Analysis of FvPDS Gene Using CRISPR/Cas9 System in Strawberry

doi:10.16420/j.issn.0513-353x.2024-0612

Acta Horticulturae Sinica ›› 2025, Vol. 52 ›› Issue (7): 1687-1695.doi: 10.16420/j.issn.0513-353x.2024-0612

• Genetic & Breeding·Germplasm Resources·Molecular Biology • Next Articles

Analysis of FvPDS Gene Using CRISPR/Cas9 System in Strawberry

LIU Lifeng¹^,², SONG Yanhong¹^,², ZHAO Xia¹, LI Gang¹^,², and ZHOU Houcheng¹^,^*()

¹ Zhengzhou Fruit Research Institute，Chinese Academy of Agricultural Sciences，Zhengzhou 450009，China
² Zhongyuan Research Center Chinese Academy of Agricultural Sciences，Xinxiang，Henan 453003，China

Received:2024-10-16 Revised:2025-05-22 Online:2025-07-23 Published:2025-07-23
Contact: and ZHOU Houcheng

Abstract

Abstract:

The phytoene desaturase gene（FvPDS）in Fragaria vesca was selected as the marker gene. This gene encodes a key enzyme in carotenoid biosynthesis. Because of its mutants with visible albino phenotypes，FvPDS has been served as a model gene for CRISPR/Cas9 mediated targeted gene editing. In this study a gene knockout vector was constructed using the CRISPR/Cas9 system，different types of mutations were detected in F. vesca through stable transformation of strawberries leaves including HLJ-005 with red fruits and HLJ-004 with white fruits. Through Agrobacterium mediated transformation 2 250 explants from HLJ-005 were transformed，147 kanamycin resistant lines were obtained through resistance screening. Of theses，24 transgenic lines（16.32%）were confirmed as positive transgenic lines by T-DNA specific PCR. Sequencing detection revealed that 5 transgenic lines had different types of mutations at the two target sites with an editing efficiency of 20.8%. For the other F. vesca HLJ-004，2 305 leaves were genetically transformed，234 kanamycin resistant lines were screened and 6 transgenic lines（2.56%）were confirmed as positive transgenic lines by T-DNA specific PCR. Sequencing detection showed that 3 lines had different types of mutations with an editing efficiency of 50%. Among these，mutations at two editing sites resulted in the deletion of single or multiple amino acids，leading to albino and dwarf phenotypes. These results demonstrate that the CRISPR/Cas9 system enables simultaneous double-site knockout in the woodland strawberry varieties HLJ-004 and HLJ-005 via stable expression. HLJ-004 and HLJ-005 can serve as suitable genetic transformation materials for strawberry gene editing，and the study validated a CRISPR/Cas9 system appropriate for editing strawberry genes.

Key words: strawberry, CRISPR/Cas9, FvPDS, gene editing

LIU Lifeng, SONG Yanhong, ZHAO Xia, LI Gang, and ZHOU Houcheng. Analysis of FvPDS Gene Using CRISPR/Cas9 System in Strawberry[J]. Acta Horticulturae Sinica, 2025, 52(7): 1687-1695.

Figures/Tables 6

Table 1 The prime names and sequences

引物名称 Prime name	引物序列（5′-3′） Sequences of primes
U626F	TGTCCCAGGATTAGAATGATTAGGC
U629R	AGCCCTCTTCTTTCGATCCATCAAC
FvPDS-F	GCATTATCTTCGTCTTGCGTGAT
FvPDS-R	GACTCCTCAATCTAGCCTAAACAC
FvPDS-BsF	ATATATGGTCTCGATTGTTCTGCCAGCACCCTTAAAGTT
FvPDS-F0	TGTTCTGCCAGCACCCTTAAAGTTTTAGAGCTAGAAATAGC
FvPDS-R0	AACAGAATTGCAGGCACAAGTCCAATCTCTTAGTCGACTCTAC
FvPDS-BsR	ATTATTGGTCTCGAAACAGAATTGCAGGCACAAGTCCAA

Fig. 1 Schematic representation of FvPDS with two target sites（a）and pKSE401 expression cassettes（b）

Fig. 2 Agrobacterium-mediated genetic transformation of the Fragaria vesca A：The leaves were pre-cultured on induction medium；B：The adventitious buds were on the screening medium；C：The cluster buds were elongated on the screening medium；D：After 1 month，the cluster buds continued to grow on the screening medium；E：Chimeric albino plant were cultured on the screening medium

