Animal cytogenetics

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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, 2017. �hal-02785183�

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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

probe

Resolution > 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

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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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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 année

Nombre d'analyses Nombres d'anomalies de structure

Results of French chromosomal control

•Cattle : 1700 per year

•By January 2017, 36 000 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)

•182 structural chromosomal abnormalities

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II. Chromosomal abnormalities

- Clinical cytogenetics

- Some examples in horse, pig and cattle

•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

•1700 animals analyzed annualy

•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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II. Chromosome abnormalities

- Clinical cytogenetics

- Some examples in horse, pig and cattle

•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 BTA1-29

Rate 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)

Sperm FISH studies with inversions

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)

Synaptonemal complexes studies

(Codina-Pascual et al, 2006) (Sun et al, 2006)

red SC

Blue Centromere

Yellow Crossing over

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

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III. Cytogenetic mapping : lessons from chicken

- The chicken genome

- High resolution mapping

- Chromosome polymorphism

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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numéro de

chromosome groupe de liaison

Genetic

sizes (cM) 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 29-30-31-32

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 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 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

- Chromosome polymorphism

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

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Valérie Fillon / ECA 2014 10 / 03 / 2014

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III. Cytogenetic mapping : lessons from chicken

- The chicken genome

- High resolution mapping

-

Chromosome polymorphism

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).

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 87E13 GGA1 GGA3 insertion breakpoint

Naked 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

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III. Cytogenetic mapping : lessons from chicken

- The chicken genome

- High resolution mapping

- Chromosome polymorphism

* 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

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.

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

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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 r

Chicken

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 - NGS

In 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., 1999

Very 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 map

Robust 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 CAM163_sca736A 364.8_381.8 sca2558 970.0 sca1445 1421.4 sca3847 1641.3 sca1434C_sca1434B 2170.2_2644.6 CAM166 3587.6 sca1434A_sca2840 3595.1_3698.4 sca368 3958.5 CAM167_sca5376 3970.5_4381.8 sca498A 4479.8 CAM170 4495.8 sca498B_sca903 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 sca597D_sca769 17420.1_17930.7 sca5519_sca469 18403.7_18759.1 CAM180_sca586 19057.1_19678.5 sca51 19941.7 sca3004_sca577 20505.4_20770.8 CAM183B_sca1481 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

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, 1990

Some 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 !

.0147

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 !

.0151

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

.0152

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 ?

.0157

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

.0165

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

.0166

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 Gourichon

Saint-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

Thank you for

your