Animal Cytogenetics Valerie Fillon
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Valerie Fillon. Animal Cytogenetics. Master. European Advanced Postgraduate Course in Classical and Molecular Cytogenetics, 2018. hal-02788100
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Valérie Fillon
GENPHYSE – INRA Toulouse - France
.01 ECA 10 mars 2014 GENPHYSE – Cytogenomic team
Institut de Recherche National Agronomique
*The laboratory is involved in the structural and functional analysis of the genome of farm animal Toulouse species
*Cytogenetic Platform
.02 I. General overview
II. Chromosomal abnormalities
III. Cytogenetic mapping : lessons from
chicken
IV. Comparative mapping and evolution
.03 Valérie Fillon / ECA 2014 10 / 03 / 2014 I. General overview
- History
- Technical aspects
- Some animal karyotypes
.04 Valérie Fillon / ECA 2014 10 / 03 / 2014 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
.05 I. General overview
- History
- Technical aspects
- Some animal karyotypes
.06 Valérie Fillon / ECA 2014 10 / 03 / 2014 Preparation of Chromosome Slides
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 !
.07 Fluorescent In Situ Hybridization FISH
Localisation directly on the chromosomes of a genomic sequence
.08 Probes Chromosome preparations
- Cell cultures - DNA (>10 kb) : BAC - Metaphase arrest - Labelling (biotine) - Cellular treatment - purification, resuspension - Spreading
Hybridation In situ - denaturation - hybridization (24h) B B probe - washing B - staining chromosome Analysis under the microscope
10 Mb probe
Resolution > 1 Mb
Chromosome assignation
Measurment
Quail Microdissection
1 2 3 4
5 5 6 7 8 Inv(8)(p1.1;q2.5)
Chromosome arms 8p and 8q generated by microdissection
A. Pinton (unpublished data) Toward CGH array...
But - Most of the abnormalities are equilibrate - Quality of the reference genome - Cost I. General overview
- History
- Technical aspects
- Some animal karyotypes
.014 Valérie Fillon / ECA 2014 10 / 03 / 2014 Chromosomes numbers of different species
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 Rattus norvegicus 42 Souris Mus musculus 40
Dog Canis familiaris 78 Cat Felis domesticus 38
.015 Trypsin digestion G banded Pig karyotype 2n=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 Goat 2n=60 Cattle 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)
.017 Indian Munjac
2n = 6 2n = 7
Reeves's Munjac
2n = 46
.018 Horse 2n = 64
C bands for sex chromosomes
.019 Chicken karyotype 2n=78
Females are heterogametic ZW Presence of 30 pairs of microchromosomes = 30% of the genome 50% of the genes (GC rich) Harmonia axyridis 2n = 16
Asian ladybeetle
Direct technique from growing eggs
.021 http://compcytogen.pensoft.net/ II. Chromosomal abnormalities
- Clinical cytogenetics
- Some examples in horse, pig and cattle
- Segregation during meiosis
.023 Valérie Fillon / ECA 2014 10 / 03 / 2014 CLINICAL CYTOGENETICS
•As in Human, chromosomal abnormalities can be responsible of congenital abnormalities, embryonic loss, infertility, cancer
significant economic losses
•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) •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 Results of French chromosomal control
•Cattle : 700 per year
3.5 % with Rob (1/29)
•Pigs : 1700 to 2000 per year
Nombre d'analyses Nombres d'anomalies de structure 3000 18 16 2500 14 2000 12 10 1500 8 1000 6 4 500 2 0 0
1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 année2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 •By January 2018, 37 800 pigs have been analyzed
•182 structural chromosomal abnormalities (de novo) •86% reciprocal translocation
