CopyrightÓ2011 by the Genetics Society of America DOI: 10.1534/genetics.110.124081
Note
Trans-Centromere Effects on Meiotic Recombination in the Zebrafish
Bradley L. Demarest,* Wyatt H. Horsley,* Erin E. Locke,* Kenneth Boucher,*
,†David J. Grunwald
‡,1and Nikolaus S. Trede*
,§,1,2*Department of Oncological Sciences,†Biostatistics Core at Huntsman Cancer Institute,‡Department of Human
Genetics, and§Department of Pediatrics, University of Utah, Salt Lake City, Utah 84112
Manuscript received September 23, 2010 Accepted for publication October 14, 2010
ABSTRACT
We report that lack of crossover along one chromosome arm is associated with high-frequency occurrence of recombination close to the opposing arm’s centromere during zebrafish meiotic recombination. Our data indicate that recombination behavior on the two arms of a chromosome is linked. These results inform mapping strategies for telomeric mutants.
T
HE zebrafish (Danio rerio) provides the unique op-portunity to recover two of the four haploid genomes produced from a single meiosis using a tech-nique known as early pressure (EP) parthenogenesis (Streisingeret al.1981, 1986). Recombination events can be reconstructed accurately from the genetic analysis of EP parthenogenotes (Streisingeret al.1986; Johnson et al.1995) (supporting information,Figure S1). Analysis of these products can reveal how the occurrence of one recombination event may affect subsequent chromo-some behaviors at meiosis.In this study we investigate recombination behavior of two zebrafish chromosomes for which telomeric recessive mutations are available. In the absence of recombination or following even numbers of crossovers along the length of the relevant chromosome arms, the early pressure (EP) half-tetrad progeny produced from heterozygous females will be homozygous for telomeric alleles. Half of such progeny will express the mutant phenotype (Figure S1). We analyzed mutant EP half-tetrad progeny in detail to identify recombination events that produced sister chromatid pairs homozygous for telomeric markers.
RESULTS AND DISCUSSION
A total of 1243 EP larvae were produced fromasm1/ heterozygous F1 females that were generated in a
mating between a uniqueasm1/P0 founder male and
a uniqueasm1/1
P0 female from the wild-type (WT) Tu strain (Figure 1A and Table S1). As illustrated by the representative genotyping analysis (Figure S2), EP par-thenogenesis produced 5-day-postfertilization (dpf) off-spring in which homologous pairs of chromosomes were derived from sister chromatid pairs. Allasmmutants were genotyped using nine chromosome 18 simple sequence repeat (SSR) markers that were polymorphic between the parentalasmandasm1chromosomes (Figure S3).
On the basis of analysis of these nine markers, 34 mutants lacked evidence of any recombination between the centromere and the asm locus. Two mutants arose from two-strand double crossovers (dco) (Figure S3C), and 3 mutants arose from four-stranddco(Figure S3D). Additionally, one exceptional individual was heterozy-gous for markers that flank the centromere and there-fore likely was not derived only from a single sister chromatid pair (Figure S3E). In sum, 5/39 (13%) EP half-tetrad mutant progeny arose from sister chromatid pairs that experienceddcos. The products ofdcos would be anticipated by established linkage maps of zebrafish, which exceed 100 cM in many cases ( Johnsonet al.1995; Kauffman et al. 1995). Consistent with our genetic analyses, a recent study using the chiasma-specific MLH1 antibody indicated the occasional presence of at least two chiasmata involving a single chromosome arm during zebrafish female meiosis (Kochakpour and Moens 2008). The MLH1-staining did not distinguish between two-, three-, or four-strand dco and at what frequency they occurred. The data presented here show unambiguously that two-strand and four-stranddcooccur at measurable frequency in zebrafish.
Among mutant half-tetrad offspring, nonrecombi-nant along the right (asm) arm of chromosome 18, we
Supporting information is available online athttp://www.genetics.org/ cgi/content/full/genetics.110.124081/DC1.
1These authors contributed equally to this work.
