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Animal Cytogenetics
Valerie Fillon
To cite this version:
Valerie Fillon. Animal Cytogenetics. Master. European Advanced Postgraduate Course in Classical and Molecular Cytogenetics, 2018. �hal-02788100�
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Animal Cytogenetics
GENPHYSE – INRA Toulouse - France
Valérie Fillon
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Toulouse
GENPHYSE – Cytogenomic team
I
nstitut de
R
echerche
N
ational
A
gronomique
*The laboratory is involved in the structural and functional analysis
of the genome of farm animal species
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Valérie Fillon / ECA 2014 10 / 03 / 2014
I. General overview
II. Chromosomal abnormalities
III. Cytogenetic mapping : lessons from
chicken
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Valérie Fillon / ECA 2014 10 / 03 / 2014
I. General overview
- History
- Technical aspects
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History :
•Beginning in the 1960’s
•1964 identification of the Rob 1/29 and a reciprocal translocation
in pigs (Gustavsson et Rockborn, 1964) (Enricsson et Rockborn, 1964)
•In the 1970’s development of the banding techniques and
establishment of the first standardized karyotypes
•Association between the chromosomal abnormalities and
reproduction troubles → establishment of animal cytogene cs
laboratories (mainly in Europe)
•Since the beginning of the 90’s → decline of clinical animal
ac vi es → gene mapping
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Valérie Fillon / ECA 2014 10 / 03 / 2014
I. General overview
- History
- Technical aspects
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Cell cultures with a high mitotic rate
Fibroblastes Lymphocytes
Arrested at the metaphase stage with colcemid Hypotonic treatment of the cells
0,075 M KCl
Fetal calf serum : water (1:5)
Fixation acetic acid : ethanol (1:3) (methanol) Preparation of slides : high quality preparations !
Preparation of
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Fluorescent In Situ Hybridization
FISH
Hybridation
In situ
Probes
Chromosome preparations
- DNA (>10 kb) : BAC - Labelling (biotine) - purification, resuspension - Cell cultures - Metaphase arrest - Cellular treatment - Spreading - denaturation - hybridization (24h) - washing - staining B B B chromosome probe
10 Mb
probeResolution > 1 Mb
Chromosome assignation Measurment
Analysis under the microscope
1 2 3 4
5
6 7 8
5
Chromosome arms 8p and 8q generated by microdissection
A. Pinton (unpublished data) Inv(8)(p1.1;q2.5)
Toward CGH array...
But - Most of the abnormalities are equilibrate
- Quality of the reference genome
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Valérie Fillon / ECA 2014 10 / 03 / 2014
I. General overview
- History
- Technical aspects
.015
Species Scientific name 2N
Human Homo sapiens 46
Rhesus Monkey Macaca mulata 42
Bovine Bos taurus 60
Pig Sus scrofa domestica 38
Horse Equus cabalus 64
Donkey Aquus asinus 62
Chicken Gallus domesticus 78 Rabbit Oryctolagus cuniculus 44 Rat
Souris Rattus norvegicusMus musculus 4240
Dog Canis familiaris 78
Cat Felis domesticus 38
A L I M E N T A T I O N A G R I C U L T U R E
E N V I R O N N E M E N T
Trypsin digestion
.017
Cattle 2n=60 Goat 2n=60 Sheep 2n=54
International System for Chromosome
Nomenclature of Domestic Bovids (ISCNDB 2000)
D. Di Berardino, G.P. Di Meo, D.S. Gallagher, H. Hayes, L. Iannuzzi(coordinator) Cytogenet Cell Genet 92:283–299 (2001)
.018 Indian Munjac Reeves's Munjac 2n = 46 2n = 6 2n = 7
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Horse 2n = 64
Chicken karyotype 2n=78
Females are heterogametic ZW
Presence of 30 pairs of microchromosomes = 30% of the genome
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Harmonia axyridis 2n = 16
Asian ladybeetle
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Valérie Fillon / ECA 2014 10 / 03 / 2014
II. Chromosomal abnormalities
- Clinical cytogenetics
- Some examples in horse, pig and cattle
- Segregation during meiosis
•Few laboratories principally in Europe carry out systematic
