Creating Pink Fruit and Fruit Without Green Shoulder Using CRISPR/ Cas9 Technology in Tomato

doi:10.16420/j.issn.0513-353x.2022-0273

Acta Horticulturae Sinica ›› 2023, Vol. 50 ›› Issue (5): 985-999.doi: 10.16420/j.issn.0513-353x.2022-0273

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

Creating Pink Fruit and Fruit Without Green Shoulder Using CRISPR/ Cas9 Technology in Tomato

LI Guobin, CAI Liangyu, XIAO Licheng, WANG Jiafa, ZHANG Dedi, ZHANG Junhong()

National Key Laboratory for Germplasm Innovation and Utilization for Fruit and Vegetable Horticultural Crops， College of Horticulture and Forestry Sciences，Huazhong Agricultural University，Wuhan 430070，China

Received:2022-11-27 Revised:2023-01-16 Online:2023-05-25 Published:2023-05-31
Contact: ZHANG Junhong E-mail:zhangjunhng@hzau.edu.cn

Abstract

Abstract:

The key quality-regulating genes SlGLK2 and SlMYB12 were knocked out in tomato cultivars（lines）Ailsa Craig（AC），B5 and B9 using CRISPR/Cas9 binary expression vector system. Nine CRISPR/Cas9 transgenic plants for each of SlGLK2 and SlMYB12 in the AC，three CRISPR/Cas9 transgenic plants of SlGLK2 in the B5，and four CRISPR/Cas9 transgenic plants of SlMYB12 in the B9 were obtained. By sequencing the target sites，it was found that different forms of base deletion，insertion and replacement occurred at 3-4 bp upstream of PAM，mainly 1-10 bp deletion as well as single base G and T insertion. In addition，a large deletions produced between the two targets is 334 bp. Knockout of SlMYB12 in AC showed pink fruits. Knockout of SlGLK2 gene in AC showed the green shoulders of fruits were obviously lighter，and knockout of SlGLK2 gene in B5 showed no‘Green Shoulder’in tomato fruits comparing to the corresponding wild type lines. In this study，SlGLK2 and SlMYB12 gene editing plants were successfully generated in different tomato materials using CRISPR/Cas9 technology，which would provide excellent germplasm for tomato quality breeding.

Key words: tomato, genome editing, SlGLK2, SlMYB12, pink fruit, fruit with green shoulder

CLC Number:

S641.2

LI Guobin, CAI Liangyu, XIAO Licheng, WANG Jiafa, ZHANG Dedi, ZHANG Junhong. Creating Pink Fruit and Fruit Without Green Shoulder Using CRISPR/ Cas9 Technology in Tomato[J]. Acta Horticulturae Sinica, 2023, 50(5): 985-999.

Figures/Tables 13

References 29

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基因 Gene	sgRNA 靶标序列 Target sequence of sgRNA
SlGLK2-sgRNA1	TGGTGTCGATTAAATTCCCA
SlGLK2-sgRNA2	TGAATAGCAGGTTCTTCACA
SlMYB12-sgRNA1	AACACCTTGTTGTGAAAAA
SlMYB12-sgRNA2	CCAAGAGCCTTCTCCATTAG

基因 Gene	sgRNA 靶标序列 Target sequence of sgRNA
SlGLK2-sgRNA1	TGGTGTCGATTAAATTCCCA
SlGLK2-sgRNA2	TGAATAGCAGGTTCTTCACA
SlMYB12-sgRNA1	AACACCTTGTTGTGAAAAA
SlMYB12-sgRNA2	CCAAGAGCCTTCTCCATTAG

引物 Primer	用途 Purpose	序列（5′-3′） Sequence
SlGLK2-CRISPR	构建载体Construction vector	F：GAATCTAACAGTGTAGTTTGTGGTGTCGATTAAATTCCCAGTTT TAGAGC TAGAAATAG
		R：GCTATTTCTAGCTCTAAAACTGAATAGCAGGTTCTTCACACAA ACTACACTG TTAGATT
SlMYB12-CRISPR	构建载体Construction vector	F：GAATCTAACAGTGTAGTTTGAACACCTTGTTGTGAAAAAGGTT TTAGAGCT AGAAATAG
		R：GCTATTTCTAGCTCTAAAACCCAAGAGCCTTCTCCATTAGCAA ACTACACTG TTAGATT
SlGLK2	检测靶点Detection target	F：GATGCTGGCATTTGTGTGATTC
		R：TTAATTACGACTAGAACCGTCAACG
SlMYB12	检测靶点Detection target	F：TAACTAGACTAAAGTGAAGAGCGGG
		R：CTAATTACCTGTTACCCAAAGTTGC

引物 Primer	用途 Purpose	序列（5′-3′） Sequence
SlGLK2-CRISPR	构建载体Construction vector	F：GAATCTAACAGTGTAGTTTGTGGTGTCGATTAAATTCCCAGTTT TAGAGC TAGAAATAG
		R：GCTATTTCTAGCTCTAAAACTGAATAGCAGGTTCTTCACACAA ACTACACTG TTAGATT
SlMYB12-CRISPR	构建载体Construction vector	F：GAATCTAACAGTGTAGTTTGAACACCTTGTTGTGAAAAAGGTT TTAGAGCT AGAAATAG
		R：GCTATTTCTAGCTCTAAAACCCAAGAGCCTTCTCCATTAGCAA ACTACACTG TTAGATT
SlGLK2	检测靶点Detection target	F：GATGCTGGCATTTGTGTGATTC
		R：TTAATTACGACTAGAACCGTCAACG
SlMYB12	检测靶点Detection target	F：TAACTAGACTAAAGTGAAGAGCGGG
		R：CTAATTACCTGTTACCCAAAGTTGC

