Effective Strategies for Creating Self-Compatible B. rapa by Introgression of Mutated SRK and SCR/SP11 Genes from B. napus

Effective Strategies for Creating Self-Compatible B. rapa by Introgression of Mutated SRK and SCR/SP11 Genes from B. napus | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report Effective Strategies for Creating Self-Compatible B. rapa by Introgression of Mutated SRK and SCR/SP11 Genes from B. napus Xueli Zhang, Shuangping Heng, Chunxiu Xiao, Cong Liu, Liping Song, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4381643/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Jul, 2024 Read the published version in Molecular Breeding → Version 1 posted 5 You are reading this latest preprint version Abstract SI (Self-incompatibility) is one of the major obstructions for the development and propagation of inbred lines in most Brassica vegetable crops. The manipulation of SI genes is an effective way to create Self-compatible (SC) materials. A mutated pollen recognition specificity gene BnSP11-1 on the A genome was responsible for the self-compatibility of the B. napus (AACC) cultivar ‘Westar’. In this study, site-specific manipulation of stigma recognition specificity gene BnSRK-1 was carried out using the CRISPR/Cas9 system in ‘Westar’. Then the mutated SI genes (BnSP11-1 and BnSRK-1) were transferred to B.rapa by distant hybridization, by continuous back cross and self pollination, 3 elite and completely self-compatible lines were obtained. The mutated SI genes can be applied widely in Brassica vegetable crops with AA genome (2n=20), such as Chinese cabbage, Pak choi, Purple flowering stalks and Chinese flowering cabbage to accelerate the breeding process. Figures Figure 1 Full Text SI (Self-incompatibility) is widespread in Brassica vegetable crops, such as Chinese cabbage, pak choi, cabbage, cauliflower, broccoli, etc, which can promote outcrossing and increase the genetic diversity of these species. However, in the breeding process, SI is one of the main obstructions to the development and propagation of inbred lines. In recent years, most Brassica vegetable crops applied the male sterility system to produce the hybrids. As the male sterility line and the maintainer line share the same SI allele, which usually causes the failure of sterile line propagation. The manipulation of SI genes is an effective way to create self-compatible (SC) materials in Brassica . Self-incompatibility in the Brassicaceae is controlled by the multi-allelic 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, it is also called the ‘ S haplotype’ (Vekemans et al. 2014). Several researches showed 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). The 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) and broccoli (Cunfa et al. 2023). However, the simultaneous mutation of two non-allelic 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 non-autonomous Helitron transposon in the promoter of the pollen recognition specificity 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 current study, we used CRISPR/Cas9 technology to mutate the BnSRK-1 gene in B. napus cultivar‘Westar’, then employed distant hybridization to introduce the mutated BnSRK-1 genes into B. rapa , ultimately creating 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,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, both the K1-a and K1-b target sites contain a single A insertion. Chimeric mutations and the entire fragment deletions between the two target sites (including 10 bp insertion and 106 bp deletion, 20 bp insertion and 106 bp deletion, 39 bp insertion and 108 bp deletion) were found in T0-11 (Fig 1C). To investigate the self-compatibility on the stigma side of T0-4, a transgenic self-incompatible line ‘W-3’ that 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 that of T0-4 (CCAT deletion in the K1-a target site and 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 