cytogenetics controls (see Ducos et al., 2008)
•These controls concern mainly the bovine and pig species (Ducos et
al., 2008)
•As in Human, chromosomal abnormalities can be responsible of
congenital abnormalities, embryonic loss, infertility, cancer
significant economic losses
•Between 8,000 to 10,000 chromosomal analyses carried out each
year in livestock species ( meanly cattle, pigs)
•These analyses generally concern phenotypically normal
individuals
•Abnormal individuals are eliminated by the breeders
•Chromosomal abnormalities are generally balanced
•reproduction troubles
•Spermatogenesis impairments oligo- azoospermia
•Reproductive failure due to imbalanced gametes
in pigs, decrease of 41% on average of the litter size for
reciprocal translocations
•Pigs : 1700 to 2000 per year
0 2 4 6 8 10 12 14 16 18 0 500 1000 1500 2000 2500 3000 19 91 19 92 19 93 19 94 19 95 19 96 19 97 19 98 19 99 20 00 20 01 20 02 20 03 20 04 20 05 20 06 20 07 20 08 20 09 20 10 20 11 annéeNombre d'analyses Nombres d'anomalies de structure
Results of French chromosomal control
•Cattle : 700 per year
•By January 2018, 37 800 pigs have been analyzed
>90% young purebred boars controled before reproduction (AI)
Prevalence of structural chromosomal rearrangement: 0.47% 1/200 boars
(Pinton et al., 2011)
•182 structural chromosomal abnormalities (de novo)
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Valérie Fillon / ECA 2014 10 / 03 / 2014
II. Chromosomal abnormalities
- Clinical cytogenetics
- Some examples in horse, pig and cattle
- Segregation during meiosis
•Sex chromosome abnormalities
X Y
Y
63, X0 / 65, XYY mosaicism in a case of equine male pseudohermaphroditism
Paget et al. (2001)
N der(3) N der(16) N der(3) N der(16) N der(3) N der(16) N N der(16) N N N der(16) N
Offspring with unbalanced karyotype
2n=38, XX (ou XY), der16 t(3;16)(q23;q22)
Translocated boar
t(3;16)(q23;q22) and
palatoschisis (cleft palate) Ducos et al., 2004
•Rcp(6;8)(10;18)
•Rcp(Y;1)
Inv(2)(q1.3;q2.5) q1.1 q2.1 q2.4 q2.6 inverted normal Normal SSC 2 Inverted SSC 2
K. Massip (unpublished data)
•Cattle
Rob 1/29 translocation
0 0,05 0,1 0,15 0,2 0,25 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 year 1/ 29 fr eq ue nc y•700 animals analyzed annualy
•Genotyping test
•Cattle
Other abnormalities
•Rcp(8;9)
•Rcp(1;15)
•Rcp(7;7) mosaic 10.7%•Inv (29)
•2n=60,XY; 61,XXY (16%)
BAC IDVGA7 on BTA29
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Valérie Fillon / ECA 2014 10 / 03 / 2014
II. Chromosome abnormalities
- Clinical cytogenetics
- Some examples in horse, pig and cattle
- Segregation during meiosis
•Male meiosis : SpermFISH (hybridization of probes on
decondensed sperm nuclei)
Analysis of the meiotic segregation pattern : estimation of the
effects on the reproduction (decrease of fertility or prolificacy),
and production of new knowledge
These approaches have been applied to analyze the meiotic
segregation patterns of different chromosomal abnormalities in
pigs and cattle
•Male meiosis : analysis of synaptonemal complexes
Segregation during meiosis
alternate segregation
adjacent II segregation adjacent I segregation
alternate segregation
balanced gametes
adjacent II segregation adjacent I segregation
Rcp(3;15)
Rcp(12;14)
unbalanced balanced
der(13;17)
13 17
Alternate Adjacent 3:0
1/29 translocation
balanced gametes 97.21% 74% unbalanced gametes 2.75% 4% dipoid gametes 0.04% 22% spz 1er GP Métaphase II BTA29 BTA1 BTA1-29Rate of unbalanced gametes for the heterozygote cows:
•
Smaller than expected according to the Human data•
Coherent with the decrease of fertility (5% in average)•
2 times more than in male gametes♂
♀
Paracentric inversion
balanced acentric dicentric Diploid other types
Pericentric inversion
balanced Dup(p)/del(q) Dup(q)/del(p) Diploid
(Anton et al., 2002)
Analysis of the early stages of meiosis
Analysis of the chromosome pairing and recombination at
the pachytene stage throughout immunocytology approach
•Development of antibodies specific of some meiotic
proteins :
Synaptonemal complex: SCP1, SCP3
Recombination: MLH1, MLH3….