Fig. 3 The positive plants were identified by pCR of HLJ-005（A）and HLJ-004（B） M：Marker；WT：Wild type；P：Plasmid used as positive control

Table 2 CRISPR/Cas9 mediated editing of FvPDS in strawberry mutant types

种质 Germplasm	外植体数 Number of explants	抗性植株 Kan-resistant plants	转化植株 Transgenic plan		突变植株 Mutant plant		杂合突变 M-he		等位突变 M-bi
HLJ-005	2 250	147		24（16.32）		5（20.83）		3（12.50）	5（20.83）
HLJ-004	2 305	234		6（2.56）		3（50.00）		1（16.67）	3（50.00）

Fig. 4 Different types of mutations in the two edited germplasms after CRISPR/Cas9 mediated gene editing A：Mutation types at the first target site of PDS gene；B：Mutation types at the second target site of PDS gene

References 27

[1]	Cui Xia, Zhang Shuaibin. 2017. The utilization and prospect of genome editing in horticultural crops. Acta Horticulturae Sinica, 44 (9):1787-1795. (in Chinese)
	崔霞, 张率斌. 2017. 基因编辑技术及其在园艺作物中的应用和展望. 园艺学报, 44 (9):1787-1795.
[2]	Guo Ye, Wan Dongyan, Chai Zhuangzhuang, Wang Yuejin, Wen Yingqiang. 2019. Knock-out analysis of VviPDS1 gene using CRISPR/Cas 9 in grapevine. Acta Horticulturae Sinica, 46 (4):623-634. (in Chinese)
	郭晔, 万东艳, 柴壮壮, 王跃进, 文颖强. 2019. 利用 CRISPR/Cas9敲除葡萄VviPDS1基因的研究. 园艺学报, 46 (4):623-634. doi: 10.16420/j.issn.0513-353x.2018-0626
[3]	Ji X, Zhang H W, Zhang Y, Wang Y P, Gao C X. 2019. Establishing a CRISPR-Cas-like immune system conferring DNA virus resistance in plants. Nature Plants, 1 (10):1-4.
[4]	Ma Cunfa, Wu Ting, Zhao Hui, Zhang Tianpei, Zhao Lijian, Yu Yonghui, Xiao Jiancheng, Li Jun, Wu Shuiqin. 2024. Creation of self-compatible broccoli lines by knocking out BoSP11 gene through CRISPR/Cas 9 technology. Acta Horticulturae Sinica, 51 (3):509-519. (in Chinese)
	马存发, 武婷, 赵辉, 张天培, 赵立坚, 余永辉, 肖建成, 李军, 巫水钦. 2024. 利用 CRISPR/Cas9技术敲除青花菜BoSP11创制自交亲和系. 园艺学报, 51 (3):509-519.
[5]	Ma X L, Liu Y G. 2016. CRISPR/Cas9-based multiplex genome editing in monocot and dicot plants. Current Protocols in Molecular Biology,115:31.6.1-31.6.21.
[6]	Nakajima I, Ban Y, Azuma A, Onoue N, Moriguchi T, Yamamoto T, Toki S, Endo M. 2017. CRISPR/Cas9-mediated targeted mutagenesis in grape. PLoS ONE, 12 (5):e0177966.
[7]	Nishitani C, Hirai N, Komori S, Wada M, Okada K, Osakabe K, Yamamoto T, Osakabe Y. 2016. Efficient genome editing in apple using a CRISPR/Cas 9 system. Scientific Reports,6:1-8.
[8]	Oosumi T H A, Gruszewski L A, Blischak A J, Baxter P A W, Shuman R E, Veilleux V S. 2006. High-efficiency transformation of the diploid strawberry(Fragaria vesca)for functional genomics. Planta,223:1219-1230.
[9]	Qin G J, Gu H Y, Ma L G, Peng Y B, Deng X W, Chen Z L, Qu L J. 2007. Disruption of phytoene desaturase gene results in albino and dwarf phenotypes in Arabidopsis by impairing chlorophyll,carotenoid,and gibberellin biosynthesis. Cell Research, 17 (5):471-482.
