G13–44) that was homozygous for the WSB/EiJ haplotype across the entire candidate interval but produced offspring that were segregating for the copy number gain ( Fig. 4 A ). This was confirmed by estimating R2d copy number in each of 27 G3 females and 16 G4 progeny that were heterozygous for a WSB/EiJ haplotype ( Fig. 4 B ; S5 Fig. ). We determined the TR in 825 progeny of G3 dams mated to FVB/NJ sires. The TRs among the 27 G3 dams were significantly different (p = 4.9x10 –12 ). In the progeny of the 15 G3 dams with high copy number there was significant TRD in favor of the WSB/EiJ allele (TR = 0.78, p = 2x10 –30 ; Fig. 4 C ). In contrast, we found absence of TRD in the 12 G3 dams that inherited the low-copy allele (TR = 0.53, p = 0.234). A genome scan for TRD as a binary trait demonstrated that presence or absence of TRD in this pedigree maps uniquely to the candidate interval ( Fig. 4 D, E ).

Analysis of individual mice with recombinant chromosomes in the candidate interval revealed that the copy number gain maps to a 900 kb interval (the R2d2 locus; Chr 2 83,631,096–84,541,308; Fig. 2 ; Fig. 3 A, B ). Specifically, the CAST/EiJ copy number gain (R2d2 CAST ; one additional copy of R2d) is located distal to the transition from the CAST/EiJ to the NZO/HILtJ haplotypes found in mice OR3172m10 and OR3172f9 because both mice have low hybridization intensity consistent with a single copy, hence they lack R2d2 CAST ( Fig. 3 A ; S4A Fig. ). Similarly, the WSB/EiJ copy number gain (R2d2 WSB ; 33 additional copies of R2d) is located proximal to the transition from the WSB/EiJ to the CAST/EiJ haplotype found on DO mouse DP2–446, because it had high hybridization intensity consistent with the presence of R2d2 WSB ( Fig. 3 B ; S4B Fig. ). These results demonstrate that R2d2 is not located immediately adjacent to R2d1 but approximately 6 Mb distal to it. The distal location of the copy number gain is confirmed by the analysis of the sum intensity of the three MegaMUGA probes that track R2d in two backcrosses involving the SPRET/EiJ inbred strain [ 26 , 41 ] ( S4C Fig. ).

A) Distribution of sum-intensity for the 34 probes in R2d present on the Mouse Diversity Array (MDA) for mice with a non-recombinant CAST/EiJ haplotype (green), a non-recombinant WSB/EiJ haplotype (purple) and non-CAST/EiJ/non-WSB/EiJ haplotypes (grey) is shown at the top of the panel. The sum intensity and recombinant haplotypes in six mice defining the boundaries of copy-number gain in the CAST/EiJ strain are shown below. B) Distribution of sum-intensity across three probes in R2d on the MegaMUGA array for mice with non-recombinant CAST/EiJ haplotype (green), a non-recombinant WSB/EiJ haplotype (purple) and non-CAST/EiJ/non-WSB/EiJ haplotypes (grey) is shown at the top of the panel. The sum intensity and recombinant haplotypes in six mice defining the boundaries of copy-number gain in the WSB/EiJ strain are shown below. C) QTL scan for the R2d2 copy number gain using MDA sum-intensity as the phenotype in 330 CC G2:F 1 mice. D) QTL scan for the R2d2 copy number gain using MegaMUGA sum-intensity as the phenotype in 96 (FVB/NJx(WSB/EiJxPWK/PhJ)F1)G2 offspring. E) Superposition of LOD curves from panels (C) and (D) on chromosome 2. The R2d2 candidate interval is shaded in yellow.

Many structural variants identified from whole-genome sequencing reads have uncertain genomic positions due to the challenge of mapping large variants that are absent from the reference genome. To determine the position of the copy number gain associated with R2d, we mapped the WSB/EiJ and CAST/EiJ alleles using segregating populations that have been genotyped at medium (MegaMUGA) or high (Mouse Diversity Array, MDA) density [ 26 , 40 ]. In the CC founder strains, probes located in R2d have hybridization intensities correlated with the number of copies estimated from aligned read depth and TaqMan CNV assays ( Fig. 2 A, B ). The MDA provides robust discrimination between the reference (one copy), CAST/EiJ (two copies) and WSB/EiJ alleles (34 copies; Fig. 3 A ). MegaMUGA is able to identify mice carrying the WSB/EiJ allele with little ambiguity ( Fig. 3 B ). Using the sum intensities of the informative probes as a quantitative trait, we mapped the WSB/EiJ and CAST/EiJ copy number gains in two independent populations and platforms. A genome scan identified a single, broad, highly significant peak on Chr 2 in each population, and those peaks overlap with each other and with the initial candidate interval for TRD ( Fig. 3 C-E ). We conclude that the copy number gain is closely linked to R2d1. This location is consistent with the large copy number gain being the causative allele. Note that both genome scans ( Fig. 3 C, D ) demonstrate that all the extra R2d copies found in WSB/EiJ are located in this interval because no other significant peak is observed in either scan. QTL mapping using TaqMan readout as the phenotype confirmed this result ( Fig. 3 D, E ).

