Recombination Map of the Common Shrew, Sorex araneus ...(Eulipotyphla, Mammalia) Pavel M. Borodin,*, ... 2 system of sex determination. It is an important mammalian model of chromosomal

Copyright � 2008 by the Genetics Society of AmericaDOI: 10.1534/genetics.107.079665

Recombination Map of the Common Shrew, Sorex araneus(Eulipotyphla, Mammalia)

Pavel M. Borodin,*,† Tatyana V. Karamysheva,* Nadezhda M. Belonogova,*,†

Anna A. Torgasheva,*,† Nikolai B. Rubtsov*,† and Jeremy B. Searle‡,1

*Institute of Cytology and Genetics, Russian Academy of Sciences, Siberian Department, Novosibirsk 630090, Russia,†Department of Cytology and Genetics, Novosibirsk State University, Novosibirsk 630090, Russia and

‡Department of Biology, University of York, York YO10 5YW, United Kingdom

Manuscript received July 29, 2007Accepted for publication October 29, 2007

ABSTRACT

The Eurasian common shrew (Sorex araneus L.) is characterized by spectacular chromosomal variation,both autosomal variation of the Robertsonian type and an XX/XY1Y2 system of sex determination. It is animportant mammalian model of chromosomal and genome evolution as it is one of the few species with acomplete genome sequence. Here we generate a high-precision cytological recombination map for thespecies, the third such map produced in mammals, following those for humans and house mice. Weprepared synaptonemal complex (SC) spreads of meiotic chromosomes from 638 spermatocytes of 22males of nine different Robertsonian karyotypes, identifying each autosome arm by differential DAPIstaining. Altogether we mapped 13,983 recombination sites along 7095 individual autosomes, usingimmunolocalization of MLH1, a mismatch repair protein marking recombination sites. We estimated thetotal recombination length of the shrew genome as 1145 cM. The majority of bivalents showed a highrecombination frequency near the telomeres and a low frequency near the centromeres. The distancesbetween MLH1 foci were consistent with crossover interference both within chromosome arms and acrossthe centromere in metacentric bivalents. The pattern of recombination along a chromosome arm was afunction of its length, interference, and centromere and telomere effects. The specific DNA sequencemust also be important because chromosome arms of the same length differed substantially in theirrecombination pattern. These features of recombination show great similarity with humans and mice andsuggest generality among mammals. However, contrary to a widespread perception, the metacentricbivalent tu usually lacked an MLH1 focus on one of its chromosome arms, arguing against a minimumrequirement of one chiasma per chromosome arm for correct segregation. With regard to autosomalchromosomal variation, the chromosomes showing Robertsonian polymorphism display MLH1 foci thatbecome increasingly distal when comparing acrocentric homozygotes, heterozygotes, and metacentrichomozygotes. Within the sex trivalent XY1Y2, the autosomal part of the complex behaves similarly to otherautosomes.

MEIOTIC recombination involves breakage and re-joining of DNA between homologous chro-

mosomes. It plays a central role in the evolution ofeukaryotes generating individual genetic variation,decreasing mutational load, and ensuring the geneticunity of species (Otto and Lenormand 2002). Recom-bination is crucially important for the orderly segregationof meiotic chromosomes and production of balancedgametes (Roeder 1997).

Meiotic recombination has been studied extensivelyboth genetically and cytologically. Genetic linkage stud-ies provide precise estimates of recombination betweeneven closely linked genes, but they require large datasets involving well-controlled crosses or well-characterizedpedigree records. The frequency of chiasmata along

bivalents at diakinesis–metaphase I provides an estimateof the global rate of recombination. However, for recom-bination mapping, basic cytological studies are limitedby difficulties in identification of individual bivalents, inmeasuring the position of chiasmata accurately and incombining data from cells that show different degreesof condensation of the chromosomes.

Recently, new methods of cytological recombinationmapping have been developed, on the basis of thelocalization of recombination sites along the synapto-nemal complex (SC) using fluorescently labeled anti-bodies to MLH1, a mismatch repair protein of maturerecombination nodules (Sherman and Stack 1995;Baker et al. 1996; Barlow and Hulten 1998; Anderson

et al. 1999; Froenicke et al. 2002; Koehler et al. 2002;Lynn et al. 2002). So far, these methods have been used toanalyze the frequency and distribution of recombinationevents for only two species of mammal, humans andhouse mice.

1Corresponding author: Department of Biology, University of York, P.O.Box 373, York YO10 5YW, United Kingdom. E-mail: [email protected]

Genetics 178: 621–632 (February 2008)

Here we present detailed MLH1 recombination mapsfor all chromosomes of a third mammal, a small insec-tivore, the Eurasian common shrew (Sorex araneus L.:Soricidae, Eulipotyphla) and compare our results withthose obtained for mice and humans. The commonshrew is a good model for such studies for several reasons:

1. The efficiency of MLH1 mapping depends cru-cially on reliable chromosome identification for eachbivalent (indeed, for metacentrics, identification ofthe individual chromosome arms). A characteristicfeature of the common shrew is the ease in whichDAPI patterns along chromosomes can be revealedin SC spreads (Belonogova et al. 2006).

