Comparison of the Haplotype Block Densities between Syntenic Regions of Rat, Mouse, and Human (Same Genome Segments as Shown in Figure 1)

<p>The scatter plots show log₁₀ of the amount of haplotype blocks per 100-kb bin in rat (horizontal) against log₁₀ of the amount of haplotype blocks seen in syntenic region of mouse (A) and human (B) genome (vertical). Data points for gene-containing and intergenic genomic bins are shown as closed and open blocks, respectively. Observed correlations of haplotype block densities are significant in linear (<i>r</i> = +0.5530; <i>p</i> < 0.0001 [A] and <i>r</i> = +0.4563; <i>p</i> = 0.0005 [B]) as well as in log-transformed space (<i>r</i> = +0.6795; <i>p</i> < 0.0001 [A] and <i>r</i> = +0.3209; <i>p</i> = 0.0180 [B]).</p

Analysis of LD Decay for Functionally Different Segments of the Human Genome

<div><p>(A) The graph shows average values of |D′| and their confidence limits (± standard deviation) as a function of the physical distance between SNPs for the following categories: (1) both SNPs reside in the same gene (blue line), (2) the SNPs reside in two different genes (green line), (3) both SNPs reside in the same intergenic region (red line), (4) one SNP resides in the gene and the other in the 30 kb upstream region of the same gene (purple line), and (5) one SNP resides in the gene and the other in the 30 kb downstream region of the same gene (gray line).</p><p>(B) Frequency distribution spectrum of |D′| values for SNP pairs at 100-kb distance. High |D′| values (>0.8) are overrepresented for equally spaced SNPs in a gene and its flanking regions as compared to intergenic regions.</p><p>(C) Frequency distribution of high LD values (|D′| > 0.5) for SNP pairs at 450-kb distance. Higher LD values are observed between a gene and its upstream region.</p><p>(D) Frequency distribution of high LD values (|D′| > 0.5) for SNP pairs at 650-kb distance. Higher LD values are observed between a gene and its upstream region.</p><p>The bin with |D′| = 1 is isolated to a separate bin in panels (B–D) as there is a considerable frequency bias for this |D′| value. Similar graphs plotted for separate human populations are available as <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020121#pgen-0020121-sg003" target="_blank">Figures S3</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020121#pgen-0020121-sg004" target="_blank">S4</a>, and S5.</p></div

Patterns of LD for Orthologous Genomic Segments of Approximately 5 Mb in Rat, Human, and Mouse

<p>LD plots for orthologous genomic segments in rat (A), human (B), and mouse (C) are shown. For each panel, the following information is shown: LD plot (top), haplotype blocks in SNP coordinates (middle), and physical map and haplotype blocks in physical coordinates (bottom). The haplotype map has a gradient representation for |D′| values that assists visual comparison of haplotype structure. Haplotype blocks were built with stringent criteria, sometimes resulting in splitting of visually recognized blocks. Three characteristic haplotype blocks that are conserved cross-species have been color-coded and are discussed in the text. Similar plots for a second mouse set, two other human populations, and the combined human set are available as <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020121#pgen-0020121-sg001" target="_blank">Figures S1</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020121#pgen-0020121-sg002" target="_blank">S2</a>.</p

Centrobin expression in epididymal sperm.

<p><b>A</b>: RT-PCR with primers flanking the junction between exon 10 and 11. Mutants show products with higher molecular weight due to the insertion of an endogenous retrovirus <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060859#pone.0060859-Wang1" target="_blank">[33]</a>. (<b>green arrowhead</b>). Wild-type product (<b>blue arrowhead</b>) can be amplified both from endogenous locus transcripts as well as from the transgenic transcripts. The “mutant” product can be also seen in the rescued males, although during PCR, the product can be masked by preferential amplification of the smaller molecular weight amplicon coming from the transgene. <b>B</b>: β-actin is shown as endogenous control. Robust expression of centrobin can be seen in sperm both from caput (less mature) and cauda (more mature) epididymis. <b>C</b>: Western blot with an antibody against C-terminal part of centrobin. There is no signal in mutants because they lack the C-terminal portion of centrobin. Note a lower molecular weight species of centrobin from from caput and cauda epididymidis sperm of rescued rats that may correspond to proteolytic processing or degradation. This can be seen in the wild-type sperm too with higher exposition time (not shown). <b>D</b>: Western blot with an N-terminal-specific antibody (middle panel) shows similar results as seen with the C-terminal antibody, 55 kDa mutant truncated protein is also identifiable (<b>green arrowhead</b>). Note: the last lane had to be developed separately due to excess signal. <b>E</b>: β-actin served as the loading control.</p

Characteristics of spermiation and sperm maturation in control, <i>hd/hd</i> mutant and rescued <i>Tg+ hd/hd</i> rats.