Using the TaqMan assay, we also found that the M16i inbred strain has a high number of copies of R2d ( Fig. 2 B ). We conclude that a large increase (> 20-fold) in R2d copy number is found exclusively in strains with TRD (WSB/EiJ, SPRET/EiJ, HR8 and M16i) and that TRD consistently favors the transmission of the allele with the copy number gain.

We used two additional methods to assay the copy number of R2d. First, we identified sets of probes on two different genotyping arrays for which the sum hybridization intensity was highly correlated with the copy numbers estimated from sequencing read depth (34 probes in MDA and 3 probes in MegaMUGA; S3 and S4 Tables, respectively). Second, we used real-time quantitative PCR to estimate the R2d copy number ( Fig. 2 B ) using TaqMan assays internal to exons of the single protein-coding gene within R2d, Cwc22 ( Fig. 2 C ). Using that gene as a proxy for the copy number gain, we found that the copy number estimates from all three methods were highly concordant for the 28 sequenced strains/individuals.

We used the normalized per-base read depth from whole-genome sequence alignments generated by the Sanger Mouse Genomes Project [ 31 , 32 , 37 , 39 ] and the HR8 selection line to estimate the number of copies of R2d in 18 inbred strains (see Materials and Methods ). Similar to C57BL/6J, 15 of the 18 strains, including 5 additional CC founder strains (A/J, 129S1/SvImJ, NOD/ShiLtJ, NZO/HlLtJ and PWK/PhJ) were copy number one (i.e., a single haploid copy), and CAST/EiJ was copy number two. In contrast, WSB/EiJ had an estimated copy number of 34, and SPRET/EiJ had an estimated copy number of 36, resulting in ~4.4 Mb of additional DNA in those strains ( Fig. 2 A ). We sequenced 10 individuals from the HR8 selection line (for which Chr 2 TRD was also observed when mated to C57BL/6J [ 31 , 32 , 40 ]) to a total depth of 125x and aligned the reads to the reference genome. All 10 individuals had evidence of a copy number gain with the same boundaries as in WSB/EiJ and SPRET/EiJ ( Fig. 2 A ; mean copy number 24.5 +/- 1.4, equating to ~3 Mb of additional DNA).

A) Read depth in 100 bp windows, normalized by the genome-wide mean read depth for each strain, for R2d1, a 158 kb region within the 9.3 Mb candidate interval defined in Fig. 1 . R2d1 includes a single (non-contiguous) copy of R2d. Strains are represented by the colors shown in the inset. Inbred strains included under the heading “classical” are A/J, 129S1/SvImJ, C57BL/6JN, NOD/ShiLtJ and NZO/HILtJ. The four large gaps represent LINEs that were inserted in the unique copy found in the reference genome after initial duplication. B) R2d copy number estimated by TaqMan assays for Cwc22. Normalized cycle threshold (ΔC t ; see Methods ) is proportional to absolute copy number on the log scale. Strains are colored as in panel A. The (M16ixL6)F2 samples shown are known to be homozygous for the M16i allele based on genotypes from the MegaMUGA array. C-F) The yellow boxes highlight the 158 kb region depicted in panel A (R2d1) and the 900 kb R2d2 locus mapping interval. Vertical dashed lines indicate the boundaries of the 9.3 Mb candidate interval. C) Locations of Ensembl genes in the NCBI/37 reference genome within the interval. The locations of the Cwc22 gene and of seven Cwc22 pseudogenes (Gm13695), are shown. D) Recombination frequency based on Liu et al. (2014), normalized by physical distance (Mb) and log10-transformed. The red line indicates the mean recombination frequency for Chr 2. E) Frequency distribution measured in 1 kb windows of SNPs with shared alleles among the three strains with TRD (WSB/EiJ, SPRET/EiJ and HR8; gray line), and with alleles perfectly consistent between strains with TRD and strains without TRD (A/J, 129S1/SvImJ, C57BL/6JN, NOD/ShiLtJ, NZO/HILt, CAST/EiJ and PWK/PhJ; red line). Lines are smoothed. Black circles indicate windows in which the strains with TRD share an allele for at least 90% of SNPs. F) Frequency distribution of reported SNPs in the candidate interval. G) The location and the number of copies of R2d that are present in R2d1 and R2d2.