2. The common shrew and related species show im-pressive diversification involving chromosomal rear-rangements. Robertsonian fusions and whole-armreciprocal translocations (WARTs) appear to havebeen involved in speciation in the S. araneus group(Searle and Wojcik 1998; Zima et al. 1998; Basset

et al. 2006) and the influence of the rearrangementson recombination may have been crucial to this,following the model of Rieseberg (2001). Thecommon shrew itself shows some of the most re-markable chromosomal variation in mammals. Todate, 68 chromosome races have been described(Wojcik et al. 2003) and the actual number of distinctraces probably goes far beyond 100. While thenumber of autosomal arms is constant within thecommon shrew (FNa¼ 40), the diploid chromosomenumber (2n) varies from 20 to 33. The source of thischromosomal variation is Robertsonian fusions, al-most certainly with further modification by WARTs(Searle and Wojcik 1998). This high degree ofchromosomal variation within the common shrewprovides plenty of opportunity to study how Robert-sonian fusions and WARTs affect recombination.

3. The common shrew has an XX/XY1Y2 sex chromo-some system. The ‘‘X’’ in S. araneus represents atandem fusion between the true mammalian X andan autosome (Sharman 1956; Fredga 1970; Pack

et al. 1993). Since the XY pair in mammals differs inits meiotic behavior from the autosomal bivalents(Ashley 2002), it is of value to determine the meioticbehavior of the autosomal arm of the sex trivalent inmale common shrews.

4. The basic karyotype of the common shrew is rathersimilar to the human karyotype. Comparative chro-mosome mapping indicates that the introduction ofonly 18 breaks in the human karyotype generatessegments that can be fused to give the karyotype ofthe common shrew (Ye et al. 2006).

5. The common shrew is one of only a small number ofspecies for which a complete genome sequence isavailable (http://www.broad.mit.edu/mammals/). Agenetic map is clearly of value for future comparisonwith the physical map.

MATERIALS AND METHODS

Animals: Twenty-two adult male common shrews were usedin this study (Table 1). They were trapped at the beginning ofthe breeding season in 2006 in the hybrid zone between theOxford and Wirral chromosome races (Zima et al. 1996),which is a continuation of the Oxford–Hermitage hybrid zone(Searle 1986a). The animals were karyotyped by the analysisof bone marrow chromosome spreads prepared accordingto Searle (1986a) and G-band stained (Seabright 1971).Chromosome nomenclature followed Searle et al. (1991) andSearle (1993), with chromosome arms represented by itali-cized letters of the alphabet and bi-armed chromosomes by asequence of two letters (the first is the long arm, the secondthe short arm). Simple Robertsonian heterozygotes for ametacentric and twin acrocentric chromosomes, for exampleko and k, o, are described as k/o. All animals used in this studywere homozygous for the chromosomes af, bc, gm, hi, pr, and tu,and had the sex trivalent de(X)/s(Y1), dv(Y2). The individualkaryotypes with respect to the chromosomes j, l, k, n, o, and qare listed in Table 1. Metacentric chromosomes kq and no werecharacteristic of the Oxford race and ko of the Wirral race; jlwas found in both races. Homozygotes for the acrocentricchromosomes k, n, o, and q were often observed in the centerof the hybrid zone and can be considered to represent a zone-specific ‘‘acrocentric race’’ (Searle 1986a).

Regarding the relationship of the races studied here, thecommon shrew is subdivided into several ‘‘karyotypic groups’’of chromosomally related races (Searle and Wojcik 1998)and the Oxford, Wirral, and acrocentric races all belong tothe ‘‘West European karyotypic group’’ (Searle 1984, 1986a;Searle and Wilkinson 1987; Searle and Wojcik 1998). It isreasonable to assume that these races are also closely relatedgenically, although there are no direct data on this. The racesare believed to have a common ancestry in a glacial refugiumin southeastern Europe at the last glacial maximum, 20,000years ago (Searle 1984; Bilton et al. 1998).

Here we study individuals homozygous for only metacentricchromosomes, individuals homozygous for a variety of meta-centric and acrocentric chromosomes, and individuals that aresimple heterozygotes for either one or two arm combinations.This range of karyotypes is commonly found elsewhere in thecommon shrew, often in hybrid zones between chromosomalraces or other areas of Robertsonian polymorphism (Searle

and Wojcik 1998). The particular situation that we havestudied in Britain where two races characterized by differentmetacentrics are separated by a third acrocentric race has alsobeen described in Sweden and Poland (Searle and Wojcik

TABLE 1

Karyotypes of common shrews used in this study

Karyotypea 2n RaceNo. of

specimensNo. of cells

analyzed

jl, kq, no 21 Oxford 2 23jl, kq, n, o 23 Oxford 1 35jl, k/q, n/o 23 Oxford 3 106jl, k/q, n, o 24 Oxford 2 54jl, k, n/o, q 24 Oxford 3 91j/l, k, n/o, q 25 Oxford 1 27jl, ko, n, q 23 Wirral 2 43jl, k/o, n, q 24 Wirral 2 53jl, k, n, o, q 25 Acrocentric 6 206

Total 22 638

a Only the variable chromosomes are given; see text.

622 P. M. Borodin et al.

1998). Therefore, we are studying a range of karyotypes that istypical for the common shrew and of wide comparative value;simple Robertsonian heterozygotes and their associated ho-mozygotes have been found in many other mammals (Searle

1993).Immunostaining, identification, and measurement of

meiotic chromosomes: Spermatocyte spreads were preparedfrom both left and right testes using the technique of Peters

et al. (1997). The immunostaining protocol was similar to thatof Anderson et al. (1999). The slides were incubated for 2 hr at37� with a rabbit polyclonal antibody against rat lateral ele-ment protein SCP3 (a gift from C. Heyting) diluted to a con-centration of 1:1000, a mouse monoclonal antibody to humanmismatch repair protein MLH1 (Pharmingen, San Diego) at1:50 dilution, and a human ANA-C antibody against centro-meric proteins (Sigma-Aldrich, St. Louis) at a 1:100 dilution in3% bovine serum albumin (BSA) in phosphate buffered saline(PBS). Slides were washed in 13 PBS and incubated for 40min at 37� with donkey anti-rabbit Cy3-conjugated antibodies( Jackson, West Grove, PA) at 1:200 dilution, goat anti-mouseFITC-conjugated antibodies ( Jackson) at 1:400 dilution andgoat anti-human FITC-conjugated antibodies (Vector Labora-tories, Burlingame, CA) at 1:100 dilution. Slides were washedwith PBS, rinsed briefly with distilled water, dried, andmounted in Vectashield with DAPI (Vector Laboratories) tostain DNA and reduce fluorescence fading.

