Brief CommuniCationhttps://doi.org/10.1038/s41477-018-0218-6

1The CAAS-YNNU Joint Academy of Potato Sciences, Yunnan Normal University, Kunming, China. 2Genome Analysis Laboratory of the Ministry of Agriculture, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen, China. 3College of Horticulture, Northwest Agriculture and Forest University, Yangling, China. 4Key Laboratory of Biology and Genetic Improvement of Horticultural Crops of the Ministry of Agriculture, Sino-Dutch Joint Laboratory of Horticultural Genomics, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing, China. 5These authors contributed equally to this work: Mingwang Ye, Zhen Peng. *e-mail: zhangchunzhi01@caas.cn; huangsanwen@caas.cn

Re-domestication of potato into an inbred line-based diploid crop propagated by seed represents a promising alternative to traditional clonal propagation of tetraploid potato, but self-incompatibility has hindered the development of inbred lines. To address this problem, we created self-compatible diploid potatoes by knocking out the self-incompatibility gene S-RNase using the CRISPR–Cas9 system. This strategy opens new avenues for diploid potato breeding and will also be use-ful for studying other self-incompatible crops.

Potato (Solanum tuberosum) is the most important tuber crop worldwide. Unlike other major crops, potato is an autotetraploid plant that is propagated from tubers. Potato researchers and breeders have long been challenged by the tetrasomic inheritance of potato and the need for vegetative propagation. The genetic gain from population improvement in major crops such as maize is approximately 1% per year1. By contrast, breeding has done little to increase yields in potato. Some century-old potato varieties, such as Russet Burbank (released in 1902)2 and Bintje (bred in 1904)3, are still widely cultivated. Thus, an increasing number of studies has focused on reinventing potato as an inbred line-based diploid crop that is propagated by seed4–6. Indeed, diploids were very prevalent among wild species and landra-ces, accounting for 70% of the potato germplasm7. Re-domestication of these diploid potatoes using modern agricultural technologies would represent a breakthrough in potato breeding. However, most tuber-bearing diploid potato species are self-incompatible, which has hampered the development of inbred lines.

Self-incompatibility in potato is controlled by the highly poly-morphic S-locus8. The identification of a dominant S-locus inhibitor (Sli), originating from the wild potato species Solanum chacoense, has paved the way for exploring diploid breeding in potato9,10. This gene has been widely used in genetic research and breeding4,11–13. However, wild S. chacoense accessions produce long stolons, approximately 1 m in length14, which will lead to excessive loss of energy in stolon growth and difficulty in harvesting. Moreover, the tubers of S. chacoense contain high levels of toxic steroidal glycoal-kaloids14,15. Owing to these undesirable traits, a viable alternative to the deployment of the Sli gene is required. Thus, in the current study, we used clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein 9 (Cas9) to create a self-compatible diploid potato line without introducing any wild DNA fragments.

The S-RNase gene, which controls self-incompatibility in potato, has high allelic diversity, with amino acid similarity varying from

32.9% to 94.5%16, and is highly expressed in styles17. The traditional method used to obtain the full-length sequences of genes such as S-RNase is rapid amplification of cDNA ends. However, high rates of polymorphism make it difficult to design the appropriate primers for successful amplification by rapid amplification of cDNA ends. The presence of relatively conserved domains in S-RNase and its tissue-specific expression pattern motivated us to isolate the full-length S-RNase sequence by RNA sequencing (RNA-seq).

