Crops ›› 2018, Vol. 34 ›› Issue (6): 36-42.doi: 10.16035/j.issn.1001-7283.2018.06.006

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Breeding Fragrant Rice Zhengdao19 Using CRISPR/Cas9 Mediated Gene Editing Technology

Wang Fuhua1,Xue Huazheng1,Wang Ya1,Wang Shengxuan1,Wang Yuetao1,Fu Jing1,Yang Wenbo1,Bai Tao1,Li Junzou2,Yin Haiqing1   

  1. 1 Institute of Cereal Crop, Henan Academy of Agricultural Sciences, Zhengzhou 450002, Henan, China
    2 Agronomy College, Henan Agricultural University, Zhengzhou 450002, Henan, China
  • Received:2018-08-24 Revised:2018-11-06 Online:2018-12-15 Published:2018-12-06

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Abstract:

Fragrant rice is favored by Chinese consumers. In this study, Fragrance rice mutant lines were developed with CRISPR/CAS9 by editing gene Badh2, a key gene determining rice fragrance of Zhengdao 19, a rice variety suitable for direct-sowing as transformation donor. Totally, 9 fragrant mutants were obtained out of 11 positive transgenic T0 plant individuals. And twenty-two individuals of six T1 lines were confirmed by editing region sequencing. Seven mutation types were discovered, including five kinds of delete, two kinds of insert. Totally, three biallelic mutation lines (T1-2, T1-3 and T1-7) and ten homozygous mutants were found. Meanwhile sequencing twenty-eight T2 individuals revealed twenty-one homozygous mutants. Utilization of selectable marker screening to T2 progeny (from T1 lines T1-2, T1-6 and T1-7) eight marker-free homozygous mutants were found, and three were from T1-2, five from T1-7, respectively. Compared with the wild type, the 2-acetyl-1-pyrroline content increased from 0.003μg/g (in the wild type) to 1.259±0.072μg/g for T0 mutants planted in field, from 0.002μg/g (in the wild type) to 0.537±0.111μg/g for T1 mutants planted in greenhouse, without significant alternation in mainly agronomic traits. All selected marker-free homozygous individuals was available to be applied in germplasm enhancement and fragrance rice breeding practice.

Key words: Rice, Zhengdao19, Fragrance, Badh2, CRISPR/CAS9

Fig.1

Target region sequencing & identification Sequence in bracket indicate target region,Sequences in box are primer pair for target region screening"

Table 1

Primer pairs used in this study"

引物名称Primer name 引物序列Primer sequence 用途Usage
Badh identify_F CCATCGGTACCCTCCTCTTC 靶点序列扩增Amplification of target region
Badh_identif_R ATCGATCGATTTGGGGCTCA
Vector_identify_F CGATTCCGGAAGTGCTTGAC 载体验证 Vector identification
Vector_identify_R CGTCTGCTGCTCCATACAAG

Table 2

Analysis of mutation in target sequences"

T1株系号
No. of
T1 line		 T1测序株Sequenced of T1 T2测序株Sequenced of T2 靶点序列
Target sequence		 突变情况
Mutation
总株数/突变体
Total/Mutant		 纯合/杂合
Homozygote/Heterzygote
in mutants		 总株数/突变体
Total/Mutant		 纯合/杂合
Homozygote/Heterzygote
in mutants
WT CAAGTACCTCCGCGCAATCGCGGCC
T1-2 3/3 0/3 6/6 6/0 CAAGTACCTCCGCGC--TCGCGGCC -2
CAAGTACCTCCGCG-----CGGCC -5
T1-3 3/3 2/1 6/6 3/3 CAAGTACCTCCGCG--------GCC -8
CAAGTACCTCCGCGCA-TCGCGGCC -1
T1-6 5/4 2/2 5/4 2/2 CAAGTACCTCCGCGCAAaTCGCGGCC +1
T1-7 5/5 3/2 5/5 5/0 CAAGTACCTCCGC------------ -12
CAAGTACCTCCGCGCAAaTCGCGGCC +1
T1-8 3/2 0/2 3/3 3/0 CAAGTACCTCCGCGCAAacacttgat +9
T1-9 3/3 3/0 3/2 2/0 CAAGTACCTCCGCGCAAaTCGCGGCC +1
小计Total 22/20 10/10 28/26 21/5

Fig.2

Genotype of edited site and vector segregation in T1 & T2 generation"

Fig.3

2-acetyl-1-pyrroline (2-AP) content of T0 and T1 lines **: Significant difference at P=0.01"

Fig.4

Phenotype and performance of agronomic traits related to yield in desirable T1 line"

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