For the first time in the domestic pig, meiotic recombination along the 18 porcine autosomes was directly studied by immunolocalization of MLH1 protein. In total, 7,848 synaptonemal complexes from 436 spermatocytes were analyzed, and 13,969 recombination sites were mapped. Individual chromosomes for 113 of the 436 cells (representing 2,034 synaptonemal complexes) were identified by immunostaining and fluorescence in situ hybridization (FISH). The average total length of autosomal synaptonemal complexes per cell was 190.3 µm, with 32.0 recombination sites (crossovers), on average, per cell. The number of crossovers and the lengths of the autosomal synaptonemal complexes showed significant intra- (i.e. between cells) and inter-individual variations. The distributions of recombination sites within each chromosomal category were similar: crossovers in metacentric and submetacentric chromosomes were concentrated in the telomeric regions of the p- and q-arms, whereas two hotspots were located near the centromere and in the telomeric region of acrocentrics. Lack of MLH1 foci was mainly observed in the smaller chromosomes, particularly chromosome 18 (SSC18) and the sex chromosomes. All autosomes displayed positive interference, with a large variability between the chromosomes.
Citation: Mary N, Barasc H, Ferchaud S, Billon Y, Meslier F, Robelin D, et al. (2014) Meiotic Recombination Analyses of Individual Chromosomes in Male Domestic Pigs (Sus scrofa domestica). PLoS ONE 9(6): e99123. https://doi.org/10.1371/journal.pone.0099123
Editor: Qinghua Shi, University of Science and Technology of China, China
Received: March 19, 2014; Accepted: May 9, 2014; Published: June 11, 2014
Copyright: © 2014 Mary et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All data are included within the manuscript.
Funding: The authors have no support or funding to report.
Competing interests: The authors have declared that no competing interests exist.
During meiosis, recombination between homologous chromosomes generates two kinds of recombination products: crossovers (CO) and non-crossovers (NCO). NCO result in the unidirectional transfer of short genomic segments (gene conversion) and therefore have a limited impact on genetic diversity. Conversely, CO result in the reciprocal exchange of large chromosome segments between homologues and play a major role in the genetic variability of populations. CO are also necessary for the correct segregation of chromosomes during meiosis-I . Lack of CO can result in chromosomal non-disjunction, leading to the production of aneuploid gametes . In the most severe cases, low levels of CO can be associated with impaired spermatogenesis , .
Recombination sites are not distributed homogeneously along the chromosomes. Indeed, two COs very rarely occur near to one another. This phenomenon, known since 1916, has been termed “interference”  and different models have been proposed to explain it , . Moreover some chromosomal regions, known as recombination hotspots , are preferentially affected by recombination. In Humans, 23,000 crossover hotspots, 1–2 kb in length and spaced approximately every 50–100 kb, have been identified –. They exhibit different recombination activities and are located in genic as well as in intergenic regions. Recently, the PR domain zinc finger protein 9 (PRDM9) has been shown to play a major role in the specification of such recombination hotspots in mice, humans and pigs , . PRDM9 encodes a histone methyl transferase that allows trimethylation of the H3K4 histone. Active hot spots in mice are enriched for H3K4me3 . Moreover, the DNA sequence matching the predicted PRDM9 binding site is present in 40% of the hot spots identified by linkage disequilibrium .
Historically, meiotic recombination studies have relied on the physical localization of chiasmatas , or on linkage analysis . The discovery of proteins involved in CO formation (especially MLH1 and MLH3 observed in late recombination nodules) allowed the direct study of recombination using immunocytological approaches . Such approaches have also been used to analyse CO interference, for example by fitting the distribution of inter-CO distances to the gamma model . Until now, immunocytological techniques have been used to study recombination patterns in various mammalian species such as mouse , Man , cattle , cat , shrew , mink , and dog , as well as 3 species of primates , but not in pigs.
The domestic pig karyotype (2n = 38) is composed of 2 sex chromosomes and 18 pairs of autosomes (5 metacentrics, 7 submetacentrics and 6 acrocentrics) . This karyotypic structure is relatively similar to that of Humans. For this reason, the pig species is a much better animal model for meiotic recombination studies than other mammalian species, like dogs or mice, whose karyotypes contain only acrocentric chromosomes.
In this paper, for the first time in the domestic pig, we present unique data on the meiotic recombination of males with normal karyotypes, obtained by direct immunocytological approach. Distribution of the MLH1 foci and the estimated strength of interference on the 18 porcine autosomal pairs were determined by identifying each autosome by multiple specific loci in situ hybridization.
Materials and Methods
Our experiments were conducted in accordance with the European Directive 2010/63/EU on the protection of animals used for scientific purposes, and validated by the Ethic Committee for Animal Experimentation of the Poitou Charentes region (France) (N°CE2012-2). The experimentation agreement number for the experimental farm in which the animals were raised was A-17-661.
Testicular samples were collected by surgical hemi-castration of 4 boars of different genotype (Large White, Meishan, minipig and crossbred between Large White and Creole) and age (195, 360, 695 and 307 days, respectively). Classical cytogenetic analyses (GTG banding karyotyping) were carried out according to Ducos et al. (1998) . Histopathological analyses were carried out as described by Barasc et al. (2014) . Analyses of the boar karyotypes and gonads did not reveal any alteration. Moreover, the seminal parameters of the boars (concentration, mobility, and morphological parameters) were within the normal limits.
