Using a candidate gene approach to identify the restorer-of-fertility Rf4 gene for maize CMS-C

Abstract only availableCytoplasmic male sterility (CMS) is a maternally inherited trait that prevents normal pollen development. Although CMS is a mitochondrial mutation, there are nuclear genes known as restorer-of-fertility genes (Rf) that restore normal pollen development. This makes CMS particularly useful in performing controlled crosses for plant breeding programs and seed production, where the desired female carries a CMS mutation and the male contains the nuclear Rf gene. In maize, CMS type C (CMS-C) can be restored to fertility by the Rf4 gene. Identification of Rf4 can provide insight into the cause of CMS-C pollen abortion, and expand our knowledge of nuclear-mitochondrial communication. Our approach for identifying Rf4 combines genetic information with the physical maps and emerging DNA sequence data. Previous studies have genetically mapped the Rf4 gene to the short arm of a chromosome 8 (Sisco, 1991). Our goal is to identify a candidate gene for Rf4 in the correct region of chromosome 8. Genes in this region were categorized based on the probability of the proteins to be targeted to the mitochondria using MitoPROT and Predotar. The genes were also analyzed for PPR motifs using TPRpred. We used the criteria of mitochondrial targeting and a probable PPR functional motif because most Rf genes identified to date are targeted to mitochondria and have a PPR motif in their sequence. Primers have been designed for candidate genes, and they will be amplified and sequenced from six different maize inbred lines: three with the fertility restoring allele Rf4 and the non-restoring allele rf4. The sequenced genes will be then analyzed for nucleotide polymorphisms that correlate with the Rf4 and rf4 alleles.NSF Plant Genomics Internship @ M

Altered nuclear gene expression in response to mitochondrial mutations in maize [abstract]

Abstract only availableMaize non-chromosomal stripe (NCS) plants are defective plants with mutations within mitochondrial DNA (mtDNA) resulting in deficiencies in the electron transfer chain (ETC). It has been shown the cells will synthesize nuclear proteins in response to the mitochondrial mutations. All NCS mutants are known to up-regulate several stress proteins in response to defective mitochondria, including alternative oxidases and heat shock proteins. Changes in RNA levels can indicate which proteins differ in expression. The NCS2 has a mutation within complex I of the ETC. Using microarrays, total RNA samples of the NCS2 mutant are being compared to those of relatives with normal mitochondria. So far, many differences in RNA levels have been observed. Data is still being collected and being analyzed to determine how many of the apparent differences are statistically significant.MU Monsanto Undergraduate Research Fellowshi

Investigations into the cause of pollen abortion in maize CMS-C

Title from PDF of title page (University of Missouri--Columbia, viewed on September 10, 2010).The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file.Dissertation advisor: Dr. Kathleen Newton.Vita.Ph. D. University of Missouri--Columbia 2009.Cytoplasmic male sterility type C (CMS-C) is atypical compared to other CMS types as no novel chimeric transcript or protein has been found correlating with the phenotype. However, a tassel-specific decrease for both ATP6 and ATP9, which are mitochondrially encoded components of the ATP synthase F0 subunit, was discovered in CMS-C. This decrease in levels was not observed when the restorer of fertility gene (Rf) was present. Other components of the ATP synthase complex analyzed did not appear to be altered in CMS-C, indicating that the cause of pollen abortion in CMS-C is affecting the stability or formation of F0. The CMS-C genome has two copies of the atp9 gene. One is the normal copy of atp9 (atp9-1), which is present in other maize cytotypes but is transcribed [about]20 fold less in CMS-C. The other copy of atp9 (atp9-2) is the predominant atp9 transcript in CMS-C and has a chimeric 5'UTR resulting in the use of a different promoter and a larger transcript (4kb instead of 1kb). We propose that the atp9-2 transcript is inefficiently translated. When synthesis demand is high, as in the tapetum layer of an anther, the mitochondria are unable to compensate and ATP9 levels decrease. This results in the formation of fewer F0 subunits and ATP synthase complexes, ultimately causing a reduction in ATP levels and abortion of the developing pollen.Includes bibliographical reference

Circular and linear mitochondrial genomes in cytoplasmic male sterile maize [abstract]

Abstract only availableCytoplasmic male sterility (CMS) is a maternally inherited condition in which a plant has an inability to produce viable pollen. It is usually due to the production of a toxic chimeric protein within the mitochondria during the maturation of pollen grains. In maize (Zea mays), there are three types of CMS: CMS-T, CMS-C and CMS-S. The S-type of cytoplasmic male sterility (CMS-S) in maize is associated with the expression of a rearranged mitochondrial DNA region. This CMS-S-specific region includes two co-transcribed chimeric open reading frames, orf355 and orf77. The nuclear restorer-of-fertility gene, Rf3, cleaves all transcripts containing both orfs, including the CMS-S-specific linear 1.6 kb mRNA; this results in male fertility. The Lancaster Surecrop-derived inbred line A619 carries a different and weaker restorer called Rf9. Fertility restoration by Rf3 and Rf9 was compared for their effects upon the CMS-associated region of mitochondrial DNA. Unlike Rf3, Rf9 affects the organization of the CMS-S-specific region. It appears to do this by affecting recombination between linear "S" plasmids and the CMS-S-specific region of the main mitochondrial genome, which produces a linear end from which transcripts for the 1.6 kb mRNA are initiated. By reducing the amount of recombination, Rf9 reduces the amount of linear template available for transcribing the S-associated 1.6 kb RNA. A reduction in this transcript is associated with an increase in pollen survival. We have studied the effects of the two restorer-of-fertility genes from several different inbred lines on the amounts of integrated and linearized orf355/orf77 genes within CMS-S mtDNA.MU Monsanto Undergraduate Research Fellowshi

Identification of chloroplast DNA insertions in nuclear chromosomes of maize B73 line using the FISH procedure

Abstract only availableIt is known that chloroplast DNA can incorporate itself into the nuclear genome of plants. However, the sites of chloroplast (ct) DNA integration into chromosomes of maize have not yet been analyzed. This project is the first attempt to find the location of the ctDNA on the maize chromosomes. Fluorescent in situ hybridization is a technique that has proved useful in karyotyping and chromosomal mapping in maize. The FISH procedure is being used in this study to discover the location of the ctDNA in the nuclear genome of the inbred line B37. In order to develop ctDNA “probes” for FISH analysis, we have used the polymerase chain reaction (PCR) to produce fragments of ctDNA. Primers were chosen to amplify fragments of 10 kb or larger. The amplified DNAs were purified and labeled with fluorescent dyes and these probes were subsequently hybridized to chromosomes. The probes recognize and bind to the corresponding DNA sequences within the chromosomes. Root tip cells were used to prepare the slides for hybridization. Because the cells are collected during the metaphase stage of division, the chromosomes are compact and more easily visible. Chromosomes that contain ctDNA can be detected using a compound microscope with fluorescent attachments. The location of the ctDNA on the chromosomes is made visible by the fluorescent labeling of the probe. Eight of eleven regions of the chloroplast genome of the B73 line have been specifically amplified and have been observed under the microscope for FISH analysis. This information will contribute to an understanding of the extent and mechanism of transfer of organellar genomes to the nucleus.MU Monsanto Undergraduate Research Fellowshi