Characterization of interspecific hybrids between Chinese cabbage (Brassica rapa) and red cabbage (Brassica oleracea)

Characterization of interspecific hybrids between Chinese cabbage (Brassica rapa) and red cabbage (Brassica oleracea)

Scientia Horticulturae 250 (2019) 33–37 Contents lists available at ScienceDirect Scientia Horticulturae journal homepage:

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Scientia Horticulturae 250 (2019) 33–37

Contents lists available at ScienceDirect

Scientia Horticulturae journal homepage:

Characterization of interspecific hybrids between Chinese cabbage (Brassica rapa) and red cabbage (Brassica oleracea) Yunxiao Wei, Fei Li, Shujiang Zhang, Shifan Zhang, Hui Zhang, Haiyun Qiao, Rifei Sun


Department of Chinese Cabbage, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing, Zhongguancun, Nandajie No. 12, Haidian District, Beijing, 100081, People’s Republic of China



Keywords: Chinese cabbage Red cabbage Synthetic Brassica napus Distant hybridization Trait separation

Interspecific hybridization is an excellent way to create germplasm resources and is considered an important driver of genome evolution. We selected one Chinese cabbage variety and one red cabbage variety for hybridization. Heterologous haploid offspring were obtained by embryo rescue, and heterologous diploids were obtained by colchicine-induced chromosomal doubling. The field traits and simple sequence repeat markers of 98 F2 plants were investigated. The result showed that the parental traits were separated in the hybrids, with traits tending to shift from those of Chinese cabbage to those of red cabbage, and trait values showed normal distributions. Simple sequence repeat patterns varied, with the number of missing bands being significantly greater than that of novel bands in hybrids. Thus, the phenotypes of the early formed allopolyploids were unstable and accompanied by dramatic changes in the genome. This study not only increased the genetic resources available for B. napus but also laid a theoretical foundation for exploring trait segregation in early formed allopolyploids.

1. Introduction Chinese cabbage (Brassica rapa L. ssp. pekinensis) (Ma et al., 2015), an economically important vegetable in Asia, belongs to the family Cruciferae, species Brassica. In addition, Chinese cabbage, Originated in China, is the vegetable crop with the largest cultivation area. The annual planting area of the country is nearly 2.67 million hm2 (40 million mu), with an output value of more than 60 billion yuan (Zhang et al., 2017). Red cabbage (Brassica oleracea convar. capitata var. rubra DC) (Park et al., 2014), have significant uses and considerable stability in the United States and Europe, also belongs to the family Cruciferae, species Brassica.Moreover, Cabbage is an important imperative vegetable crop and its consumption improves human health. It acts as a central food source because it contained many bioactive compounds (Volden et al., 2008; Singh et al., 2006). Distant hybridization can synthesize parental advantages or create heterosis, promoting intergenic exchanges and riching germplasm resources (Cicin, 1954). Germplasm resources are the genetic material basis for variety improvement. Distant hybridization is one of the important ways to create new plant species and breed new varieties (Whitney et al., 2010; Chen et al., 2018). In addition, based on the “U-triangle” (1935) theory and Brassica genome sequencing data, B. napus was generated from a cross ⁎

between B. rapa and B. oleracea approximately ˜7500 years ago (Chalhoub et al., 2014). Hybridization between the two species readily produces offspring. The distant hybridization between B. rapa and B. oleracea can not only extend the germplasm resources of B. napus, but also can rich the resources of the parent by backcross. Hybridization between Chinese cabbage and red cabbage could result in allopolyploid. Early formed allopolyploid is unstable. Newly synthesized B. napus has abundant variability in many characteristics, such as flower size, flowering time, waxy layer characteristics and leaf shape and size (Gaeta et al., 2007). However, cultivated varieties must be genetically stable germplasm resources, and the trait segregation of newly synthesized allopolyploids hinders further utilization. (Mestiri et al., 2010; Tian et al., 2010). Furthermore, distant hybridization and polyploidization are important factors in promoting sequence evolution and in inducing tandem repeat variation (Tang et al., 2009). The novel and disappearance of large numbers of repetitive DNA sequences are very common among synthetic allopolyploid (Tang et al., 2008; Zhang et al., 2016). Therefore, exploring the trait and sequence variation of selfing offspring of synthetic B. napus can help us understand the mechanism of phenotypic stability in newly synthesized allopolyploids. There have been few reports on the hybridization between Chinese cabbage and red cabbage (Zhang et al., 2004; Gaeta et al., 2007; Karim et al., 2014; Zhang et al., 2016; Shen et al., 2017). Here, one Chinese

Corresponding author. E-mail address: [email protected] (R. Sun). Received 1 December 2018; Received in revised form 23 January 2019; Accepted 26 January 2019 0304-4238/ © 2019 Published by Elsevier B.V.