Meiotic cells were prepared as previously described by Massip et al. (2010) . The synaptonemal complex proteins 3 (SCP3) and 1 (SCP1), MutL homolog 1 protein (MLH1) and centromeres were detected using the following primary antibodies: rabbit anti-SCP3 (1∶1000; ABCAM, Cambridge, UK), rabbit anti-SCP1 (2∶1000; ABCAM, Cambridge, UK), mouse anti-MLH1 (2∶100; Becton Dickinson, Francklin Lakes, NJ), Human anti-kinetochore (1∶100; Antibodysuits Incorporated, Davis), respectively, and prepared in a solution of PBT (PBS +0.16% BSA +0.1% Tween). The secondary antibodies consisted of Alexa 594 conjugated donkey anti-rabbit (1∶100, Molecular Probes), Alexa 488 conjugated goat anti-mouse (1∶100, Molecular Probes), and AMCA conjugated donkey anti-human (1∶100, Jackson, Grove, PA, USA). Spermatocytes were captured and analyzed using the Cytovision FISH imaging system (Leica Microsystems, Nanterre, France).
Fluorescent In Situ Hybridization (FISH)
FISH experiments were performed on the same slides, according to Massip et al. (2010) , with slight modifications. The 18 autosomal bivalents were identified by 3 successive hybridizations of BAC probe combinations, as presented in Figure 1. These BAC probes were selected in the telomeric regions of the chromosomes  and were obtained from the Biological Resources Center-GADIE (http://www-crb.jouy.inra.fr/) . Probes were labeled with biotin or digoxygenin using the bioprime labeling system (Invitrogen) and revealed using Alexa 594 conjugated to Streptavidin (Molecular Probes, Eugene, OR, USA) and FITC conjugated mouse anti-digoxygenin antibodies (Sigma, St Louis, MO).
1st column: set of 7 BACs used for each hybridization with their positions and labeling nucleotide. 2nd column: raw image of the capture of the 4 BACs labeled with biotin and revealed in red. 3rd column: raw image of the capture of the 3 BACs labeled with digoxigenin and revealed in green. 4th column: identification of chromosome arms on spermatocyte after immunolocalization of SCP3 (red), MLH1 (green) and kinetochores (blue).
Image and Statistical Analyses
After identification of each bivalent, the images were analyzed to determine the length of the synaptonemal complexes (SC), and the positions of the centromere and CO, using MicroMeasure 3.3 software . The relative positions of the MLH1 signals and centromeres were expressed as percentages of the total SC lengths, starting from the end of the q arms. The distances between adjacent MLH1 foci (relative distances expressed as % of total SC length) were used to estimate the interference for each chromosome. The strength of interference was estimated by fitting the frequency distribution of the inter-CO distances to the gamma model, as explained in Broman and Weber (2000)  and Lian et al (2008) . Maximum likelihood estimates of the υ parameter of the gamma function were obtained using the free online Wessa software .
Differences in the length of SC and the number of MLH1 foci per cell, between the 4 boars, were examined by one-way analyses of variance (ANOVA) and subsequent multiple comparison analyses (Tukey test). The same tests were used to compare the relative lengths between mitotic and meiotic chromosomes. Differences in the number of MLH1 foci per chromosome between two individuals were examined by Student’s t-test. Pearson correlation analyses were used to investigate the relationships between the number of MLH1 foci and the length of autosomal SCs per cell and per chromosome, the relationships between the two sets of distances from the centromere to the nearest COs on the p- and q-arms, and the relationships between the meiotic vs mitotic chromosome length differences and the percentage of GC content.
Results and Discussion
Analyses of Synaptonemal Complex Lengths
It has been demonstrated that the physical SC lengths are continuously varying along the pachytene substages , . Therefore, knowledge of the physical (absolute) SC lengths per se is of relatively limited interest. However, frequencies of recombination are frequently expressed in the literature in CO/µm. Consequently, such results are also given and discussed below.
In this study, immunocytological techniques were used to analyze 436 pachytene cells from 4 normal boars of different ages and breeds. The average total autosomal SC lengths per cell for these 4 boars are presented in Table 1. High intra-individual variability was observed, (the largest for the Meishan boar, ranging from 146 µm to 268 µm). Nevertheless, the between-boar differences for this trait were highly significant (P<0.001). The difference between the two extremes (179.6 µm for Mini 1 and 199.3 µm for Meishan, respectively) represented 10% of the overall mean for this trait (190.3 µm). This value is slightly higher than the 161.5 µm already published for swine . However, this latter study was conducted on a small number of spermatocytes, using electron microscopy and, as previously mentioned, high intra-individual variability could explain this difference.
