Juxtaposition of heterozygous and homozygous regions causes reciprocal crossover remodelling via interference during Arabidopsis meiosis

  1. Piotr A Ziolkowski
  2. Luke E Berchowitz
  3. Christophe Lambing
  4. Nataliya E Yelina
  5. Xiaohui Zhao
  6. Krystyna A Kelly
  7. Kyuha Choi
  8. Liliana Ziolkowska
  9. Viviana June
  10. Eugenio Sanchez-Moran
  11. Chris Franklin
  12. Gregory P Copenhaver
  13. Ian R Henderson  Is a corresponding author
  1. University of Cambridge, United Kingdom
  2. Adam Mickiewicz University, Poland
  3. University of North Carolina at Chapel Hill, United States
  4. University of North Carolina School of Medicine, United States
  5. University of Birmingham, United Kingdom
8 figures, 9 tables and 1 additional file

Figures

Testing for crossover modification by Arabidopsis natural variation.

(A) Historical crossover frequency (red, cM/Mb) and sequence diversity (π, blue) along the physical length of the Arabidopsis thaliana chromosomes (Mb) (Cao et al., 2011; Choi et al., 2013). Mean …

https://doi.org/10.7554/eLife.03708.003
Figure 2 with 1 supplement
High-throughput measurement of crossover frequency using image analysis of fluorescent seed.

(A) Combined red and green, red alone and green alone fluorescent micrographs of seed from a self-fertilized 420/++ plant. (B) CellProfiler output showing identification of seed objects by green …

https://doi.org/10.7554/eLife.03708.007
Figure 2—source data 1

420 crossover frequency measured via manual or automated scoring of seed fluorescence.

https://doi.org/10.7554/eLife.03708.008
Figure 2—figure supplement 1
Distinguishing 420 RFP-GFP/++ vs RFP-+/+-GFP recombinant individuals.

(A) Genetic diagram illustrating generation of F2 plants heterozygous for the 420 fluorescent transgenes, annotated as in Figure 1C. F2 plants heterozygous for the fluorescent transgenes can occur …

https://doi.org/10.7554/eLife.03708.009
Variation in F1 hybrid crossover frequency.

(AE) Genetic distance (cM) measurements for fluorescent crossover intervals I1b, I1fg, I2f, 420 and CEN3 with individual replicates (black dots) and mean values (red dots) for the crosses labelled …

https://doi.org/10.7554/eLife.03708.010
Figure 3—source data 1

I1b F1 flow cytometry count data.

https://doi.org/10.7554/eLife.03708.011
Figure 3—source data 2

I1b F1 flow cytometry count data.

https://doi.org/10.7554/eLife.03708.012
Figure 3—source data 3

I1b F1 flow cytometry count data.

https://doi.org/10.7554/eLife.03708.013
Figure 3—source data 4

I1b F1 flow cytometry count data.

https://doi.org/10.7554/eLife.03708.014
Figure 3—source data 5

CEN3 F1 flow cytometry count data.

https://doi.org/10.7554/eLife.03708.015
Figure 4 with 1 supplement
Modification of crossover frequency by juxtaposition of heterozygosity and homozygosity.

(A) Diagram illustrating chromosome 3 genotypes (black = Col, red = Ct) in RG/++ F1 individuals and their F2 progeny. A single chromosome is shown for simplicity. Gametes or progeny are analysed for …

https://doi.org/10.7554/eLife.03708.017
Figure 4—source data 1

420 Col/Ct F2 fluorescent seed count data.

https://doi.org/10.7554/eLife.03708.018
Figure 4—source data 2

I2f Col/Ct F2 fluorescent seed count data.

https://doi.org/10.7554/eLife.03708.019
Figure 4—source data 3

CEN3 Col/Ct F2 flow cytometry count data.

https://doi.org/10.7554/eLife.03708.020
Figure 4—figure supplement 1
Modification of crossover frequency by juxtaposition of heterozygosity and homozygosity.

(A) CEN3 cM from Col/Ct CEN3/++ F2 (black), Col/Col homozygotes (red) and Col/Ct F1 (blue) individuals. Horizontal dotted lines indicate mean value. See Figure 4—source data 3. (B) Chromosome 3 …

https://doi.org/10.7554/eLife.03708.021
Figure 5 with 1 supplement
Juxtaposition of heterozygous and homozygous regions triggers reciprocal crossover remodelling.