2Corresponding author:Huntsman Cancer Institute, University of Utah,
2000 Circle of Hope, Salt Lake City, UT 84112. E-mail: [email protected]
Figure1.—(A) Pedigree of early pressure (EP) gynogenetic half-tetrad progeny that were analyzed. Fish lines: asm was induced with ethylnitrosourea and mutants were dis-tinguished from wild-type by morphology (small, protruding eyes and small head phe-notypes) or by in situ hybridization (using an antisense RNA probe for the T cell-specific kinase lck) at 5 day postfertilization (dpf)
(Tredeet al.2008). Correspondence between
lack of T cells and head morphology is 100% (data not shown). The heterozygousasm mu-tation was induced in the WIK background and a mapping hybrid cross was generated for the present study by crossing a single
asm1/ heterozygote with a single wild-type Tu individual (Figure 1A,Figure S2).asm1/ F1individuals were identified by progeny test crosses.OG076was identified in the Tu¨bingen 2005 screen by its lack of macrophages and neutrophils (P. Herbomeland M. Redd, per-sonal communication). It was generated on the Tu background and crossed to WIK for mapping purposes. EP parthenogenesis: Sperm collection, egg extrusion, in vitro fer-tilization, and timing of pressure treatment were performed as described previously (Streisingeret al.1981). Sperm was UV-treated in a Stratagene stratalinker set to deliver 73
104 mJ. Pressures and hydraulic press equip-ment used were described previously (Gestl
et al.1997). Generation of mutant offspring:
EP parthenogenotes were generated from het-erozygous F1females and phenotypically clas-sified as either mutant (asm) or wild type (WT). For analysis of recombination affecting theasmchromosome, EP was performed on multiple occasions on eggs derived from a total of 20asm1
F1females (Figure 1A,Figure S2). Resulting gynogenetic diploid half-tetrad larvae were raised to 5 dpf whenasmmutants are viable: among.4000 offspring produced from matings between heterozygotes,25% were mutant at 5 dpf (data not shown). For analysis of theOG076chromosome, eggs from four heterozygous females were subjected to EP, larvae were fixed at 4 dpf and assayed for presence of neutrophils by staining with Sudan Black (LeGuyaderet al. 2008). (B) Nonindepen-dence of recombination on the two arms of chromosome 18. Primers and PCR: Centromere-linked microsatellite marker loci were obtained from published studies (Shimoda et al. 1999; Mohideen et al. 2000). https://wiki.zfin.org/display/prot/MGH-CVRC+Mapping+Resources. PCR products were resolved by gel electrophoresis using 3% metaphor agarose. Recombination events in 34 mutant and 23 wild-type sibling EP offspring fromasm1
heterozygous females were analyzed using the chromosome 18 SSR markers depicted in the bar graph on the left. Half-tetrad EP offspring were divided into two groups: mutant offspring whose chromosomes 18 were nonrecombinant along the right arm (no exchange on right arm), and wild-type offspring that har-bored a single chromosome 18 that was recombinant along the right arm (exchange on right arm). Because recombination af-fecting either of the two left arms of the sister chromatid pair could produce a heterozygous half-tetrad, we would expect that 7.4% of the EP progeny would be heterozygous for marker Z9194. Chromosomes with a right arm exchange exhibited a frequency of exchange in the left pericentric interval very close to expectations on the basis of the published map distance. In contrast, the frequency of exchange in the left pericentric interval appeared aberrantly high among larvae lacking an exchange on the right arm of chromosome 18. The two groups differed significantly with respect to the occurrence of recombination in the left peri-centric interval. *These larvae constituted allasmmutant EP progeny lacking exchanges on the right arm of chromosome 18 (Figure S3B).†These larvae were selected at random from the phenotypically wild-type EP progeny and were genotyped to verify that a right arm exchange had occurred. (C) Nonindependence of recombination on the two arms of chromosome 14. Recom-bination events in 14 mutant and 47 wild-type EP offspring ofOG0761
heterozygous females were analyzed using the markers depicted in the bar graph on the left. Half-tetrad EP offspring were divided into two groups: mutant offspring whose chromosomes 14 were nonrecombinant along the left arm and wild-type offspring that harbored a single chromosome 14 that was recombinant along the left arm. Because recombination affecting either of the two right arms of the sister chromatid pair would produce a heterozygous half-tetrad, we would expect that 8.8% of the EP progeny would be heterozygous for marker Z22094. Chromosomes with a left arm exchange exhibited a frequency of exchange in the right pericentric interval very close to expectations. In contrast, the frequency of exchange in the right pericentric interval appeared aberrantly high among larvae lacking an exchange on the left arm of chromosome 14. The two groups differed significantly with respect to the occurrence of recombination in the right peri-centric interval. *These larvae were homozygous mutant EP progeny and presumably most lacked exchanges on the left arm of chromosome 14.†These larvae were selected at random from the phenotypically wild-type EP progeny and genotyped to verify that a left arm exchange was present.