Among the eight CC founder strains, the candidate interval has 5,018 SNPs, 1,286 small insertions/deletions and 35 structural variants that are private to the WSB/EiJ strain [ 37 – 39 ]. Although this very large number of variants would typically make it difficult to confidently identify and prioritize candidates, one large structural variant has several unique features that made it a strong candidate causative allele for the TRD phenotype. That structural variant is a copy number gain of a 127 kb-long genomic DNA segment (herein referred as R2d for responder to drive). In the reference genome, R2d is composed of nine non-contiguous sections that, in total, span 158 kb (see R2d1 locus; Chr 2 77,707,014–77,865,265; Fig. 2 A ; S2 Table ).

We found that dams carrying eight of the ten recombinant chromosomes exhibited significant TRD in the Chr 2 interval (TR range 0.69–1.0, p ≤ 2.1x10 –5 ; Fig. 1 A ), but dams carrying two other recombinant chromosomes did not (TR = 0.48 and 0.37, p ≥ 0.72; Fig. 1 B ). These results are consistent with our conclusion that heterozygosity on Chr 2 is required but not sufficient for TRD; therefore, dams with Mendelian transmission ratios were not used for mapping the locus subject to TRD. Dams with TRD in favor of the WSB/EiJ allele were all heterozygous for a 9.3 Mb interval (the candidate interval; boxed in Fig. 1 A ). The proximal boundary of the candidate interval is defined by the recombination found in the CC strain CC039/Unc (i.e., the most distal SNP inconsistent with a WSB/EiJ haplotype). The distal boundary of the candidate interval is defined by the recombination found in DO-732 and DO-832 females (i.e., the most proximal SNP inconsistent with a WSB/EiJ haplotype). Those SNPs define the maximum boundaries of the locus subject to TRD, Chr 2 76,860,362–86,117,205 (all positions from NCBI/37 unless otherwise noted).

CC and DO mice were crossed to generate G1 dams, which were then crossed to FVB/NJ sires to determine the TR in their progeny. Each G1 dam carries a chromosome that is recombinant for the WSB/EiJ haplotype (shown under the heading cis) and a non-WSB/EiJ chromosome (the haplotype on the homologue is shown at far right under the heading trans). Dams with the same diplotype in the central region of Chr 2 were grouped together to define ten unique diplotypes. The aggregate number of WSB/EiJ and non-WSB/EiJ alleles transmitted by dams of each diplotype are shown for dams A) with TRD and B) without TRD. Significance of TR deviation from Mendelian expectation of 0.5 was computed using one-sided binomial exact test (p-value). The contribution from the eight founders of the CC and DO are shown in different colors. Thick purple bars indicate the extent of WSB/EiJ contributions, and thin bars indicate the extent of contributions from all other strains. The black box indicates the boundaries of the R2d candidate interval as determined by the region that is WSB/EiJ in all dams with TRD.

To define the boundaries of the locus subject to TRD, we screened 61 CC lines and 378 DO mice that had been genotyped with MegaMUGA for recombinations involving the WSB/EiJ haplotype in the 75–90 Mb interval of Chr 2. We identified five DO females (DO-600, DO-681, DO-732, DO-832 and DO-OCA45) and two CC strains (CC039/Unc and CC042/GeniUnc) that each had at least one informative recombination ( Fig. 1 ). Next, we mated four of the DO females (all except DO-OCA45 that was already heterozygous) and the two CC strains to one of two additional CC lines (CC001/Unc and CC005/TauUnc) that had no contribution from WSB/EiJ on Chr 2, to obtain heterozygous G1 hybrid females. Each hybrid female was genotyped with MegaMUGA and mated to FVB/NJ males (total of 35 crosses; S1 Table ).

We also conclude that the grandparental origin of the WSB/EiJ allele has no influence on TRD because the TR levels were not significantly different between three pairs of reciprocal F1 dams (compare crosses 7 and 8, 9 and 10 and 17 and 18 in Table 1 ; p = 0.53, 0.11 and 0.59, respectively).