(A) Schematic diagram illustrating the physical location of 420 and I3bc transgenes expressing fluorescent proteins in seed and pollen. Beneath are diagrams illustrating the locations of Col/Col …

https://doi.org/10.7554/eLife.03708.024
Figure 5—source data 1

Three colour I3bc FTL flow cytometry count data.

https://doi.org/10.7554/eLife.03708.025
Figure 5—source data 2

Three colour I3bc FTL flow cytometry count data–measurement of crossover interference.

https://doi.org/10.7554/eLife.03708.026
Figure 5—figure supplement 1
Analysis of I3bc recombination using three-colour flow cytometry.

Flow cytometry plots are shown measuring pollen for the indicated colour of fluorescent protein. In the upper plot total hydrated pollen is divided into blue and non-blue populations using polygonal …

https://doi.org/10.7554/eLife.03708.027
Figure 6 with 1 supplement
Genetic requirements of crossover remodelling via juxtaposition of heterozygous and homozygous regions.

(AD) Replicate measurements of 420 (red) and CEN3 (blue) genetic distances (cM) are plotted in wild type, fancm and fancm zip4. See Figure 6—source data 1, 2. Chromosome 3 genotypes of the plants …

https://doi.org/10.7554/eLife.03708.029
Figure 6—source data 1

420 fluorescent seed count data from wild type, fancm and fancm zip4 individuals with varying heterozygosity.

https://doi.org/10.7554/eLife.03708.030
Figure 6—source data 2

CEN3 flow cytometry count data from wild type, fancm and fancm zip4 individuals with varying heterozygosity.

https://doi.org/10.7554/eLife.03708.031
Figure 6—figure supplement 1
Generation of wild type, fancm or fancm zip4 420-CEN3 individuals with varying patterns of Col/Ct heterozygosity.

Diagram showing the crossing scheme used to generate plants to test the requirement of recombination pathways in crossover remodelling. At relevant points the genotype of chromosome 3 is illustrated …

https://doi.org/10.7554/eLife.03708.032
Total chiasmata frequencies are stable between Col, Ct and recombinant lines.

(AE) Metaphase-I chromosome spreads from anthers from (A) Col/Col 420, (B) Ct/Ct, (C) Col × Ct F1, (D) a Col × Ct 420 (HOM-HET) cold recombinant line and (E) a Col × Ct 420 hot (HET-HOM) …

https://doi.org/10.7554/eLife.03708.033
Figure 8 with 1 supplement
Crossover interference increases when heterozygous and homozygous regions are juxtaposed.

(AD) Replicate measurements of I3b and I3c genetic distances (cM), and I3bc crossover interference are plotted in wild type, fancm, fancm zip4 and zip4. Black dots represent replicate measurements …

https://doi.org/10.7554/eLife.03708.035
Figure 8—source data 1

I3bc fluorescent seed count data from wild type, fancm and fancm zip4 individuals with varying heterozygosity.

https://doi.org/10.7554/eLife.03708.036
Figure 8—source data 2

Calculation of I3bc interference from wild type, fancm and fancm zip4 individuals with varying heterozygosity.

https://doi.org/10.7554/eLife.03708.037
Figure 8—source data 3

I3bc fluorescent seed count data from wild type and zip4 individuals with varying heterozygosity.

https://doi.org/10.7554/eLife.03708.038
Figure 8—source data 4

Calculation of I3bc interference in wild type and zip4.

https://doi.org/10.7554/eLife.03708.039
Figure 8—figure supplement 1
Generation of wild type, fancm, zip4 or fancm zip4 I3bc/++ plants with varying patterns of Col/Ct heterozygosity.

Diagram showing the crossing scheme used to generate plants to investigate the impact of the Col/Ct heterozygosity on crossover interference. Genotypes differing in polymorphism pattern for crosses …

https://doi.org/10.7554/eLife.03708.040

Tables

Table 1

Correlations between historical recombination and sequence diversity at varying physical scales

https://doi.org/10.7554/eLife.03708.004
Scale (π)Chr1Chr2Chr3Chr4Chr5
5 kb0.5210.3010.5450.5750.541
10 kb0.5560.3050.5650.6020.562
50 kb0.6570.3810.5790.6920.619
100 kb0.6990.5630.6010.7440.646
500 kb0.7410.5280.6150.8410.653
1 Mb0.6390.5040.6830.8460.624
Scale (θ)Chr1Chr2Chr3Chr4Chr5
5 kb0.5370.2980.5570.5850.553
10 kb0.5690.3030.5760.6100.572
50 kb0.6620.3820.5920.6990.623
100 kb0.7100.5730.6170.7520.650
500 kb0.7540.5340.6350.8440.655
1 Mb0.6470.5040.6970.8490.635
  1. Spearman's rank correlation between historical crossover frequency estimates from LDhat and sequence diversity (θ and π) at varying physical scales (Cao et al., 2011; Choi et al., 2013). Adjacent windows of the indicated physical size were used for correlations.

