Generation of polymorphic markers tightly linked to the thymus enlargement loci by phenotype-directed representational difference analysis.
To obtain genetic markers linked to a specific genetic locus, genomic subtraction with a DNA pool of backcross or F2 intercross animals with a specific genotype at the locus is known to be effective. To determine whether the pooling strategy is also effective for isolation of genetic markers linked to a quantitative phenotype that can potentially be controlled by multiple genetic loci, we tested the ability of representational difference analysis (RDA) to isolate genetic markers linked to the thymus enlargement observed in the BUF/Mna (BUF) rat. This is known to be controlled by single major and minor genes, Ten1 and Ten2, on Chromosomes (Chrs) 1 and 13, respectively, both of which have dose effects on the normal WKY/Ncj (WKY) allele. DNA from an inbred WKY rat was used as the tester, and the driver was prepared from a DNA pool of 12 (WKY x BUF)F1 x BUF backcross rats with high thymus ratios (thymus weight/body weight), expected to have dominance of the BUF allele in the responsible loci. By two RDA series with the restriction enzymes BglII and BamHI, respectively, 28 polymorphic markers were isolated, and 8 of them were shown to be linked to Ten1, and one to Ten2. One of the 8 markers linked to Ten1 demonstrated no recombination in 18 rats with high thymus ratios. RDA with a DNA pool based on a quantitative phenotype (phenotype-directed RDA) can thus be considered an efficient approach for direct isolation of polymorphic markers linked to a quantitative trait.
Toyota
M
M
Carcinogenesis Division, National Cancer Center Research Institute, Tokyo 104, 5-1-1, Tsukiji, Chuo-ku, Japan.
Ushijima
T
T
Suzui
M
M
Murakumo
Y
Y
Imai
K
K
Sugimura
T
T
Matsuyama
M
M
D000818:Animals; D015139:Blotting, Southern; D002874:Chromosome Mapping / Q000379:methods; D003001:Cloning, Molecular; D004247:DNA / Q000032:analysis; D004587:Electrophoresis, Agar Gel; D008040:Genetic Linkage; D005819:Genetic Markers; D005838:Genotype; D007178:Inbreeding; D008126:Lod Score; D009693:Nucleic Acid Hybridization; D010641:Phenotype; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D051381:Rats; D011915:Rats, Inbred BUF; D011916:Rats, Inbred F344; D011917:Rats, Inbred Lew; D011920:Rats, Inbred WF; D011921:Rats, Inbred WKY; D013952:Thymus Hyperplasia / Q000235:genetics*
D005819:Genetic Markers; D004247:DNA