found that the left arm pericentric marker Z9194 was heterozygous more often (20 of 34¼59%) than would be predicted from the published female map distance of 3.7 cM for the Z9194–centromere interval (Figure 1B). As recombination affecting either of the two left arm sister chromatid strands would produce a heterozygous half-tetrad, 7.4% of the EP progeny are expected to be heterozygous. We analyzed 23 randomly chosen pheno-typic wild-type half-tetrad siblings (all recombinant on the right arm of chromosome 18) and found that only 2 of 23 (8.7%) were recombinant in the left interval, very close to the expected fraction. The difference in the occurrence of recombination within the left pericentric interval was highly significant between the group of EP progeny produced from meioses that had experienced crossover along the right arm and the group that had not (P¼2.13104, Fisher’s exact test, two tailed).
To test whether effects on crossover behavior that extend beyond the centromere are a general feature in zebrafish, we examined recombination in an indepen-dent line harboring the telomeric mutationOG076. We mapped the mutant locus (W. H. Horsley and N. S. Trede, unpublished data) to the left arm of chromo-some 14, adjacent (0.6 cM) to Z65389, located93 cM from the centromere. HeterozygousOG0761/females were generated and their EP progeny analyzed for recombination events (Figure 1C). Fourteen OG076 half-tetrad mutants and 47 wild-type half-tetrad siblings were examined at three markers. All mutants main-tained parental linkage with the centromere marker and were presumably nonrecombinant on the left arm. Twelve of the 14 mutants (86%) had an exchange in the right pericentric interval (4.4 cM between markers Z26376 and Z22094). In contrast, all 47 of the wild-type half-tetrads were recombinant on the left arm, and only 5 of these (11%) were recombinant in the right peri-centric interval, close to the expected frequency of 8.8% for this interval. In sum, absence of recombination on one arm is coupled with significant differences in the probability of exchange in the pericentric region of the opposing arm on chromosome 14 (P ¼ 2.6 3 107, Fisher’s exact test, two tailed).
We note there is conflicting data whether recombi-nation behavior on one chromosome arm is linked with that on the opposing arm (Mather 1936; Colombo and Jones1997; Bromanand Weber2000; Falqueet al. 2007). The different findings may reflect differences in experimental designs to detect trans-centromere asso-ciations or species-specific differences in the occurrence of such associations.
We considered the possibility that the evidence for pericentric recombination reflected the unexpected acquisition of markers from paternal DNA from the ‘‘UV-treated’’ sperm used to activate parthenogenetic development, creating aneuploid half-tetrad progeny carrying multiple pericentric alleles. This model ap-peared unlikely in that the 5-dpf-EP progeny
homozy-gous for the telomeric mutations appeared uniform and distinctive in phenotype, as expected for normal diploid mutants. We directly examined this possibility by using genotyped parents and our standard EP method to produce gynogenetic offspring. Embryos were tested at 3 dpf for the presence of detectable levels of a paternal allele at a marker locus on chromosome 18. Among 132 half-tetrad progeny generated with two independent batches of UV-treated sperm, we failed to detect any pa-ternal contribution (Figure S4). Similarly, Streisinger et al.(1981) reported no genetic contribution from UV-treated sperm in.600 gynogenetic embryos. We con-clude that the vast majority of cases in which embryos were heterozygous for pericentromeric markers arose frombona fiderecombination events.
A second possibility is that recombination events affecting each arm of a chromosome occurred inde-pendently, but that some combinations of crossovers were not recovered in the 5-dpf-EP progeny we analyzed, leading to the enriched recovery of half-tetrads with absence of recombination on one arm and pericentric recombination on the other arm of a chromosome. Selective recovery of half-tetrad progeny could have occurred in two ways to produce the results we observed. First, it is possible that meiotic products arising from a combination of pericentric recombination on one arm and a crossover anywhere on the opposing arm were selectively lost. The combination of crossovers might have increased the likelihood of aberrant chromosome segregation during meiosis, yielding aneuploid EP progeny that failed to develop and were lost from analysis (Buonomo et al. 2000; Rockmill et al. 2006; Subramanianand Bickel2008). In this scenario, lack of crossover along one entire arm is the permissive condition, and analysis of the EP offspring that lacked crossover on theasmorOG076arms (Figure 1, B and C) would indicate the true frequencies with which re-combination occurred on the opposing arms. This interpretation would require that well over half of the half-tetrads in which anasmorOG076arm experienced a crossover were lost prior to analysis. By extrapolation, pericentric recombination on any chromosome arm would frequently contribute to the production of aneuploid gametes, a prediction that is incompatible with the very high viability (often .95%) that accom-panies most fertilization events with zebrafish in the laboratory.