The preparations were visualized with an Axioplan 2 imag-ing microscope (Carl Zeiss) equipped with a CCD camera (CVM300, JAI), CHROMA filter sets, and ISIS4 image-processingpackage (MetaSystems GmbH). Brightness and contrast of allimages were enhanced using PaintShopPro 7.0.

Only cells containing complete sets of chromosomes wereanalyzed. The number of cells studied for each karyotype islisted in Table 1. Each chromosome arm was identified by itsspecific DAPI pattern according to Belonogova et al. (2006).The centromere position for each SC was identified by anANA-C focus. Although we used the same fluorochrome fordetection of the ANA-C and MLH1 antibodies, ANA-C focidiffered from MLH1 foci by their brighter and more diffusestaining (Figure 1). MLH1 signals were only scored if they werelocalized on an SC. The length of the SC of each chromosomearm was measured in micrometers using MicroMeasure 3.3(Reeves 2001) and the positions of MLH1 foci in relation tothe centromere were recorded.

To generate recombination maps, we calculated the abso-lute position of each MLH1 focus multiplying the relativeposition of each focus by the average absolute length for theappropriate chromosome arm. These data were pooled foreach arm and graphed to represent a recombination map(Figures 2 and 3).

The absolute distances between neighboring foci weremeasured from the images. The relative distances betweenthe foci across the centromere were calculated as fractions ofchromosome length; relative distances within each arm werecalculated as fractions of the arm length. The data on MLH1foci for each arm were fitted to gamma distributions by amaximum-likelihood method using STATISTICA 6 (StatSoft,2001) and the shape parameter (y) was used as a measure ofthe strength of interference (De Boer et al. 2006). TheSTATISTICA package was also used for ANOVA, correlations,and other statistical analysis.

RESULTS AND DISCUSSION

Characteristics of the SCs: We found a close corre-spondence between the average relative length of SCscompared with mitotic chromosomes (Spearman rank

correlation, rs ¼ 0.95, P , 0.001). However, several SCswere noticeably shorter (bc, d) or longer (hi, jl, k, n, tu)than would be expected on the basis of their relativemitotic lengths (Table 2). We also found substantial dif-ferences between SCs and mitotic chromosomes in thearm ratio of metacentrics. For example, arms p and twere the long arms in mitotic chromosomes pr and tuand the short arms in the SC. This confirms that it isdangerous to extrapolate from mitotic chromosomelength for the identification of SCs and emphasizes theimportance of using DAPI banding for this purpose.

The mean (6 SD) total length of the autosomal SCs(including the autosomal arm d of the sex trivalent) was142.8 6 18.8 mm. An ANOVA revealed significant effectsof individual (F1,19 ¼ 14.1, P , 0.001) and race (F1,2 ¼9.0, P , 0.001) on the variation for this trait. Studies onhumans have also revealed significant individual varia-tion (Lynn et al. 2002; Sun et al. 2004, 2005, 2006a,b).The causes of this variation are unclear but Lynn et al.(2002) suggested that allelic variation in loci encod-ing the proteins involved in chromosome pairing and

Figure 1.—SC spread from a shrew spermatocyte at pachy-tene, stained with DAPI (blue) and immunolabeled with anti-bodies to SCP3 (red), MLH1 (green), and centromereproteins (green). Bar, 5 mm. Chromosome arms (indicatedby letters next to their telomeres) were identified by DAPIbanding. Centromeres (indicated by arrows) differ fromMLH1 foci by their brighter and more diffuse staining. Notethat the centromeres on the af bivalent and on the d arm ofthe sex trivalent are misaligned and therefore generate weak-er signals than aligned centromeres.

Recombination Map of the Common Shrew 623

recombination (such as SPO11, MRE11, RAD51, andDMC1) might mediate differences in SC length.

The number of MLH1 foci: The mean (6 SD) num-ber of MLH1 foci over all autosomes (including theautosomal arm d of the sex trivalent) was 21.9 6 2.0, witha range of 15–30 foci per cell. This is in accordance withthe chiasma count per late diplotene/early diakinesiscell, estimated earlier for common shrews from theOxford–Hermitage hybrid zone (21.8 6 1.7, with arange between 18 and 28; Searle 1986b). We found nosignificant differences in number of MLH1 foci per cellbetween the Oxford (22.2 6 2.0), Wirral (21.6 6 1.9),and acrocentric (21.7 6 1.9) shrews (F2,635 ¼ 2.4, P ¼0.09).

To estimate the recombination length of each chromo-some in centimorgans, the average number of MLH1foci for the chromosome was multiplied by 50 MU (onerecombination event ¼ 50 cM). The genetic length ofeach arm, calculated in this way, is shown in Table 3. Weestimated the total autosomal map length for the malecommon shrew as 1095 cM. To estimate the total map

length for the male genome we added 50 cM for theobligate crossover in the XY1 pairing region, giving 1145cM. As the shrew genome is 2850 Mb (Vinogradov

1998), 1 cM of the shrew genetic map is equal to �2.5Mb of sequence. This is rather close to the estimatesobtained by MLH1 mapping for male mice (2.75;Anderson et al. 1999) but twice that for human males(1.20; Sun et al. 2006a).

