Functional analysis and development of a CRISPR/Cas9 allelic series for a CPR5 ortholog necessary for proper growth of soybean trichomes

Benjamin W. Campbell; Jacob W. Hoyle; Bruna Bucciarelli; Adrian O. Stec; Deborah A. Samac; Wayne A. Parrott; Robert M. Stupar

doi:10.1038/s41598-019-51240-7

Functional analysis and development of a CRISPR/Cas9 allelic series for a CPR5 ortholog necessary for proper growth of soybean trichomes

Benjamin W. Campbell, Jacob W. Hoyle, Bruna Bucciarelli, Adrian O. Stec, Deborah A. Samac, Wayne A. Parrott, Robert M. Stupar

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21 Scopus citations

Abstract

Developments in genomic and genome editing technologies have facilitated the mapping, cloning, and validation of genetic variants underlying trait variation. This study combined bulked-segregant analysis, array comparative genomic hybridization, and CRISPR/Cas9 methodologies to identify a CPR5 ortholog essential for proper trichome growth in soybean (Glycine max). A fast neutron mutant line exhibited short trichomes with smaller trichome nuclei compared to its parent line. A fast neutron-induced deletion was identified within an interval on chromosome 6 that co-segregated with the trichome phenotype. The deletion encompassed six gene models including an ortholog of Arabidopsis thaliana CPR5. CRISPR/Cas9 was used to mutate the CPR5 ortholog, resulting in five plants harboring a total of four different putative knockout alleles and two in-frame alleles. Phenotypic analysis of the mutants validated the candidate gene, and included intermediate phenotypes that co-segregated with the in-frame alleles. These findings demonstrate that the CPR5 ortholog is essential for proper growth and development of soybean trichomes, similar to observations in A. thaliana. Furthermore, this work demonstrates the value of using CRISPR/Cas9 to generate an allelic series and intermediate phenotypes for functional analysis of candidate genes and/or the development of novel traits.

Original language	English (US)
Article number	14757
Journal	Scientific reports
Volume	9
Issue number	1
DOIs	https://doi.org/10.1038/s41598-019-51240-7
State	Published - Dec 1 2019

Bibliographical note

Funding Information:
The authors are grateful to Yung-Tsi Bolon and Jeff Roessler for identifying the R59C46 soybean mutant, and the authors are grateful to Amy D’Arcey for her assistance providing somatic embryo, T0 and T1 plant photographs. Microscopy imaging and analysis was performed at the University Imaging Centers, University of Minnesota, and the authors would like to specifically thank Gail Celio for her assistance with the SEM imaging. This work was supported in part by the Minnesota Soybean Research and Promotion Council (Grant No. 19-16C), the United Soybean Board (Grant No. 1520-532-5603), the United States Department of Agriculture (Specific Cooperative Agreement No. 58-5062-7-010), the National Science Foundation (Grant No. IOS-1444581), and the National Science Foundation Postdoctoral Research Fellowship in Biology (Grant No. IOS-1710790). Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. USDA is an equal opportunity provider and employer.

Publisher Copyright:
© 2019, The Author(s).

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title = "Functional analysis and development of a CRISPR/Cas9 allelic series for a CPR5 ortholog necessary for proper growth of soybean trichomes",

abstract = "Developments in genomic and genome editing technologies have facilitated the mapping, cloning, and validation of genetic variants underlying trait variation. This study combined bulked-segregant analysis, array comparative genomic hybridization, and CRISPR/Cas9 methodologies to identify a CPR5 ortholog essential for proper trichome growth in soybean (Glycine max). A fast neutron mutant line exhibited short trichomes with smaller trichome nuclei compared to its parent line. A fast neutron-induced deletion was identified within an interval on chromosome 6 that co-segregated with the trichome phenotype. The deletion encompassed six gene models including an ortholog of Arabidopsis thaliana CPR5. CRISPR/Cas9 was used to mutate the CPR5 ortholog, resulting in five plants harboring a total of four different putative knockout alleles and two in-frame alleles. Phenotypic analysis of the mutants validated the candidate gene, and included intermediate phenotypes that co-segregated with the in-frame alleles. These findings demonstrate that the CPR5 ortholog is essential for proper growth and development of soybean trichomes, similar to observations in A. thaliana. Furthermore, this work demonstrates the value of using CRISPR/Cas9 to generate an allelic series and intermediate phenotypes for functional analysis of candidate genes and/or the development of novel traits.",

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N2 - Developments in genomic and genome editing technologies have facilitated the mapping, cloning, and validation of genetic variants underlying trait variation. This study combined bulked-segregant analysis, array comparative genomic hybridization, and CRISPR/Cas9 methodologies to identify a CPR5 ortholog essential for proper trichome growth in soybean (Glycine max). A fast neutron mutant line exhibited short trichomes with smaller trichome nuclei compared to its parent line. A fast neutron-induced deletion was identified within an interval on chromosome 6 that co-segregated with the trichome phenotype. The deletion encompassed six gene models including an ortholog of Arabidopsis thaliana CPR5. CRISPR/Cas9 was used to mutate the CPR5 ortholog, resulting in five plants harboring a total of four different putative knockout alleles and two in-frame alleles. Phenotypic analysis of the mutants validated the candidate gene, and included intermediate phenotypes that co-segregated with the in-frame alleles. These findings demonstrate that the CPR5 ortholog is essential for proper growth and development of soybean trichomes, similar to observations in A. thaliana. Furthermore, this work demonstrates the value of using CRISPR/Cas9 to generate an allelic series and intermediate phenotypes for functional analysis of candidate genes and/or the development of novel traits.

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Functional analysis and development of a CRISPR/Cas9 allelic series for a CPR5 ortholog necessary for proper growth of soybean trichomes

Abstract

Bibliographical note

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HItachi S3500N Variable Pressure SEM

University Imaging Centers

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Functional analysis and development of a CRISPR/Cas9 allelic series for a CPR5 ortholog necessary for proper growth of soybean trichomes

Abstract

Bibliographical note

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Electron Microscopy

HItachi S3500N Variable Pressure SEM

University Imaging Centers

Cite this