demonstrate 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 the self-compatibility into B. rapa . To transfer the self-compatibility from B. napus to B. rapa , the transgenic line T1-4-5 was crossed with a self-incompatible line ‘QR44’ that contains a recessive S allele BrS-44 from non-heading Chinese cabbage and the F 1 hybrid was obtained (Fig 1E, S2). BC 1 progenies were obtained through backcross of the interspecific hybrid as female parents and with ‘QR44’ as male parents. S locus specific molecular marker SPeS1-7/8 was used to select the plants containing S allele BnS-1 , and 20 out of 37 plants were selected (Fig S3). Furthermore, BC 2 progenies were obtained through the backcross of one BC 1 plant containing S allele BnS-1 as the female parent and with ‘QR44’ as the male parent. In the BC 2 generation, 9 out of 13 plants contained S allele BnS-1 (Fig S4), all the plants were self-pollinated and the SCI index was calculated. Of the 9 plants that contain the S allele BnS-1 , 5 plants showed self-compatibility phenotypes with the SCI index >2, and the rest 4 plants are 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 (BC 2 F 2 ) of the SC plants, 3 elite and completely self-compatible lines that contain homozygous S allele BnS-1 (Fig 1F) were obtained. In summary, we successfully performed site-specific manipulation of the stigma recognition specificity gene BnSRK-1 using the CRISPR/Cas9 system in B. napus cultivar ‘Westar’ that contains the mutated pollen recognition specificity gene BnSP11-1 . Furthermore, we transferred both 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 AA genome (2n=20), such as Chinese cabbage, purple flowering stalks, and Chinese flowering cabbage to accelerate the breeding process. Declarations Supplementary Information The online version contains supplementary material available at Author Contributions Xueli Zhang, Shuangping Heng, Chunxiu Xiao, Cong Liu and Liping Song performed 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 authors read and approved the final manuscript. Corresponding authors Correspondence to Aihua Wang and Changbin Gao. Funding This research was supported by grants from the Wuhan knowledge innovation special project (2022020801010413). Author Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Availability of data and materials Not applicable. Competing interests The authors declare no competing interests. References Bateman AJ (1955) Self-incompatibility systems in angiosperms: III. Cruciferae. Heredity 9 (1):53-68. doi:10.1038/hdy.1955.2 Chen F, Yang Y, Li B, Liu Z, Khan F, Zhang T, Zhou G, Tu J, Shen J, Yi B (2019) Functional analysis of M-locus protein kinase revealed a novel regulatory mechanism of self-incompatibility in Brassica napus L. International journal of molecular sciences 20 (13):3303 Cunfa M, Ting W, Hui Z, Tianpei Z, Lijian Z, Yonghui Y, Jiancheng X, Jun L, Shuiqin W (2023) Creation of Self-compatible Broccoli Lines by Knocking out BoSP11 Gene Through CRISPR/Cas9 Technology. 园艺学报. doi:10.16420/j.issn.0513-353x.2023-0065 Gao C, Zhou G, Ma C, Zhai W, Zhang T, Liu Z, Yang Y, Wu M, Yue Y, Duan Z, Li Y, Li B, Li J, Shen J, Tu J, Fu T (2016) Helitron-like transposons contributed to the mating system transition from out-crossing to self-fertilizing in polyploid Brassica napus L. Scientific Reports 6 (1):33785. doi:10.1038/srep33785 Kachroo A, Schopfer CR, Nasrallah ME, Nasrallah JB (2001) Allele-specific receptor-ligand interactions in Brassica self-incompatibility. Science 293 (5536):1824-1826 Ma C, Zhu C, Zheng M, Liu M, Zhang D, Liu B, Li Q, Si J, Ren X, Song H (2019) CRISPR/Cas9-mediated multiple gene editing in Brassica oleracea var. capitata using the endogenous tRNA-processing system. Horticulture Research 6 (1):20. doi:10.1038/s41438-018-0107-1 Murase K, Shiba H, Iwano M, Che F-S, Watanabe M, Isogai A, Takayama S (2004) A membrane-anchored protein