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To cite this version:
Valerie Fillon. Animal Cytogenetics. European Advanced Postgraduate Course in Classical and
Molecular Cytogenetics, 2016. �hal-02801267�
.01
Animal Cytogenetics
GENPHYSE – INRA Toulouse - France
Valérie Fillon and Alain Pinton
ECA 10 mars 2014 .02
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 *Cytogenetic Platform
.03
Valérie Fillon / ECA 2014 10 / 03 / 2014
I. General overview
II. Chromosomal abnormalities
III. Cytogenetic mapping
IV. Comparative mapping and evolution
.04
Valérie Fillon / ECA 2014 10 / 03 / 2014
I. General overview
- History
- Technical aspects
- Some animal karyotypes
.05
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
cytogenetics laboratories (mainly in Europe)
•Since the beginning of the 90’s → decline of clinical
animal activities-> gene mapping
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Valérie Fillon / ECA 2014 10 / 03 / 2014
I. General overview
- History
- Technical aspects
.07
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
Chromosome Slides
.08
Fluorescent In Situ Hybridization
FISH
Localisation directly on the chromosomes of a genomic sequence
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 probe Resolution > 1 Mb Chromosome assignation Measurment
Analysis under the microscope
1 2 3 4
5
6 7 8
5
Microdissection
Chromosome arms 8p and 8q generated by microdissection
A. Pinton (unpublished data) Inv(8)(p1.1;q2.5)
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Valérie Fillon / ECA 2014 10 / 03 / 2014
I. General overview
- History
- Technical aspects
- Some animal karyotypes
.014
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 norvegicus Mus musculus 42 40
Dog Canis familiaris 78
Cat Felis domesticus 38
Chromosomes numbers of different species
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
G banded Pig karyotype 2n=38
.016
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)
.017
Horse 2n = 64
Chicken karyotype 2n=78
Females are heterogametic ZW
Presence of 30 pairs of microchromosomes = 30% of the genome 50% of the genes (GC rich)
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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
- Cytogenetic polymorphism : chicken
•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
CLINICAL CYTOGENETICS
•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 0 2 4 6 8 10 12 14 16 18 0 500 1000 1500 2000 2500 3000 1 9 9 1 1 9 9 2 1 9 9 3 1 9 9 4 1 9 9 5 1 9 9 6 1 9 9 7 1 9 9 8 1 9 9 9 2 0 0 0 2 0 0 1 2 0 0 2 2 0 0 3 2 0 0 4 2 0 0 5 2 0 0 6 2 0 0 7 2 0 0 8 2 0 0 9 2 0 1 0 2 0 1 1 année
Nombre d'analyses Nombres d'anomalies de structure
Results of French chromosomal control
•By January 2014, 30 000 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) •160 structural
chromosomal abnormalities
•86% reciprocal translocation
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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
- Cytogenetic polymorphism : chicken
Principal chromosomal abnormalities identified in Pig, Cattle and Horse.
•Sex chromosome abnormalities
X Y
Y
63, X0 / 65, XYY mosaicism in a case of equine male pseudohermaphroditism
Paget et al.(2001)
Gonads: testis like structure (no spermatozoa)
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
•Reciprocal translocations
•Rcp(6;8)(10;18)•Reciprocal translocations
•Rcp(Y;1)•Reciprocal translocations
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)
•Inversions
•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 /2 9 f re q u e n c y•1700 animals analyzed annualy
Statutory obligation for all young bulls→AI
•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 BAC IDVGA7 on BTA29 BAC IDVGA7 on BTA29 BAC IDVGA7 on BTA29
A. Garnier (unpublished data)
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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
- Cytogenetic polymorphism : chicken
•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
Sperm FISH studies with rcp
Rcp(3;15)
Rcp(12;14)
Sperm FISH studies with rcp
