Herman Silva

The genome of woodland strawberry ( Fragaria vesca )

The woodland strawberry, Fragaria vesca (2n = 2x = 14), is a versatile experimental plant system. This diminutive herbaceous perennial has a small genome (240 Mb), is amenable to genetic transformation and shares substantial sequence identity with the cultivated strawberry (Fragaria × ananassa) and other economically important rosaceous plants. Here we report the draft F. vesca genome, which was sequenced to ×39 coverage using second-generation technology, assembled de novo and then anchored to the genetic linkage map into seven pseudochromosomes. This diploid strawberry sequence lacks the large genome duplications seen in other rosids. Gene prediction modeling identified 34,809 genes, with most being supported by transcriptome mapping. Genes critical to valuable horticultural traits including flavor, nutritional value and flowering time were identified. Macrosyntenic relationships between Fragaria and Prunus predict a hypothetical ancestral Rosaceae genome that had nine chromosomes. New phylogenetic analysis of 154 protein-coding genes suggests that assignment of Populus to Malvidae, rather than Fabidae, is warranted.

The high-quality draft genome of peach (Prunus persica) identifies unique patterns of genetic diversity, domestication and genome evolution

Rosaceae is the most important fruit-producing clade, and its key commercially relevant genera (Fragaria, Rosa, Rubus and Prunus) show broadly diverse growth habits, fruit types and compact diploid genomes. Peach, a diploid Prunus species, is one of the best genetically characterized deciduous trees. Here we describe the high-quality genome sequence of peach obtained from a completely homozygous genotype. We obtained a complete chromosome-scale assembly using Sanger whole-genome shotgun methods. We predicted 27,852 protein-coding genes, as well as noncoding RNAs. We investigated the path of peach domestication through whole-genome resequencing of 14 Prunus accessions. The analyses suggest major genetic bottlenecks that have substantially shaped peach genome diversity. Furthermore, comparative analyses showed that peach has not undergone recent whole-genome duplication, and even though the ancestral triplicated blocks in peach are fragmentary compared to those in grape, all seven paleosets of paralogs from the putative paleoancestor are detectable.

NPR1 differentially interacts with members of the TGA/OBF family of transcription factors that bind an element of the PR-1 gene required for induction by salicylic acid.

NPR1 is a critical component of the salicylic acid (SA)-mediated signal transduction pathway leading to the induction of defense genes, such as the pathogenesis-related (PR)-1 gene, and enhanced disease resistance. Using a yeast two-hybrid screen, we identified several NPR1-interacting proteins (NIPs). Two of these NIPs are members of the TGA/OBF family of basic leucine zipper (bZIP) transcription factors; this family has been implicated in the activation of SA-responsive genes, including PR-1. Six TGA family members were tested and shown to differentially interact with NPR1: TGA2 and TGA3 showed strong affinity for NPR1; TGA5 and TGA6 exhibited weaker affinity; and TGA1 and TGA4 displayed little or no detectable interaction with NPR1, respectively. Interestingly, the amino-termini of these factors were found to decrease their stability in yeast and differentially affect their apparent affinity toward NPR1. The interacting regions on NPR1 and the TGA factors were also defined. Each of four point mutations in NPR1 that disrupt SA signaling in Arabidopsis completely blocked interaction of NPR1 with TGA2 and TGA3. TGA2 and TGA3 were also found to bind the SA-responsive element of the Arabidopsis PR-1 promoter. These results directly link NPR1 to SA-induced PR-1 expression through members of the TGA family of transcription factors.

A rapid and efficient method for purifying high quality total RNA from peaches (Prunus persica) for functional genomics analyses

Prunus persica has been proposed as a genomic model for deciduous trees and the Rosaceae family. Optimized protocols for RNA isolation are necessary to further advance studies in this model species such that functional genomics analyses may be performed. Here we present an optimized protocol to rapidly and efficiently purify high quality total RNA from peach fruits (Prunus persica). Isolating high-quality RNA from fruit tissue is often difficult due to large quantities of polysaccharides and polyphenolic compounds that accumulate in this tissue and co-purify with the RNA. Here we demonstrate that a modified version of the method used to isolate RNA from pine trees and the woody plant Cinnamomun tenuipilum is ideal for isolating high quality RNA from the fruits of Prunus persica. This RNA may be used for many functional genomic based experiments such as RT-PCR and the construction of large-insert cDNA libraries.

