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

Authors: CAMPBELL, BENJAMIN - University Of Minnesota; HOYLE, JACOB - University Of Wisconsin; BUCCIARELLI, BRUNA - University Of Minnesota; STEC, ADRIAN - University Of Minnesota; Samac, Deborah - Debby; PARROTT, WAYNE - University Of Georgia; STUPAR, ROBERT - University Of Minnesota

Submitted to: Scientific Reports
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/25/2019
Publication Date: 10/14/2019
Citation: Campbell, B.W., Hoyle, J.W., Bucciarelli, B., Stec, A.O., Samac, D.A., Parrott, W.A., Stupar, R.M. 2019. Functional analysis and development of a CRISPR/Cas9 allelic series for a CPR5 ortholog necessary for proper growth of soybean trichomes. Scientific Reports. 9:14757. https://doi.org/10.1038/s41598-019-51240-7.
DOI: https://doi.org/10.1038/s41598-019-51240-7

Interpretive Summary: A proven method for improving crop plants for desired agronomic traits is irradiation mutagenesis that causes novel genetic variation through deletions and rearrangements of DNA. However, the large multiple deletions generated often make it difficult to identify the causative gene or genes underlying a trait. This study used a soybean fast neutron irradiation mutant with altered leaf hair morphology and used traditional genetic mapping approaches to identify a set of possible candidate genes underlying the mutant phenotype. To substantiate the role of the candidate gene in the short leaf hair trait, the DNA sequence of the gene in a normal soybean line was edited in order to generate plants with mutations in the gene. The gene-edited plants displayed the same altered leaf hair morphology as the fast neutron induced mutant, validating the candidate gene. This research provided the proof-of-concept test for using gene editing in combination with traditional mapping approaches to overcome technical challenges involved in identifying underlying genes in soybean fast neutron mutants. This approach can be used by the plant research community as an improved pipeline for mapping and validating genes for important agronomic traits.

Technical Abstract: Recent developments in genomic and genome editing technologies have facilitated the mapping, cloning, and validation of genetic variants that underlie trait variation. In this study, a combination of bulked-segregant analysis, array comparative genomic hybridization, and CRISPR/Cas9 methodologies were used to rapidly identify a CPR5 ortholog that is essential for proper trichome growth and nuclear DNA endoreduplication in soybean (Glycine max). The mutant line used for mapping was derived from fast neutron mutagenesis and exhibited morphological differences compared to its parent line, including substantially shorter trichomes with smaller trichome nuclei compared to the 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 CPR5. Five T0 plants were generated carrying a CRISPR/Cas9 transgene targeting the CPR5 ortholog, and these plants harbored a total of four different putative knockout alleles and two in-frame alleles of the CPR5 ortholog. Segregation analysis of the knockout and in-frame alleles provided validation of the candidate gene, wherein T1 plants carrying homozygous or heterozygous in-frame alleles exhibited nearly normal trichome development, and T1 plants with homozygous knockout alleles exhibited short trichomes. These findings demonstrate that the CPR5 ortholog is essential for proper growth and development of soybean trichomes and phenocopies the Arabidopsis cpr5 mutant trichome phenotype. We thus named the soybean ortholog GmCPR5, an abbreviation of G. max CPR5. Furthermore, this work demonstrates the capacity and value of using CRISPR/Cas9 to generate an allelic series for functional analysis of candidate genes and/or the development of novel traits.