Genome data uncover four synergistic key regulators for extremely small body size in horses

Abstract Background Miniature size in horses represents an extreme reduction of withers height that originated after domestication. In some breeds, it is a highly desired trait representing a breed- or subtype-specific feature. The genomic changes that emerged due to strong-targeted selection towards this distinct type remain unclear. Results Comparisons of whole-genome sequencing data from two Miniature Shetland ponies and one standard-sized Shetland pony, performed to elucidate genetic determinants for miniature size, revealed four synergistic variants, limiting withers height to 34.25 in. (87 cm). Runs of homozygosity regions were detected spanning these four variants in both the Miniature Shetland ponies and the standard-sized Shetland pony. They were shown to be characteristic of the Shetland pony breed, resulting in a miniature type under specific genotypic combinations. These four genetic variants explained 72% of the size variation among Shetland ponies and related breeds. The length of the homozygous regions indicate that they arose over 1000 years ago. In addition, a copy number variant was identified in DIAPH3 harboring a loss exclusively in ponies and donkeys and thus representing a potential height-associated variant. Conclusion This study reveals main drivers for miniature size in horses identified in whole genome data and thus provides relevant candidate genes for extremely short stature in mammals

Additional file 1 of Genome data uncover four synergistic key regulators for extremely small body size in horses

Mapping statistics of whole-genome sequencing data. In total 32 samples of equids of different populations were analyzed in this study. Sequence read achieve ID, sequencing parameters and mean coverage are shown. (DOCX 15 kb

Additional file 12 of Genome data uncover four synergistic key regulators for extremely small body size in horses

Primers and assays for validation of variants in ROH regions. SNVs in ROH regions specifically filtered for their functional effects were genotyped using Kompetitive Allele Specific PCR (KASP). Primer sequences, annealing temperature and number of PCR cycles are shown. (DOCX 23 kb

Additional file 3 of Genome data uncover four synergistic key regulators for extremely small body size in horses

Comparison of ROH regions with Fst. The highest 1% Fst investigated for ROHs in the same region found in three Shetland ponies. In total 439 overlapping regions were identified. (XLSX 20 kb

Additional file 7 of Genome data uncover four synergistic key regulators for extremely small body size in horses

Characterization of potential Shetland pony-specific CNV regions. The size of the 97 CNV regions identified in the intersection of all four CNV detection runs for Shetland pony-specific CNVs is shown in base pairs (bp) per chromosome. The number and size of all CNV regions varies among the chromosomes. On eleven chromosomes, no CNV region could be found in the overlapping results. (TIFF 16611 kb

Additional file 11 of Genome data uncover four synergistic key regulators for extremely small body size in horses

General linear model analysis testing for genotypic and allelic effects on cannon bone circumference. The effect on cannon bone circumference is shown for all three polymorphisms genotyped in 52 Shetland ponies whose cannon bone circumference measurements were available. (DOCX 13 kb

Additional file 13 of Genome data uncover four synergistic key regulators for extremely small body size in horses

Primers for validation of two homozygous deletions found in Shetland ponies. Multiplex-PCR reverse primers located in the deletion and proximal of the deletion were used for targeting the two deletions. Product sizes and annealing temperatures are displayed. (DOCX 12 kb

Additional file 5 of Genome data uncover four synergistic key regulators for extremely small body size in horses

Functional classification of genes. Variants identified in ROH regions in the two Miniature Shetland ponies and one standard sized Shetland pony, which were estimated to be deleterious (SIFT prediction) were investigated for their genomic positions in genes. In addition, variants, which showed an exclusively homozygous mutant genotype in all three Shetland ponies, were added to the analysis. Human orthologue genes were functionally classified using DAVID Functional Annotation Tool. (XLSX 23 kb

Additional file 8 of Genome data uncover four synergistic key regulators for extremely small body size in horses

Intersection of potential Shetland pony-specific CNV regions (CNVRs). The overlap of four CNV detection results for Shetland pony-specific CNVRs shows 97 CNVR harboring 14 different genes. (DOCX 18 kb