unbalanced balanced
der(13;17)
13 17
Alternate Adjacent 3:0
Sperm FISH studies with rob
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♂
♀
Sperm FISH studies with rob
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…. Synaptonemal complexes studies
SC electronique microscopy
Synaptonemal complexes studiesImmunocytological staining
(B. de Massy, 2005) Synaptonemal complexes studies
Zygotene Pachytene
Maternal sister chromatids
Paternal sister chromatids
Axial element central element
(B. de Massy, 2005) Synaptonemal complexes studies
(Codina-Pascual et al, 2006)
(Sun et al, 2006)
red SC
Blue Centromere
Yellow Crossing over
Synaptonemal complexes studies
Visualisation of SC : anti SCP3 et SMC3 Visualisation of Recombination nodules : anti LMH1
MLH1-focus mapping in birds shows equal recombination between sexes and diversity of crossover patterns
P. L. Caldero´n & M. I. Pigozzi* Chromosome Research (2006) 14:605–612
spermatogenesis arrest at the spermatocyte stage
(Barasc et al., 2012) Synaptonemal complexes studies
Effects of chromosomal rearrangements
SSC13 SSC12 N T N T Synaptonemal complexes studies
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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
- Cytogenetic polymorphism : chicken
.052
Rose comb mutation in chicken
Plos Genetics, 2012
.053
Studied by Bateson & Punnett (1906) 2 different traits :
- Comb morphology (dominant)
- Hypofertility of males (recessif)
•Loss of motility of spermatozoas in homozygous males
GGA7 : small 38Mb macrochromosome
Cytogenetic characterization
1 2 3 4 5 6 7 8 Z W
GDR Cytogénomique Structurale et Evolutive Paris - 22 et 23 octobre 2010
Inversion of 7 Mb for R1
2 different genotypes : R1 and R2 leading to the same phenotype
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Valérie Fillon / ECA 2014 10 / 03 / 2014
III. Cytogenetic mapping
- Map integration
- Characterization of the chicken karyotype
- High resolution mapping
- Cytogenetic and New Generation Sequencing
Fluorescent In Situ Hybridization
FISH
Localisation directly on the chromosomes of a genomic sequence
Map integration
Genetic map
Cytogenetic map
Molecular markers
Large insert clones
BAC
INTEGRATIONExcept in mouse : no commercial probes
BAC ressources
Map integration
FISH
RH
Genetic map
BAC
Contigs
Sequence
CHROMOSOME 1 6 5 4 3 2 1 5 4 3 2 1 2 1 1 2 3 4 1 2 12 34 5 6 1 23 45 1 2 3 4 bw30B21 bw31B10 bw43G6 bw25G16 bw30P7 P1H9 P6D4 bw4F8 bw38E8 P3-3 P1-2 P2-9 P2-6 B3H9 P2H3 P2-5 P2-10 P4D9 B2B4 P4B11 P4D8 CHROMOSOME 2 3 21 4 3 21 2 1 1 2 1 2 3 4 5 6 12 34 5 6 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 23 4 5 bw29L12 bw13L14 P3D3 P1-9 P5A6 CHROMOSOME 4 1 1 2 43 2 1 1 2 3 12 3 4 5 bw37E19 bw62D14 bw8H20bw112C24 bw125P16 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 3 4 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
Chicken cytogenetic map
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
The genome coverage
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Valérie Fillon / ECA 2014 10 / 03 / 2014
III. Cytogenetic mapping
- Map integration
- Characterization of the chicken karyotype
- High resolution mapping
- Cytogenetic and New Generation Sequencing
.064
Characterization of the chicken karyotype
.065
Size estimation: 26-27
numéro de
chromosome groupe de liaison sizes (cM)Genetic Bac ou paint
9 E36C06W08 132 bac/paint 10 E29C09W09 120 bac/paint 11 E30C14W10 88 bac/paint 12 E16C17W22 90 bac/paint 13 E48C28W13W27 74 bac/paint 14 E35C18W14 + C37 77 bac/paint 15 E18C15W15 71 + 8 bac 16 Ch16 60 bac 17 E41W17 + C24 70 + 0 bac 18 E31E21C25W12 47 bac/paint 19 E52W19 40 bac/paint
20 E47W24 + E32 62 bac
21 E54 64 bac 22 E38 21 bac 23 E27C36W25W26 13 bac/paint 24 E49C20W21 58 bac/paint 25 no - paint 26 E60E04W23 67 bac/paint 27 E59C35W20 75 bac/paint 28 E53C34W16 75 bac/paint 29 E62 0 bac/paint 30 E65 11 bac/paint 31 E64 0 bac/paint 32 E25C31 21 bac/paint 33 no - paint 34 no - paint 35 no - paint 36 no - paint 37 no - paint 38 no - paint
7 chromosomes sans assignation à des groupes de liaison
?
