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.
The protocol we report here is based on biolistic delivery of the transforming DNA to tobacco leaves, selection of transplastomic clones by spectinomycin resistance and regeneration of plants with uniformly transformed plastid genomes. Because the plastid genome of Nicotiana tabacum derives from Nicotiana sylvestris, and the two genomes are highly conserved, vectors developed for N. tabacum can be used in N. sylvestris. Also, the tissue culture responses of N. tabacum cv. Petit Havana and N. sylvestris accession TW137 are similar, allowing plastid engineering protocols developed for N. tabacum to be directly applied to N. sylvestris. However, the tissue culture protocol is applicable only in a subset of N. tabacum cultivars. Here we highlight differences between the protocols for the two species. We describe updated vectors targeting insertions in the unique and repeated regions of the plastid genome as well as systems for marker excision. The simpler genetics of the diploid N. sylvestris, as opposed to the allotetraploid N. tabacum, make it an attractive model for plastid transformation.