Abstract
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.
Highlights
Maize (Zea mays L.) is an important food and feed crop worldwide
Plasmid Construction For generating the CRISPR/Cas9-mediated multi-gene mutants, the 19-bp fragment targeting the predictive genes were designed on the CRISPR-P 2.0 and evaluated on a website, respectively [26]
To establish a more efficient method for simultaneously editing homologous genic male-sterility (GMS) genes, in this study, we only transferred one CRISPR/Cas9 editing vector into maize to produce novel knockoutmutants of ZmTGA9-1/-2/-3, which further confirmed the function of the ZmTGA9 family on male fertility
Summary
Maize (Zea mays L.) is an important food and feed crop worldwide. As a fast growing C4 plant and a successful crop of heterosis utilization, it provides more than one-half of global calorie consumption [1]. The wide use of hybrid varieties has greatly increased maize yield. Manual or mechanical detasseling is required for maize hybrid seed production to prevent self-pollination. Detasseling has obvious drawbacks, e.g., it is time-consuming, labor-intensive, expensive, affects plant growth, and reduces the yield of hybrid seeds [2]. Developing a male-sterility line is critical for producing hybrid maize seed
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