kinase involved in Brassica self-incompatibility signaling. Science 303 (5663):1516-1519 Okamoto S, Odashima M, Fujimoto R, Sato Y, Kitashiba H, Nishio T (2007) Self‐compatibility in Brassica napus is caused by independent mutations in S‐locus genes. The Plant Journal 50 (3):391-400 Schopfer CR, Nasrallah ME, Nasrallah JB (1999) The male determinant of self-incompatibility in Brassica. Science 286 (5445):1697-1700 Stein JC, Howlett B, Boyes DC, Nasrallah ME, Nasrallah JB (1991) Molecular cloning of a putative receptor protein kinase gene encoded at the self-incompatibility locus of Brassica oleracea. Proceedings of the National Academy of Sciences 88 (19):8816-8820 Stone SL, Arnoldo M, Goring DR (1999) A breakdown of Brassica self-incompatibility in ARC1 antisense transgenic plants. Science 286 (5445):1729-1731 Takasaki T, Hatakeyama K, Suzuki G, Watanabe M, Isogai A, Hinata K (2000) The S receptor kinase determines self-incompatibility in Brassica stigma. Nature 403 (6772):913-916 Takayama S, Shimosato H, Shiba H, Funato M, Che F-S, Watanabe M, Iwano M, Isogai A (2001) Direct ligand–receptor complex interaction controls Brassica self-incompatibility. Nature 413 (6855):534-538 Vekemans X, Poux C, Goubet PM, Castric V (2014) The evolution of selfing from outcrossing ancestors in Brassicaceae: what have we learned from variation at the S‐locus? Journal of Evolutionary Biology 27 (7):1372-1385 Ye M, Peng Z, Tang D, Yang Z, Li D, Xu Y, Zhang C, Huang S (2018) Generation of self-compatible diploid potato by knockout of S-RNase. Nature Plants 4 (9):651-654 Supplementary Files SupplementaryMaterial.docx Table S1. The primers used in this study. Table S2. Self-Compatibility index (SCI) of the plants in the BC 2 generation. Fig. S1. PCR amplification of the Cas9 gene in the transgenic line T1-4. Fig. S2. PCR amplification of BnS-1 specific molecular marker SPeS1-7/8 in F 1 plants. Fig. S3. PCR amplification of BnS-1 specific molecular marker SPeS1-7/8 in BC 1 plants. Fig. S4. PCR amplification of BnS-1 specific molecular marker SPeS1-7/8 in BC 2 plants. Cite Share Download PDF Status: Published Journal Publication published 01 Jul, 2024 Read the published version in Molecular Breeding → Version 1 posted Editorial decision: Correct before final acceptance 21 Jun, 2024 Reviewers agreed at journal 12 May, 2024 Reviewers invited by journal 11 May, 2024 Editor assigned by journal 09 May, 2024 First submitted to journal 08 May, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4381643","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":301450110,"identity":"52196df9-249f-42f1-ad4f-3d2ff3f17f81","order_by":0,"name":"Xueli Zhang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Xueli","middleName":"","lastName":"Zhang","suffix":""},{"id":301450111,"identity":"a0ed6cc7-8b20-443b-92f5-7a0278ee9b12","order_by":1,"name":"Shuangping Heng","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Shuangping","middleName":"","lastName":"Heng","suffix":""},{"id":301450112,"identity":"51a3fa71-4c5e-4983-8f94-3e1ee3c91413","order_by":2,"name":"Chunxiu Xiao","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Chunxiu","middleName":"","lastName":"Xiao","suffix":""},{"id":301450113,"identity":"df5c1008-cf15-458a-ade6-6836f21c7b55","order_by":3,"name":"Cong Liu","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Cong","middleName":"","lastName":"Liu","suffix":""},{"id":301450114,"identity":"a4063fe6-7b4c-4818-b2d8-69d6971b2af3","order_by":4,"name":"Liping Song","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Liping","middleName":"","lastName":"Song","suffix":""},{"id":301450115,"identity":"a04d6d93-f7db-4ada-b79d-7a2db4762f83","order_by":5,"name":"Liguang Tang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Liguang","middleName":"","lastName":"Tang","suffix":""},{"id":301450116,"identity":"180c1ad3-153f-4bf0-a16d-ddb6fc5dc85e","order_by":6,"name":"Congan