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Rapid alterations of DNA sequence and cytosine methylation induced by somatic hybridization between Brassica oleracea L. var. italica and Brassica nigra (L.) Koch

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Abstract

Somatic hybrids were produced between hypocotyl protoplasts of Brassica oleracea L. var. italica (broccoli) and mesophyll protoplasts of B. nigra (black mustard) using polyethylene glycol—mediated protoplast fusion. A total of fifteen somatic hybrids derived from six calli (no. 1, 3, 8, 21, 38 and 44) were obtained. Cytological analysis showed that all the hybrids possessed 2n = 34, the sum of the parental chromosomes and the genomic in situ hybridization analysis revealed their BBCC genome constitutes. Moreover, all the hybrids exhibited different type of meiosis abnormalities, which were more usually observed in pollen mother cells at metaphase II/anaphase II (MII/AII, 16.1–39.6 %) than at metaphase I/anaphase I (MI/AI, 7.8–15.2 %). Simple sequence repeat analysis revealed that all the hybrids showed the same cytoplasmic genome as broccoli. Structure and methylation-variation of the nuclear were investigated by amplified fragment length polymorphism (AFLP) and DNA methylation-sensitive amplification polymorphism (MSAP). Our results indicated that all the hybrids mainly had the AFLP and MSAP banding patterns from the addition of two parents plus some alterations. The incidences of the AFLP polymorphic bands in the hybrids showed a range of 9.8–18.7 % while the DNA methylation alteration in the hybrid no. 38 was 4.07 %. This result suggested that somatic hybridization could induce more DNA sequence changes than methylation alterations in the early stage of allotetraploid hybrids.

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Abbreviations

PEG:

Polyethylene glycol

SRAP:

Sequence–related amplified polymorphism

GISH:

Genomic in situ hybridization

AFLP:

Amplified fragment length polymorphism

MSAP:

Methylation-sensitive amplification polymorphism

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Acknowledgments

Special thanks are given to Professor Liu for initiating our somatic hybridization research and for the gift of fusion materials. This work was financially supported by Natural Science Foundation of China (31201629), Science and Technology Department of Zhejiang Province, and Zhejiang Academy of Agricultural Sciences.

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  1. Institute of Vegetables, Zhejiang Academy of Agricultural Sciences, Hangzhou, 310021, China

    Xiaoguang Sheng, Zhenqing Zhao, Huifang Yu, Jiansheng Wang & Honghui Gu

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  1. Xiaoguang Sheng
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Correspondence to Honghui Gu.

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Sheng, X., Zhao, Z., Yu, H. et al. Rapid alterations of DNA sequence and cytosine methylation induced by somatic hybridization between Brassica oleracea L. var. italica and Brassica nigra (L.) Koch. Plant Cell Tiss Organ Cult 115, 395–405 (2013). https://doi.org/10.1007/s11240-013-0371-6

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