Transport of UDP-galactose in plants: Identification and functional characterization of AtUTr1, an Arabidopsis thaliana UDP-galactose/UDP-glucose transporter

The synthesis of non-cellulosic polysaccharides and glycoproteins in the plant cell Golgi apparatus requires UDP-galactose as substrate. The topology of these reactions is not known, although the orientation of a plant galactosyltransferase involved in the biosynthesis of galactomannans in fenugreek is consistent with a requirement for UDP-galactose in the lumen of the Golgi cisternae. Here we provide evidence that sealed, right-side-out Golgi vesicles isolated from pea stems transport UDP-galactose into their lumen and transfer galactose, likely to polysaccharides and other acceptors. In addition, we identified and cloned AtUTr1, a gene from Arabidopsis thaliana that encodes a multitransmembrane hydrophobic protein similar to nucleotide sugar transporters. Northern analysis showed that AtUTr1 is indeed expressed in Arabidopsis. AtUTr1 is able to complement the phenotype of MDCK ricin-resistant cells; a mammalian cell line deficient in transport of UDP-galactose into the Golgi.In vitro assays using a Golgi-enriched vesicle fraction obtained from Saccharomyces cerevisiae expressing AtUTr1-MycHis is able to transport UDP-galactose but also UDP-glucose. AtUTr1- MycHis does not transport GDP-mannose, GDP-fucose, CMP-sialic acid, UDP-glucuronic acid, or UDP-xylose when expressed inS. cerevisiae. AtUTr1 is the first transporter described that is able to transport UDP-galactose and UDP-glucose. Thus AtUTr1 may play an important role in the synthesis of glycoconjugates in Arabidopsis that contain galactose and glucose.

Engineering disease and pest resistance in plants

Improvements in transformation techniques and the isolation of many genes whose transcripts or protein products either have antimicrobial or insecticidal activity or are involved in the synthesis of products with such activities have provided valuable tools for engineering resistance in plants. Future exploitation of this technology should provide an environmentally friendly alternative to traditional disease and pest control measures.

Characterization of a New Arabidopsis Mutant Exhibiting Enhanced Disease Resistance

In many plant-pathogen interactions, resistance is associated with the synthesis and accumulation of salicylic acid (SA) and pathogenesis-related (PR) proteins. At least two general classes of mutants with altered resistance to pathogen attack have been identified in Arabidopsis. One class exhibits increased susceptibility to pathogen infection; the other class exhibits enhanced resistance to pathogens. In an attempt to identify mutations in resistance-associated loci, we screened a population of T-DNA tagged Arabidopsis thaliana ecotype Wassilewskija (Ws) for mutants showing constitutive expression of the PR-1 gene (cep). A mutant was isolated and shown to constitutively express PR-1, PR-2, and PR-5 genes. This constitutive phenotype segregated as a single recessive trait in the Ws genetic background. The mutant also had elevated levels of SA, which are responsible for the cep phenotype. The cep mutant spontaneously formed hypersensitive response (HR)-like lesions on the leaves and cotyledons and also exhibited enhanced resistance to virulent bacterial and fungal pathogens. Genetic analyses of segregating progeny from outcrosses to other ecotypes unexpectedly revealed that alterations in more than one gene condition the constitutive expression of PR genes in the original mutant. One of the mutations, designated cpr20, maps to the lower arm of chromosome 4 and is required for the cep phenotype. Another mutation, which has been termed cpr21, maps to chromosome 1 and is often, but not always, associated with this phenotype. The recessive nature of the cep trait suggests that the CPR20 and CPR21 proteins may act as negative regulators in the disease resistance signal transduction pathway.

Construction and Comparative Analyses of Highly Dense Linkage Maps of Two Sweet Cherry Intra-Specific Progenies of Commercial Cultivars