Table 2

Fluorescent crossover reporter intervals

https://doi.org/10.7554/eLife.03708.005
IntervalChrMethodT-DNA 1T-DNA 2MbLocationcM/Mb (Col-0)cM/Mb (F1)Heterozygosity
I1b1Pollen3,905,441-YFP5,755,618-dsRed21.85Interstitial4.254.051.93 (3.16)
I1c1Pollen5,755,618-dsRed29,850,022-CFP4.09Interstitial4.55N/A2.80 (3.16)
I1fg1Pollen24,645,163-YFP25,956,590-dsRed21.31Interstitial6.206.022.52 (3.16)
I2a2Pollen12,640,092-CFP13,226,013-YFP0.59Interstitial5.19N/A2.33 (3.30)
I2b2Pollen13,226,013-YFP14,675,407-dsRed21.45Interstitial3.09N/A1.53 (3.30)
I2f2Pollen18,286,716-dsRed218,957,093-YFP0.67Sub-telomeric13.0217.411.43 (3.30)
4203Seed256,516-GFP5,361,637-dsRed25.11Sub-telomeric3.702.931.19 (3.37)
CEN33Pollen11,115,724-YFP16,520,560-dsRed25.40Centromeric2.112.386.69 (3.37)
I3b3Pollen498,916-CFP3,126,994-YFP2.63Sub-telomeric5.99N/A1.11 (3.37)
I3c3Pollen3,126,994-YFP4,319,513-dsRed21.19Sub-telomeric4.01N/A1.64 (3.37)
I5c5Pollen2,372,623-CFP3,760,756-YFP1.39Interstitial4.01N/A1.01 (3.27)
I5d5Pollen3,760,756-YFP5,497,513-dsRed21.74Interstitial3.20N/A1.56 (3.27)
  1. The interval name is listed together with chromosome, method of scoring and location of the flanking T-DNAs together with the fluorescent proteins they encode. Interval cM/Mb values from Col-0 homozygous are listed (Col-0), in addition to the mean cM/Mb observed across all F1 crosses (F1). Heterozygosity values were calculated using pairwise comparison of polymorphism data from the 19 genomes project to the Col reference (Gan et al., 2011), and the mean value for the interval shown, in addition to the mean chromosome value in parentheses.

Table 3

Genetic distance in F1 heterozygotes

https://doi.org/10.7554/eLife.03708.006
AccessionLocationI1bI1fgI2f420CEN3TotalP
Tsu-0Tsushima, Japan6.66.36.914.59.443.7<2.00 × 10−16
Hi-0Hilversum, Netherlands6.86.96.913.69.643.8<2.00 × 10−16
Wil-2Vilnius, Lithuania6.16.96.115.910.145.0<2.00 × 10−16
Kn-0Kaunas, Lithuania7.46.68.015.58.746.2<2.00 × 10−16
Ler-0Gorzow, Poland6.68.27.612.311.946.6<2.00 × 10−16
Ws-0Vassilyevichy, Belarus6.77.710.213.09.046.6<2.00 × 10−16
No-0Nossen, Germany7.47.96.714.111.447.4<2.00 × 10−16
Wu-0Wurzburg, Germany7.66.39.514.011.448.8<2.00 × 10−16
Zu-0Zurich, Switzerland7.57.113.412.29.950.10.0438
Po-0Poppelsdorf, Germany7.27.99.115.810.951.00.000484
Ct-1Catania, Italy7.88.77.215.912.151.79.27 × 10−08
Oy-0Oystese, Norway7.78.48.515.712.552.80.969
Rsch-4Rschew, Russia7.96.810.715.212.453.10.505
Col-0Columbia, USA8.08.28.818.011.554.5
Sf-2San Feliu, Spain8.28.87.418.612.355.30.724
KasKashmir, India6.98.613.213.813.355.8<2.00 × 10−16
KondPugus, Tajikistan7.18.115.813.711.456.2<2.00 × 10−16
Edi-0Edinburgh, Scotland8.08.013.413.313.656.3<2.00 × 10−16
Bay-0Bayreuth, Germany8.68.311.318.611.558.3<2.00 × 10−16
Mt-0Martuba, Libya9.67.813.220.69.660.8<2.00 × 10−16
ShaPamiro-Alaya, Tajikistan7.87.520.07.018.660.90.0012
C24Columbia, USA8.88.518.512.114.161.9<2.00 × 10−16
Bur-0Burren, Ireland6.79.121.914.717.870.2<2.00 × 10−16
Cvi-0Cape Verde Islands9.110.011.312.627.670.7<2.00 × 10−16
Can-0Las Palmas, Canary Isles7.88.522.112.431.482.2<2.00 × 10−16
CoCoimbra, Portugal11.113.8
Nw-0Neuweilnau, Germany14.714.4
Mh-0Szczecin, Poland14.910.1
Wl-0Wildbad, Germany17.09.5
Bu-0Burghaun, Germany28.98.8
CIBC5Ascot, United Kingdom13.211.3
RRS7North Liberty, USA17.211.7
F1 cM mean7.67.911.515.012.954.8
cM StDev0.80.94.83.64.99.3
  1. The accessions crossed to are listed with their geographic location. Genetic distance (cM) data is shown for the five fluorescent intervals, in addition to a summed total. Also shown are the mean and standard deviation for all F1s. A generalized linear model (GLM) was used to test for significant differences between total recombinant vs non-recombinant counts between replicate groups of Col-0 homozygotes and F1 heterozygotes. Tests were performed for genotypes where data from all five tested intervals had been collected.

