Cytoplasmic male sterile (CMS) lines in maize have been classified by their response to specific restorer genes into three categories: cms-C, cms-S, and cms-T. A mitochondrial genome representing each of the CMS cytotypes has been sequenced and male sterility in the cms-S and cms-T cytotypes is linked to chimeric mitochondrial genes. To identify markers for plastid genotyping, we sequenced the plastid genomes (ptDNA) of three fertile maize lines (B37, B73, A188) and the B37 cms-C, cms-S, and cms-T cytoplasmic substitution lines. We found that the plastid genomes of B37 and B73 lines are identical. Furthermore, the fertile and CMS plastid genomes are conserved, differing only by 0-3 single nucleotide polymorphisms (SNPs) in coding regions and 8-22 SNPs and 10-21 short insertions/deletions in noncoding regions. To gain insight into the origin and transmission of the cms-T trait, we identified three SNPs unique to the cms-T plastids, and tested the three diagnostic SNPs in 27 cms-T lines, representing the HA, I, Q, RS and T male sterile cytoplasms. We report that each of the tested 27 cms-T group accessions have the same three diagnostic plastid SNPs indicating a single origin and maternal co-transmission of the cms-T mitochondria and plastids to the seed progeny. Our data exclude exceptional pollen transmission of organelles or multiple horizontal gene transfer events as the source of the urf13-T gene in the cms-T cytoplasms. Plastid genotyping enables a reassessment of evolutionary relationships of cytoplasms in cultivated maize.
Uniform transformation of the thousands of plastid genome (ptDNA) copies in a cell is driven by selection for plastid markers. When each of the plastid genome copies is uniformly altered, the marker gene is no longer needed. Plastid markers have been efficiently excised by site-specific recombinases expressed from nuclear genes either by transforming tissue culture cells or introducing the genes by pollination. Here we describe a protocol for the excision of plastid marker genes directly in tobacco (Nicotiana tabacum) plants by the Cre recombinase. Agrobacterium encoding the recombinase on its T-DNA is injected at an axillary bud site of a decapitated plant, forcing shoot regeneration at the injection site. The excised plastid marker, the bar au gene, confers a visual aurea leaf phenotype; thus marker excision via the flanking recombinase target sites is recognized by the restoration of normal green color of the leaves. The bar au marker-free plastids are transmitted through seed to the progeny. PCR and DNA gel blot (Southern) protocols to confirm transgene integration and plastid marker excision are also provided herein.