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.

Fig. 1.

Fluorescent markers in pollen tetrads. (A and B) An Agrobacterium T-DNA construct (A) containing ECFP, EYFP, or DsRed driven by the LAT52 promoter and a selectable marker conferring resistance to either kanamycin (npt II) or glufosonate (Bar) was used to transform qrt1 Arabidopsis seed resulting in T₁ plants expressing the fluorescent protein in their pollen (B shows pollen from three different plants). (C–E) Crossing lines with differently colored transgenes on the same chromosome (C and D) enables detection of CO events in the interval between the transgenes (E, merge of C and D with arrow indicating recombinant tetrad).

Fig. 2.

Map of fluorescent transgenes. The chromosomal (green bars) insertion site of the transgene carried by each FTL lines is indicated by a red (DsRED), yellow (EYFP), or cyan (ECFP) circle. The genetic intervals (I1, I3, I5a, and I5b) used in this study are delineated with brackets, and the locus used to detect GC event (GC) is also marked. The Arabidopsis Information Resource “chromosome map tool” was used to place T-DNA insertion points on the physical map (15).

Fig. 3.

Genetic analyses using the visual assay system. (Top) Map distances in centimeters were measured by using flowers from the primary bolt (1), branches of the primary bolt (2), and branches of branches (3) in both I1 (Left) and I3 (Right). (Middle) As a control for flower age, map distances were also measured in individual flowers, beginning with the first and ending with the last, produced by the primary axis in I1 (Left) and I3 (Right). (Bottom) The influence of temperature during flowering was measured in I1 (Left) and I3 (Right) by using flowers from the primary axis. Error bars (Top and Bottom) are based on standard error.

Fig. 4.

Measuring interference. DsRed (red circle), EYFP (yellow circle), and ECFP transgene (cyan circle) define two adjacent genetic intervals (I5a and I5b; see text) on chromosome 5. The four chromatids present after replication can experience no COs (A), single COs in either interval (B and C), and DCOs in the combined interval including two-strand DCOs (D), both kinds of three-strand DCOs (E and F), and four-strand DCOs (G) in the two intervals. Each of these events can be distinguished by observing the segregation of the transgenes.

Fig. 5.

Detecting GC. A heterozygous plant with one fluorescent EYFP allele (yellow circle) and one mutant nonfluorescent eyfp allele (black circle) can be used to detect meiotic GC events in pollen tetrads.