SC electronique microscopy
Immunocytological staining
(B. de Massy, 2005)
Synaptonemal complexes studies
Zygotene Pachytene
Maternal sister chromatids
Paternal sister chromatids
Axial element central element
(B. de Massy, 2005)
Effects of chromosomal rearrangements
SSC13 SSC12 N T N T
Synaptonemal complexes studies
red SC
Blue Centromere
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Valérie Fillon / ECA 2014 10 / 03 / 2014
III. Cytogenetic mapping : lessons from chicken
- The chicken genome
- High resolution mapping
The chicken = Typical bird organisation
The chicken = Typical bird organisation
2- diploïd number : 2n = 76 to 82
3- microchromosomes : 60 à 64
1- sex chromosomes ZZ and ZW
The challenge : microchromosomes 2n = 78
60 microchr
Standard karyotype : Ladjali et al 1999 Sequenced : 2004
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Painting probes Bac clones
Maps integration
FISH
RH
Genetic map
BAC
Contigs
Sequence
Genetic map
Cytogenetic map
Molecular markers
Large insert clones
BAC
CHROMOSOME 1 6 5 4 3 2 1 5 4 3 2 1 2 1 1 2 3 4 1 2 1 2 3 4 5 6 1 2 3 4 5 1 2 3 4 bw30B21 bw31B10 bw43G6 bw25G16 bw30P7 P1H9 P6D4bw4F8 bw38E8 P3-3 P1-2 P2-9 P2-6 B3H9 P2H3 P2-5 P2-10 P4D9 B2B4 P4B11 P4D8 CHROMOSOME 2 3 2 1 43 21 2 1 1 2 1 2 3 4 5 6 1 2 3 4 56 7 3 2 1 1 2 3 bw26B13 bw14J6 bw41C2 bw6D24 bw55L19 P2E4 bw107K17 bw26A22 CHROMOSOME 3 P5D4 1 1 2 3 2 1 1 2 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 bw29L12 bw13L14 P3D3 P1-9 P5A6 CHROMOSOME 4 1 1 2 4 3 2 1 1 2 3 1 23 4 5 bw37E19 bw62D14 bw8H20bw112C24bw125P16 bw118M14 bw33G16 bw12C6 bw3K18 bw37H20 CHROMOSOME 5 1 1 2 1 1 2 3 4 1 2 3 4 5 P6C6 bw9B13 CHROMOSOME 6 1 1 2 3 4 5 6 7 1 1 bw27G19 SCD1, B1E7 bw30C21 bw124H24 bw10J13 P4G2 bw69P21 bw27C3 CHROMOSOME 7 1 1 2 1 1 2 3 4 5 6 CHROMOSOME 7 1 1 2 1 1 2 3 4 5 6 bw26M16, P4G2 bw69P21 bw27C3 bw21P13 bw8F6 CHROMOSOME 8 2 1 1 2 34 1 1 bw40L3 bw29C17 bw60M16 CHROMOSOME Z 3 4 3 21 2 1 1 2 1 2 3 4 2 1 1 2 3 bw83N24 bw71017 bw79C4 bw13E2
A L I M E N T A T I O N A G R I C U L T U R E
E N V I R O N N E M E N T
Integration of genetic and cytogenetic maps
Microchromosomes Macro-M b se qu en ce Improvement in 2016 Genome sequenced in 2004
Partialy sequenced
Microchromosomes
6 Missing and unidentified
New !
60 microchromosomes : 3 to 20 Mb (30%) GC and gene rich (50%)
24/30 with FISH markers Still ongoing !
