Lorinda Anderson Colorado State University

Fort Collins, CO

Meiotic Crossover (CO) AssaysMeiotic Crossover (CO) AssaysGenetic Cytological

chiasmata MLH1 foci

RecombinationNodules (RNs)

Lateralelement

Centralelement

Transversefilaments

Chromatinloops

Recombination nodule (RN)

RN = CO

Crossing Over During MeiosisCrossing Over During Meiosis1. Each pair of homologous chromosomes

(bivalent) needs at least one crossover to assure proper segregation at anaphase I. Obligate crossover.

maize

Crossing Over During MeiosisCrossing Over During Meiosis1. Each pair of homologous chromosomes (bivalent) needs at least one

crossover to assure proper segregation at anaphase I.

2. Crossovers are not distributed evenly on chromosomes.

Tomato SC1 RN distribution

Chromosome (SC) length (um)

0 2 4 6 8 10 12 14 16 18 20 22 24 26

RN

freq

uenc

y

0

2

4

6

8

10

12

14

16

cum

ulat

ive

cM

0

20

40

60

80

100

120

Chang et al. 2007 Genetics

Crossing Over During MeiosisCrossing Over During Meiosis1. Each pair of homologous chromosomes (bivalent) needs at least one crossover to

assure proper segregation at anaphase I.

2. Crossovers are not distributed evenly on chromosomes.

3. Crossovers display genetic interference (Sturtevant 1915; Muller 1916). The presence of one crossover reduces the likelihood of another crossover nearby.

X X X X

X X X X

Distance between COs

Without Interference

With Interferencefr

eq

ue

ncy

fre

qu

en

cy

Crossing Over During MeiosisCrossing Over During Meiosis1. Each pair of homologous chromosomes (bivalent) needs at least one crossover to

assure proper segregation at anaphase I.

2. Crossovers are not distributed evenly on chromosomes.

3. Crossovers display genetic interference (Sturtevant 1915; Muller 1916). The presence of one crossover reduces the likelihood of another crossover nearby.

X X X X

X X X X

Distance between COs

Without Interference

With Interferencefr

eq

ue

ncy

fre

qu

en

cy

Crossing Over During Meiosis

1. Each pair of homologous chromosomes (bivalent) needs at least one crossover to assure proper segregation at anaphase I.

2. Crossovers are not distributed evenly on chromosomes.

3. Crossovers display genetic interference. The presence of one crossover reduces the likelihood of another crossover nearby.

4. There are several pathways for crossing over. In plants, animals, and yeast:

- Major pathway = interfering Class I COs - MLH1

- Minor pathways = non-interfering Class II COs - MUS81

Mouse spermatocyteSCs = whiteMLH1 = red

Tomato microsporocyteSCs = redMLH1 = green

Arabidopsis (Pradillo et al., 2014) 100’s of DSBs

10’s of COs

Arabidopsis (Pradillo et al., 2014) 100’s of DSBs

10’s of COs

Aberrant intermediates

1. Mutants:- Arabidopsis mlh3 mutants have residual crossovers (Jackson et al. 2006).

- Arabidopsis mus81 mutants have increased interference (Berchowitz et al. 2007)

Double strand breaks

Pathway 1 (80%) COsInterferingProduces the obligatory CO

Pathway 2 (20%) COsNon-interfering

MUS81MLH1/MLH3

2. Mathematical modeling: - Linkage maps in Arabidopsis (Copenhaver et al. Genetics 2002)

- RN positions in maize (Falque et al. Plant Cell 2009)

Den

sity

of

pairs

Two-pathways model

Single-pathway model

No interference model

Maize chr. 10

Distance between successive COs

Modeling infers about 15% of COs in Maize are Class II

3. Cytological observations: RN maps available for tomato (Sherman and Stack 1995). MLH1 foci mapped and compared to RN map (Lhuissier et al. 2007).

Average number of MLH1 foci is lower than the average number of RNs.

kc

SCMLH1

kc

This comparison indicated that most, but not all, RNs are MLH1-positive in tomato.

Other RNs = class II COs?

3. Cytological observations: Lhuissier et al. 2007EM: Immunogold shows some RNs with MLH1 and

some RNs without MLH1

Gold beads mark MLH1 protein No gold beads on

this RN

Remaining quantitative questions in wild-type organisms:

-How are the two classes of crossovers (especially class II COs)

specifically distributed along chromosomes?

-Do the two CO classes interact with each other? If so, how does

this affect interference relationships?