Prevalence of structural chromosomal rearrangement: 0.47% 1/200 boars (Pinton et al., 2011)
>90% young purebred boars controled before reproduction (AI) II. Chromosomal abnormalities
- Clinical cytogenetics
- Some examples in horse, pig and cattle
- Segregation during meiosis
.029 Valérie Fillon / ECA 2014 10 / 03 / 2014 Principal chromosomal abnormalities identified in Pig, Cattle and Horse. •Sex chromosome abnormalities
63, X0 / 65, XYY mosaicism in a case of equine male pseudohermaphroditism
Paget et al. (2001)
Gonads: testis like structure (no spermatozoa)
Y
Y X •Reciprocal translocations t(3;16)(q23;q22) and palatoschisis (cleft palate)
Ducos et al., 2004
Offspring with unbalanced karyotype Translocated boar 2n=38, XX (ou XY), der16 t(3;16)(q23;q22)
N der(3) N N
N der(16) N der(16) •Reciprocal translocations
•Rcp(6;8)(10;18) •Reciprocal translocations
•Rcp(Y;1) •Inversions
Inv(2)(q1.3;q2.5)
Normal SSC 2 Inverted SSC 2
q1.1 q2.1
q2.4 q2.6 normal inverted K. Massip (unpublished data) •Cattle
Rob 1/29 translocation
Statutory obligation for all young bulls→AI
0,25
0,2
0,15
0,1 1/29 frequency1/29
0,05
0 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 year
•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 A. Garnier (unpublished data) II. Chromosome abnormalities
- Clinical cytogenetics
- Some examples in horse, pig and cattle
- Segregation during meiosis
.038 Valérie Fillon / ECA 2014 10 / 03 / 2014 Segregation during meiosis
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 : SpermFISH (hybridization of probes on decondensed sperm nuclei)
•Male meiosis : analysis of synaptonemal complexes Sperm FISH studies with rcp
alternate segregation
adjacent I segregation adjacent II segregation Sperm FISH studies with rcp
alternate segregation balanced gametes
adjacent I segregation adjacent II segregation Sperm FISH studies with rcp
Rcp(3;15)
Rcp(12;14) Sperm FISH studies with rob
der(13;17)
13 17
Alternate Adjacent 3:0
balanced unbalanced Sperm FISH studies with rob 1/29 translocation
BTA1 ♂ ♀
balanced gametes 97.21% 74% BTA29 unbalanced gametes 2.75% 4% 1er GP
dipoid gametes 0.04% 22% BTA1-29
Métaphase II Rate of unbalanced gametes for the heterozygote cows: spz • Smaller than expected according to the Human data • Coherent with the decrease of fertility (5% in average) • 2 times more than in male gametes Sperm FISH studies with inversions
(Anton et al., 2002)
Paracentric inversion balanced acentric dicentric Diploid other types
Pericentric inversion
balanced Dup(p)/del(q) Dup(q)/del(p) Diploid Sperm FISH studies with inversions Synaptonemal complexes studies
Analysis of the early stages of meiosis
•Development of antibodies specific of some meiotic proteins :
Synaptonemal complex: SCP1, SCP3 Recombination: MLH1, MLH3….
Analysis of the chromosome pairing and recombination at the pachytene stage throughout immunocytology approach Synaptonemal complexes studies
SC electronique microscopy Synaptonemal complexes studies
Immunocytological staining
Zygotene Pachytene
Maternal sister chromatids
Axial element
central element
Paternal sister chromatids
(B. de Massy, 2005) Synaptonemal complexes studies
(B. de Massy, 2005) Synaptonemal complexes studies
Effects of chromosomal rearrangements SSC13 SSC12 N T N T
red SC Blue Centromere Yellow Crossing over III. Cytogenetic mapping : lessons from chicken
- The chicken genome
- High resolution mapping
- Chromosome polymorphism
.052 Valérie Fillon / ECA 2014 10 / 03 / 2014 The chicken = Typical bird organisation
1- sex chromosomes ZZ and ZW
2- diploïd number : 2n = 76 to 82