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Valérie Fillon / ECA 2014 10 / 03 / 2014
III. Cytogenetic mapping : lessons from chicken
- The chicken genome
-
High resolution mapping
Lampbrush chromosomes
Chromosome Structure and Function Laboratory
Biological Research Institute
Saint-Petersburg State University
During spermatogenesis
Y chromosome of Drosophila transforms into lampbrush
Lampbrush chromosomes can be found in oocytes of:
fishes
amphibians
reptilia
birds
insects
Lampbrush chromosomes can be found during
Meiosis I
prophase
anaphase
telophase
leptotene
zygotene
pachytene
DIPLOTENE
diakinesis
metaphase
Lampbrush chromosomes were first seen by
Flemming W (1882)
in sections of salamander (Ambystoma mexicanum) oocytes
Rückert J (1892)
in sections of dogfish (Squalus acanthias) oocytes
Duryee WR (1937)
removal of lampbrush chromosomes from living oocytes
Kropotova EV, Gaginskaya ER (1984)
Making lampbrush chromosomes
- nucleus removal from oocytes and
adherent yolk cleaning
- lampbrush chromosome removal from nucleus
- chromosome spreading for 30 min at +4°C
Lampbrush slide- centrifugation for 15 min at 3500 g
Chicken lampbrush chromosome 1
Chicken mitotic chromosome 1
185 mm
6 mm
Lampbrush chromosomes
Chicken LBC4 and mitotic chicken chromosome 4 (100x)
Lampbrush chromosome structure
Chiasmas
Chromomeres
bivalent
Chromatides
DNA loops
Transcription units
L K J I H G F E D C 1 2 3 4 5 1 2 1 2 3 1 2 1 2 1 2 1 2 1 2 B A
Chiasma distribution along the chicken lampbrush chromosome 1
Ch
ias
m
a f
re
qu
en
cy
100%Chromosome 4 poule
High resolution cytogenetic maps
Fillon et al., 1996
B
Y
B
Y et rDNA
Microchromosome 16 is the MHC and NOR
bearing chromosome
GGA16 genetic map
Partialy sequenced
Microchromosomes
6 Missing and unidentified
New !
60 microchromosomes : 3 to 20 Mb (30%) GC and gene rich (50%)
Cytogenetic high resolution map
NOR
: near the centromere, covers 40% of the chromosome
B@
at the q end
Intégration des cartes
SEQ0097 0,0 SEQ0366 34,2 GCT1819 46,1 GCT2019 53,7 GCT1823 61,5 GCT2022 84,6 SEQ0069 112,8 SEQ0113 134,2 SEQ0464 196,2 GCT2046 204,5 SEQ0368 216,5 SEQ0367 253,5 GGA16 (cR)RH map
Cytogenetic map
GGA16 organisation
Y@
N
OR
B@
Map Integration
B@ NOR Centromère.083
Valérie Fillon / ECA 2014 10 / 03 / 2014
III. Cytogenetic mapping : lessons from chicken
- The chicken genome
- High resolution mapping
Accumulation of visible variations since chicken domestication Causal mutations ?
Naked neck phenotype (locus Na):
- reduction of body feathering,
- complete lack of feather on the neck,
- increased heat tolerance.
Mendelian trait, incomplete dominance
(Davenport, 1914).
Mapped on GGA3 (Pitel et al., 2000).
Inverse PCR on flanking borders:
Putative insertion of 73 kb
of GGA1 into GGA3.
Selection of BACs from
GGA1 and GGA3 to confirm
this insertion.
105H13 87E13 GGA1 GGA3 insertion breakpointNaked neck
3 Na 3 wt 1 1 insertion Decondensation of the terminal chromatine
Insertion from GGA1 (green spot) in the Na locus (red spot) on GGA3 clearly visible
87E13, GGA3 105H13, GGA1 Focus on GGA3 Na
Naked neck caused by a 73 kb insertion from GGA1 in GGA3, next to BMP12 : Increased BMP12 expression in skin (activating regulatory elements
from GGA1 or disruption of repressive elements from GGA3).
Neck skin more sensitive (retinoic acid production) to BMP signaling. Specific suppression of feathers on the neck (cryptic pattern).
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Studied by Bateson & Punnett (1906)
2 different traits :
- Comb morphology (dominant)
- Hypofertility of males (recessif)
• Loss of motility of spermatozoas in homozygous males
- Fine mapping in the region identified on GGA7: no recombination in 7 Mb in linkage map. inversion ?
- WGS with mate-pair library: 7.4 Mb inversion
- Selection of BACs from GGA7 to confirm this inversion: 2 on breakpoints, 2 inside inversion
7 Mb 24B23 27C3 95H11 Wild-type allele 5G3 R1 allele GGA7
Rose comb
Inversion of 7 Mb for R1
Rose comb caused by a 7.4 Mb inversion on GGA7 :
Comb phenotype: expression of MNR2 (breakpoint 1) in embryo’s comb
Reduced fertilty: disruption of CCDC108 (breakpoint 2), flagellar protein
2 different genotypes : R1 and R2 leading to the same phenotype
Conclusion
Accumulation of visible variations since chicken domestication: Many causal genes/variants are now identified.