Comparison between Mitotic and Meiotic Chromosome Lengths
The relative lengths of mitotic and meiotic chromosomes (identified by FISH) were compared for the LW1.0 and Meish boars (Table 2). Some SC were significantly longer than the corresponding mitotic chromosomes (SSC3, SSC6 and SSC14; P<0.001), whereas others were significantly shorter (SSC1, SSC4, SSC5, SSC8, SSC9, and SSC11; P<0.001). Nevertheless, the correlation between the two measures was very high for both individuals (r = 0.953 for LW1.0 and r = 0.952 for Meish). Similarly, for some chromosomes, the positions of the centromeres between the SC and mitotic chromosomes differed significantly (Table 2). These results demonstrated that, for a given SC, the variation in length (between the mitotic chromosome and SC) on a chromosome arm was independent of the variation in length on the other arm. Similar results were obtained in Humans and mice , . Differences in GC content and gene density between chromosomes have been proposed to explain this phenomenon.
Number of MLH1 Foci Per Cell
The number of MLH1 foci per spermatocyte was estimated by analysing 436 meiocytes from the 4 boars (Table 1). The overall distribution was Gaussian with an average number of 32.0±4.5 MLH1 foci per cell.
Considerable intra-individual variability (between cells) was again observed in the number of MLH1 foci per spermatocyte (from 21 to 40 MLH1 foci per cell for the Meishan boar, for example). Large differences were also observed between individuals. Nevertheless, only the value obtained for the minipig boar (Mini 1) differed significantly from the others. Similar results had already been obtained in Humans –, mice  and in former pig studies based on genetic (linkage) analyses , . In the pig study by Vingborg et al. (2009) , the estimated total recombination length was 1,441 cM (centi Morgan, genetic map unit), i.e. 18% lower than the 1,760 cM found in the study by Tortereau et al. (2012) . Considering that one recombination event corresponds to 50 cM, the total recombination length estimated in our study ranged from 1,509 cM to 1,866 cM (for LW 1.0 and Mini 1, respectively). These results are consistent with those of Vingborg et al. (2009)  and Tortereau et al. (2012) .
Number of MLH1 Foci Per Autosome
By combining immunostaining and FISH experiments, we were able to determine the number of MLH1 foci per individual chromosome for two animals (LW1.0 and Meish; Table 3). One hundred and thirteen cells representing 2034 bivalents were analyzed. As expected, the average number of MLH1 signals was higher for the large chromosomes than for the small ones. Comparisons between the two boars did not reveal any difference in the number of MLH1 foci per chromosome (P>0.05) except for SSC 15. These results suggest that, for 2 individuals with a comparable total number of MLH1 foci per cell, the number of foci per chromosome remains identical.
Lack of MLH1 Foci on Some Bivalents
The lack of MLH1 signals on some bivalents (autosome or sex bivalent) was noted (Table 1). Sex bivalents never presented more than one MLH1 signal, and an absence of MLH1 signal was particularly frequent (from 26 to 39%) on this particular bivalent as compared with the other chromosomes (from 1.2 to 2.8%). Comparable results were obtained in human studies, which also revealed great variability between individuals (9 to 44% of sex chromosomes without MLH1 foci ). It has been suggested that the sex bivalent XY is predisposed to nondisjunction due to a frequent lack of recombination in the pseudoautosomal region , . This is consistent with the higher rates of aneuploidy estimated for sex chromosomes as compared with autosomes . However, the proportions of XY bivalents lacking MLH1 signals are much higher than the aneuploidy rates of sex chromosomes reported in Man and pigs (in sperm as well as in newborn , –). This suggests that a certain proportion of germ cells lacking the MLH1 signal on the XY bivalent are eliminated during spermatogenesis. On the other hand, these high rates of XY bivalents without any MLH1 signal observed in our study, as well as in previous human studies, could also be due, in part, to technical problems (limited accessibility of the MLH1 antibodies to this specific bivalent due to their particular shape and molecular environment, higher rate of hybridization failure due to the limited size of the pseudoautosomal region, …). This specific point has not been documented so far, and should be investigated further.
In contrast to results obtained in humans , we did not observe any obvious relationship between the frequency of spermatocytes lacking MLH1 signals in the XY bivalents and the total number of MLH1 signals per cell (Table 1). However, it is very difficult to document a relationship between two variables using only 4 points (individuals). Complementary data are required to document this point more thoroughly. We also compared, for each individual, the total numbers of autosomal MLH1 signals per cell: (a) in cells with one MLH1 signal on the XY bivalent, and (b) in cells without any MLH1 signal on the XY bivalent. The (a - b) difference was positive in all cases, but low and statistically different from zero for 2 boars only: (a–b) = 1.6 (P = 0.06) for LW1.0; (a–b) = 0.6 (P = 0.39) for LWxCr; (a–b) = 1.3 (P = 0.03) for Meish and (a–b) = 2.9 (P = 0.01) for Mini1.
The frequencies of autosomes without any MLH1 signal were much lower, and varied from 1.2% to 2.8% between individuals, as indicated previously (Table 1). These proportions are comparable to those observed in mice (4%, ), dog (0.5%, ) and shrew (0.7%, ). The lack of MLH1 signal mainly concerned the small chromosomes, with 87% of them being observed on SSC5, SSC10, SSC11, SSC12, SSC16, SSC17 and SSC18. The smallest pig autosome (SSC 18) aggregated on its own 29% of the cases. Similar results have been reported in humans , where the absence of MLH1 signal was most frequently observed for one of the smallest chromosomes (HSA 22).
