Rapid and visual monitoring of alien sequences using crop wild relatives specific oligo-painting: The case of cucumber chromosome engineering

Abstract

Wild species related to domesticated crops (crop wild relatives, or CWRs) represent a high level of genetic diversity that provides a practical gene pool for crop pre-breeding employed to address climate change and food demand challenges globally. Nevertheless, rapid identifying and visual tracking of alien chromosomes and sequences derived from CWRs have been a technical challenge for crop chromosome engineering. Here, a species-specific oligonucleotide (oligo) pool was developed by using the reference genome of Cucumis hystrix (HH, 2n = 2x = 24), a wild species carrying many favorable traits and interspecific compatibility with cultivated cucumber (C. sativus, CC, 2n = 2x = 14). These synthetic double-stranded oligo probes were applied to validate the assembly and characterize the chromosome architectures of C. hystrix, as well as to rapidly identify C. hystrix-chromosomes in diverse C. sativus-hystrix chromosome-engineered germplasms, including interspecific hybrid F1 (HC), synthetic allopolyploids (HHCC, CHC, and HCH) and alien additional lines (CC-H). Moreover, a ∼2Mb of C. hystrix-specific sequences, introduced into cultivated cucumber, were visualized by CWR-specific oligo-painting. These results demonstrate that the CWR-specific oligo-painting technique holds broad applicability for chromosome engineering of numerous crops, as it allows rapid identification of alien chromosomes, reliable detection of homoeologous recombination, and visual tracking of the introgression process. It is promising to achieve directed and high-precision crop pre-breeding combined with other breeding techniques, such as CRISPR/Cas9-mediated chromosome engineering.

Plant phenotyping relevance

CWR特異的オリゴペインティングによる染色体構造・外来染色体・導入配列の可視化を開発し、複数の育種材料で検証している。染色体状態の画像取得・判定が研究の中心であり、単なる分子測定や生物学的実験の補助測定ではない。

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