Phased T2T horse and donkey assemblies from a mule reveal peculiar equid centromere evolution.

Research Overview

• This study presents complete, telomere-to-telomere genome assemblies of a Thoroughbred horse and a donkey obtained from sequencing their mule offspring.
• The new assemblies deliver unprecedented resolution of complex genomic regions and provide deep insights into the unusual evolution of centromeres in equids (horse family).

Background and Objectives

• Equids include horses, donkeys, and their hybrids like mules, which have interesting genomic characteristics, especially regarding centromeres—the chromosome regions essential for proper cell division.
• Centromeres in equids are unusual because their function can be uncoupled from satellite DNA, repetitive sequences typically found at centromeres in other species.
• Previous equid genome assemblies had gaps, especially in repetitive and complex regions like satellite arrays, duplications, and telomeres, limiting understanding of centromere structure and evolution.
• The objective was to produce complete telomere-to-telomere (T2T) genome assemblies of a horse and donkey from their mule offspring in order to resolve these challenging regions and study centromere biology in detail.

Methodology

• Sequencing was done on a mule, which carries both horse and donkey chromosomes, enabling phased genome assembly of the parental species.
• The assemblies were constructed from telomere-to-telomere for all chromosomes, meaning no gaps remained even in highly repetitive or duplicated sequences.
• Subsequent annotation was performed to identify regions such as:
  • Satellite DNA arrays
  • Duplications
  • Telomeres
  • Centromeres (both satellite-based and satellite-free)
  • Non-centromeric satellite loci
• Detailed molecular characterization focused on centromere proteins, specifically CENP-A (an epigenetic marker that determines centromere identity) and CENP-B (a centromere-associated protein), including their binding domains and interactions.
• Comparative genomic analyses were conducted to explore differences in satellite DNA and centromere positioning between horse and donkey chromosomes, illuminating evolutionary processes.

Key Findings

• These assemblies are the first complete T2T references accepted by NCBI for horse and donkey, filling in previously inaccessible or ambiguous genomic regions.
• Both satellite-based centromeres (containing repetitive satellite DNA) and satellite-free centromeres were identified, demonstrating centromere plasticity—meaning centromere structure and sequence can vary considerably among equids.
• Non-centromeric satellite DNA loci were also annotated, providing a more complete map of the repetitive landscape in equid genomes.
• The characteristic association between CENP-A and CENP-B proteins observed in many model organisms was absent in equids:
  • CENP-A binding domains were highly variable, suggesting epigenetic factors alone may drive centromere identity more than specific DNA sequences.
  • CENP-B protein, typically implicated in binding satellite DNA and stabilizing centromeres, was not associated with CENP-A in these species, highlighting unique centromeric biology in horses and donkeys.
• Comparisons of satellite repeats and centromere positions revealed an accelerated rate of chromosomal rearrangements and karyotypic reshuffling in the equid lineage, contributing to their genomic diversity and evolution.

Significance and Implications

• The complete genome references provide foundational resources for equine genomics research, aiding studies from veterinary medicine to evolutionary biology.
• The findings uncover novel aspects of centromere biology, including the flexibility of centromere composition and function in mammals, challenging existing paradigms based mainly on traditional model organisms.
• Understanding equid centromere evolution can shed light on chromosome stability, hybrid fertility (like mules), and species adaptation mechanisms.
• The assemblies support large-scale projects such as the Equine Pangenome Project by providing high-quality reference genomes for multiple equid species.