Using Fluorescence in situ hybridization to study maize lines genetically predicted to have chromosomal abnormalities [abstract]

Abstract only availableSince the 1960s, genetic evidence has indicated chromosome damage and nondisjunction in lines of maize containing knob heterochromatin-bearing chromosomes and at least two B chromosomes. However, at that time researchers lacked the technology to visualize these occurrences. Now, using Fluorescence in situ Hybridization to "paint" and photograph the chromosomes, it is possible to accurately karyotype and identify broken, missing, or extra chromosomes. A line with a very large number of heterochromatic knobs had been crossed with another line containing supernumerary B chromosomes. This F1 hybrid that had been self pollinated (B73+B/K10) was chosen for study by the FISH method because it contains both knobs and B chromosomes, as well as exhibiting abnormalities such as irregular rows, ovule abortion, and defective kernels. This material combined a high knob number with B chromosomes and exhibited properties suggestive of chromosome breakage or nondisjunction. Metaphase spreads from the root tips were prepared and hybridized to fluorescent probes. Spreads were observed using fluorescence microscopy. The majority of the plants studied possessed the normal content of 20 A chromosomes plus varying numbers of B chromosomes. One individual was found with 21 chromosomes that might have resulted from nondisjunction. No chromosomal breakage was evident in this background. FISH proved to be a powerful cytogenetic tool in observing these plants; however, further research on this topic is needed to provide insight into the cause of the genetic abnormalities

Construction and applications of engineered minichromosomes in plants

The use of genetically modified crops is constantly finding new areas of application, including the production of compounds with therapeutic value. Current technology for producing transgenic crops relies on random integrations that can have variable expression and could potentially disrupt the endogenous genes. Also combining multiple transgenes requires a lengthy crossing scheme and can bring along linked genes from one variety into another. The current invention developed by researchers at the University of Missouri is a technology that will allow continued addition of transgenes as the need arises in the future using engineered plant minichromosomes. Artificial chromosome platforms in maize were produced by telomere-mediated truncation while simultaneously adding sequences that will permit amendments to the chromosome indefinitely. Such engineered minichromosomes have the potential to be used as a vector for efficient stacking of multiple genes for insect, bacterial and fungal resistances together with herbicide tolerance and crop quality traits unlinked to endogenous genes in a circumstance that would foster faithful expression. The collection of transgenes on minichromosomes might be combined with haploid breeding techniques to facilitate their transfer among diverse lines of a crop. A toolkit of lines that will permit additions and subtractions of genes from engineered minichromosomes is being assembled. Because of the near universality of the telomere sequence in the plant kingdom, engineered minichromosomes should be able to be produced easily in most plant species by this technique. Potential Areas of Applications: * Stack multiple transgenes on an independent chromosome with potentially no limit to number. * Facilitate transfer of transgenes into different varieties of a crop species by combining them with haploid breeding procedures

Retension of knobs in chromosome tips in maize

Abstract only availableKnobs are deeply staining chromosomal sites on maize chromosomes. Molecularly, they are composed of a 180 base pair repeat. Their positions on the chromosomes are variable but usually internal in maize. In relatives, the knobs are usually found on the tips of chromosomes. They have been observed for a long time, yet their function remains a mystery. Knobless maize lines do not appear to have knobs. I used fluorescence in situ hybridization (FISH) to test whether cryptic knob sequences exist at the chromosome tips in maize but have avoided normal detection. Long exposure time detects weak signals near the ends of most chromosomes and some cryptic internal sites. Knobless lines are ideal because they do not have the large knobs which can make such detection difficult, if not impossible. I found the Knobless Tama Flint and Knobless Wilbur Flint lines to have cryptic knobs on most chromosomes. Zea diploperennis exhibited knobs on every chromosome, usually at the tips. Thus, although knobs as usually detected in maize are internal, maize has cryptic knob sequences at the ends of most chromosomes in a similar situation as its relatives suggesting a conserved function at chromosome termini.NSF-REU Program in Biological Sciences & Biochemistr

A phenotypic comparison of heterosis and inbreeding depression across different ploidy levels of maize

Abstract only availableIt is hypothesized that dosage-dependent gene regulators controlling the quantitative traits contribute to heterosis and inbreeding depression in maize. Heterosis, also known as hybrid vigor, refers to the phenomenon that the F1 progeny derived from crosses between diverse inbred varieties exhibit greater biomass, development rate, and fertility than the better of the two parents. Inbreeding depression, however, is the reverse of heterosis in which case the offspring have inferior traits to that of the parents. The study of heterosis and inbreeding depression at different ploidy levels will help us to test this hypothesis. Maize was planted in completely randomized blocks replicated three times. We will be analyzing nine phenotypic traits to help us determine if there is a significant difference between the diploid and triploid hybrid plants in heterosis as well as diploid and tetraploid hybrid plants for the rate of inbreeding depression. This will be done by looking at both the hybrids and their corresponding inbred parents of all ploidy levels. The most recent trait available to be analyzed is the height at four weeks. Statistical analysis of the plant height at four weeks will be used to analyze heterosis in each hybrid at different ploidy levels and determine if gene dosage is indeed a factor in inbreeding depression and heterosis.NSF-REU Program in Biological Sciences & Biochemistr

Analysis of the mtDNA insertion site on chromosome 9L in maize inbreds using fluorescence in situ hybridization