To verify the utility of this method, we generated RNA-seq data using RNA from the styles of the diploid potato clone S. tuberosum group Phureja S15-65. We carried out de novo assembly of the RNA-seq data using Trinity (v2.2.0) and calculated the expression levels of all transcripts using RSEM (v1.2.9). In total, 83,545 transcripts were obtained, which greatly exceeded the number of genes (39,000) in the potato reference genome, as some genes had different isoforms or their complete transcripts were divided into different fragments. Using the sequence of the S-RNase protein from the potato reference genome DM as a query, five target sequences were found among the assembled transcripts, all of which were highly expressed in the style (Supplementary Table 1). The target sequences contained two 5′ untranslated regions and two 3′ untranslated regions; thus, we designed PCR primers to amplify the full-length S-RNase genes (Supplementary Table 2). We ultimately obtained two complete S-RNase alleles (Sp3 and Sp4, named in accordance with the nam-ing conventions proposed by Dzidzienyo et al.16) (Supplementary Fig. 1). The amino acid sequences of Sp3 and Sp4 share a similarity of 59%, indicating that they are different S-RNase alleles. Phylogenetic analysis showed that they are located in the same clade as known functional S-RNase genes (Fig. 1a). We also validated their expres-sion by quantitative PCR (Fig. 1b). Our results demonstrate that de novo assembly of RNA-seq data is an efficient, low-cost way to clone genes, especially for genes with high diversity in a population.

The self-incompatibility mechanism is conserved in Solanaceae8,18. Although most wild tomatoes are self-incompatible, cultivated tomato is self-compatible due to the loss of function of S-RNase and other self-incompatibility-related genes18. Thus, we reasoned that we could re-invent self-incompatible diploid potato by knocking out the S-RNase alleles using the CRISPR–Cas9 system. To induce mutations in S-RNase, we designed a CRISPR–Cas9 con-struct targeting the first exon of S-RNase. The single guide RNA was designed to target the relatively conserved domain of the S-RNase protein and could target Sp3 and Sp4 simultaneously (Supplementary Fig. 1). We generated 96 plantlets that grew on medium containing

Generation of self-compatible diploid potato by knockout of S-RNaseMingwang Ye1,2,5, Zhen Peng1,2,5, Die Tang2, Zhongmin Yang3, Dawei Li3, Yunmei Xu1, Chunzhi Zhang2,4* and Sanwen Huang   2,4*

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kanamycin (50 mg l−1). To evaluate the efficiency of the mutations, we amplified and sequenced the full-length S-RNase sequences in all T0 plants. In total, we obtained 17 clones containing mutations in Sp3 or Sp4. However, some mutations were heterozygous in Sp3 or Sp4, which is not possible in diploids unless the plants are chimeric.

Moreover, the corresponding in vitro-grown plants were much stronger than the wild-type clones. Thus, the ploidy of these plants might have doubled during tissue culture, which is a common phenomenon in potato19. Flow cytometry analysis revealed that some plants were tetraploid (Supplementary Fig. 2); these clones

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Fig. 1 | Knockout of S-RNase overcomes self-incompatibility in potato. a, Phylogenetic tree of S-RNases and S-like RNases. Fungal RNase T2 from Aspergillus oryzae was included as an outgroup. N. ala, Nicotiana alata; N. glu, Nicotiana glutinosa; N. tab, Nicotiana tabacum; S. lyc, Solanum lycopersicum; S. oka, Solanum okadae; S. phu, S. tuberosum group Phureja; S. ste, S. tuberosum group Stenotomum. The different colours indicate the different types of RNase. b, The relative expression levels of two S-RNase alleles in the style. The y axis represents the S-RNase expression level that related to the internal control gene EIF-3e. The black dots represent the expression levels of three biological replicates, and the error bars represent the standard error of three biological replicates. c, The gene structure of S-RNase and the mutation patterns in T0 transgenic plants. sgRNA, single guide RNA; WT, wild type. d, Pollen tube growth in pollinated styles. At least five stigmas were observed for each line. The experiments were repeated twice and the same results were obtained. Scale bar, 400 μ m. e, Fruit setting in wild-type and transgenic plants. Five cuttings of each T0 line were grown in the greenhouse and artificially pollinated; all of the plants produced fruits. Scale bars, 0.5 cm. f, Segregation of the Cas9-free seedlings in the T1 generation. The y axis represents the number of seedlings. The grey and white columns indicate the number of tested T1 seedlings and the Cas9-free T1 seedlings, respectively.

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were excluded from further analysis. We ultimately obtained five mutant diploid clones, each containing mutations in at least one S-RNase allele (Fig. 1c and Supplementary Table 3). As S-RNase is a co-dominant gene, the mutation of either allele would enable pollen containing the same S-haplotype to extend into the style, thereby resulting in self-compatibility.

