DOI: 10.5061/dryad.gb5mkkwx5
title: Data from: Male mouse recombination maps for each autosome identified by chromosome painting

description: Linkage maps constructed from genetic analysis of gene order and crossover
 frequency provide few clues to the basis of the genomewide distribution of
 meiotic recombination, such as chromosome structure, that influences
 meiotic recombination. To bridge this gap, we have generated the first
 cytological recombination map that identifies individual autosomes in the
 male mouse. We prepared meiotic chromosome (synaptonemal complex [SC])
 spreads from 110 mouse spermatocytes, identified each autosome by
 multicolor fluorescence in situ hybridization of chromosome- specific DNA
 libraries, and mapped 12,000 sites of recombination along individual
 autosomes, using immunolocalization of MLH1, a mismatch repair protein
 that marks crossover sites. We show that SC length is strongly correlated
 with crossover frequency and distribution. Although the length of most SCs
 corresponds to that predicted from their mitotic chromosome length rank,
 several SCs are longer or shorter than expected, with corresponding
 increases and decreases in MLH1 frequency. Although all bivalents share
 certain general recombination features, such as few crossovers near the
 centromeres and a high rate of distal recombination, individual bivalents
 have unique patterns of crossover distribution along their length. In
 addition to SC length, other, as-yet-unidentified, factors influence
 crossover distribution leading to hot regions on individual chromosomes,
 with recombination frequencies as much as six times higher than average,
 as well as cold spots with no recombination. By reprobing the SC spreads
 with genetically mapped BACs, we demonstrate a robust strategy for
 integrating genetic linkage and physical contig maps with mitotic and
 meiotic chromosome structure.
description: <em>SC Spreads and Immunostaining</em>
 Three juvenile (20–21 d old) C57BL/6J mice (the same line
 analyzed by the Mouse Genome Sequencing Project) were used to prepare and
 immunolabel the SC spreads, as described elsewhere (Anderson et al. 1999).
 Complete sets of SCs in which the SCs were well separated but not
 obviously stretched or broken and that had ≥ 19 MLH1 foci were selected
 for analysis. Three fluorescent images (4, 6-diamino-2-phyenylindole
 [DAPI], SCP3, and MLH1) were captured for each SC set.
 <em>mFISH</em> After image
 acquisition of the immunofluorescence signals, the spermatocyte
 preparations were subjected to two or three rounds of denaturation and
 FISH. To identify each autosome, chromosome-specific painting probes
 (Rabbitts et al. 1995) were combinatorially labeled with fluorescein
 isothiocyanate (FITC)–2-deoxyuridine 5-tri phosphate (dUTP), Cy5-dUTP
 (both from Amersham), or 6-carboxytetramethylrhodamine (TAMRA)-dUTP
 (Applied Biosystems) and were combined to form two different probe pools
 (table 1). The probes were fluorescently labeled by the incorporation of
 FITC-dUTP, Cy5-dUTP (both from Amersham), or TAMRA-dUTP (Applied
 Biosystems) during the PCR reaction. For labeling, the 2- deoxythymidine
 5-triphosphate (dTTP) concentration in the PCR reaction mixture was
 reduced to 120 mM (2- deoxyadenosine 5-triphosphate [dATP],
 2-deoxycytidine 5_-triphosphate [dCTP], and 2-deoxyguanosine
 5-triphosphate [dGTP] 160 mMeach), and either 30 mMFITCdUTP, 30 mM
 Cy5-dUTP (both from Amersham), or 10 mMTAMRA-dUTP was added. The painting
 probes were precipitated together with 20 mg of mouse Cot-1 DNA
 (Invitrogen/Life Technologies) and were dissolved in 15 ml of
 hybridization solution (50% formamide; 2#sodium saline citrate (SSC), pH
 7.0; and 10% dextran sulfate). Five genetically mapped BAC clones
 (Osoegawa et al. 2000; Genetic and Physical Maps of the Mouse Chromosome
 Web site; Roswell Park Mouse Screening Project Web site) were hybridized
 to SC spreads in a third round of FISH, after chromosomes were identified
 using the two chromosome-painting hybridizations described above. The BAC
 DNA was labeled by nick translation with Cy5-dUTP (Invitrogen/Life
 Technologies). For each BAC, 400 ng of labeled DNA and 4 mg of mouse Cot-
 1 DNA were precipitated together and dissolved in 15 ml of hybridization
 solution. The BACs were mapped to mitotic metaphase chromosomes, using the
 same hybridization solutions and standard procedures. To allow FAL
 analysis and sequential hybridizations on the same nuclei, the FISH
 protocol (Muller et al. 2002) was modified as follows: After imaging the
 antibody- labeled cells, the antifade solution was removed by washing the
 slides twice in 4# SCC/0.1% Tween-20 at 37C for 15 min, and the slides
 were dehydrated in an alcohol series. The first round of denaturation was
 7 min, and in situ hybridization was performed for 48 h at 37C. After
 separate image acquisition of the three different colors of FISH signals,
 the next round of FISH (including antifade removal, dehydration,
 denaturation, and hybridization) was performed, as described above, except
 that the denaturation time was reduced to 2 min.