Despite the agronomical importance and high synteny with other Prunus species, breeding improvements for cherry have been slow compared to other temperate fruits, such as apple or peach. However, the recent release of the peach genome v1.0 by the International Peach Genome Initiative and the sequencing of cherry accessions to identify Single Nucleotide Polymorphisms (SNPs) provide an excellent basis for the advancement of cherry genetic and genomic studies. The availability of dense genetic linkage maps in phenotyped segregating progenies would be a valuable tool for breeders and geneticists. Using two sweet cherry (Prunus avium L.) intra-specific progenies derived from crosses between ‘Black Tartarian’ × ‘Kordia’ (BT×K) and ‘Regina’ × ‘Lapins’(R×L), high-density genetic maps of the four parental lines and the two segregating populations were constructed. For BT×K and R×L, 89 and 121 F1 plants were used for linkage mapping, respectively. A total of 5,696 SNP markers were tested in each progeny. As a result of these analyses, 723 and 687 markers were mapped into eight linkage groups (LGs) in BT×K and R×L, respectively. The resulting maps spanned 752.9 and 639.9 cM with an average distance of 1.1 and 0.9 cM between adjacent markers in BT×K and R×L, respectively. The maps displayed high synteny and co-linearity between each other, with the Prunus bin map, and with the peach genome v1.0 for all eight LGs (LG1–LG8). These maps provide a useful tool for investigating traits of interest in sweet cherry and represent a qualitative advance in the understanding of the cherry genome and its synteny with other members of the Rosaceae family.

Isolation and functional characterization of cold-regulated promoters, by digitally identifying peach fruit cold-induced genes from a large EST dataset

BackgroundCold acclimation is the process by which plants adapt to the low, non freezing temperatures that naturally occur during late autumn or early winter. This process enables the plants to resist the freezing temperatures of winter. Temperatures similar to those associated with cold acclimation are also used by the fruit industry to delay fruit ripening in peaches. However, peaches that are subjected to long periods of cold storage may develop chilling injury symptoms (woolliness and internal breakdown). In order to better understand the relationship between cold acclimation and chilling injury in peaches, we isolated and functionally characterized cold-regulated promoters from cold-inducible genes identified by digitally analyzing a large EST dataset.ResultsDigital expression analyses of EST datasets, revealed 164 cold-induced peach genes, several of which show similarities to genes associated with cold acclimation and cold stress responses. The promoters of three of these cold-inducible genes (Ppbec1, Ppxero2 and Pptha1) were fused to the GUS reporter gene and characterized for cold-inducibility using both transient transformation assays in peach fruits (in fruta) and stable transformation in Arabidopsis thaliana. These assays demonstrate that the promoter Pptha1 is not cold-inducible, whereas the Ppbec1 and Ppxero2 promoter constructs are cold-inducible.ConclusionThis work demonstrates that during cold storage, peach fruits differentially express genes that are associated with cold acclimation. Functional characterization of these promoters in transient transformation assays in fruta as well as stable transformation in Arabidopsis, demonstrate that the isolated Ppbec1 and Ppxero2 promoters are cold-inducible promoters, whereas the isolated Pptha1 promoter is not cold-inducible. Additionally, the cold-inducible activity of the Ppbec1 and Ppxero2 promoters suggest that there is a conserved heterologous cold-inducible regulation of these promoters in peach and Arabidopsis. These results reveal that digital expression analyses may be used in non-model species to identify candidate genes whose promoters are differentially expressed in response to exogenous stimuli.

Genetic structure based on EST–SSR: a putative tool for fruit color selection in Japanese plum (Prunus salicina L.) breeding programs

Prunus salicina is one of the most economically important stone fruits. However, there is scarce genetic information available, which makes it difficult to implement marker-assisted selection (MAS) in genetic improvement programs. Recently, next-generation sequencing has greatly enhanced breeding program strategies, generating information associated with the identification of expressed sequence tag–simple sequence repeats (EST–SSRs) and single-nucleotide polymorphisms (SNPs), two of the most used molecular markers in MAS. Few studies have focused on developing EST–SSR markers considering both gene expression levels of contrasting phenotypes and specific transcription factors of metabolic pathways. This study investigated the transcriptome profile of P. salicina in fruits with contrasting skin colors, obtaining 54,224 unique contigs. From this dataset, 44 EST–SSRs have been generated, considering gene expression levels of contrasting phenotypes and specific transcription factor from three metabolic pathways: citric acid, carbohydrate metabolism and flavonoid pathways. Three EST–SSR markers developed from the putative flavonoid pathway transcription factors PsMYB10, PsMYB1 and PsbHLH35 were selected to determine genetic structure in 29 cultivars. This structure was contrasted with the genetic structure generated using genomic SNPs obtained by genotyping-by-sequencing (GBS). The analysis using SNPs identified two groups, while the use of selected EST–SSRs identified three. In contrast to the structure given by the SNPs, the EST–SSRs grouped all the yellow cultivars in one cluster, which was composed mainly of cultivars of this color. The EST–SSRs developed in this study may be considered as candidate markers to be evaluated in MAS strategies in genetic improvement programs.