Different approaches (mutants, mathematical modeling, cytology) are all consistent plants have two classes of COs with different characteristics.

Remaining quantitative questions in wild-type organisms:

-How are the two classes of crossovers (especially class II COs)

specifically distributed along chromosomes?

-Do the two CO classes interact with each other? If so, how does

this affect interference relationships?

Different approaches (mutants, mathematical modeling, cytology) are all consistent plants have two classes of COs with different characteristics.

Answering these questions requires mapping

individual COs by class in the same samples.

1.Linkage mapping – cannot distinguish between the 2 types of COs.

2.Mutants – relative proportions (and distributions) can be

different if one or the other pathway is missing. e.g. mus81-/-

mice have increases in MLH1 foci without a corresponding

change in chiasma counts (Holloway et al. 2008).

3.Cytological – very labor-intensive, limited sample sizes, but still…

1.Lhuissier et al. 2007 compared numbers and distributions of MLH1 foci (class I COs) with those of RNs (all COs), but on different samples from different labs.

2.To investigate interference between class I and class II COs, both types must be observed on the same SC samples.

1. Plan: Label with antibodies to MUS81 and MLH1 at the same time.

MUS81

1. Plan: Label with antibodies to MUS81 and MLH1 at the same time.

No correspondence between MUS81 foci and RNs

MUS81

1. Plan: Label with antibodies to MUS81 and MLH1 at the same time.

Immunogold: - Perfect for a few examples - Too laborious and expensive for quantitative analyses - Lack of sensitivity for weak foci

RN labeled with MLH1 & 5 nm gold

Cytological approach:Cytological approach:

MLH1 immunolabeling to identify Class I COs and EM to identify all COs (RNs). Unlabeled RNs ~ Class II COs

Immunogold: - Perfect for a few examples - Too laborious and expensive for quantitative analyses - Lack of sensitivity for weak foci

RN labeled with MLH1 & 5 nm gold

Cytological approach:Cytological approach:

We developed a new correlative approach that uses both immunofluorescence LM and EM.

MLH1 immunolabeling to identify Class I COs and EM to identify all COs (RNs). Unlabeled RNs ~ Class II COs

Faster, more sensitiveMLH1+ RNs = Class I

All RNs

- Cherry Tomato microsporocytes collected at pachytene of Meiosis I, and SC spreads prepared on plastic-coated slides.

- Fluorescent immunolabeling of SMC1 (marks lateral element of SC in red) and MLH1 (marks class I COs in green).

- Imaging with fluorescence light microscopy (LM).

Correlative microscopy (LM to EM) procedureCorrelative microscopy (LM to EM) procedure

SMC1MLH1

- Cherry Tomato microsporocytes collected at pachytene of Meiosis I, and SC spread on plastic-coated slides.

- Fluorescent immunolabeling of SMC1 (marks lateral element of SC in red) and MLH1 (marks class I COs in green).

- Imaging with fluorescence light microscopy (LM).

- Cover glass removed, and slides stained with phosphotungstic acid (PTA).

- Phase contrast microscopy used to identify the correct SC spreads and place grids over them, then plastic lifted from the slides onto grids for EM.

- Electron microscope (EM) imaging of the same SCs at 3,000× magnification. Annotation of RNs and kinetochores (to identify SCs).

Correlative microscopy (LM to EM) procedureCorrelative microscopy (LM to EM) procedure

kk

k

k

k

k

k

k

k

k

kk

k = kinetochoreR = recombination nodule

twists

RN CE

LEs

- Cherry Tomato microsporocytes collected at pachytene of Meiosis I, and SC spread on plastic-coated slides.

- Fluorescent immunolabeling of SMC1 (marks lateral element of SC in red) and MLH1 (marks class I COs in green).

- Imaging with fluorescence light microscopy (LM).

- Cover glass removed, and slides stained with phosphotungstic acid (PTA).

- Phase contrast microscopy used to identify the correct SC spreads and place grids over them then plastic lifted from the slides onto grids for EM.

- Electron microscope (EM) imaging of the same SCs at 3,000× magnification. Annotation of RNs and kinetochores (to identify SCs).

- Fluorescent image layered over the EM image.

- Each previously identified RN was assessed for MLH1 fluorescent signal.

