Draft genome data of Prunus avium cv 'Stella'.

Prunus avium cv. 'Stella' total cellular DNA was isolated from emerging leaf tissue and sequenced using Roche 454 GS FLX Titanium, and Illumina HiSeq 2000 High Throughput Sequencing (HTS) technologies. Sequence data were filtered and trimmed to retain nucleotides corresponding to Phred score 30, and assembled with CLC Genomics Workbench v.6.0.1. A total of 107,531 contigs were assembled with 185 scaffolds with a maximum length of 132,753 nucleotides and an N50 value of 4,601. The average depth of coverage was 135.87 nucleotides with a median depth of coverage equal to 31.50 nucleotides. The draft 'Stella' genome presented here covers 77.8% of the estimated 352.9Mb P. avium genome and is expected to facilitate genetics and genomics research focused on identifying genes and quantitative trait loci (QTL) underlying important agronomic and consumer traits.

Transcriptome Analysis Reveals Potential Mechanisms for Ethylene-Inducible Pedicel-Fruit Abscission Zone Activation in Non-Climacteric Sweet Cherry (Prunus avium L.).

The harvesting of sweet cherry (Prunus avium L.) fruit is a labor-intensive process. The mechanical harvesting of sweet cherry fruit is feasible; however, it is dependent on the formation of an abscission zone at the fruit-pedicel junction. The natural propensity for pedicel-fruit abscission zone (PFAZ) activation varies by cultivar, and the general molecular basis for PFAZ activation is not well characterized. In this study, ethylene-inducible change in pedicel fruit retention force (PFRF) was recorded in a developmental time-course with a concomitant analysis of the PFAZ transcriptome from three sweet cherry cultivars. In 'Skeena', mean PFRF for both control and treatment fruit dropped below the 0.40 kg-force (3.92 N) threshold for mechanical harvesting, indicating the activation of a discrete PFAZ. In 'Bing', mean PFRF for both control and treatment groups decreased over time. However, a mean PFRF conducive to mechanical harvesting was achieved only in the ethylene-treated fruit. While in 'Chelan' the mean PFRF of the control and treatment groups did not meet the threshold required for efficient mechanical harvesting. Transcriptome analysis of the PFAZ region followed by the functional annotation, differential expression analysis, and gene ontology (GO) enrichment analyses of the data facilitated the identification of phytohormone-responsive and abscission-related transcripts, as well as processes that exhibited differential expression and enrichment in a cultivar-dependent manner over the developmental time-course. Additionally, read alignment-based variant calling revealed several short variants in differentially expressed genes, associated with enriched gene ontologies and associated metabolic processes, lending potential insight into the genetic basis for different abscission responses between the cultivars. These results provide genetic targets for the induction or inhibition of PFAZ activation, depending on the desire to harvest the fruit with or without the stem attached. Understanding the genetic mechanisms underlying the development of the PFAZ will inform future cultivar development while laying a foundation for mechanized sweet cherry harvest.

Evaluation of multiple approaches to identify genome-wide polymorphisms in closely related genotypes of sweet cherry (Prunus avium L.).

Identification of genetic polymorphisms and subsequent development of molecular markers is important for marker assisted breeding of superior cultivars of economically important species. Sweet cherry (Prunus avium L.) is an economically important non-climacteric tree fruit crop in the Rosaceae family and has undergone a genetic bottleneck due to breeding, resulting in limited genetic diversity in the germplasm that is utilized for breeding new cultivars. Therefore, it is critical to recognize the best platforms for identifying genome-wide polymorphisms that can help identify, and consequently preserve, the diversity in a genetically constrained species. For the identification of polymorphisms in five closely related genotypes of sweet cherry, a gel-based approach (TRAP), reduced representation sequencing (TRAPseq), a 6k cherry SNParray, and whole genome sequencing (WGS) approaches were evaluated in the identification of genome-wide polymorphisms in sweet cherry cultivars. All platforms facilitated detection of polymorphisms among the genotypes with variable efficiency. In assessing multiple SNP detection platforms, this study has demonstrated that a combination of appropriate approaches is necessary for efficient polymorphism identification, especially between closely related cultivars of a species. The information generated in this study provides a valuable resource for future genetic and genomic studies in sweet cherry, and the insights gained from the evaluation of multiple approaches can be utilized for other closely related species with limited genetic diversity in the breeding germplasm.