He","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Congan","middleName":"","lastName":"He","suffix":""},{"id":301450117,"identity":"9711f647-e916-43a4-8d25-1d1a52572699","order_by":7,"name":"Bincai Wang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Bincai","middleName":"","lastName":"Wang","suffix":""},{"id":301450118,"identity":"8af3037f-5c03-4707-956d-881e2e0885d7","order_by":8,"name":"Aihua Wang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Aihua","middleName":"","lastName":"Wang","suffix":""},{"id":301450119,"identity":"b992b871-8418-449d-8513-eeca5b0dd83a","order_by":9,"name":"Changbin Gao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxklEQVRIiWNgGAWjYJACZgYDGzl59sbGhx+I11KQZmzYc7jZWIJ4LR8OJzbcSG8T4CFGubz74YOfCwwOGzPOfNjGIMFgJ6fbQECL4Zm0ZOkZBuly7NKJbQ8KGJKNzQ4Q0tKQY8bMY2BtzDg7sd1AguFA4jaCWvrfgLQwJzbcPNgmwUOMFnkJsC3OQO8zEqnFQOIZyC+gQE4EBrIBEX6R708GhtgfUFQef/jwQ4WdHEEtBqgKDAgoB9vSQISiUTAKRsEoGOEAAPCrQQKFt9QCAAAAAElFTkSuQmCC","orcid":"","institution":"","correspondingAuthor":true,"prefix":"","firstName":"Changbin","middleName":"","lastName":"Gao","suffix":""}],"badges":[],"createdAt":"2024-05-07 08:56:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4381643/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4381643/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11032-024-01487-4","type":"published","date":"2024-07-01T12:10:53+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":56912179,"identity":"d70059d4-a650-43c3-8ea2-e9edca30a9dd","added_by":"auto","created_at":"2024-05-22 05:26:36","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1495712,"visible":true,"origin":"","legend":"\u003cp\u003eTargeted mutagenesis of\u003cem\u003e BnSRK-1\u003c/em\u003e and introgression of the mutated \u003cem\u003eBnS-1 \u003c/em\u003eallele from \u003cem\u003eB. napus \u003c/em\u003eto \u003cem\u003eB. rapa\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e(A) Schematic illustrating the \u003cem\u003eBnSRK-1\u003c/em\u003e gene with the two target sites. (B) Schematic diagram of the construction of the expression vector pKSE401. (C) Site-specific mutations of \u003cem\u003eBnSRK-1\u003c/em\u003e. The PAM is in red. Deletions are denoted by black dashes.\u003c/p\u003e\n\u003cp\u003e(D) Pollen pollination assays of ‘W-3’.\u003c/p\u003e\n\u003cp\u003e(E) Pollen pollination assays of ‘W-3’.\u003c/p\u003e\n\u003cp\u003e(F) F1 hybrid lines obtained from‘Westar’ב QR44’.\u003c/p\u003e\n\u003cp\u003e(G) F1 hybrid lines obtained from‘T0-4 בQR44’.\u003c/p\u003e\n\u003cp\u003e(H) Pods in the incompatible pollination lines.\u003c/p\u003e\n\u003cp\u003e(I) Pods in the self-compatible pollination lines.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4381643/v1/9b3186bd3a8a55888c492774.jpg"},{"id":60581788,"identity":"c2fd54b7-be28-4dc4-84f4-1035e1a4a3fb","added_by":"auto","created_at":"2024-07-18 12:11:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1776039,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4381643/v1/631a0330-f9d6-4ad5-ac79-f5789c9a8f06.pdf"},{"id":56912180,"identity":"7ee24d04-793b-4198-9cb1-6d4a5ca00f4d","added_by":"auto","created_at":"2024-05-22 05:26:37","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":393967,"visible":true,"origin":"","legend":"\u003cp\u003eTable S1. The primers used in this study.\u003c/p\u003e\n\u003cp\u003eTable S2. Self-Compatibility index (SCI) of the plants in the BC\u003csub\u003e2\u003c/sub\u003e generation.\u003c/p\u003e\n\u003cp\u003eFig. S1. PCR amplification of the Cas9 gene in the transgenic line T1-4.\u003c/p\u003e\n\u003cp\u003eFig. S2. PCR amplification of \u003cem\u003eBnS-1 \u003c/em\u003especific molecular marker SPeS1-7/8 in F\u003csub\u003e1\u003c/sub\u003e plants.\u003c/p\u003e\n\u003cp\u003eFig. S3. PCR amplification of \u003cem\u003eBnS-1 \u003c/em\u003especific molecular marker SPeS1-7/8 in BC\u003csub\u003e1\u003c/sub\u003e plants.