<p><b>A:</b> Histologic section of a control seminiferous epithelium showing the release of mature spermatids during stage IX of spermatogenesis. <b>B:</b> Histologic section of a <i>hd/hd</i> mutant displaying abnormally shaped heads (<b>arrowheads</b>) and severed flagella (<b>arrows</b>) of mature spermatids seen in a similar spermatogenic stage IX. Note that round spermatids display normal structure. <b>C:</b> Histologic section of a rescued <i>Tg+ hd/hd</i> rat showing heads (<b>arrowheads</b>) separated from the flagella seen in a similar spermatogenic stage IX. The <b>inset</b> illustrates a thin cytoplasmic bridge (<b>pointer</b>) connecting the spermatid head to its developing flagellum. The number of decapitated mature spermatids released at spermiation is larger in the rescued rat as compared to the mutant. Round spermatids display normal features. <b>D:</b> Histologic section of the tail region of the epididymis of a control rat showing well-developed sperm in the lumen and the epididymal epithelium with normal characteristics. <b>E:</b> In the <i>hd/hd</i> mutant, the epididymal lumen contains multiple compact whorls (<b>dashed circle</b>) each consisting of entangled flagella seen in the <b>inset</b> at higher magnification. The height of epididymal epithelium is reduced. F: In the rescued <i>Tg+ hd/hd</i> rat epididymis, flagellar whorls coexist with numerous spherical bodies (<b>dashed box</b>). The <b>arrow</b> indicates a decapitated sperm. The height of epididymal epithelium is reduced. <b>G–H:</b> Phase contrast microscopy (panel <b>G</b>) and immunofluorescent localization of ODF2 (panel <b>H</b>) in decapitated sperm and spherical bodies harvested from the epididymal cauda of a rescued <i>Tg+ hd/hd</i> rat. The <b>pointers</b> in panel <b>H</b> indicate immunoreactive ODF2. <b>I:</b> Electron microscopy of a spherical body and sperm flagella in the epididymal lumen of a <i>Tg+ hd/hd</i> rat. The <b>dashed circle</b> indicates aggregates of outer dense fibers in the spherical body equivalent to those seen in panels <b>G</b> and <b>H</b>. The <b>dashed boxes</b> indicate cross-sections of sperm flagella (middle piece) each with an intact axoneme and surrounded by fragmented outer dense fibers (<b>ODFs</b>). The <b>arrow</b> indicates two fused sperm flagella (principal piece). Scale bar in all panels and inset is 5 µm.</p

Detection of centrobin in testis by Western blotting.

<p><b>A</b>: Western blot using centrobin C-terminal antibody that recognizes both endogenous and transgenic wild-type centrobin. <i>hd</i>-allele specific truncated form cannot be detected. Lack of centrobin in mutants is supplemented to approximately normal level in the rescued males. <b>B</b>: Centrobin N-terminal antibody confirms presence of wild-type protein and the <i>hd</i>-specific truncated protein (lower amount in heterozygote controls). <b>C</b>: We used β-actin as a loading control. <b>D</b>: Relative amount of full-length centrobin as determined from densitometry of the Western blot in B. The proportion is different among groups with full-length centrobin (ANOVA p = 10⁻⁵), post-hoc comparison by Tukey’s test shows significant difference between the adjacent infertile and fertile groups (p = 4.45 10⁻⁴). Numbers labeling the lanes identify individual animals.</p

Limb phenotype, growth retardation rescue and improvement of reproductive phenotype.

