Expansion of a chromosomal repeat in Escherichia coli: roles of replication, repair, and recombination functions

<p>Abstract</p> <p>Background</p> <p>Previous studies of gene amplification in <it>Escherichia coli </it>have suggested that it occurs in two steps: duplication and expansion. Expansion is thought to result from homologous recombination between the repeated segments created by duplication. To explore the mechanism of expansion, a 7 kbp duplication in the chromosome containing a leaky mutant version of the <it>lac </it>operon was constructed, and its expansion into an amplified array was studied.</p> <p>Results</p> <p>Under selection for <it>lac </it>function, colonies bearing multiple copies of the mutant <it>lac </it>operon appeared at a constant rate of approximately 4 to 5 per million cells plated per day, on days two through seven after plating. Expansion was not seen in a <it>recA </it>strain; null mutations in <it>recBCD </it>and <it>ruvC </it>reduced the rate 100- and 10-fold, respectively; a <it>ruvC recG </it>double mutant reduced the rate 1000-fold. Expansion occurred at an increased rate in cells lacking <it>dam</it>, <it>polA, rnhA</it>, or <it>uvrD </it>functions. Null mutations of various other cellular recombination, repair, and stress response genes had little effect upon expansion. The <it>red </it>recombination genes of phage lambda could substitute for <it>recBCD </it>in mediating expansion. In the <it>red</it>-substituted cells, expansion was only partially dependent upon <it>recA </it>function.</p> <p>Conclusion</p> <p>These observations are consistent with the idea that the expansion step of gene amplification is closely related, mechanistically, to interchromosomal homologous recombination events. They additionally provide support for recently described models of RecA-independent Red-mediated recombination at replication forks.</p

DEVELOPMENT OF GENOMIC RESOURCES AND IDENTIFICATION OF MARKER-TRAIT ASSOCIATIONS IN STRAWBERRY

Crosses were performed and progeny populations were developed in diploid and octoploid strawberry (Fragaria) species for the purposes of genetic analysis and breeding. A high throughput genotyping platform - the Affymetrix IStraw90® Axiom® strawberry SNP array - was evaluated and employed for these purposes. Phenotyping was conducted with respect to several traits of interest, including flower color, flower and fruit pigment compositions, and verticillium wilt resistance, for the ultimate purpose of identifying marker-trait associations useful in breeding. In the ancestral diploid species Fragaria iinumae, an F2 mapping population was developed from a cross between two accessions previously collected in Hokkaido, Japan. High-throughput genotyping platforms, the IStraw90 SNP Array and Genotyping-by-Sequencing (GBS), were utilized to develop high density linkage maps for F. iinumae. A linkage map based on 21 of the F2 generation plants genotyped on both platforms consists of 4,110 markers, while the second map based 85 F2 plants genotyped on only GBS consists of 895 markers. The linkage maps will used in a parallel research project to anchor an F. iinumae genomic sequence assembly, with the aim of providing an additional reference genome for Fragaria. A 455 member Closed Pedigree Set of octoploid strawberry plants was developed with diverse flower colors and progress was made on improvement of fruit quality through selection. The range of floral cyanidin and pelargonidin contents determined by HPLC in the progeny exceeded the range of contents of the founding parents, and thus exhibited transgressive segregation. The non-white (NW) flower trait was found to be a completely dominant to white, and no true-breeding, non-white flowered plants were found. The IStraw90 SNP Array was utilized to genotype 41 members of the Closed Pedigree Set and among a total of 5,674 segregating markers, 35 markers predictive of NW flower color were identified on chromosome 5. Additional genotyping will be required to further characterize the marker-trait association for color and to identify quantitative trait loci for hue. Fragaria diploid and polyploid species were phenotyped for verticillium wilt resistance, and disease resistance ratings of the octoploid cultivars were compared to the results of previous published studies and presented in a pedigree format to help identify sources of resistance. The results will be used to identify germplasm for developing populations for high throughput genotyping on the IStraw90 Array for the ultimate goal of identifying marker-trait associations for the purpose of marker-assisted breeding for resistance