The TRs among maternally segregating crosses were significantly different (p = 2.4x10 –90 ), demonstrating that TRD depends on genetic background (i.e., TRD is under genetic control). The 12 crosses using F1 hybrid dams can be divided into three classes based on the observed TR ( S3 Fig. ). F1 hybrid dams derived from crosses between WSB/EiJ and CAST/EiJ or PWD/PhJ showed no distortion (crosses 7–10 in Table 1 ; aggregate TR = 0.485, 95% CI = 0.46–0.51, p = 0.23). Moderate but significant distortion was present in F1 hybrid dams derived from crosses between WSB/EiJ and A/J, 129S1/SvImJ, NZO/HILtJ or NOD/ShiLtJ; and in (CC042/GeniUncxCC001/Unc)F1 hybrid dams (crosses 11–15 in Table 1 ; aggregate TR = 0.645, 95% CI = 0.61–0.68, p = 8.3x10 –19 ). Finally, extreme distortion was observed in reciprocal (WSB/EiJxC57BL/6J)F1 hybrid dams and in (CC001/UncxCC039/Unc)F1 hybrid dams (crosses 16–18 in Table 1 ; aggregate TR = 0.943, 95% CI = 0.93–0.96, p = 9.6x10 –193 ). We conclude that heterozygosity for the WSB/EiJ allele in the central region of Chr 2 is necessary but not sufficient to observe TRD, because TR was consistent with Mendelian inheritance in some dams that met that criterion.

TRs in six paternally segregating crosses (rows 1–6 in Table 1 ) were as expected under the null hypothesis of Mendelian segregation (range 0.482–0.524, p ≥ 0.37). In contrast, the mean TR in maternally segregating crosses (rows 7–18 in Table 1 ) was 0.666 and deviated significantly from the null hypothesis (p = 3.4x10 –89 ). We conclude that, in the genetic backgrounds tested, TRD in favor of the WSB/EiJ allele on Chr 2 is restricted to the progeny of heterozygous dams.

To determine the parental origin of the TRD, we analyzed 5,499 offspring from 18 experimental crosses in which exactly one parent was heterozygous for the WSB/EiJ allele in an interval spanning the region of maximum distortion on Chr 2 (75–90 Mb) [ 33 – 35 , 37 , 38 ]. In all cases the heterozygous parent was an F1 hybrid derived either from an intercross between the WSB/EiJ inbred strain and one of eight other inbred strains (the seven founder strains of the CC or PWD/PhJ), or from two CC strains, of which one was homozygous for the WSB/EiJ allele on Chr 2 and the other was homozygous for a non-WSB/EiJ allele. F1 hybrids were mated to either C57BL/6J or FVB/NJ mice, and their progenies were euthanized at birth and genotyped using genetic markers located in the region of maximum distortion. For each cross, we computed the TR of the WSB/EiJ allele and the non-WSB/EiJ allele using the aggregate genotypes across all litters from parents with identical genotypes ( Table 1 ).

To test whether TRD of the WSB/EiJ allele in Chr 2 is present in the DO, we analyzed 1,175 animals from DO generation 8 (G8) that were genotyped using two related genotyping arrays (MUGA or MegaMUGA, see Materials and Methods ). We sampled the genotypes of each individual at 1 Mb intervals along Chr 2 and then computed the overall frequencies of the eight founder alleles at each position. The WSB/EiJ allele was over-represented relative to the other seven founder alleles across a roughly 100 Mb region in the middle of Chr 2 ( S2 Fig. ). However, there was a striking difference in the level of distortion observed in the CC and the DO, with the WSB/EiJ allele frequency reaching a maximum of 0.22 in the CC compared to 0.55 in the DO. This result indicates that the additional outcrossing in the DO is associated with higher levels of TRD. We conclude that TRD favoring the WSB/EiJ allele is a general feature of crosses in the CC genetic background; however, the level of TRD may vary widely depending on the number of generations of outbreeding.