Altogether, 7095 autosomal SCs were analyzed (in-cluding SCs for the autosomal arm d of the sex tri-valent). Of these, 51 (0.7%) lacked an MLH1 focus;these were found in 48 of the 638 cells scored (7.5%).These frequencies are within the limits of variationfound in studies on mice and humans. Koehler et al.(2002) showed that 0.1% of autosomal SCs in males offour inbred mouse strains had 0 foci, while in the studyof Anderson et al. (1999) involving C57BL males, thefrequency of such bivalents was 4%. Sun et al. (2006b)found substantial variation in the frequency of autoso-mal SCs lacking MLH1 among 10 normal men, althoughthe overall mean frequency of achiasmate autosomal

Figure 2.—Distribution of MLH1 focialong the arms of the Robertsonian-invariantchromosomes, i.e., those showing no Robert-sonian polymorphism. One individual ana-lyzed was a j/l heterozygote but it providedinsufficient data for analysis and here we showthe data from jl metacentric homozygotes.The x-axis shows the position of MLH1 foci;the marks on this axis are separated by1 mm. Letters indicate the telomeric ends ofthe arms; arrows show where the centromeresare located. The y-axis indicates the frequencyof MLH1 foci in each 0.5-mm interval (notethe different scaling for each chromosome).The blue line shows the frequency for thearms containing a single MLH1 focus, thered line shows two MLH1 foci, and the greenline shows three MLH1 foci. The black lineshows the overall frequencies of MLH1 foci.Due to a very low frequency of arms contain-ing more than one MLH1 focus per arm forthe bivalents pr and tu, only the overall fre-quencies are shown for these bivalents.


bivalents was rather low (0.3%). The frequency of cellscontaining one or more such bivalents varied amongindividuals between 1 and 11%. The studies on humansand mice demonstrate that small bivalents suffer a muchhigher risk of recombination failure than large bivalents(Anderson et al. 1999; Koehler et al. 2002; Sun et al.2006b). In our study, achiasmate bivalents were pre-dominantly small metacentrics and acrocentrics; 24were chromosome tu, 11 q, 6 o, 4 k, 3 n, 2 gm, and 1 pr.The cells containing achiasmate bivalents did notappear to be exceptional (e.g., at late pachytene or

poorly stained) and did not differ significantly fromthe whole sample in their total SC length (147.5 6

20.6 mm). Searle (1986b) analyzed the frequency ofautosomal univalents at diakinesis–metaphase I in com-mon shrews from the Oxford–Hermitage hybrid zone.He found a similar frequency of achiasmate bivalents asdetected in this study (0.4–1.0%). Thus, althoughachiasmate bivalents might be expected to trigger apachytene checkpoint leading to apoptosis (Roeder

and Bailis 2000), there is no evidence for this in thecommon shrew.

Rather often we observed metacentric bivalents withno MLH1 foci at one arm and one or more foci in theother arm (overall frequency among metacentric biva-lents, 15.4%). This was fairly common in pr and a typicalfeature of chromosome tu (Table 3), supporting pre-vious chiasma data (Searle 1986b). Only 10.8% of tubivalents had MLH1 foci on both arms. Chromosometu is the smallest and the most ancient among theautosomal metacentrics of the common shrew (Searle

and Wojcik 1998). Interestingly, it evolved by a centricshift or pericentric inversion from an acrocentricchromosome while all other autosomal metacentricsare derived from Robertsonian fusions (Wojcik andSearle 1988). Presumably the acrocentric ancestral totu was characterized by a single chiasma. The conversionof that acrocentric into a metacentric did not lead to arequirement for separate chiasmata in each arm. Theseresults are of interest because it is a common perceptionthat there is a minimum requirement of one chiasmaper chromosome arm for correct segregation (Dumas

and Britton-Davidian 2002 and references therein).The number of MLH1 foci that we observed per arm

of chromosomes involved in Robertsonian polymor-phism did not depend on the karyotype (homozygousmetacentric, homozygous acrocentric, and heterozy-gous) (Table 3). The effect of karyotype was nonsig-nificant for all arms tested: k (F2,635 ¼ 1.2, P ¼ 0.31),n (F2,635 ¼ 0.9, P ¼ 0.42), o (F2,635 ¼ 1.8, P ¼ 0.16), andq (F2,635 ¼ 0.7, P ¼ 0.49). These results contrast to thechiasma data obtained by Bidau et al. (2001) and Dumas

and Britton-Davidian (2002) for the house mouse,which showed a significant decrease in recombinationfrequency in the arms of metacentric chromosomescompared to twin acrocentrics. In the mouse the de-crease in recombination frequency in the arms ofmetacentric chromosomes compared to twin acrocen-trics was determined mainly by a reduction in doublechiasmata (Bidau et al. 2001; Dumas and Britton-Davidian 2002). In the common shrew all the arms ofthe variable chromosomes were rather small and veryrarely accommodated more than one MLH1 focus evenin the acrocentric state.

We also did not find significant differences in thenumber of MLH1 foci for any particular chromosomearm in a metacentric, comparing between different armcombinations. Thus, arm k had a similar number of foci

Figure 3.—Distribution of MLH1 foci along the arms ofthe Robertsonian-variable chromosomes, i.e., those showingRobertsonian polymorphism. The x-axis shows the positionof MLH1 foci; the marks on this axis are separated by1 mm. Letters indicate the telomeric ends of the arms; arrowsshow where the centromeres are located. The y-axis indicatesthe frequency of MLH1 foci in each 0.5-mm interval. The linesrepresent the overall frequency of MLH1 foci in each interval.The blue line shows the distribution in acrocentric homozy-gotes, the green line shows that in metacentric homozygotes,and the red line shows that in heterozygotes.


in kq and ko bivalents (t1,99 ¼ 1.4, P ¼ 0.16), and armo had a similar number of foci in ko and no bivalents(t1,64 ¼ 0.7, P ¼ 0.48).