 <em>Fluorescence Microscopy, Image
 Acquisition,</em> <em>and
 Analysis</em> The location of each imaged,
 immunolabeled SC spread was recorded using a software-controlled automatic
 stage (Maerzhaeuser) so that it could be accurately relocated on the slide
 for mFISH. Digital images were obtained using a cooled CCD camera, Quantix
 series (Photometrics), coupled to a Zeiss Axioplan II microscope. Each
 color signal was acquired as a black-and white image, using appropriate
 filter sets (Chroma Technologies), and was merged with SmartCaptureVP
 software (DigitalScientific). <em>Analysis of
 MLH1 Distribution on SCs</em> After the identity
 of each bivalent was determined by use of mFISH, the images of the
 corresponding SC spreads were analyzed for MLH1 distribution. For each SC,
 the position of each MLH1 focus was recorded as a relative distance
 (percentage of total length) from the centromere, using MicroMeasure
 (Reeves 2001). The centromeric end of each SC was identified by the
 surrounding DAPI-bright AT-rich heterochromatin. MLH1 foci were mapped if
 the MLH1 and SCP3 signals overlapped. A total of 110 sets were analyzed,
 and 70 SCs (3% of the total of 2,090 autosomal SCs) were eliminated from
 the analysis because of SC overlap near an MLH1 focus, possible SC
 stretching, or inconclusive FISH identification. To obtain an average
 absolute length for each identified SC, the average relative length of
 each SC was determined, then multiplied by the average absolute length of
 a complete set of mouse SCs (164.5 µm = 163.7 ÷ 0.995, to correct for
 rounding errors) (table 2) (Anderson et al. 1999). The relative position
 of each focus was multiplied by the average absolute length for the
 appropriate SC to obtain the absolute (micrometer) position of each focus.
 The data for each of the autosomal SCs were pooled and graphed in
 histogram form to demonstrate the pattern of MLH1 distribution for each
 SC. The 0.2-µm intervals used for graphing correspond to ∼3.44 Mb of DNA
 (calculated from the size, in megabases, of the autosomal fraction of the
 mouse genome; National Center for Biotechnology Information). The
 interference distance between two foci on the same SC was calculated as a
 percentage of the euchromatic length of the SC, as described by Anderson
 et al. (1999). To convert MLH1 foci to distances in centimorgans, the
 number of MLH1 foci in each 0.2-µm SC interval was divided by the total
 number of SCs observed, then multiplied by 50 map units per MLH1 focus
 (one crossover p 50 cM). The average number of MLH1 foci per 0.2-µm
 interval was determined for each autosomal SC in our sample. For the
 larger SCs, the average was about three; for the shorter SCs this number
 was slightly higher. To identify potential “hot” and “cold” regions of
 recombination, we added or subtracted one focus to identify regions of
 high (&gt;14 for longer SCs, as high as 6 for the shorter SCs) and low
 (&lt;2 for longer SCs and &lt;4 for shorter SCs) recombination
 frequency, respectively.
description: # Male mouse recombination maps for each autosome identified by chromosome
 painting ## Description of
 the data and file structure The data are presented in an Excel spreadsheet
 with 22 sheets.  Sheet 1 (karyotype-absolute positi calc) defines the
 average length of each mouse SC, after identification using
 chromosome-specific DNA probes.  Sheet 2 (Notes) contains definitions of
 the headings used for Sheet 3 (raw data sorted by SC) and Sheets 4 through
 22 ("SC1 abs" through "SC19 abs"). Sheet 2 also
 contains explanations for how the karyotype was derived, and two
 references in which this data was used for publication are also
 presented.  Sheet 3 contains the positions of all MLH1 foci observed on
 all of the SCs with each MLH1 focus position expressed as a fraction of SC
 length from the centromere.  Sheets 4 – 22 (labeled as "SC1
 abs", "SC2 abs", "SC3 abs", "SC4 abs",
 SC5 abs, "SC6 abs", "SC7 abs", "SC8 abs",
 "SC9 abs", "SC10 abs", "SC11 abs",
 "SC12 abs", "SC13 abs", "SC14 abs",
 "SC15 abs", "SC16 abs", SC17 abs, "SC18
 abs",  and "SC19 abs") contain the positions of all MLH1
 foci observed on each identified SC with each MLH1 focus position
 expressed both as a fraction of SC length from the centromere and as an
 absolute (µm) position from the centromere. Most mouse SCs have one or two
 MLH1 foci, although a few SCs were observed with no foci.  In cases where
 an SC has only one or no MLH1 foci, an asterisk (*) has been inserted in
 the appropriate cells of columns headed F1, F2, absF1 or absF2.