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Positional cloning in sheep and goat : Available tools and perspectives

Positional cloning in sheep and goat : Available tools and perspectives. Laurent SCHIBLER INRA, Génétique animale Laboratoire de Génétique Biochimique et de Cytogénétique. sub chromosomal location. Biochemical knowledge. Comparative data from other species. Gene product. Functional assay.

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Positional cloning in sheep and goat : Available tools and perspectives

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  1. Positional cloning in sheep and goat :Available tools and perspectives Laurent SCHIBLER INRA,Génétique animale Laboratoire de Génétique Biochimiqueet de Cytogénétique

  2. sub chromosomallocation Biochemical knowledge Comparative data from other species Geneproduct Functionalassay Positionalcloning Functionalcloning : caseins, scrapie Positionalcandidateapproaches Candidate genes approaches Strategies for gene identification Unidentifiedgene

  3. Positional cloning strategy ACTATTATG ACTGTTATG Gene Inventory Regional Cloning GeneticLocalization Mutation Screening

  4. Positional cloning strategy ACTATTATG A CTGTTATG Families Genetic markers A1A2 A3A4 A1A3 A5A6 A1A5 A3A6 A1A5 A3A5 A1A6 A1A5 Large insert libraries Comparative Mapping FISH RH panels Genome Sequence

  5. Genetic mapping • The sheep genetic map Crawford et al 1995 de Gortari et al 1998 Maddox et al 2001 • 26 pairs of autosomes (3 metacentric) • 1062 loci • 121 markers developedfrom genes • 3530 cM • Average spacing : 3.4 cM • International database

  6. Genetic mapping 1 1 1 2 1 3 1 4 2 1 2 2 2 3 2 4 2 5 3 1 3 2 3 3 3 4 3 5 3 6 4 1 4 2 4 3 4 4 4 5 4 6 CHI1 • The goat genetic map Vaiman et al 1996 Schibler et al. 1998 • 29 pairs of acrocentric autosomes • 307 microsatellite loci • 23 genes • 2737 cM on autosomes • Average spacing : 14.5 cM • International database

  7. Genetic mapping • Linkage disequilibrium (LD) • Domestication results in a drastic choice of a limited number of individuals  bottleneck in reproductive populations. • LD mapping has the potential to position loci to small chromosomal segments.

  8. Genetic mapping These approaches need to test simultaneously for linkage and linkage disequilibrium to minimize the risk of type I error. In ruminants, LD mapping might be a viable approach for QTL discovery using the available maps of medium density, although fine-mapping resolution may be limited. • Linkage disequilibrium (LD) McRae et al. 2002 • A study in Coopworth and Romney sheep populations • Very high LD and LD decreased as a function of distance. • But substantial LD between pairs of unlinked markers • Same results than in cattle (Farnir et al. 2000)

  9. Genetic mapping LSCV098 LSCV095 LSCV082 LSCV086 1 LodScore unit interval (0.05cM) LSCV203 LSCV202 3 LodScore unit interval (0.09cM) • Linkage disequilibrium Schibler et al. 2000

  10. Physical mapping Clone distribution Part 1 25 20 150 kb 15 110 kb % of clones 10 50 kb 5 7 kb 0 100 100 110 110 120 120 130 130 140 140 150 150 160 160 200 200 Part 4 Size (kb) 200 kb 25 150 kb 20 110 kb 15 % of clones 50 kb 10 5 7 kb 0 30 30 80 80 100 100 120 120 140 140 160 160 180 180 200 200 Size (kb) • The Goat Bacterial Artificial Chromosome library. • 61.440 clones arrayed in 40 Superpools • Two size ranges : 140 and 180 kb, with a mean insert size of 153 kb Schibler et al. 1998

  11. Physical mapping • Sheep large insert libraries. • Yeast Artificial Chromosome : YAC Library • 29.000 clones with a mean insert size of 680 kb (6X cov). • Available as Filters for hybridization and Pools for PCR screening Broom et al. 1994 • Bacterial Artificial Chromosome : BAC Library • 90.000 clones with a mean insert size of 123 kb (3X cov). • Available as Nylon Filters and 39 SuperPools. Vaiman et al. 1999

  12. Physical mapping LSCV73 LSCV35 LSCV66 LSCV77 LSCV76 LSCV79 LSCV78 LSCV81 LSCV45 LSCV75 LSCV74 LSCV82 LSCV69 LSCV68 LSCV67 LSCV56 LSCV119 LSCV120 LSCV115 LSCV116 NCK RBP1 TFDP2 ATP1B WI9573 WI7255 400 kb 1000 kb 900 kb 800 kb STSG3182 STSG9571 • Use in regional contig building : example of PIS PIS PIS PIS

  13. Comparative genomics 7 MA Suiformes Swine 60 MA Artiodactyla Goat 45 MA 15 MA 80 MA Caprinae Ruminants Sheep Bovidae Bovinae Cattle 120 MA Perissodactyla Horse 180 MA Eutheriens Carnivora Cat Primates Human Rodents Mice

