<i>De Novo</i> Centromere Formation and Centromeric Sequence Expansion in Wheat and its Wide Hybrids

Centromeres typically contain tandem repeat sequences, but centromere function does not necessarily depend on these sequences. We identified functional centromeres with significant quantitative changes in the centromeric retrotransposons of wheat (CRW) contents in wheat aneuploids (Triticum aestivum) and the offspring of wheat wide hybrids. The CRW signals were strongly reduced or essentially lost in some wheat ditelosomic lines and in the addition lines from the wide hybrids. The total loss of the CRW sequences but the presence of CENH3 in these lines suggests that the centromeres were formed de novo. In wheat and its wide hybrids, which carry large complex genomes or no sequenced genome, we performed CENH3-ChIP-dot-blot methods alone or in combination with CENH3-ChIP-seq and identified the ectopic genomic sequences present at the new centromeres. In adcdition, the transcription of the identified DNA sequences was remarkably increased at the new centromere, suggesting that the transcription of the corresponding sequences may be associated with de novo centromere formation. Stable alien chromosomes with two and three regions containing CRW sequences induced by centromere breakage were observed in the wheat-Th. elongatum hybrid derivatives, but only one was a functional centromere. In wheat-rye (Secale cereale) hybrids, the rye centromere-specific sequences spread along the chromosome arms and may have caused centromere expansion. Frequent and significant quantitative alterations in the centromere sequence via chromosomal rearrangement have been systematically described in wheat wide hybridizations, which may affect the retention or loss of the alien chromosomes in the hybrids. Thus, the centromere behavior in wide crosses likely has an important impact on the generation of biodiversity, which ultimately has implications for speciation.着丝粒（centromere）通常携带串联重复序列（tandem repeat sequences），但着丝粒功能未必依赖此类序列。本研究在普通小麦（Triticum aestivum）非整倍体（aneuploids）以及小麦远缘杂交后代中，鉴定出了小麦着丝粒反转录转座子（CRW，centromeric retrotransposons of wheat）含量发生显著数量变化的功能性着丝粒。在部分小麦端体二体系（ditelosomic lines）以及远缘杂交附加系（addition lines）中，CRW信号显著减弱甚至完全消失。此类材料中CRW序列完全缺失但仍存在CENH3，这表明着丝粒可从头形成（de novo）。对于基因组庞大复杂或尚未完成测序的小麦及其远缘杂交材料，本研究单独采用CENH3染色质免疫沉淀-斑点印迹（CENH3-ChIP-dot-blot），或联合CENH3染色质免疫沉淀测序（CENH3-ChIP-seq）技术，鉴定出了新生着丝粒处存在的异位基因组序列（ectopic genomic sequences）。此外，新生着丝粒处鉴定出的DNA序列转录水平显著上调，提示对应序列的转录可能与新生着丝粒形成相关。在小麦-长穗偃麦草（Th. elongatum）杂交后代中，观察到由着丝粒断裂诱导产生的、携带2个和3个CRW序列区域的稳定外源染色体（alien chromosomes），但其中仅1个区域为功能性着丝粒。在小麦-黑麦（Secale cereale）杂交材料中，黑麦着丝粒特异性序列沿染色体臂扩散，可能引发着丝粒扩张（centromere expansion）。本研究系统阐述了小麦远缘杂交过程中，通过染色体重排（chromosomal rearrangement）导致着丝粒序列频繁发生显著数量改变的现象，该现象可能影响杂交中外源染色体的保留或丢失。因此，远缘杂交中的着丝粒行为可能对生物多样性的产生具有重要影响，最终对物种形成（speciation）具有潜在意义。