Complementation with a CaMV 35S::MYC::AtRAD51 Construct. The AtRAD51 cDNA was cloned by PCR amplification of a wild-type young inflorescence cDNA sample with the gene-specific primers oMC858 and oMC859, verified by sequencing, and inserted into a binary MYC vector (pROK2-MYC vector). An Agrobacterium C3581 strain containing the confirmed construct was used to transform AtRAD51/atrad51-1 plants. Transgenic plants were selected with kanamycin resistance, and plants that carried the atrad51-1 allele but not the wild-type AtRAD51 allele were identified by using PCR. For the wild-type allele, we used the AtRAD51 gene-specific primers oMC1446 (5'-TGCCGTATGCTCAACAGGAGGT-3') and oMC1447 (5'-GAACGCTATTGTGATCTCATGTGTGTTACA-3'), which were located in introns flanking the T-DNA (portion of the tumor-inducing plasmid that is transferred to plant cells) insertion. For the atrad51-1 allele, we used the AtRAD51 primer oMC858 and a T-DNA-specific primer oMC1286 (5'-ATATTGACCATCATACTCATTGC-3'). The primer pair oMC859 and oMC1479 (5'-CGCCATTTCCCTCCACTCTC-3') was used to test for AtRAD51 expression, and the primer pair oMC859 and oMC1478 (5'-ATGGAGCAAAAGCTCATTTCTGAAG-3', a primer specific for the tagged 6´ MYC sequence upstream of the AtRAD51 cDNA in the 35S::MYC::AtRAD51 construct) was used to examine the expression of the MYC::AtRAD51 transgene. Expression of the constitutive APT1 gene was examined as a control (1).

Generation of the atrad51-1 atspo11-1 Double Mutant. A cross was performed between AtSPO11-1/atspo11-1 and AtRAD51/atrad51-1 plants, and resulting F₁ double-heterozygous plants (AtRAD51/atrad51-1; AtSPO11-1/atspo11-1) were identified by using PCR with gene-specific primers. Progeny of the double-heterozygous plants were analyzed to identify double mutants by PCR with the following gene-specific primers: (i) for AtRAD51 and atrad51-1 alleles: oMC1446, oMC1447, and oMC1286; (ii) for AtSPO11-1 wild-type and mutant alleles: oMC703 (5'-ACGTATCGGGCCTAAATTCC-3'), oMC704 (5'-TTTGGAGATCTTCCTTCAGCC-3'), and oMC705 (5'-ACTGGGATTCGTCTTGGACA-3'). Amplification with the primers oMC1446 and oMC1447 can generate a 379-bp band if one or two wild-type copies of AtRAD51 are present, and oMC1446 and oMC1286 can be used to detect the presence of a T-DNA insertion. oMC703 and oMC704 can produce a 404-bp band if a wild-type AtSPO11-1 copy is present, and oMC703 and oMC705 can be used to detect the T-DNA insertion.

Transmission Electron Microscopy. For transmission electron microscopy, the fixation and infiltration procedures were according to ref. 2 with minor modifications. Entire inflorescences were submerged in a fixative (2.8% glutaraldehyde in 0.1 M Hepes buffer, pH 7.2/0.02% [lsqb]vol/vol[rsqb] Triton X-100) for 2 h at room temperature and then transferred into fresh fixative for 1 day at 4° C. Samples were rinsed three times in the same buffer for 15 min each wash, postfixed in 1% OsO₄ in Hepes buffer for 1 day at 4° C, rinsed again, dehydrated through a graded acetone series with a 10% increment for 15 min in each, and infiltrated in Spurr’s resin. Before embedding, all buds were dissected from individual inflorescences and numbered according to their sizes. To determine appropriate meiotic stages, thick cross sections through the buds were cut, collected on glass slides, stained with 0.1% toluidine blue in 1% sodium tetraborate, and observed with a light microscope. Ultrathin sections were prepared with a diamond knife on a Reichert Jung Ultracut microtome, placed on copper-mesh grids, stained with 2% uranyl acetate in 50% ethanol for 16 min and with Reynold’s lead citrate for 12 min, and examined with a JEOL 1200 EXII transmission electron microscope at 80 kV. At least 50 ultrathin sections were analyzed for each bud. Images were captured by using a charge-coupled device camera.

1. Moffatt, B. A., McWhinnie, E. A., Agarwal, S. K. & Schaff, D. A. (1994) Gene 143, 211–216.

2. Marsh, T. C., Cole, E. S., Stuart, K. R., Campbell, C. & Romero, D. P. (2000) Genetics 154, 1587–1596.