A second selective loss model that could account for our observed results involves loss of half-tetrad progeny that failed to experience recombination on one arm and failed to experience recombination in the pericentric interval on the opposing arm. Such selective loss would produce an apparent inflation of the proportion of half-tetrads with no crossover on one arm and pericentric recombination on the other arm. Applying this interpre-tation to the results we observed for chromosomes 18 and 14 (Figure 1, B and C), in which pericentric
tion was recovered in 8- to 10-fold excess above the expected frequency for the pericentric interval, would require loss of 85–90% of the half-tetrads in which an asm or OG076 arm failed to experience a crossover. Once again, if we extrapolate the selective loss of products to meioses in whichany of the 50 chromo-some arms failed to experience a crossover, we would predict a very high fraction of nonviable gametes to be produced under normal conditions, a prediction in-compatible with the observed high viability of zygotes in the laboratory.
A third possibility is that our analyses have uncovered a bona fide linkage between crossover behaviors on opposing chromosome arms. The mechanism underly-ing this association is not revealed by our experiments. It is possible that pericentromeric exchanges exert an inhibitory effect that propagates across the centromere and blocks the generation of additional crossovers. Alternatively, there may be mechanisms that work to ensure the occurrence of a crossover so that absence of crossing over along a chromosome arm is causally linked with the generation of a nearby pericentromeric event. Whether crossovers arise independently but then are subsequently subject to selection, or mechanisms that operate across centromeres coordinate the generation of multiple chiasmata, the net result is the transmission in the zebrafish of meiotic products with a highly nonrandom distribution of crossovers.
Our data have implications for using EP for mapping purposes. First, given the high frequency of crossovers close to the centromere opposite nonrecombinant chromosome arms, markers on both sides of the cen-tromere need to be used. Second, the occurrence of four-stranddcoimplies that a homozygous mutant with the opposing centromere marker allele does not ex-clude linkage to that chromosome. Third, telomere markers should be used to confirm linkage.
We thank Kent Golic and Frank Stahl for helpful comments and discussions. N.S.T. was supported by National Institutes of Health (NIH) National Heart, Lung, and Blood Institute awards K08 HL004233 and 1R21HD060310 and by the Huntsman Cancer Foun-dation. Huntsman Cancer Institute core facilities, supported by grant P30 CA042014, and the University of Utah supported Centralized Zebrafish Animal Resource facility contributed to this work. D.J.G. was supported by NIH PO1 HD048886.
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Communicating editor: E. Alani
GENETICS
Supporting Information
http://www.genetics.org/cgi/content/full/genetics.110.124081/DC1
Trans-Centromere Effects on Meiotic Recombination in the Zebrafish
Bradley L. Demarest, Wyatt H. Horsley, Erin E. Locke, Kenneth Boucher,
David J. Grunwald and Nikolaus S. Trede
Copyright
Ó
2011 by the Genetics Society of America
B. L. Demarest et al.
2 SI
oogenesis
early pressure early pressure B No recombination
A Maternal genotype
C Two-strand single crossover
asm asm+
asm asm asm+ asm+ asm asm asm+ asm+
asm asm+ asm asm+
asm asm asm+ asm+
early pressure early pressure D Two-strand double crossover
E Four-strand double crossover
asm asm asm+ asm+
asm+ asm+ asm asm
asm asm asm+ asm+
asm asm asm+ asm+
FIGURE S1
FIGURE S1.—Genotypes of EP parthenogenetic offspring of asm+/- females. Co-segregation of the asm mutation and its parental centromere can be disrupted by recombination. (A) The maternal genotype. (B-E) The left side of each panel shows diagrams of recombination at the four-strand bivalent stage of Meiosis I, with newly replicated sister chromatids joined by a shared centromere; the right side of each panel shows the genotypes of the half-tetrad offspring that could be produced following the recombinations. (B) In the absence of crossovers between the locus of the mutation and its centromere, all heterozygous markers in the interval will segregate at Meiosis I and only homozygous half-tetrads, asm–/– mutant or asm+/+ wild-type, will be produced. Resulting mutant EP offspring will carry only the parentally linked allele at the centromere, representing a Parental Ditype (PD). (C) Single crossover produces only asm+/– half-tetrads, which may be homozygous for either centromere.