3- microchromosomes : 60 à 64
2n = 78 60 microchr
Standard karyotype : Ladjali et al 1999 The challenge : microchromosomes Sequenced : 2004 Gagal5 : 2016 Characterization of the chicken karyotype
.054 Painting probes Bac clones Maps integration
Sequence
Genetic map RH
BAC Contigs
FISH Genetic map Cytogenetic map INTEGRATION
Molecular markers Large insert clones BAC 6 bw30P7 P3-3 3 bw55L19 5 3 4 2 1 2 3 bw30B21 4 2 P1-2 2 3 P4D9 1 21 5 4 P2-9 3 bw31B10 2 bw26B13 2 P2-6 1 P2-5 1 bw43G6 1 1 1 2 bw29L12 B3H9 B2B4 1 P5A6 1 bw14J6 1 bw25G16 1 P4D8 1 2 1 P2E4 2 1 2 1 3 4 bw112C24 1 4 P1H9 1 3 1 2 1 2 bw8H20 1 bw37H20 P1-9 1 bw125P16 1 2 3 3 4 1 2 P2H3 bw41C2 5 2 4 2 2 6 P3D3 1 bw118M14 3 bw4F8 2 5 7 1 4 P6C6 1 8 2 2 6 P2-10 9 1 bw3K18 3 P6D4 10 P5D4 bw33G16 4 3 2 2 5 1 bw6D24 3 3 2 1 6 2 1 bw62D14 4 3 2 4 P4B11 3 bw12C6 5 2 1 5 3 4 2 3 bw9B13 3 3 6 4 bw13E2 4 7 bw107K17 5 5 4 5 bw38E8 bw13L14 bw37E19 CHROMOSOME 5 bw26A22 CHROMOSOME 1 CHROMOSOME 2 CHROMOSOME 3 CHROMOSOME 4
4 bw83N24 3 2 2 1 2 bw29C17 3 2 1 1 2 2 bw69P21 1 1 1 bw69P21 1 1 1 1 1 1 bw60M16 bw21P13 2 1 1 bw27G19 1 1 1 3 bw40L3 4 1 2 2 2 2 1 3 3 bw30C21 1 3 bw27C3 CHROMOSOME 8 4 1 3 bw27C3 4 bw8F6 bw71017 4 SCD1, B1E7 4 5 1 5 bw10J13 5 6 2 bw79C4 6 bw26M16, P4G2 2 3 6 P4G2 bw124H24 7 CHROMOSOME 7 CHROMOSOME Z CHROMOSOME 7 CHROMOSOME 6
Chicken cytogenetic map Integration of genetic and cytogenetic maps
The genome coverage
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 Nature, 2004 Genome sequenced in 2004 Improvement in 2016 Hillieret al, Nature, 2004 Warren et al, G3, nov2016 Mb sequence Mb
Macro- Microchromosomes 60 microchromosomes : 3 to 20 Mb (30%) GC and gene rich (50%)
Partialy sequenced 6 Missing and unidentified
24/30 with FISH markers Still ongoing !
New !
Microchromosomes III. Cytogenetic mapping : lessons from chicken
- The chicken genome
- High resolution mapping
- Chromosome polymorphism
.063 Valérie Fillon / ECA 2014 10 / 03 / 2014 High resolution mapping
Lampbrush chromosomes
Chromosome Structure and Function Laboratory Biological Research Institute Saint-Petersburg State University Lampbrush chromosomes can be found in oocytes of:
fishes amphibians reptilia birds insects
During spermatogenesis Y chromosome of Drosophila transforms into lampbrush plants (Acetabularia acetabulum) Lampbrush chromosomes can be found during leptotene zygotene prophase pachytene DIPLOTENE diakinesis
metaphase Meiosis I
anaphase
telophase 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) isolation of lampbrush chromosomes from bird oocytes Making lampbrush chromosomes
- nucleus removal from oocytes and adherent yolk cleaning
- lampbrush chromosome removal from nucleus
Lampbrush slide - chromosome spreading for 30 min at +4°C
- centrifugation for 15 min at 3500 g
- fixation in 2% formaldehyde for 5 min and 50% and 70% ethanol Chicken mitotic chromosome 1
6 mm
185 mm
Chicken lampbrush chromosome 1 Lampbrush chromosomes
Chicken LBC4 and mitotic chicken chromosome 4 (100x)
Derjusheva et al., 2003 Lampbrush chromosome structure
DNA loops
Chromatides
Chromomeres
Chiasmas
Transcription units
bivalent 100% Chiasma distribution along the chicken lampbrush chromosome 1 Chiasma Chiasma frequency
1 2 3 4 5 1 2 1 2 3 1 2 1 2 1 2 1 2 1 2 A B C D E F G H I J K L
Cytological recombination maps
High resolution cytogenetic maps Chromosome 4 poule
Galkina et al 2006
Microchromosome 16 is the MHC and NOR bearing chromosome
B B Y Y et rDNA
Fillon et al., 1996 GGA16 genetic map
Groenen et al., 2000 60 microchromosomes : 3 to 20 Mb (30%) GC and gene rich (50%)
Partialy sequenced 6 Missing and unidentified
New !
