Creation of Self-compatible Broccoli Lines by Knocking out BoSP11 Gene Through CRISPR/Cas9 Technology

doi:10.16420/j.issn.0513-353x.2023-0065

Acta Horticulturae Sinica ›› 2024, Vol. 51 ›› Issue (3): 509-519.doi: 10.16420/j.issn.0513-353x.2023-0065

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

Creation of Self-compatible Broccoli Lines by Knocking out BoSP11 Gene Through CRISPR/Cas9 Technology

MA Cunfa¹, WU Ting¹, ZHAO Hui¹, ZHANG Tianpei¹, ZHAO Lijian¹, YU Yonghui¹, XIAO Jiancheng¹, LI Jun²^,^*(), WU Shuiqin¹^,^*()

¹ Zhejiang MITSUO Seed Co.，Ltd，Jiaxing，Zhejiang 314023，China
² School of Modern Agriculture，Jiaxing Vocational & Technical College，Jiaxing，Zhejiang 314036，China

Received:2023-04-11 Revised:2023-08-28 Online:2024-03-25 Published:2024-03-22
Contact: LI Jun, WU Shuiqin

Abstract

Abstract:

By using CRISPR/Cas9 technology to knock out SRK gene or SP11 gene，the self- incompatible Brassica oleracea vegetables can be directly transformed into self-compatible materials. In this study，a double target CRISPR/Cas9 gene editing vector was constructed based on the sequence of broccoli（Brassica oleracea var. italic）BoSP11 gene obtained by whole genome sequencing，and then used Agrobacterium mediated genetic transformation to knock out the BoSP11 gene. The results showed that 23 of the 29 transgenic plants had gene mutation，and the editing efficiency was 79.3%. The results of TA clone sequencing showed that there were four homozygous mutations and 19 heterozygous mutations. The statistics of the seed setting of T₀ generation of homozygous mutant plants showed that the self-compatibility index at flowering stage was as high as 6.82，which was the highest compatibility index increase reported so far. The T₁generation plants were pollinated by bees in the isolated greenhouses, and the average seed yield per plant was 24.8 g，which completely conducted the self-pollination of parent materials for seed production without any manual involvement.

Key words: broccoli, cabbage, CRISPR/Cas9, gene editing, BoSP11, self-compatible lines

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.

Figures/Tables 8

References 29

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用途 Usage	引物名称 Primer name	引物序列（5′-3′） Primer sequence	产物大小/bp Product size
BoSP11克隆 Cloning the BoSP11	2MSP11-F1	TTTCTGCGAGTAAAAGAGAATC	1 029
BoSP11克隆 Cloning the BoSP11	2MSP11-R1	CTATTTACAATCGCAAGAATAAGTACC
靶序列合成 Construction of target site	sgBoSP11-F1	TGCATGTTTCATATTCATCGTTTC	20
	sgBoSP11-R1	AAACGAAACGATGAATATGAAACA
	sgBoSP11-F2	TGCAGGTAAGCGCAAGAAGCCTTC	20
	sgBoSP11-R2	AAACGAAGGCTTCTTGCGCTTACC
靶位点序列扩增 Amplification of target region	BoSP11-F1	TCTGCGAGTAAAAGAGAATCTTTA	540
	BoSP11-R1	TAGTGATATTGGAAGCGGTACAGG
	BoSP11-F2	AGTAACCAATCGGATGAACTATGT	459
	BoSP11-R2	TCGCAAGAATAAGTACCCATTTGA
转基因检测 Detection of the transformation	Cas9-F1	CAAGTACGTGAACTTCCTCTACC	404
	Cas9-R1	GCTGGGAAAGGTCGATACGAGTC
	Cas9-F2	GACGTCGTCCAATTCTGCTAACCA	464
	Cas9-R2	CGCCGATGAGGTTCTTCTTGATGG

用途 Usage	引物名称 Primer name	引物序列（5′-3′） Primer sequence	产物大小/bp Product size
BoSP11克隆 Cloning the BoSP11	2MSP11-F1	TTTCTGCGAGTAAAAGAGAATC	1 029
BoSP11克隆 Cloning the BoSP11	2MSP11-R1	CTATTTACAATCGCAAGAATAAGTACC
靶序列合成 Construction of target site	sgBoSP11-F1	TGCATGTTTCATATTCATCGTTTC	20
	sgBoSP11-R1	AAACGAAACGATGAATATGAAACA
	sgBoSP11-F2	TGCAGGTAAGCGCAAGAAGCCTTC	20
	sgBoSP11-R2	AAACGAAGGCTTCTTGCGCTTACC
靶位点序列扩增 Amplification of target region	BoSP11-F1	TCTGCGAGTAAAAGAGAATCTTTA	540
	BoSP11-R1	TAGTGATATTGGAAGCGGTACAGG
	BoSP11-F2	AGTAACCAATCGGATGAACTATGT	459
	BoSP11-R2	TCGCAAGAATAAGTACCCATTTGA
转基因检测 Detection of the transformation	Cas9-F1	CAAGTACGTGAACTTCCTCTACC	404
	Cas9-R1	GCTGGGAAAGGTCGATACGAGTC
	Cas9-F2	GACGTCGTCCAATTCTGCTAACCA	464
	Cas9-R2	CGCCGATGAGGTTCTTCTTGATGG

编号 No.	蕾期自交 Self-pollinated at bud stage			花期自交 Self-pollinated at flowering stage
编号 No.	种荚数 Number of seed pods	种子数 Number of seeds	自交亲和指数 Self-compatibility index	种荚数 Number of seed pods	种子数 Number of seeds	自交亲和指数 Self-compatibility index
#7	144	1 172	8.14	187	1 276	6.82
#16	156	1 084	6.95	378	2 071	5.48
#24	96	645	6.72	132	638	4.84
WT	83	387	4.66	122	21	0.17

编号 No.	蕾期自交 Self-pollinated at bud stage			花期自交 Self-pollinated at flowering stage
编号 No.	种荚数 Number of seed pods	种子数 Number of seeds	自交亲和指数 Self-compatibility index	种荚数 Number of seed pods	种子数 Number of seeds	自交亲和指数 Self-compatibility index
#7	144	1 172	8.14	187	1 276	6.82
#16	156	1 084	6.95	378	2 071	5.48
#24	96	645	6.72	132	638	4.84
WT	83	387	4.66	122	21	0.17

株系 Line	检测株数 Number of detected plants	无转基因成分株数 Number of transgenic free plants	定植株数 Number of plants planted	采种质量/g Seed harvest weight	平均单株收种质量/g Average seed weight per plant
#7	447	111	86	2 130	24.8
#16	450	107	79	1 580	20.0
#24	444	94	65	1 050	16.2
总计Total	1 341	312	230	4 760	20.7

Creation of Self-compatible Broccoli Lines by Knocking out BoSP11 Gene Through CRISPR/Cas9 Technology

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