  14. Comparative genomics 3 8 11 3 centric fusions BTA 1 + 3BTA 2 + 8BTA 5 + 11 1 1 2 2 3 5 4 4 5 7 6 6 7 10 8 9 9 14 X chromosomes are different 10 12 11 19 12 16 13 13 14 18 15 15 16 20 17 17 18 21 19 22 20 23 Bovinae and Caprinae differ by a small 9-14 translocation YY 21 29 22 26 23 24 24 25 26 27 25 28 Sheep Catlle X X • Banding patterns showed chromosome similarities • Comparative mapping in ruminants. Cattle and sheep karyotypes BTA 2n= 60 OAR 2n= 54

  15. Comparative genomics • Comparative mapping in ruminants. • Interspecific use of microsatellites • About 30 % of cattle microsatellites are polymorphic in sheep and goats. • About 40 % of sheep microsatellites are polymorphic in goats. • 243 markers in common between cattle and goat. • 209 markers in common between sheep and goat. • 572 markers in common between cattle and sheep

  16. Comparative genomics • Comparative mapping with man • At the Cytogenetic level : Chromosome painting • Human and sheep probes on muntjac • Human probes on sheep chromosomes • Identification of 48 conserved segments Burkin et al. 1997 Yang et al. 1997 Iannuzzi et al. 1999

  17. Comparative genomics • Comparative mapping with man. • At the level of gene order : FISH and RH mapping • The first integrated comparative cytogenetic map for the ruminants, established in goats. (schibler et al. 1998) • Integration of genetic and cytogenetic maps by FISH mapping of 141 microsatellites. • Comparative mapping with human and mouse by FISH mapping of 202 BACs containing identified type I loci. • A ruminant map with 262 genes and 15 ESTs cytogenetically mapped, if results from cattle and sheep are included. • 3 to 20 genes/chromosome, 8,5 genes/chromosome on average • 1 gene every 18 cM on average • 54 conserved syntenic segments, in agreement with ZOO FISH. • 107 segments of about 27 cM with conserved gene order.

  18. Comparative genomics CHI 20 • Comparative mapping with human. • Global conservation

  19. Comparative genomics CHI 7 • Comparative mapping with human. • Synteny breakage with intrachromosomal rearrangements

  20. Comparative genomics CHI 10 • Comparative mapping with human. • Patchwork of conserved segments

  21. Comparative genomics • Comparative mapping with human. • At the gene order level : FISH and RH mapping • The first integrated comparative cytogenetic map for the ruminant, established on goats. (schibler et al. 1998) • Sheep comparative genomics (Iannuzzi et al. 1999-2003) • FISH mapping of goat BACs on sheep. • Sheep RH panel • 130 clones at 12.000 rad produced at INRA (LGBC, LGC) • 96 best cell lines still under characterization.

  22. Comparative genomics • Similarities between different species, and similar gene order along part of the chromosome. • Take advantage of the dense maps of human, mouse and rat genomes and especially of the draft sequence of the human genome • Characterisation of a gene, or a whole genome, in one species could provide important clues about gene function in other species, and about the nature and function of individual genes more widely

  23. Comparative genomics PIS CHI1 • Use in positional cloning : Example of PIS • Identification of human orthologous region

  24. Comparative genomics CHI 22q23-23 PIS • Use in positional cloning : Example of PIS • Fine mapping of human orthologous region

  25. Comparative genomics LSCV73 LSCV35 LSCV66 LSCV77 LSCV76 LSCV81 LSCV79 LSCV78 LSCV75 LSCV45 LSCV74 LSCV82 LSCV67 LSCV69 LSCV68 LSCV56 LSCV119 LSCV120 LSCV115 LSCV116 NCK RBP1 TFDP2 ATP1B WI7255 WI9573 800 kb 900 kb 400 kb 1000 kb STSG3182 STSG9571 • Use in positional cloning : Example of PIS • Chromosome walking using multiple comparative entry points PIS

  26. Tools available in other species • The cattle BAC based physical maps • Ordered set of overlapping clones (contig assembly) • Immediate access to any segment of the genome that can be defined genetically • Starting point for studying the large-scale organization of the genome • First step toward sequencing the genome

  27. Tools available in other species A B C D E F G V V • The cattle BAC based physical maps. • The INRA bovine BAC based physical map • fingerprinting using the double digest method. • recognize overlapping patterns through a subset of short restriction fragments. Schibler et al. 2004 Electrophoresisafter clone digestion Identification of overlapping clones through the presence of common restriction fragments Iterative processfor contig assembly(FPC6 software)

  28. Tools available in other species Examples of Restriction profiles ET900 1 2 3 • The cattle BAC based physical maps. • The INRA bovine BAC based physical map

  29. Tools available in other species • The cattle BAC based physical maps. • The INRA bovine BAC based physical map • 81.250 clones from the INRA BAC library (Eggen et al. 2001) • 19.600 clones from the CHORI-240 BAC library (www.chori.org/bacpac). • 5.990 contigs from 2 to 164 clones (15 in mean) covering about 400 kb on average. • About 2770 Mb covered by contigs, ie > 90 % of the bovine genome. • 747 anchored contigs thanks to 1300 PCR screened markers. • 1400 contigs anchored to the human genome (2200 BES)

  30. Tools available in other species Example of Contigs : the polled locus on BTA 01 • The cattle BAC based physical maps.

