Genomics

In the last few years a considerable effort has yielded the complete genome sequence of four key Rosaceae species: apple, strawberry, peach, and pear with more to come. With this genomic information it is now possible to identify gene family members rapidly and through other genomic technologies such as mRNA-seq and test gene function. This has greatly accelerated the pace at which research is done. As currently only a few of the 30 - 60,000 predicted genes in the genome have been described in each of these species, the Rosaceae community has a unique opportunity to standardise gene names within and across the Rosaceae species. The Rosaceae Gene Name Standardization Subcommittee has been formed to propose a naming guideline around naming genes in Rosaceae. This poster details the issues around naming genes and proposes a common nomenclature standard for species gene, and splice variation. Curated gene database in GDR (Genome Database for Rosaceae) will contain user-submitted gene names in addition to those curated from NCBI nr database. GDR will also host gene family submission site, as well as gene name submission site, to maintain a user-curated list of gene families. To avoid confusion in future publications, the subcommittee recommends researchers to register their gene names and gene family data to GDR prior to publication to facilitate the effort on the gene naming standardization.

GDR (www.rosaceae.org) is a curated and integrated web-based relational database providing access to publicly available genomic, genetic and breeding data for Rosaceous crops. GDR contains annotated whole genome sequences, transcripts, markers, trait loci, genetic maps, genes, taxonomy, germplasm, publications and communication resources for the Rosaceae community. In this update we report on new functionality and data including access the addition of RNASeq and GBS data viewable through implementation of the JBrowse genome viewer, as well as new genome, trait, map and marker data, and new or improved search tools. The accepted community database for Rosaceae research, GDR was accessed by 14,227 users from 127 countries in 2013, with over 170,000 pages accessed.

Disease resistance (R) genes from different Rosaceae species have been identified by map-based cloning for resistance breeding, and most disease resistance genes encode NBS-LRR protein. However, reports describing evolutionary pattern of R-genes in Rosacese species are rare because several Rosaceae genome sequences have only become available in last several years. We performed a systematic genome-wide survey of NBS-LRR genes among five Rosaceae plants, including Fragaria vesca (woodland strawberry), Malus × domestica (apple), Pyrus bretschneideri (Asian pear), Prunus persica (peach) and Prunus mume (mei, or Japanese apricot) with 144, 748, 469, 354 and 352 NBS-LRR genes. The high proportions of multi-genes and similar Ks peaks (Ks = 0.1- 0.2) of gene families in the four woody genomes indicate that recent duplications played an important role in the four woody perennial Rosaceae species. Subsequently, 385 species-specific duplicate clades were dominant in the phylogenetic tree constructed by all 2067 NBS-LRR genes. High percentages of NBS-LRR genes involved in species-specific duplication were found among the five genomes (54.86% in woodland strawberry, 68.05% in apple, 57.56% in pear, 44.07% in peach and 45.74% in mei). It might be inferred that species-specific duplication mainly contributes to the expansion of NBS-LRR genes in the five Rosacese species. In addition, the Ks and Ka/Ks values of TIR-NBS-LRR (TNL) were significantly greater than those of non-TIR-NBS-LRR (non-TNL), suggesting that rapidly evolved TNLs have different evolutionary patterns to adapt to different pathogens compared with non-TNL genes. In addition, some of the RPW8 domain-containing NBS-LRRs had Ka/Ks ratios less than 1, suggesting that they were driven by positive selection.

Bacterial spot, caused by Xanthomonas arboricola pv. pruni (Xap), is the most dangerous bacterial disease for Prunus. 'Redkist' peach variety is spot resistant whereas 'J.H. Hale' is susceptible. In order to characterize the molecular mechanisms underlying the incompatible and the compatible interaction between peach and Xap, we performed RNA sequencing for differential gene expression profiling on both varieties during very-early phases of interaction: 30 min, 1 h, 3h post-infection (hpi). Over 550 million reads provided an extensive view on the defense responses in both genotypes. RNA-Seq analysis detected 20,837 expressed genes in leaf, representing 75% of peach predicted genes. A total of 803 and 838 differentially expressed genes (DEGs) were identified as spot responsive genes in 'Redkist' and 'J.H. Hale' respectively during all the time points. The former regulated 88.9% of DEGs at 3 hpi while the latter only 53.4%. Both varieties induced genes involved in signal transduction, hormone biosynthesis, secondary metabolism, ROS burst, defense proteins, transcription factors. Also, GO term enrichment analysis resulted in terms including ‘immune system process’, ‘defense response’, ‘cell death’. Finally, the validation by Q-PCR confirmed the relative gene expression to be consistent with RNA-Seq data analysis. This study revealed very similar molecular responses to Xap in 'Redkist' and 'J.H. Hale' after bacterial infection and are consistent with previous studies. Between the two, crucial differences were detected in the expression of transcription factors, salicylic acid biosynthetic genes, and ROS burst, shedding light on the effective disease response exerted by a resistant host relative to a susceptible