Discussion

A large copy number variant causes maternal TRD and reduces the average litter size in heterozygous dams After demonstrating that TRD occurs only through the germline of F1 female mice, we were faced with two major obstacles in our efforts to map the causative locus. First, although heterozygosity for the WSB/EiJ allele is required, it is not sufficient for meiotic drive (Table 1; S1 Table). Therefore, we initially mapped the responder by determining the minimum region of overlap for the WSB/EiJ haplotype only in dams with TRD (Fig. 1). This yielded a 9.3 Mb candidate interval. Second, the candidate interval spans a recombination-cold region [37,40,42], and the frequency of recombination is three-fold lower than expected in the CC (Fig. 2 D). Although this likely contributes to the overall deficit in recombinant chromosomes (none observed versus an expected 23 in the 378 DO females and 4 in 61 CC lines), the complete lack of recombinants involving the WSB/EiJ haplotype is striking, and, for the purposes of this study, a major impediment to the precise mapping the responder. Within the candidate interval, a single variant (R2d2) stands out as the most likely cause of TRD. R2d2 consists of one or more copies of a 127 kb sequence (R2d). High copy number (≥ 24) is present in all four strains with reported TRD and low copy number (≤ 2) is present in all eight strains without TRD (Fig. 2 A, C). The expansion in copy number leads to an increase of at least 3 Mb in DNA content within the allele favored by maternal TRD. Among CC founders, only WSB/EiJ has a high copy number allele. As the reference genome is based on a single classical inbred strain, C57BL/6J, copy number gains in other strains or wild mice may be located in a different physical location. Fortunately, the presence of a third allele in CAST/EiJ (which exhibited a twofold enrichment of sequencing reads) combined with the fact that recombinations involving the CAST/EiJ haplotype are not suppressed within the 9.3 Mb candidate interval, enabled us to map the physical location of R2d2 to a 900 kb region located 6 Mb distal to R2d1, the locus where the sequencing reads mapped in the reference genome (Fig. 3). Importantly, the mapping of R2d2 was enabled by the availability of deep sequence data for each of the strains used in our experiments [25–27,37,42,43 and this study] and by combining the results of experiments completed 20 years apart [25–27,43,44]. We determined the number and spatial distribution of SNPs in the 9.3 Mb candidate interval that partition the ten inbred strains with whole genome sequence in a pattern consistent with the TRD phenotype (three strains with TRD: WSB/EiJ, SPRET/EiJ and HR8; and seven strains without TRD: A/J, C57BL6/J, 129S1/SvImJ, NOD/ShiLtJ, NZO/HILtJ, CAST/EiJ and PWK/PhJ). Compared to a genome-wide mean of 1 consistent SNP every ~3.2 kb, within the 900 kb region where we mapped R2d2 there was a mean of 1 consistent SNP every 883 bp (p < 1.0x10–4, one-sided Student’s t-test; Fig. 2 E). This reduction in diversity is not due to undercalling of SNPs in the R2d2 candidate interval (Fig. 2 F). The fact that consistent SNPs are rare in most of the genome but are common within the 900 Kb region in which R2d2 maps supports the hypothesis that R2d2 is the causative allele for TRD. Most importantly, we identified a DO female (DO-G13–44) that was homozygous for the WSB/EiJ haplotype across the entire R2d candidate interval but was heterozygous for R2d2 alleles with different copy numbers (Fig. 4). We generated a three-generation pedigree and analyzed the R2d copy number, the Chr 2 haplotype and TR in the progeny of heterozygous dams with different copy numbers. This analysis revealed perfect correlations between the inheritance of R2d2WSBdel1 and complete absence of TRD in favor of the WSB/EiJ allele, and between the inheritance of R2d2WSB and presence of TRD. This experiment demonstrates that the reduction in copy number from 33 to 11 is sufficient to restore Mendelian segregation, and that R2d2 is the causative allele for maternal TRD. Further evidence that TRD requires an R2d2 allele with copy number of above 11 is provided by the NU/J inbred strain. This strain has intermediate copy number (7, estimated by TaqMan) but no TRD in the progeny of (NU/JxC57BL/6J)F1 female hybrids (0.55, p = 0.55; S12 Fig.).