To investigate the self-compatibility of the transgenic plants, we transferred all mutant clones to the greenhouse under a 16-h light/8-h dark cycle. We performed artificial pollinations at the open flower stage, with at least 50 flowers self-pollinated per line. We observed the growth of pollen tubes in styles by aniline blue fluorochrome staining. In wild-type styles, pollen tube growth was suppressed and was restricted to the upper part of the style, whereas in mutant styles, many pollen tubes extended to the ovary (Fig. 1d). After self-pollination, all mutant lines produced fruits (Fig. 1e). Although the rate of fruit setting varied among lines, the number of seeds per fruit was sufficient for further propagation, ranging from 67 to 288 seeds per fruit. These results demonstrate that induc-ing loss-of-function mutations in S-RNase genes resulted in self- compatibility in potato. The growth vigour and plant morphology of the mutant lines did not differ from those of the wild type, indi-cating that they can directly be used for breeding.

To determine whether the mutations could be transmitted to the T1 generation, we sowed the T1 seeds on the MS medium. The ger-mination rate was > 95% for all five families. The ratio of T1 plants without the Cas9 cassette varied from 3.6% to 24.5% (Fig. 1f and Supplementary Table 3). The Chi-square test showed that lines 42 and 44 contained a single copy of the Cas9 cassette and lines 32 and 57 contained two copies. However, as the ratio of the Cas9-free T1 plants of line 66 is lower than 1/4 but higher than 1/16, we infer that the number of T1 plants used for the test might not be enough or the genomic region harbouring the Cas9 cassette might exhibit segregation distortion in the selfed progeny. We detected mutations in the S-RNase genes in all Cas9-free plants. The chimeric muta-tions in lines 32 and 44 did not inherit into the T1 generation and only a single mutation of Sp4 in line 57 was transmitted to the T1 generation (Supplementary Table 3), suggesting that the mutations in the leaves and germ cells were different. All T1 plants harboured mutations in at least one S-RNase allele (Supplementary Table 3), indicating that the pollen containing the wild-type S-RNase was rejected during selfing.

In summary, we designed an efficient approach for overcoming self-incompatibility in potato without introducing any exogenous DNA. Using the same method, we also obtained S-RNase mutants using two S. tuberosum group Phureja clones (S15-47 and S15-76) and two S. tuberosum group Stenotomum clones (S15-48 and S15-107) (Supplementary Table 2). More self-compatible clones are needed to increase the genetic diversity of the S-locus to avoid a potential genetic bottleneck in future diploid breeding efforts. These self-compatible diploid potatoes can be used to produce inbred lines, which can be further used to generate mutant librar-ies, recombinant inbred lines, near isogenic lines and introgression lines. These populations will be a powerful tool to accelerate basic research and genetic improvement in potato. Furthermore, the strategy presented should be beneficial for researchers and breeders of other self-incompatible crops, such as plants in the Cruciferae and Rosaceae families, allowing them to make more rapid progress in genetic improvement.

MethodsDe novo assembly of RNA-seq data. Total RNA was isolated from potato styles using an RNAprep Pure Plant Kit (polysaccharides and polyphenolics rich) (Tiangen). The RNA was subjected to whole-genome resequencing using the Illumina HiSeq X Ten platform (reads length = 150 bp) (Novogene). The insert size of the library was 300 bp and 13 million reads were generated. The RNA-seq data were de novo assembled using Trinity (v2.2.0) without a reference guide with default parameters. Transcript qualification and analysis of the differential

expression of genes and isoforms in the samples were performed using RSEM (v1.2.9). The assembled transcripts were built as the local database and known S-RNase protein was aligned to the database using the tblastn program of BLAST (v2.5.0) with default parameters on the Linux system; those with a low E-value (< 1 × 10−5) and a high FPKM (fragments per kilobase of transcript per million mapped reads) value (> 200) were identified as candidate S-RNase sequences.

