Generation of a New Glutinous Photothermosensitive Genic-Male-Sterile (PTGMS) Line by CRISPR/Cas9-Directed Mutagenesis of Wx in Rice (Oryza sativa L.)
Abstract
:1. Introduction
2. Materials and Methods
2.1. Experimental Material, Rice Material Planting, and Growth Environment
2.2. Construction of pYLCRISPR/Cas9-Wx Vector
2.2.1. Target Site Selection and Vector Construction
2.2.2. Construction of pYLCRISPR/Cas9-Wx Vector
2.3. Genotyping, Phenotyping, and Screening of T-DNA-Free Plants
2.4. Grain Phenotype of Endosperm and Iodine Staining
2.5. Determination of Amylose Content by Dual-Wavelength Method
2.6. Pollen Fertility Survey
2.7. Data Analysis
3. Results
3.1. Vector Construction
3.2. Genotyping and Analysis of Mutation Types in T0 Plants
3.3. Screening of T-DNA-Free Plants
3.4. Determination of Amylose Content in T2 Generation
3.5. Plant Type, Fertility Transformation, and Appearance Analysis of Grain and Rice Kernels of T2 Homozygous Mutant Lines
3.6. Analysis of the Genetic Background of Y58S
4. Discussion
5. Conclusions
Author Contributions
Funding
Data Availability Statement
Conflicts of Interest
Abbreviations
CRISPR/Cas9 | Clustered regularly interspaced short palindromic repeats/Cas9 |
AC | Amylose content |
PAM | Protospacer adjacent motif |
QTL | Quantitative trait loci |
PTGMS | Photothermo-sensitive genic-male-sterile |
WT | Wild type |
Wx | Waxy |
T-DNA | Transfer DNA |
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Primer Name | Primer Sequence 5’-3’ |
---|---|
Wx-text-F | TCCGCCACGGGTTCCAG |
Wx-text-R | CTCCTACCTCAGCCACAACG |
U-F | CTCCGTTTTACCTGTGGAATCG |
gR-R | CGGAGGAAAATTCCATCCAC |
Wx-U6a-1F | TGTGTGCTTACAGCCATGGCGTTTTAGAGCTAGAAAT |
Wx-U6a-1R | GCCATGGCTGTAAGCACACACGGCAGCCAAGCCAGCA |
Pps-GGL | TTCAGAGGTCTCTCTCGACTAGTATGGAATCGGCAGCAAAGG |
Pgs-GGR | AGCGTGGGTCTCGACCGACGCGTATCCATCCACTCCAAGCTC |
PB-R | GCGCGCGGTCTCTACCGACGCGTATCC |
PB-L | GCGCGCgGTCTCGCTCGACTAGTATGG |
HPT-F | ATTTGTGTACGCCCGACAGT |
HPT-R | GTGCTTGACATTGGGGAGTT |
CAS9-F | CTGACGCTAACCTCGACAAG |
CAS-R | CCGATCTAGTAACATAGATGACACC |
Types | Heterozygous | Bi-Allelic | Homozygous | Total |
---|---|---|---|---|
Number | 5 | 7 | 6 | 18 |
Transformation Material | Mutation | Type |
---|---|---|
W-T | GCCA_TGGCTGTAAGCACACA | |
W-1 | GCCTCTGGATGGAAGCACACA | +1/+3substitution |
W-4 | GCCAATGGCTGTAAGCACACA | +1 |
W-6 | GCCA_TGGGCTGTAAGCACACA | +1 |
T2 Line | AC (%) |
---|---|
W-4-H-3 | 0.8 |
W-4-H-5 | 1.0 |
W-1-B-2 | 1.8 |
W-1-B-5 | 0.6 |
W-1-B-10 | 0.9 |
Y58S (control) | 13.8 |
T2 Homozygous Mutation Lines | Plant Height (cm) | No. of Panicle/Plant | Flag Leaf Length (cm) | Flag Leaf Width (cm) | Length of Panicle (cm) | Grain no. per Spike | Grain Set Rate (%) |
---|---|---|---|---|---|---|---|
W-4-H-3 | 83.3a | 6 | 45.3 a | 1.5 a | 25.0 a | 183 a | 84.6 a |
W-4-H-5 | 83.5 a | 6 | 45.5 a | 1.5 a | 26.2 a | 184 a | 85.0 a |
W-1-B-2 | 82.7 a | 6 | 46.2 a | 1.6 a | 25.8 a | 181 a | 85.2 a |
W-1-B-5 | 81.9 a | 7 | 46.5 a | 1.5 a | 25.8 a | 180 a | 85.0 a |
Y58S (CK) | 82.7 a | 7 | 46.8 a | 1.6 a | 26.2 a | 186 a | 85.5 a |
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Teng, K.; Wang, X.; Guo, X.; Liu, Y.; Li, R. Generation of a New Glutinous Photothermosensitive Genic-Male-Sterile (PTGMS) Line by CRISPR/Cas9-Directed Mutagenesis of Wx in Rice (Oryza sativa L.). Agriculture 2021, 11, 1044. https://doi.org/10.3390/agriculture11111044
Teng K, Wang X, Guo X, Liu Y, Li R. Generation of a New Glutinous Photothermosensitive Genic-Male-Sterile (PTGMS) Line by CRISPR/Cas9-Directed Mutagenesis of Wx in Rice (Oryza sativa L.). Agriculture. 2021; 11(11):1044. https://doi.org/10.3390/agriculture11111044
Chicago/Turabian StyleTeng, Kaichong, Xin Wang, Xinying Guo, Yaoguang Liu, and Rongbai Li. 2021. "Generation of a New Glutinous Photothermosensitive Genic-Male-Sterile (PTGMS) Line by CRISPR/Cas9-Directed Mutagenesis of Wx in Rice (Oryza sativa L.)" Agriculture 11, no. 11: 1044. https://doi.org/10.3390/agriculture11111044
APA StyleTeng, K., Wang, X., Guo, X., Liu, Y., & Li, R. (2021). Generation of a New Glutinous Photothermosensitive Genic-Male-Sterile (PTGMS) Line by CRISPR/Cas9-Directed Mutagenesis of Wx in Rice (Oryza sativa L.). Agriculture, 11(11), 1044. https://doi.org/10.3390/agriculture11111044