\u003c/p\u003e\n\u003cp\u003eFig. S4. PCR amplification of \u003cem\u003eBnS-1 \u003c/em\u003especific molecular marker SPeS1-7/8 in BC\u003csub\u003e2\u003c/sub\u003e plants.\u003c/p\u003e","description":"","filename":"SupplementaryMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-4381643/v1/69c4493e1e85e34c5db7b820.docx"}],"financialInterests":"","formattedTitle":"Effective Strategies for Creating Self-Compatible B. rapa by Introgression of Mutated SRK and SCR/SP11 Genes from B. napus","fulltext":[{"header":"Full Text","content":"\u003cp\u003eSI (Self-incompatibility) is widespread in \u003cem\u003eBrassica \u003c/em\u003evegetable crops, such as Chinese cabbage, pak choi, cabbage, cauliflower, broccoli, etc, which can promote outcrossing and increase the genetic diversity of these species. However, in the breeding process, SI is one of the main obstructions to the development and propagation of inbred lines. In recent years, most \u003cem\u003eBrassica \u003c/em\u003evegetable crops applied the male sterility system to produce the hybrids. As the male sterility line and the maintainer line share the same SI allele, which usually causes the failure of sterile line propagation. The manipulation of SI genes is an effective way to create self-compatible (SC) materials in \u003cem\u003eBrassica\u003c/em\u003e. Self-incompatibility in the \u003cem\u003eBrassicaceae\u003c/em\u003e is controlled by the multi-allelic 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, it is also called the \u0026lsquo;\u003cem\u003eS\u003c/em\u003e haplotype\u0026rsquo; (Vekemans et al. 2014). Several researches showed that mutations in genes involved in female specificity (\u003cem\u003eSRK\u003c/em\u003e gene), male specificity (\u003cem\u003eSP11/SCR\u003c/em\u003e gene), or downstream signaling pathways (\u003cem\u003eMLPK\u003c/em\u003e, \u003cem\u003eARC1\u003c/em\u003e) 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). \u003c/p\u003e\n\u003cp\u003eThe 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) and broccoli (Cunfa et al. 2023). However, the simultaneous mutation of two non-allelic 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 \u003cem\u003eBrassica\u003c/em\u003e, the basic diploid species\u003cem\u003e B. rapa\u003c/em\u003e (AA, 2n= 20) and \u003cem\u003eB. oleracea\u003c/em\u003e (CC, 2n= 18) are self-incompatible, but the cultivated allotetraploid \u003cem\u003eB. napus\u003c/em\u003e (AACC, 2n= 38) is self-compatible. Insertion of a non-autonomous \u003cem\u003eHelitron\u003c/em\u003e transposon in the promoter of the pollen recognition specificity gene \u003cem\u003eBnSP11-1\u003c/em\u003e in the A genome was responsible for the self-compatibility of the \u003cem\u003eB. napus\u003c/em\u003e cultivar\u0026lsquo;Westar\u0026rsquo;(Gao et al. 2016). Thus, in the current study, we used CRISPR/Cas9 technology to mutate the \u003cem\u003eBnSRK-1\u003c/em\u003e gene in \u003cem\u003eB. napus\u003c/em\u003e cultivar\u0026lsquo;Westar\u0026rsquo;, then employed distant hybridization to introduce the mutated \u003cem\u003eBnSRK-1\u003c/em\u003e genes into \u003cem\u003eB. rapa\u003c/em\u003e, ultimately creating SC materials.\u003c/p\u003e\n\u003cp\u003eTo induce mutations in the \u003cem\u003eBnSRK-1\u003c/em\u003e gene, we designed a CRISPR-Cas9 construct that targets \u003cem\u003eBnSRK-1\u003c/em\u003e. Two guide RNAs (named K1-a and K1-b) were designed to target the first exon of \u003cem\u003eBnSRK-1\u003c/em\u003e (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 \u003cem\u003eBnSRK-1\u003c/em\u003e gene. Finally, 4 transgenic plants (T0-4, T0-10,T0-11,T0-25) exhibited mutations in the target region of the \u003cem\u003eBnSRK-1\u003c/em\u003e 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, both the K1-a and K1-b target sites contain a single A insertion. Chimeric mutations and the entire fragment deletions between the two target sites (including 10 bp insertion and 106 