<i>Spo11^+/+</i> and <i>Spo11^+/−</i> animals.

<p>(<b>A</b>) Mice heterozygous for a null allele of <i>Spo11</i> exhibited a significant decrease in RAD51 foci (a marker of DSBs) by comparison to wildtype littermates. However, (<b>B</b>) the mean number of MLH1 foci (a marker of COs), (<b>C</b>) mean SC lengths and (<b>D</b>) mean DNA loop sizes were not different between the two genotypes.</p

Inter-strain variation in early- and mid-stage meiotic recombination proteins.

<p>The number and location of foci for proteins acting upstream of MLH1 in the recombination pathway [i.e., (<b>A</b>) RAD51 (<b>B</b>) DMC1 (<b>C</b>) MSH4] or anti-recombination pathway [i.e., (<b>D</b>) BLM] were determined for each of the three strains and inter-strain values compared. For each of the four proteins, mean numbers of foci per cell varied significantly among the three strains. Data for each protein were based on 4–6 animals per strain, and a minimum of 60 cells per strain (see Supplemental Tables 2–5 for data).</p

Inter-strain variation in SC length and DNA loop size.

<p>(<b>A</b>) Mean autosomal SC lengths varied in direct relationship to MLH1 values; i.e., the strain with the lowest mean MLH1 values (CAST) had the shortest SCs, while the strain with the highest MLH1 values (B6) had the longest SCs. Data are based on analyses of mid-pachytene spermatocytes from 2 CAST (n = 8 cells), 3 C3H (n = 11 cells) and 2 B6 (n = 21 cells) males. (<b>B</b>) DNA loop sizes for individual chromosomes were calculated as the average of the width of the FISH signal at three points along the SC: at the centromere, the midpoint, and the telomere; image shows example for chromosome 12. (<b>C</b>) Chromosomes 1, 12, and 19 were examined and for each, we observed an inverse relationship between strain-specific mean MLH1 values and mean DNA loop sizes.</p

Inter-strain variation in mean MLH1 values.

<p>(<b>A</b>) Pachytene cell from B6 male immunostained with antibodies to MLH1 (green) and SYCP3 (red). The number of MLH1 foci per cell were counted and used as a surrogate for meiotic recombination events. (<b>B</b>) The mean number of MLH1 foci per spermatocyte for 5 CAST (n of cells = 105), 7 C3H (n of cells = 209) and six B6 (n of cells = 110) males varied significantly among the three inbred strains. (<b>C, D</b>) For each strain, slides were hybridized with FISH probes for chromosomes 19 and 1 and the positions of MLH1 foci were calculated as a percent of SC length (centromere = 0%; telomere = 100%). (<b>C</b>) On chromosomes 19 with a single MLH1 focus, the focus was typically medially or distally placed; data are based on analysis of 50 chromosomes/strain. (<b>D</b>) On chromosomes 1 with two MLH1 foci, typically one was proximally located and the other distally placed, consistent with positive interference. Data based on analysis of 50 cells/strain.</p

Combined mapping for <i>Hipp1a</i> and <i>Hipp9a.</i>

<p>This figure shows mapping data for the hippocampus weight loci <i>Hipp1a</i> and <i>Hipp9a</i> using 34 BXD strains (BXD; shaded line) and 679 advanced intercross animals (AIL, thin solid line) as well as the composite map using the described method (thick solid line). The genome-wide adjusted composite P = 0.05 threshold is −log P = 3.5 (dark solid horizontal line). Since 5000 permutations were used for each data set, the maximum −log P<3.7 (graphed as −log P = 3.7 for convenience) for each individual data set, so increasing the number of permutations might increase the peak combined value and slightly improve the range of the combined interval. Bars underneath the peaks are labeled AIL, BXD, and combined to indicate the l-LOD support interval of these mapping populations.</p

The need for locus-specific P-values.

<p>The 95% LOD score (the LOD score equivalent to a locus-specific P = 0.05) was calculated using 10,000 permutations for markers on Chr. 1 for body weight in several different populations. Each marker is indicated by a dot with connecting lines interpolated between adjacent markers. TJL BXD are BXD strains available from The Jackson Laboratory (The BXD strains developed by Taylor and colleagues <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001036#pone.0001036-Taylor1" target="_blank">[26]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001036#pone.0001036-Taylor2" target="_blank">[27]</a>). New BXD are the recently developed BXD strains currently resident at UTHSC. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001036#pone.0001036-Peirce2" target="_blank">[18]</a> Note that the maximum and minimum values of the 95^th percentile LOD score vary considerably for the AIL population, somewhat for the RI (New BXD and TJL BXD) populations, (predicted by missing data pattern) and very little for the 183 member F2 population tested. (There are only three widely spaced markers genotyped for the F2 population on Chr. 1, so the interpolation between points should not be interpreted as a meaningful line. However, markers on all chromosomes were very similar, between a 95% LOD of 1.2 and 1.4.)</p

Chromosome-specific MLH1 localization patterns in males and females.

