Recently, the research team at Peking University Institute of Advanced Agricultural Sciences has made significant progress in the field of cucumber genome and EMS mutant library research. Their findings, published in Molecular Plant on March 4, 2025, provide crucial support for cucumber functional genomics and lay a solid foundation for developing superior cucumber varieties.

Cucumber is a globally important vegetable crop, with pickling cucumbers being one of the main cultivated types widely grown in regions such as the Americas, Europe, and Asia. However, the previously released genome assembly of the American pickling cucumber was of inadequate quality, severely hindering progress in cucumber functional genomics research and variety breeding. The crop genetics and breeding platform team led by Zhang Xingping and Deng Yun, in collaboration with the bioinformatics platform team led by He Hang at Peking University Institute of Advanced Agricultural Sciences, selected an excellent Russian-type pickling cucumber inbred line, CUK2021. Using high-precision ONT ultra-long reads and Hi-C sequencing technology, they successfully assembled a high-quality cucumber reference genome, CUK2021, which includes complete centromeres and 13 telomeres, with a total length of 326.24 Mb. This genome exhibits outstanding completeness and accuracy, with excellent metrics such as sequencing data mapping rate, genome coverage, and BUSCO scores.

By comparing the CUK2021 genome with the Chinese long cucumber v4 genome, researchers discovered a large number of structural and local variations, including inversions, translocations, and unaligned regions. These variations are mainly concentrated in the centromeric regions, with genes in the unaligned regions primarily related to homologous recombination, the citric acid cycle, and cysteine and methionine metabolism. Additionally, the study revealed the important role of transposons in gene duplication. Through the analysis of long terminal repeat retrotransposons (LTR-RTs), researchers found that the Copia and Gypsy superfamilies have undergone significant expansion over the past 500,000 years, promoting gene duplication and evolution.

To advance cucumber functional genomics research, the team constructed a mutant library for CUK2021 using EMS pollen mutagenesis. The phenotypic mutation rate in the M1 generation was 1.39%, increasing to 12% in the M2 generation. Several valuable mutants have been identified from the library, including those with strong male characteristics, shortened internodes, sparse spines, and altered fruit shapes. These mutants not only provide new perspectives for cucumber functional gene research but also offer valuable genetic stocks for developing new pickling cucumber varieties. Sequencing analysis revealed that repeated EMS mutagenesis on already mutagenized materials does not significantly increase the mutation rate. The genome assembly data and mutant library sequencing data have been submitted to the National Genomics Data Center for global sharing by researchers.

Figure 1: Gap-free cucumber reference genome and pollen EMS-induced mutant library

Finally, the researchers conducted an in-depth study on a mutant (m96) exhibiting yellowing young leaves and apoptotic phenotypes in mature leaves and fruits. Through BSA analysis, RNA-seq analysis, and a series of experiments, they found that the m96 phenotype is conferred by a semi-dominant gene and identified the candidate gene Csa1G074800, which enhances resistance to cucumber mosaic virus. Preliminary functional validation of this gene was achieved through virus-induced gene silencing (VIGS) experiments.

This groundbreaking research provides valuable resources for the genetic improvement of cucumbers, promising to advance the global cucumber industry and offer consumers more delicious and high-quality cucumbers. With the continuous efforts of researchers, the future of cucumber breeding holds many more surprises.

This achievement is another significant advancement in cucurbit crops by the crop breeding and bioinformatics platforms at Peking University Institute of Advanced Agricultural Sciences, following the decoding of the cultivated watermelon T2T genome (Molecular Plant, 2022), the watermelon T2T super pan-genome (Nature Genetics, 2024), and the watermelon mutant library. Tian Yao, Li Kui, and Li Tonghui are the co-first authors of the paper, with Zhang Xingping, Deng Yun, and He Hang as the corresponding authors. The research also received guidance from Academician Deng Xing Wang and Researcher Xue Yan. This study was supported by the Shandong Provincial Science and Technology Innovation Program, the Ningbo Science and Technology Innovation Program, and the Weifang Seed Industry Innovation Team Project.

Authers: from left to right Yun Deng, Hang He, Xingping Zhang, Tonghui Li, Yao Tian and Kui Li