De novo copy number variations in cloned dogs from the same nuclear donor

Abstract

Background: Somatic mosaicism of copy number variants (CNVs) in human body organs and de novo CNV event in monozygotic twins suggest that de novo CNVs can occur during mitotic recombination. These de novo CNV events are important for understanding genetic background of evolution and diverse phenotypes. In this study, we explored de novo CNV event in cloned dogs with identical genetic background.

Results: We analyzed CNVs in seven cloned dogs using the nuclear donor genome as reference by array-CGH, and identified five de novo CNVs in two of the seven clones. Genomic qPCR, dye-swap array-CGH analysis and B-allele profile analysis were used for their validation. Two larger de novo CNVs (5.2 Mb and 338 Kb) on chromosomes X and 19 in clone-3 were consistently validated by all three experiments. The other three smaller CNVs (sized from 36.1 to 76.4 Kb) on chromosomes 2, 15 and 32 in clone-3 and clone-6 were verified by at least one of the three validations. In addition to the de novo CNVs, we identified a 37 Mb-sized copy neutral de novo loss of heterozygosity event on chromosome 2 in clone-6.

Conclusions: To our knowledge, this is the first report of de novo CNVs in the cloned dogs which were generated by somatic cell nuclear transfer technology. To study de novo genetic events in cloned animals can help understand formation mechanisms of genetic variants and their biological implications.

Figure 1

Whole-genome plot of CNVs and qPCR validation. (A) Whole-genome plot of CNVs identified between Labrador retriever and Boxer genomes. Nine CNV regions (7 CNV-Gs, red arrow; 2 CNV-Ls, green arrow) were randomly selected for qPCR validation. The X-axis represents individual chromosomes and the Y-axis represents signal intensity ratios (Labrador retriever/Boxer) on a log2 scale. (B) Log2ratio plot around the nine selected CNV regions. Black bars represent primer position for qPCR validation. The X-axis represents genomic position (Mb) and the Y-axis represents signal intensity ratios (Labrador retriever/Boxer) on log2 scale. (C) Three primer sets located in the CNV-L regions detected copy loss, and the other eight primer sets located in the CNV-G regions detected copy gain. The red and green line represent detection criterion for copy gain and copy loss, respectively. The Y-axis represents fold change based on the Boxer as the calibrator.

Figure 2

Dye-swap analysis for the clone-3 genome. (A) Genome-wide signal intensity plot from clone-3. Four CNV regions were identified in the clone-3 genome (red arrow). The X-axis represents individual chromosomes and the Y-axis represents signal intensity ratio (clone-3/donor) on a log2 scale. (B) Log2ratio plots around the four CNV regions in clone-3. All CNV-Gs identified in clone-3 (red line) show flipped signal intensity plots (green line) in the dye-swap hybridization. The X-axis represents genomic position (Mb), and the Y-axis represents signal intensity ratio on a log2 scale.

Figure 3

Examples of allelic imbalance in clone-3. (A) Upper panel, Log2ratio plot on chromosome X in clone 3. A 5.2-Mb copy number gain region on chromosome X was identified in clone-3 (black arrow). The X-axis represents genomic position (Mb) and the Y-axis represents signal intensity ratio on a log2 scale. Lower panel, B-allele plot of the proximal part of chromosome X (dashed box of upper panel). A heterozygous cluster was detected in chromosome X (black arrow), and this allelic imbalance has the same genomic position as the copy gain region. (B) Upper panel, Log2ratio plot on chromosome 19 in the clone-3. A 338-Kb copy number gain region on chromosome 19 was identified in clone-3 (black arrow). Lower panel, B-allele plot of the dashed box on chromosome 19. A heterozygous cluster was detected on chromosome 19 (black arrow), and this allelic imbalance has same position as the copy gain region.

Figure 4

Example of the LOH event in clone-6. A 37-Mb de novo LOH event was identified on the distal end of chromosome 2 in clone-6 that was not detected in the donor or other clones. The X-axis represents genomic position (Mb), and the Y-axis represents B-allele intensity.

Figure 5

Multiple-point genomic qPCR validation. Left, Log2ratio plot on chromosome X (A), chromosome 19 (B), chromosome 15 (D), chromosome 32 (E) in clone 3 and chromosome 2 (C) in clone-6. Red and green bars represent primer position within the CNV-Gs and CNV-Ls, respectively. Black bars represent primer position outside the expected CNV breakpoint. The X-axis represents genomic position (Mb) and the Y-axis represents signal intensity ratio on a log2 scale. Right, multiple-point genomic qPCR validation. The Y-axis represents fold-change based on the nuclear donor as calibrator.