Most eukaryotic centromeres contain long arrays of tandem repeats, with unit lengths of 150 - 300 bp. We searched for such repeats in the functional centromeres of the model legume Medicago truncatula ( Medicago) accession Jemalong A17. To this end three repeats, MtR1, MtR2 and MtR3, were identified in 20 Mb of a low-pass, whole genome sequencing data set generated by a random shotgun approach. The nucleotide sequence composition, genomic organization and abundance of these repeats were characterized. Fluorescent in situ hybridization of these repeats on chromosomes at meiosis I showed that only the MtR3 repeat, encompassing stretches of 450 kb to more than 1.0 Mb, is located in the functional portion of all eight centromeres. MtR1 and MtR2 occupy distinct regions in pericentromeric heterochromatin. We also studied the presence and distribution of MtRs in Medicago accession R108-1, a genotype with a genome that is 20% smaller than that of Jemalong A17. We determined that while MtR3 is also centromeric on all pachytene bivalents in R108-1, MtR1 and MtR2 are not present in the R108 genome
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