A large part of these variants are structural (and regulating) changes: Naked neck is caused by a 73 kb insertion from GGA1 in GGA3 Rose comb is caused by a 7.4 Mb inversion on GGA7
Many other traits are the consequence of other structural changes as CNV (Pea comb), large duplications (Duplex comb, Late feathering, Muffs and beard), large deletions (Db), retrovirus insertion (Recessive white, Blue egg)…
Combine with genomics and sequencing, cytogenetic approach is very useful to confirm these structural changes.
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Valérie Fillon / ECA 2014 10 / 03 / 2014
IV. Comparative mapping and evolution
- Evolution studies in birds
Sibley et J.E. Ahlquist (1990) TURNICIFORMES PICIFORMES GALBULIFORMES BICEROTIFORMES UPUPIFORMES TROGONIFORMES CORACIIFORMES COLIIFORMES CUCULIFORMES PSITTACIFORMES APODIFORMES TROCHILIFORMES MUSOPHAGIFORMES STRIGIFORMES COLUMBIFORMES GRUIFORMES CICONIIFORMES PASSERIFORMES Millions years 125 100 75 50 25 0 STRUTHIONIFORMES TINAMIFORMES CRACIFORMES GALLIFORMES ANSERIFORMES Hybridations DNA/DNA Ratites = paleognathous neoaves Monophyletic group
10% of the 10000 species have been karyotyped Christidis, 1990
2/3 of species : 76 to 82 chromosomes
Diploid number Sp ec ies nu m be rChicken
Quail
Turkey
Duck
The chicken = Typical bird organisation
The chicken = Typical bird organisation
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How to investigate
the comparison
of bird genomes ?
2 challenges :
- Numerous indistinguishable microchromosomes
- High conservation
Finding rearrangments ?
- Giemsa staining - Banding studies - Painting - BAC-FISH - NGSIn Galliformes :
Stock et Bunch, 1982
G-banding homologies
Strong conservation of the 3 first macrochromosome pairs
Simple rearrangments between related species : Strigidae, Owls, Pigeons, Gulls, Parrots, Galliformes
Heterologous Painting
Chicken
painting
probes
Bird
metaphases
* Chr 4 Chr4 + micro Shetty et al., 1999Very good conservation
Simple chromosome correspondances More than 40 species investigated
Shibusawa et al, 2004 Very good conservation
Simple chromosome correspondances
Zoo FISH
Chicken
BAC clones
Other species
metaphases
Establishment of comparative cytogenetic maps
Cytogenetic map Microchr Bac clones Wageningen library
Avianome European project (1998-2000)
Addressing the microchromosomes for the first time
Quail Turkey Duck
GGA18
qter
BW1D2 BW19B13
Fillon et al, 2007
Addressing the microchromosomes for the first time
Chicken
Quail
GGA4
Neocentromere in quail
Centromere Centromere
Galkina et al, 2006
Chicken
Duck
Chicken genome as a reference
200 chicken Bac mapped (Fillon et al, 2007; Skinner et al, 2009)
Duck genome sequenced in 2009
NGS : Illumina Genome AnalyzerII
• 78,487 scaffolds
• largest: 5.9Mb (GGA1: 200Mb) • N50 scaffold: 1.2Mb • 1100 Mb 100bp 1kb 10kb 100kb 1Mb 10Mb Length N50 (Huang et al, 2013)No chromosomal assignation
Most of the scaffolds are small
0 8H20 1400133 23C4 1548681 112C24 3459649 24P2 7476691 23I6 8241461 18H15 13614218 13I5 31735940 22J17 40337012 23K3 43440240 36E8 46471164 21J21 49415052 62D14 70062288 75K23* 83467896 13E2* 84542448 37E19* 88368544 18I11 89400280 94230400 Sca811- sca637 Sca720 Sca919 sca168 sca400 sca347 sca2530 sca1205 sca405 sca376* sca1075 sca229 sca1335
GGA4
Assignation of some duck scaffolds to chromosomes Lack of precision of the cytogenetic comparative map
Integration of the cytogenetic map to the sequence
Use of Narcisse software (Courcelle et al, 2008) Fillon and Griffin data
Only simple and rare interchromosomal rearrangment
fusion/fission events
Potential intrachromosomal rearrangments ?
Lack of precision of the comparative cytogenetic map High resolution mapping strategy : RH mapping and NGS
- To build dense RH comparative maps : the principle is to
fractionnate the genome by irradiation in hybrid cells and to
detect by genotyping the presence or absence of markers in
irradiated hybrid cells. Closer the markers are in the genome,
higher is the probability to find them together in the same
hybrid cell.