Groupe 1
Groupe 2
Groupe 3
19-21-22-23 24-26-27-28 29-30-31-32Hybridation against the FISH tags
.070 0 20 40 60 80 100 120 140 160 180 200 12 3 45 6 78 9 10 11 12 13 14 151617 18 19 20 21 22 23 24 25 * 26 27 28 29 * 30 * 31 * 32 33 * 34 * 35 * 36 * 37 * 38 * W ZU Mb Chromosomes
Sequence :
933
Mb and
120
« unknown »
400 Kb !!!!
30 microchromosome pairs from 7 to 23 Mb
*
7 microchromosomes absent from the sequence
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Valérie Fillon / ECA 2014 10 / 03 / 2014
III. Cytogenetic mapping
-
Map integration
- Characterization of the chicken karyotype
-
High resolution mapping
Lampbrush chromosomes
Chromosome Structure and Function Laboratory
Biological Research Institute
Saint-Petersburg State University
High resolution mapping
During spermatogenesis
Y chromosome of Drosophila transforms into lampbrush
Lampbrush chromosomes can be found in oocytes of:
fishes
amphibians
reptilia
birds
insects
plants (Acetabularia acetabulum)
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)
isolation of lampbrush chromosomes from bird oocytes
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
- fixation in 2% formaldehyde for 5 min and 50% and 70% ethanol
Chicken lampbrush chromosome 1
Chicken mitotic chromosome 1
185
µ
m
6
µ
m
Les chromosomes en écouvillon
Chicken LBC4 and mitotic chicken chromosome 4 (100x)
Derjusheva et al., 2003Lampbrush chromosome structure
Chiasmas Chromomeres bivalent Chromatides DNA loops Transcription units L K J I H G F E D C 12 3 4 5 1 2 1 2 3 1 2 1 2 12 1 2 1 2 B A
Chiasma distribution along the chicken lampbrush chromosome 1
C
h
ia
sm
a
f
re
q
u
e
n
cy
100%Cytological recombination maps
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
Groenen et al., 2000 Nature, 2004 0 20 40 60 80 100 120 140 160 180 200 1 23 4 56 7 89 10 11 12 13 14 151617 18 19 20 21 22 23 24 25
*
26 27 28 29*
30*
31*
32 33*
34*
35*
36*
37*
38*
W ZU Mb ChromosomesSéquence : 933 Mb et 120 « unknown »
450 Kb !!!!
30 paires de microchr. de 7 à 23 Mb GGA16 : 10 Mb estiméCarte cytogénétique haute résolution
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@Intégration des cartes
B@
NOR
Centromère
.092
III. Cytogenetic mapping
-
Map integration
- Characterization of the chicken karyotype
- High resolution mapping
- Cytogenetic and New Generation Sequencing
*10 smallest
*GGA16 : bearing MHC and NOR
7 Microchromosomes absent of the assembly
60 microchromosomes : 3 to 20 Mb (30%)
GC and gene rich (50%) PO41 specific repeated sequence
ChrUn 2n=78
Identifying and sequencing the missing microchromosomes
*Collaboration : S. Galkina (St-Petersburg University)
*International synergy : Wes Warren
The full chicken genome (since 2004)
Sangers sequencing
Identifying the missing microchromosomes
1-Description of microchromosomes
Saifitdinova A., Daks A., V. Fillon, E. Gaginskaya, S. Galkina. Cytogenetic description of chicken microchromosomes at the lampbrush phase // Chromosome Research. 2014. V.22. P. 411-412.
St-Petersburg
Sequencing the missing microchromosomes
Microdissection of LBC
Whole genome amplification MALBAC
(Multiple Annealing and Looping Based Amplification Cycles)
FISH control NGS
sequencing 2-Microgenomics
8
Pyrophage
Whole genome amplification MALBAC
(Multiple Annealing and Looping Based Amplification Cycles)
Go-Taq
MALBAC utilizes special primers that allow amplicons to have complementary ends and therefore to loop, preventing DNA from being copied exponentially
Sequencing the missing microchromosomes
3-Microgenomics
Preliminary results on microdissected pig chromosome SSC1
Alain Pinton
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Valérie Fillon / ECA 2014 10 / 03 / 2014
IV. Comparative mapping and evolution
- evolution studies in birds
- what about reptiles ?