Correlation between SC length and number ofMLH1 foci: As for previous studies in mice and humans(Anderson et al. 1999; Sun et al. 2004, 2006a), we alsofound in common shrews a very strong positive corre-lation between the average length of SCs of individualchromosome arms and the number of MLH1 foci onthose arms (Spearman rank correlation, rs ¼ 0.95, P ,

0.001). On the other hand, the correlation between thetotal length of SCs and number of MLH1 foci per cellwas much weaker (rs ¼ 0.17, P , 0.001) as found formale mice (Froenicke et al. 2002), but in contrast to thestrong correlation found in human males (Lynn et al.2002).

Crossover interference: The occurrence of a cross-over usually decreases the probability that another willoccur close by. This phenomenon is called positiveinterference (see Jones and Franklin 2006 for areview). The strength of interference can be estimatedfrom the distances between adjacent recombinationsites. Table 4 shows the average distances betweenneighboring MLH1 foci within chromosome arms andacross centromeres in metacentric bivalents. The abso-lute distance varied from 1.0 to 15.6 mm within an armand from 1.2 to 27.1 mm across the centromere, with thetranscentromere distances significantly larger (P ,

0.001). These results indicate that there may be cross-over interference across centromeres as well as withinchromosome arms as suggested for a variety of organ-isms including humans (Colombo and Jones 1997;Broman and Weber 2000; Drouaud et al. 2006). Thereis the following expectation with transcentromere in-

terference, which we tested for metacentrics in thecommon shrew: The most proximal MLH1 focus on onechromosome arm should influence the most proximalfocus on the other arm, such that their distances fromthe centromere will be inversely correlated. We didindeed find inverse correlations in the shrew metacen-trics af, bc, gm, hi, jl, and pr (r ¼ �0.14, �0.28, �0.12,�0.23, �0.10, �0.10, respectively; P , 0.05). In otherwords, transcentromere interference explains 1–8% oftotal variance in distances between the centromeres andthe most proximal foci in these bivalents. For theremaining metacentrics the correlations were not sig-nificant, but in all cases this is reasonably ascribed tosmall sample sizes.

Transcentromere absolute distance is greater onaverage for the larger bivalents (Table 4). This isassociated with another phenomenon, the prominentmisalignment of centromeres, which is most frequentlyseen on large configurations such as af, bc, and the sextrivalent (Figure 1). Recombination events are thoughtto occur at sites of initiation of synapsis (Bishop andZickler 2004; Henderson and Keeney 2004) and,therefore, DNA homology is required at this stage.However, synapsis may proceed in between recombina-tion sites without a homology check (Ashley 1988;Zickler and Kleckner 1999). The larger the regionthat may synapse without a homology check, the greaterthe likelihood that prominent misalignment may occur,explaining why such misalignments tend to be observedon large configurations. As well as misalignment ofcentromeres, sometimes DAPI patterns between twoMLH1 foci within a chromosome arm were misaligned.

The absolute distances between two foci separatedby the centromere were usually larger in metacentric

TABLE 2

Comparison between the lengths of synaptonemal complexes and mitotic metaphase chromosomes in the common shrew

Absolute length of SC in mm Relative length of SCa Relative lengthof mitotic

chromosomesb

% difference fromexpected relative

length ratiocChromosome Mean SD Mean SD

af 24.7 3.8 17.3 1.0 17.3 0.0bc 24.9 4.0 17.5 1.0 20.5 �14.9d 11.1 1.9 7.8 0.7 9.4 �17.2gm 14.1 2.0 9.9 0.6 9.2 7.6hi 17.0 2.7 11.9 0.8 10.8 10.1jl 14.6 2.2 10.2 0.7 8.9 15.1kd 7.7 1.2 5.4 0.5 4.5 20.0nd 5.6 0.8 4.0 0.4 3.4 16.8od 5.2 0.8 3.6 0.4 3.6 0.3pr 7.7 1.1 5.4 0.4 5.8 �6.9qd 4.3 0.7 3.0 0.3 3.0 �0.3tu 5.8 0.9 4.1 0.4 3.4 19.4

a Percentage of total autosomal SC length.b Percentage of total autosomal mitotic length (calculated from the data of Kral and Radjabli 1974).c Calculated as ½(relative SC length/relative mitotic chromosome length) � 1� 3 100.d The data for the arms of the chromosomes showing Robertsonian variation were averaged for acrocentric homozygotes, meta-

centric homozygotes of various arm combinations, and heterozygotes.


homozygotes for chromosomes showing Robertsonianpolymorphism compared to the corresponding hetero-zygotes (jl, t1,645¼ 2.1, P , 0.05; kq, t1,206¼ 4.3, P , 0.01;no, t1,236¼ 2.9, P , 0.01; although ko, t1,84¼ 1.2, P¼ 0.12;see also Table 4). This can be interpreted as a weakeningof transcentromere interference in Robertsonian het-erozygotes relative to metacentric homozygotes. It iswell known that discontinuity of the SC or the switchingof pairing partners decreases the strength of interfer-ence (see Zickler and Kleckner 1999 for a review).This can also be interpreted in terms of the ‘‘stressrelief’’ model of crossover interference suggested byKleckner et al. (2004). According to this model, recom-bination is induced by mechanical stress. A crossoverleads to local stress relief and therefore to inhibition offurther crossovers nearby. Since the SC mediates theinterference, its interruption at the centromeres of the

twin-acrocentrics in a Robertsonian trivalent may di-minish transcentromere interference in heterozygotescompared to metacentric homozygotes.