The comparative mapping strategy using RH mapping
A
C
B
D
E
F
H1 H2 H3 H4 H5 A-B C F D E 1 1 1 0 0 1 1 1 0 0 1 1 1 1 0 0 1 0 0 1 0 1 0 1 1 1 0 0 0 0- To build dense RH comparative maps : the principle is to
fractionnate the genome by irradiation in hybrid cells and to
detect by genotyping the presence or absence of markers in
irradiated hybrid cells. Closer the markers are in the genome,
higher is the probability to find them together in the same
hybrid cell.
- Ordering the scaffolds using them as markers to improve
the duck genome assembly
Obtention of RH maps corresponding to
whole duck chromosomes sequences
aligned against the chicken genome to
establish precised comparative maps
Ordering the scaffolds using them as markers to improve the
duck genome assembly
Obtention of RH maps corresponding to whole
duck chromosomes sequences aligned against
the chicken genome to establish precised
comparative maps
The comparative mapping strategy using RH mapping
0 23I2 5313873 19L16 30906752 26E13 31298204 40A2 37125732 41H16 39748208 82N9 41159436 15I23 46289664 40I2* 50105552 B2B4* 50818964 18L21* 52061632 41G5** 58331284 41E24 78193872 41C2 101587968 26A22 154839280 154873760
GGA2
sca681 sca783 sca2213 sca581 sca9452 sca1153 sca316* sca868** sca1034 sca129 sca1521 59561388 9L1*** sca356***• orange: inversion • pink: translocation • blue: inversion APL2 GGA2
Example of chromosome 2
Many complex
intrachromosomal
rearrangements
Full mapRobust reliable maps
Physical anchorage
Sca74_1
Sca1034_1
GGA2 APL2
Example of chromosome 2: inversion checked by FISH
Detecting rearrangements on micros : inversion on GGA11
APL12 GGA11 cR sca1191 0.0 sca1176 20 CAM172 32 sca743 46 sca903 80 sca498B 94 CAM170 114 sca498A 123 sca5376 132 CAM167 139 sca368 164 sca2840 201 sca1434A 206 CAM166 210 sca1434B 217 sca1434C 238 sca3847 304 sca2558 349 sca736A 402 CAM163 434 sca736B 441 CAM174 458 CAM175 473 sca597A 485 sca597B 499 CAM185 516 sca597C 528 sca597D 551 sca769 568 CAM180 590 sca469 607 sca5519 614 sca586 640 sca51 649 sca577 684 CAM183B 688 sca1481 695 sca2156 728 CAM163sca736A 364.8381.8 sca2558 970.0 sca1445 1421.4 sca3847 1641.3 sca1434Csca1434B 2170.22644.6 CAM166 3587.6 sca1434Asca2840 3595.13698.4 sca368 3958.5 CAM167sca5376 3970.54381.8 sca498A 4479.8 CAM170 4495.8 sca498Bsca903 6455.26556.8 sca3849 6998.4 CAM172 7674.6 sca1176 7684.6 sca1191 8523.3 sca743 9541.0 sca736B 10601.1 CAM174 11601.1 CAM175 11901.1 sca597A 13630.9 CAM185 14330.8 sca597B 14920.2 sca597C 16120.9 sca597Dsca769 17420.117930.7 sca5519sca469 18403.718759.1 CAM180sca586 19057.119678.5 sca51 19941.7 sca3004sca577 20505.420770.8 CAM183Bsca1481 20886.621044.6 sca2156 21582.7Zebra Finch is the outgroup : passeriformes
Generation of comparative maps by aligning whole chromosome
sequences from different sequenced species : galloanserae
Finding breakpoint regions and lineage
specific rearrangements
APL2 TGU2 APL2 MGA M GA 6 M GA 3 53 .2 5M b 10 0, 4M b APL2 GGA2
RH mapping and avian comparative maps
TGU2 GGA2
Lineage specific rearrangements : Chromosome 2
Duck lineage
Passeriformes Galliformes Galliformes Anseriformes Zebra finch Turkey Chicken Duck 0 50 100 MYA MGA13 GGA11 TGU11 APL12
RH mapping and avian comparative maps
Lineage specific rearrangements : GGA11
GGA11 GGA11
Determination of 7 conserved blocks outgroup
Array CGH and Copy Number Variation
Fewer CNV compare to mammals
CNV hotspots shared by distant species
Possible link with some cytogenetic rearrangements ?