- sex chromosomes
Typical bird organisation
Typical bird organisation
2- diploïd number : 2n = 76 to 82
3- microchromosomes : 60 à 64
1- sex chromosomes ZZ and ZW
10% of the 9000 speciesWho belongs microchromosomes ?
Who belongs microchromosomes ?
FISH (Sturgeon)
Salamanders
Ophidiens (snakes)
Boa VipèreEvolution in birds
Evolution in birds
- Monophyletic group
- Closest groupe: crocodilians
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
Poule
Quail
Dinde
Duck
Avian karyotypes share the same organisation
In Galliformes :
Stock et Bunch, 82Chromosome correspondances
ZooFISH Chromosomes of a different speciesProbes
reference species
Ex : chicken probes.0111
Numerous intrachromosomal
rearrangements in spite of high synteny
conservation between duck and chicken
GENPHYSE – INRA Toulouse - France
Fillon Valérie, Rao Man, Faraut Thomas, Griffin Darren,
Morisson Mireille and Vignal Alain
(Sibley and 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
80 millions years of evolution between duck and chicken
Chicken
Duck
2- diploïd number : 2n = 76 to 82
3- microchromosomes : 60 à 64
1- sex chromosomes ZZ and ZW
Typical bird organisation
Chicken genome as a reference : Sequenced in 2004 (Sangers) Closed to the ancestral kayotype
Zoo FISH
Chicken
BAC clones
Duck
metaphases
More than 200 BAC clones mapped
(Fillon et al, 2007 ; Skinner et al, 2009)
Bird karyotypes are very stable compare to mammals
No interchromosomal rearrangements (except GGA4)
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
Duck Genome Assembly (2009, BGI)
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)
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.
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
The comparative mapping strategy using RH mapping
•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
Full map sca1452 sca1221 sca1452 sca1221 GGA3 APL3 Full map
Example of chromosome 3 : inversion and breakpoint
sca691 sca691
GGA5
APL5
Full map
Example of chromosome 5 : insertion
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 CAM163 364.8 sca736Asca2558 381.8 970.0 sca1445 1421.4 sca3847 1641.3 sca1434C 2170.2 sca1434B 2644.6 CAM166 3587.6 sca1434A 3595.1 sca2840 3698.4 sca368 3958.5 CAM167 3970.5 sca5376 4381.8 sca498A 4479.8 CAM170 4495.8 sca498Bsca903 6455.2 6556.8 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 sca5519 18403.7 sca469 18759.1 CAM180 19057.1 sca586 19678.5 sca51 19941.7 sca3004sca577 20505.4 20770.8 CAM183Bsca1481 20886.6 21044.6 sca2156 21582.7
Zebra 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
Passeriformes Galliformes Galliformes Anseriformes Zebra finch Turkey Chicken Duck 0 50 100 MYA MGA13 GGA11 TGU11 APL12RH mapping and avian comparative maps
Lineage specific rearrangements : GGA11
GGA11 GGA11
Detemination of 7 conserved blocks
outgroup APL2 TGU2 APL2 MGA M G A 6 M G A 3 5 3 .2 5 M b 1 0 0 ,4 M b APL2 GGA2
RH mapping and avian comparative maps TGU2
GGA2
Lineage specific rearrangements : Chromosome 2
Duck lineage
2- Pour les microchromosomes ...
No interchromosomal rearrangments except one splitting GGA4
Numerous intrachromosomal complex rearrangements :
- lineage specific
- number of rearrangements consistents with the
divergence times.
CONCLUSION
Evidence for evolutionnary breakpoints
Bird karyotypes are very stable compare to mammals
Typical avian karyotype
Typical avian karyotype
2- diploïd number: 2n = 76 à 82
3- microchromosomes : 60 à 64
1- Sex chromosomes ZZ et ZW
10% of the avian species
Diploïd number
Diploïd number
2/3 of species : 76 to 82 chromosomes
Some exceptions ...