材料 Material	单果质量/g Fruit weight	果实纵径/mm Fruit length	果实横径/mm Fruit diameter	果形指数 Index of fruit shape	果实硬度/（× 10⁵ Pa） Fruit hardness
AC	28.95 ± 0.83 b	34.51 ± 0.37 c	38.02 ± 0.77 c	0.90 ± 0.03 a	8.14 ± 0.37 b
SlMYB12-AC-3	28.82 ± 0.75 b	34.45 ± 0.76 c	37.79 ± 0.64 c	0.91 ± 0.02 a	7.86 ± 0.52 b
SlMYB12-AC-4	29.46 ± 1.02 b	34.04 ± 0.25 c	38.03 ± 0.92 c	0.89 ± 0.02 a	8.32 ± 0.41 b
SlMYB12-AC-7	28.03 ± 0.62 b	34.26 ± 0.43 c	37.65 ± 0.43 c	0.91 ± 0.01 a	8.22 ± 0.26 b
SlGLK2-AC-3	27.97 ± 0.69 b	33.52 ± 0.21 c	37.63 ± 0.26 c	0.89 ± 0.03 a	8.47 ± 0.67 b
SlGLK2-AC-4	28.84 ± 1.25 b	33.96 ± 0.64 c	38.12 ± 0.71 c	0.89 ± 0.02 a	7.92 ± 0.85 b
SlGLK2-AC-5	28.88 ± 0.48 b	34.62 ± 0.41 c	38.46 ± 0.65 c	0.90 ± 0.01 a	8.08 ± 0.56 b
B5	80.82 ± 0.67 a	41.42 ± 1.21 ab	53.96 ± 1.84 ab	0.76 ± 0.04 b	11.33 ± 1.08 a
SlGLK2-B5-1	79.94 ± 1.23 a	39.61 ± 1.73 b	52.75 ± 2.17 b	0.75 ± 0.06 b	10.76 ± 0.84 a
SlGLK2-B5-2	81.89 ± 1.06 a	43.30 ± 1.55 a	55.53 ± 1.49 a	0.77 ± 0.03 b	11.59 ± 0.86 a
SlGLK2-B5-3	81.03 ± 1.35 a	41.42 ± 1.83 ab	55.02 ± 2.27 a	0.75 ± 0.05 b	10.94 ± 1.23 a

Creating Pink Fruit and Fruit Without Green Shoulder Using CRISPR/ Cas9 Technology in Tomato

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材料 Material	可溶性固形物/% Soluble solid	可滴定酸/% Titrable acid	固酸比 S/T	总维生素C/（mg · g^-1） Total vitamin C	还原维生素C/（mg · g^-1） Reduced vitamin C
AC	5.7 ± 0.34 a	0.62 ± 0.21 b	9.19 ± 0.87 a	0.09 ± 0.01 b	0.09 ± 0.01 c
SlMYB12-AC-3	5.3 ± 0.27 b	0.69 ± 0.17 b	7.68 ± 1.21 b	0.08 ± 0.02 b	0.08 ± 0.01 c
SlMYB12-AC-4	5.5 ± 0.61 a	0.64 ± 0.13 b	8.59 ± 0.96 ab	0.12 ± 0.02 b	0.09 ± 0.01 c
SlMYB12-AC-7	5.8 ± 0.29 a	0.63 ± 0.24 b	9.21 ± 1.35 a	0.10 ± 0.01 b	0.09 ± 0.01 c
SlGLK2-AC-3	5.9 ± 0.53 a	0.68 ± 0.30 b	8.67 ± 1.07 ab	0.11 ± 0.01 b	0.10 ± 0.01 c
SlGLK2-AC-4	5.6 ± 0.28 a	0.66 ± 0.27 b	8.48 ± 0.67 ab	0.11 ± 0.02 b	0.09 ± 0.01 c
SlGLK2-AC-5	5.5 ± 0.36 a	0.63 ± 0.18 b	8.73 ± 1.26 a	0.11 ± 0.02 b	0.09 ± 0.01 c
B5	6.0 ± 0.27 a	1.10 ± 0.22 a	5.45 ± 0.93 c	0.20 ± 0.03 a	0.19 ± 0.02 a
SlGLK2-B5-1	6.0 ± 0.34 a	1.04 ± 0.17 a	5.77 ± 0.72 c	0.16 ± 0.04 a	0.15 ± 0.03 b
SlGLK2-B5-2	5.6 ± 0.51 a	1.08 ± 0.31 a	5.18 ± 0.64 c	0.20 ± 0.03 a	0.17 ± 0.02 a
SlGLK2-B5-3	5.8 ± 0.43 a	1.21 ± 0.37 a	4.79 ± 1.37 d	0.18 ± 0.03 a	0.16 ± 0.03 ab

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