Chromosome painting reveals conserved centromere oligonucleotide sequences and comparable karyotypes across diverse Citrus species

Abstract

The centromere plays a pivotal role in the karyotype of citrus chromosomes. However, the development of markers capable of distinguishing individual chromosomes remains a challenge. Oligo-FISH provides an efficient method for generating citrus centromere-specific markers. Accurate identification of centromere positions is a prerequisite for marker development. In this study, centromere locations on each chromosome were recalibrated using previously published ChIP-seq data of CsCENH3, aligned with the high-quality sweet orange genome (SWO v3). A total of 16,827 45-nucleotide oligos spanning nine centromeric regions were screened, with each region containing between 868 and 5,965 oligos, yielding an approximate density of one oligo per kilobase. Oligos from the centromere regions of chromosomes 1 (Chr1) and 4 (Chr4) were randomly selected to synthesize centromere-specific probes. Dual-color FISH assays on mitotic and meiotic chromosomes revealed distinct signals from each probe on homologous chromosomes. These signals coincided with those from the previously identified centromeric marker CL34contig88, demonstrating the ability of the probes to differentiate centromeres of individual chromosomes. Furthermore, chromosome painting was conducted across several citrus species, including Citrus sinensis, C. reticulata, C. limon, C. grandis, Fortunella japonica, and Poncirus trifoliata, with centromeric signals for Chr1 and Chr4 observable in all species. Chr1 and Chr4 exhibited characteristics of submetacentric and metacentric chromosomes, respectively, based on arm ratios, reflecting the conserved karyotypic structure of these chromosomes across citrus species and their consistent centromeric oligo sequences. These findings underscore the potential of centromere-specific probes in advancing citrus cytology and provide a robust foundation for exploring centromeric sequence evolution in citrus.

Keywords

Citrus / Centromere marker / Oligo-FISH / Karyotype / Homologous chromosome

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Funding

National Natural Science Foundation of China(32202432)

Natural Science Foundation of Hubei Province(2022CFB675)

Agriculture Research System of China(CARS-26)

National Natural Science Foundation of China(U23A20203)

National Key Research & Development Program of China(2024YFD1200501)

RIGHTS & PERMISSIONS

The Author(s)