Large-scale detection and characterization of interchromosomal rearrangements in normozoospermic bulls using massive genotype and phenotype data sets

Abstract

In this paper, we developed a highly sensitive approach to detect interchromosomal rearrangements in cattle by searching for abnormal linkage disequilibrium patterns between markers located on different chromosomes in large paternal half-sib families genotyped as part of routine genomic evaluations. We screened 5571 families of artificial insemination sires from 15 breeds and revealed 13 putative interchromosomal rearrangements, 12 of which were validated by cytogenetic analysis and long-read sequencing. These consisted of one Robertsonian fusion, 10 reciprocal translocations, and the first case of insertional translocation reported in cattle. Taking advantage of the wealth of data available in cattle, we performed a series of complementary analyses to define the exact nature of these rearrangements, investigate their origins, and search for factors that may have favored their occurrence. We also evaluated the risks to the livestock industry and showed significant negative effects on several traits in the sires and in their balanced or aneuploid progeny compared with wild-type controls. Thus, we present the most comprehensive and thorough screen for interchromosomal rearrangements compatible with normal spermatogenesis in livestock species. This approach is readily applicable to any population that benefits from large genotype data sets, and will have direct applications in animal breeding. Finally, it also offers interesting prospects for basic research by allowing the detection of smaller and rarer types of chromosomal rearrangements than GTG banding, which are interesting models for studying gene regulation and the organization of genome structure.

Figure 1.

A novel approach to detect interchromosomal rearrangements (IRs) using SNP array genotypes in large half-sib families. (A) Regression curve of linkage disequilibrium (LD) significance thresholds (P = 0.001) obtained from series of 10,000 simulations. Blue dots indicate the results of simulations with 10 progeny group sizes ranging from 30 to 1000. Orange dots point to the maximal significant LD observed for 13 paternal half-sib families. (B) Circos plot (Yu et al. 2018; yimingyu.shinyapps.io/shinycircos/) representing the 13 rearrangements found. (C) Whole-genome LD map for a bull assumed to have an IR between BTA3 and BTA8. (D) Detail of the LD map between markers from these two chromosomes.

Figure 2.

Validation of 12 IRs by cytogenetic analyses. (A) FISH mapping on cultured fibroblasts from four daughters of Nt with BAC clones located in the translocated segment from BTA4 (labeled in red) and in the centromeric region of BTA8 (labeled in green). From left to right, these animals show a normal karyotype, a balanced rearrangement, a partial BTA4 trisomy, and a partial BTA4 monosomy. (B) Schematic representation of the rearranged chromosomes based on GTG-banding karyotypes. Chromosomes with a loss or gain of material are shown in yellow and green, respectively. The schematics of the original chromosomes are available in Supplemental Figures S1D–S13D. (C) Location of the breakpoints on a theoretical chromosome. Coordinates are expressed as a percentage of chromosome length. Note the presence of 13 out of 24 breakpoints in the third quarter, whereas only six would be expected by chance.

Figure 3.

Analysis of conception rate, nonreturn rate (NRR), and juvenile mortality. (A) Conception rate of first artificial inseminations (1^st AI) of 2900 bulls with nulliparous females, conventional semen, expressed as SD from the mean to allow comparison between bulls from different breeds. Arrowheads indicate carriers of the following IRs: red for reciprocal translocations, yellow for insertional translocation, gray for reciprocal with derivative loss, and green for Robertsonian translocation. (B) NRR for five affected Holstein bulls (i.e., the proportion of females that were not reinseminated after an insemination with the semen of the bull in question) and mean NRR of 2152 Holstein bulls. Two extreme bulls, Qu and Ma, are highlighted in red and yellow. (C) Mortality rates over the first year of life among the daughters of 2854 bulls considered, expressed as the SD from the mean of their respective breeds (same color code as in vignette A). (D) Distribution of mortality rates at 1 yr of age for Nt's progeny (n below bars). (*) Fisher comparison with wild-type group P < 0.05, (**) P < 0.01.

Figure 4.

Comparison of the performance of daughters with normal and abnormal karyotypes among the progeny of nine IR-affected bulls. Cohort sizes are given below each bar. Significance levels refer to comparisons with wild type groups. Student's t-test for first AI, Fisher's test for the other traits): (*) P < 0.1; (**) P < 0.05; (***) P < 0.01.

Figure 5.

Contribution of PacBio CLR sequencing to the characterization of IR break points and fusion points. (A) Sequence coverage plots around the breakpoints of t(3;8) supporting a heterozygous deletion of ∼2 Mb on BTA3 of bull Ma. (B) Sequence coverage plots around the breakpoints of t(24;29),-der29 supporting a loss of the derivative chromosome of bull Ou. For vignettes A and B, control alignments are provided in Supplemental Figures S6I and S10I. (C) Read alignment around the second breakpoint of inv ins(8,4) at 76.6 Mb on BTA4 (bull Nt). Split reads that also map to BTA8 and to BTA4 around position 65.9 Mb are colored in green and yellow, respectively. Note that the breakpoint resulted in the disruption of CCM2. (D, top) Read alignment around the breakpoint of t(1;11) on BTA11 with split read aligning to BTA1 colored in light green (bull Ja). (D, bottom) Details of the genes and putative TADs (according to Wang et al. 2018) located around the breakpoint of t(1;11) on BTA11.