Development of a Traceability System Based on a SNP Array for Large-Scale Production of High-Value White Spruce (Picea glauca).

Biological material is at the forefront of research programs, as well as application fields such as breeding, aquaculture, and reforestation. While sophisticated techniques are used to produce this material, all too often, there is no strict monitoring during the "production" process to ensure that the specific varieties are the expected ones. Confidence rather than evidence is often applied when the time comes to start a new experiment or to deploy selected varieties in the field. During the last decade, genomics research has led to the development of important resources, which have created opportunities for easily developing tools to assess the conformity of the material along the production chains. In this study, we present a simple methodology that enables the development of a traceability system which, is in fact a by-product of previous genomic projects. The plant production system in white spruce (Picea glauca) is used to illustrate our purpose. In Quebec, one of the favored strategies to produce elite varieties is to use somatic embryogenesis (SE). In order to detect human errors both upstream and downstream of the white spruce production process, this project had two main objectives: (i) to develop methods that make it possible to trace the origin of plants produced, and (ii) to generate a unique genetic fingerprint that could be used to differentiate each embryogenic cell line and ensure its genetic monitoring. Such a system had to rely on a minimum number of low-cost DNA markers and be easy to use by non-specialists. An efficient marker selection process was operationalized by testing different classification methods on simulated datasets. These datasets were generated using in-house bioinformatics tools that simulated crosses involved in the breeding program for which genotypes from hundreds of SNP markers were already available. The rate of misidentification was estimated and various sources of mishandling or contamination were identified. The method can easily be applied to other production systems for which genomic resources are already available.

The landscape of nucleotide polymorphism among 13,500 genes of the conifer picea glauca, relationships with functions, and comparison with medicago truncatula.

Gene families differ in composition, expression, and chromosomal organization between conifers and angiosperms, but little is known regarding nucleotide polymorphism. Using various sequencing strategies, an atlas of 212k high-confidence single nucleotide polymorphisms (SNPs) with a validation rate of more than 92% was developed for the conifer white spruce (Picea glauca). Nonsynonymous and synonymous SNPs were annotated over the corresponding 13,498 white spruce genes representative of 2,457 known gene families. Patterns of nucleotide polymorphisms were analyzed by estimating the ratio of nonsynonymous to synonymous numbers of substitutions per site (A/S). A general excess of synonymous SNPs was expected and observed. However, the analysis from several perspectives enabled to identify groups of genes harboring an excess of nonsynonymous SNPs, thus potentially under positive selection. Four known gene families harbored such an excess: dehydrins, ankyrin-repeats, AP2/DREB, and leucine-rich repeat. Conifer-specific sequences were also generally associated with the highest A/S ratios. A/S values were also distributed asymmetrically across genes specifically expressed in megagametophytes, roots, or in both, harboring on average an excess of nonsynonymous SNPs. These patterns confirm that the breadth of gene expression is a contributing factor to the evolution of nucleotide polymorphism. The A/S ratios of Medicago truncatula genes were also analyzed: several gene families shared between P. glauca and M. truncatula data sets had similar excess of synonymous or nonsynonymous SNPs. However, a number of families with high A/S ratios were found specific to P. glauca, suggesting cases of divergent evolution at the functional level.

Development of high-density SNP genotyping arrays for white spruce (Picea glauca) and transferability to subtropical and nordic congeners.

High-density SNP genotyping arrays can be designed for any species given sufficient sequence information of high quality. Two high-density SNP arrays relying on the Infinium iSelect technology (Illumina) were designed for use in the conifer white spruce (Picea glauca). One array contained 7338 segregating SNPs representative of 2814 genes of various molecular functional classes for main uses in genetic association and population genetics studies. The other one contained 9559 segregating SNPs representative of 9543 genes for main uses in population genetics, linkage mapping of the genome and genomic prediction. The SNPs assayed were discovered from various sources of gene resequencing data. SNPs predicted from high-quality sequences derived from genomic DNA reached a genotyping success rate of 64.7%. Nonsingleton in silico SNPs (i.e. a sequence polymorphism present in at least two reads) predicted from expressed sequenced tags obtained with the Roche 454 technology and Illumina GAII analyser resulted in a similar genotyping success rate of 71.6% when the deepest alignment was used and the most favourable SNP probe per gene was selected. A variable proportion of these SNPs was shared by other nordic and subtropical spruce species from North America and Europe. The number of shared SNPs was inversely proportional to phylogenetic divergence and standing genetic variation in the recipient species, but positively related to allele frequency in P. glauca natural populations. These validated SNP resources should open up new avenues for population genetics and comparative genetic mapping at a genomic scale in spruce species.

The dog and rat olfactory receptor repertoires.

BACKGROUND: Dogs and rats have a highly developed capability to detect and identify odorant molecules, even at minute concentrations. Previous analyses have shown that the olfactory receptors (ORs) that specifically bind odorant molecules are encoded by the largest gene family sequenced in mammals so far. RESULTS: We identified five amino acid patterns characteristic of ORs in the recently sequenced boxer dog and brown Norway rat genomes. Using these patterns, we retrieved 1,094 dog genes and 1,493 rat genes from these shotgun sequences. The retrieved sequences constitute the olfactory receptor repertoires of these two animals. Subsets of 20.3% (for the dog) and 19.5% (for the rat) of these genes were annotated as pseudogenes as they had one or several mutations interrupting their open reading frames. We performed phylogenetic studies and organized these two repertoires into classes, families and subfamilies. CONCLUSION: We have established a complete or almost complete list of OR genes in the dog and the rat and have compared the sequences of these genes within and between the two species. Our results provide insight into the evolutionary development of these genes and the local amplifications that have led to the specific amplification of many subfamilies. We have also compared the human and rat ORs with the human and mouse OR repertoires.

Potential value of Plasmodium falciparum-associated antigen and antibody detection for screening of blood donors to prevent transfusion-transmitted malaria.

BACKGROUND: Malaria antibody detection is a valuable tool in the prevention of transfusion-transmitted malaria in countries with a high proportion of donors with travel exposure to malaria. The immunofluorescent antibody test (IFAT) is still the reference method, but it is not suitable for screening of blood donors. ELISA would be an interesting alternative to the IFAT, but it lacks sensitivity. STUDY DESIGN AND METHODS: To evaluate the potential value of a combined screening strategy based on malaria antigen and antibody detection, plasma samples from 203 patients infected with Plasmodium falciparum were tested with an ELISA for the detection of malaria antibodies (Malaria IgG CELISA, Cellabs) and a P. falciparum histidine-rich protein-2 kit (Malaria P.f., ICT Diagnostics) for the detection of malaria antigens. RESULTS: Among patients with positive IFAT results, CELISA had a sensitivity of 71 percent, whereas the combined screening tests (CELISA and Malaria P.f.) had a sensitivity of 88 percent (p < 0.001). Sequential samples from 50 patients were tested. The combined screening tests shortened the detection of seroconversion from 11.4 +/- 1.6 to 5.3 +/- 1.1 days (p < 0.001). CONCLUSION: Combined malaria antigen and antibody detection, with methods compatible with mass screening, may constitute an attractive alternative to the IFAT for blood donor screening.