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Fig. 1. Chromosome number of haploids (AC) and allopolyploids (AACC).

plant height, leaf length, leaf width, petiole length, leaf color, leaf edge wavy, leaf edge serration, foliar shrinkage, degree of purple and blade hair. In addition, the traits of each F2 plant and parent were recorded.

cabbage variety and one red cabbage variety were selected for hybridization. Heterologous haploids were obtained by embryo rescue, and the heterologous diploids were obtained by colchicine-induced chromosomal doubling. The offspring could serve as ornamental-type rapeseed materials that combine the advantages of the two species. In addition, the field traits and simple sequence repeat (SSR) markers of 98 F2 plants were investigated. This research laid a theoretical foundation for exploring trait separation in the allopolyploid polyploidization process.

2.4. Genomic DNA isolation and PCR analysis Genomic DNA was isolated from leaves of the parents and 98 F2 hybrids using a modified CTAB method (Kidwell and Osborn, 1992). Fourteen SSR markers were randomly chosen from the Brassica Database (Supplementary Table 1) and used for PCR amplification. The PCR primers were purchased from Qingke Co, Beijing, China. The amplifications were performed in 5-μl volumes containing 4–8 ng of template DNA, 0.4 μl of forward and reverse primers (0.2 μl each), 2.5 μl 2× super Taq mix, and water to make up the volume. The thermal cycling conditions were 94 °C for 5 min; 30 cycles of 94 °C for 30 s, 55 °C for 30 s and 72 °C for 30 s; and, finally, 72 °C for 10 min. The PCR products were separated on an 8% polyacrylamide gel by electrophoresis and visualized with silver staining (Zhang et al., 2016).

2. Materials and methods 2.1. Plant materials The commercial variety of Chinese cabbage, jinfeng, was the female parent, and the commercial variety of red cabbage, zixing, was the male parent. The plant materials were provided by the Cabbage Laboratory of the Institute of Vegetable and Flowers, Chinese Academy of Agricultural Sciences, Beijing, China.

3. Results 2.2. Embryo rescue and colchicine-induced doubling 3.1. Obtaining hybrid offspring We planted parental materials in glasshouses. The ovaries of plants at 12–14 d after pollination were treated with 75% ethanol for 30 s, sterilized with 7% NaClO for 15 min, and finally washed with sterile water three times. The ovules in the ovary were stripped with tweezers and inoculated into MS medium. The inoculated ovules were cultured at 25 ± 2 °C with 10 h light d−1 at an intensity of 2000 lx for 35–55 d (Sharma et al., 1996; Ayotte et al., 1987). The seedlings of the live ovules were grown until four or five leaves appeared after differentiation and rooting. Then, the roots of seedlings were soaked in 2% colchicine for 48 h to obtain allopolyploids (AACC) before they were planted in a greenhouse (Zhao et al., 1996).

Haploid (AC) hybridization between Chinese cabbage and red cabbage was performed by embryo rescue. Then, allopolyploids (AACC) were obtained by colchicine-induced chromosomal doubling. All heterogeneous polyploids were identified by the number of apical chromosomes, with one hybrid having 38 chromosomes. Haploids (AC) have 19 chromosomes and allopolyploids (AACC) have 38 chromosomes (Fig. 1). Seeds were collected by allopolyploid (AACC) bud selfpollination. The 98 F2 and 6 parental (three each) plants were grown in the Shunyi base of the Chinese Academy of Agricultural Sciences’ Institute of Vegetables and Flowers (Beijing, China).

2.3. Determining ploidy and characteristics

3.2. Morphological characteristics

The root tips of parents and hybrids were cut, incubated in saturated p-dichlorobenzene for 2.5 h at room temperature, and then fixed in Carnoy’s solution (methanol: acetic acid = 3:1) for 24–48 h. They were stored in 70% ethanol at 4℃. From the root, 1–2 mm of tip was cut and placed in a 0.075-mol/L KCl hypotonic solution for 30 min and then rinsed with distilled water three times. Tips were digested with a mixture of 2.5% cellulase and 2.5% pectinase at 37 °C for 55 min. The enzyme solution was removed by washing with distilled water at 4 °C for 30 min. Finally, the root tip was placed on a slide, the fixative as added prior to flame drying, and the sample was observed under a phase-contrast microscope. Slides showing chromosomal dispersion and dissociation were stained with DAPI and photographed under a fluorescence microscope (Lou et al., 2017). The morphological traits investigated included plant expansion,

The comprehensive performance of hybrids was between that of the two parents (Fig. 2a). The wax powder content gradually increased (Fig. 2b). The shape of leaves gradual changed from Chinese cabbage to red cabbage (Fig. 2c). In summary, traits tended to shift from those of the Chinese cabbage to those of red cabbage. Moreover, based on data from our field surveys, we found that the leaf color was mostly light green and grayish green, the leaf edge wavy was mostly small, leaf edge serrations were mostly complex, leaf wrinkles were mostly characterized by a slight wrinkle and a lot of wrinkles. The degree of purple was mostly a little purple, a few were purple-free and purple on the stem veins. The leaf hairs were mostly non-existent (Supplementary Table 2). In addition, the values of plant expansion, plant height, leaf length, leaf width and petiole length varied among the 98 F2 plants. The averages of plant expansion, leaf width and petiole length were higher than the 34

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Fig. 2. The morphology of parents and interspecific hybrids plants.

