E. Sánchez-Morán and J.L. Santos acted as joint senior authors.
Together yes, but not coupled: new insights into the roles of RAD51 and DMC1 in plant meiotic recombination
Article first published online: 15 DEC 2011
© 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd
The Plant Journal
Volume 69, Issue 6, pages 921–933, March 2012
How to Cite
Pradillo, M., López, E., Linacero, R., Romero, C., Cuñado, N., Sánchez-Morán, E. and Santos, J. L. (2012), Together yes, but not coupled: new insights into the roles of RAD51 and DMC1 in plant meiotic recombination. The Plant Journal, 69: 921–933. doi: 10.1111/j.1365-313X.2011.04845.x
- Issue published online: 16 MAR 2012
- Article first published online: 15 DEC 2011
- Accepted manuscript online: 8 NOV 2011 09:46AM EST
- Received 20 July 2011; revised 1 November 2011; accepted 2 November 2011; published online 15 December 2011.
Figure S1. FISH images of different zygotene spreads of Atrad51-2 PMCs. The arrows point the two T-DNA signals (pGKB5 vector), corresponding to the homologous chromosomes. Bar represents 5 μm.
Figure S2. Comparison of the AtRAD51 cDNA sequences in Col and Ws accessions. The total length is 1026 bp. The start and stop codons and the three base changes are highlighted in blue, whereas the asterisks indicate matched nucleotides. (1: ACA=ACG=Thr; 2: UUG=UUA=Leu; 3: GUU=GUC=Val).
Figure S3. Cisplatin sensitivity analyses in plants of Col, Atrad51-2 and Atrad51-3. The pictures were captured 13 days after sowing. 1: Col; 2: Atrad51-2; 3: Atrad51-3. (a) Plants grown on 15 mm cisplatin-containing medium. (b) Plants grown on 30 mm cisplatin-containing medium. (c) Plants grown on 50 mm cisplatin-containing medium.
Figure S4. Double immunolocalization of ZYP1 and ASY1 on Atrad51-2 zygotene-pachytene spread nuclei. In the upper cell we observed foci but not initiation of polymerization of ZYP1, whereas in the lower one there are some ZYP1 stretches (arrows). The location of the AE associated ASY1 protein is similar to that observed in wild-type plants. Bar represents 5 μm.
Figure S5. Different stages of female meiosis in the Atrad51-2 mutant. (a) Zygotene. Arrows indicate synapsed regions. (b) Diplotene with bivalents (arrows). (c) Metaphase I with univalents and bivalents (arrow). (d) Telophase I with some fragments (arrows). (e) Prophase II with unbalanced nuclei and sister chromatids arising from the equational segregation of a univalent. Bars represent 5 μm.
Figure S6. Meiotic stages in PMCs of the Atspo11-1-5 mutant. (a) Zygotene-pachytene. Unsynapsed chromosomes. (b) Metaphase I with ten univalents. (c) Telophase I and (d) metaphase II with unbalanced nuclei. (e) Abnormal anaphase II in which chromatids are dispersed throughout the cell. (f) Tetrad with size variation among nuclei. Bars represent 5 μm.
Figure S7. Meiotic stages in PMCs of the Atspo11-1-5/Atrad51-2 double mutant. (a) Absence of synapsis at zygotene-pachytene. (b) Diakinesis and (c) metaphase I with ten univalents. There are not chromosome fragments. (d) Prophase II and (e) metaphase II with three nuclei. (f) Telophase II with slight chromosome fragmentation. (g) Polyad. Bars represent 5 μm.
Table S1 Co-segregation analysis of the mutant phenotype and the T-DNA insertion. As homozygous mutant plants are fully sterile, three Atrad51-2 heterozygous mutant plants were self-fertilized to generate three sets of F1 progeny. No significant differences from the expected segregation (in parentheses), 3 kanamycin resistant:1 kanamycin sensitive (3 KmR:1 KmS), were obtained when the seeds of each offspring were germinated to test antibiotic resistance. These KmR plants did not present any difference with the expected ratio 2 fertile:1 sterile. Hence, the observed data matched with the expected data for the existence of a single T-DNA insertion and for a genetic link between the mutation and the T-DNA insertion. Furthermore, nine sets of F2 seeds from KmR and heterozygous plants (confirmed by PCR) were selected to repeat the cosegregation analysis, and again no differences between the observed and the expected data were found
Table S2 Segregation analysis of self-fertilized Atrad51-2 plants 16 days after exposure to 200 Gy. Data indicate that there were not significant differences between the observed and expected values (in parentheses) for a 1 kanamycin sensitive:2 kanamycin resistant with four leaves (heterozygous): 1 kanamycin resistant with two leaves (homozygous) segregation
Table S3 Mean leaf number comparisons by Student’s t-tests between Ws and the progeny of self-fertilizated Atrad51-2 after 14 days on different cisplatin-containing media. The progenies of Atrad51-2 heterozygous plants were sowed on kanamycin-containing media to select heterozygous and homozygous Atrad51-2 plants (wild-type plants died as consequence of their sensitivity to the antibiotic). The percentage of non germinated seeds is also indicated. Wild-type and Atrad51-2 plants produced different number of leaves on 15 and 30 μm cisplatin-containing media. However, they produced the same numbers of leaves on 50 and 75 μm cisplatin-containing media, probably because the number of DSBs is so high that they cannot be repaired even when normal AtRAD51 protein is present. We detected differences between wild-type and Atrad51-2 plants on 0 μm cisplatin-containing media, perhaps as consequence of the kanamycin presence in the Atrad51-2 Petri plates
Table S4 Segregation analysis of self-fertilizated Atrad51-2 plants after 14 days on 30 μm cisplatin-containing medium. Data indicate that there were not significant differences between the observed and expected values (in parentheses) for a 1 kanamycin sensitive:2 kanamycin resistant with four leaves (heterozygotes): 1 kanamycin resistant with two leaves (homozygotes) segregation
Table S5 Mean leaf number comparisons by Student’s t-tests between Col, Atrad51-2 and Atrad51-3 plants after 13 days on different cisplatin-containing media. Data corresponding to Atrad51-2 and Atrad51-3 refers to the progeny of self-fertilized heterozygous plants
Table S6 Duplicated region percentages shared by the different Arabidopsis chromosomes
Table S7 Comparison between the numbers of observed and expected non-homologous associations considering the amount of segmental duplications shared by the different chromosomes. 97 metaphase I bivalents were analyzed. Chromosomes 1 and 3 were grouped because in the Ws accession we were unable to differentiate them in accuracy. Expected values are indicated in parentheses
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