Relationship between the Number of MLH1 Foci and the SC Length
Relationships between the total number of MLH1 foci per cell and the total SC length per cell were analyzed for each individual (Figure 2). The two variables were positively, albeit (very) moderately, correlated (the correlation was positive but not statistically different from 0 for the LW1.0 boar). Comparable results have been obtained in human studies (correlation values ranging from 0.02 to 0.69 ).
x-axis: total length of autosomal SC per cell (µm); y-axis: total number of MLH1 foci per cell.
In our study, the average total autosomal SC length per cell was 190.3 µm, for an average of 32.0 MLH1 foci per cell. This gives, on average, one MLH1 signal every 5.9 µm which is comparable to the results obtained in humans (1 focus/6.0 µm, ), dogs (1 focus/6.2 µm, ), mink (1focus/6.3 µm, ) and shrew (1 focus/6.5 µm, ) but quite different from those obtained for the cat (1foci/4.6 µm, ) and mouse (1 foci/7.1 µm, ). However, considerable variability was observed between individuals (which were of different genotypes), ranging from 4.8/µm for Mini 1 to 6.5/µm for Meish. Studies carried out in mice  suggested that the between-strain differences in crossover levels were associated with a variation in the length of the meiotic axes (smaller DNA loops and longer SCs in the strains exhibiting large numbers of MLH1 foci per cell). Our results (Table 1) do not support this hypothesis since boars with the lowest total SC lengths per cell (Mini1 and LWxCr) presented the highest rates of recombination (37.33 and 31.37 MLH1 foci per cell, respectively). Thus, the variability in the total number of MLH1 foci observed between the four boars cannot be fully explained by the variability in the total autosomal SC lengths, and vice versa. Another explanation for the between-boar difference would be that the overall genetic background could regulate SC length independently of CO formation. As mentioned in the introduction, allelic variation for PRDM9 has been associated with hotspot activity in mice and humans , , . Variation of RNF212 alleles also affects genome-wide recombination in humans –. Such allelic variations could explain part of the inter-individual variation of the MLH1 foci number observed in our study. No data supporting this hypothesis could be found in the literature for the pig species. This should be investigated in the near future.
The relationships between the number of MLH1 foci and the absolute SC length (in µm) were also analyzed using the data obtained for each bivalent independently (Table 3). As expected, the two variables were highly correlated in the two studied animals (r = 0.936 and r = 0.912 for LW 1.0 and Meish, respectively). A similar relationship has already been observed in other mammals , . In order to determine whether this relationship was comparable in acrocentric and non-acrocentric (i.e. metacentric and submetacentric) chromosomes, data from the two individuals (LW 1.0, and Meish) were pooled (to increase the number of observations per chromosome category). This pooling of data from two different individuals obliged us to consider relative SC lengths instead of absolute SC lengths in the subsequent analyses. The obtained correlations were slightly higher when acrocentric and non-acrocentric chromosomes were considered separately (r = 0.957 and r = 0.943, respectively) as compared with r = 0.924 when all the autosomes were considered simultaneously. As shown in Figure 3, the linear regression lines of the number of MLH1 foci (Y) on the length of SC (X) for the two kinds of chromosomes presented similar slopes (P = 0.89) but significantly different intercepts (higher for non-acrocentric; P<0.05). This indicates that, in general, metacentric and submetacentric chromosomes present higher recombination rates than acrocentrics. However, results obtained in other species indicate a different situation when the metacentric chromosome results from a fusion between 2 acrocentrics. Indeed, studies of individuals from different mice species or from bovidae differing only in the presence of Robertsonian translocations, revealed a reduction of recombination on the fused acrocentric chromosomes (which became metacentric), as compared to the free initial acrocentric chromosomes , . Different Robertsonian translocations have been identified in several young boars controlled in our laboratory . This should provide us with an opportunity to more thoroughly document this point in the near future.
Distributions of the MLH1 Foci on Individual Chromosomes
The distributions of MLH1 foci for some representative chromosomes of LW1.0 are presented in Figure 4, and for the 18 autosomes of the two analyzed boars in Figure S1. Overall, the MLH1 foci distributions were not uniform. As already reported in the literature, the CO were concentrated in specific areas known as recombination hotspots. The two main hotspots for non-acrocentric chromosomes were located in telomeric regions, one on the p-arm, and the other on the q-arm. Conversely, the frequencies of MLH1 foci in the centromeric regions of non-acrocentric chromosomes were generally low (even an absence was noted for some chromosomes, SSC 11 for instance). Significantly different distributions were observed for acrocentrics. Two hotspots were frequently observed in this kind of chromosomes, as in non-acrocentrics, but one was located close to the centromeres. Similar recombination patterns were obtained in pigs by Tortereau et al. (2012) using high-density linkage map analysis . These results seemed to indicate that, in acrocentric chromosomes, the centromere does not hinder the formation of CO in the porcine male, in contrast to other mammalian species like dogs . For these latter, it was proposed that recombination in the centromeric regions might interfere with kinetochore assembly, and that a reduction of recombination in these regions could prevent this phenomenon. However, it has been shown that the swine DNA sequences in the centromeric regions differ between acrocentric and non-acrocentric chromosomes. More precisely, the acrocentric swine subgenome presents a higher degree of DNA sequence homogenization (nature of sequences and copy number) than the metacentric one , , . This could provide an explanation for the difference observed between the two species.