Fig. 3. The result of field characteristics.

3.3. Molecular characterization

parents, while the averages of plant height and leaf length were between that of the two parents (Fig. 3a). Then, all trait values were divided into 10 levels, and the number of plants belonging to each level was determined. The result showed that the trait values showed a normal distribution (Fig. 3b). A small number of individual plants were highly variable.

Fourteen SSR primer pairs (Supplementary Table 1) were used to detect the sequence variation of hybrids and parents. Novel bands existed in the hybrids, while some parental bands were missing (Fig. 4b). The number of missing bands was significantly greater than the number of novel bands. Different primers produced varied results, as did different plants. In addition, the number of missing AA bands was 35

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Fig. 4. The result of ploidy identification and molecular analysis.

observed in the hybrids (Raskina et al., 2008). There were differences in the statistical results among the F2 plants, but there was no regularity (He et al. 2017). Furthermore, the number of missing AA bands was significantly greater than the number of missing CC bands, which proved that the two genomes show difference when response to wholegenome duplication events and that the AA genome is more active than the CC genome (Nicolas et al., 2008). The result confirmed that the high mutation rate of tandem repeats may play an important role in early stage of allopolyploid formation. Therefore, exploring the effects of SSR sequence variation on the traits is necessary. In addition, with further developments in sequencing technology, the SSR variation mechanism across the whole genome need further exploration. It is also necessary to further explore the relationship between SSR variation and trait separation.

significantly greater than the number of missing CC bands (Fig. 4a). This indicates that, genetic information has been mutated, and the AA genome was more active than the CC genome in response to “genomic shock”.

4. Discussion The newly formed allopolyploid is unstable (Leitch and Leitch, 2008; Jackson and Chen, 2010). The newly synthesized B. napus has a rich variation in self-crossing progeny, and some important genetic types that have disease-resistance potential were used for breeding (Eickermann et al., 2011). In this experiment, Chinese cabbage served as the female parent and red cabbage served as the male parent. The hybrids displayed significant variation in traits, and variant plants with purple on the stem veins, epicuticular wax powder and many wrinkled leaves were found. The hybrids with desirable variation in important agronomical traits could serve as breeding materials for B. napus by selfing or backcrossing. Additionally, backcrosses between newly formed allopolyploid and parents could rich the genetic resources of Chinese cabbage and red cabbage. Furthermore, some hybrid offspring showed the character of non-heading (Fig. 2b), like kale (Brassica oleracea var. sabellica L.), which could provide powerful support for hybrids becoming commercially ornamental vegetables. Gaeta et al. investigated the field haracters of 50 new synthetic B. napus by single seed descent, and there was variation in leaf morphology and cuticular waxiness among polyploid lines (Gaeta et al., 2007). In addition, the backcross progeny of new synthetic B. napus also displayed extensive morphological variation (Zhang et al., 2016). However, the field variation of F2 population, which came from the same strain F1, been rarely studied. In our investigation of the field characteristics of synthetic B. napus, we observed that leaf color followed a yellow green to dark green gradient, accompanied by an increase in purple and changes in the presence of wax powder. Thus, traits tended to shift from those of Chinese cabbage to those of red cabbage. In addition, the trait values showed a normal distribution, with a small number of individual plants being highly variable. Therefore, breeders could expand the population of self-bred offspring to screen for desirable variations in future breeding work. A single plant could be selected for a purpose, and a stable genetic line could be obtained from multi-generation self-breeding. Tandem repeats play important roles in chromatin formation and the evolution, functions and structures of genes (Zhang et al., 2006; Gemayel et al., 2010; Yaakov and Kashkush, 2011). The loss of parental sequences and/or the appearance of novel sequences at the initial stage of allopolyploidization are common events(Gaeta et al., 2007; Xiong et al., 2011). In our study, novel and missing SSR sequences were

Conflict of interest The authors declare that they have no conflict of interest. Contribution of authors R.S. and Y.W designed the experimental design and wrote the article. FL, SJZ, SZ, and HZ contributed to the contributed to the interpretation of the results and coordinated the study. Y.W. performed the experiment. All the authors read and approved the final manuscript. Acknowledgments This research was funded by the National Key Research and Development Program of China (grant number 2017YFD0101802) and the Fundamental Research Funds for Central Non-profit Scientific Institution (grant number IVF-BRF2018003). The experiment was performed at the Key Laboratory of Biology and Genetic Improvement of Horticultural Crops, Ministry of Agriculture, Beijing, China. We thank Robbie Lewis, MSc, PhD, from LiwenBianji, Edanz Group China (www., for editing the English text of a draft of this manuscript. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi: References Ayotte, R., Harney, P.M., Machado, V.S., 1987. The transfer of triazine resistance from Brassica napus L. to B. Oleracea L. I. Production of F1 hybrids through embryo rescue.


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