The mean intra-arm distances between adjacentMLH1 foci in the shrew varied from 4.3 to 6.4 mm,according to the chromosome arm, with greater dis-tances for larger arms (Table 4). In the mouse, whichhas a similar overall MLH1 focus density as the shrew,similar mean distances between MLH1 foci (4.1–7.0mm) and a similar between-arm variation were found forarms of comparable sizes (Anderson et al. 1999). Re-cently De Boer et al. (2006, 2007) estimated the strengthof crossover interference in the mouse using parametersof the gamma distribution. The gamma distributiondescribes the probability of the distances that wouldoccur if MLH1 focus precursors were randomly placedalong the bivalent and only every nth precursor would

TABLE 3

Number of MLH1 foci per chromosome arm

No. of MLH1 fociGenetic

length (cM)

No. of cells containing

Arm Karyotypea Mean SD 0 foci 1 focus 2 foci 3 foci

a af 2.09 0.50 104.5 3 46 481 108b bc 1.72 0.49 86.0 1 188 438 11c bc 1.69 0.52 84.5 4 202 418 14d de/s, dv 1.83 0.44 91.5 0 128 493 17f af 1.14 0.38 57.0 6 534 98 0g gm 1.22 0.45 61.0 7 483 147 1h hi 1.19 0.43 59.5 11 495 132 0i hi 1.09 0.35 54.5 12 556 70 0j jl 1.06 0.28 53.0 6 561 44 0j j/l 1.04 0.19 52.0 0 26 1 0k ko 1.05 0.21 52.5 0 41 2 0k k/o 0.94 0.30 47.0 4 48 1 0k kq 0.98 0.23 49.0 2 55 1 0k k/q 1.06 0.32 53.0 4 143 13 0k k 1.06 0.29 53.0 4 299 20 1l jl 1.03 0.25 51.5 10 572 29 0l j/l 0.96 0.19 48.0 1 26 0 0m gm 0.96 0.23 48.0 31 602 5 0n no 0.96 0.21 48.0 1 22 0 0n n/o 1.00 0.09 50.0 1 222 1 0n n 1.00 0.14 50.0 4 383 4 0o ko 0.98 0.15 49.0 1 42 0 0o k/o 0.91 0.30 45.5 5 48 0 0o no 1.00 0.00 50.0 0 23 0 0o n/o 0.97 0.17 48.5 7 217 0 0o o 0.98 0.15 49.0 6 288 1 0p pr 0.94 0.26 47.0 42 593 3 0q kq 0.98 0.13 49.0 1 57 0 0q k/q 0.96 0.21 48.0 7 153 0 0q q 0.97 0.17 48.5 12 407 1 0r pr 0.90 0.30 45.0 64 573 1 0t tu 0.53 0.50 26.5 297 341 0 0u tu 0.54 0.50 27.0 296 342 0 0

a A karyotype ko will produce a metacentric bivalent, k/o will produce a trivalent (metacentric ko and acro-centrics k and o), and k will produce an acrocentric bivalent. The same is true for other chromosomes. de/s, dv isthe sex trivalent.


result in a focus. The higher the n value, the stronger theinterference. The n value can be estimated via the shapeparameter (y) of a gamma distribution. We determinedthe y-value for which the observed frequency distribu-tion of interfocus distances fitted best to a gammadistribution. The y-values estimated for the chromosomearms of the male common shrew varied between 11.1and 15.6 (Table 4) and were similar to those obtainedfor mouse bivalents of comparable sizes (y ¼ 13.7–14.4,De Boer et al. 2006; y ¼ 8.9–11.7, De Boer et al. 2007).These estimates suggest that the strength of crossoverinterference is rather conservative among mammalswhen arms of similar SC size and recombination densityare compared.

Recombination maps of Robertsonian invariantchromosomes: Individual distributions of MLH1 focifor each of the chromosomes not involved in Robertso-nian variation differed little among representatives ofthe two parental races and acrocentric individuals fromthe center of the hybrid zone. Of all distributions cross-tested between the races (24 tests) only one testcomparing values for each 0.5-mm interval gave a P-value ,0.01 (between the acrocentric shrews and theOxford shrews for bivalent tu: x2

11 ¼ 24:8, P , 0.01). Forthis reason we pooled the data from all karyotypes andplotted recombination maps for each arm of the in-variant bivalents (Figure 2).

Bivalents in the common shrew reproduce a generaldistribution of recombination events that is similaracross a wide variety of vertebrates such as fish (Moens

2006), birds (Pigozzi and Solari 1999; Pigozzi 2001;Calderon and Pigozzi 2006), mice (Anderson et al.

1999; Froenicke et al. 2002), and humans (Barlow andHulten 1998; Lynn et al. 2002; Tease et al. 2002; Sun

et al. 2004, 2006a): a pronounced recombination peaknear to the telomere, a deficiency near to the centro-mere, and a bi- or a multimodal distribution along thechromosome arms. (Note that where we use the term‘‘telomere’’ in this article, we always mean the structureat the distal end of a chromosome or a chromosomearm; we disregard telomeres that are situated next to thecentromere in single-armed chromosomes.)

Suppression of recombination in the pericentromericarea of the mouse chromosomes has been interpretedas an effect of the blocks of centromeric heterochro-matin that reside there (Froenicke et al. 2002). In thecommon shrew there is only a very small amount ofcentromeric C-heterochromatin (Schmid et al. 1982;Belonogova et al. 2006) and it appears less likely thatthis causes the recombination suppression.