Gene content and sequence composition of R2d The presence of R2d sequences at two distinct locations (Fig. 2 G) indicates an initial duplication of this segment in the ancestor of CAST/Eij, WSB/EiJ, SPRET/EiJ and Hsd:ICR. R2d spans a highly expressed protein coding gene (Cwc22; Fig. 2 C) that is implicated in RNA splicing [38,44], a predicted gene of unknown function that overlaps with the last exon of Cwc22 (Gm13727) and a pseudogene (Gm13726). DNA copy number variation for Cwc22 has been described previously [38,45]. Cwc22 is highly expressed in mouse oocytes and fertilized eggs [45,46]. The Cwc22 gene is a known eQTL in mouse: allele-specific RNA-seq of brain tissue from reciprocal crosses between WSB/EiJ, PWK/PhJ and CAST/EiJ showed extreme differential expression, with the WSB/EiJ allele more highly expressed than the other two [46,47]. Apart from its size and repetitive nature, an important feature of the R2d2 locus is its remarkable uniformity between three divergent genetic backgrounds that are separated by ~1 million years of evolution: WSB/EiJ, SPRET/EiJ and HR8 [47–49]. For example in WSB/EiJ and SPRET/EiJ the genome-wide mean is 1 SNP every ~60 bp [37] and the mean SNP frequency within R2d is significantly reduced to 1 SNP every 1,342 bp (t-test, p = 3.9x10–58). Further analysis will be required to determine the respective ages of the duplication and the copy number change(s), and whether interspecific introgression [48–51] is required to explain the unlikely degree of sequence conservation between M. m. domesticus and M. spretus. We note that, while unlikely given the results of our QTL mapping (Fig. 3), it is possible that there have been additional duplication events that have also inserted R2d in other chromosomes. Additionally, the causal allele may incorporate additional DNA sequences, including some that may be absent in the reference genome (similar to the origin of the sequence on maize chromosome Ab10 that causes meiotic drive in that species). If that is the case, the causal allele may be much larger than 4.4 Mb. For example, HSR alleles as large as 200 Mb have been described [50–52].

How do meiotic drive and embryonic lethality contribute to TRD at R2d? A second focus of our study was to discriminate among the many mechanisms [29,52] that could give rise to TRD at R2d2, and to rule out as many as possible. First, the fact that TRD is only observed through the maternal germline rules out both spermatogenesis-mediated processes and sperm competition. Second, the presence of TRD at birth rules out differential survival of offspring. Third, the fact that distortion was independent of the maternal granddam precludes cytoplasmic effects. The remaining plausible explanations are differential fertilization based on the oocyte genotype, embryonic lethality and/or meiotic drive. The first two mechanisms should reduce the average litter size proportionally to TR (black line in S7 Fig.), while the average absolute number of offspring inheriting the favored genotype (R2d2WSB) per litter remains constant. The number of resorbed embryos observed in pregnant females could distinguish the two mechanisms because it should be greater in the second than in the first scenario. In contrast, if meiotic drive is solely responsible for TRD then the following should be true: 1) average litter size is independent of TRD, 2) the average absolute number of offspring inheriting the favored genotype (R2d2WSB) per litter is higher in dams with TRD than in dams with Mendelian segregation, and 3) the level of embryonic lethality is independent of the presence and level of distortion. The data shown in the Results section are most consistent with the combined action of embryonic lethality and meiotic drive. Specifically, meiotic drive is required to explain both the fact that the observed average litter size in the DO-G13 and DO-G16 dams, in the DO-G13–44 pedigree and in the (NZO/HILtJxWSB/EiJ)F1 dams is greater than predicted based on TR (S7 Fig.), and that the average absolute number of offspring inheriting the R2d2WSB genotype per litter is greater in dams with TRD (Fig. 5). Note that some p-values in comparisons involving (NZO/HILtJxWSB/EiJ)F1 crosses failed to reach statistical significance due to the small sample size, but the trends were always consistent with those in DO dams with TRD. An alternative explanation that does not involve meiotic drive would require the combined presence of increased ovulation in dams with TRD and pre- or post-implantation genotype-dependent competition between embryos favoring the allele with the high copy number at R2d2. Genotyping at R2d2 and re-analysis of 159 F2 females from the M16ixL6 intercross [29] confirms an overdominant effect of the R2d2 genotype in the number of live and dead embryos at day 16 of gestation, as predicted under the meiotic drive and embryo competition scenarios, but shows no effect of the R2d2 locus on ovulation rates (S9 Fig.). This result is not due to a lack of power, as we have 80% power (at a = 0.5) to detect a difference in the mean ovulation rate du to an effect of the R2d2 genotype and QTLs for ovulation rate were identified in the original study [29,53–55]. In summary, the effect of the R2d2 genotype on reproductive phenotypes is most consistent with the meiotic drive hypothesis. However, the possibility remains that the genotype-associated difference in number of live embryos may be due to differential fertilization or implantation. Additional breeding experiments and genotyping of pre-implantation embryos will resolve the remaining questions concerning the mechanisms involved in TRD at R2d2. It is interesting to speculate about the types of embryonic lethality that are consistent with our data and with previous reports of TRD on Chr 2. Lethality is associated with distortion at R2d2, and thus the simplest explanation is preferential death of embryos inheriting maternal R2d2NotWSB alleles. However, such a scenario would require parent-of-origin-dependent death of embryos with maternal C57BL/6J, 129S1/SvImJ, NOD/ShiLtJ and NZO/HILtJ R2d2 alleles in crosses involving F1 females (Table 1) and CAST/EiJ, PWK/PhJ and A/J R2d2 alleles in the CC/DO females (S10 Fig.). The lack of evidence of TRD and parent-of-origin lethality in dozens of crosses involving these alleles [53–55], combined with the lack of evidence for imprinted genes in the central region of Chr 2 [2–4,46,56], appears to rule out this explanation. Specifically, the Cwc22 gene present in R2d is not imprinted in brain, kidney, lung and liver in crosses involving the WSB/EiJ, PWK/PhJ and CAST/EiJ strains [46]. A more likely explanation for the joint and correlated presence of meiotic drive and lethality is that the unequal segregation of chromosomes and/or chromatids that leads to TRD in euploid embryos may also lead to increased Chr 2 aneuploidy, and thus to embryonic death (all autosomal aneuploidy is embryonic-lethal in the mouse). This would also explain the slight increase in the number of resorbed embryos observed at mid-gestation (S8 Fig.; S1 Table). This hypothesis makes the testable prediction that Chr 2 should be especially affected by aneuploidy in some dams with TRD. Importantly, co-segregation of a deletion allele of R2d2 and increased litter size in the DO-G13–44 pedigree demonstrates that lethality is mediated by an element within the R2d repeat.

