lorinda anderson colorado state university fort collins, co
TRANSCRIPT
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
Fre
quen
cy
0
5
10
15
20
25
cum
ulat
ive
freq
uenc
y
0.0
0.2
0.4
0.6
0.8
1.0
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
0.6
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
5
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