For each autosome, the x-axis indicates the position of the signals on the SC, from the q (left) arm to the p (right) arm. This axis is divided into a number of intervals proportional to the length of the SC. The y-axis indicates the number of MLH1 foci in each interval. The vertical line in bold represents the centromere and the dotted line the average number of MLH1 signals per SC. For each autosome, the columns (from lighter to darker blue) indicate bivalent with 1, 2, 3 or 4 MLH1 foci.
Positive correlations between sequence parameters (GC content, repetitive elements content and short sequence) and recombination rate have been reported in humans , mice , dogs  and pigs . Considering that the relationship between the SC length and CO frequency is also positive (Figure 3), this would imply that chromosomes with high GC content would present longer SC than expected from the corresponding mitotic chromosomes, as well as high levels of recombination. Our results, presented in Figure 5, confirm these predictions. Indeed, SSC3, SSC6 and SSC14 have a high GC content and are longer than their corresponding mitotic chromosomes, in contrast to SSC1 and SSC8. Moreover, SSC3 and SSC6, for example, present higher levels of recombination than SSC 1 (0.68 cM/Mb, 0.66 cM/Mb and 0.42 cM/Mb respectively).
The difference in relative length between meiotic and mitotic chromosomes is expressed as a percentage (% difference = relative SC length/relative mitotic chromosome length × 100). GC content of the porcine chromosomes was obtained from the porcine sequence 10.2 (http://www.ncbi.nlm.nih.gov/genome/84?project_id=28993).
Analysis of Interference
The interference parameter for the different porcine autosomes was estimated from the pooled results of the 2 individuals analyzed (Figure 6). The MLH1 interfoci distances were expressed as the distances between two adjacent MLH1 foci in percentages of the SC lengths. As for numerous other organisms , , the gamma model provides a good fit to the inter-foci distribution for our recombination data. The estimated υ parameter, for all the chromosomes, was significantly greater than 1 (ranging from 4.2 for SSC1 to 44.3 for SSC10), which demonstrates a positive interference as observed in humans , or mice . The level of interference differed significantly between chromosomes, the strength of interference being globally lower for large chromosomes than for small ones. This is consistent with results obtained in humans and mice , , , which indicated that the strength of interference is modulated by the SC length.
Data obtained from the pooled data of the 2 individuals studied using FISH. There is no value for SSC18 because this chromosome rarely presented more than one MLH1 signal.
The average relative distance between adjacent MLH1 foci, in all bivalents presenting only two foci (n = 1099), was 67.5% of the bivalent length (range 7.5–92.8%). This result is very close to the values reported in humans (68%, ), and mice (70%, ).
Effects of the Centromere on Interference
Evidence from several species suggests that interference acts across the centromere , , –. To document this point, the relationship between (x-axis) the distances between the centromere and the nearest MLH1 foci measured on the p arm [d (P)], and (y-axis) the distances between the centromere and the nearest MLH1 foci on the q arm [d (Q)], was analyzed using data from SC with at least one MLH1 signal on each arm (Figure 7). The distances between the centromeres and the MLH1 foci were expressed as percentages of the SC lengths. The analysis revealed a significant negative correlation between the two distances (r = −0.510; P<0.0001), confirming that interference runs across the centromere in swine chromosomes.
This article reports original and unique data concerning the direct analysis of meiotic recombination and interference in the pig species, a major agricultural as well as an interesting model species for chromosome research. The domestic pig is only the third species, after humans and mice, to have been analyzed by immunolocalization combined with chromosome specific in situ hybridization, which allowed a direct analysis of crossover frequency and distribution, as well as an estimation of the interference strength on each individual SC. Moreover, use of the immunolocalization approach made it possible to study recombination in the sexual chromosomes for the first time in pigs. Some important results already obtained in humans and/or mice have been confirmed, whereas others were more specific to pig chromosomes, as for instance the difference in recombination rates between acrocentric and non-acrocentric chromosomes (lower rate for acrocentrics). This work provides us with reference data concerning meiotic recombination and interference in normal pigs. Further work will be carried out in our group to i) produce comparable data for the female meiosis, and ii) document the impact of different kinds of chromosomal rearrangements on recombination and interference.
Distribution of MLH1 foci for all autosomes from the LW 1.0 and Meish. For each autosome, the x-axis indicates the position of the signals on the SC, from the q (left) arm to the p (right) arm. This axis is divided into a number of intervals proportional to the length of the SC. The Y-axis indicates the number of MLH1 foci in each interval. The vertical line in bold represents the centromere and the dotted line the average number of MLH1 signals per SC. For each autosome, the columns (from lighter to darker blue) indicate bivalent with 1, 2, 3 or 4 MLH1 foci.
Conceived and designed the experiments: SF YB AP AD. Performed the experiments: NM HB AC ALD NB FM. Analyzed the data: NM DR. Contributed to the writing of the manuscript: NM MY HA AD AP.