- 2955 RNs from more than 1800 SCs were identified by class and mapped onto SCs that were identified by relative length and arm ratio

Correlative microscopy (LM to EM) procedureCorrelative microscopy (LM to EM) procedure

MLH1-MLH1+

20

15

10

5

0

Num

ber

of R

Ns

per

set

Mean = 15.3 (82%)

Mean = 3.5 (18%)

Proportions of MLH1+ and MLH1- RNsProportions of MLH1+ and MLH1- RNsN = 150 nuclei (complete sets of SCs)

Average = 18.8 RNs per nucleus 15.3 MLH1+ RNs (82%) 3.5 MLH1- RNs (18%)

~ 75% of all SCs have no MLH1- RNs

MLH1+ RNs and MLH1- RNs and ChiasmataMLH1+ RNs and MLH1- RNs and Chiasmata

Predict chiasma frequency from RNs:

1 RN = 1 CO = 1 chiasma1 or more RNs per arm ~ 1 chiasma

Rod = 1Ring = 2

MLH1+ and MLH1- RNs and ChiasmataMLH1+ and MLH1- RNs and Chiasmata

Chiasmata per nucleus

Observed = 16.0

Predicted

All RNs = 16.4

MLH1+ = 14.6

Predict chiasma frequency from RNs:

1 RN = 1 CO = 1 chiasma1 or more RNs per arm ~ 1 chiasma

Rod = 1Ring = 2

MLH1+ and MLH1- RNs and ChiasmataMLH1+ and MLH1- RNs and Chiasmata

Chiasmata per nucleus

Observed = 16.0

Predicted

All RNs = 16.4

MLH1+ = 14.6

Predict chiasma frequency from RNs:

1 RN = 1 CO1 or more RNs per arm = 1 chiasma

Rod = 1Ring = 2

Confirms:MLH1- RNs = CO

5

1 2

3

4

6

Are there other differences between R+ and R-?Are there other differences between R+ and R-?

1 2

3

4

5

6

RN size comparison

Length (m)

0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35

Wid

th ( m

)

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

0.22MLH1-positive RNsMLH1-negative RNs

Size difference between MLH1+ and MLH1- RNsSize difference between MLH1+ and MLH1- RNs

RN Class No. Obs.

Ave. Length (nm)

Ave. Width (nm)

MLH1 + 669 157 ± 36 93 ± 24

MLH1 - 140 127 ± 41 81 ± 23

t-test p < 0.001 p < 0.001

tomato SC1

Fraction of SC length

0.0 0.2 0.4 0.6 0.8 1.0

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quen

cy

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Distribution of MLH1+ and MLH1- RNsDistribution of MLH1+ and MLH1- RNs158 SCs332 RNs

2.1 RNs / SC 1.7 MLH1+ 0.4 MLH1-

(tot SC length=31 µm)

The shapes of the distributions for MLH1+ and MLH1- RNs are significantly different for all chromosomes Kolmogorov-Smirnov test p < 0.04

tomato SC1

Fraction of SC length

0.0 0.2 0.4 0.6 0.8 1.0

Fre

quen

cy

0

5

10

15

20

25

cum

ulat

ive

freq

uenc

y

0.0

0.2

0.4

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0.8

1.0

Distribution of MLH1+ and MLH1- RNs158 SCs332 RNs

2.1 RNs / SC 1.7 MLH1+ 0.4 MLH1-

(tot SC length=31 µm)

~45%

~22%

For most tomato chromosomes: MLH1- RNs are disproportionately located

in pericentromeric regions (heterochromatin) and in short arms.

MLH1+ RNs are disproportionately observed in long arms.

MLH1- RNs are disproportionately located in pericentromeric regions and in short arms of acrocentric tomato chromosomes.

Distribution of MLH1+ and MLH1- RNs

~60%

~10%

Why is this important?

fancm and recQ A/B mutants increase class II COs in Arabidopsis – (Crismani et al. 2012; Seguela-Arnaud et al. 2015)Plant breeding applications: It may be possible to specifically increase CO in low recombination regions to mobilize genes present around the centromere.

SC length (2% intervals)

SC5/12

0.0 0.2 0.4 0.6 0.8 1.00

5

10

15

20

25

0.0

0.2

0.4

0.6

0.8

1.0SC11

0.0 0.2 0.4 0.6 0.8 1.00

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10

15

20

25

0.0

0.2

0.4

0.6

0.8

1.0

Metacentric chromosomes differ from acrocentric chromosomes.Less difference between MLH1+ and MLH1- distributions.

What could explain differences in arm preferences?What could explain differences in arm preferences?

Chr. No. obs.