Microchromosomes Cytogenetic high resolution map
NOR : near the centromere, covers 40% of the chromosome
B@ at the q end
Chiasma between Y and B complexes, but in different position
IntégrationMap Integration des cartes
GGA16 organisation GGA16 (cR) B@
SEQ0097 0,0 B@ SEQ0366 34,2 GCT1819 46,1 GCT2019 53,7 GCT1823 61,5
GCT2022 84,6
SEQ0069 112,8 NOR Y@ SEQ0113 134,2
N SEQ0464 196,2 GCT2046 204,5 OR SEQ0368 216,5
Centromère SEQ0367 253,5
Cytogenetic map RH map Description of LBC microchromosomes III. Cytogenetic mapping : lessons from chicken
- The chicken genome
- High resolution mapping
- Chromosome polymorphism
.083 Valérie Fillon / ECA 2014 10 / 03 / 2014 Accumulation of visible variations since chicken domestication Causal mutations ? Naked neck
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). Naked neck
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
insertion GGA1
GGA3 breakpoint
87E13 Hybridization of 87E13 (GGA3) and 105H13 (GGA1) on heterozygous Na/wt
87E13, GGA3 105H13, GGA1 Focus on GGA3 Na
1
3 wt Decondensation of the terminal chromatine 3 Na insertion Insertion from GGA1 1 (green spot) in the Na locus (red spot) on GGA3 clearly visible Naked neck
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). Rose comb locus R
Studied by Bateson & Punnett (1906)
2 different traits :
- Comb morphology (dominant)
- Hypofertility of males (recessif) • Loss of motility of spermatozoas in homozygous males
.089 Rose comb
- Fine mapping in the region identified on GGA7: 7 Mb 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
95H1124B23 27C3 5G3 Wild-type allele
R1 allele
GGA7 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
Inversion of 7 Mb for R1 2 different genotypes : R1 and R2 leading to the same phenotype
R2 have rose comb phenotype but restored fertility… 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. IV. Comparative mapping and evolution
- Evolution studies in birds
- Bird among vertebrates
.095 Valérie Fillon / ECA 2014 10 / 03 / 2014 Monophyletic group Ratites = paleognathous STRUTHIONIFORMES TINAMIFORMES CRACIFORMES GALLIFORMES ANSERIFORMES TURNICIFORMES PICIFORMES GALBULIFORMES BICEROTIFORMES UPUPIFORMES TROGONIFORMES CORACIIFORMES COLIIFORMES CUCULIFORMES PSITTACIFORMES APODIFORMES TROCHILIFORMES MUSOPHAGIFORMES STRIGIFORMES neoaves COLUMBIFORMES GRUIFORMES CICONIIFORMES PASSERIFORMES 125 100 75 50 25 0
Millions years Hybridations DNA/DNA Discrepancies : diurnal bird of preys Sibley et J.E. Ahlquist (1990) 10% of the 10000 species have been karyotyped
Christidis, 1990 pce number Species
Diploid number
2/3 of species : 76 to 82 chromosomes The chicken = Typical bird organisation
Chicken Turkey
Quail Duck How to investigate the comparison of bird genomes ? 2 challenges :
- Numerous indistinguishable microchromosomes
- High conservation From Classical work to NGS
- Giemsa staining - Banding studies Finding rearrangments ? - Painting - BAC-FISH - NGS
.099 G-banding homologies
Only a few studies, impossible for the microchromosomes Strong conservation of the 3 first macrochromosome pairs Simple rearrangments between related species : Strigidae, Owls, Pigeons, Gulls, Parrots, Galliformes
In Galliformes :
Stock et Bunch, 1982 Heterologous Painting
Chicken Bird painting metaphases probes
Shetty et al., 1999
* Chr 4 Chr4 + micro (Pheasant, Emu, Owl, Duck…) More than 40 species investigated (Review in Griffin et al, 2007)
