Animal cytogenetics

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To cite this version:

Valerie Fillon. Animal cytogenetics. Master. European Advanced Postgraduate Course in Classical

and Molecular Cytogenetics, 2014. �hal-02793566�

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.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 bird

evolution

.04

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

.06

I. General overview

- History

- Technical aspects

(3)

.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A. Pinton (unpublished data) Inv(8)(p1.1;q2.5)

(4)

.013

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

LE CARYOTYPE DU POULET : typique de l’organisation des génomes aviaires

Females are heterogametic ZW

Presence of 30 pairs of microchromosomes = 30% of the genome

(5)

.019

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

(6)

.025

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

(7)

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)

.034

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

(8)

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 BTA1-29

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

♂

♀

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)

(9)

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 studies

Immunocytological staining

(B. de Massy, 2005)

Zygotene Pachytene

Maternal sister chromatids

Paternal sister chromatids

Axial element central element

(B. de Massy, 2005)

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

red SC

Blue Centromere Jaune (vert) 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 Synaptonemal complexes studies

(10)

Analysis of recombination 0 10 20 30 40 50 -1 -0,8 -0,6 -0,4 -0,2 +0,2 +0,4 +0,6 +0,8 +1 nbre de CO bras Q bras P 3 CO 2 CO 1 CO metacentrics t c t Synaptonemal complexes studies

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

.052

II. Chromosome abnormalities

- Clinical cytogenetics

- Some examples in horse, pig and cattle

- Segregation during meiosis

- Cytogenetic polymorphism : chicken

.053

Rose comb mutation in chicken

Plos Genetics, 2012

.054

Studied by Bateson & Punnett (1906) 2 different traits :

- Comb morphology (dominant)

- Hypofertility of males (recessif)

(11)

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

.057

III. Cytogenetic mapping

- Map integration

- Characterization of the chicken karyotype

- High resolution mapping

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

INTEGRATION

Except in mouse : no commercial probes

(12)

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

.064

III. Cytogenetic mapping

- Map integration

- Characterization of the chicken karyotype

- High resolution mapping

.065

Characterization of the chicken karyotype

(13)

How to identify a new microchromosome ?

Size estimation: 26-27

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 Hybridation against the FISH tags

.071 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 Chromosomes

Sequence : 933

Mb and

120 « unknown »

400 Kb !!!!

30 microchromosome pairs from 7 to 23 Mb

(14)

.073

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

(15)

Chicken lampbrush chromosome 1

Chicken mitotic chromosome 1

185 µ

m

6 µ

m

Chicken lampbrush chromosome 1

Les chromosomes en écouvillon

Chicken LBC4 and mitotic chicken chromosome 4 (100x)

Derjusheva et al., 2003

Lampbrush 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

(16)

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 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

Séquence : 933 Mb et 120 « unknown »

450 Kb !!!!

30 paires de microchr. de 7 à 23 Mb GGA16 : 10 Mb estimé

Carte cytogénétique haute résolution

NOR: near the centromere, covers 40% of the chromosome

B@at the q end

(17)

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

.093

IV. Comparative mapping and

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

Poule

Quail

Dinde

Duck

Avian karyotypes share the same organisation

In Galliformes :

(18)

Chromosome correspondances

ZooFISH _{Chromosomes of} a different species

Probes

reference species

Ex : chicken probes

(19)

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

Cytogenetic data on APL2

•orange: inversion •pink: translocation •blue: inversion 106 APL2(ref:GGA) GGA2 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***

(kindly provided by V.Fillon and D.Griffin) RH mapping and avian comparative maps

APL2

Confirmation

by FISH

Sca616_1 Sca1008_0 Sca713 Sca616_0 Sca1034_1 Sca74_1 56.56Mb 56.80Mb 57.10Mb 63.23Mb 67.26Mb 78.11Mb GGA2 APL2(ref:GGA2) centromere 107 RH mapping and avian comparative maps

GGA2 APL2

Detecting rearrangements

108 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 sca736A_sca2558 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 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 18403.7 sca469 18759.1 CAM180 19057.1 sca586 19678.5 sca51 19941.7 sca3004_sca577 20505.4 20770.8 CAM183B_sca1481 20886.6 21044.6 sca2156 21582.7kb RH mapping and avian comparative maps

(20)

Rearrangement confirmed by FISH

sca2558

sca1176

RH mapping and avian comparative maps

2- Pour les microchromosomes ...

No interchromosomal rearrangments except some splittings

Only a few intrachromosome rearrangments

Bird karyotypes are very stable compare to mammals

ZOO FISH Sscr

14

11 12 13 14 15 16 21 22 23 24 25 26 27 28 29 SFTPC SYK* GALT NOS1 COMT ACTA1* RYR2* PLAU* RBP3 CHAT* ACTA2 DNTT DAO* UBC* LPL* IGL* OAT PAX2 CYP17 22 22 12 9 9 8 12q24.1 22q11.2 8p21 9p13 8q22 17q25 17q26 28q19 28q17 28q13-14 26q21 26q23 8p22 9q22.3 12q24.2-24.31 10q11.2 10q11.2 10q22-24 10q24 12q24.31-24.33 22q11.2 1q42.1 1q42.1-43 10q24 10q24.3-25.1 10q24.3 10q26

Chir

Hsap

Anchorage markers localization •Pig species

Evolution in birds

- Monophyletic group

- Closest groupe: crocodilians

- Commun ancestor with mamals: 300 millions d ’années

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 species

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

(21)

Chromosomes sexuels ZZ et ZW :

The case of Ratites

Hardly differenciated sex schromosomes

Who belongs microchromosomes ?

FISH (Sturgeon)

Salamanders

Ophidiens (snakes)

Boa Vipère 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

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

(22)

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 !

Tortues actuelles (300 sp) serpents et lézards (8200 sp) crocodiles _{(23 sp)} Oiseaux (9000 sp)

220 My between crocodiles and birds

Boa

Poulet

What about reptiles ?

(23)

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 !

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

No homology between human XY and ZW chicken 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 ?

(24)

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

(25)

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

*Sex determination ??? DMRT1 ???

*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

(26)

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 chromsomes :

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.

(27)

Through mammals !

Genome research, 2008

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