Long arm synapsed

first

Expected ratio (L/S)

Observed ratio(L/S)

9 31 77% 1.8 3.4

10 124 80% 2.1 4.0*

12 54 56% 1.05 1.25

ShortArmLong Arm

Ch 9 Ch 12

Synaptic patterns:Which arm synapses first?

What could explain differences in arm preferences?What could explain differences in arm preferences?ShortArmLong Arm

Ch 9 Ch 12

Long arms synapse first – higher frequency of MLH1+ RNsShort arms synapse later – higher frequency of MLH1- RNs

Heterochromatin synapses last – higher frequency of MLH1- RNs

Interference: Interference: Numbers of MLH1+ and MLH1- RNs per nucleus

Observed and Expected (Poisson) frequencies of MLH1-positive and MLH1-negative RNs per SC set

Number of MLH1-positive RNs per SC set

0 5 10 15 20 25 30

Fre

quen

cy

0.00

0.05

0.10

0.15

0.20

0.25MLH1+ LNs - obsMLH1+ LNs - expMLH1- LNs - obsMLH1- LNs - exp

Observed and expected (if no interference) frequencies of MLH1+ and MLH1- RNs per SC set

Number of RNs per nucleus

ML

H1

-po

sitiv

e R

Ns

ML

H1

-ne

gativ

e R

Ns

nu = 1 No significant interference

nu > 1 Significant positive interference

The parameter nu is a quantitative measurement of interference strength

[nu-Inf ; nu-Sup] : 95% confidence interval

InterferenceInterference: Using distance between COs to evaluate interference

(Gamma model)

InterferenceInterference: Among pairs of MLH1+ RNs

Inhibition up to about 13 µm

Same data shuffled to remove interference effects

Den

sity

of i

nter

-CO

dis

tanc

es

Interference due to MLH1+ / MLH1+ interactions

Inhibition up toabout 8 µm

Den

sity

of i

nter

-CO

dis

tanc

es

Inhibition up to about 13 µm

Den

sity

of i

nter

-CO

dis

tanc

es

Interference due to MLH1+ / MLH1+ interactions

Interference due to MLH1+ / MLH1- interactions

InterferenceInterference:

SummarySummary

In wild-type tomato, MLH1+ (class I COs) and MLH1- RNs (class II COs) differ in:

1.Numbers: MLH1-dependent COs represent about 82% of all COs.

2.Size: MLH1-positive RNs are larger than MLH1- RNs. Protein composition.

3.Distribution: Class I COs are disproportionately located in long arms while Class II COs are disproportionately located in pericentromeric regions (heterochromatin) and in short arms.

a. Associated with patterns of synapsis

b. Possible importance to plant breeding: fancm and recQ A/B mutants increase class II COs in Arabidopsis

4.Interference:

1. Class I COs interfere with each other (up to ~13 µm SC).

2. Class II COs do not interfere between themselves.

3. Class I COs and class II COs interfere but less strongly (up to ~8 µm SC).

RN

Inactivated recomb.Intermed. - NCO

DSB

recombination Intermediate

cent

rom

eres

CO

MLH1+ CO (I)

MLH1- CO (II)

Short arm Long arm

Long arm/Short arm differences

“Shotgun” model for crossing over

Protein complex (EN)

“Shotgun” model for crossing over

RNs

Inactivated recomb.Intermed. - NCO

DSB

recombination Intermediate

cent

rom

eres

CO

MLH1+ CO (I)

MLH1- CO (II)

Aberrant recomb.Intermed.

Short arm Long arm

Heterochromatin difference

Protein complex (EN)

Funding :

Quantitative Genetics and Evolution – Le Moulon Gif-sur-Yvette, France

Sayantani Basu-Roy Matthieu Falque Olivier Martin

Dept of Biology, Colorado State UniversityFort Collins, CO

Lorinda K. AndersonLeslie D. LohmillerXiaomin TangD. Boyd HammondLauren JavernickLindsay Shearer

Stephen Stack

Thank you for your attention…

For SCs with only two RNs:

Distribution of MLH1+ and MLH1- RNs differs from distribution of two MLH1+ RNs

MLH1-negative RNs represent class II COsMLH1-negative RNs represent class II COs

MLH1-negative RN

MLH1-positive RN

Most SCs (75%) have no MLH1- RNs.

MLH1 focus size variability is not related to RN size.

Zakharyevich et al. 2012 (yeast)

MLH1

FANCM

MLH1-MLH3Complex

Class I COs

MUS81Complex-

Class II COs

SMC1MLH1

LM to EM

Lateral element

Recombination nodule (RN)

on the synaptonemal complex (SC) RN = CO