Genomic analyses of wild argali, domestic sheep, and their hybrids provide insights into chromosome evolution, phenotypic variation, and germplasm innovation

Abstract

Understanding the genetic mechanisms of phenotypic variation in hybrids between domestic animals and their wild relatives may aid germplasm innovation. Here, we report the high-quality genome assemblies of a male Pamir argali (O ammon polii, 2n = 56), a female Tibetan sheep (O aries, 2n = 54), and a male hybrid of Pamir argali and domestic sheep, and the high-throughput sequencing of 425 ovine animals, including the hybrids of argali and domestic sheep. We detected genomic synteny between Chromosome 2 of sheep and two acrocentric chromosomes of argali. We revealed consistent satellite repeats around the chromosome breakpoints, which could have resulted in chromosome fusion. We observed many more hybrids with karyotype 2n = 54 than with 2n = 55, which could be explained by the selfish centromeres, the possible decreased rate of normal/balanced sperm, and the increased incidence of early pregnancy loss in the aneuploid ewes or rams. We identified genes and variants associated with important morphological and production traits (e.g., body weight, cannon circumference, hip height, and tail length) that show significant variations. We revealed a strong selective signature at the mutation (c.334C > A, p.G112W) in TBXT and confirmed its association with tail length among sheep populations of wide geographic and genetic origins. We produced an intercross population of 110 F₂ offspring with varied number of vertebrae and validated the causal mutation by whole-genome association analysis. We verified its function using CRISPR-Cas9 genome editing. Our results provide insights into chromosomal speciation and phenotypic evolution and a foundation of genetic variants for the breeding of sheep and other animals.

Figure 1.

Research framework, hybridization scheme, karyotype and chromosomal painting, statistics of homologs and gene families, phylogenetic tree, and synteny landscape. (A) Roadmap of initiation, technical systems, analysis and experiments, the results, and purposes of this study. (B) The wild (argali, O. ammon)-domestic sheep hybridization scheme. The F₁-hybrid is subsequently backcrossed to Tibetan sheep to produce the progenies, and later hybrid generations are produced by intercrossing and backcrossing. (C) Karyotype and chromosomal painting of Tibetan sheep, argali, and the F₁-hybrid. DNA probes (GNAQ: green signal; STK39: red signal) were used to label the long arm and short arm of Chromosome 2 and two acrocentric chromosomes. DNA was stained with 4,6-diamidino-2-phenylindole (DAPI; blue). (D) Number of homologs among the Marco Polo subspecies of argali (O. ammon polii), sheep (O. aries), and goat (Capra hircus). (E) Proportions of different gene numbers in 19,917 gene families in the nine mammal species. (F) Phylogenetic tree of nine mammal species based on 10,043 single-copy orthologous genes. Numbers in green to the right of nodes are the divergence times and their 95% confidence intervals (95% CIs). Values next to the branches represent the numbers of gene family expansions/contractions. (MYA) Million years ago. (G) Genome-wide syntenic relationship among goat (C. hircus), sheep (O. aries), the Marco Polo subspecies of argali (O. ammon polii), and the argali-domestic sheep F₁-hybrid. Syntenic blocks involved in chromosome fusion are marked with colored ribbons.

Figure 2.

Evolution of three metacentric chromosomes of domestic sheep and the proposed molecular basis of fertility for the F₁-hybrid. (A) Repeated sequences surrounding the breakpoints on the chromosomes involved in chromosome fusions in goat (C. hircus), argali (O. ammon polii), and domestic sheep (O. aries). (B) Schematic representation of chromosomal segregation and pairing (metacentric Chromosome 2 of domestic sheep and two acrocentric pseudochromosomes LG04 and LG07 from argali) during hybridization between argali (O. ammon polii) and domestic sheep (O. aries). Six different types of zygotes can be produced by the F₁-hybrid, giving only one normal and one balanced embryo after fertilization with domestic sheep. (C) Proposed molecular mechanisms underlying the preference of F₁-hybrid oocytes to generate gametes with 27 chromosomes rather than 28 chromosomes. That is, selfish centromeres (red/yellow circles) prefer the egg side when attached to the metaphase I spindle (purple) (Nikalayevich and Verlhac 2021). Chromosomes are in blue/pink, oocyte cytoplasm is in gray, and the zone of cortical proximity is in darker gray.