CisSERS: Customizable In Silico Sequence Evaluation for Restriction Sites.

High-throughput sequencing continues to produce an immense volume of information that is processed and assembled into mature sequence data. Data analysis tools are urgently needed that leverage the embedded DNA sequence polymorphisms and consequent changes to restriction sites or sequence motifs in a high-throughput manner to enable biological experimentation. CisSERS was developed as a standalone open source tool to analyze sequence datasets and provide biologists with individual or comparative genome organization information in terms of presence and frequency of patterns or motifs such as restriction enzymes. Predicted agarose gel visualization of the custom analyses results was also integrated to enhance the usefulness of the software. CisSERS offers several novel functionalities, such as handling of large and multiple datasets in parallel, multiple restriction enzyme site detection and custom motif detection features, which are seamlessly integrated with real time agarose gel visualization. Using a simple fasta-formatted file as input, CisSERS utilizes the REBASE enzyme database. Results from CisSERS enable the user to make decisions for designing genotyping by sequencing experiments, reduced representation sequencing, 3'UTR sequencing, and cleaved amplified polymorphic sequence (CAPS) molecular markers for large sample sets. CisSERS is a java based graphical user interface built around a perl backbone. Several of the applications of CisSERS including CAPS molecular marker development were successfully validated using wet-lab experimentation. Here, we present the tool CisSERS and results from in-silico and corresponding wet-lab analyses demonstrating that CisSERS is a technology platform solution that facilitates efficient data utilization in genomics and genetics studies.

Defects in the Expression of Chloroplast Proteins Leads to H2O2 Accumulation and Activation of Cyclic Electron Flow around Photosystem I.

We describe a new member of the class of mutants in Arabidopsis exhibiting high rates of cyclic electron flow around photosystem I (CEF), a light-driven process that produces ATP but not NADPH. High cyclic electron flow 2 (hcef2) shows strongly increased CEF activity through the NADPH dehydrogenase complex (NDH), accompanied by increases in thylakoid proton motive force (pmf), activation of the photoprotective qE response, and the accumulation of H2O2. Surprisingly, hcef2 was mapped to a non-sense mutation in the TADA1 (tRNA adenosine deaminase arginine) locus, coding for a plastid targeted tRNA editing enzyme required for efficient codon recognition. Comparison of protein content from representative thylakoid complexes, the cytochrome bf complex, and the ATP synthase, suggests that inefficient translation of hcef2 leads to compromised complex assembly or stability leading to alterations in stoichiometries of major thylakoid complexes as well as their constituent subunits. Altered subunit stoichiometries for photosystem I, ratios and properties of cytochrome bf hemes, and the decay kinetics of the flash-induced thylakoid electric field suggest that these defect lead to accumulation of H2O2 in hcef2, which we have previously shown leads to activation of NDH-related CEF. We observed similar increases in CEF, as well as increases in H2O2 accumulation, in other translation defective mutants. This suggests that loss of coordination in plastid protein levels lead to imbalances in photosynthetic energy balance that leads to an increase in CEF. These results taken together with a large body of previous observations, support a general model in which processes that lead to imbalances in chloroplast energetics result in the production of H2O2, which in turn activates CEF. This activation could be from either H2O2 acting as a redox signal, or by a secondary effect from H2O2 inducing a deficit in ATP.

Application of Cydia pomonella expressed sequence tags: Identification and expression of three general odorant binding proteins in codling moth.

