|Nam, Young woo|
|Lee, Jeong ran|
|Song, Ki hoon|
|Lee, Mi kyung|
Submitted to: Journal of Theoretical and Applied Genetics
Publication Type: Peer reviewed journal
Publication Acceptance Date: 2/15/2005
Publication Date: 8/20/2007
Citation: Nam, Y., Lee, J., Song, K., Lee, M., Staub, J.E. 2007. Construction of two BAC libraries from cucumber (Cucumis sativus l.) and identification of clones linked to yield component quantitative trait loci. Journal of Theoretical and Applied Genetics. 111:150-161. Interpretive Summary: Cucumber has a narrow genetic base (there are not very many genetic differences among cucumber cultivars). This can present a large problem since as the same genetic cultivars are used to make new cultivars, they become increasingly susceptible to large disease epidemics since all cultivars have the same genes. There is a great need to increase the genetic diversity of cucumber by introducing new genes from exotic sources (wild cucumber populations) into cucumber. In order to determine which sources are most diverse DNA analysis is required (DNA is found in all cells and controls the genetics and development of plants to maturity). It is important to develop biotechnological tools to analyzed DNA. One such tool is the creation of genetic maps which pin-point the location of genes (bits of DNA that provide genetic information for controlling specific traits) on chromosomes (physical structures made up of genes that house genetic information). One way of doing this is to try to get all the DNA lined up in a straight order and count the number of genes. The genes can be ordered by using bacterial artifical chromosomes (BAC; structures that contain DNA that are similar to the plant being analyzed). In this experiment we used BAC to get the genes lined up in cucumber. This alignment allowed us to locate the genes for sex and plant stature (dwarf) in cucumber. It is now possible to analyze cultivars and exotic cucumber plants to determine if they have the same DNA for sex and plant stature. This is the first step to identifying the location and characteristics of other genes of economical importance (e.g., disease resistance). As more genes are identified scientists will be able to more precisely build strategies to incorporate genes from exotic cucumber plants to broaden the genetic base of cucumber. This will reduce to occurance of disease epidemics in cucumber culture, and thus allow the U.S. grower to be more competitive in the global market.
Technical Abstract: Two bacterial artificial chromosome (BAC) libraries were constructed from an inbred line derived from a recombinant cultivar of cucumber (Cucumis sativus L.). Intact nuclei were isolated and embedded in agarose plugs, and high-molecular-weight DNA was subsequently partially digested with BamHI or EcoRI. Ligation of double size-selected DNA fragments with the pECBAC1 vector yielded two libraries containing 23,040 BamHI and 18,432 EcoRI clones. The two libraries together represent approximately 10.8 haploid cucumber genomes. The average BamHI and EcoRI insert sizes were estimated to be 107.0 kb and 100.8 kb, respectively, and the BAC clones lacking inserts comprised 1.3% and 14.5% of the BamHI and EcoRI libraries, respectively. Hybridization with a C0t-1 DNA probe revealed that approximately 36% of BAC clones likely carried repetitive, sequence-enriched DNA. The frequencies of BAC clones carrying chloroplast or mitochondrial DNA range from 0.20% to 0.47%. Four sequence characterized amplified region (SCAR), four simple sequence repeat (SSR), and a random amplified polymorphic DNA (RAPD) marker linked with yield component quantitative trait loci (QTL) were used either as probes to hybridize high-density colony filters prepared from both libraries or as primers to screen an ordered array of pooled BAC DNA prepared from the BamHI library. Positive BAC clones were identified in predicted numbers, as screening by PCR amplification effectively overcame the problems associated with an over abundance of positives from hybridization with two SCAR markers. The BAC clones identified herein that are closely linked to de (determinate habit) or F (gynoecy) will be useful for positional cloning of these economically important genes. These BAC libraries will also facilitate physical mapping of the cucumber genome and comparative genome analyses with other plant species.