[10]	Schaeffer S M, Nakata P A. 2016. The expanding footprint of CRISPR/Cas 9 in the plant sciences. Plant Cell Rep,35:1451-1468.
[11]	Shan Q W, Wang Y P, Li J, Gao C X. 2014. Genome editing in rice and wheat using the CRISPR/Cas system. Nat Protoc, 9 (10):2395-2410. doi: 10.1038/nprot.2014.157 pmid: 25232936
[12]	Shu Haiyan, Wang You, Xian Shuyun, Li Keming, Zhan Rulin, Chang Shenghe. 2022. Phytoene desaturase gene Aco-PDS 1 was edited in pineapple genome. Molecular Plant Breeding, 20 (22):7446-7452. (in Chinese)
	舒海燕, 王悠, 冼淑云, 李科明, 詹儒林, 常胜合. 2022. 菠萝八氢番茄红素脱氢酶基因Aco-PDS1编辑株系的构建. 分子植物育种, 20 (22):7446-7452.
[13]	Shulaev V, Sargent D J, Crowhurst R N, Mockler T C, Folkerts O, Delcher A L, Jaiswal P, Mockaitis K, Liston A, Mane S P, Burns P, Davis T M, Slovin J P, Bassil N, Hellens R P, Evans C, Harkins T, Kodira C, Desany B, Crasta O R, Jensen R V, Allan A C, Michael T P, Setubal J C, Celton J M, Rees D J, Williams K P, Holt S H, Ruiz Rojas J J, Chatterjee M, Liu B, Silva H, Meisel L, Adato A, Filichkin S A, Troggio M, Viola R, Ashman T L, Wang H, Dharmawardhana P, Elser J, Raja R, Priest H D, Bryant D W, Jr Fox S E, Givan S A, Wilhelm L J, Naithani S, Christoffels A, Salama D Y, Carter J, Lopez Girona,E, Zdepski A, Wang W, Kerstetter R A, Schwab W, Korban S S, Davik J, Monfort A, Denoyes-Rothan B, Arus P, Mittler R, Flinn B, Aharoni A, Bennetzen J L, Salzberg S L, Dickerman A W, Velasco R, Borodovsky M, Veilleux R E, Folta K M. 2011. The genome of woodland strawberry(Fragaria vesca). Nature Genetics, 43 (2):109-116.
[14]	Tian S W, Jiang L J, Gao Q, Zhang J, Zong M, Zhang H Y, Ren Y, Guo S G, Gong G Y, Liu F, Xu Y. 2017. Efficient CRISPR/Cas9-based gene knockout in watermelon. Plant Cell Reports, 36 (3):399-406. doi: 10.1007/s00299-016-2089-5 pmid: 27995308
[15]	Wilson F M, Harrison K, Armitage A D, Simkin A J, Harrison R J. 2019. CRISPR/Cas9-mediated mutagenesis of phytoene desaturase in diploid and octoploid strawberry. Plant Methods, 15 (45):1-13.
[16]	Xing H L, Dong L, Wang Z P, Zhang H Y, Han C Y, Liu B, Wang X C, Chen Q J. 2014. A CRISPR/Cas9 toolkit for multiplex genome editing in plants. BMC Plant Biol,14:317.
[17]	Xing S N, Jia M R, Wei L Z, Mao W W, Abbasi U A, Zhao Y Y, Chen Y T, Cao M L, Zhang K, Dai Z G, Dou Z C, Jia W S, Li B B. 2018. CRISPR/Cas9-introduced single and multiple mutagenesis in strawberry. Journal of Genetics and Genomics,45:685-687.
[18]	Xing S N, Kunling Chen K L, Zhu H C, Rui Zhang R, Zhang H W, Li B B, Gao C X. 2020. Fine-tuning sugar content in strawberry. Gemome Biology, 21 (230):1-14.
