Genetic characterization of novel and CRISPR-Cas9 gene edited maize brachytic 2 alleles

• Published in: Plant gene 2020-03, Vol.21, p.100198, Article 100198
• Main Authors: Bage, Shannon A., Barten, Ty J., Brown, Alana N., Crowley, James H., Deng, Mingqi, Fouquet, Romain, Gomez, Jose R., Hatton, Thomas W., Lamb, Jonathan C., LeDeaux, John R., Lemke, Bryce M., Manjunath, Siva, Marengo, Matthew S., Morales, Elisa Y., Garcia, Manuel Oyervides, Peevers, Jeanette M., Pellet, Jean-Luc, Avendano, Adan Rojas, Rymarquis, Linda A., Sridharan, Krishnakumar, Valentine, Michelle F., Yang, Dennis H., Cargill, Edward J.
• Format: Article
• Language: English
• Subjects: adenosine triphosphate; alleles; auxins; Brachytic; Corn; CRISPR-Cas systems; CRISPR-Cas9; DNA repair; enzymes; exons; gene editing; genetic variation; germplasm; introns; missense mutation; mutants; phenotype; plant height; Polymorphism; retrotransposons; stop codon; terminal repeat sequences; transcription (genetics); Transposon; transposons; Zea mays
• ISSN: 2352-4073; 2352-4073
• DOI: 10.1016/j.plgene.2019.100198

Maize plants with mutations at brachytic 2 (br2) reduce plant height through internode shortening while maintaining the rest of the plant's relative size. The gene product of Br2 encodes an ATP binding cassette type B (ABCB) auxin transporter. Several br2 mutations have been previously reported, notably independent characterizations of 8 bp and 241 bp deletions (br2-23 and br2-qpa1, respectively), a single missense mutation (br2-qph1), and a MITE transposon insertion (br2-NC238). Two new br2 mutations (designated br2-7081 and br2-7861) have now been characterized with different genetic mechanisms than prior reports providing further examples of functional genetic variation in maize. A gene edited br2 allele (designated br2-1005) has also been generated through use of CRISPR-Cas9 technology. Both novel br2 transposon mutants in this report (br2-7081, br2-7861) arose spontaneously, independently and were identified in proprietary maize germplasm; both contain insertions that result in frameshifts with presence of premature stop codons. The br2-7081 allele contains a 4.7 kb insertion in exon 5, which is identified as a Ty1-copia family long terminal repeat (LTR) retrotransposon. The br2-7861 allele contains a 579 bp insertion in intron 4, which is identified as a partial Sirevirus LTR retrotransposon, and encodes a transcribed exon of 190 bp. A gene edit in br2 (br2-1005) was generated when a CRISPR-Cas9-induced double strand break was repaired by the plant via non-homologous end joining, causing a 1 bp frameshift resulting in a premature stop codon in exon 5. Also, of note in this study is the 8 bp deletion reportedly characterizing the br2-23 mutant allele was unexpectedly found in wild-type Br2 (non-brachytic) germplasm indicating the 8 bp deletion by itself likely does not result in a brachytic phenotype. The novel mutant alleles reported here provide further examples of functional genetic variation in maize br2, either through inherent genetic variation mechanisms or generated using site-directed nuclease technology. The br2-1005 allele highlights the utility of gene editing to phenocopy naturally occurring mutations, and all three alleles in this report provide additional opportunities to research the brachytic semi-dwarf phenotype in maize.