Very good conservation Simple chromosome correspondances Shibusawa et al, 2004 Very good conservation Simple chromosome correspondances Intrachromosomal rearrangments ? Establishment of comparative cytogenetic maps
Avianome European project (1998-2000)
Chicken Other species BAC clones Zoo FISH metaphases
Quail Cytogenetic map Turkey Microchr Bac clones Duck Wageningen library
Addressing the microchromosomes for the first time Addressing the microchromosomes for the first time
GGA18 BW19B13 qter
BW1D2 Fillon et al, 2007
Microchromosomes conservation Griffin et al, 2008 Quail Chicken
2006 GGA4
Galkina et al 2006 Neocentromere in quail Galkina et al, 2006
Centromere
Centromere
Red-legged partridge chr4 : Kasaï et al, 2003 Chicken Duck
Chicken genome as a reference 200 chicken Bac mapped (Fillon et al, 2007; Skinner et al, 2009) Duck genome sequenced in 2009
80 millions years of evolution Duck Genome Assembly (2009, BGI)
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 No chromosomal assignation
Most of the scaffolds are small
(Huang et al, 2013) Integration of the cytogenetic map to the sequence
0 Sca720 1400133 8H20 Sca811- sca637 1548681 23C4 Sca919 3459649 112C24 sca168 24P2 7476691 sca400 8241461 23I6 13614218 18H15 sca347 31735940 13I5 40337012 22J17 sca2530 43440240 23K3 sca1205 46471164 36E8 sca229 49415052 21J21 sca1335 70062288 62D14 83467896 75K23* sca405 84542448 13E2* sca376* 88368544 37E19* 89400280 18I11 sca1075 94230400 GGA4 Assignation of some duck scaffolds to chromosomes Lack of precision of the cytogenetic comparative map
Fillon and Griffin data Use of Narcisse software (Courcelle et al, 2008) 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 The comparative mapping strategy using RH mapping The comparative mapping strategy using RH mapping
- 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.
H1 H2 H3 H4 H5
A 1 1 1 0 0
B 1 1 1 0 0 A-B C 1 1 1 1 0 C D 0 1 0 0 1 D E 0 1 0 1 1 E 1 0 0 0 0 F F The comparative mapping strategy using RH mapping
- 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 The comparative mapping strategy using RH mapping
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
Rao et al, 2012 0 5313873 23I2 sca681
30906752 19L16 sca783 sca2213 31298204 26E13 sca581 37125732 40A2 sca9452 39748208 41H16 sca1153 41159436 82N9 46289664 15I23 sca316* 50105552 40I2* sca868** B2B4* 50818964 sca356*** 52061632 18L21* 58331284 41G5** 59561388 9L1*** 78193872 41E24 sca1034 101587968 41C2 sca129
154839280 26A22 sca1521 154873760
GGA2 Example of chromosome 2
• orange: inversion
• pink: translocation
• blue: inversion
Many complex intrachromosomal rearrangements
Robust reliable maps
Full map Physical anchorage GGA2 APL2 Example of chromosome 2: inversion checked by FISH
Sca74_1
APL2 GGA2
Sca1034_1 Full map Detecting rearrangements on micros : inversion on GGA11
CAM163 364.8 sca736A 381.8 0.0 sca1191 sca2558 970.0 20 sca1176 sca1445 1421.4 32 CAM172 sca3847 1641.3 sca1434C 2170.2 46 sca743 sca1434B 2644.6 80 sca903 CAM166 3587.6 94 sca498B sca1434A 3595.1 sca2840 3698.4 114 CAM170 sca368 3958.5 123 sca498A CAM167 3970.5 132 sca5376 sca5376 4381.8 sca498A 4479.8 139 CAM167 CAM170 4495.8 164 sca368 sca498B 6455.2 201 sca2840 sca903 6556.8 sca1434A sca3849 6998.4 206 CAM172 7674.6 210 CAM166 sca1176 7684.6 217 sca1434B sca1191 8523.3 238 sca1434C 304 sca3847 sca743 9541.0 349 sca2558 402 sca736A sca736B 10601.1 434 CAM163 CAM174 11601.1 441 sca736B CAM175 11901.1 458 CAM174 473 CAM175 485 sca597A sca597A 13630.9 499 sca597B CAM185 14330.8 516 CAM185 sca597B 14920.2 528 sca597C 551 sca597D sca597C 16120.9 568 sca769 590 CAM180 sca597D 17420.1 607 sca469 sca769 17930.7 614 sca5519 sca5519 18403.7 sca469 18759.1 640 sca586 CAM180 19057.1 649 sca51 sca586 19678.5 684 sca577 sca51 19941.7 sca3004 20505.4 688 CAM183B sca577 20770.8 695 sca1481 CAM183B 20886.6 728 sca2156 sca1481 21044.6 cR sca2156 21582.7 APL12 GGA11 Generation of comparative maps by aligning whole chromosome sequences from different sequenced species : galloanserae
