|Ponce De Leon, F|
Submitted to: Animal Genetics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/17/1999
Publication Date: N/A
Citation: Interpretive Summary: It has long been known that producers who try to improve the lean meat content in their pigs by breeding can inadvertantly breed for porcine stress syndrome. Pigs with this syndrome will be very lean but when stressed will sometimes die. This trait is known to map on swine chromosome 6. As scientists attempt to map quantitative traits in pigs they have focused on this chromosome and have attempted to develop modern molecular markers for this part of swine chromosome 6 (the q arm). This paper reports the use of microdissection techniques to cut out, under a microscope, a portion of swine chromosome 6 to develop additional molecular, or microsatellite markers, in this area of chromosome 6. Scientists know that the gene for the porcine skeletal muscle ryanodine receptor gene (RYR1), a mutation in which causes porcine stress syndrome, maps there. Using microdissection, a genomic library was generated from material only on this q arm of swine chromosome 6, as verified by fluorescen hybridization techniques. Using standard molecular tools microsatellites containing DNA repeat units of the nucleotides C and A, or (CA)n repeat sequences, were identified and primers developed for amplification of many of these (CA)n repeat-containing clones. Eventually, 22 of these microsatellite markers were used to genotype pigs from the University of Illinois Yorkshire x Meishan swine reference population. These tests verified that all 22 of these markers were assigned within a very small (50 CM) region of the swine chromosome 6 linkage map, indicating that microdissection was an excellent technique to use to develop chromosome region specific markers. These 22 markers can now be used to try to identify pigs with lean carcass traits that do not bear the mutation which causes porcine stress syndrome.
Technical Abstract: To develop additional microsatellite (MS) markers in the region of the porcine skeletal muscle ryanodine receptor gene (RYR1), a microdissected genomic library was generated from the proximal half of the q arm of swine chromosome 6. Purified DNA was restriction enzyme-digested, ligated to oligonucleotide adaptors and amplified by PCR using primers complementary to the adaptor sequences. The purity of the amplified products and boundaries of the microdissected chromosomal region were verified by fluorescence in situ hybridization. (CA)n-containing sequences were then identified in a small insert genomic library generated from the PCR- amplified microdissected DNA. Oligonucleotide primers were developed for the PCR amplification of 30 of the 46 (CA)n repeat-containing clones, which were subsequently used to amplify DNA isolated from unrelated pigs of different breeds to determine the informativeness of these MS markers. Twenty-two of these MS markers were genotyped on the University of Illinoi Yorkshire x Meishan swine reference population. These 22 markers were all assigned within a 50.7-CM region of the swine chromosome 6 linkage map, indicating the specificity of the microdissected library.