Root hypoxia limits stone fruit tree (SFTs) development. To overcome this problem, SFTs are grafted on rootstocks with different degrees of tolerance to root hypoxia. However, the molecular base of such variability is largely unknown. Here we performed a transcriptome analysis of roots from rootstocks with contrasting responses to hypoxia using Illumina deep sequencing. Plants of the genotypes ‘Mariana 2624’ and 'Mazzard F12/1', tolerant and sensitive respectively, were subjected to hypoxic conditions by waterlogging. Transcriptomes from root samples collected from control (without waterlogging) and hypoxia stressed plants (6, 24 and 72h) were compared. The 81% of generated sequences were mapped to the reference genome of P. persica (L.). Analysis of the differentially expressed genes (DEG) revealed that most of the genes were down-regulated in the waterlogging stages. Regarding their identities and mode of regulation (up or down regulated) among DEGs, there was a high number of common DEG between the two genotypes for each sampling time, but also a high number of exclusive DEG was detected per genotype at each time. A few DEG presented opposite modes of regulations between the two genotypes throughout the hypoxia treatment. The most differentially affected functional categories are “oxidation reduction”, “carbohydrate metabolic process” and “oxidoreductase activity” at both genotypes, but only the susceptible genotype showed significant alteration of genes involved in responses to “oxidative stress”, “oxidoreductase activity”, “peroxidase activity” and “antioxidant activity”. This research represents a valuable source of information for further studies to identify the mechanism and genes that define tolerance to hypoxia in Prunus. Acknowledgment: CEAF_R08I1001 – CONICYT, FONDECYT 1121117, FONDECYT11110079 (RA).

For over 75 years the United States black raspberry industry has been in steady decline due in large part to a lack of adapted and disease resistant cultivars. Recently, potential health benefits associated with black raspberry’s high concentration of bioactive compounds have sharply increased interest in breeding and production of new cultivars. To facilitate and enhance breeding efforts, we have developed a wide set of genomic resources based on deep sequencing of the black raspberry genome and broad sampling of the transcriptome. Further, we have performed reduced representation genotyping-by-sequencing (GBS) for the ORUS 4305 mapping population that was propagated and planted in growers’ and research fields across five US production areas. A high-quality genome assembly was generated through high-throughput genome sequencing of a highly homozygous accession (~2200 scaffolds, ~240 Mbp, ~353kb N50, 0.06% SNP). Over 50% of the genomic sequence was placed onto linkage groups based on our GBS analyses. RNA-seq data from seven replicated libraries were assembled by de novo and reference-guided approaches, forming the basis for our empirically-based structural annotation (~30,000 transcription units). Associated functional predictions are in progress. These broad genomic resources are poised to enable marker-assisted breeding efforts and lead to development of improved black raspberry cultivars.

Peach/nectarine varieties exhibit unique features that are the basis for distinctive traits involved in fruit quality. The repertoire of genes that each variety expresses at a given time, is a key issue in acquiring the characteristics that distinguish them. In order to get further insights in the molecular aspects that are important for fruit quality and postharvest life, we carried out a deep transcriptomic analysis of four Prunus persica varieties. Two early-season and two late-season varieties were analyzed for different traits such as firmness, color, solid soluble, acidity as well as their susceptibility to mealiness and response to postharvest treatments aimed to control the postharvest disorders. The late season varieties resulted to be more susceptible to chilling injury than the early season varieties. In addition, the comparison between the late-season varieties showed a differential response to treatments such as controlled atmosphere and conditioning. The transcriptomic analysis during ripening, cold storage and postharvest treatments using an Illumina-based RNA-Seq approach, showed clear differences in the repertoire of genes that are expressed among varieties during ripening, cold storage and postharvest treatments. The expressed genes were identified using the Prunus persica reference genome and those differentially expressed were mapped to metabolic networks. Our results indicate that there are set of genes that are differentially expressed in each of the condition analyzed. Their relationship to the traits analyzed in the present study will be discussed. Supported by: Fondap-CRG 15090007, P10-062-F, PB-16, DS Conicyt D-21090737, UNAB DI-64-12/1 and Fondecyt 3140294

