Stable minichromosome and functional neocentromere derived from rye 7R chromosome arm

The study of newly formed centromere with stable transmission ability can provide theoretical guidance for the construction of artificial chromosomes. More neocentromeres are needed to study the mechanisms of their formation.

In this study, a minichromosome 7RLmini was derived from the progeny of wheat-rye 7R monosomic addition line. The minichromosome 7RLmini contained subtelomeric tandem repeats pSc119.2 and rye-specific pSc200, and it came from the distal region of the long arm of 7R chromosome. A neocentromere was formed in this minichromosome, and it did not contain centromeric repetitive sequences CCS1 and pAWRC.1. CENH3 ChIP-seq and ssDRIP-seq data confirmed that a 2.4 Mb segment from the rye 7R chromosome was involved in the neocentromere formation and enrichment of R-loops in this region. Within the 2.4 Mb segment, the GC content was higher that of AT, and a major binding position of CENH3 nucleosomes was identified on a 6 kb unknown LTR retrotransposon TE00002448. This unknown LTR retrotransposon was rye-specific and distributed through all the arms of rye chromosomes. The minichromosome exhibited stable generational transmission.

A minichromosome from rye 7R with neocentromere was obtained in this study and the neocentromere was formed at the position far away from its native equivalent. This minichromosome provides additional material for the research on the mechanism of neocentromere formation. We theorize that R-loops and transposable element might be involved in the positioning of CENH3 nucleosomes in a functional neocentromere.