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

Abstract

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.