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Animal Cytogenetics Valerie Fillon

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Animal Cytogenetics Valerie Fillon Animal Cytogenetics Valerie Fillon To cite this version: Valerie Fillon. Animal Cytogenetics. Master. European Advanced Postgraduate Course in Classical and Molecular Cytogenetics, 2015. hal-02793565 HAL Id: hal-02793565 https://hal.inrae.fr/hal-02793565 Submitted on 5 Jun 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Animal Cytogenetics Valérie Fillon and Alain Pinton 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 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 cytogenetics laboratories (mainly in Europe) •Since the beginning of the 90’s → decline of clinical animal activities → 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) I. General overview - History - Technical aspects - Some animal karyotypes .013 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 .014 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) .016 Horse 2n = 64 C bands for sex chromosomes .017 Chicken karyotype 2n=78 Females are heterogametic ZW Presence of 30 pairs of microchromosomes = 30% of the genome 50% of the genes (GC rich) II. Chromosomal abnormalities - Clinical cytogenetics - Some examples in horse, pig and cattle - Segregation during meiosis - Cytogenetic polymorphism : chicken .019 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 •Pigs 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 2014, 30 000 pigs have been analyzed •160 structural chromosomal abnormalities •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 - Cytogenetic polymorphism : chicken .025 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 inverted normal 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 1/29 frequency 0,05 0 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 year •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 A. Garnier (unpublished data) II. Chromosome abnormalities - Clinical cytogenetics - Some examples in horse, pig and cattle - Segregation during meiosis - Cytogenetic polymorphism : chicken .034 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 (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 Synaptonemal complexes studies
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