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Additional file 1:. The estimated breeding value (EBV) distribution of 791 cows’ 1–180-day-old calves that died

Additional file 3: RNAseq data of the C1RL transcript (LOC112446756, XM_024992686.1) from National Center for Biotechnology Information Bos taurus Annotation release 106. Available from URL: https://www.ncbi.nlm.nih.gov/gene/?term=XM_024992686.1

cDNA sequence of bovine ANXA10. The boundaries between exons 1 and 2 and between exons 6 and 7 are indicated by arrows (magenta). Sequences of cDNA from CNVR_322 carrier animals were deleted exons 2 to 6 (yellow marked sequences). Sequencing of the 300-bp RT-PCR product from the CNVR_322 carrier animal shows a 90 bp fragment, which corresponds to the entire intron (414,996 bp to 415,085 bp) between the 7th and 8th exon of ANXA10, inserted into the deleted-transcript of ANXA10 at the 75 bp position (bracket). (PDF 130 kb)

Expression of ANXA10 protein in Anxa10 +/+, Anxa10 +/-, and Anxa10 -/- mice. (a) Western blotting for stomach from Anxa10 +/+, Anxa10 +/-, and Anxa10 -/- female mice using antibody to full length (AF3544) and to internal region (sc-135234) of the ANXA10 protein. Recombinant mouse ANXA10 protein were expressed in HeLa cells by pCAGGS-Anxa10 expression plasmid and used as positive control. A 30-kDa band was reduced in Anxa10 +/- female mouse that was not detected in Anxa10 -/- female mouse (arrow). (b) Localization of ANXA10 in the stomach from Anxa10 +/+ (b1, b2) and Anxa10 -/- (b3, b4) female mice. The section was counterstained with DAPI (blue). ANXA10 immunoreactivities were localized at the apical membrane of mucous cells (green), which were detected only in Anxa10 +/+ (b1, b2). Scale bars: b1, 100 μm; b2 50 μm. (PDF 4572 kb)

Relative ANXA10 expression levels in Japanese Black cattle. Tissues and cells are indicated on the y-axis. Total RNA was extracted from tissues (1 -16) and primary dermal fibroblasts (17) of two female Japanese Black cattle. Relative gene expression levels in the different tissues are shown as the means of the quantities relative to the value for liver (dotted line). (PDF 40 kb)

Ovaries and placentas in Anxa10 +/+ and Anxa10 -/- female mice. (a) Histological analysis of the ovaries at E15.5 from Anxa10 +/+ (a1) and Anxa10 -/- female mice (a2). The corpora lutea and the follicles are indicated by asterisks and arrows, respectively. (b) Sections from the placenta at E12.5 from Anxa10 +/+ (b1) and Anxa10 -/- female mice (b2) were immunostained with anti-integrin beta 3 antibody (green) and counterstained with DAPI (blue). lab, labyrinth; jz, junctional zone; dec, decidua. (c) Histological analysis of the placenta at E15.5 from Anxa10 +/+ (c1) and Anxa10 -/- female mice (c2). Nucleus fetal erythrocytes (arrows) and maternal erythrocytes in the labyrinth zone of placenta (c1, c2). Scale bars: a1, c2 100 μm; b1, 50 μm. (PDF 16202 kb)

Relative Anxa10 expression levels in mice. Tissues are indicated on the y-axis. Total RNA was extracted from tissues (1â 20) of mice at 15.5Â days of pregnancy, except for uterus (3) from non-pregnant mice. Relative gene expression levels in the different tissues are shown as the means of the quantities relative to the value for liver (dotted line). (PDF 42 kb)

Distribution of ANAI 4 in 1,433 cows for the replication study. A sample population (nâ =â 1,433) was derived from the remainder of the cohort from the same farm used for the GWAS. (PPTX 47Â kb)

Distribution of ANAI 4 in 10,399 cows in this study. (PDF 31Â kb)

Relative expression of ACVR2 in cow tissues and cells. Relative ACVR2A expression levels in tissues and cells are indicated on the Y-axis. Total RNA was extracted from tissues (1â 16), primary dermal fibroblasts (17) from 2 female Japanese Black cattle, or from bovine primary endometrial epithelial cells (18). Relative gene expression levels in the different tissues are shown as mean quantities relative to the value observed in ovarian tissue (dotted line). (PPTX 50Â kb)

Expression of pCAGGS- ACVR2A in LβT2 cells. (A) To determine whether the pCAGGS-ACVR2A plasmid was expressed in LβT2 cells, we transfected 2 × 105 cells per well in a 24-well plate with a mixture of 200 ng of the FSHB promoter-reporter plasmid, pCAGGS-ACVR2A (the amount of each plasmid is indicated in the lanes) and 10 ng of pRL-TK Renilla. HA-ACVR2A expression was confirmed by western blot analysis using an anti-HA antibody. Unstained Precision Plus protein standards were used as a marker (BioRad, Cat. #161-0363). HA-ACVR2A expression was detected with multiple bands (~57.8 kDa) because TGF-beta type-II receptors are normally modified by phosphorylation and glycosylation, causing then to migrate heterogeneously in sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels. (B) Relative ACVR2A band intensities were measured using the ImageQuant TL Analysis Toolbox. The amounts of loaded proteins were calibrated by Coomassie Brilliant Blue-stained bands between 37 and 75 kDa. The bars represent the mean ± SEM observed in triplicate from 3 independent experiments. (PPTX 706 kb)

Quantile-quantile plots of the GWAS results for ANAI 4 . The red dots represent the observed − log10 P values, and the straight line represents the expected − log10 P values under the null hypothesis. (PPTX 68 kb)

Transfection and co-transfection efficiencies of LβT2 cells. (A) To examine the transfection efficiency of LβT2 cells, we used the pCAGGS-EGFP and pCAGGS-mCherry vectors. At 24 h post-transfection, double fluorescence-positive cells were counted using ImageJ software. The data shown represent the transfection efficiencies of LβT2 cells in 5 experiments. The average transfection efficiency was 8.8 ± 1.62 % (3,368 cells). (B–D) To examine co-transfection efficiency in LβT2 cells, we used the pCAGGS-EGFP and pCAGGS-mCherry vectors. At 24 h post-transfection, double fluorescence-positive cells (B, magenta) were counted using ImageJ software. All GFP positive cells (C, green) were mCherry-positive (D, red), representing 100 % of 3,368 cells. (PPTX 6646 kb)