|Quigley, Charles - Chuck|
Submitted to: The Plant Genome
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/1/2008
Publication Date: 11/1/2008
Citation: Gaitan-Solis, E., Choi, I., Quigley, C.V., Cregan, P.B., Tohme, J. 2008. Single nucleotide polymorphisms in common bean: their discovery and genotyping using a multiplex detection system. The Plant Genome. 1:125-134. Interpretive Summary: DNA markers serve as genetic landmarks and are interspersed among the genes throughout the genome of higher organisms including the common bean. If a marker is located near a gene of interest, the marker can be used to select for the desired form of the gene. For example, the common bean breeder can use a DNA marker to identify plants that carry the form of the gene that gives resistance to a disease rather than the form that leads to susceptibility. It was the objective of this work to find Single-nucleotide Polymorphism (SNP) DNA markers in the genes and in non-genic DNA regions surrounding genes. The frequency of SNPs was assessed in 47 DNA fragments of common bean DNA from each of 10 different common beans including both cultivated and wild bean varieties from Central and South America. A total of 239 SNP DNA markers were found in the 47 DNA fragments via the comparison of the DNA sequences of the 10 common bean varieties. In addition, these SNPs were analyzed using the Luminex-100 flow cytometer and it was determined that this system provided rapid and accurate SNP marker analysis. This information will be of use to common bean geneticists and breeders who can use these DNA markers and the Luminex-100 analysis system in the genetic analysis of common bean for purposes of assessing genetic diversity, for creating genetic maps and in marker assisted selection to create disease resistant and higher quality common bean varieties.
Technical Abstract: Single-nucleotide Polymorphism (SNP) markers are by far the most common form of DNA polymorphism in a genome. The objectives of this study were to discover SNPs in common bean comparing sequences from coding and non-coding regions obtained from Genbank and genomic DNA and to compare sequencing results with that were obtained using single base extension (SBE) assays on the Luminex-100 system for use in high throughput germplasm evaluation. We assessed the frequency of SNPs in 47 fragments of common bean DNA, using SBE as the evaluation methodology. We conducted a sequence analysis of 10 genotypes of cultivated and wild beans belonging to the Mesoamerican and Andean genetic pools of P. vulgaris. For the 10 genotypes evaluated, a total of 20,964 bp of sequence was analyzed in each genotype and compared, resulting in the discovery of 239 SNPs and 133 InDels, giving an average SNP frequency of one per 88 bp and an InDel frequency of one per 157 bp. This is the equivalent of a nucleotide diversity (theta) of 6.27 x 10-3. Comparisons with the SNP genotypes previously obtained by direct sequencing showed that the SBE assays on the Luminex-100 were accurate, with 2.5% being miscalled and 1% showing no signal. These results indicate that the Luminex-100 provides a high-throughput system that can be used to analyze SNPs in large samples of genotypes both for purposes of assessing diversity and also for mapping studies.