- 1. Baudat F, Imai Y, de Massy B (2013) Meiotic recombination in mammals: localization and regulation. Nat Rev Genet. 14(11): 794–806.
- 2. Hassold T, Hunt P (2001) To err (meiotically) is human: the genesis of human aneuploidy. Nat Rev Genet. 2(4): 280–91.
- 3. Egozcue J, Templado C, Vidal F, Navarro J, Morer-Fargas F, et al. (1983) Meiotic studies in a series of 1100 infertile and sterile males. Hum Genet. 65(2): 185–8.
- 4. Tempest HG (2011) Meiotic recombination errors, the origin of sperm aneuploidy and clinical recommendations. Syst Biol Reprod Med. 57(1–2): 93–101.
- 5. Muller HJ (1916) The mecanism of crossing over. Am. Nat. 50: 193–221.
- 6. Berchowitz LE, Copenhaver GP (2010) Genetic interference: don’t stand so close to me. Curr Genomics. 11(2): 91–102.
- 7. Hulten MA (2011) On the origin of crossover interference: A chromosome oscillatory movement (COM) model. Mol Cytogenet. 4: 10.
- 8. Petes TD (2001) Meiotic recombination hot spots and cold spots. Nat Rev Genet. 2(5): 360–9.
- 9. McVean GA, Myers SR, Hunt S, Deloukas P, Bentley DR, et al. (2004) The fine-scale structure of recombination rate variation in the human genome. Science. 304(5670): 581–4.
- 10. Myers S, Bottolo L, Freeman C, McVean G, Donnelly P (2005) A fine-scale map of recombination rates and hotspots across the human genome. Science. 310(5746): 321–4.
- 11. de Massy B (2013) Initiation of meiotic recombination: how and where? Conservation and specificities among eukaryotes. Annu Rev Genet. 47: 563–99.
- 12. Baudat F, Buard J, Grey C, Fledel-Alon A, Ober C, et al. (2010) PRDM9 is a major determinant of meiotic recombination hotspots in humans and mice. Science. 327(5967): 836–40.
- 13. Tortereau F, Servin B, Frantz L, Megens HJ, Milan D, et al. (2012) A high density recombination map of the pig reveals a correlation between sex-specific recombination and GC content. BMC Genomics. 13: 586.
- 14. Grey C, Barthes P, Chauveau-Le Friec G, Langa F, Baudat F, et al. (2011) Mouse PRDM9 DNA-binding specificity determines sites of histone H3 lysine 4 trimethylation for initiation of meiotic recombination. PLoS Biol. 9(10): e1001176.
- 15. Myers S, Freeman C, Auton A, Donnelly P, McVean G (2008) A common sequence motif associated with recombination hot spots and genome instability in humans. Nat Genet. 40(9): 1124–9.
- 16. Hulten M (1974) Chiasma distribution at diakinesis in the normal human male. Hereditas. 76(1): 55–78.
- 17. Mikawa S, Akita T, Hisamatsu N, Inage Y, Ito Y, et al. (1999) A linkage map of 243 DNA markers in an intercross of Gottingen miniature and Meishan pigs. Anim Genet. 30(6): 407–17.
- 18. Anderson LK, Reeves A, Webb LM, Ashley T (1999) Distribution of crossing over on mouse synaptonemal complexes using immunofluorescent localization of MLH1 protein. Genetics. 151(4): 1569–79.
- 19. Lian J, Yin Y, Oliver-Bonet M, Liehr T, Ko E, et al. (2008) Variation in crossover interference levels on individual chromosomes from human males. Hum Mol Genet. 17(17): 2583–94.
- 20. Froenicke L, Anderson LK, Wienberg J, Ashley T (2002) Male mouse recombination maps for each autosome identified by chromosome painting. Am J Hum Genet. 71(6): 1353–68.
- 21. Sun F, Oliver-Bonet M, Liehr T, Starke H, Ko E, et al. (2004) Human male recombination maps for individual chromosomes. Am J Hum Genet. 74(3): 521–31.
- 22. Vozdova M, Sebestova H, Kubickova S, Cernohorska H, Vahala J, et al. (2013) A comparative study of meiotic recombination in cattle (Bos taurus) and three wildebeest species (Connochaetes gnou, C. taurinus taurinus and C. t. albojubatus). Cytogenet Genome Res. 140(1): 36–45.
- 23. Borodin PM, Karamysheva TV, Rubtsov NB (2008) [Immunofluorescent analysis of meiotic recombination and interference in the domestic cat]. Tsitologiia. 50(1): 62–6.
- 24. Borodin PM, Karamysheva TV, Belonogova NM, Torgasheva AA, Rubtsov NB, et al. (2008) Recombination map of the common shrew, Sorex araneus (Eulipotyphla, Mammalia). Genetics. 178(2): 621–32.
- 25. Borodin PM, Basheva EA, Zhelezova AI (2009) Immunocytological analysis of meiotic recombination in the American mink (Mustela vison). Anim Genet. 40(2): 235–8.