[19]	Xiong Wenwen, Liu Huimin, Chen Kaiyuan, Du Juan, Song Botao, Jing Shenglin. 2023. Optimization of gene editing system based on heat-treated CRISPR/Cas 9 potato plants. Acta Horticulturae Sinica, 50 (12):2591-2600. (in Chinese) doi: 10.16420/j.issn.0513-353x.2022-1168
	熊文汶, 刘慧敏, 陈凯园, 杜鹃, 宋波涛, 景晟林. 2023. 基于热处理CRISPR/Cas9马铃薯植株的基因编辑体系优化. 园艺学报, 50 (12):2591-2600. doi: 10.16420/j.issn.0513-353x.2022-1168
[20]	Yang Liang, Liu Huan, Ma Yanqin, Li Ju, Wang Hai’e, Zhou Yujie, Long Haicheng, Miao Mingjun, Li Zhi, Chang Wei. 2024. Creating high lycopene fruit using CRISPR/Cas 9 technology in tomato. Acta Horticulturae Sinica, 51 (2):253-265. (in Chinese)
	杨亮, 刘欢, 马燕勤, 李菊, 王海娥, 周玉洁, 龙海成, 苗明军, 李志, 常伟. 2024. 利用CRISPR/Cas9技术创制高番茄红素番茄新材料. 园艺学报, 51 (2):253-265.
[21]	Yang W H, Ren J Q, Liu W R, Liu D, Xie K D, Zhang F, Wang P W, Guo W W, Wu X M. 2023. An efficient transient gene expression system for protein subcellular localization assay and genome editing in citrus protoplasts. Horticultural Plant Journal, 9 (3):425-436.
[22]	Yu X X, Yu J T, Lu Y, Li W J, Huo G Z, Zhang J, Li Y, Zhao J J, Li J. 2024. An efficient and universal protoplast-based transient gene expression system for genome editing in Brassica crops. Horticultural Plant Journal, 10 (4):983-994.
[23]	Yuan Xuening, Yao Fengge, An Yi, Jiang Cheng, Chen Ningning, Huang Lichao, Lu Mengzhu, Zhang Jin. 2025. Application of CRISPR/Cas genome editing in woody plant trait improvement. Chinese Science Bulletin, 70 (16):2509-2525. (in Chinese)
	袁雪宁, 姚凤鸽, 安轶, 江成, 陈宁宁, 黄李超, 卢孟柱, 张进. 2025. CRISPR/Cas基因组编辑在木本植物性状改良中的应用. 科学通报, 70 (16):2509-2525.
[24]	Zhang Y, Zhou P, Bozorov T A, Zhang D Y. 2021. Application of CRISPR/Cas 9 technology in wild apple(Malus sieverii)for paired sites gene editing. Plant Methods, 17 (1):79. doi: 10.1186/s13007-021-00769-8 pmid: 34281579
[25]	Zhou J H, Wang G M, Liu Z C. 2018. Efficient genome editing of wild strawberry genes,vector development and validation. Plant Biotechnology Journal,16:1868-1877.
[26]	Zhou Xiangchun, Xing Yongzhong. 2016. The application of genome editing in identification of plant gene function and crop breeding. Hereditas, 38 (3):227-242. (in Chinese)
	周想春, 邢永忠. 2016. 基因组编辑技术在植物基因功能鉴定及作物育种中的应用. 遗传, 38 (3):227-242.
[27]	Zuo Xin, Li Mingming, Li Xinrong, Miao Chunyan, Li Yanfang, Yang Xu, Zhang Zhongyi, Wang Fengqing. 2022. CRISPR/Cas 9 technology for RcPDS1 gene editing in Rehmannia chingii. Acta Horticulturae Sinica, 49 (7):1532-1544. (in Chinese) doi: 10.16420/j.issn.0513-353x.2021-0475
	左鑫, 李铭铭, 李欣容, 苗春妍, 李炎枋, 杨旭, 张重义, 王丰青. 2022. CRISPR/Cas9技术在天目地黄RcPDS1基因编辑中的应用. 园艺学报, 49 (7):1532-1544. doi: 10.16420/j.issn.0513-353x.2021-0475

