Improvement of Rice Blast Resistance by Pyramiding the R Gene Pigm and the Non-R Gene bsr-d1

doi:10.7668/hbnxb.20193133

Abstract

Abstract:

In order to improve the blast resistance of Shuijing 3,an excellent food-flavor rice variety,CRISPR/Cas9 gene editing technology combined with gene chip technology were used to pyramid the R gene Pigm and the non-R gene bsr-d1 into Shuijing 3.Firstly,Bsr-d1 was selected as the target gene to construct a recombinant expression vector using the CRISPR/Cas9 gene editing system,and transformed into the excellent food-flavor rice Shuijing 3 by Agrobacterium-mediated method.The homozygous bsr-d1 mutant lines without T-DNA elements,including five mutation types as T insertion,G insertion,GA deletion,CGCA deletion and CGCAGA deletion,were screened out.The japonica line Jinyu 1 containing a broad-spectrum blast resistance gene Pigm was used as the gene donor parent to cross with the homozygous bsr-d1 mutant lines without transgenic components.The Pigm gene was introduced into bsr-d1 mutant lines by cross,backcross and self-cross combing molecular breeding chip to simultaneously perform Pigm gene and background-assisted selection.The improved lines SJ3-G1,SJ3-G2,SJ3-G3,SJ3-G4,SJ3-G5,which were homozygous for the disease resistance genes(carrying both bsr-d1 and Pigm genes)and whose background recovery rates were all above 96%,were finally obtained.The improved strains of Shuijing 3 displayed enhanced leaf blast resistance compared with the wild type in inoculated identification test using Magnaporthe grisea strain GUY11.After inoculation with M.oryzae,the POD activities in the improved strains of Shuijing 3 were significantly lower than that of the wild-type control,while the H₂O₂ contents were significantly higher than that of the wild-type control.The improved Shuijing 3 lines with blast resistance carrying both bsr-d1 and Pigm genes are obtained by CRISPR/Cas9 gene editing technology combined with gene chip technology.

Key words: Rice, Pigm, bsr-d1, Blast resistance, CRISPR/Cas9

WANG Ya, WANG Yuetao, SHEN Guanwang, WANG Fuhua, WANG Shengxuan, BAI Tao, YIN Haiqing. Improvement of Rice Blast Resistance by Pyramiding the R Gene Pigm and the Non-R Gene bsr-d1[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(5): 157-165. doi: 10.7668/hbnxb.20193133.

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http://www.hbnxb.net/EN/Y2022/V37/I5/157

Figures/Tables 7

Fig.1 Functional locus of Bsr-d1 gene promoter in different rice varieties(A) and schematic information of the CRISPR-Cas9-Bsr-d1 recombinant vector(B)

Fig.2 PCR detection of Hyg and Bsr-d1 in 34 independent transformants in T₀ generation Marker.2000 bp DNA Marker;+.Positive control of Hyg;-.ddH₂O; 1—34.34 independent transformants in T₀ generation.

Fig.3 DNA/amino acid sequences alignment of wild type and mutants of Shuijing 3 A.Five types of homozygous mutants of Shuijing 3 in T₀ generation;B.Bsr-d1 gene amino acid sequences in the wild type and five homozygous mutant lines: SJ3.Wild type,SJ3+T.T base insertion,SJ3+G.G base insertion,SJ3-2.GA bases deletion,SJ3-4.CGCA bases deletion,SJ3-6.CGCAGA bases deletion.

Fig.4 PCR detection of Hyg in partial bsr-d1 homozygous mutant plants in T₁ generation Marker.2000 bp DNA Marker;1—24.24 independent plants in T₁ generation.

Fig.5 Genetic background recovery of Shuijing 3 bsr-d1 homozygous mutant(SJ3+T)carrying Pigm gene Blue.Heterozygous gene locus;Red.Homozygous Pigm locus.

Fig.6 Identification of improved strains of Shuijing 3 and the wild-type control after inoculation with M.coryza

Fig.7 POD activities and H₂O₂contents in leaves of improved strains of Shuijing 3 and the wild-type control inoculated with M.oryzae at different stages Different lowercase letters above the bars indicate significant difference between the wild type and the improved lines at the 0.05 levels by t-test at the same stage.

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