Quantitative PCR. The RNA was reverse transcribed using a FastQuant RT Kit (with gDNase) (Tiangen). Gene-specific oligonucleotides were designed using the Primer Premier 5 software (Applied Biosystems). EIF-3e was used as the internal control gene. PCRs were performed on a BIO-RAD CFX96 using SYBR Premix (Roche) according to the manufacturer’s instructions. The relative gene expression levels were calculated using the 2−ΔCt method20.

Plasmid construction and transformation. The 19-nt single guide RNA sequence for S-RNase in the diploid clone S15-65 was selected manually from the conserved domain and used for BLAST analysis against the potato reference genome version 4.03 (ref. 21); no other potential matches in the genome were identified. The target sequence was incorporated into the CRISPR–Cas9 vector pKSE401 (ref. 22) The diploid self-incompatible S. tuberosum group Phureja S15-65 clone was used in this study. Three-week-old plantlets were used for transformation. Agrobacterium-mediated transformation of potato internodes was conducted as previously described with some modifications23: after 2 days of pre-culture, the explants were co-cultured with Agrobacterium harbouring pKSE401 with the target sequence for 2 days in the presence of 2 mg l−1 α -naphthaleneacetic acid and 1 mg l−1 zeatin, followed by callus induction and regeneration mediated by 0.01 mg l−1 α -naphthaleneacetic acid and 2 mg l−1 zeatin until shoot proliferation. Positive transformants were screened based on growth on the medium containing 50 mg l−1 kanamycin.

Detection of mutations in the transgenic lines. To detect the mutations in S-RNase, the full-length S-RNase sequences were amplified from all regenerated plantlets and sequenced. As chromosome doubling occurred at high frequency during potato callus regeneration19, the ploidy of the mutated plants was determined by flow cytometry (BD FACSDiva v7.0 Software).

Aniline blue fluorochrome staining of pollen tubes. Pollinations were performed using mature self-pollen. Carpels were collected 48 h post-pollination and immediately placed in 3:1 95% ethanol:glacial acetic acid. After fixation, the carpels were submerged in 5 M NaOH softening solution for 24 h. After washing with double-distilled water, the carpels were incubated in 0.005 mg ml−1 aniline blue fluorochrome in 0.1 M K2HPO4 (pH 10) buffer for 24 h and viewed under a fluorescence microscope.

Analysis of selfed progeny. The T1 seeds were sown in modified MS medium (2.2 g l−1 MS powder, 10 g l−1 sucrose and 6 g l−1 agar (pH 5.8)). Four weeks after sowing, DNA was extracted from fresh leaves. The detection of the Cas9 cassette was performed for all T1 plants. The two S-RNase alleles in all clones lacking the Cas9 cassette were amplified and sequenced.

Reporting Summary. Further information on experimental design is available in the Nature Research Reporting Summary linked to this article.

Data availability. The RNA-seq data have been deposited in the NCBI with the accession number SRX3979417.

Received: 27 February 2018; Accepted: 11 July 2018; Published: xx xx xxxx

references 1. Duvick, D. N. Adv. Agron. 86, 83–145 (2005). 2. Bachem, P. C. et al. Am. J. Potato Res. 91, 594–609 (2014). 3. Ramulu, K. S., Dijkhuis, P. & Roset, S. Theor. Appl. Genet. 65, 329–338 (1983). 4. Lindhout, P. et al. Potato Res. 54, 301–312 (2011). 5. Li, Y., Li, G., Li, C., Qu, D. & Huang, S. Chinese Potato 27, 96–99 (2013). 6. Jansky, S. H. et al. Crop Sci. 56, 1412–1422 (2016). 7. Spooner, D. M., Ghislain, M., Simon, R., Jansky, S. H. & Gavrilenko, T.