Analysis of FvPDS Gene Using CRISPR/Cas9 System in Strawberry

RichHTML

PDF

Knowledge

Abstract

Cite this article

share this article

Figures/Tables 6

References 27

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	SONG Qianna, SONG Lushuai, DUAN Yonghong, BAI Xiaodong, and FENG Ruiyun. Establishment a System to Obtain CRISPR/Cas9 Genome Editing Plants Based on Hairy Root Transformation in Potatoes [J]. Acta Horticulturae Sinica, 2025, 52(7): 1758-1768.
[2]	YANG Lei, LU Peng, ZHANG Jianjun, YANG Li, FAN Jingfang, LI Li, and FENG Jia. A New Strawberry Cultivar‘Jixiang’ [J]. Acta Horticulturae Sinica, 2025, 52(7): 1935-1936.
[3]	FENG Jia, LU Peng, LI Li, YANG Li, and YANG Lei. Genome Wide Identification and Functional Characterization of Strawberry Pectate Lyases Related to Fruit Softening [J]. Acta Horticulturae Sinica, 2025, 52(6): 1441-1450.
[4]	WANG Wenlong, LIU Zhaokun, LU Wenjun, WANG Yaolong, LI Xiaofeng, ZHU Hongfang, LIU Tongkun, LI Ying, HOU Xilin, ZHANG Changwei. Enhancing Ascorbic Acid Content in Non-heading Chinese Cabbage Using Gene Editing Technology [J]. Acta Horticulturae Sinica, 2025, 52(5): 1317-1325.
[5]	ZHANG Zhiyuan, WANG Dan, XI Zhumei, WANG Xianhang. Advances in Plant Promoter Research [J]. Acta Horticulturae Sinica, 2025, 52(4): 1065-1092.
[6]	WANG Zhongyi, LIU Yi, HU Bowen, ZHU Fan, LIU Feng, YANG Sha, XIONG Cheng, OU Lijun, DAI Xiongze, ZOU Xuexiao. Construction of a High-Efficiency Genetic Transformation System in Pepper Leveraging RUBY and CaREF1 [J]. Acta Horticulturae Sinica, 2025, 52(4): 1093-1104.
[7]	YIN Tao, DONG Junxiao, SHAO Yongchun, ZHANG Cuiling, ZHANG Ruifen, HOU Junhe, LI Shaoxuan, YU Fushun, LIU Fangxin, SUN Hongtao. A New Strawberry Cultivar‘Chunsha’ [J]. Acta Horticulturae Sinica, 2025, 52(1): 259-260.
[8]	LIU Yuxiang, HAN Fengqing, ZHAO Xinyu, LIU Yumei, LI Zhansheng, $\boxed{\hbox{FANG Zhiyuan}}$. Cloning and Functional Analysis of Lateral Branching Regulatory Gene BoBRC1 in Broccoli [J]. Acta Horticulturae Sinica, 2024, 51(9): 1997-2007.
[9]	LI Wenjing, SHI Shuo, ZHANG Hao, ZHANG Jiangyan, SONG Sihao, YANG Aizhen, SHEN Yuanyue, GUO Jiaxuan, GAO Fan. Effects of Exogenous Nitric Oxide and Putrescine on Drought Resistance of Strawberry Seedlings [J]. Acta Horticulturae Sinica, 2024, 51(9): 2131-2142.
[10]	WANG Chenyu, LIU Mengjun, WANG Lixin, LIU Zhiguo. Research Progress of CRISPR/Cas9 Technology and Its Application in Horticultural Plants [J]. Acta Horticulturae Sinica, 2024, 51(7): 1439-1454.
[11]	JIANG Rui, ZHOU Shengjun, ZHU Yuqiang, WANG Xin, TAN Jihong, WANG Huasen, ZHANG Peng. Research Progress of CRISPR/Cas9 Gene Editing Techniques in Melon Crops [J]. Acta Horticulturae Sinica, 2024, 51(7): 1683-1694.
[12]	MA Xingyun, FAN Bingli, TANG Guangcai, JIA Zhiqi, LI Ying, XUE Dongqi, ZHANG Shiwen. Preliminary Study on the Mechanism of DXR Regulating Chloroplast Development Flower Color and Fruit Coloring in Tomato [J]. Acta Horticulturae Sinica, 2024, 51(6): 1241-1255.
[13]	WANG Wenjiao, XING Junjie, SHEN Chengcheng, LI Bin. Screening and Functional Analysis of CsCOL5，the Upstream Regulator of CsCER1，in Cucumber Pericarp Wax Synthesis [J]. Acta Horticulturae Sinica, 2024, 51(5): 1005-1016.
[14]	MA Cunfa, WU Ting, ZHAO Hui, ZHANG Tianpei, ZHAO Lijian, YU Yonghui, XIAO Jiancheng, LI Jun, WU Shuiqin. Creation of Self-compatible Broccoli Lines by Knocking out BoSP11 Gene Through CRISPR/Cas9 Technology [J]. Acta Horticulturae Sinica, 2024, 51(3): 509-519.
[15]	YANG Juanbo, GUO Lili, LU Shixiong, GOU Huimin, WANG Shuaiting, ZENG Baozhen, MAO Juan. Cloning and Functional Analysis of FaGH3.17 Gene in Strawberry [J]. Acta Horticulturae Sinica, 2024, 51(11): 2483-2494.