- 26. Basheva EA, Bidau CJ, Borodin PM (2008) General pattern of meiotic recombination in male dogs estimated by MLH1 and RAD51 immunolocalization. Chromosome Res. 16(5): 709–19.
- 27. Garcia-Cruz R, Pacheco S, Brieno MA, Steinberg ER, Mudry MD, et al. (2011) A comparative study of the recombination pattern in three species of Platyrrhini monkeys (primates). Chromosoma.
- 28. Ford CE, Pollock DL,Gustavsson I (1980) Proceedings of the first international conference for the standardization of banded karyotype of domestic animals.
- 29. Ducos A, Berland HM, Pinton A, Guillemot E, Seguela A, et al. (1998) Nine new cases of reciprocal translocation in the domestic pig (Sus scrofa domestica L.). J Hered. 89(2): 136–42.
- 30. Barasc H, Ferchaud S, Mary N, Cucchi MA, Lucena AN, et al. (2014) Cytogenetic analysis of somatic and germinal cells from 38,XX/38,XY phenotypically normal boars. Theriogenology. 81(2): 368–72 e1.
- 31. Massip K, Yerle M, Billon Y, Ferchaud S, Bonnet N, et al. (2010) Studies of male and female meiosis in inv(4)(p1.4;q2.3) pig carriers. Chromosome Res. 18(8): 925–38.
- 32. Mompart F, Robelin D, Delcros C, Yerle-Bouissou M (2013) 3D organization of telomeres in porcine neutrophils and analysis of LPS-activation effect. BMC Cell Biol. 14: 30.
- 33. Rogel-Gaillard C, Bourgeaux N, Billault A, Vaiman M, Chardon P (1999) Construction of a swine BAC library: application to the characterization and mapping of porcine type C endoviral elements. Cytogenet Cell Genet. 85(3–4): 205–11.
- 34. Reeves A (2001) MicroMeasure: a new computer program for the collection and analysis of cytogenetic data. Genome. 44(3): 439–43.
- 35. Broman KW, Weber JL (2000) Characterization of human crossover interference. Am J Hum Genet. 66(6): 1911–26.
- 36. Wessa P (2013) Free Statistics Software, Office for Research Development and Education.
- 37. Villagomez DA (1993) Zygotene-pachytene substaging and synaptonemal complex karyotyping of boar spermatocytes. Hereditas. 118(1): 87–99.
- 38. Vranis NM, Van der Heijden GW, Malki S, Bortvin A (2010) Synaptonemal complex length variation in wild-type male mice. Genes (Basel). 1(3): 505–20.
- 39. Schwarzacher T, Mayr B, Schweizer D (1984) Heterochromatin and nucleolus-organizer-region behaviour at male pachytene of Sus scrofa domestica. Chromosoma. 91(1): 12–9.
- 40. Codina-Pascual M, Campillo M, Kraus J, Speicher MR, Egozcue J, et al. (2006) Crossover frequency and synaptonemal complex length: their variability and effects on human male meiosis. Mol Hum Reprod. 12(2): 123–33.
- 41. Lynn A, Koehler KE, Judis L, Chan ER, Cherry JP, et al. (2002) Covariation of synaptonemal complex length and mammalian meiotic exchange rates. Science. 296(5576): 2222–5.
- 42. Sun F, Oliver-Bonet M, Liehr T, Starke H, Turek P, et al. (2006) Variation in MLH1 distribution in recombination maps for individual chromosomes from human males. Hum Mol Genet. 15(15): 2376–91.
- 43. Baier B, Hunt P, Broman KW, Hassold T (2014) Variation in genome-wide levels of meiotic recombination is established at the onset of prophase in mammalian males. PLoS Genet. 10(1): e1004125.
- 44. Vingborg RK, Gregersen VR, Zhan B, Panitz F, Hoj A, et al. (2009) A robust linkage map of the porcine autosomes based on gene-associated SNPs. BMC Genomics. 10: 134.
- 45. Sun F, Oliver-Bonet M, Liehr T, Starke H, Turek P, et al. (2006) Analysis of non-crossover bivalents in pachytene cells from 10 normal men. Hum Reprod. 21(9): 2335–9.
- 46. Hassold TJ, Sherman SL, Pettay D, Page DC, Jacobs PA (1991) XY chromosome nondisjunction in man is associated with diminished recombination in the pseudoautosomal region. Am J Hum Genet. 49(2): 253–60.
- 47. Shi Q, Spriggs E, Field LL, Rademaker A, Ko E, et al. (2002) Absence of age effect on meiotic recombination between human X and Y chromosomes. Am J Hum Genet. 71(2): 254–61.
- 48. Templado C, Uroz L, Estop A (2013) New insights on the origin and relevance of aneuploidy in human spermatozoa. Mol Hum Reprod. 19(10): 634–43.
- 49. Hall H, Hunt P, Hassold T (2006) Meiosis and sex chromosome aneuploidy: how meiotic errors cause aneuploidy; how aneuploidy causes meiotic errors. Curr Opin Genet Dev. 16(3): 323–9.
- 50. Villagomez DA, Parma P, Radi O, Di Meo G, Pinton A, et al. (2009) Classical and molecular cytogenetics of disorders of sex development in domestic animals. Cytogenet Genome Res. 126(1–2): 110–31.
