Selection signatures in goats reveal copy number variants underlying breed-defining coat color phenotypes

Abstract

Domestication and human selection have formed diverse goat breeds with characteristic phenotypes. This process correlated with the fixation of causative genetic variants controlling breed-specific traits within regions of reduced genetic diversity, so called selection signatures or selective sweeps. Using whole genome sequencing of DNA pools (pool-seq) from 20 genetically diverse modern goat breeds and bezoars, we identified 2,239 putative selection signatures. In two Pakistani goat breeds, Pak Angora and Barbari, we found selection signatures in a region harboring KIT, a gene involved in melanoblast development, migration, and survival. The search for candidate causative variants responsible for these selective sweeps revealed two different copy number variants (CNVs) downstream of KIT that were exclusively present in white Pak Angora and white-spotted Barbari goats. Several Swiss goat breeds selected for specific coat colors showed selection signatures at the ASIP locus encoding the agouti signaling protein. Analysis of these selective sweeps revealed four different CNVs associated with the white or tan (AWt), Swiss markings (Asm), badgerface (Ab), and the newly proposed peacock (Apc) allele. RNA-seq analyses on skin samples from goats with the different CNV alleles suggest that the identified structural variants lead to an altered expression of ASIP between eumelanistic and pheomelanistic body areas. Our study yields novel insights into the genetic control of pigmentation by identifying six functionally relevant CNVs. It illustrates how structural changes of the genome have contributed to phenotypic evolution in domestic goats.

Fig 1. Manhattan plots showing −ZHp values from 20 diverse goat breeds.

The red horizontal line indicates the chosen significance threshold of −ZHp = 4. Each dot represents a 150 kb window. Each plot contains 29 autosomes and two unplaced scaffolds representing the X chromosome. Selection signatures co-localizing with known coat color genes are marked with arrows.

Fig 2. CNVs at the KIT locus.

The coverage plot of bezoars (BEZ) does not show any copy number variation and represents the wildtype allele. In the Pak Angora breed (ANG), the coverage plot shows a triplication of ~100 kb downstream of the KIT gene. In the Barbari breed (BAR), the same region is duplicated. The Barbari allele shows a complex rearrangement involving the insertion of a ~23 kb genome segment originating at 89.2 Mb into the duplicated sequence at ~70.9 Mb with the simultaneous deletion of ~16 kb of KIT sequence. Please note that the coverage at ~89.2 Mb corresponds to three times the average. One genome equivalent corresponds to the wildtype sequence at ~89.2 Mb. Read-pair information indicated that the other two genome equivalents are inserted into the duplicated sequence at ~70.9 Mb (S4 Fig). The dashed red line indicates the average coverage across the whole genome of each pool-seq dataset.

Fig 3. CNVs at the ASIP locus.

A Coverage plots of the ASIP locus in different goat breeds reveal four different CNVs. The bezoar (BEZ) coverage plot shows uniform coverage and is characteristic for the wildtype allele (A^bz). Underneath, four different mutant ASIP alleles associated with different CNVs are illustrated. The line on top of each plot schematically indicates the most likely configuration of these mutant alleles derived from the available short-read sequence information (S4 Fig). The dashed red line indicates the average coverage across the whole genome of each breed. B Schematic drawings and C representative photographs illustrating the coat color phenotypes of the studied breeds. The photo of the bezoar was obtained during summer, when the dark stripes at the collar and the belly are much less pronounced than in the winter coat. Note that some of the patterns show an exactly inverse distribution of eumelanin and pheomelanin. For example, goats with the A^sm allele have white (pheomelanistic) facial stripes and legs, while goats with the A^b or A^pc alleles have black (eumelanistic) facial stripes and legs.

Fig 4. ASIP mRNA expression and identified transcripts in skin.

A Representative photographs of the five sampled goat breeds. The biopsy sites are numbered and indicated by red circles. B Trimmed mean of M (TMM) values of ASIP mRNA expression were determined from RNA-seq data for each sample. The colors of the bars correspond to the pigmentation of the skin samples. Please note that the Valais Blackneck goat (VAG) has a black base color that is independent of the ASIP gene. This goat has a white spotting phenotype and lacks melanocytes in its caudal half. The low ASIP expression in the unpigmented white skin sample of this goat underscores the difference to the pheomelanistic pale white pigmentation in other goats. C ASIP transcript isoforms in pheomelanistic skin samples from goats with different ASIP alleles. Transcript isoforms X1 and X2 correspond to the RefSeq accessions XM_018057735.1 and XM_018057736.1. CNV breakpoints of the A^b, A^Wt, and A^pc alleles are indicated.