Effective strategies for creating self-compatible B. rapa by introgression of mutated SRK and SCR/SP11 genes from B. napus

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Self-incompatibility (SI) is widespread in Brassica vegetable crops, such as Chinese cabbage, pak choi, cabbage, cauliflower, and broccoli, which can promote outcrossing and increase the genetic diversity of these species. However, in the breeding process, SI is one of the main obstacles to the development and propagation of inbred lines. In recent years, most Brassica vegetable crops have applied the male sterility system to produce hybrids. Both the male sterile line and the maintainer line share the same SI allele, which usually causes propagation failure. The manipulation of SI genes is an effective way to create self-compatible (SC) materials in Brassica. Self-incompatibility in Brassicaceae is controlled by the multiallelic S locus, which mainly contains one pollen recognition specificity gene, SP11/SCR (S-locus protein 11/S-locus cysteine rich protein), and one stigma recognition specificity gene, SRK (S-locus receptor kinase) (Bateman 1955; Schopfer et al. 1999; Stein et al. 1991; Takasaki et al. 2000; Takayama et al. 2001). Once the stigma receives pollen with the same S haplotype, a complex signaling cascade is elicited to reject self-pollen (Takayama et al. 2001; Kachroo et al. 2001). The S locus genes are transmitted to the progeny as one unit, which is also called the ‘S haplotype’ (Vekemans et al. 2014). Several studies have shown that mutations in genes involved in female specificity (SRK gene), male specificity (SP11/SCR gene), or downstream signaling pathways (MLPK, ARC1) could cause the loss of SI (Okamoto et al. 2007; Chen et al. 2019; Stone et al. 1999; Murase et al. 2004; Gao et al. 2016).

Recently, characterized CRISPR/Cas9 technology has attracted great attention and has been applied for the creation of SC plants by manipulating one of the SI genes, such as potato (Ye et al. 2018), cabbage (Ma et al. 2019) or broccoli (Ma et al. 2023). However, the simultaneous mutation of two nonallelic self-incompatibility recognition genes is more practically valuable in production, as it ensures compatibility in both self-pollination and use as either the female or male parent. In Brassica, the basic diploid species B. rapa (AA, 2n = 20) and B. oleracea (CC, 2n = 18) are self-incompatible, but the cultivated allotetraploid B. napus (AACC, 2n = 38) is self-compatible. Insertion of a nonautonomous Helitron transposon in the promoter of the pollen recognition-specific gene BnSP11-1 in the A genome was responsible for the self-compatibility of the B. napus cultivar ‘Westar’ (Gao et al. 2016). Thus, in the present study, we used CRISPR/Cas9 technology to mutate the BnSRK-1 gene in the B. napus cultivar ‘Westar’ and then employed distant hybridization to introduce the mutated BnSRK-1 gene into B. rapa, ultimately resulting in the generation of SC materials.

To induce mutations in the BnSRK-1 gene, we designed a CRISPR-Cas9 construct that targets BnSRK-1. Two guide RNAs (named K1-a and K1-b) were designed to target the first exon of BnSRK-1 (Fig. 1A, 1B; Table S1). A total of 26 transgenic plants were generated, PCR products of target sites were amplified, and then T-A cloning and Sanger sequencing were used to test whether the CRISPR/Cas9 construct could properly edit the BnSRK-1 gene. Finally, 4 transgenic plants (T0-4, T0-10, T0-11 and T0-25) exhibited mutations in the target region of the BnSRK-1 gene (Fig. 1C, Fig S1). In T0-4, homozygous mutations were identified at the K1-a target site (ATGG deletion) and K1-b target site (CA deletion). T0-10 and T0-25 showed the same heterozygous mutations, and both the K1-a and K1-b target sites contained a single A insertion. Chimeric mutations and deletions of the entire fragment between the two target sites (including 10 bp insertions and 106 bp deletions, 20 bp insertions and 106 bp deletions, and 39 bp insertions and 108 bp deletions) were found in T0-11 (Fig. 1C).

Fig. 1.

