GENOME ORGANIZATION AS REVEALED BY GENOME MAPPING WHY MAP GENOMES? HOW TO MAP GENOMES?

Why map genomes? Understand genome organization Understand genome evolution (comparative mapping) Find and clone important genes Marker assisted selection of important traits

Exploring Genomes: Mapping Genetic mapping Linkage mapping Requires allelic variation and meiotic products Pedigree analysis, LOD scores Physical mapping In situ hybridization Somatic cell genetics Radiation hybrids Contig assembly

LINKAGE MAPPING Genetic mapping. Based on recombination in meiosis Requires allelic variation for detection Can be done experimentally with crosses or retrospectively from family studies

Allelic Variation Phenotypic Pigment Disease Immunologic Blood type Tissue antigens Protein Electrophoretic variation

Allelic Variation, DNA Restriction Fragment Length Polymorphisms (RFLPs) Microsatellites= Simple Tandem Repeats (STRs) Single Nucleotide Polymorphisms (SNPs)

Pedigree Analysis: AABB x aabb AaBb x aabb 5 children: AaBb, AaBb, aabb, aabb, aaBb Are genes A and B linked? Probability - assume no linkage: assume  =.10 gametes: AB (.25) ab (.25) aB (.25) Ab (.25) AB (.45) ab (.45) aB (.05) Ab (.05) gametes liklihood ratio (  =.10) = (.45) (.45) (.45) (.45) (.05) (.25) (.25) (.25) (.25) (.25) = = 2.06 Z = 0.31

Linkage Map: 1 centiMorgan=1% recombination a b c d __|________|____________|____|_____ _ 10cM 15cM 5cM

Linkage map: human chr 12

Interesting notes about linkage maps Different for males and females (in mammals) Female map = 1.5x male map Maps are usually presented as sex averaged Different across species Human linkage map = 3200 cM total Mouse linkage map = 1600 cM total Different in regions of the same chromosome Not proportional across same physical distance On the average, however: 1cM = 1 megabase DNA (human) 1cM= 2 megabases DNA (mice)

Somatic Cell Genetics Mapping using interspecific hybrid cells Advantages Allelic variation not required Rapid development of a map for any species Disadvantages Gene order is difficult to establish Limited to characters expressed in cell culture Initially expensive and labor intensive

Amino Acids Simple Sugars blocked by Aminopterin de novo nucleotide synthesis MAJOR PATHWAY TK HGPRT Pyrimidine bases nucleosides Purine bases nucleosides SALVAGE PATHWAY Hypoxanthine Thymidine Phosphorylated Thymidine Guanine Nucleotides HGPRT TK DNA

Synteny: the occurrence of two loci on the same chromosome, independent of the distance that separates them. Linkage: non-random distribution of alleles in gametogenesis. Tendency of parental combinations of alleles to stay together.

Radiation Hybrids Advantages Ordered maps without polymorphism Ordered comparative maps EST maps Integrate EST maps with linkage maps Disadvantages Cost of panel construction Differential sensitivity of genotyping markers

Selectable marker Cattle cells lethally irradiated, ouabain sensitive Non-recombinant rodent cells die in HAT Diploid cattle cell lineRecipient rodent cell line Fusion of parental cell lines in PEG Radiation hybrid containing Cattle chromosomal fragments on a rodent genomic background Irradiation of donor cells breaks chromosomes Mutant

Ordered Contigs and Sequencing, the Ultimate Physical Map Large Insert Libraries, BACs and YACs, 200 kb- 1mb Smaller Insert Libraries Phage, Plasmids, Cosmids, 20 – 45 kb