Transfer of chromosomes through haploid induction in maize (Zea mays)
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[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] The B chromosome (B) of maize (Zea mays) is a supernumerary chromosome with no essential genes, no phenotypic effect at low copy number, and survives by a selfish drive mechanism in the male reproductive lineage. The selfish drive is accomplished by nondisjunction of the B chromosome centromere at the second pollen mitosis of a microspore with a single B chromosome, to produce one sperm carrying two B chromosomes and one carrying none. This nondisjunction is followed by preferential fertilization of the egg by the sperm carrying B chromosomes. Engineered minichromosomes (minis) are made by truncating chromosomes with a transgene carrying a selectable marker and telomere repeat. Maize B chromosomes are used as a target for this truncation because they have no vital genes, so truncation of a B chromosome has no phenotypic effect beyond that caused by expression of the transgenes. Truncation of a B chromosome will be selected over the truncation of an A chromosome, because the truncation of an A chromosome causes large deletions that are generally detrimental to the plant. Lines derived from Stock 6 produce maternal haploids when crossed as a male. Though the primary gene controlling Stock 6 haploid induction was recently identified by a different group, the process by which haploids are produced is still unknown. Whole B chromosomes transferred from a paternal inducer to maternal haploids show that fertilization does occur. The transfer of whole or broken A chromosomes to haploids show that transfer is not restricted to the B chromosome. Sectors of color observed in kernels pollinated by a Stock 6 inducer show that the paternal chromosomes are present in some tissue, but absent in other tissue that is derived from the same fertilization event. This suggests that chromosome elimination is occurring subsequent to fertilization. Minichromosomes and B chromosomes were backcrossed into a Stock 6-derived line, RWS, then tested for transfer to maternal haploids. B chromosomes were successfully transferred to maternal haploids. This served as a proof of concept for the rapid introduction of minis to new lines, a technique that could significantly reduce the scale and time required for breeding transgenes into new lines. 13 cases of B chromosomes transferring to haploids were visualized by fluorescence in-situ hybridization (FISH), and in each case the B chromosomes were found as a pair. Minichromosomes were not recovered in a haploid, even though more haploids derived from crosses with RWS + minichromosomes were screened than from RWS + B chromosomes. The minichromosomes had comparable frequency of inheritance to the B chromosomes in non-haploid materials from the same crosses. Aneuploidies close to the diploid or haploid state were discovered in the putative haploid materials and characterized by karyotyping with FISH. Broken chromosomes were common among these aneuploidies; this shows that part of the mechanism of haploid induction by Stock 6 lines produces broken chromosomes. A novel rearrangement of the B chromosome, "Scrambled B Long arm" (ScramBL) was recovered in an otherwise maternal haploid plant when screening for transferred B chromosomes. This rearrangement appears to be derived from the distal portion of the B long arm, likely with duplications of the distal tip, and a de novo centromere. It is probable that the formation of ScramBL involved chromosome breakage. When plants with a normal B chromosome and a ScramBL were used as a male in a cross, about half the progeny had no supernumerary chromosomes, while the majority of the other half possessed a single ScramBL. Two new derivatives of the B and ScramBL also emerged in the 57 progeny tested, suggesting an interaction between the B and ScramBL sometime during pollen formation. This dissertation also describes the development of two new, rapid-cycling, miniature lines of maize: Fast-Flowering Mini-Maize A and B, produced to accelerate maize research (McCaw et al., 2016c). These lines can complete a seed-to-seed cycle in 60 days, making them competitive with popular model systems like Arabidopsis thaliana and Brachypodium distachyon. Fast-Flowering Mini-Maize maintains the benefits inherent to maize as a model organism, such as ease of crossing due to separate male and female flowers, and the ability to determine genotype from seed color markers, but requires less space than traditional maize lines, and can produce five or more generations per year instead of three. The genome of Fast-Flowering Mini-Maize-A was sequenced and aligned to the B73 reference genome; seed color markers were also introgressed into the line. The two lines, A and B, show hybrid vigor when crossed together, and the flowering time of each individual line and the hybrid are influenced by greenhouse conditions; it may be possible to speed up their generation time or performance by optimizing growth conditions.
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