Abstract only availableAlmost all eukaryotic nuclear genomes show evidence of organellar DNA insertions originating from mitochondrial DNA (mtDNA) and chloroplast DNA (cpDNA). While the precise mechanisms of incorporation remain unknown, the phenomenon is frequent and ongoing in many species. In Zea mays, mtDNA insertions differ among inbred lines. A very large mtDNA insertion is found near the centromere of the long arm of chromosome 9 in the B73 inbred. This insertion contains the majority of the mitochondrial genome, while a similarly positioned insertion in the Mo17 inbred line is much smaller. We used recombinant inbred lines from the intermated B73 x Mo17 (IBM) population to determine if the insertions are indeed at the same position. We selected lines with recombination in this region of chromosome 9L. Using two mtDNA probes present in the insertions in both B73 and Mo17, we applied a chromosome painting technique called fluorescence in situ hybridization (FISH) to root-tip metaphase chromosomes and looked for the presence of the mtDNA site on chromosome 9L in the selected IBM lines. If the mtDNA insertion sites in B73 and Mo17 are at different locations, then at least one of the recombinant IBM lines should not display a mtDNA insertion at the chromosome 9 location. However, all of the recombinant IBM lines examined displayed the mtDNA insertion site on chromosome 9L. This indicates that the Mo17 and B73 insertions likely occupy the same region on the chromosome. Furthermore, this suggests that the large mtDNA insertion occurred recently in B73 at a pre-existing site present in both B73 and Mo17.NSF-REU Program in Biological Sciences & Biochemistr

Mitochondrial DNA insertion into nuclear chromosomes of maize

Abstract only availableEvery mitochondrion contains its own DNA separate from the nucleus. Over evolutionary time, most of the mitochondrial genes have moved to the nucleus so that now mitochondria require nuclear DNA to function. This type of transfer is an apparently ongoing process based on our observations that large pieces of the mitochondrial genome have been transferred to the nucleus. The focus of this study was to find the locations of mitochondrial DNA (mtDNA) on nuclear chromosomes in the B73 line of maize and compare these locations to other lines, using fluorescence in situ hybridization (FISH). First, cosmids previously made from a normal mtDNA genotype (NB) were maxi-prepped and then direct labeled with fluorescent tags. Next we prepared slides of B73 root tips and hybridized the labeled cosmids as well as marker probes to the cells. After hybridization, the slides were viewed and chromosome spread pictures were taken showing the location of the cosmids on the chromosomes. This process was then performed on root tip chromosomes from the Mo17, Black Mexican Sweet (BMS), and B37 lines. Twelve cosmids, representing about 71% of the mitochondrial genome, were examined and 8 different nuclear insertion sites were identified. These dispersed locations were predominantly near centromeres or telomeres on chromosomes 2 and 9. These new findings will make understanding the B73 nuclear genome sequence easier because now researchers will know what to expect at these locations and provide new information about the mechanism of mitochondrial genome transfer to the nucleus.NSF-REU Biology & Biochemistr

Analysis of mitochondrial DNA insertions into a nuclear chromosome of the maize B73 line

Abstract only availableMitochondrial DNA (mtDNA) is known to have integrated into the nuclear DNA of plants and animals. The purpose of this project is to investigate the on-going migration of mtDNA into the nuclear DNA of maize plants. Specific objectives are to discover the amount of DNA incorporated, whether it is the whole mitochondrial genome or sections, and to see if it has replicated after migration. The maize inbred line B73 has a particularly large mt DNA insert on chromosome 9. Using the fluorescent in situ hybridization (FISH) method, the arrangement of inserted mitochondrial DNA was examined. The FISH method uses fluorescently labeled mtDNA as probes for hybridization to chromosomes. Regions of the chromosomes that contain mtDNA can then be detected using a compound microscope with fluorescent attachments. Locations that contain more mtDNA are brighter. Three combinations of probes that cover different parts of the mitochondrial genome were employed. In order to analyze the arrangement of the DNA, the chromosomes were prepared from a stage of meiosis called pachynema in which the chromosomes are elongated and have not yet begun to condense. The results have confirmed the presence of all three probes within the large insertion of mtDNA on chromosome 9 of B73. The data suggest that either different parts of the mitochondrial genome are incorporated preferentially or that there is selective replication of portions of the mitochondrial genome after incorporation.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

Identification of chloroplast DNA insertions in B73 nuclear chromosomes [abstract]

Abstract only availableIt is known that DNA from mitochondria and chloroplasts migrates to the nucleus and incorporates into the nuclear genome. It is unknown how often this transfer and integration process occurs or if specific sites in the chromosomes are preferred. We have evidence that chloroplast DNA has integrated into multiple sites within chromosomes in the B73 inbred line of maize. This has been shown with a procedure called FISH, Fluorescence in situ Hybridization, in which fluorescent probes created from DNA hybridize with chromosomes in places where there is a match. Probes for this project were generated from chloroplast DNA. The chloroplasts were separated by centrifugation and lysed to obtain the DNA. The chloroplast genome was subdivided into 15 regions. Pieces of chloroplast DNA corresponding to 14 of the 15 regions were amplified using PCR (the polymerase chain reaction). The amplified DNA was purified and labeled with fluorescent dyes to create probes. These probes were subsequently hybridized to metaphase chromosome spreads on slides. The probes recognized and bound to DNA sequences in the chromosomes. With the completion of the chloroplast DNA mapping, the B73 inbred maize line will have a complete diagram of the locations of major chloroplast DNA insertions.MU Monsanto Undergraduate Research Fellowshi

Life sciences elevator pitches

The slides for some of the panelists' presentations can be found by going to Presentations collection for the Missouri Technology Expo 2010: https://mospace.umsystem.edu/xmlui/handle/10355/9623This video presents the elevator pitches given in the field of life sciences. Each elevator pitch consists of a presentation from the faculty/student innovator, followed by questions/answers from the audience