bp deletion, 20 bp insertion and 106 bp deletion, 39 bp insertion and 108 bp deletion) were found in T0-11 (Fig 1C). \u003c/p\u003e\n\u003cp\u003eTo investigate the self-compatibility on the stigma side of T0-4, a transgenic self-incompatible line \u0026lsquo;W-3\u0026rsquo; that contains a functional \u003cem\u003eBnSP11-1\u003c/em\u003e gene in \u003cem\u003eB. napus\u003c/em\u003e was used (Gao et al. 2016). Pollination assays showed that the stigma of T0-4 was compatible with the pollen of \u0026lsquo;W-3\u0026rsquo;, while the stigma of wild type \u0026lsquo;Westar\u0026rsquo; was incompatible with the pollen of \u0026lsquo;W-3\u0026rsquo; (Fig 1D). Then in the T1 generation, 20 plants were obtained, and all of them showed the same mutations at the target sites as that of T0-4 (CCAT deletion in the K1-a target site and TG deletion in the K1-b target site). The stigmas of all the plants were compatible with the pollen of \u0026lsquo;W-3\u0026rsquo;, and 4 plants (T1-4-2, T1-4-4, T1-4-14, and T1-4-18) were Cas9-free (Fig S1). All the results demonstrate that inducing loss-of-function mutations in the \u003cem\u003eBnSRK-1 \u003c/em\u003egene resulted in self-compatibility in the stigma of \u003cem\u003eB. napus\u003c/em\u003e. Both the pollen side and stigma side of T0-4 have lost self-incompatibility and can be used to transfer the self-compatibility into \u003cem\u003eB. rapa\u003c/em\u003e. \u003c/p\u003e\n\u003cp\u003eTo transfer the self-compatibility from \u003cem\u003eB. napus\u003c/em\u003e to \u003cem\u003eB. rapa\u003c/em\u003e, the transgenic line T1-4-5 was crossed with a self-incompatible line \u0026lsquo;QR44\u0026rsquo; that contains a recessive \u003cem\u003eS\u003c/em\u003e allele \u003cem\u003eBrS-44\u003c/em\u003e from non-heading Chinese cabbage and the F\u003csub\u003e1\u003c/sub\u003e hybrid was obtained (Fig 1E, S2). BC\u003csub\u003e1\u003c/sub\u003e progenies were obtained through backcross of the interspecific hybrid as female parents and with \u0026lsquo;QR44\u0026rsquo; as male parents. \u003cem\u003eS\u003c/em\u003e locus specific molecular marker SPeS1-7/8 was used to select the plants containing \u003cem\u003eS\u003c/em\u003e allele \u003cem\u003eBnS-1\u003c/em\u003e, and 20 out of 37 plants were selected (Fig S3). Furthermore, BC\u003csub\u003e2\u003c/sub\u003e progenies were obtained through the backcross of one BC\u003csub\u003e1\u003c/sub\u003e plant containing \u003cem\u003eS\u003c/em\u003e allele \u003cem\u003eBnS-1\u003c/em\u003e as the female parent and with \u0026lsquo;QR44\u0026rsquo; as the male parent. In the BC\u003csub\u003e2\u003c/sub\u003e generation, 9 out of 13 plants contained \u003cem\u003eS\u003c/em\u003e allele \u003cem\u003eBnS-1 \u003c/em\u003e(Fig S4), all the plants were self-pollinated and the SCI index was calculated. Of the 9 plants that contain the \u003cem\u003eS\u003c/em\u003e allele \u003cem\u003eBnS-1\u003c/em\u003e, 5 plants showed self-compatibility phenotypes with the SCI index \u0026gt;2, and the rest 4 plants are 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 (BC\u003csub\u003e2\u003c/sub\u003eF\u003csub\u003e2\u003c/sub\u003e) of the SC plants, 3 elite and completely self-compatible lines that contain homozygous \u003cem\u003eS\u003c/em\u003e allele \u003cem\u003eBnS-1 \u003c/em\u003e(Fig 1F) were obtained. \u003c/p\u003e\n\u003cp\u003eIn summary, we successfully performed site-specific manipulation of the stigma recognition specificity gene \u003cem\u003eBnSRK-1\u003c/em\u003e using the CRISPR/Cas9 system in \u003cem\u003eB. napus\u003c/em\u003e cultivar \u0026lsquo;Westar\u0026rsquo; that contains the mutated pollen recognition specificity gene \u003cem\u003eBnSP11-1\u003c/em\u003e. Furthermore, we transferred both mutated SI genes to \u003cem\u003eB. rapa\u003c/em\u003e 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 AA genome (2n=20), such as Chinese cabbage, purple flowering stalks, and Chinese flowering cabbage to accelerate the breeding process.