Figure 3.

Genetic structure and genetic diversity of Tibetan sheep, argali, and the hybrids. (A) Plots of principal component analysis (principal components 1 and 2) for 425 individuals. (B) Nucleotide diversity of argali, Tibetan sheep, and the hybrids. (C) Population genetic structure of argali, Tibetan sheep, and the hybrids inferred using the ADMIXTURE program. The vertical solid line indicated the separations between argali, Tibetan sheep, and the hybrids.

Figure 4.

Identification of candidate genes related to the tail length and body weight of domestic sheep. (A,D) Manhattan and quantile–quantile (Q-Q) plots of association signals for the traits of tail length and body weight. The dashed lines colored purple represent the significant thresholds with –log(P-value) = 8.88. SNPs near the peaks with different significant values are marked in red (maximum –log₁₀(P)) and brown (–log₁₀(P) ≥ 7). Functional genes surrounding the peaks are indicated by green boxes. (B,E) Linkage disequilibrium (LD) plots in the regions surrounding the TBXT and HMGN1 genes. (C,F) Boxplots for the tail length associated with SNP (Chr 8: 88,341,610 C/A) and for the body weight associated with SNP (Chr 17: 26,391,397 T/G). The lines in boxes denote the median values; box limits are the upper and lower quartiles, and whiskers show the range of the data; n indicates the number of individuals with the same genotype. Significance of differences between the phenotypic values: (*) P < 0.05, (**) P < 0.01, determined by the Mann–Whitney U test.

Figure 5.

Genome-wide selection sweep test, genome-wide association study (GWAS) analysis, and functional validation of the TBXT gene. (A) Computerized tomography (CT) scanning of the caudal vertebrae number for fat-rumped (Kazakh, Duolang, Bashibai, and Bayinbuluke) and fat-tailed (Hetian and Cele Black) sheep. (B) Boxplots for the number of caudal vertebrae between the fat-rumped and fat-tailed sheep breeds. (C) Genome-wide selection sweep tests (the F_ST-based and π-ratio-based methods) for the tail configuration between fat-rumped and fat-tailed sheep breeds. (D) Calculation of Tajima's D-values and π and F_ST values for SNPs in the candidate genomic region Chr 8: 87.56–87.88 Mb between the fat-rumped and fat-tailed sheep breeds. (E) Amino acid (i.e., p. G112W in the TBXT gene) alternation among different mammalian species. (F) Manhattan and Q-Q plots of association signals for the tail length and the number of caudal vertebrae in domestic sheep. The dashed line represents the significance threshold (–log₁₀(0.05/total SNPs) = 8.56). The gene structure of the TBXT gene is shown in green, and the exon regions are shown in blue at the bottom. (G) Boxplots for the tail length and the number of caudal vertebrae associated with the three genotypes of c.334C > A in TBXT in the F₂ intercross population of 110 individuals. (H) Different phenotypes in the tail configuration for individuals with the CC and AA genotypes of c.334C > A in TBXT; picture credit: Wen-Rong Li. (I) Sequences of sgRNA targeting exon 2 of the TBXT gene and 123-bp single-strand DNA oligonucleotides (ssODNs) for homologous recombination-mediated repair in CRISPR-Cas9 experimentation. (J) Target sequences in wild-type (WT) and 19 mosaic mutant merino sheep. Target mutations are indicated in the red bold font. (K) Different phenotypes of the tail configuration for WT and mutant merino sheep (GM079); picture credit: Wen-Rong Li. (L) CT scanning of the tail configuration for WT, GM079, and the offspring of GM079. (M) Boxplots for the tail length and the number of caudal vertebrae of the 19 WT sheep, 14 C334A target mutation (TM) merino sheep, and six sheep with a short indel (KO) in the TBXT gene. (N) Boxplots for the number of caudal vertebrae of the 19 WT sheep and 24 offspring of GM079 (i.e., 19 heterozygotes with the C334A target [TM/+] and five heterozygotes with an 8-bp deletion [KO/+]).