Remapping of the belted phenotype in cattle on BTA3 identifies a multiplication event as the candidate causal mutation

Background: It has been known for almost a century that the belted phenotype in cattle follows a pattern of dominant inheritance. In 2009, the approximate position of the belt locus in Brown Swiss cattle was mapped to a 922-kb interval on bovine chromosome 3 and, subsequently, assigned to a 336-kb haplotype block based on an animal set that included, Brown Swiss, Dutch Belted (Lakenvelder) and Belted Galloway individuals. A possible candidate gene in this region i.e. HES6 was investigated but the causal mutation remains unknown. Thus, to elucidate the causal mutation of this prominent coat color phenotype, we decided to remap the belted phenotype in an independent animal set of several European bovine breeds, i.e. Gurtenvieh (belted Brown Swiss), Dutch Belted and Belted Galloway and to systematically scan the candidate region. We also checked the presence of the detected causal mutation in the genome of belted individuals from a Siberian cattle breed. Results: A combined linkage disequilibrium and linkage analysis based on 110 belted and non-belted animals identified a candidate interval of 2.5 Mb. Manual inspection of the haplotypes in this region identified four candidate haplotypes that consisted of five to eight consecutive SNPs. One of these haplotypes overlapped with the initial 922-kb interval, whereas two were positioned proximal and one was positioned distal to this region. Next-generation sequencing of one heterozygous and two homozygous belted animals identified only one private belted candidate allele, i.e. a multiplication event that is located between 118,608,000 and 118,614,000 bp. Targeted locus amplification and quantitative real-time PCR confirmed an increase in copy number of this region in the genomes of both European (Belted Galloway, Dutch Belted and Gurtenvieh) and Siberian (Yakutian cattle) breeds. Finally, using nanopore sequencing, the exact breakpoints were determined at 118,608,362 and 118,614,132 bp. The closest gene to the candidate causal mutation (16 kb distal) is TWIST2. Conclusions: Based on our findings and those of a previously published study that identified the same multiplication event, a quadruplication on bovine chromosome 3 between positions 118,608,362 and 118,614,132 bp is the most likely candidate causal mutation for the belted phenotype in cattle

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Additional file 5. Nanopore sequencing results. (a) Nanopore reads mapped (minimap2) to the belted candidate region are shown. The shaded parts of the reads highlight unmapped portions of the read that were mapped as a secondary alignment in a separate read. These secondary alignments are highlighted by blue borders. (b) Split-alignment of breakpoint-spanning read visualized by Ribbon [52]. The highlighted read (bold blue line) is shown as a zoom in the lower panel, showing that the beginning of the read is found at the end of the repeated region and the end is found at the beginning, thus illustrating the concatenation of the repeat units found in belted cattle. (c) Exact breakpoints were identified by inspection of the partially mapped reads. The right breakpoint at 118,614,132 bp shows that the unmapped portion of the split-aligned reads starts with a sequence that is located at 118,608,362 bp, which thus defines the left breakpoint

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Additional file 8. Haplotypes of the Russian Yakutian animals checked by qPCR. This file shows 42-SNP haplotypes of the 10 Russian Yakutian animals that were checked by qPCR. As in Additional file 1, SNPs that were excluded from the mapping procedure (MAF < 0.025) are marked in grey color in the first line, and the first five haplotypes represent the most common and extended haplotypes of the European breeds Belted Galloway (BGAhap1 and BGAhap2), Dutch Belted (DBEhap) and Gurtenvieh (GUVhap1 and GUVhap2). Red boxes again indicate common parts of these five haplotypes and represent the four inner candidate haplotypes (IC-Hap1-4, Table 3). Below these common haplotypes, the haplotypes of the 10 Siberian Russian Yakutian animals are grouped as follows: the first seven animals were belted according to phenotype and qPCR, the next animal was belted according to phenotype but non-belted in the qPCR, and the last two animals were non-belted according to phenotype and qPCR. Interestingly, the belted Russian Yakutian (RUY) animals do not share a common haplotype within IC-Hap4, which carries the candidate mutation BeltMulti6kb

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Additional file 2. Alignment of CH240-104M22 and the reference sequence. Pairwise alignment of the BAC-clone CH240-104M22 with the bosTaurus6 reference sequence of the 6-kb candidate region showing almost complete identity

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Additional file 6. Alignment of the repetitive elements at the beginning of the 6-kb candidate segment according to bosTaurus6 and bosTaurus8. The pairwise alignment of the reference sequence of the SINE element ART2A (bosTau6) and the LINE element BovB (bosTau8) at the beginning of the 6-kb candidate segment shows that ART2A is part of BovB

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Additional file 3. TLA results. The genomic region chr3:118,590,000–118,632,000 (bosTau8) is displayed. The arrows indicate the position of the primer sets used for TLA. For the belted animal (GLW54-2), an increased copy number (3 to 4.5 times) was detected in the region indicated by the red rectangle. The y-axis is limited to max. 1000X

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Additional file 7. Gene interaction network. This figure illustrates the interactions between KIT (causal for the belt in pigs), ADAMTS20 (causal for the belt in mice) and TWIST2 (most likely causal for the belt in cattle) in mice. Interaction line colors are as follows: orange: predicted functional relationship, red: physical interactions, purple: co-expression; grey: phenotype (based on mouse genome informatics) and blue: participation in the same reaction within a pathway

MOESM1 of Remapping of the belted phenotype in cattle on BTA3 identifies a multiplication event as the candidate causal mutation

Additional file 1. Inner candidate haplotypes detected by manual analysis of the extended confidence interval. This file shows the 60-SNP haplotypes of the extended candidate interval for all 110 animals that were used for remapping of the belt locus. SNPs that were excluded from the mapping procedure (MAF < 0.025) are marked with grey color in the first line. The black box indicates the 336-kb interval identified by Drögemüller et al. [13]. The first five haplotypes represent the most common and extended haplotypes for Belted Galloway (BGAhap1 and BGAhap2, shown in bright and dark blue), Dutch Belted (DBEhap, shown in beige) and Gurtenvieh (GUVhap1 and GUVhap2, shown in dark and bright green). Red boxes indicate common parts of these five haplotypes and represent the four inner candidate haplotypes (IC-Hap1-4, Table 3). The haplotypes of the animals that were used for remapping are shown below in the following order: (i) Belted Galloway, (ii) Dutch Belted, (iii) Gurtenvieh, (iv) a belted cross between Gurtenvieh and Pinzgauer cattle and (v) non-belted control animals