Fig 1.
Pedigree and identical-by-descent (IBD) analysis.
(A) A pedigree of CW-3-segregating (I-VII) and related sires. Bold lines indicate inheritance of the Q haplotype. (B) Haplotypes of CW-3-segregating sires and related animals. Sire B did not segregate CW-3 nor produced affected offspring, strongly suggesting that the telomeric ends of the critical regions are at MS087. Marker information is shown in S7 Table. ND, not determined.
Fig 2.
Association analyses using imputed BovineHD genotypes (A), segments of the identical-by-descent (IBD) Q haplotype (B), and candidate causative variations (C).
Association with carcass weight was analyzed by a variance component approach using EMMAX software [39] with adjustments for age, slaughterhouse, and year as covariates and fixed effects. (A) Red and blue dots represent p values of imputed BovineHD genotypes in–log10 scale before and after conditioning, respectively. A conditioned analysis was performed by including an imputed genotype of BovineHD0800025437 as a covariate in the model. (B) Light blue and black lines represent p values of approximately 1-Mb and 500-kb Q haplotypes in–log10 scale, respectively. (C) Brown dots, a red triangle, and blue squares represent p values of Bovine50K genotypes, experimentally validated BovineHD0800025437 genotype, and the genotypes of candidate causative variations in–log10 scale, respectively.
Fig 3.
Clinical features of skeletal dysplasia.
Affected calf (A) and cow (B) are presented. Red circles show the symptoms of thin neck, extended shoulder, thick joints of the limbs, and hip bone-enlargement. (C) Tibias from cows with FGD3 Cys-171 homozygous (NLBC-1 and K) and a non-carrier control (T) cows. They were 7.1–8.3 years old. Withers height (WH), shank circumference (SC), tibia length (TL), and circumference of tibia shaft (CT) are as follows: NLBC-1, 135.6 cm (WH), 20.4 cm (SC), 38.8 cm (TL), 13.6 cm (CT); K, 140 cm (WH), 18.5 cm (SC), 38.4 cm (TL), 13.0 cm (CT); T, 128.8 cm (WH), 17.8 cm (SC), 36.6 cm (TL), 12.0 cm (CT). The arrows indicate medial malleolus and intercondylar eminence. Dashed lines were drawn to facilitate observation.
Fig 4.
Mapping of homozygosity and autozygosity for skeletal dysplasia.
(A) The results of the genome-wide homozygosity and autozygosity mapping of skeletal dysplasia using 14 affected and 34 normal animals from three families. Black horizontal bars mark the limits between the 29 autosomes. ASSHOM and ASSIST were used for mapping [14]. (B) BTA8 (C) Schematic representation of homozygous regions on BTA8 in 29 affected animals.
Fig 5.
Effects of CW-3 Q or risk allele on skeletal measurements.
(A) Growth curves of heterozygous (n = 98, solid line) and homozygous q-steers (n = 165, broken line). The non-synonymous SNP in PTPDC1 was used as a marker for CW-3. The average and standard error (S.E.) are shown. Left, withers height; middle, body weight; right, chest circumference. (B) Distribution of skeletal measurements of calves with respective genotypes. Offspring steers from Sire R were genotyped with non-synonymous SNPs encoding His-171-Cys in FGD3. The number of non-carrier, carrier, and risk allele-homozygous animals were 165, 159, and 9, respectively. Upper left, withers height; upper right, chest circumference; lower left, body weight; lower right, abdominal circumference. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
Fig 6.
Amino acid sequence alignment around His-171, GEF activity, and gene expression of FGD3.
(A) His-171 of bovine FGD3 and the flanking region is well conserved among mammalian species. The RhoGEF domain corresponds to the region from His-164 to Ala-344 of bovine FGD3. (B) NIH3T3 cells were transfected with the bicistronic expression plasmid encoding HA-tagged wild or mutant bovine FGD3(SA) and V5-tagged Cdc42. FGD3(SA) denotes FGD3 with two serines in the destruction motif (Ser-83 and Ser-87 of bovine FGD3) replaced by alanines [24]. After 48 hours, cell extracts were prepared and submitted to the GST–CRIB pull-down assay. A portion of cell extracts and the pull-down products were subjected to SDS-PAGE followed by immunoblotting to detect Cdc42-V5 and FGD3(SA)-HA. The experiment was repeated two more times, and the results are shown in S4 Fig. (C) RNA was extracted using RNAiso Plus (Takara) from tissues of a 1-month-old Holstein calf. Primary chondrocytes were prepared from the ear cartilage and cultured as micromass. Five days later, RNA was extracted from the micromass using an RNeasy kit (QIAGEN, Valencia, CA, USA). RT-PCR was performed using a standard method and PCR primers given in S8 Table. PCR products were resolved in a 2% agarose gel. Lane M, 100-bp DNA ladder; lane 1, bone marrow just under the growth plate of a femur; lane 2, growth plate cartilage of the femur; lane 3, ear cartilage; lane 4, micromass culture from the ear cartilage. (D) In situ hybridization of Fgd3 on the femur of a 3-week-old C57BL/6 mouse. Sense and antisense RNA probes correspond to nt. 129–956 of the mouse Fgd3 cDNA (NM_015759).
Table 1.
Deviations of the skeletal measurements of the FGD3 Cys-171 homozygotes and a non-carrier cow from normal growth curves.
Table 2.
Effect of the risk allele on carcass traits.