Steven A. Thompson

Mapping of the loci controlling oleic and linolenic acid contents and development of fad2 and fad3 allele-specific markers in canola (Brassica napus L.).

The quality of canola oil is determined by its constituent fatty acids such as oleic acid (C18:1), linoleic acid (C18:2) and linolenic acid (C18:3). Most canola cultivars normally produce oil with about 55–65% oleic acid and 8–12% linolenic acid. High concentrations of linolenic acid lead to oil instability and off-type flavor, while high levels of oleic acid increase oxidative stability and nutritional value of oil. Therefore, development of canola cultivars with increased oleic acid and reduced linolenic acid is highly desirable for canola oil quality. In this study, we have mapped one locus that has a major effect and one locus that has a minor effect for high oleic acid and two loci that have major effects for low linolenic acid in a doubled haploid population. The major locus for high C18:1 was proven to be the fatty acid desaturase-2 (fad2) gene and it is located on the linkage group N5; the minor locus is located on N1. One major QTL for C18:3 is the fatty acid desaturase-3 gene of the genome C (fad3c) and it is located on N14. The second major QTL resides on N4 and is the fad3a gene of the A genome. We have sequenced genomic clones of the fad2 and fad3c genes amplified from an EMS-induced mutant and a wild-type canola cultivar. A comparison of the mutant and wild-type allele sequences of the fad2 and fad3c genes revealed single nucleotide mutations in each of the genes. Detailed sequence analyses suggested mechanisms by which both the mutations can cause altered fatty acid content. Based on the sequence differences between the mutant and wild-type alleles, two single nucleotide polymorphism (SNP) markers, corresponding to the fad2 and fad3c gene mutations, were developed. These markers will be highly useful for direct selection of desirable fad2 and fad3c alleles during marker-assisted trait introgression and breeding of canola with high oleic and low linolenic acid.

Development of highly polymorphic SNP markers from the complexity reduced portion of maize [Zea mays L.] genome for use in marker-assisted breeding

The duplicated and the highly repetitive nature of the maize genome has historically impeded the development of true single nucleotide polymorphism (SNP) markers in this crop. Recent advances in genome complexity reduction methods coupled with sequencing-by-synthesis technologies permit the implementation of efficient genome-wide SNP discovery in maize. In this study, we have applied Complexity Reduction of Polymorphic Sequences technology (Keygene N.V., Wageningen, The Netherlands) for the identification of informative SNPs between two genetically distinct maize inbred lines of North and South American origins. This approach resulted in the discovery of 1,123 putative SNPs representing low and single copy loci. In silico and experimental (Illumina GoldenGate (GG) assay) validation of putative SNPs resulted in mapping of 604 markers, out of which 188 SNPs represented 43 haplotype blocks distributed across all ten chromosomes. We have determined and clearly stated a specific combination of stringent criteria (>0.3 minor allele frequency, >0.8 GenTrainScore and >0.5 Chi_test100 score) necessary for the identification of highly polymorphic and genetically stable SNP markers. Due to these criteria, we identified a subset of 120 high-quality SNP markers to leverage in GG assay-based marker-assisted selection projects. A total of 32 high-quality SNPs represented 21 haplotypes out of 43 identified in this study. The information on the selection criteria of highly polymorphic SNPs in a complex genome such as maize and the public availability of these SNP assays will be of great value for the maize molecular genetics and breeding community.

Improved high-throughput sunflower and cotton genomic DNA extraction and PCR fidelity

The extraction of high-quality genomic DNA for PCR amplification from sunflower (Helianthus annuus) and cotton (Gossypium spp.) is challenging because of the presence of polysaccharides, secondary metabolites, and polyphenolics in the tissues. A high-throughput DNA extraction protocol was needed in our laboratory for simple sequence repeats (SSR)-marker screening and other molecular analyses that do not require organic extraction steps of phenol or chloroform. Here we describe 2 improved highthroughput protocols for DNA extraction and in-PCR modification that result in successful PCR amplification of sunflower and cotton. While the sunflower DNA extraction protocol uses reducing agents such as sodium metabisulfite and dithiothreitol (DTT), the cotton protocol uses polyvinylpyrrolidone (PVP) in PCR reactions and reducing agents in the DNA extraction procedure.

Mapping of the Ogura fertility restorer gene Rfo and development of Rfo allele-specific markers in canola (Brassica napus L.)

Ogura cytoplasmic male sterility (CMS) and its corresponding nuclear fertility restorer gene, Rfo, have been introduced from radish to Brassica species by interspecific crosses. Rfo restores male fertility by altering the translational expression of Orf138, a mitochondrial gene, whose expression results in the male sterile phenotype. This system has been extensively investigated and breeding restorer lines for the Ogura CMS has become a major objective for hybrid seed production in many canola breeding programs. In this study, we have sequenced genomic clones of Rfo amplified from a canola restorer line R2000, licensed from INRA, France, and a Dow AgroSciences non-restorer line Nexera 705 using primers designed from the radish Rfo sequence (GenBank accession AJ550021). Sequence alignment revealed three homologous sequences of Rfo. Two of the sequences were present in both R2000 and Nexera 705 but the third one was present only in R2000. These results suggested that the first two sequences could be the homoeologous sequences of Rfo already existing in the canola genome and the third one could be the radish Rfo introduced into canola. Based on the sequence differences between the restorer and non-restorer lines, Rfo allele-specific PCR markers were developed. We also developed a high throughput, Rfo allele-specific Invader® assay through Third Wave Technologies. Linkage analysis revealed a co-segregation between the allele-specific marker and the phenotypes for fertility restoration. This allele-specific marker has been mapped in the linkage group N19 and proved to be very useful for direct selection of Rfo alleles for fertility restoration during marker-assisted introgression of the Ogura restorer for hybrid development in canola.

An improved enrichment procedure to develop multiple repeat classes of cotton microsatellite markers

The availability of a large number of molecular markers is a prerequisite, especially in cotton, for identifying a sufficient number of informative markers for mapping and genetic analysis. Despite the global importance of the cotton crop, few informative microsatellite markers are available, primarily because of the cost associated with their development. This report describes an improved and cost-effective strategy for developing microsatellite markers. Genomic DNA was randomly sheared with nitrogen gas to obtain unbiased representation of the genome, and the fragments containing microsatellites were captured by using biotinylated oligos and streptavidin-based recovery. Six libraries enriched for 14 microsatellite motifs were constructed and screened. Nearly 4900 simple sequence repeat (SSR)-containing sequences were identified, leading to the development of more than 1200 markers in a small amount of time.