Single nucleotide polymorphism discovery in elite north american potato germplasm

BACKGROUND: Current breeding approaches in potato rely almost entirely on phenotypic evaluations; molecular markers, with the exception of a few linked to disease resistance traits, are not widely used. Large-scale sequence datasets generated primarily through Sanger Expressed Sequence Tag projects are available from a limited number of potato cultivars and access to next generation sequencing technologies permits rapid generation of sequence data for additional cultivars. When coupled with the advent of high throughput genotyping methods, an opportunity now exists for potato breeders to incorporate considerably more genotypic data into their decision-making. RESULTS: To identify a large number of Single Nucleotide Polymorphisms (SNPs) in elite potato germplasm, we sequenced normalized cDNA prepared from three commercial potato cultivars: 'Atlantic', 'Premier Russet' and 'Snowden'. For each cultivar, we generated 2 Gb of sequence which was assembled into a representative transcriptome of (~)28-29 Mb for each cultivar. Using the Maq SNP filter that filters read depth, density, and quality, 575,340 SNPs were identified within these three cultivars. In parallel, 2,358 SNPs were identified within existing Sanger sequences for three additional cultivars, 'Bintje', 'Kennebec', and 'Shepody'. Using a stringent set of filters in conjunction with the potato reference genome, we identified 69,011 high confidence SNPs from these six cultivars for use in genotyping with the Infinium platform. Ninety-six of these SNPs were used with a BeadXpress assay to assess allelic diversity in a germplasm panel of 248 lines; 82 of the SNPs proved sufficiently informative for subsequent analyses. Within diverse North American germplasm, the chip processing market class was most distinct, clearly separated from all other market classes. The round white and russet market classes both include fresh market and processing cultivars. Nevertheless, the russet and round white market classes are more distant from each other than processing are from fresh market types within these two groups. CONCLUSIONS: The genotype data generated in this study, albeit limited in number, has revealed distinct relationships among the market classes of potato. The SNPs identified in this study will enable high-throughput genotyping of germplasm and populations, which in turn will enable more efficient marker-assisted breeding efforts in potato

Maize (Zea mays L.) Genome Diversity as Revealed by RNA-Sequencing

Maize is rich in genetic and phenotypic diversity. Understanding the sequence, structural, and expression variation that contributes to phenotypic diversity would facilitate more efficient varietal improvement. RNA based sequencing (RNA-seq) is a powerful approach for transcriptional analysis, assessing sequence variation, and identifying novel transcript sequences, particularly in large, complex, repetitive genomes such as maize. In this study, we sequenced RNA from whole seedlings of 21 maize inbred lines representing diverse North American and exotic germplasm. Single nucleotide polymorphism (SNP) detection identified 351,710 polymorphic loci distributed throughout the genome covering 22,830 annotated genes. Tight clustering of two distinct heterotic groups and exotic lines was evident using these SNPs as genetic markers. Transcript abundance analysis revealed minimal variation in the total number of genes expressed across these 21 lines (57.1% to 66.0%). However, the transcribed gene set among the 21 lines varied, with 48.7% expressed in all of the lines, 27.9% expressed in one to 20 lines, and 23.4% expressed in none of the lines. De novo assembly of RNA-seq reads that did not map to the reference B73 genome sequence revealed 1,321 high confidence novel transcripts, of which, 564 loci were present in all 21 lines, including B73, and 757 loci were restricted to a subset of the lines. RT-PCR validation demonstrated 87.5% concordance with the computational prediction of these expressed novel transcripts. Intriguingly, 145 of the novel de novo assembled loci were present in lines from only one of the two heterotic groups consistent with the hypothesis that, in addition to sequence polymorphisms and transcript abundance, transcript presence/absence variation is present and, thereby, may be a mechanism contributing to the genetic basis of heterosis

mRNA-Seq Analysis of the Pseudoperonospora cubensis Transcriptome During Cucumber (Cucumis sativus L.) Infection

Pseudoperonospora cubensis, an oomycete, is the causal agent of cucurbit downy mildew, and is responsible for significant losses on cucurbit crops worldwide. While other oomycete plant pathogens have been extensively studied at the molecular level, Ps. cubensis and the molecular basis of its interaction with cucurbit hosts has not been well examined. Here, we present the first large-scale global gene expression analysis of Ps. cubensis infection of a susceptible Cucumis sativus cultivar, ‘Vlaspik’, and identification of genes with putative roles in infection, growth, and pathogenicity. Using high throughput whole transcriptome sequencing, we captured differential expression of 2383 Ps. cubensis genes in sporangia and at 1, 2, 3, 4, 6, and 8 days post-inoculation (dpi). Additionally, comparison of Ps. cubensis expression profiles with expression profiles from an infection time course of the oomycete pathogen Phytophthora infestans on Solanum tuberosum revealed similarities in expression patterns of 1,576–6,806 orthologous genes suggesting a substantial degree of overlap in molecular events in virulence between the biotrophic Ps. cubensis and the hemi-biotrophic P. infestans. Co-expression analyses identified distinct modules of Ps. cubensis genes that were representative of early, intermediate, and late infection stages. Collectively, these expression data have advanced our understanding of key molecular and genetic events in the virulence of Ps. cubensis and thus, provides a foundation for identifying mechanism(s) by which to engineer or effect resistance in the host

Cryptic Chlorination by a Non-haem Iron Enzyme During Cyclopropyl Amino Acid Biosynthesis

Enzymatic incorporation of chlorine, bromine or iodine atoms occurs during the biosynthesis of more than 4,000 natural products1. Halogenation can have significant consequences for the bioactivity of these products so there is great interest in understanding the biological catalysts that perform these reactions. Enzymes that halogenate unactivated aliphatic groups have not previously been characterized. Here we report the activity of five proteins—CmaA, CmaB, CmaC, CmaD and CmaE—in the construction of coronamic acid (CMA; 1-amino-1-carboxy-2-ethylcyclopropane), a constituent of the phytotoxin coronatine synthesized by the phytopathogenic bacterium Pseudomonas syringae2. CMA derives from l-allo-isoleucine, which is covalently attached to CmaD through the actions of CmaA, a non-ribosomal peptide synthetase module, and CmaE, an unusual acyltransferase. We show that CmaB, a member of the non-haem Fe2+, α-ketoglutarate-dependent enzyme superfamily, is the first of its class to show halogenase activity, chlorinating the γ-position of l-allo-isoleucine. Another previously undescribed enzyme, CmaC, catalyses the formation of the cyclopropyl ring from the γ-Cl-l-allo-isoleucine product of the CmaB reaction. Together, CmaB and CmaC execute γ-halogenation followed by intramolecular γ-elimination, in which biological chlorination is a cryptic strategy for cyclopropyl ring formation