The codling moth, Cydia pomonella, is one of the most important pests of pome fruits in the world, yet the molecular genetics and the physiology of this insect remain poorly understood. A combined assembly of 8 341 expressed sequence tags was generated from Roche 454 GS-FLX sequencing of eight tissue-specific cDNA libraries. Putative chemosensory proteins (12) and odorant binding proteins (OBPs) (18) were annotated, which included three putative general OBP (GOBP), one more than typically reported for other Lepidoptera. To further characterize CpomGOBPs, we cloned cDNA copies of their transcripts and determined their expression patterns in various tissues. Cloning and sequencing of the 698 nt transcript for CpomGOBP1 resulted in the prediction of a 163 amino acid coding region, and subsequent RT-PCR indicated that the transcripts were mainly expressed in antennae and mouthparts. The 1 289 nt (160 amino acid) CpomGOBP2 and the novel 702 nt (169 amino acid) CpomGOBP3 transcripts are mainly expressed in antennae, mouthparts, and female abdomen tips. These results indicate that next generation sequencing is useful for the identification of novel transcripts of interest, and that codling moth expresses a transcript encoding for a new member of the GOBP subfamily.

Comparative genomics analysis in Prunoideae to identify biologically relevant polymorphisms.

Prunus is an economically important genus with a wide range of physiological and biological variability. Using the peach genome as a reference, sequencing reads from four almond accessions and one sweet cherry cultivar were used for comparative analysis of these three Prunus species. Reference mapping enabled the identification of many biological relevant polymorphisms within the individuals. Examining the depth of the polymorphisms and the overall scaffold coverage, we identified many potentially interesting regions including hundreds of small scaffolds with no coverage from any individual. Non-sense mutations account for about 70 000 of the 13 million identified single nucleotide polymorphisms (SNPs). Blast2GO analyses on these non-sense SNPs revealed several interesting results. First, non-sense SNPs were not evenly distributed across all gene ontology terms. Specifically, in comparison with peach, sweet cherry is found to have non-sense SNPs in two 1-aminocyclopropane-1-carboxylate synthase (ACS) genes and two 1-aminocyclopropane-1-carboxylate oxidase (ACO) genes. These polymorphisms may be at the root of the nonclimacteric ripening of sweet cherry. A set of candidate genes associated with bitterness in almond were identified by comparing sweet and bitter almond sequences. To the best of our knowledge, this is the first report in plants of non-sense SNP abundance in a genus being linked to specific GO terms.

Rootstock scion somatogenetic interactions in perennial composite plants.

The ancient plant production practice of grafting which instantly imparts new physiological properties to the desirable scion still remains shrouded in mystery. Yet, grafting remains a widely used technique in the production of several horticultural species. In a composite grafted plant, rootstocks control many aspects of scion growth and physiology including yield and quality attributes as well as biotic and abiotic stress tolerance. Broadly, physical, physiological, biochemical and molecular mechanisms have been reviewed to develop an integrated understanding of this enigmatic process that challenges existing genetic paradigms. This review summarizes the reported mechanisms underlying some of the economically important traits and identifies several key points to consider when conducting rootstock scion interaction experiments. Study of the somatogenetic interactions between rootstock and scion is a field that is ripe for discovery and vast improvements in the coming decade. Further, utilization of rootstocks based on a better understanding of the somatogenetic interactions is highly relevant in the current agricultural environment where there is a need for sustainable production practices. Rootstocks may offer a non-transgenic approach to rapidly respond to the changing environment and expand agricultural production of annual and perennial crops where grafting is feasible in order to meet the global food, fiber and fuel demands of the future.

Genetic diversity and relatedness of sweet cherry (prunus avium L.) cultivars based on single nucleotide polymorphic markers.

Most previous studies on genetic fingerprinting and cultivar relatedness in sweet cherry were based on isoenzyme, RAPD, and simple sequence repeat (SSR) markers. This study was carried out to assess the utility of single nucleotide polymorphism (SNP) markers generated from 3' untranslated regions (UTR) for genetic fingerprinting in sweet cherry. A total of 114 sweet cherry germplasm representing advanced selections, commercial cultivars, and old cultivars imported from different parts of the world were screened with seven SSR markers developed from other Prunus species and with 40 SNPs obtained from 3' UTR sequences of Rainier and Bing sweet cherry cultivars. Both types of marker study had 99 accessions in common. The SSR data was used to validate the SNP results. Results showed that the average number of alleles per locus, mean observed heterozygosity, expected heterozygosity, and polymorphic information content values were higher in SSRs than in SNPs although both set of markers were similar in their grouping of the sweet cherry accessions as shown in the dendrogram. SNPs were able to distinguish sport mutants from their wild type germplasm. For example, "Stella" was separated from "Compact Stella." This demonstrates the greater power of SNPs for discriminating mutants from their original parents than SSRs. In addition, SNP markers confirmed parentage and also determined relationships of the accessions in a manner consistent with their pedigree relationships. We would recommend the use of 3' UTR SNPs for genetic fingerprinting, parentage verification, gene mapping, and study of genetic diversity in sweet cherry.