<p>The chromosomal locations of MLH1 foci were determined using the same cells as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085075#pone-0085075-g002" target="_blank">Figure 2</a> for ten representative large, medium and small chromosomes. Each chromosome arm was arbitrarily divided into five equal regions – centromeric, proximal, interstitial, distal, and telomeric – and the distribution of MLH1 foci recorded for both chromosome arms for metacentric and sub-metacentric chromosomes or for the q-arm only of acrocentric chromosomes. The distribution differed significantly between females (black) and males (white) for seven of the ten chromosomes: 1 (χ²=24.9; p<0.005), 6 (χ²=24.8; p<0.005), 13 (χ²=13.8; p=0.01), 16 (χ²=32.1; p<0.0001), 18 (χ²=47.7; p<0.0001), 21 (χ²=22.3; p<0.0001), and 22 (χ²=20.8; p<0.0001). However, sex-specific differences were not evident for chromosomes 9 (χ²=15.1; p=0.088), 14 (χ²=5.3; p=0.262), or 15 (χ²=7.8; p=0.101).</p

Spacing between adjacent MLH1 foci in males and females.

<p>Inter-focal distances, calculated as the percent of the length of the synaptonemal complex between adjacent MLH1 foci, were determined using the same cells as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085075#pone-0085075-g002" target="_blank">Figure 2</a>; male data are depicted in white, female data in black. To obtain sufficient numbers of cells for direct male:female comparisons, we restricted our analysis to chromosomes having the same number of MLH1 foci in males and females; i.e., for chromosome 1 we analyzed cells in which the chromosome exhibited four MLH1 foci and for chromosomes 13, 14, 16, 18 and 22, cells in which the relevant chromosome exhibited two MLH1 foci. Thus, for chromosome 1, we made three measurements of inter-focal distances per cell, while for chromosomes 13, 14, 16, 18 and 22 we made a single measurement of inter-focal distance per cell. For chromosomes 6, 9, 15 and 21 we had a limited number of cells with the same number of MLH1 foci in both sexes; thus, these chromosomes were excluded from the analysis. For each chromosome, inter-focal distances were binned (by % value) into ten groups. The distribution of categories of inter-focal distances was significantly different between males and females for each of the six chromosomes: 1 (χ²=51.7; p<0.0001), 13 (χ²=26.7; p<0.0005), 14 (χ²=30.6; p<0.0001), 16 (χ²=31.9; p<0.0001), 18 (χ²=50.0; p<0.0001), and 22 (χ²=48.8; p<0.0001).</p

Genome-wide mean MLH1 values in human males and females.

<p>MLH1 foci were used as a marker for meiotic recombination events. (A) shows a representative pachytene oocyte and (B) a representative pachytene spermatocyte, that were immunostained for SYCP3 (in red), a component of the synaptonemal complex; CREST (in blue), detecting centromeric regions; and MLH1 foci (in green), detecting crossovers. (C) In total 4660 spermatocytes from 56 males (white) and 2038 oocytes from 63 females (black) were examined. Mean MLH1 counts (± S.E.) were significantly lower in males than females (49.09 ± 0.07 vs. 69.25 ± 0.29; t=92.5, p<0.0001) and the range was narrower in males than females (30-66 vs. 27-119). (D) Mean number (± S.E.) of MLH1 foci per cell for individual male and female samples, demonstrating the lack of overlap between the sexes, and the increased variation in individual female cases by comparison with males.</p

Comparison of SC length and DNA loop size in males and females.

<p>Male data are in white, female data in black. (A) Chromosome-specific SC lengths were determined for cells scored in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085075#pone-0085075-g002" target="_blank">Figure 2</a> and striking sex-specific differences were evident on all ten chromosomes analyzed: 1 (t=8.9; p<0.0001), 6 (t=23.1; p<0.0001), 9 (t=12.8; p<0.0001), 13 (t=26.7; p<0.0001), 14 (t=30.8; p<0.0001), 15 (t=24.1; p<0.0001), 16 (t=21.7; p<0.0001), 18 (t=28.6; p<0.0001), 21 (t=36.4; p<0.0001), and 22 (t=35.4; p<0.0001). (B, C) Three individual chromosomes (1, 16 and 21) were analyzed for DNA loop size, using the deflection of FISH paint probes from the SC as a surrogate for loop size. For chromosomes 1 and 16, we measured the width of the FISH signal at the centromere and three points on each chromosome arm, and averaged the seven values. For chromosome 21, loop size was taken as the average of three measurements, one at the centromere and two on the long arm. (B) Blow-up image of a portion of a representative pachytene stage oocyte, labeled with DAPI (blue) and a chromosome 1 paint probe (red). White bars represent the seven individual DNA loop measurements, three from each chromosome arm and one at the centromere. The centromere was identified using CREST prior to FISH. (C) DNA loop size means were significantly greater in males for each chromosome; i.e., for chromosome 1 (2 males, n of cells=24; 3 females, n=23; t=15.2; p<0.0001), for 16 (2 males, n=39; 3 females, n=32; t=20.8; p<0.0001) and for 21 (2 males, n=37; 2 females, n=43; t=16.0; p<0.0001).</p