Characterization of pearl millet mitochondrial DNA fragments rearranged by reversion from cytoplasmic male sterility to fertility

Cloned pearl millet [Pennisetum glaucum (L.) R. Br.] mitochondrial (mt) DNA fragments rearranged by spontaneous reversion from cytoplasmic male sterility (cms) to fertility were characterized by restriction mapping, hybridization with maize mt genes, and transcription analyses. The clones characterized were a 4.7-kb fragment found only in the male-sterile cytoplasm and lost upon reversion to fertility, a 10.9-kb fragment found in all cytoplasms and not changed by reversion, a 13.6-kb fragment found in the male-sterile and -fertile normal cytoplasms and lost in seven of the eight revertants studied, and a 9.7-kb fragment not found in the male-sterile cytoplasm but produced by reversion from male sterility to fertility. The restriction maps verified that the four cloned pearl millet fragments contained two sets of repeated sequences, one on the 4.7-, 10.9-, and 13.6-kb fragments, the other on the 13.6- and 9.7-kb fragments. The rrn18, rrn5, and coxI genes were located in the repeated regions of the 4.7-, 10.9-, and 13.6-kb cloned fragments. The correlation of reversion (eight independent events) with the loss of fragments containing the rrn18, rrn5, and coxI genes suggests that those lost fragments and their gene content could be responsible for the expression of cms. Transcriptional analyses using both Northern blots and end-labeled mtRNA probes verified that transcripts homologous to the rrn18 and coxI genes were present in pearl millet total mtRNA. However, no transcript differences were detected among cms, revertant, and fertile normal cytoplasms, suggesting that the reversion process involves mutational changes that may not affect transcript size. Transcript analyses indicated that the 10.9-kb clone contained an unidentified gene on the end opposite the rrn18 gene; however, since it was present in all cytoplasms, it is not believed to be involved in cms.