Concurrent modifications in the three homeologs of Ms45 gene with CRISPR-Cas9 lead to rapid generation of male sterile bread wheat (Triticum aestivum L.)
Hexaploid bread wheat is not readily amenable to traditional mutagenesis approaches. In this study, we show efficient utilization of CRISPR-Cas system and Next Generation Sequencing for mutant analysis in wheat.
Identification and manipulation of male fertility genes in hexaploid bread wheat is important for understanding the molecular basis of pollen development and to obtain novel sources of nuclear genetic male sterility (NGMS). The maize Male sterile 45 (Ms45) gene encodes a strictosidine synthase-like enzyme and has been shown to be required for male fertility. To investigate the role of Ms45 gene in wheat, mutations in the A, B and D homeologs were produced using CRISPR-Cas9. A variety of mutations in the three homeologs were recovered, including a plant from two different genotypes each with mutations in all three homeologs. Genetic analysis of the mutations demonstrated that all three wheat Ms45 homeologs contribute to male fertility and that triple homozygous mutants are required to abort pollen development and achieve male sterility. Further, it was demonstrated that a wild-type copy of Ms45 gene from rice was able to restore fertility to these wheat mutant plants. Taken together, these observations provide insights into the conservation of MS45 function in a polyploid species. Ms45 based NGMS can be potentially utilized for a Seed Production Technology (SPT)-like hybrid seed production system in wheat.
KeywordsWheat Ms45 Pollen development Male sterility CRISPR-Cas
The authors thank Kara Califf for valuable comments and editorial assistance in preparation of this manuscript. The authors also thank Tracey Fisher, Maria Federova, and Jim Gaffney for their critical review of the manuscript. Technical help provided by Myeong-Je Cho and Sergei Svitashev for plant transformation, Wang-Nan Hu for QRT-PCR, and Katherine Thilges for microscopy is gratefully acknowledged.
Conceptualization: Mark Cigan, Manjit Singh, Josh Young, Investigation: Manjit Singh, Manish Kumar, Josh Young, Writing—original draft: Manjit Singh, Mark Cigan, Writing—review & editing: Manjit Singh, Mark Cigan, Marc Albertsen.
Compliance with ethical standards
Conflict of interest
All authors are current or former employees of DuPont Pioneer. MS and AMC are inventors on a provisional patent application on this work and are current or former employees of DuPont Pioneer, who owns the patent application.
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