- 51. Pinton A, Barasc H, Raymond Letron I, Bordedebat M, Mary N, et al. (2011) Meiotic studies of a 38,XY/39,XXY mosaic boar. Cytogenet Genome Res. 133(2–4): 202–8.
- 52. Codina-Pascual M, Oliver-Bonet M, Navarro J, Campillo M, Garcia F, et al. (2005) Synapsis and meiotic recombination analyses: MLH1 focus in the XY pair as an indicator. Hum Reprod. 20(8): 2133–9.
- 53. Pan Z, Yang Q, Ye N, Wang L, Li J, et al. (2012) Complex relationship between meiotic recombination frequency and autosomal synaptonemal complex length per cell in normal human males. Am J Med Genet A. 158A(3): 581–7.
- 54. Berg IL, Neumann R, Lam KW, Sarbajna S, Odenthal-Hesse L, et al. (2010) PRDM9 variation strongly influences recombination hot-spot activity and meiotic instability in humans. Nat Genet. 42(10): 859–63.
- 55. Parvanov ED, Petkov PM, Paigen K (2010) Prdm9 controls activation of mammalian recombination hotspots. Science. 327(5967): 835.
- 56. Chowdhury R, Bois PR, Feingold E, Sherman SL, Cheung VG (2009) Genetic analysis of variation in human meiotic recombination. PLoS Genet. 5(9): e1000648.
- 57. Fledel-Alon A, Leffler EM, Guan Y, Stephens M, Coop G, et al. (2011) Variation in human recombination rates and its genetic determinants. PLoS One. 6(6): e20321.
- 58. Kong A, Thorleifsson G, Stefansson H, Masson G, Helgason A, et al. (2008) Sequence variants in the RNF212 gene associate with genome-wide recombination rate. Science. 319(5868): 1398–401.
- 59. Yang Q, Zhang D, Leng M, Yang L, Zhong L, et al. (2011) Synapsis and meiotic recombination in male Chinese muntjac (Muntiacus reevesi). PLoS One. 6(4): e19255.
- 60. Bidau CJ, Gimenez MD, Palmer CL, Searle JB (2001) The effects of Robertsonian fusions on chiasma frequency and distribution in the house mouse (Mus musculus domesticus) from a hybrid zone in northern Scotland. Heredity (Edinb). 87(Pt 3): 305–13.
- 61. Ducos A, Revay T, Kovacs A, Hidas A, Pinton A, et al. (2008) Cytogenetic screening of livestock populations in Europe: an overview. Cytogenet Genome Res. 120(1–2): 26–41.
- 62. Pita M, García-Casado P, Toro MA, Gosálvez J (2008) Differential expansion of highly repeated DNA sequences in the swine subgenomes. J Zool Syst Evol Res. 46: 186–189.
- 63. Rogel-Gaillard C, Hayes H, Coullin P, Chardon P, Vaiman M (1997) Swine centromeric DNA repeats revealed by primed in situ (PRINS) labeling. Cytogenet Cell Genet. 79(1–2): 79–84.
- 64. Kong A, Gudbjartsson DF, Sainz J, Jonsdottir GM, Gudjonsson SA, et al. (2002) A high-resolution recombination map of the human genome. Nat Genet. 31(3): 241–7.
- 65. Shifman S, Bell JT, Copley RR, Taylor MS, Williams RW, et al. (2006) A high-resolution single nucleotide polymorphism genetic map of the mouse genome. PLoS Biol. 4(12): e395.
- 66. Wong AK, Ruhe AL, Dumont BL, Robertson KR, Guerrero G, et al. (2010) A comprehensive linkage map of the dog genome. Genetics. 184(2): 595–605.
- 67. de Boer E, Stam P, Dietrich AJ, Pastink A, Heyting C (2006) Two levels of interference in mouse meiotic recombination. Proc Natl Acad Sci U S A. 103(25): 9607–12.
- 68. de Boer E, Dietrich AJ, Hoog C, Stam P, Heyting C (2007) Meiotic interference among MLH1 foci requires neither an intact axial element structure nor full synapsis. J Cell Sci. 120(Pt 5): 731–6.
- 69. Petkov PM, Broman KW, Szatkiewicz JP, Paigen K (2007) Crossover interference underlies sex differences in recombination rates. Trends Genet. 23(11): 539–42.
- 70. Broman KW, Rowe LB, Churchill GA, Paigen K (2002) Crossover interference in the mouse. Genetics. 160(3): 1123–31.
- 71. Colombo PC, Jones GH (1997) Chiasma interference is blind to centromeres. Heredity (Edinb). 79 (Pt 2): 214–27.
- 72. Brown PW, Judis L, Chan ER, Schwartz S, Seftel A, et al. (2005) Meiotic synapsis proceeds from a limited number of subtelomeric sites in the human male. Am J Hum Genet. 77(4): 556–66.
- 73. Drouaud J, Mercier R, Chelysheva L, Berard A, Falque M, et al. (2007) Sex-specific crossover distributions and variations in interference level along Arabidopsis thaliana chromosome 4. PLoS Genet. 3(6): e106.