Zebra Finch is the outgroup : passeriformes
Finding breakpoint regions and lineage specific rearrangements Lineage specific rearrangements : Chromosome 2 MGA APL2 GGA2 TGU2 GGA2 APL2 TGU2 APL2 53.25Mb MGA6 100,4Mb MGA3
Duck lineage
RH mapping and avian comparative maps Lineage specific rearrangements : GGA11
outgroup Zebra finch TGU11 Passeriformes
GGA11
Turkey Galliformes MGA13 GGA11
Chicken Galliformes
GGA11 APL12 Duck Anseriformes
MYA
100 50 0 Determination of 7 conserved blocks Similar findings : Skinner and Griffin,RH 2012 mapping and avian comparative maps Array CGH and Copy Number Variation
Griffin et al, 2008 Skinner et al, 2009
16 CNV 32 CNV
Fewer CNV compare to mammals CNV hotspots shared by distant species Possible link with some cytogenetic rearrangements ? 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
Bird karyotypes are very stable compare to mammals Some exceptions !
Diploïd number
2/3 of species : 76 to 82 chromosomes Some exceptions ... Nombred’espèces
Nombre diploide
Christidis, 1990 Hoopoe Burhinus oedicnemus 2n = 40 Upupa epops 2n = 126
Falco peregrinus 2n = 50 King fisher 2n = 138 Heterogeneous diploïd number within orders
Charadriiformes 40 à 98
Falconiformes Cathartidae : 80 Accipitridae : 66 à 68 Falconinae : 50 à 54 Accipitridae case
STRUTHIONIFORMES TINAMIFORMES CRACIFORMES GALLIFORMES ANSERIFORMES TURNICIFORMES PICIFORMES GALBULIFORMES BICEROTIFORMES UPUPIFORMES TROGONIFORMES CORACIIFORMES COLIIFORMES CUCULIFORMES PSITTACIFORMES APODIFORMES TROCHILIFORMES MUSOPHAGIFORMES STRIGIFORMES COLUMBIFORMES GRUIFORMES CICONIIFORMES PASSERIFORMES Phylogeny of Accipitridae
Modern birds
Struthioniformes Tinamiformes Craciformes Galliformes Anseriformes Turniciformes Piciformes Galbuliformes Bucerotiformes Upupiformes Trogoniformes Buteoninae (Buzzards) Coraciiformes Milvinae (Kites) Coliiformes Haliaeetinae (Sea-eagles) Cuculiformes Accipitrinae (Hawks) Psittaciformes Aquilinae (Eagles) Apodiformes Storks and Circaetinae (Short-toed eagles) Trochiliformes diurnal raptors Aegypiinae (Large vultures) Musophagiformes Gypaetinae (Small vultures) Strigiformes Perninae (Honey buzzards) Accipitridae Columbiformes Pandionidae (Osprey) Sagittariidae Gruiformes Elanidae (Small kites) Falconidae Ciconiiformes Cathartidae Passeriformes Ciconiidae Bed’hom et al, 2004 But some exceptions ! Accipitridae case
Atypical karyotype of Accipitridae
Egyptian vulture karyotype (Neophron percnopterus)
Moderate diploid number (66)
Few microchromosomes
Bed’hom et al, 2004 Mapping of BACs
Gga Npe
Gga (1p) Npe (25)
Gga (2q) Npe (23)
Gga (3q) Npe (24)
Gga (4q) Npe (1q) Egyptian vulture (Neophron)
Gga (Zp) Npe (Zq)
Use of chicken cytogenetic map Gga (12) Npe (21) Only Z markers on similar localizations
Bed’hom and Fillon 2004 Chromosome painting
1 2 3 4
5 6 7 8
Chicken (Gallus)
Chicken painting probes using flow-sorted Osprey (Pandion) chromosomes
Hybridization on Osprey metaphase
Few conserved segments A lot of interchromosomal rearrangments !