B. L. Demarest et al. 3 SI
5 female p17 female
p18 female 28 female
32 female
41 female
42 female 44 female 50 female
45 female 51 female
54 female 57 female
58 female 61 female 65 female
67 female
70 female 71 female
72 female 74 female 75 female 80 female 82 female
EP mut 67.1 EP mut 67.2 EP mut 67.3 EP mut 67.4 EP mut 67.5 EP mut 67.6 EP mut 57.1 EP mut 57.2 EP mut 57.3 EP mut 57.4 EP mut p17.1 EP mut p17.2 EP mut p17.3 EP mut 109.1 EP mut 109.2 EP mut 109.3 EP mut 30.1 EP mut 30.2 EP mut 71.1 EP mut 71.2 EP mut 41.1 EP mut 41.2 EP mut 41.3 EP mut 41.4 EP mut 41.5 EP mut 41.6 EP mut 41.7 EP mut 32.1 EP mut 32.2 EP mut 32.3 EP mut 32.4 EP mut 50.1 EP mut 50.2 EP mut 50.3 EP mut 82.1 EP mut 82.2 EP mut 82.3 EP mut 28.1 EP mut 51.1 EP mut 65.1 EP WT 67.1 EP WT 67.2 EP WT 67.3 EP WT 67.4 EP WT 67.5 EP WT 67.6 EP WT 57.1 EP WT 57.2 EP WT 57.3 EP WT 57.4 EP WT p17.1 EP WT p17.2 EP WT p17.3 EP WT 109.1 EP WT 109.2 EP WT 109.3 EP WT 30.1 EP WT 30.2 EP WT 71.1 EP WT 71.2 EP WT 28.1 EP WT 51.1 EP WT 65.1 F1
asm mutant phenotype
asm mutant phenotype
WT phenotype asm+/– 4-strand dco aberrant non-EP 4-strand dco 4-strand dco FIGURE S2 B
p14-408 male Tu 002 female
P0 F1 F2 (EP)
asm+/–
asm mutants WT siblings
asm+/+
asm+/–
EP Parthenogenesis
A
p14-408 male Tu
002 female
P0
FIGURE S2.— Genotypic analysis of the transmission of a centromere marker. (A) Genotyping of representative
individuals from the pedigree described in Fig. 1A indicates inheritance of two alleles of the centromeric microsatellite marker Z11257 through the pedigree. (B) The asm/+ P0 male 14-408 and the wild-type P0 female Tu 002 were each homozygous for a different allele of Z11257. Whereas all F1 females were heterozygous for z11257, a centromere-linked simple sequence repeat (SSR) marker on chromosome 18 (https://wiki.zfin.org/display/prot/MGH-CVRC+Mapping+Resources), all 40 EP half-tetrad mutant offspring (with the exception of mut 30.2) and all 23 EP wild-type offspring, derived from designated (*) F1 females, carried only one allele of the centromere marker. One exceptional mutant (EP mut 30.2) could not have been derived from a sister chromatid pair; this mutant and its presumed wild-type counterpart were eliminated from further quantitative analyses. Genotypic analysis of EP half-tetrad asm mutant and WT offspring is shown, where EP mut 41.1 indicates a mutant
B. L. Demarest et al. 4 SI
B. L. Demarest et al. 5 SI
asm asm+
A Genotype of asm+/ F1 females
Z10008 47.2 cM
Z9154 65.3 cM
Z7961 75.4 cM Z9094 60.7 cM Z11257 45.1 cM
Z9194 43.4 cM 1.7 cM 3.7 cM
30.3 cM 56.4 cM
Z22032 65.3 cM
Z63731 78.1 cM Z10729 71.9 cM
B Non-recombinant mutants (n = 34)
(n = 14)
(n = 1) (n = 1)
(n = 1) (n = 1) (n = 1)
(n = 1)
(n = 20) Z10008 47.2 cM
Z9154 65.3 cM
Z7961 75.4 cM Z9094 60.7 cM Z11257 45.1 cM Z9194 43.4 cM
Z22032 65.3 cM
Z63731 78.1 cM Z10729 71.9 cM
asm asm
D 4-strand double crossover mutants (n = 3)
Z10008 47.2 cM
Z9154 65.3 cM
Z7961 75.4 cM Z9094 60.7 cM Z11257 45.1 cM Z9194 43.4 cM
Z22032 65.3 cM
Z63731 78.1 cM Z10729 71.9 cM
asm
asm asm asm asm asm
C 2-strand double crossover mutants (n = 2)
Z10008 47.2 cM
Z9154 65.3 cM
Z7961 75.4 cM Z9094 60.7 cM Z11257 45.1 cM Z9194 43.4 cM
Z22032 65.3 cM
Z63731 78.1 cM Z10729 71.9 cM
asm asm asm asm
E Non-early pressure genotype mutant (n = 1)