Nombre diploide N o m b re d ’e sp èc es Christidis, 90Oedicnème criard 2n = 40
King fisher 2n = 138
Upupa epops 2n = 126
Falco peregrinus 2n = 50
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 ANSERIFORMESAccipitridae 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 CiconiidaeCircaetinae (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
Bed’hom et al
Atypical karyotype of Accipitridae
Moderate diploid number (66) Middle-sized macrochromosomes Few microchromosomes Limits of comparative cytogenetics 1 2 3 4 5 Z W 7 8 9 1 0 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 2 0 2 1 2 2 2 3 2 4 2 5 2 6 2 7 2 8 2 9 3 0 3 1 3 2 1 1 6
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 Bed’hom et al, 2004
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 interchromosomic rearrangments !
.0140
Valérie Fillon / ECA 2014 10 / 03 / 2014
IV. Comparative mapping and evolution
- evolution studies in birds
- what about reptiles ?
- sex chromosomes
Boa
Chicken
What about reptiles ?
Turtles (300 sp)
Snakes and lizards
(8200 sp) crocodiles (23 sp)
Birds (9000 sp)
220 My between crocodiles and birds
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 ! .0148
Valérie Fillon / ECA 2014 10 / 03 / 2014
IV. Comparative mapping and evolution
- evolution studies in birds
- what about reptiles ?
- origin of sex chromosomes
.0149
The origin of sex chromosomes
-In Mammals : X and W chromosomes -In Birds : Z and W chromosomes
No homology between human XY and ZW chicken sex chromosomes
Nanda et al.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 Hybridations ADN/ADN Ratatites = paleognathous Carinates = neognathous
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
Does it exist a common origin of the sex chromosomes
between reptiles and birds ?
Chinese soft-shelled turtle
(Pelodiscus sinensis)
Japonese four-striped rat snake
(Elaphe quadrivirgata)
Siam crocodile
(Crocodylus siamensis)
Cytogenetic investigations in 3 reptile species - identification of sex chromosome - comparative mapping of homologues of chicken Z linked genes
Identification of the sex-chromosomes in the Chinese soft-shelled turtle
Conventional Giemsa staining
C-banding and Comparative Genomic Hybridization FISH mapping of 18S-28S rRNA and Ag-NOR staining
ZZ/ZW sex chromosome in this turtle : Z is a microchr. NOR bearing sex chromosome
Inactivated Z
FISH mapping of 5 chicken Z-linked genes Molecular cloning : extraction of total RNA from cells
RT-PCR to obtain cDNA Amplification of homologous genes purification of the band cloning in E. coli sequencing
FISH mapping of 5 chicken Z-linked genes
Synteny conservation in chicken and reptiles Gene order conservation in reptiles (CS13).
Neocentromere ?
*Sex chromosome differentiation could be caused by
- loss of euchromatin
- accumulation of repeated DNA (absence of recombination)
*High conservation of the Z-linked gene
in the bird and reptile ancestor
*Confirmation of the phylogeny of Sauropsidae
*Comparison with the Ostrich paleognathous :
order of genes conserved wtith these Reptiles
The Z of paleognathous bird is the ancestral bird Z chromosome*GGAZ : well conserved among birds despite intrachromosomic
rearrangments
*Conservation of the avian Z is lost in mammals
*Some previous studies have demonstrated the synteny
conservation between GGAZ and some reptiles chromosomes
Investigation of the evolution of the bird Z chromosome
in Squamates
- FISH painting
- comparative mapping
Description of new karyotypes no observation of heteromorphic chr.
caméléon
gekko
Un autre gekko
- flow sorted GGAZ chromosome
- flow sorted chromosome of C. niloticus
- flow sorted chromosome of S. scincus
Gene mapping by PCR of chicken Z-linked genes on Reptiles flow sorted chromosomes :
synteny conservation
FISH painting DOP-PCR
Reciprocal painting of SSC2 on GGA Z and W (difficult !).
These results are against the hypothesis of
avian-like sex determination in the squamates
Conservation of synteny with the GGAZ :
- but different chromosomes are labelled depending on the species according to the phylogeny:
ex : p arm of Chr. 2 (15 species) ex : acrocentric chr. (12 species) - but it is not syntenic with ZW or XY sex chromosome in Reptiles when identified.
Through mammals !
Genome research, 2008