Supernumerary B chromosomes are optional additions to the basic set of A chromosomes, and occur in all eukaryotic groups. They differ from the basic complement in morphology, pairing behavior, and inheritance and are not required for normal growth and development. The current view is that B chromosomes are parasitic elements comparable to selfish DNA, like transposons. In contrast to transposons, they are autonomously inherited independent of the host genome and have their own mechanisms of mitotic or meiotic drive. Although B chromosomes were first described a century ago, little is known about their origin and molecular makeup. The widely accepted view is that they are derived from fragments of A chromosomes and/or generated in response to interspecific hybridization. Through next-generation sequencing of sorted A and B chromosomes, we show that B chromosomes of rye are rich in gene-derived sequences, allowing us to trace their origin to fragments of A chromosomes, with the largest parts corresponding to rye chromosomes 3R and 7R. Compared with A chromosomes, B chromosomes were also found to accumulate large amounts of specific repeats and insertions of organellar DNA. The origin of rye B chromosomes occurred an estimated ∼1.1–1.3 Mya, overlapping in time with the onset of the genus Secale (1.7 Mya). We propose a comprehensive model of B chromosome evolution, including its origin by recombination of several A chromosomes followed by capturing of additional A-derived and organellar sequences and amplification of B-specific repeats.

Significance: The generation of haploids is the most powerful means to accelerate the plant-breeding process. We elucidated whether point mutations in the centromere-specific histone H3 variant CENH3 could be harnessed for the induction of haploids. We identified plants with impaired centromere loading caused by a mutation in the centromere-targeting domain (CATD). The same mutation results in reduced loading of CENH3 in transgenic Arabidopsis and sugar beet. Arabidopsis plants carrying this single point mutation in wild-type CENH3 were used as haploid inducers. Because the identified mutation site is highly conserved and because point mutations can be generated by mutagenesis or genome editing, the described method offers opportunities for application in a wide range of crop species.

B chromosomes (Bs) are supernumerary components of the genome and do not confer any advantages on the organisms that harbor them. The maintenance of Bs in natural populations is possible by their transmission at higher than Mendelian frequencies. Although drive is the key for understanding B chromosomes, the mechanism is largely unknown. We provide direct insights into the cellular mechanism of B chromosome drive in the male gametophyte of rye (Secale cereale). We found that nondisjunction of Bs is accompanied by centromere activity and is likely caused by extended cohesion of the B sister chromatids. The B centromere originated from an A centromere, which accumulated B-specific repeats and rearrangements. Because of unequal spindle formation at the first pollen mitosis, nondisjoined B chromatids preferentially become located toward the generative pole. The failure to resolve pericentromeric cohesion is under the control of the B-specific nondisjunction control region. Hence, a combination of nondisjunction and unequal spindle formation at first pollen mitosis results in the accumulation of Bs in the generative nucleus and therefore ensures their transmission at a higher than expected rate to the next generation.

Summary: B chromosomes (Bs) are supernumerary chromosomes that vary in number among individuals of the same species. Because of their dispensable nature, their non‐Mendelian inheritance and their origin from A chromosomes (As), one might assume that Bs followed a different evolutionary pathway from As, this being reflected in differences in their high‐copy DNA constitution. We provide detailed insight into the composition and distribution of rye (Secale cereale) B‐located high‐copy sequences. A‐ and B‐specific high‐copy sequences were identified in silico. Mobile elements and satellite sequences were verified by fluorescence in situ hybridization (FISH). Replication was analyzed via EdU incorporation. Although most repeats are similarly distributed along As and Bs, several transposons are either amplified or depleted on the B. An accumulation of B‐enriched satellites was found mostly in the nondisjunction control region of the B, which is transcriptionally active and late‐replicating. All B‐enriched sequences are not unique to the B but are also present in other Secale species, suggesting the origin of the B from As of the same genus. Our findings highlight the differences between As and Bs. Although Bs originated from As, they have since taken a separate evolutionary pathway.