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Use of CRISPR/Cas9-Based Gene Editing to Simultaneously Mutate Multiple Homologous Genes Required for Pollen Development and Male Fertility in Maize

by 1,†, 1,†, 1,†, 1, 1, 1,2, 1,2, 1,2, 1,2,* and 1,2,*
1
Zhongzhi International Institute of Agricultural Biosciences, Shunde Graduate School, Research Center of Biology and Agriculture, University of Science and Technology Beijing (USTB), Beijing 100024, China
2
Beijing Engineering Laboratory of Main Crop Bio-Tech Breeding, Beijing International Science and Technology Cooperation Base of Bio-Tech Breeding, Beijing Solidwill Sci-Tech Co., Ltd., Beijing 100192, China
*
Authors to whom correspondence should be addressed.
These authors contributed equally to this work.
Academic Editors: Francesco Carimi and Laurens Pauwels
Cells 2022, 11(3), 439; https://doi.org/10.3390/cells11030439
Received: 7 December 2021 / Revised: 22 January 2022 / Accepted: 25 January 2022 / Published: 27 January 2022
(This article belongs to the Special Issue Pollen Development)
Male sterility represents an important trait for hybrid breeding and seed production in crops. Although the genes required for male fertility have been widely studied and characterized in many plant species, most of them are single genic male-sterility (GMS) genes. To investigate the role of multiple homologous genes in anther and pollen developments of maize, we established the CRISPR/Cas9-based gene editing method to simultaneously mutate the homologs in several putative GMS gene families. By using the integrated strategies of multi-gene editing vectors, maize genetic transformation, mutation-site analysis of T0 and F1 plants, and genotyping and phenotyping of F2 progenies, we further confirmed gene functions of every member in ZmTGA9-1/-2/-3 family, and identified the functions of ZmDFR1, ZmDFR2, ZmACOS5-1, and ZmACOS5-2 in controlling maize male fertility. Single and double homozygous gene mutants of ZmTGA9-1/-2/-3 did not affect anther and pollen development, while triple homozygous gene mutant resulted in complete male sterility. Two single-gene mutants of ZmDFR1/2 displayed partial male sterility, but the double-gene mutant showed complete male sterility. Additionally, only the ZmACOS5-2 single gene was required for anther and pollen development, while ZmACOS5-1 had no effect on male fertility. Our results show that the CRISPR/Cas9 gene editing system is a highly efficient and convenient tool for identifying multiple homologous GMS genes. These findings enrich GMS genes and mutant resources for breeding of maize GMS lines and promote deep understanding of the gene family underlying pollen development and male fertility in maize. View Full-Text
Keywords: CRISPR/Cas9; gene editing; multiple homologous genes; pollen development; genic male sterility; maize CRISPR/Cas9; gene editing; multiple homologous genes; pollen development; genic male sterility; maize
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MDPI and ACS Style

Liu, X.; Zhang, S.; Jiang, Y.; Yan, T.; Fang, C.; Hou, Q.; Wu, S.; Xie, K.; An, X.; Wan, X. Use of CRISPR/Cas9-Based Gene Editing to Simultaneously Mutate Multiple Homologous Genes Required for Pollen Development and Male Fertility in Maize. Cells 2022, 11, 439. https://doi.org/10.3390/cells11030439

AMA Style

Liu X, Zhang S, Jiang Y, Yan T, Fang C, Hou Q, Wu S, Xie K, An X, Wan X. Use of CRISPR/Cas9-Based Gene Editing to Simultaneously Mutate Multiple Homologous Genes Required for Pollen Development and Male Fertility in Maize. Cells. 2022; 11(3):439. https://doi.org/10.3390/cells11030439

Chicago/Turabian Style

Liu, Xinze, Shaowei Zhang, Yilin Jiang, Tingwei Yan, Chaowei Fang, Quancan Hou, Suowei Wu, Ke Xie, Xueli An, and Xiangyuan Wan. 2022. "Use of CRISPR/Cas9-Based Gene Editing to Simultaneously Mutate Multiple Homologous Genes Required for Pollen Development and Male Fertility in Maize" Cells 11, no. 3: 439. https://doi.org/10.3390/cells11030439

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