Skinner et al, 2009 Griffin et al, 2008
2- Pour les microchromosomes ...
Interchromosomal rearrangments ex : the splitting GGA4
- fusion/fission events
Numerous intrachromosomal complex rearrangements :
- lineage specific
- number of rearrangements consistents with the
divergence times.
Evidence for evolutionnary breakpoints
Diploïd number
Diploïd number
2/3 of species : 76 to 82 chromosomes
Some exceptions ...
Nombre diploide No m br e d ’e sp èc es Christidis, 1990Some exceptions !
Some exceptions !
Burhinus oedicnemus 2n = 40
King fisher 2n = 138
Hoopoe
Upupa epops 2n = 126
Heterogeneous diploïd number within orders
Charadriiformes 40 à 98
Falconiformes
Cathartidae : 80
Accipitridae : 66 à 68
Falconinae : 50 à 54
TURNICIFORMES PICIFORMES GALBULIFORMES BICEROTIFORMES UPUPIFORMES TROGONIFORMES CORACIIFORMES COLIIFORMES CUCULIFORMES PSITTACIFORMES APODIFORMES TROCHILIFORMES MUSOPHAGIFORMES STRIGIFORMES COLUMBIFORMES GRUIFORMES CICONIIFORMES PASSERIFORMES STRUTHIONIFORMES TINAMIFORMES CRACIFORMES GALLIFORMES ANSERIFORMES
Accipitridae case
Ciconiiformes Gruiformes Struthioniformes Tinamiformes Craciformes Galliformes Anseriformes Turniciformes Piciformes Galbuliformes Bucerotiformes Upupiformes Trogoniformes Coraciiformes Coliiformes Cuculiformes Psittaciformes Apodiformes Trochiliformes Musophagiformes Strigiformes Columbiformes Passeriformes Accipitridae Sagittariidae Falconidae Cathartidae Ciconiidae
Circaetinae (Short-toed eagles) Aquilinae (Eagles)
Buteoninae (Buzzards) Accipitrinae (Hawks) Haliaeetinae (Sea-eagles) Milvinae (Kites)
Aegypiinae (Large vultures) Gypaetinae (Small vultures) Perninae (Honey buzzards) Pandionidae (Osprey) Elanidae (Small kites)
Modern birds
Storks and diurnal raptors
Phylogeny of Accipitridae
Atypical karyotype of Accipitridae
Moderate diploid number (66) Few microchromosomes
Egyptian vulture karyotype (Neophron percnopterus)
Bed’hom et al, 2004
Mapping of BACs
Use of chicken cytogenetic map
Only Z markers on similar localizations Egyptian vulture (Neophron)
Npe (25) Npe (23) Npe (24) Npe (1q) Npe (Zq) Npe (21) Gga (1p) Gga (2q) Gga (3q) Gga (4q) Gga (Zp) Gga (12) Gga Npe
Chromosome painting
1 2 3 4
5 6 7 8
Chicken (Gallus)
Osprey (Pandion) Chicken painting probes using flow-sorted
chromosomes
Hybridization on Osprey metaphase Few conserved segments
Bed’hom et al, 2004 A lot of interchromosomal rearrangments !
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The Phylogenomic Avian Project
http://avian.genomics.cn/en/index.html Consortium :
- Erich Jarvis - Duke University, - Guojie Zhang - BGI,
- Tom Gilbert - University of Copenhagen Part of G10K :
- 2009
- 10 000 vertebrate species 50 papers
48 birds sequenced
Large scale phylogenomic analysis Comparative genomics
- avian genome evolution - sex chromosome evolution - molecular basis of fligth, loss of teeth, vocal learning New bioinformatic tools
48 birds sequenced
Large scale phylogenomic analysis Comparative genomics
- avian genome evolution - sex chromosome evolution - molecular basis of fligth, loss of teeth, vocal learning Genome scale phylogeny of birds
-Remarkable evolutionary stasis - Incomplete resolution of the lineage tree
2014
Avian
Phylogenomics Consortium ‘Bird 10K’ project :
to generate draft genome
sequences for about 10,500 extant bird species over the next 5 years Take off June 2015 (Zhang 2015, Nature)
250 species sequenced !