  31. Tools available in other species • The cattle BAC based physical maps. • The International bovine BAC based physical map • International effort funded by USDA and Alberta Science and Research Authority (ASRA). • Improved agarose gel complete digest technology (Marra et al. 1997) • 280.000 Fingerprints generated at the Genome Science Center in Vancouver (www.bgsc.ca) from the CHORI-240 BAC library • Contigs are available through Internet Contig Explorer (iCE) • 193.900 clones • 11.950 contigs. • 640 markers. (www.chori.org/bacpac).

  32. Tools available in other species • Integration of bovine BAC based physical maps.

  33. Tools available in other species • The genome sequences. • Comparative genomics based on the genome sequence will help the identification of candidate genes and regulatory sequences. • Human and mouse sequence are now completed. • A Zebra Fish draft sequence release covering 96 % of the genome is available. • A public draft genome assembly covering more than 90% of the estimated 2.8 Gb genome is available for Rat.

  34. Tools available in other species • The genome complete sequences. • Many databases and Bioinformatic tools are available

  35. Tools available in other species • The genome complete sequences. • The bovine genome sequencing effort • High priority to complete the genome sequence by NIH • Not a complete and 99% accurate sequence but a first draft to be used to compare with human, mouse, rat sequences • 145.000 CHORI-240 BAC clones have been end sequenced • 266.320 BAC ends (BESs) in genbank. • 17.500 INRA clones will be end sequenced • 25.000 INRA BESs to date.

  36. Tools available in other species • The genome complete sequences. • The bovine genome sequencing effort • Towards a cattle physical map anchored to the human genome (Larkin et al 2003). • A preliminary analysis of 60.547 BES using BLASTN • 30 % significant hits with human • 10 % significant hits with mouse • 60 % of cattle BES with significant hits in both human and mice are located within known genes.

  37. Tools available in other species Genetic localization Contig Map Human sequence 0 Mb 0 BES M6 QTL M5 M4 Human Homologous region M3 M2 M1 BES 120 Mb • Use of cattle physical map and sequences to speed up positional cloning in small ruminants. BES matching Human sequence QTLLocalization

  38. Tools available in other species 0 M6 M5 M’ M4 M3 M’’ M2 M1 • Use of cattle physical map and sequences to speed up positional cloning in small ruminants. Genetic localization Contig Map Human sequence 0 Mb BES Identification of new markersin silico Identification of new contigs QTL BES Inventory in the region BES 120 Mb

  39. Tools available in other species • Use of cattle physical map and sequences to speed up positional cloning in small ruminants. • Increased marker density • Development of mapping methods exploiting simultaneously linkage and linkage disequilibrium (Farnir, 2002 Meuwissen 2002 Perez-Enciso 2003).

  40. Tools available in other species LSCV02 LSCV13 LSCV12 LSCV10 LSCV09 LSCV07 LSCV15 LSCV04 LSCV03 LSCV01 OAR18 ~30 cM BMS2815 GMSV36 TGLA122 TGLA337 BMC5221 BM7243 ILSTS92 ILSTS16 DIK64 BTA21 HSA14q32 HSA14q21 HSA14q12-q13 HSA15q13 CHRNA7 PRKCM DDX24 PNN • Example of contig building on OAR18 – BTA21.

  41. Tools available in other species • Phenotypic data in human and mouse • OMIM in Man • Systematic determination of gene function. • Screening for dominant mutation after mutagenesis in mouse • Wealth of data, available through international databases

  42. Conclusion • Major genes have been identified by positional cloning • SLS (Beever 1998), Inverdale (Galloway 2000), Booroola (Mulsant 2001), Callypige (Freking 2002) • PIS (Vaiman 2001) • Many QTL have been localized • Marker Assisted Selection is currently implemented but • risk of recombination between the markers and the gene • within family validity of markers Identification of the gene of interest and the causal mutation would thus represent a great advantage

  43. Conclusion Genome  Gene(s) Intron Exon transcription (maturation) STOP ATG Transcriptome mARN(s)  Exon AAAAA translation Protein(s)  Proteome  Glycosilation Phosphorylation • The complete sequence is not sufficient • Lack of precise phenotypic data limits however mapping resolution • Need to dissect the phenotype in biological « simpler » entities • Qualitative and quantitative Analysis of gene expression - in different organs, tissues or cell types, - for different development stages, - for different physio-pathological status

  44. Thank you for your attention !

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