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Development of a novel allele-specific Rfo marker and creation of Ogura CMS fertility-restored interspecific hybrids in Brassica oleracea

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A novel allele-specific Rfo marker was developed and proved to be effective for MAS of Rfo gene in B. oleracea background and six Ogu-CMS fertility-restored interspecific hybrids were created for the first time.

Abstract

Ogura cytoplasmic male sterility (Ogu-CMS) has been extensively used for Brassica oleracea hybrid production. However, because of maternal inheritance, all the hybrids produced by CMS lines are male sterile and cannot be self-pollinated, which prohibits germplasm maintenance and innovation. This problem can be overcome by using the Ogu-CMS restorer line, but restorer material is absent in B. oleracea crops. Here, Rfo, a fertility-restored gene of Ogu-CMS, was transferred from rapeseed restorer lines into a Chinese kale Ogu-CMS line using interspecific hybridization combined with embryo rescue. Nine interspecific, triploid plant progenies were identified at morphological and ploidy level, with phenotypes intermediate between those of rapeseed and Chinese kale. Because the Rfo marker (Hu et al., Mol Breeding 22:663–674, 2008) cannot distinguish the Rfo and its homologies under a B. oleracea background, a novel allele-specific Rfo marker was developed based on the BLAST analysis of highly homologous Rfo sequences in B. oleracea. Screening using the novel Rfo marker found that six interspecific hybrids carrying Rfo were also fertile, although fertility varied during different flowering periods. Furthermore, BC1 offsprings with the Rfo gene were selected with the allele-specific Rfo marker and showed restored fertility. These results indicated that the novel allele-specific marker could be used for the MAS of Rfo gene in B. oleracea, and this study lays the foundation for the development of Ogu-CMS restorer material in cabbage and its related other subspecies.

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Acknowledgments

The authors thank Prof. Jianjun Lei, Prof. Yunchang Li, Prof. Hanzhong Wang, and Prof. Wei Qian for providing materials. This work was supported by grants from the National Natural Science Foundation of China (31272180), the Major State Basic Research Development Program (973 Program, 2012CB113906), the National High Technology Research and Development Program of China (863 Program, 2012AA100102), the Key Projects in the National Science and Technology Pillar Program during the Twelfth Five-Year Plan Period (2012BAD02B01), and the earmarked fund for the Modern Agro-Industry Technology Research System, China (nycytx-35-gw01). This work was performed in the Key Laboratory of Biology and Genetic Improvement of Horticultural Crops (North China), Ministry of Agriculture, Beijing 100097, People’s Republic of China.

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  1. Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, No. 12 ZhongGuanCun South St., Beijing, 100081, People’s Republic of China

    Hai-long Yu, Zhi-yuan Fang, Yu-mei Liu, Li-mei Yang, Mu Zhuang, Hong-hao Lv, Zhan-sheng Li, Feng-qing Han, Xiao-ping Liu & Yang-yong Zhang

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  1. Hai-long Yu
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Communicated by Richard G.F. Visser.

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122_2016_2728_MOESM1_ESM.jpg

Supplementary Fig. S1 Multiple sequence alignment of Rfo sequences with Rfo-homologous sequences in B.oleracea, B.napus, B.rapa (JPEG 2678 kb)

122_2016_2728_MOESM2_ESM.jpg

Supplementary Fig. S2 Sequence alignment of Rfo fragment with Rfo-like fragment amplified by primers Con-F/Con-R. The red arrows represent the location of the primers BnRFO-AS2F/BnRFO-NEW-R (JPEG 4826 kb)

122_2016_2728_MOESM3_ESM.jpg

Supplementary Fig. S3 Embryo development during the embryo rescue process. (a) Freshly removed pods. (b) Excised embryos. (c) Collapsed embryos. (d) Development of pods during the embryo rescue process. (e) Development of embryos in the early period (< 15 days after pollination). (f) Development of embryos in the late period (> 25 days after pollination) (JPEG 631 kb)

122_2016_2728_MOESM4_ESM.jpg

Supplementary Fig. S4 Fertility performance of (a) YL2, and (b) YL5 at different days after flowering (DAF). The red arrows represent the variation of pollen at different DAFs. Left panels: pollen performance; Right panels: pollen viability (JPEG 290 kb)

122_2016_2728_MOESM5_ESM.jpg

Supplementary Fig. S5 (a) PCR amplification in partial BC1 progenies using the novel allele-specific Rfo marker. Lanes 1-8: non-restored BC1 progenies, lanes 9-16: restored- fertility BC1 progenies. Pollen performance in non-restored BC1 progenies (b) and restored- fertility BC1 progenies (c,d) (JPEG 146 kb)

122_2016_2728_MOESM6_ESM.jpg

Supplementary Fig. S6 Protein sequence alignment of Rfo with Rfo homologous sequences in B.oleracea, B.napus, B.rapa (JPEG 2342 kb)

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Yu, Hl., Fang, Zy., Liu, Ym. et al. Development of a novel allele-specific Rfo marker and creation of Ogura CMS fertility-restored interspecific hybrids in Brassica oleracea . Theor Appl Genet 129, 1625–1637 (2016). https://doi.org/10.1007/s00122-016-2728-9

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