Fruit crop species, and particularly apple (Malus x domestica), are an important component of human diet. Most apples can usually be stored for several months before consumption in controlled environments (cold temperature, controlled atmosphere), thus slowing down the developmental processes associated with maturation. During this storage period, one of the most significant modification occurs to the quality of the fruit texture. Indeed, some varieties are known to develop undesirable characteristics such as softness, loss of crispiness, or mealiness. Physiological and biochemical studies are currently being performed in several research groups to understand the basis of these changes. However, it is only recently that genomic approaches have been used to investigate fruit maturation.
To understand the molecular bases of fruit maturation, we developed a transcriptomic approach using a 120k AryANE microarray (1). Within the frame of the EU-FP7-Fruitbreedomics project, 16 varieties were characterised with sensory, instrumental and transcriptomic analyses over four months of cold storage, during two consecutive years. The objective is to identify differentially expressed genes and gene networks associated (i) with fruit maturation, (ii) fruit texture changes. Results from this study will provide us with an overall picture of the gene networks involved in fruit maturity development, and may allow us to identify early markers associated with fruit texture deterioration.
(1)Celton et al., New Phytol, 2014. In Press.

The Chilean strawberry (Fragaria chiloensis) has emerged as a new berry with excellent organoleptic characteristics, however its fast softening is a limiting step for commercialization. F. chiloensis is a non-climacteric fruit and its ripening seems to be coordinated by hormones like auxins (Aux) and abscisic acid (ABA). During ripening Aux levels decrease while that of ABA increases. Softening is related to cell wall dissembling and several related enzymes have been described to take part during softening of F. chiloensis fruit, including polygalacturonase (PG), xyloglucan endotransglycosylase/ hydrolase (XTH1) and expansin 2 (Exp2). The level of their transcripts increases during fruit softening. When the fruit is subjected to several hormonal treatments (Aux, ABA, GA3, ethylene, 1-MCP) in order to establish their effects on the expression of these genes, significant variations are recorded. An activator effect of auxin on the expression of XTH1 and a repressive effect on PG is found. ABA induces the expression of XTH1 and PG, GA3 displays an activator effect on XTH1, Exp2 and PG, and finally 1-MCP represses the expression of PG and Exp2 while induces that of XTH1. To explain this regulatory mechanism, the promoter regions of these genes were obtained by Genome walker, cloned and in silico analyzed to reveal putative cis elements using PlantCARE. The promoters contained several regulatory elements responding to hormones which could explain their differential responsiveness. This provides evidences to clarify the hormonal regulation of key genes during ripening of F. chiloensis fruit. Acknowledgments to Anillo ACT-1110 and Fondecyt 1110792 Projects.

Fruit quality is built on complex traits such as taste, texture, colours and aromas, developed during fruit maturation and cold storage. The genetic, molecular and ecophysiological bases of these traits are still mainly unknown.
Apple fruit development is characterized by a complex network of sink-source interactions due to concomitant vegetative and bourse shoots development during the growing season. Several studies are pointing out a possible direct relationship between dry mater accumulation and high quality texture (1, 2). In order to study the impact of trophic competitions on fruit quality, the sink-source equilibrium was modulated at the level of the fruit bearing branch or the inflorescence by defoliation treatments.
The experiments were carried out on four apple varieties with contrasting sensory phenotypes. Apple quality was investigated with sensory, morphometric, mechanical, biochemical and transcriptomic analyses. The impact on fruit development and quality is depending on the timing of the defoliation treatment and on the variety. If late defoliation did not impact much fruit quality, data suggest an important role of bourse leaves in early stages of fruit development. Fruits transcriptome analyses also revealed a differential stress response to cold storage, probably due to increased sunlight exposition which depend on tree architecture. A drastic effect was observed on developing buds with a total fall during the next winter.
(1) Palmer et al., 2010 Journal of the Science of Food and Agriculture 90(15).
(2) Saei et al., 2011 Scientia Horticulturae 130(1).