Maternal TRD at R2d is an oligogenic trait Overall, we assessed TR at R2d2 in hundreds of females carrying a single WSB/EiJ allele in at least nine distinct genetic backgrounds (Table 1; S1 Table). The presence of significantly different TR levels among F1 hybrid dams, combined with the fact that we observe both extreme TRD and no distortion in the progeny of females with A/J, C57BL/6J, 129S1/SvImJ, NOD/ShiLtJ, CAST/EiJ and PWK/PhJ alleles in trans at R2d2 (S10 Fig.), demonstrates that TRD is under genetic control of at least one additional locus (i.e., there is at least one unlinked distorter locus that is genetically variable in the CC and DO mice). Furthermore, the presence of at least two significantly different levels of distortion among F1 hybrid dams (Table 1; S3 Fig.) indicates either that more than one distorter locus is involved or that an allelic series exists at a single distorter locus. Further evidence that TRD is under control of one or more unlinked distorters was provided by 15 female DO-G13–44 G1 offspring that inherited the high-copy allele. Those dams had significantly different levels of TRD (p = 9.8x10–5). Note that there was no correlation between the presence or level of TRD and the paternally inherited allele (one-way ANOVA, F = 2.21 on 1 and 23 df, p = 0.15; Fig. 3). In the DO-G13–44 pedigree, females that inherited the R2d2WSBdel1 allele had copy number 11 (S11 Fig.), indicating a partial rather than complete deletion of the expansion. Using the TaqMan assay, we identified two additional DO females (DO-G13–49 and DO-G16–107; S4 Fig.; S11 Fig.) that had results consistent with a copy number loss in the WSB/EiJ haplotype. The presence of the deletion in the respective germlines was confirmed by the TaqMan assay in their progenies (S4 Fig.). Importantly, each one of the three deletions appears to be independent because these females are not closely related, their WSB/EiJ haplotypes in Chr 2 are different and the copy number present in each female is also different (S12 Fig.). The deletions appear to be internal to R2d2 based on the analysis of the MegaMUGA genotypes and intensities [57] at all surrounding markers. The repeated observation of independent deletions indicates that R2d2 is rather unstable and may explain the fact that, despite its presence in laboratory strains and wild mice, it has not led (yet) to a complete selective sweep. Known meiotic drive systems (S1 Fig.) consist of one or more responder loci (a locus subject to preferential segregation during meiosis) and a single distorter (the effector locus required for drive at the responder). In meiotic drive systems that are stable in natural populations, responder and distorter loci are tightly linked and are typically protected from decoupling by factors that inhibit recombination, such as structural variation [7,11,14]. Although R2d2 resides within a recombination-cold region, the distorter is not closely linked to R2d2 based on the TR observed and the diplotypes present in F1 hybrid and DO dams (Fig. 1; S8 Fig.). Therefore, at least one unlinked distorter is required to explain the observed variability in TRD. These observations indicate that the maternal TRD phenotype has a complex genetic architecture. Specifically, a minimum number of copies of R2d are required in heterozygosity at R2d2 for TRD to be observed. Therefore, it can be classified as overdominant, restricted to the female germline and caused by structural variation. Similar characteristics have been recently reported for the Xce locus that controls X-inactivation choice; notably, characterization of Xce relied on the analysis of a genetically diverse set of F1 hybrid mice [58]. In addition, multiple alleles at unlinked loci interact to determine whether distortion occurs at R2d2, and to what extent. This is unique among meiotic drive systems (S1 Fig.) and has important implications for the natural history of the system and for the ease of genetic dissection. We hypothesize that variation in TR levels at R2d2 results from the interaction of alleles originating from multiple taxa, and thus the use of inter-specifc and inter-subspecific mouse populations was key to the characterization of this system. Wild-derived strains and wild-caught mice have enabled important biological discoveries [4,59], and we echo previous encouragements of a more prominent role for these resources in biological and biomedical research [60,61].