Bot. Rev. 80, 283–383 (2014). 8. Kao, T. H. & McCubbin, A. G. Proc. Natl Acad. Sci. USA 93,

12059–12065 (1996). 9. Hosaka, K. & Hanneman, R. E. Jr. Euphytica 99, 191–197 (1998). 10. Hosaka, K. & Hanneman, R. E. Jr. Euphytica 103, 265–271 (1998). 11. Phumichai, C., Mori, M., Kobayashi, A., Kamijima, O. & Hosaka, K. Genome

48, 977–984 (2005). 12. Endelman, J. B. & Jansky, S. H. Theor. Appl. Genet. 129, 935–943 (2016). 13. Duangpan, S., Zhang, W., Wu, Y., Jansky, S. H. & Jiang, J. Plant Physiol. 163,

21–29 (2013). 14. Jansky, S. H., Chung, Y. S. & Kittipadukal, P. J. Plant Regist. 8,

195–199 (2014).

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15. Leisner, C. P. et al. Plant J. 94, 562–570 (2018). 16. Dzidzienyo, D. K., Bryan, G. J., Wilde, G. & Robbins, T. P. Theor. Appl. Genet.

129, 1985–2001 (2016). 17. Anderson, M. A. et al. Nature 321, 38–44 (1986). 18. Kondo, K. et al. Plant J. 29, 627–636 (2002). 19. Karp, A., Jones, M. G. K., Foulger, D., Fish, N. & Bright, S. W. J. Am. Potato J.

66, 669–684 (1989). 20. Livak, K. J. & Schmittgen, T. D. Methods 25, 402–408 (2001). 21. Sharma, S. K. et al. G3 3, 2031–2047 (2013). 22. Xing, H. et al. BMC Plant Biol. 14, 327 (2014). 23. Ducreux, L. J., Morris, W. L., Taylor, M. A. & Millam, S. Plant Cell Rep. 24,

10–14 (2005).

acknowledgementsThis work was supported by Advanced Technology Talents in Yunnan Province (2013HA025) and the National Natural Science Foundation of China (31601360 to C.Z.). This work was also supported by the Chinese Academy of Agricultural Science (ASTIP-CAAS) and the Shenzhen municipal and Dapeng district governments.

author contributionsS.H. and C.Z. conceived and designed the experiments. M.Y. and Z.Y. performed the potato transformation. Z.P. and Y.X. conducted the genotyping and phenotyping of T0 and T1 plants. D.T. performed the bioinformatics analyses. D.L. made the CRISPR–Cas9 construct. C.Z., Z.P., M.Y. and S.H. wrote the manuscript.

Competing interestsThe authors declare no competing interests.

additional informationSupplementary information is available for this paper at https://doi.org/10.1038/s41477-018-0218-6.

Reprints and permissions information is available at www.nature.com/reprints.

Correspondence and requests for materials should be addressed to C.Z. or S.H.

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Corresponding author(s): Professor Sanwen Huang

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Data collection No software was used.

Data analysis Trinity (v2.2.0) was used for de novo assembly of RNA-seq data. RSEM (v1.2.9) was used to calculate the expression of genes and isoforms. The program tblastn of BLAST (v2.5.0) was used to search for the S-RNase homologs. BD FACSDiva (v7.0) was used to analyze the results generated by flow cytometry.

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Sample size Based on our previous studies, there are usually one or two copies of Cas9-cassette transformed into the T0 regenerated plants in potato. According to the Mendelian genetic law, the segregation of Cas9-containing and Cas9-free plants in T1 generation should be 3:1 or 15:1. To screening the Cas9-free T1 plants, we screened 136 or 192 T1 seedlings for each T0 family, and obtained the Cas9-free plants for all families.

Data exclusions No data was excluded for analysis.

Replication In this study, we developed a method to generate self-compatible diploid potato. We tested this method in five other diploid potato clones, and it worked well for all of them.

Randomization This is not relevant to our study. No allocation was involved in this study.

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ChIP-seq

Flow cytometry

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Flow CytometryPlots

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Methodology

Sample preparation The fresh leaves from in vitro plants were chopped and incubated in lysate. Then the cell nucleus were collected by centrifugation.

Instrument BD LSRFortessa™ X-20

Software BD FACSDiva v7.0 Software

Cell population abundance The proportion of target cell was higher than 70%.

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Gating strategy We used known diploid and tetraploid potato clones as control to deduce the ploidy of transgenic plants.

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