Analysis of copy number variations among diverse cattle breeds

Abstract

Genomic structural variation is an important and abundant source of genetic and phenotypic variation. Here, we describe the first systematic and genome-wide analysis of copy number variations (CNVs) in modern domesticated cattle using array comparative genomic hybridization (array CGH), quantitative PCR (qPCR), and fluorescent in situ hybridization (FISH). The array CGH panel included 90 animals from 11 Bos taurus, three Bos indicus, and three composite breeds for beef, dairy, or dual purpose. We identified over 200 candidate CNV regions (CNVRs) in total and 177 within known chromosomes, which harbor or are adjacent to gains or losses. These 177 high-confidence CNVRs cover 28.1 megabases or approximately 1.07% of the genome. Over 50% of the CNVRs (89/177) were found in multiple animals or breeds and analysis revealed breed-specific frequency differences and reflected aspects of the known ancestry of these cattle breeds. Selected CNVs were further validated by independent methods using qPCR and FISH. Approximately 67% of the CNVRs (119/177) completely or partially span cattle genes and 61% of the CNVRs (108/177) directly overlap with segmental duplications. The CNVRs span about 400 annotated cattle genes that are significantly enriched for specific biological functions, such as immunity, lactation, reproduction, and rumination. Multiple gene families, including ULBP, have gone through ruminant lineage-specific gene amplification. We detected and confirmed marked differences in their CNV frequencies across diverse breeds, indicating that some cattle CNVs are likely to arise independently in breeds and contribute to breed differences. Our results provide a valuable resource beyond microsatellites and single nucleotide polymorphisms to explore the full dimension of genetic variability for future cattle genomic research.

Figure 1.

Cattle copy number variation and segmental duplication regions display a local tandem distribution pattern. CNV regions (177 events, 28 Mb, ∼1% of the bovine genome) reported by 90 array CGH experiments are shown above the chromosomes in green (gain), red (loss), and dark blue (both). The bar height represents their frequencies: short (appeared in 1 sample), median (≥2 samples), and tall (≥5 samples). Segmental duplications (94.4 Mb, 3.1% of the bovine genome) predicted by two independent computational approaches are illustrated on the chromosomes in red (WSSD), blue (WGAC), or purple (both). The patterns are depicted for all duplications for ≥5 kb in length and ≥90% sequence identity. The gaps in the assembly are represented on the chromosomes as white ticks. For clarity, distribution patterns with the unassigned sequence contigs (chrUnAll) are shown separately in Supplemental Figure S1.

Figure 2.

FISH confirmation. Examples of interphase two-color FISH include three BAC clones. Clones 338M16, 117G16, and 259A1 (red) were identified in CNVR31, 56 and 70, corresponding to WC1.1, ULBP17, and ULBP21, and ATP-binding cassette transporter C4, respectively. Increased signal intensity was confirmed using cohybridization with a unique control BAC clone (297K6, blue) in the same nucleus. These BACs produced variable signal count and/or intensity, 338M16: 3, 2, and 2 and 3; 117G16: 2, 2 and 3, and 2 and 259A1: 2, 3, and 3 in these three cell lines, respectively (for summary, see also Supplemental Table S8). Tandem distribution patterns were most frequently observed. The results of all FISH experiments are available online at http://bfgl.anri.barc.usda.gov/cattleCNV/.

Figure 3.

Colocalization analysis of cattle CNV regions and segmental duplications. Relationships between flanking distances and numbers of cattle CNV regions overlapped with all SDs (A) or 1020 high-confidence SD regions (B).

Figure 4.

A detailed analysis of CNVR56 corresponding to the major ULBP gene cluster (chr9:90,150,000–90,550,000). On the chromosome, cattle SDs are predicted by WSSD (brown) and WGAC (blue and red represent intra- and interchromosomal WGAC duplications). Above chromosome are gain events (green = three copies and dark green = four copies), while below are loss events (one copy) of corresponding regions arranged vertically according to their relative log₂ ratios from the chromosomal baseline. Limousin9 displays both loss and gain events (labeled as blue) within this region. The UCSC gene and expression tracks are shown at the bottom. Five light blue vertical lines represent potential breakpoint regions. ANG, Angus; BAN, Brangus; CHL, Charolais; GIR, Gir; GLB, Gelbvieh; GZR, Guzerat; HFD, Hereford; HOL, Holstein; LMS, Limousin; NDA, N'Dama; RAN, Red Angus; ROM, Romosinuano; and SMT, Simmental.