Crossover and Noncrossover Pathways in Mouse Meiosis Hélène Guillon, Frédéric Baudat, Corinne Grey, R. Michael Liskay, Bernard de Massy  Molecular Cell  Volume 20, Issue 4, Pages 563-573 (November 2005) DOI: 10.1016/j.molcel.2005.09.021 Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 1 Strategy for the Parallel Detection of CRs and NCRs Selected at the BsrFI Site (A) Genomic DNA from mice heterozygous at Pmsb9 (B6xR209) contains both parental genomes plus a low amount of potential recombinant molecules. Polymorphic sites are shown as black and white circles. Examples of a B6-R209 CR and of an NCR on B6 are depicted. A first round of PCR, PCRI, is performed on small pools of genomic DNA with P1 (allele-specific primer) and P2 (nonspecific primer). The PCR products are then subjected to a second round (PCRII) of three independent PCRs with P3/P4, P5/P6, or P3/P7. Primers P4 and P5 are on opposite strands and specific for the same marker, the BsrFI site. The presence or absence of amplified products allows determination of whether a given pool contains a gene conversion event at BsrFI without CR, a CR to the left of BsrFI, or to the right of BsrFI. (B) Detection of CRs and NCRs among pools of testis genomic DNA. Forty-eight pools containing 191 amplifiable genomes extracted from whole testis of 18-day-old B6xR209 mice were analyzed according to the strategy described in (A). PCRI was done with primer 269U/3457L. PCRII were done with primers 269U/2107L16R209 (P3/P4), 2092U16R209/3402L20 (P5/P6), and 402U/3402L20b (P3/P7). The products of PCRII were separated on agarose gel electrophoresis. Molecular Cell 2005 20, 563-573DOI: (10.1016/j.molcel.2005.09.021) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 2 Kinetics of CR and NCR Formation during the First Wave of Spermatogenesis in Wt Mice The frequencies of CRs and NCRs were determined at 11, 14, 15, 17, 18, and 21 days after birth during the first wave of spermatogenesis in B6xR209 hybrids. The frequencies of CRs are the sum of the frequencies of B6-R209 and R209-B6 molecules. The frequencies of B6-R209 and R209-B6 CRs were equivalent as expected. The frequencies of NCRs correspond to events detected at BsrFI on B6 parental genome. Similar NCR frequencies on R209 genome were obtained when tested (spermatocytes at day 15 and oocytes). The timing of progression of the first wave of meiosis through the stages of prophase, leptotene (L), zygotene (Z), pachytene (P), diplotene (D), and metaphase I (MI) is indicated below the abscissa. Diamonds represent CRs, and squares represent NCRs. Frequencies are given ± two SDs. Molecular Cell 2005 20, 563-573DOI: (10.1016/j.molcel.2005.09.021) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 3 Mapping of Exchange Points among Simple CR Products The region where most CRs occur is divided in four intervals according to polymorphic markers (TaqI/BsrFI, BsrFI/StyI, StyI/SphI, and SphI/Eco57I, see positions on Figure 4). The relative CR density is calculated by dividing the proportion of CRs within each interval (relative to all CRs between TaqI and Eco57I) by its length. (A) Distribution of CRs in wt male mice: 101 CRs isolated from testis DNA of 17, 18, and 21 day B6xR209 mice (diamonds) and 69 CRs from sperm DNA of adult B10xR209 mice (Guillon and de Massy, 2002; squares, gray line). (B) Distribution of CRs in wt oocytes. Forty-one CRs isolated from newborn ovaries were mapped (black circles). (C) Distribution of CRs in Mlh1−/− male mice: 109 CRs with a single exchange point in the TaqI/Eco57I interval, isolated from testis DNA of 17, 18, and 21 day mice (black triangles). Molecular Cell 2005 20, 563-573DOI: (10.1016/j.molcel.2005.09.021) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 4 Mapping of Recombinant Molecules (A) Mapping of conversion tracts in NCR products. The positions of all polymorphisms and primers used for PCR in the amplified interval are shown. Primer numbers correspond to coordinates on the DNA sequence. Forty-five NCR products from spermatocytes and 14 from oocytes were analyzed in wt mice (+/+) and 37 and 29 in Mlh1−/− mice. The events were selected at BsrFI. The XbaI, BsphI, BseRI, TaqI, StyI, and SphI polymorphisms were tested by restriction digest. Polymorphisms between BsrFI and TaqI were analyzed by sequencing. The dashed line represents the segment not analyzed. (B) Types of CR products in male mice. Five types of CR molecules have been identified: one simple exchange (“A”), mixed strands best explained by two simple exchanges (“B”), one single distal exchange (left of TaqI) (“C”), mosaic product with more than one exchange point (“D”), and complex molecules containing mixed strands and exchange points to the left of the TaqI polymorphism (“E”). (C) Detailed mapping data on 18 C, D, or E type CRs detected in Mlh1−/− male mice. Molecular Cell 2005 20, 563-573DOI: (10.1016/j.molcel.2005.09.021) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 5 Timing of CR and NCR Formation in Wt and Mlh1−/− Mice during the First Wave of Spermatogenesis. (A) CR frequencies were obtained both by the direct selection of CRs and the parallel CR/NCR assay (wt, diamonds; Mlh1−/−, triangles, dotted line). Values plotted are the sum of the frequencies of B6-R209 and R209-B6 CR products. Frequencies are given ± two SDs. (B) NCR frequencies were determined by the parallel CR/NCR procedure outlined in Figure 1 (wt, squares; Mlh1−/−, circles, dotted line). Values plotted are the frequencies of products detected at BsrFI on B6 parental genome. Frequencies are given ± two SDs. Molecular Cell 2005 20, 563-573DOI: (10.1016/j.molcel.2005.09.021) Copyright © 2005 Elsevier Inc. Terms and Conditions