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Conclusion
- microchromosomes = the main features of bird karyotypes - Simple interchromosomal rearrangments : fusion/fission
- Numerous intrachromosomal rearrangments than expect first - Rapid evolution of accipitrids karyotypes
Remarkable stability of avian genome structure
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Valérie Fillon / ECA 2014 10 / 03 / 2014
IV. Comparative mapping and evolution
- Evolution studies in birds
- Bird among vertebrates
- Significance of microchromosomes
- Birds and reptiles
Who belongs microchromosomes ?
Who belongs microchromosomes ?
FISH (Sturgeon)
Salamanders
Ophidiens (snakes)
Boa
Vipère
Chicken EST absent from the chicken assembly Sequence similarity with HSA19
Synteny conservation
Evolutionary significance of microchromosomes
• 30% of the genome – 50% genes – GC rich
• Main feature of avian karyotypes
• Related to the genome compaction ?
• Increasing the recombination rates
• Physiological adaptation (flight) ?
• Ancestral synteny conservation
• Appeared by fission of the ancestral vertebrate karyotype ?
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Valérie Fillon / ECA 2014 10 / 03 / 2014
IV. Comparative mapping and evolution
- Evolution studies in birds
- Bird among vertebrates
-
Significance of microchromosomes
- Birds and reptiles
Boa
Chicken
Turtles (300 sp)
Snakes and lizards
(8200 sp) crocodiles (23 sp)
Birds (9000 sp)
Trachemys scripta elegans
The GGA karyotype is closer to the turtle karyotype than to the Crocodile karyotype Chicken and turtle probably share some caracteristics of the ancestral sauropsidae karyotype
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Valérie Fillon / ECA 2014 10 / 03 / 2014
IV. Comparative mapping and evolution
- Evolution studies in birds
- Bird among vertebrates
- Significance of microchromosomes
- Birds and reptiles
.0154
The origin of sex chromosomes
-In Birds : Z and W chromosomes
-In reptiles : Z/W or XY
No homology between human XY and ZW chicken sex chromosomes
Sibley et J.E. Ahlquist (1990) TURNICIFORMES PICIFORMES GALBULIFORMES BICEROTIFORMES UPUPIFORMES TROGONIFORMES CORACIIFORMES COLIIFORMES CUCULIFORMES PSITTACIFORMES APODIFORMES TROCHILIFORMES MUSOPHAGIFORMES STRIGIFORMES COLUMBIFORMES GRUIFORMES CICONIIFORMES PASSERIFORMES Millions d’années 125 100 75 50 25 0 STRUTHIONIFORMES TINAMIFORMES CRACIFORMES GALLIFORMES ANSERIFORMES DNA/DNA hybridisations Ratatites = paleognathous Carinates = neognathous
Sex chromosomes ZZ and ZW :
The case of Ratites
Temperature-dependent sex determination system :
- All Crocodiles
- Tuatara (iguanes)
- most turtles
- some lizards
Genetic sex determination system :
- All snakes
- most lizards
- a few turtles
Sex chromosomes : ZZ/ZW or XX/XY
ex : all snakes have female heterogamety ZZ/ZW
ex : in lizards and turtles both ZZ/ZW and XX/XY
Unindistinguishable sex chromosomes
In Reptiles : karyotype similarities between bird, snakes and turtles
- microchromosomes
- ZZ/ZW
Does it exist a common origin of the sex chromosomes
between reptiles and birds ?
Does it exist a common origin of the sex chromosomes
between reptiles and birds ?
Sex chromosomes : ZZ/ZW or XX/XY but Undistinguishable
ex : all snakes have female heterogamety ZZ/ZW ex : in lizards and turtles both ZZ/ZW and XX/XY
Pokornà et al, 2011 FISH painting of GGAZ
Synteny conservation in chicken and reptiles Gene order conservation in reptiles (CS13).
*High conservation of the Z-linked gene
in the bird and reptile ancestor
*Conservation of synteny with GGAZ
But it is not syntenic with ZW or XY sex
chromosomes in Reptiles when identified
Different origin of sex chromosomes
in birds and reptiles
Acknowledgments
INRA Toulouse Valérie Fillon Alain Vignal Frédérique Pitel Mireille Morisson INRA Jouy-en-Josas Michèle Tixier-Boichard Bertrand Bed’Hom INRA Nouzilly David GourichonSaint-Petersburg State University
Chromosome Sructure and Function lab Svetlana Galkina Alsu Sayfitdinova Elena Gaginskaya And colleagues Wageningen University Martien Groenen Richard Crooijmans Genome Institut Wes Warren University of Kent Darren Griffin