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSupplementary\u003c/strong\u003e \u003cstrong\u003eInformation\u003c/strong\u003e The online version contains supplementary material available at\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e Xueli Zhang, Shuangping Heng, Chunxiu Xiao, Cong Liu and Liping Song\u0026nbsp;performed experiments and analyzed the data.\u0026nbsp;Xueli Zhang and Shuangping Heng\u0026nbsp;wrote the manuscript.\u0026nbsp;Aihua Wang and Changbin Gao designed the experiments. Liguang Tang, Congan He, Bincai Wang, Aihua Wang and Changbin Gao\u0026nbsp;contributed materials and reagents.\u0026nbsp;All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding authors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence to Aihua Wang and Changbin Gao.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e This research was supported by grants from the Wuhan knowledge innovation special project (2022020801010413).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBateman AJ (1955) Self-incompatibility systems in angiosperms: III. Cruciferae. Heredity 9 (1):53-68. doi:10.1038/hdy.1955.2\u003c/li\u003e\n\u003cli\u003eChen F, Yang Y, Li B, Liu Z, Khan F, Zhang T, Zhou G, Tu J, Shen J, Yi B (2019) Functional analysis of M-locus protein kinase revealed a novel regulatory mechanism of self-incompatibility in Brassica napus L. International journal of molecular sciences 20 (13):3303\u003c/li\u003e\n\u003cli\u003eCunfa M, Ting W, Hui Z, Tianpei Z, Lijian Z, Yonghui Y, Jiancheng X, Jun L, Shuiqin W (2023) Creation of Self-compatible Broccoli Lines by Knocking out BoSP11 Gene Through CRISPR/Cas9 Technology. 园艺学报. doi:10.16420/j.issn.0513-353x.2023-0065\u003c/li\u003e\n\u003cli\u003eGao C, Zhou G, Ma C, Zhai W, Zhang T, Liu Z, Yang Y, Wu M, Yue Y, Duan Z, Li Y, Li B, Li J, Shen J, Tu J, Fu T (2016) Helitron-like transposons contributed to the mating system transition from out-crossing to self-fertilizing in polyploid Brassica napus L. Scientific Reports 6 (1):33785. doi:10.1038/srep33785\u003c/li\u003e\n\u003cli\u003eKachroo A, Schopfer CR, Nasrallah ME, Nasrallah JB (2001) Allele-specific receptor-ligand interactions in Brassica self-incompatibility. Science 293 (5536):1824-1826\u003c/li\u003e\n\u003cli\u003eMa C, Zhu C, Zheng M, Liu M, Zhang D, Liu B, Li Q, Si J, Ren X, Song H (2019) CRISPR/Cas9-mediated multiple gene editing in Brassica oleracea var. capitata using the endogenous tRNA-processing system. Horticulture Research 6 (1):20. doi:10.1038/s41438-018-0107-1\u003c/li\u003e\n\u003cli\u003eMurase K, Shiba H, Iwano M, Che F-S, Watanabe M, Isogai A, Takayama S (2004) A membrane-anchored protein kinase involved in Brassica self-incompatibility signaling. Science 303 (5663):1516-1519\u003c/li\u003e\n\u003cli\u003eOkamoto S, Odashima M, Fujimoto R, Sato Y, Kitashiba H, Nishio T (2007) Self‐compatibility in Brassica napus is caused by independent mutations in S‐locus genes. The Plant Journal 50 (3):391-400\u003c/li\u003e\n\u003cli\u003eSchopfer CR, Nasrallah ME, Nasrallah JB (1999) The male determinant of self-incompatibility in Brassica. Science 286 (5445):1697-1700\u003c/li\u003e\n\u003cli\u003eStein JC, Howlett B, Boyes DC, Nasrallah ME, Nasrallah JB (1991) Molecular cloning of a putative receptor protein kinase gene encoded at the self-incompatibility locus of Brassica oleracea. Proceedings of the National Academy of Sciences 88 (19):8816-8820\u003c/li\u003e\n\u003cli\u003eStone SL, Arnoldo M, Goring DR (1999) A breakdown of Brassica self-incompatibility in ARC1 antisense transgenic plants. Science 286 (5445):1729-1731\u003c/li\u003e\n\u003cli\u003eTakasaki T, Hatakeyama K, Suzuki G, Watanabe M, Isogai A, Hinata K (2000) The S receptor kinase determines self-incompatibility in Brassica stigma. Nature 403 (6772):913-916\u003c/li\u003e\n\u003cli\u003eTakayama