Submitted to: Theoretical and Applied Genetics
Publication Type: Peer reviewed journal
Publication Acceptance Date: 3/7/2003
Publication Date: 6/10/2003
Citation: Chung, S., Staub, J.E. 2003. The development and evaluation of consensus chloroplast primer pairs that possess highly variable sequence regions in a diverse array of plant taxa. Theoretical and Applied Genetics. Interpretive Summary: There are biotechnologies that have been developed called molecular markers. Molecular markers allow for the characterization of the genetic constitution (nature) of organisms. They have been used in many organisms to better understand the inheritance of genes (units of DNA that control traits), determine the similarity been individuals, and for legal use in plant variety protection (PVP) and plant patenting (PP). They are most useful when there are broad genetic differences (genes are different) between individuals. Commercial cucumber varieties are extremely similar and thus it is difficult to tell varieties apart. This means that difficulties can arise when seed companies apply for plant protection instruments (PVP and PP) for protection of their research investments (development of unique varieties). It would be important therefore to develop unique molecular markers for the purposes of differentiating individuals based on their genetic composition (genes). Therefore, a series of experiments were undertaken to develop new molecular markers specific for cucumber. These experiments lead to the development and testing of 23 new and unique molecular markers useful for genetic discrimination (seeing differences between individuals) which will be useful for genetic analysis. These markers will assist the researcher in the analysis of genetic differences between closely related individuals. Since it was determined by our experimentation that these molecular markers are useful in defining very small genetic dissimilarities, they will assist U.S. seed companies in PVP and PP to protect their investments and thus improve their productivity and competitiveness. These markers will also be valuable in determining relationships between individuals to enhance the effectiveness and efficiency of plant breeding, thus reducing costs to the grower and consumer.
Technical Abstract: Although universal or consensus chloroplast primers are available, they are limited by their number and genomic distribution. Therefore, a set of consensus chloroplast primer pairs for simple sequence repeats (ccSSRs) analysis was constructed from tobacco (Nicotiana tabacum L.) chloroplast sequences. These were then tested for their general utility in the genetic analysis of a diverse array of plant taxa. In order to increase the number of ccSSRs beyond that previously reported, the target sequences for SSR motifs was set at A or T (n = 7) mononucleotide repeats. Each SSR sequence motif, along with ± 200 bp flanking sequences from the first of each mononucleotide base repeat, was screened for homologies with chloroplast DNA sequences of other plant species in GenBank databases using BLAST search procedures. Twenty-three putative marker loci those possessed conserved flanking sequence spans were selected for consensus primer pair construction using commercially available computer algorithms. All primer pairs produced amplicons after PCR employing genomic DNA from members of the Cucurbitaceae (six species), and Solanaceae (four species). Sixteen, 22, and 19 of the initial 23 primer pairs were successively amplified by PCR using template DNA from species of the Apiaceae (two species), Brassicaceae (one species), and Fabaceae (two species), respectively. Twenty of 23 primer pairs were also functional in three monocot species of the Liliaceae [onion (Allium cepa L.) and garlic (Allium sativum L.)], and the Poaceae (oat; Avena sativa L.). Sequence analysis of selected ccSSR fragments suggests that ccSSR length and sequence variation could be useful as a tool for investigating the genetic relationships within a genus or closely related taxa (i.e., tribal level). In order to provide for a marker system having significant coverage of the cucumber chloroplast genome, ccSSR primers were strategically "recombined" and named recombined consensus chloroplast primers (RCCP) for PCR analysis. Successful amplification after extended-length PCR of 16 RCCP primer pairs from cucumber (Cucumis sativus L.) DNA suggested that the amplicons detected may be representative of the cucumber chloroplast genome. These RCCP pairs, therefore, could be useful as an initial molecular tool for investigation of traits related to chloroplast gene(s) in cucumber and closely related species.