Table 4

F1 heterozygosity levels relative to Col-0

https://doi.org/10.7554/eLife.03708.016
AccessionChr 1I1bI1fgChr 2I2fChr 3420CEN3Chr 4Chr 5
Bur-03.351.863.623.601.513.581.576.203.893.16
Can-03.752.993.513.920.923.981.028.275.344.24
Ct-12.621.672.292.611.853.350.966.913.233.36
Edi-03.301.913.643.260.913.051.345.483.423.81
Hi-02.431.591.871.801.502.581.074.622.692.46
Kn-03.151.782.853.352.183.581.496.693.763.40
Ler-03.101.612.663.622.243.431.137.393.873.53
Mt-03.021.771.163.491.573.171.075.703.952.71
No-03.252.282.713.361.273.521.217.143.513.56
Oy-03.481.682.103.050.582.941.236.162.952.72
Po-02.451.781.192.360.672.870.795.992.532.59
Rsch-42.941.841.173.361.223.091.055.373.893.22
Sf-23.611.944.243.542.063.741.308.243.813.58
Tsu-03.371.682.363.691.393.981.148.783.693.05
Wil-23.561.992.453.772.113.811.567.554.443.34
Ws-03.251.933.543.681.583.301.306.653.703.41
Wu-03.132.531.953.140.673.501.227.413.363.15
Zu-03.101.852.023.831.433.190.965.843.383.64
Mean3.161.932.523.301.433.371.196.693.633.27
Correlation (cM)0.13 (p = 0.61)0.47 (p = 0.05)−0.29 (p = 0.23)0.06 (p = 0.81)0.28 (p = 0.26)
  1. Accessions sequenced as part of the 19 genomes project were analysed (Gan et al., 2011) and heterozygosity calculated as the sum of SNPs and indel lengths divided by the length of region (kb). Correlations were between heterozygosity within the interval measured and F1 cM measurements.

Table 5

Chromosome 3 genotype counts from hot and cold quartile 420/++ Col/Ct F2 individuals

https://doi.org/10.7554/eLife.03708.022
Marker coordinates (bp)Hot quartile HETHot quartile HOMCold quartile HETCold quartile HOMFDR p value
2590003403401
27180003403401
53520003403401
637500021133404.36 × 10−04
694800017173311.05 × 10−04
767400015193312.12 × 10−05
849500012223403.65 × 10−07
94040008263313.79 × 10−08
106950008263041.36 × 10−06
1164900011232774.36 × 10−04
1235600011232774.36 × 10−04
15949000132123114.48 × 10−02
19165000171721130.591
23040000132117170.591
  1. The number of 420/++ Col/Ct F2 individuals showing Col homozygosity (HOM) or Col/Ct heterozygosity (HET) for the indicated marker positions, in either the hottest or coldest F2 quartile. The p value was obtained by performing a chi square test between homozygous and heterozygous marker genotype counts in the hottest and coldest quartiles (2x2 contingency table), followed by FDR correction for multiple testing.

