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Acta Agronomica Sinica ›› 2024, Vol. 50 ›› Issue (7): 1658-1668.doi: 10.3724/SP.J.1006.2024.34196

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Establishment of large fragment knockout in pea genome by CRISPR/Cas9 technology

HUANG Shu-Xian1(), LIU Rong1, LI Guan2, SHU Qin1, XU Fei1, ZONG Xu-Xiao1,*(), YANG Tao1,*()   

  1. 1Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China
    2Institute of Crop Germplasm Resources, Shandong Academy of Agricultural Sciences, Jinan 250100, Shandong, China
  • Received:2023-11-19 Accepted:2024-01-31 Online:2024-07-12 Published:2024-06-15
  • Contact: *E-mail: yangtao02@caas.cn; E-mail: zongxuxiao@caas.cn
  • Supported by:
    National Natural Science Foundation of China(32241042);China Agriculture Research System of MOF and MARA(CARS-08-G11);State Key Laboratory of Crop Gene Resources and Breeding

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Abstract:

Pea is one of the main food legumes in the world and has been paid more and more attention due to its rich nutritional value and good ecological value. CRISPR/Cas9 as a new biology breeding tool has been widely used in many crops, however, its usage in pea is very limited. In this study, we successfully achieved large fragment deletion of the Psat01G0240600-T1, Psat03G0303300-T1, and Psat03G0304700-T1 genes in “Zhongwan 6” variety by CRISPR/Cas9 technology. The large fragment knockout ratio was 24.1%, 13.0%, and 3.6%, respectively. Further analysis revealed that there were significant differences in editing efficiency among different target sites and large fragment deletion depend on the specific target site with the lower editing efficiency between the two target sites. In this study, we explored the application of CRISPR/Cas9 by using the hairy root system in pea and achieved large fragment knockout for the first time in pea, which is of great significant for the research of peas.

Key words: pea, CRISPR/Cas9, hairy root, large fragment knockout

Table 1

Primers for detecting gene sequence"

引物名称Primer name 引物序列Primer sequences (5′-3′)
Psat01G0240600-T1-FP ATGTCAAGTGGTAGTAGAGACCCTC
Psat01G0240600-T1-RP TCATCGGCATAACCTTCTTCCACC
Psat03G0303300-T1-1-FP ATGCCTAGGAATATGGTCGATCCTC
Psat03G0303300-T1-1-RP GAGAGTGCCTATCTTGACAC
Psat03G0303300-T1-2-FP CGGTGAAAACACTCACGTGTATGTG
Psat03G0303300-T1-2-RP TTAGCATCTCCTTCCACCGCAGCCG
Psat03G0304700-T1-FP ATGGCAGGTAGTAGCAGGAATCCTC
Psat03G0304700-T1-RP TTATCTAAATGTTCTTCCACCAGAGCC

Table 2

Primers used in construction of vectors"

引物名称Primer name 引物序列Primer sequence (5′-3′)
CmYLCV FP TGCTCTTCGCGCTGGCAGACATACTGTCCCAC
Psat01G0240600-T1-gRNA1 RP TCGTCTCCAACCCTTGGTTGCTGCCTATACGGCAGTGAACCTG
Psat01G0240600-T1-gRNA1 FP TCGTCTCAGGTTATTGTTGGGTTTTAGAGCTAGAAATAGC
Psat01G0240600-T1-RNA2 RP TCGTCTCACAGGAATATCAGCTGCCTATACGGCAGTGAAC
Psat01G0240600-T1-RNA2 FP TCGTCTCACCTGCAACTACTGTTTTAGAGCTAGAAATAGC
oCsy-E RP TGCTCTTCTGACCTGCCTATACGGCAGTGAAC
Psat03G0303300-T1-gRNA1 RP TCGTCTCCAACCAACTGAGACTGCCTATACGGCAGTGAACCTG
Psat03G0303300-T1-gRNA1 FP TCGTCTCAGGTTAACCGTCCGTTTTAGAGCTAGAAATAGC
Psat03G0303300-T1-gRNA2 RP TCGTCTCATTGACAGTTGAACTGCCTATACGGCAGTGAAC
Psat03G0303300-T1-gRNA2 FP TCGTCTCATCAAAGAGAGCGGTTTTAGAGCTAGAAATAGC
Psat03G0304700-T1-gRNA1 RP TCGTCTCAGGTGACTCGGAGCTGCCTATACGGCAGTGAAC
Psat03G0304700-T1-gRNA1 FP TCGTCTCACACCTATGGTAGGTTTTAGAGCTAGAAATAGC
Psat03G0304700-T1-gRNA2 RP TCGTCTCAGTCGTGGCCTTTCTGCCTATACGGCAGTGAAC
Psat03G0304700-T1-gRNA2 FP TCGTCTCACGACCCACCTCAGTTTTAGAGCTAGAAATAGC
Check FP CTAGAAGTAGTCAAGGCGGC
MBF GTAAAACGACGGCCAGT