Reciprocal translocations evolution pattern ? Bed’hom et al, 2004 The Phylogenomic Avian Project
Consortium : - Erich Jarvis - Duke University, - Guojie Zhang - BGI, - Tom Gilbert - University of Copenhagen
Part of G10K : - 2009 - 10 000 vertebrate species
50 papers
http://avian.genomics.cn/en/index.html .0135 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 New bioinformatic tools 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 !
Need of chromosome assignation : optical maps, cytogenetics Conclusion
Remarkable stability of avian genome structure
- microchromosomes = the main features of bird karyotypes
- Simple interchromosomal rearrangments : fusion/fission
- Numerous intrachromosomal rearrangments than expect first
- Rapid evolution of accipitrids karyotypes
A lot to do to understand microchromosomes…
.0139 IV. Comparative mapping and evolution
- Evolution studies in birds
- Bird among vertebrates - Significance of microchromosomes - Birds and reptiles - origin of sex chromosomes
.0140 Valérie Fillon / ECA 2014 10 / 03 / 2014 Evolutionnary significance of microchromosomes
Who belongs microchromosomes ? FISH (Sturgeon) Salamanders
Ophidiens (snakes)
Vipère Boa Chicken EST absent from the chicken assembly Sequence similarity with HSA19 Synteny conservation
http://www.ensembl.org 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 ?
Still a lot to discover ! IV. Comparative mapping and evolution
- Evolution studies in birds
- Bird among vertebrates - Significance of microchromosomes - Birds and reptiles - origin of sex chromosomes
.0145 Valérie Fillon / ECA 2014 10 / 03 / 2014 What about reptiles ?
Chicken
Boa Snakes and lizards crocodiles Turtles (8200 sp) (23 sp) (300 sp) Birds (9000 sp)
220 My between crocodiles and birds And crocodiles ????
Trachemys scripta elegans
Crocodylus niloticus Reciprocal heterologous painting 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
Microchromosomes are yet to investigate ! IV. Comparative mapping and evolution
- Evolution studies in birds
- Bird among vertebrates - Significance of microchromosomes - Birds and reptiles - origin of sex chromosomes
.0153 Valérie Fillon / ECA 2014 10 / 03 / 2014 The origin of sex chromosomes
-In Birds : Z and W chromosomes
-In reptiles : Z/W or XY
-In Mammals : X and Y chromosomes
.0154 Nanda et al., 1999
No homology between human XY and ZW chicken sex chromosomes Ratatites = paleognathous STRUTHIONIFORMES TINAMIFORMES CRACIFORMES GALLIFORMES ANSERIFORMES TURNICIFORMES PICIFORMES GALBULIFORMES BICEROTIFORMES UPUPIFORMES TROGONIFORMES CORACIIFORMES COLIIFORMES CUCULIFORMES Carinates = PSITTACIFORMES neognathous APODIFORMES TROCHILIFORMES MUSOPHAGIFORMES STRIGIFORMES COLUMBIFORMES GRUIFORMES CICONIIFORMES PASSERIFORMES 125 100 75 50 25 0
Millions d’années
DNA/DNA hybridisations Sibley et J.E. Ahlquist (1990) Sex chromosomes ZZ and ZW :
The case of Ratites
Hardly differenciated sex schromosomes 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
Ex : Boa
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
FISH painting of GGAZ
Pokornà et al, 2011 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 Through mammals !
Genome research, 2008 Acknowledgments Saint-Petersburg State University Chromosome Sructure and Function lab Svetlana Galkina Alsu Sayfitdinova Elena Gaginskaya INRA Toulouse And colleagues Valérie Fillon Alain Vignal Wageningen University Frédérique Pitel Martien Groenen Mireille Morisson Richard Crooijmans
INRA Jouy-en-Josas Genome Institut Michèle Tixier-Boichard Bertrand Bed’Hom Wes Warren
INRA Nouzilly University of Kent David Gourichon Darren Griffin Thank you for your attention… !