S, Shimosato H, Shiba H, Funato M, Che F-S, Watanabe M, Iwano M, Isogai A (2001) Direct ligand\u0026ndash;receptor complex interaction controls Brassica self-incompatibility. Nature 413 (6855):534-538\u003c/li\u003e\n\u003cli\u003eVekemans X, Poux C, Goubet PM, Castric V (2014) The evolution of selfing from outcrossing ancestors in Brassicaceae: what have we learned from variation at the S‐locus? Journal of Evolutionary Biology 27 (7):1372-1385\u003c/li\u003e\n\u003cli\u003eYe M, Peng Z, Tang D, Yang Z, Li D, Xu Y, Zhang C, Huang S (2018) Generation of self-compatible diploid potato by knockout of S-RNase. Nature Plants 4 (9):651-654\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-breeding","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"molb","sideBox":"Learn more about [Molecular Breeding](https://www.springer.com/journal/11032)","snPcode":"11032","submissionUrl":"https://submission.nature.com/new-submission/11032/3","title":"Molecular Breeding","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4381643/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4381643/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"SI (Self-incompatibility) is one of the major obstructions for the development and propagation of inbred lines in most Brassica vegetable crops. The manipulation of SI genes is an effective way to create Self-compatible (SC) materials. A mutated pollen recognition specificity gene BnSP11-1 on the A genome was responsible for the self-compatibility of the B. napus (AACC) cultivar ‘Westar’. In this study, site-specific manipulation of stigma recognition specificity gene BnSRK-1 was carried out using the CRISPR/Cas9 system in ‘Westar’. Then the mutated SI genes (BnSP11-1 and BnSRK-1) were transferred to B.rapa by distant hybridization, by continuous back cross and self pollination, 3 elite and completely self-compatible lines were obtained. The mutated SI genes can be applied widely in Brassica vegetable crops with AA genome (2n=20), such as Chinese cabbage, Pak choi, Purple flowering stalks and Chinese flowering cabbage to accelerate the breeding process.","manuscriptTitle":"Effective Strategies for Creating Self-Compatible B. rapa by Introgression of Mutated SRK and SCR/SP11 Genes from B. napus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-22 05:26:32","doi":"10.21203/rs.3.rs-4381643/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Correct before final acceptance","date":"2024-06-21T21:46:35+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-05-12T04:38:23+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-05-12T03:59:37+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-05-09T07:38:46+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Breeding","date":"2024-05-08T22:51:59+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-breeding","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"molb","sideBox":"Learn more about [Molecular Breeding](https://www.springer.com/journal/11032)","snPcode":"11032","submissionUrl":"https://submission.nature.com/new-submission/11032/3","title":"Molecular Breeding","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"260fcefa-f8d2-469d-bcf5-f64e393da89c","owner":[],"postedDate":"May 22nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-07-18T12:10:53+00:00","versionOfRecord":{"articleIdentity":"rs-4381643","link":"https://doi.org/10.1007/s11032-024-01487-4","journal":{"identity":"molecular-breeding","isVorOnly":false,"title":"Molecular Breeding"},"publishedOn":"2024-07-01 12:10:53","publishedOnDateReadable":"July 1st, 2024"},"versionCreatedAt":"2024-05-22 05:26:32","video":"","vorDoi":"10.1007/s11032-024-01487-4","vorDoiUrl":"https://doi.org/10.1007/s11032-024-01487-4","workflowStages":[]},"version":"v1","identity":"rs-4381643","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4381643","identity":"rs-4381643","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}