Table 3

Hi-TOM sequencing primers"

引物名称Primer name 引物序列Primer sequence (5′-3′)
Psat01G0240600-T1-1-Hi-FP GGAGTGAGTACGGTGTGCGGCTGTGAATTCAAACCTTCT
Psat01G0240600-T1-1-Hi-RP GAGTTGGATGCTGGATGGGCATCTGGATCCACCATGATC
Psat01G0240600-T1-2-Hi-FP GGAGTGAGTACGGTGTGCGAAAATGCTATGACTATG
Psat01G0240600-T1-2-Hi-RP GAGTTGGATGCTGGATGGCAACATCATCATCACAAGC
Psat03G0303300-T1-1-Hi-FP GGAGTGAGTACGGTGTGCCTGTATCTTTGAGTGTTG
Psat03G0303300-T1-1-Hi-RP GAGTTGGATGCTGGATGGCGTTACTAGGGCTAGGTG
Psat03G0303300-T1-2-Hi-FP GGAGTGAGTACGGTGTGCGGGAAAGAGGCAGTGTTT
Psat03G0303300-T1-2-Hi-RP GAGTTGGATGCTGGATGGTTAGCATCTCCTTCCACC
Psat03G0304700-T1-1-Hi-FP GGAGTGAGTACGGTGTGCAGGAATCCTCTCGCTGTTGG
Psat03G0304700-T1-1-Hi-RP GAGTTGGATGCTGGATGGGATCGTTTCCACCAACATTC
Psat03G0304700-T1-2-Hi-FP GGAGTGAGTACGGTGTGCGGTGACTGATATTCCAGC
Psat03G0304700-T1-2-Hi-RP GAGTTGGATGCTGGATGGTTCTGTCGCCATCCTGGA

Table 4

Large fragment deletion detection primers"

引物名称Primer name 引物序列Primer sequence (5′-3′)
Psat01G0240600-T1dpdFP TGGTCCAACTTACTTGCCTTGA
Psat01G0240600-T1dpdRP GCAGTGATTCCCAATTGAGTGTA
Psat03G0303300-T1dpdFP AGACCAAACGAGGTACGGTT
Psat03G0303300-T1dpdRP TGCAATGAACTAACCCCCGC
Psat03G0304700-T1dpdFP GTCACGGACGTGAGCAAAACGACATGG
Psat03G0304700-T1dpdRP ACGACACACACATGGATAAACACTGC

Fig. 1

Conservative motifs of pea FT gene family proteins"

Fig. 2

Target sites selection Green box: exon; Black solid line: intron; Blue letter: target site; Red letter: PAM."

Fig. 3

Schematic diagrams of editing vector The green box: CmYLCV promoter; the blue box: specific endonuclease Csy4; the gray box: gRNA; the orange box: 35S terminator."

Fig. 4

Editing efficiency of a single target site in three genes"

Fig. 5

Analysis of target mutation A: the mutation analysis of six targets showed that the gray column was the deletion mutation type, and the white column was the other mutation type; B: six target deletion types and their efficiencies; C: the average efficiency of six target deletion types."

Table 5

Large fragment deletion efficiency of three genes"

基因 ID
Gene ID		 鉴定的样品数量
Number of samples
identified		 大片段缺失的数量
Number of large
fragment deletion		 突变频率
Mutation frequency (%)		 片段缺失的大小
Size of the missing
fragments (bp)
Psat01G0240600-T1 87 21 24.1 161-967
Psat03G0303300-T1 77 10 13.0 1642-1673
Psat03G0304700-T1 84 3 3.6 228-809

Fig. S1

Edited genes large fragment deletion electrophoresis maps Agarose gel electrophoresis schematic diagram of Psat01G0240600-T1, Psat03G0303300-T1, and Psat03G0304700-T1. The band pointed by the blue arrows indicate the samples with large fragments in sequencing."

Fig. S2

PCR sequencing results of large fragment deletion samples of Psat01G0240600-T1, Psat03G0303300-T1, and Psat03G0304700-T1 The blue highlight represents the target sequence, the red highlight represents the PAM site, the orange highlight represents the insertion sequence, the green highlight represents the base mutation, “—//—” represents the base omission, and “------//------” represents the fragment deletion."

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