What is the mechanism by which R2d2WSB influences its own segregation? Centromeres (i.e., the site of kinetochore formation) are remarkable loci that control, in cis, proper segregation of chromosomes during mitosis and meiosis. It is easy to envision how a responder at, or tightly linked to, a centromere can influence chromosome segregation. Recent evidence shows that kinetochore protein levels and microtubule binding are positively correlated with preferential segregation to the oocyte in mice that are heterozygous for Robertsonian fusions [62], indicating that differences in centromere “strength” lead to meiotic drive. Responders located far away from centromeres are thought to influence their own segregation in cis by becoming “neocentromeres” and taking advantage of the inherited functional polarity of the female meiotic spindle [63]. We hypothesize that R2d2 may act as a neocentromere after epigenetic activation mediated by C57BL/6J, NZO/ShiLtJ, 129S1/SvImJ, and NOD/HILtJ alleles at the distorter(s). The discovery of multiple R2d2 alleles with different copy numbers demonstrates that the presence of the distal insertion of R2d is not sufficient for meiotic drive; rather, some minimum copy number (> 11) is required for TRD. This raises the possibility that meiotic drive at R2d2 is dosage-dependent, such that fine-scale control over the level of TRD is possible by adjusting the number of copies of R2d. If R2d2 is acting as a neocentromere, this may also indicate that some minimum size and/or number of repeats is required for recognition and activation by the epigenetic machinery. The Ab10 system of maize provides examples of responders that function as neocentromeres and for which the level of meiotic drive depends on the size of the responder (i.e., knob size) [11]. The effect on the Chr 2 centromere of activating an ectopic neocentromere at R2d2 is unknown, but it might explain the moderate levels of lethality caused by aneuploidy and suggests that some coordination between the two loci is required to achieve chromosome segregation. Meiosis involving chromosomes with neocentromeres may lead to an increased rate of non-disjunction and a reduced rate of recombination.

Implications of R2d2 for the CC and DO The conclusion that a genetically complex meiotic drive system is responsible for TRD favoring the WSB/EiJ allele at R2d2 is fully consistent with the initial observations of TRD in the CC, with our prediction that positive selection of the WSB/EiJ allele occurred during outcrossing or in early inbreeding generations [35], with the presence of similar levels of TRD in extinct and extant CC lines at intermediate generations of the CC (S5 Table) and with the fact that C57BL/6J, 129S1/SvImJ, NOD/ShiLtJ and NZO/HILtJ haplotypes at R2d2 are not underrepresented among the currently completed CC strains (http://csbio.unc.edu/CCstatus/index.py). The observed levels of TRD in crosses that use DO females are consistent with presence of different alleles at the distorter(s) (S7 Fig.; S1 Table). Although the discovery and identification of TRD that emerged from the DO pseudo-randomized mating scheme offered the opportunity to characterize a novel meiotic drive responder, the existence of such a locus could negatively impact the utility of this population for genetic studies. Fortunately, the locus was discovered before complete fixation of the R2d2WSB allele. Although the candidate interval spans 900 kb, TRD affects a much larger region in the DO because the strength of selection in favor of the WSB/EiJ allele is outpacing the rate at which recombination can degrade linkage disequilibrium in the region. Ultimately, this region would become an actual or statistical ‘blind-spot’ in the DO, such that the non-WSB/EiJ allele frequencies would become too small to detect allelic effects on phenotypic variation. Efforts are underway to purge the WSB/EiJ allele from the DO breeding population at this locus or to select for mice carrying a WSB/EiJ haplotype with a low copy number for R2d2, rather than allow the region to become fixed. Using marker-assisted selection, progeny of heterozygous WSB/EiJ carrier crosses are excluded from subsequent generations. Allele frequencies and random segregation on all other chromosomes are being preserved (EJC unpublished).