We have sequenced five distinct mitochondrial genomes in maize: two fertile cytotypes (NA and the previously reported NB) and three cytoplasmic-male-sterile cytotypes (CMS-C, CMS-S, and CMS-T). Their genome sizes range from 535,825 bp in CMS-T to 739,719 bp in CMS-C. Large duplications (0.5-120 kb) account for most of the size increases. Plastid DNA accounts for 2.3-4.6% of each mitochondrial genome. The genomes share a minimum set of 51 genes for 33 conserved proteins, three ribosomal RNAs, and 15 transfer RNAs. Numbers of duplicate genes and plastid-derived tRNAs vary among cytotypes. A high level of sequence conservation exists both within and outside of genes (1.65-7.04 substitutions/10 kb in pairwise comparisons). However, sequence losses and gains are common: integrated plastid and plasmid sequences, as well as noncoding ''native'' mitochondrial sequences, can be lost with no phenotypic consequence. The organization of the different maize mitochondrial genomes varies dramatically; even between the two fertile cytotypes, there are 16 rearrangements. Comparing the finished shotgun sequences of multiple mitochondrial genomes from the same species suggests which genes and open reading frames are potentially functional, including which chimeric ORFs are candidate genes for cytoplasmic male sterility. This method identified the known CMS-associated ORFs in CMS-S and CMS-T, but not in CMS-C.
Mitochondrial DNA (mtDNA) insertions into nuclear chromosomes have been documented in a number of eukaryotes. We used fluorescence in situ hybridization (FISH) to examine the variation of mtDNA insertions in maize. Twenty overlapping cosmids, representing the 570-kb maize mitochondrial genome, were individually labeled and hybridized to root tip metaphase chromosomes from the B73 inbred line. A minimum of 15 mtDNA insertion sites on nine chromosomes were detectable using this method. One site near the centromere on chromosome arm 9L was identified by a majority of the cosmids. To examine variation in nuclear mitochondrial DNA sequences (NUMTs), a mixture of labeled cosmids was applied to chromosome spreads of ten diverse inbred lines: A188, A632, B37, B73, BMS, KYS, Mo17, Oh43, W22, and W23. The number of detectable NUMTs varied dramatically among the lines. None of the tested inbred lines other than B73 showed the strong hybridization signal on 9L, suggesting that there is a recent mtDNA insertion at this site in B73. Different sources of B73 and W23 were examined for NUMT variation within inbred lines. Differences were detectable, suggesting either that mtDNA is being incorporated or lost from the maize nuclear genome continuously. The results indicate that mtDNA insertions represent a major source of nuclear chromosomal variation.
S-type cytoplasmic male sterility (CMS-S) in maize is associated with high levels of a 1.6-kb RNA in mitochondria. This RNA contains two chimeric open reading frames (ORFs), orf355 and orf77. The previously described nuclear restorer-of-fertility allele Rf3 causes the processing of all transcripts that contain these chimeric ORFs. The Lancaster Surecrop-derived inbred line A619 carries a restorer that is distinct from Rf3 in that it selectively reduces only the CMS-S-specific 1.6-kb RNA. We have found that 10 additional Lancaster lines carry a single restoring allele traceable to either of two inbred lines, C103 and Oh40B. The C103 and Oh40B restorers are allelic to each other, but not to Rf3. Thus, this restoring allele, designated Rf9, represents a second naturally occurring CMS-S restorer in maize. Rf9 is a less effective restorer of fertility than is Rf3; its expression is influenced by both inbred nuclear background and temperature. Rf9 acts to reduce the amounts of orf355/orf77-containing linear mitochondrial subgenomes, which are generated by recombination of circular subgenomes with CMS-S-specific linear plasmids. The 1.6-kb RNA, which is transcribed only from linear ends, is correspondingly reduced.
Organellar DNA transfer into the nucleus and incorporation into chromosomes are continuing processes. We have examined chloroplast DNA insertions in maize chromosomes using fluorescence in situ hybridization (FISH). Probes to detect the nuclear-plastid sequences (NUPTs) were generated using 14 overlapping fragments of the chloroplast genome. Using current FISH methods, detection of NUPTs on mitotic metaphase chromosomes requires relatively large insertions (>3 kb) with strong sequence similarity to chloroplast DNA. In the B73 inbred line, hybridization of each fragment separately resolved a total of 49 NUPT sites; fewer sites (30) were detectable when all the probes were combined. The combined set of probes was hybridized to 10 genetically useful maize inbred lines and identified 19–30 NUPTs in each line. In all but two of the lines, a prominent NUPT was present on the long arm of chromosome 5. Collectively, the number of NUPTs exceeds the number of nuclear-mitochondrial (NUMT) sites identified within the same set of inbred lines. A majority of the NUPTs are found at sites that are different from the NUMTs. Like NUMTs, the positions of the NUPTs vary greatly among the lines, suggesting that the transfers are recent as well as frequent. Thus, insertions of large segments of chloroplast and mitochondrial DNA are components of the dynamic fraction of maize nuclear genomes.
The transfer of mitochondrial DNA (mtDNA) into nuclear genomes is a regularly occurring process that has been observed in many species. Few studies, however, have focused on the variation of nuclear-mtDNA sequences (NUMTs) within a species. This study examined mtDNA insertions within chromosomes of a diverse set of Zea mays ssp. mays (maize) inbred lines by the use of fluorescence in situ hybridization. A relatively large NUMT on the long arm of chromosome 9 (9L) was identified at approximately the same position in four inbred lines (B73, M825, HP301, and Oh7B). Further examination of the similarly positioned 9L NUMT in two lines, B73 and M825, indicated that the large size of these sites is due to the presence of a majority of the mitochondrial genome; however, only portions of this NUMT (∼252 kb total) were found in the publically available B73 nuclear sequence for chromosome 9. Fiber-fluorescence in situ hybridization analysis estimated the size of the B73 9L NUMT to be ∼1.8 Mb and revealed that the NUMT is methylated. Two regions of mtDNA (2.4 kb and 3.3 kb) within the 9L NUMT are not present in the B73 mitochondrial NB genome; however, these 2.4-kb and 3.3-kb segments are present in other Zea mitochondrial genomes, including that of Zea mays ssp. parviglumis, a progenitor of domesticated maize.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.