Plant & Animal Genome VI Conference

W80

ASSAYING CENTROMERE FUNCTIONS AND GENOME-WIDE RECOMBINATION IN ARABIDOPSIS

University of Chicago, EBC #401, 1103 E 57th Street, Chicago IL 60637

We used tetrad analysis in Arabidopsis to perform the first genome-wide measurement of genetic exchange in individual meiotic cells. Using this method we have determined centromere positions for all five chromosomes and have measured recombination frequecies across the entire genome. This work will facilitate the understanding of centromere structure and function in higher eukaryotes, and may enable the construction of artificial chromosome vectors in plants. Moreover, the ability to analyze recombinaton, at the genomic level, makes it possible to investigate the controls that regulate crossover (CO) number and distribution. This analysis is made possible by the Arabidopsis quartet mutant, which causes the products of pollen meiosis to remain attached. By crossing quartet mutants from the Columbia and Landsberg ecotypes, we generated F₁ plants with pollen tetrads that segreate hundreds of genetic markers. Individual tetrads from these F₁ plants were used to pollinate genetically marked stigmas, often generating four plants. We initially scored a set of 52 molecular markers, spaced at ~10 cM intervals, in plants from 57 terads. This survey localized each centromere to an average interval of 11 cM. Subsequently, we have expanded our set of tetrads to over 140 and identified additional molecular markers, resulting in a refined map location of ~1 cM for each centromere. Additionally we identified the recombination CO points in each meiotic chromosome for the set of 57 tetrads. Previous estimations of CO levels relied on inferring a correlation between cytologically observed chiasmata and genetic exchange, characterizingrecombination over limited regions, or measuring average recombination frequencies in populations produced by randomly assorted gametes. Such methods have not determined at a genomic level, the frequency and variation in the number of COs in individual mioses. By directly measuring the number and distribution of COs in complete meiotic tetrads we demonstrated i) surprisingly precise regulation of CO number in each meiosis, ii) considerably reduced recombination along chromosomes carrying ribosomal DNA arays iii) numerous, sex-specific recombination hot spots and iv) and an inversely proportional relationship between recombination frequencies and chromosome size.