Regarding recombination near the telomere, this wasnot precisely in the terminal segment. Instead the peakwas usually located at 2–3 mm from the telomeres. Anexcess of recombination near the telomeres is appar-ently due to early involvement of this region inchromosome pairing (Scherthan et al. 1996; Zickler

and Kleckner 1999). Recombination too close to thecentromere or telomere may be suppressed as the resultof natural selection. It has been shown in humans thatchiasmata in such locations often lead to nondisjunc-tion (Hassold and Hunt 2001).

The distribution of MLH1 foci along all the largearms of metacentric chromosomes, except a and h, wasusually bimodal with a major peak near the distal end

TABLE 4

The average absolute and relative distances between neighboring MLH1 foci including the y-value(shape parameter) of the gamma distribution for the within-arm data

Distance across centromere Distance within arms

Absolute (mm) Relativea Absolute (mm) Relativea

Chromosome N Mean SD Mean SD N Mean SD Mean SD y

af 625 10.5 3.4 0.42 0.11 788 6.4 2.2 0.42 0.13 11.1bc 634 10.0 3.3 0.40 0.12 904 6.1 1.9 0.48 0.13 13.6d 525 5.4 1.6 0.48 0.13 13.8gm 601 7.3 2.2 0.52 0.13 152 5.2 1.8 0.54 0.13 15.3hi 621 8.7 2.6 0.51 0.13 199 5.0 1.5 0.55 0.13 14.5jl 621 9.8 2.3 0.66 0.12 71 4.3 1.1 0.55 0.13 15.6j/l 26 8.8 2.4 0.60 0.13ko 42 8.8 1.7 0.65 0.11k/o 44 8.3 2.2 0.64 0.10kq 55 8.7 2.2 0.63 0.12k/q 153 7.3 1.7 0.60 0.12no 22 8.1 1.4 0.67 0.12n/o 216 7.2 1.5 0.64 0.11pr 533 5.3 1.2 0.69 0.12tu 69 3.6 0.8 0.57 0.10

a Relative interfocus distance across the centromere was calculated in terms of fractions of the chromosomelength; within a chromosome arm it was calculated as a fraction of the arm length.


and the other peak in the middle of the arm or nearerthe centromere. Arms a and h displayed clear trimodaldistributions. The arms of the chromosomes pr and tuwere too small to accommodate more than one cross-over. Each of them contained one peak.

It is interesting to compare the distributions of MLH1foci between chromosome arms of approximately equalsize in metacentric bivalents. The distributions wererather similar in the arms p and r (x2

8 ¼ 9:2, P ¼ 0.33)and drastically different between the arms b and c(x2

25 ¼ 53:3, P , 0.001), h and i (x218 ¼ 190:1, P ,

0.001), and j and l (x214 ¼ 158:7, P , 0.001).

These comparisons show that the size of the chromo-some arm is an important but not the only factor con-trolling the distribution of crossovers. Local hot andcold regions of recombination can significantly modu-late the overall crossover distribution along a chromosome.Analysis of factors controlling local recombination ratein human, rat, and mouse have demonstrated the im-portance of GC/AT ratio, CpG density, and the occur-rence of repetitive elements as well as chromosome sizeand proximity to the telomere and centromere, and therelative contribution of these factors varied betweenspecies ( Jensen-Seaman et al. 2004).

Recombination maps of Robertsonian variable chro-mosomes: For the chromosomes that do show Robert-sonian polymorphism we found a substantial differencein MLH1 distribution for the same arms between ho-mozygotes for metacentric chromosomes, homozygotesfor acrocentrics, and heterozygotes (Figure 3).

In all homozygotes for acrocentrics we observed uni-modal distributions with a peak of MLH1 foci in themiddle of the arm. In heterozygotes there was stillgenerally a single peak but the distribution was shiftedtowards a more distal localization. For arm o of k/oheterozygotes the pattern became bimodal with asecond peak occurring near to the telomere. For meta-centric homozygotes the distalization and presence of asecond peak were even more pronounced in all arms, aresult consistent with limited studies comparing chiasmaposition in trivalents and homologous metacentric biva-lents in shrews (Giagia-Athanasopoulou and Searle

2003). The data are also in accordance with those ofDumas and Britton-Davidian (2002) for the housemouse. They too found a reduction in the number ofproximal chiasmata and a more distal chiasma distribu-tion in metacentric homozygotes compared to acrocen-tric homozygotes and proposed that the suppressiveeffect of the centromere on recombination was morepronounced in metacentric chromosomes.

From an evolutionary perspective, it is of significancethat there is a greater tendency for reduced recombi-nation in proximal chromosomal regions in metacen-tric homozygotes compared with simple Robertsonianheterozygotes. This makes it difficult to argue thatrecombination characteristics of heterozygotes are caus-ing a reduction in gene flow across the hybrid zones be-

tween the Oxford, acrocentric, and Wirral races. It alsosuggests that speciation through recombination sup-pression (Rieseberg 2001) is not possible in hybridzones of the common shrew characterized by simpleheterozygotes. It would be of interest to determine therecombination characteristics of complex heterozy-gotes in the common shrew, i.e., hybrids between racesthat differ by metacentrics with a common chromosomearm and that form chain configurations of four or moreelements at meiosis I (Searle 1993). The greater po-tency of complex heterozygotes than simple heterozy-gotes in promoting reproductive isolation is shown byBasset et al. (2006) for the speciation event that sep-arated S. araneus and S. antinorii, and recombinationsuppression in complex heterozygotes has been sug-gested as a factor in a speciation event in the housemouse (Pialek et al. 2001).