Targeted mutagenesis of BnSRK-1 and introgression of the mutated BnS-1 allele from B. napus to B. rapa. A Schematic illustrating the BnSRK-1 gene with the two target sites. B Schematic diagram of the construction of the pKSE401 expression vector. C Site-specific mutations of BnSRK-1. The PAM is shown in red. Deletions are denoted by black dashes. D Pollen pollination assays of ‘W-3’. E Pollen pollination assays of ‘W-3’. F F1 hybrid lines obtained from ‘Westar’ × ‘ QR44’. G F1 hybrid lines obtained from ‘T0-4 × ‘QR44’. H Pods in the incompatible pollination lines. I Pods in the self-compatible pollination lines

To investigate self-compatibility on the stigma side of T0-4, the transgenic self-incompatible line ‘W-3’, which contains a functional BnSP11-1 gene in B. napus, was used (Gao et al. 2016). Pollination assays showed that the stigma of T0-4 was compatible with the pollen of ‘W-3’, while the stigma of wild-type ‘Westar’ was incompatible with the pollen of ‘W-3’ (Fig. 1D). Then, in the T1 generation, 20 plants were obtained, and all of them showed the same mutations at the target sites as those in T0-4 (i.e., a CCAT deletion in the K1-a target site and a TG deletion in the K1-b target site). The stigmas of all the plants were compatible with the pollen of ‘W-3’, and 4 plants (T1-4–2, T1-4–4, T1-4–14, and T1-4–18) were Cas9-free (Fig S1). All the results demonstrated that inducing loss-of-function mutations in the BnSRK-1 gene resulted in self-compatibility in the stigma of B. napus. Both the pollen side and stigma side of T0-4 have lost self-incompatibility and can be used to transfer self-compatibility into B. rapa.

To transfer self-compatibility from B. napus to B. rapa, the transgenic line T1-4–5 was crossed with the self-incompatible line ‘QR44’, which contains the recessive S allele BrS-44 from nonheading Chinese cabbage, and the F₁ hybrid was subsequently obtained (Fig. 1E, Fig. S2). BC₁ progenies were obtained through backcrossing of the interspecific hybrids as female parents and with ‘QR44’ as the male parent. The S locus-specific molecular marker SPeS1-7/8 was used to select the plants containing the S allele BnS-1, and 20 out of 37 plants were selected (Fig S3). Furthermore, BC₂ progenies were obtained through the backcrossing of one BC₁ plant containing the S allele BnS-1 as the female parent and with ‘QR44’ as the male parent. In the BC₂ generation, 9 out of 13 plants contained the S allele BnS-1 (Fig S4), all the plants were self-pollinated, and the SCI was calculated. Of the 9 plants that contained the S allele BnS-1, 5 plants showed self-compatible phenotypes with an SCI > 2, and the remaining 4 plants were self-incompatible, which may be attributed to the genomic instability of the offspring caused by distant hybridization (Table S2). By 2 generations of self-pollination (BC₂F₂) of the SC plants, 3 elite and completely self-compatible lines that contained the homozygous S allele BnS-1 (Fig. 1F) were obtained.

In summary, we successfully performed site-specific manipulation of the stigma recognition-specific gene BnSRK-1 using the CRISPR/Cas9 system in the B. napus cultivar ‘Westar’, which contains the mutated pollen recognition specificity gene BnSP11-1. Furthermore, we transferred both the mutated SI genes to B. rapa by distant hybridization and created SC inbreeding lines. SC is quite important for the breeding of most Brassica vegetable crops, improving the reproductive efficiency of breeding materials and reducing seed production costs. The mutated SI genes can also be applied in other Brassica vegetable crops with an AA genome (2n = 20), such as Chinese cabbage, purple flowering stalks, and Chinese flowering cabbage, to accelerate the breeding process.

Supplementary Information

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Author contributions

Xueli Zhang, Shuangping Heng, Chunxiu Xiao, Cong Liu and Liping Song performed the experiments and analyzed the data. Xueli Zhang and Shuangping Heng wrote the manuscript. Aihua Wang and Changbin Gao designed the experiments. Liguang Tang, Congan He, Bincai Wang, Aihua Wang and Changbin Gao contributed materials and reagents. All the authors have read and approved the final manuscript.

Funding

This research was supported by grants from the Wuhan Knowledge Innovation Special Project (2022020801010413).

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Competing interests

The authors declare no competing interests.