|Marek, Laura - ISU|
Submitted to: Genome
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: April 20, 1997
Publication Date: N/A
Interpretive Summary: The development of genetic road-maps of plant chromosomes helps researchers to pin-point the location of genes such as disease resistance genes, and to clone them. Cloning allows the genetic engineering of the genes so that they can do their job better. But, before the genetic road-maps can be used to clone the genes researchers need a way to span the great distances between road-map markers; the regions where the genes are. In this project the authors developed a special living library of DNA that allows them to span long distances on chromosomes and to clone genes. This library contains long pieces of soybean chromosomes; much longer than have been put into soybean libraries before. The authors then showed how they could extend the length of the region covered by the library clones by overlapping clones. The overlapping segments were pieced together by looking at distinctive molecular 'fingerprints' of the DNA. This work will lhelp researchers to 'walk' along chromosomes and to clone genes which are close to road-map markers.
Technical Abstract: We constructed a soybean bacterial artificial chromosome (BAC) library suitable for map-based cloning and physical mapping in soybean. This library consists of approximately 40,000 clones (four to five genome equivalents) stored individually in 384-well microtiter dishes. A random sampling of 224 clones yielded an average insert size of 150 kb, giving a 98% probability of recovering any specific sequence. We screened the library for seven single or very low copy genic or genomic sequences using the polymerase chain reaction (PRC) and found between one and seven BACs for each of the seven sequences. When testing the library with a portion of the soybean psbA chloroplast gene we found less than one percent chloroplast DNA representation. We also screened the library for eight different classes of disease resistance gene analogs (RGAs) and identified BACs containing all RGAs except for class 8. We arranged nine of the class s1 RGA BACs and six of the class 3 RGA BACs into individual contigs based o fingerprint patterns observed after Southern probing of restriction digests of the member BACs with a class-specific sequence. This resulted in the partial localization of the different multigene family sequences without precise definition of their exact positions. Using PCR-based end rescue techniques, and RFLP mapping of BAC ends, we mapped individual BACs of each contig onto linkage group J of the soybean public map. The class 1 contig mapped to the region on linkage group J containing several disease resistance genes. The arrangement of the BACs within this contig has been confirmed using PCR. The end of the class 1 contig core BAC mapped to two positions on linkage group J and cosegregated with two class 1 RGA loci suggesting that this segment is within an area of regional duplications.