Rapid gene-based SNP and haplotype marker development in non-model eukaryotes using 3'UTR sequencing.

BACKGROUND: Sweet cherry (Prunus avium L.), a non-model crop with narrow genetic diversity, is an important member of sub-family Amygdoloideae within Rosaceae. Compared to other important members like peach and apple, sweet cherry lacks in genetic and genomic information, impeding understanding of important biological processes and development of efficient breeding approaches. Availability of single nucleotide polymorphism (SNP)-based molecular markers can greatly benefit breeding efforts in such non-model species. RNA-seq approaches employing second generation sequencing platforms offer a unique avenue to rapidly identify gene-based SNPs. Additionally, haplotype markers can be rapidly generated from transcript-based SNPs since they have been found to be extremely utile in identification of genetic variants related to health, disease and response to environment as highlighted by the human HapMap project. RESULTS: RNA-seq was performed on two sweet cherry cultivars, Bing and Rainier using a 3' untranslated region (UTR) sequencing method yielding 43,396 assembled contigs. In order to test our approach of rapid identification of SNPs without any reference genome information, over 25% (10,100) of the contigs were screened for the SNPs. A total of 207 contigs from this set were identified to contain high quality SNPs. A set of 223 primer pairs were designed to amplify SNP containing regions from these contigs and high resolution melting (HRM) analysis was performed with eight important parental sweet cherry cultivars. Six of the parent cultivars were distantly related to Bing and Rainier, the cultivars used for initial SNP discovery. Further, HRM analysis was also performed on 13 seedlings derived from a cross between two of the parents. Our analysis resulted in the identification of 84 (38.7%) primer sets that demonstrated variation among the tested germplasm. Reassembly of the raw 3'UTR sequences using upgraded transcriptome assembly software yielded 34,620 contigs containing 2243 putative SNPs in 887 contigs after stringent filtering. Contigs with multiple SNPs were visually parsed to identify 685 putative haplotypes at 335 loci in 301 contigs. CONCLUSIONS: This approach, which leverages the advantages of RNA-seq approaches, enabled rapid generation of gene-linked SNP and haplotype markers. The general approach presented in this study can be easily applied to other non-model eukaryotes irrespective of the ploidy level to identify gene-linked polymorphisms that are expected to facilitate efficient Gene Assisted Breeding (GAB), genotyping and population genetics studies. The identified SNP haplotypes reveal some of the allelic differences in the two sweet cherry cultivars analyzed. The identification of these SNP and haplotype markers is expected to significantly improve the genomic resources for sweet cherry and facilitate efficient GAB in this non-model crop.

The genome of the domesticated apple (Malus × domestica Borkh.).

We report a high-quality draft genome sequence of the domesticated apple (Malus × domestica). We show that a relatively recent (>50 million years ago) genome-wide duplication (GWD) has resulted in the transition from nine ancestral chromosomes to 17 chromosomes in the Pyreae. Traces of older GWDs partly support the monophyly of the ancestral paleohexaploidy of eudicots. Phylogenetic reconstruction of Pyreae and the genus Malus, relative to major Rosaceae taxa, identified the progenitor of the cultivated apple as M. sieversii. Expansion of gene families reported to be involved in fruit development may explain formation of the pome, a Pyreae-specific false fruit that develops by proliferation of the basal part of the sepals, the receptacle. In apple, a subclade of MADS-box genes, normally involved in flower and fruit development, is expanded to include 15 members, as are other gene families involved in Rosaceae-specific metabolism, such as transport and assimilation of sorbitol.