Table 6

Chromosome 2 genotype counts from hot and cold quartile I2f/++ Col/Ct F2 individuals

https://doi.org/10.7554/eLife.03708.023
Marker coordinates (bp)Hot quartile HETHot quartile HOMCold quartile HETCold quartile HOMFDR p value
132,0009118121
2,346,0007138121
4,748,0008129111
6,789,0007131190.63
11,443,0005152006.26 × 10−05
13,036,0007132003.32 × 10−04
14,117,0009112001.30 × 10−03
15,240,0009112001.30 × 10−03
16,909,0001372000.0262
17,439,0001642000.238
18,287,0002002001
18,960,0002002001
19,311,0001822000.764
  1. The number of I2f/++ Col/Ct F2 individuals showing Col homozygosity (HOM) or Col/Ct heterozygosity (HET) for the indicated markers, in either the hottest or coldest F2 quartile. The p value was obtained by performing a chi square test between homozygous and heterozygous marker genotype counts in the hottest and coldest quartiles (2 × 2 contingency table), followed by FDR correction for multiple testing.

Table 7

Chromosome 3 genotype counts from hot and cold quartile CEN3/++ Col/Ct F2 individuals

https://doi.org/10.7554/eLife.03708.028
Marker coordinates (bp)Hot quartile HETHot quartile HOMCold quartile HETCold quartile HOMFDR P
259000161417131
2718000161418121
5352000191117131
7674000201012180.129
849500023713170.0389
940400026416140.0308
111157243003001
165205603003001
2100800027314160.00477
2207600023712180.0308
2304000024610200.00477
  1. The number of CEN3/++ Col/Ct F2 individuals showing Col homozygosity (HOM) or Col/Ct heterozygosity (HET) for the indicated markers, in either the hottest or coldest quartile. The p value was obtained by performing a chi square test between homozygous and heterozygous marker genotype counts in the hottest and coldest quartiles (2 × 2 contingency table), followed by FDR correction for multiple testing.

Table 8

Tetrad FTL cM data in Col/Col and Col/Ler backgrounds

https://doi.org/10.7554/eLife.03708.041
Col/ColCol/Ler
IntervalPDNPDTcM*PDNPDTcM*
1b397637428.05 ± 0.29439526526.58 ± 0.25
1c302211169518.62 ± 0.04315618189119.73 ± 0.04
2a678724303.06 ± 0.15592002832.28 ± 0.13
2b658226354.48 ± 0.18579604073.28 ± 0.16
3b436322255719.37 ± 0.3527582105613.99 ± 0.38
3c618557365.53 ± 0.21357622383.28 ± 0.22
5c535616665.58 ± 0.21545806765.51 ± 0.20
5d535816645.56 ± 0.21554025944.94 ± 0.20
  1. *

    Map distance in cM (±S.E.).

  2. Significant difference in map distance in the heterozygous Col/Ler background compared to the same interval in the Col/Col homozygous background.

Table 9

Tetrad FTL crossover interference data in Col/Col and Col/Ler backgrounds

https://doi.org/10.7554/eLife.03708.042
Col/ColCol/Ler
IntervalW/o adj. CO*w/ adj. CO*R1W/o adj. CO*w/ adj. CO*R2
1b10.69 ± 0.403.31 ± 0.303.239.78 ± 0.371.22 ± 0.188.04§
1c20.61 ± 0.457.92 ± 0.762.622.13 ± 0.463.52 ± 0.506.29§
2a3.20 ± 0.161.18 ± 0.302.752.42 ± 0.140.37 ± 0.216.55
2b4.65 ± 0.191.74 ± 0.442.683.41 ± 0.160.53 ± 0.306.44
3b20.84 ± 0.376.95 ± 0.822.314.73 ± 0.402.92 ± 0.765.05
3c7.65 ± 0.301.90 ± 224.034.28 ± 0.300.66 ± 0.186.46
5c5.87 ± 0.233.23 ± 0.471.825.85 ± 0.222.35 ± 0.432.49
5d5.85 ± 0.233.22 ± 0.481.825.29 ± 0.222.07 ± 0.382.56
  1. *

    Map distances in cM (±S.E.) for intervals with and without adjacent crossovers (CO).

  2. Ratios of map distances for intervals with and without adjacent crossovers in homozygous Col/Col (R1) and heterozygous Col/Ler (R2) backgrounds.

  3. Significant difference in map distances in intervals when adjacent interval does or doesn't have a CO.

  4. §

    Significant difference between R2 and R1.

Additional files

Supplementary file 1

Oligonucleotides used to genotype Col-0/Ct-1 polymorphisms.

https://doi.org/10.7554/eLife.03708.043

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