Z10008 47.2 cM
Z9154 65.3 cM
Z7961 75.4 cM Z9094 60.7 cM Z11257 45.1 cM Z9194 43.4 cM
Z22032 65.3 cM
Z63731 78.1 cM Z10729 71.9 cM
asm asm
asm asm
MGH positions
MGH sex-averaged
distances distancesfemale FIGURE S3
B. L. Demarest et al.
6 SI
Female egg donor
Gynogenetic Half-tetrad Progeny
Male sperm donor
FIGURE S4
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46
FIGURE S4.—Sperm-derived centromere alleles are not contributed to EP gynogenetic offspring. Forty-four 3 dpf EP offspring
B. L. Demarest et al. 7 SI
TABLE S1
Production of recombinant and non-recombinant Early Pressure parthenogenetic larvae
Number Percent
non-recombinant asm mutants 34 2.74
4-strand dcoasm mutants 3 0.24
2-strand dcoasm mutants 2 0.16
non-recombinant WTs (calculated) (34) 2.74
4-strand dco WTs (calculated) (3) 0.24
3-strand dco WTs (calculated) (10) 0.81
2-strand dco WTs (calculated) (2) 0.16
single recombinants WTs (calculated) (1153) 92.91
total gynogenetic diploid offspring 1241 100
All EP mutant half-tetrad progeny were genotyped directly. To estimate the numbers of meioses in which no recombination
occurred along the asm arm of chromosome 18 as well as those that experienced single or double crossover (dco) recombination
events, we start from the following considerations. First, we assume that recombination within the interval between asm and the
centromere closely approximates recombination along the entire chromosome arm. Second, the recovery of asm homozygotes is
truly representative of the production of sister chromatid pairs homozygous for telomeric markers: homozygous asm mutants
survive as well as their wild-type siblings to 5 dpf, the stage at which they were identified as mutant and harvested for analysis.
Third, the events recovered in the homozygous mutant EP progeny represent but one-half of the meiotic events that yielded sister
chromatid pairs homozygous at the asm locus. We assume reciprocal events produced homozygous wild-type offspring, which in
this study were not distinguished from their more frequent heterozygous wild-type siblings. Hence non-recombinant chromosome
arms were produced in 68 meioses, and approximately 4 instances of two-strand and 6 instances of four-strand dco occurred
among the 1241 meioses studied here. Fourth, as three-strand dcos must produce sister chromatid pairs heterozygous for telomeric
markers, we did not recover these events and measure them directly. However, assuming no chromatid interference (HAWLEY
and WALKER 2003; ZHAO and SPEED 1996) the expected ratio of 2-strand : 3-strand : 4-strand dcos is 1:2:1, thus predicting
10 three-strand dcos among the meioses recovered here. Given the rarity of dcos, we assume that meioses with three or more
crossovers are sufficiently rare as to be ignored in this analysis. In sum, among the 1241 meioses in our analysis, we estimate 68
meioses (5.5%) in which the asm arm failed to recombine (non-recombinant), 20 meioses (1.6%) in which the asm arms
experienced dco, and 1153 meioses (92.9%) in which single crossovers affecting the asm arm occurred. As noted by Streisinger et
al. (STREISINGER et al. 1986), if exchanges along a chromosome arm occurred independently of each other, then loci distant
from a centromere might assort independently among the EP offspring, and at maximum, two-thirds of the EP offspring should
B. L. Demarest et al. 8 SI
interference as has been observed previously in many organisms, including fishes (HAWLEY and WALKER 2003; JOHNSON et
al. 1995; STREISINGER et al. 1986; THORGAARD et al. 1983). The high interference does not preclude dco, and in fact three
of the five double recombinants we recovered arose from meioses in which two crossovers occurred within intervals of 12 – 15 cM
on chromosome 18 (Figure S3).
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