<p><b>A</b>: Autopods of mutants compared to transgenic rescue and controls. Left forelimb (top) and hind limb (bottom). Anterior to posterior axis from top to bottom. Normal hind limb has 5 toes, normal forelimb has 4 fingers and a rudimentary thumb. Note missing digit II and hypoplastic digit III in mutants only. <b>B</b>: Body weight. Transgenic rescue rats are indistinguishable from controls, mutants are significantly smaller (post-hoc P = 0.0039 in comparison to rescued males). <b>C</b>: Testis weight (both testes together). Transgenic rescued rats are indistinguishable from controls, mutants are significantly smaller (post-hoc P = 0.0018 in comparison to rescued males). <b>D</b>: Epididymis weight (both epididymides) Transgenic rescue rats are indistinguishable from controls, mutants are significantly smaller (post-hoc P = 0.0297 in comparison to rescued males). <b>E</b>: Sperm count (cauda epididymidis of both sides). Rescued males have more than 3 times more spermatozoa compared to mutants (post-hoc P = 0.00114). However, control males have at least 4 times more spermatozoa compared to rescued males (post hoc P = 0.000816 for rescued males compared to wild-type males). Mutants n = 3, rescued n = 7, transgenic controls n = 6, nontransgenic (wild-type = WT) controls n = 5. One-way ANOVA, with post hoc Tukey test for unequal N.</p

Transgenic construct, its localization and expression.

<p><b>A</b> Top: Transgenic construct contains CMV_IE enhancer, chicken β-actin promoter together with first noncoding exon and intron with rabbit β-globin acceptor splice site, coding sequence and rabbit β-globin 3’UTR and polyA signal. Violet arrows represent PCR primers used to amplify the insertion sites (see <b>B</b>). Red arrows depict primers used to assess expression of the transgene (see <b>D</b> and <b>E</b>). <b>A</b> Middle: structure of the insertion site region showed in detail (genome assembly Rn3.4, chr16∶43998593–44005224). Green: homology with the mouse (above zero in the nonrepetitive flanking sequence), grey rectangles: repetitive sequences; the insertion site is red. <b>A</b> Bottom: Rat chromosome 16. Red line: insertion site; purple: centromere. The approximate positions of the genes flanking the transgene are shown. <b>B:</b> Inversion PCR amplifying a fragment of chromosome 16 only in the transgenic animals. <b>C:</b> Long range PCR confirming insertion of the transgene to chromosome 16. Expected product sizes were (for wild-type and transgenic respectively): chr16F-chr16R 6632 bp and 11898 bp (impossible to be amplified with the PCR system employed); chr16F-tgR 944 bp (only transgenic); tgF-chr16R 6080 bp (only transgenic). <b>D</b>: Expression of the transgene in somatic tissues by RT-PCR. <b>E:</b> Expression of the transgene in testis by RT-PCR. <i>hd/hd</i> or <i>hd</i> = hypodactylous mutants, transgene negative; <i>Tg</i> (<i>Tg+</i>) = transgene positive; wt = wild-type; “<i>−</i>“ in <b>C</b>: water negative control; “−” RT in <b>D</b> and <b>E</b>, reverse transcriptase dropout negative control. Numbers of individual animals according to our internal system for animal identification are given in panel <b>E</b>.</p

FDR in LV, Fat, Kidney, and Adrenal Tissues

<div><p>(A) For each major eQTL detected in LV, fat, kidney, and adrenal tissue, the expected FDR was calculated and plotted against different <i>p</i>-value thresholds in the range 10⁻⁶–0.05. Insert: FDR for various <i>p</i>-values in the range 10⁻⁶–10⁻³.</p><p>(B) Vase box-plots for the FDRs of the <i>cis-</i> and <i>trans</i>-acting eQTLs detected at <i>p</i> = 0.05 in LV, fat, kidney, and adrenal tissue. Vase box-plots are box-plots where the width of the box at each point is proportional to the density of the data there. The thick line indicates the median FDR for each distribution.</p></div

Genetic Architecture of Genetic Variation in Gene Expression

<p>For each considered transcript the major eQTL was identified by linkage analysis (genome-wide significance, <i>p</i> = 0.05) and characterised as <i>cis</i> or <i>trans</i>. Additive allelic effect and heritability (<i>h</i>²_QTL) for each <i>cis-</i>eQTL (black symbol) and <i>trans-</i>eQTL (grey symbol) were plotted for LV, fat, kidney, and adrenal tissues.</p