There are other considerations relating to the meta-centric or acrocentric ‘‘condition’’ in shrews and mice.In a chromosome race defined by a metacentric theremay be the possibility of groups of epistatically interact-ing genes close to the metacentric centromere beingable to segregate as a unit because of the lack ofrecombination. This could lead to different alternativeadaptive multilocus genotypes within a population orgeographic region (‘‘coadaptive gene complexes,’’ e.g.,Burton et al. 1999; De Jong and Nielsen 2002), andresultant polymorphism or regional genetic subdivision.In an acrocentric chromosome race, such coadaptedgene complexes are less likely to evolve.

In both shrews and mice, some races are character-ized by a large number of acrocentrics and an associatedhigh diploid number, and other races by a large numberof metacentrics and associated low diploid number. Interms of number of segregating units, this is clearlyhigher in the acrocentric than in the metacentric races,so the former should show greater genic variation(Qumsiyeh 1994). Clearly, recombination also affectsgenic variability, and so the reduced centromeric re-combination in the metacentric races relative to theacrocentric races, will increase the differential in genicvariability. Qumsiyeh (1994) argues that chromosomalforms with different levels of genic variability may beadapted to different conditions, e.g., adaptation to‘‘pliable habitats’’ in the case of the acrocentric racesand adaptation to ‘‘constant or specialized habitats’’ inthe case of the metacentric races. There have, however,been no rigorous tests of this idea.

Recombination pattern of the sex trivalent: The Xchromosome of the common shrew represents a tan-dem fusion between the true mammalian X and anautosome (Sharman 1956; Fredga 1970; Pack et al.1993). At male meiosis a trivalent comprising the Xchromosome (de), the true Y (Y1 or the s chromosome),and the autosomal counterpart (Y2 or the dv chromo-some) is formed (Wallace and Searle 1990; Borodin

1991; Pack et al. 1993). We usually detected a single


MLH1 focus in the X–Y1 pairing region (Figure 1). Themean (6 SD) size of the Y1 SC was 3.4 6 0.7 mm.Although in some cells the Y1 was paired completelywith the short arm of the X chromosome, the MLH1focus was always located very close to the telomere. Itsaverage distance from the telomere was 0.3 6 0.2 mmwith a range from 0 to 0.7 mm. This indicates that thepseudoautosomal region (Burgoyne 1982) is located inthe most distal part of the true X and Y1 chromosomes.Thus, despite being fused with an autosome, thegonosomal part of the X remained autonomous in itsmeiotic behavior and showed characteristics displayedby a typical unfused mammalian X.

We did not observe MLH1 foci on the short arm vof the autosomal homolog of the sex trivalent. For theautosomal arm d of the sex trivalent we usually observedtwo MLH1 foci. Single and triple foci were rare (Table3). For sex trivalents containing only one MLH1 focuson autosomal arm d, these foci were located mainly inthe middle of the arm (Figure 2). The group with twofoci showed a trimodal distribution. In general, arm dwas rather similar to other autosomal arms of compara-ble size (b and c) in the frequency of MLH1 foci, theirdistribution, and interference parameters (Table 4).Previously we have demonstrated that arm d also actslike a typical autosome in chromosome pairing at malemeiosis and is not involved in the process of X in-activation in female somatic cells (Pack et al. 1993).

Conclusion: In summary, we have presented immu-nocytological recombination maps for every autosomein the male common shrew. Our maps demonstrate aglobal pattern rather similar to that found in humanand mouse. We observed an excess of recombination indistal parts of bivalents and its suppression near thecentromere. As for other vertebrates studied, malecommon shrews show a very strong correlation of SClength with the number of MLH1 foci, and, therefore,with genetic length of the chromosome. Interferenceestimates for the common shrew, i.e., average absoluteand relative distances between neighboring crossovers,and the shape parameter of the gamma distribution,were comparable with corresponding estimates ob-tained for mouse chromosomes. All these comparisonssuggest that a general pattern of recombination isconserved across mammals and perhaps all vertebrates.

As for other vertebrates, in the common shrew weobserved that every chromosome arm has its ownspecific pattern of recombination, which is a complexfunction of its length, centromere and telomere effects,interference, and sequence characteristics. Chromo-some arms of the same length may differ significantlyin their recombination pattern. These recombinationpatterns remain rather conservative in representativesof different chromosome races of the common shrewthat apparently have evolved independently for severalthousand years (Searle and Wilkinson 1987). Wefound no difference in recombination patterns of the

invariant chromosomes between the Oxford and Wirralraces of the common shrew.

On the other hand, the recombination pattern of anindividual chromosome arm varied substantially depend-ing on whether it is an acrocentric chromosome or part ofa metacentric chromosome. In contrast to Bidau et al.(2001) and Dumas and Britton-Davidian (2002) forthe house mouse, in common shrews we did not find asignificant decrease in numbers of MLH1 foci on thearms of metacentric chromosomes compared to acro-centrics. However, in agreement with them, we found asubstantial redistribution of the crossovers to more distalpositions in the metacentric chromosomes.

We thank C. Heyting, S. Garagna, A. Agoulnik, and N. Belyaev forthe generous gift of antibodies and the Microscopic Center of theSiberian Branch of the Russian Academy of Sciences (RAS) forgranting access to microscopic equipment. This work was supportedby research grants from The International Association for the Pro-motion of Cooperation with Scientists from the New IndependentStates of the Former Soviet Union, the Russian Foundation for BasicResearch, and the Programs of RAS ‘‘Biosphere Origin and Evolution’’and ‘‘Biodiversity.’’

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Communicating editor: T. R. Magnuson


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