Pollen tetrad-based visual assay for meiotic recombination in Arabidopsis

Abstract

Recombination, in the form of cross-overs (COs) and gene conversion (GC), is a highly conserved feature of meiosis from fungi to mammals. Recombination helps ensure chromosome segregation and promotes allelic diversity. Lesions in the recombination machinery are often catastrophic for meiosis, resulting in sterility. We have developed a visual assay capable of detecting Cos and GCs and measuring CO interference in Arabidopsis thaliana. This flexible assay utilizes transgene constructs encoding pollen-expressed fluorescent proteins of three different colors in the qrt1 mutant background. By observing the segregation of the fluorescent alleles in 92,489 pollen tetrads, we demonstrate (i) a correlation between developmental position and CO frequency, (ii) a temperature dependence for CO frequency, (iii) the ability to detect meiotic GC events, and (iv) the ability to rapidly assess CO interference.cross-over ͉ meiosis ͉ tetrad ͉ gene conversion ͉ interference

Genetic and environmental determinants of unreduced gamete production in Brassica napus, Sinapis arvensis and their hybrids

“…This may draw resources away from gamete production, increasing the number of meiotic errors. In addition, buds developing on primary, secondary and tertiary branches of Arabidopsis thaliana were shown to have different frequencies of crossover events in meiosis (Francis et al, 2007), implicating a higher level of meiotic abnormalities in later developing buds. Finally, it is not impossible that some shift in environmental conditions may have occurred over the 2-week sampling period, despite the consistent environmental controls in the Phytotron.…”

Analysis of Arabidopsis genome-wide variations before and after meiosis and meiotic recombination by resequencing Landsberg erecta and all four products of a single meiosis

“…Moreover, because the Arabidopsis qrt1 mutant meioses produce all four meiotic products as attached spores, which then develop into attached functional pollen grains, Arabidopsis offers a unique opportunity among plants and animals to carry out tetrad analysis, as was done using hundreds of DNA markers (Preuss et al 1994;Francis et al 2007). Here, we sequenced the Ler genome using high-throughput sequencing to uncover over 400,000 genome variations between Ler and Col, with functional implications.…”

Meiosis in living color: Fluorescence-based tetrad analysis in Arabidopsis

“…Second, the technique reported here will allow us to address the mechanisms that control the distribution of meiotic exchange in higher eukaryotes, and most importantly address the mechanism of cross-over interference (19). The technique presented by Francis et al (1) will facilitate the identification of mutants defective in the maintenance of interference and in other mechanisms that control cross-over distribution. These mutants will provide the keys required to understand the mechanisms by which exchange distribution is controlled.…”

“…Unfortunately, the very purpose of meiosis, distributing the four chromatids into four gametes, creates a problem for greedy geneticists who would like to be able to recover all four of the original chromatids! In this issue of PNAS, Francis et al provide an elegant and efficient solution to this problem in Arabidopsis thaliana (1).…”

“…Ideally, one wants to be able to determine the genotype of each spore by just looking at it. Francis et al achieve this goal by creating a large number of precisely mapped transgene constructs in the qrt1 mutant background (1). Each of these constructs encodes one of three fluorescent proteins (red, yellow, or cyan) under control of a pollen-specific promoter.…”