Genomics

Apple latent spherical virus (ALSV) vectors have been shown to effectively induce stable virus-induced gene silencing (VIGS) in a wide range of plant species including Rosaceous fruit tree species, such as apple and pear. In this study, we attempted to develop a VIGS-based gene evaluation system for fruit tree species in Prunus, using the ALSV viral vector system. Partial sequence of PHYTOENE DESATURASE (PDS) of apricot (Prunus armeniaca) was cloned and ligated into the T-DNA region of a binary vector pBICAL2. The T-DNA region of pBICAL2, designed based on the RNA2 of ALSV, contains a single ORF for the ALSV polyprotein under the control of CaMV35S promoter sequence. The partial ParPDS sequence was ligated in frame with the coding sequences for the movement protein and the capsid protein Vp25 flanking the cloning site. The resultant pBICAL2-ParPDS was introduced into a disarmed Agrobacterium strain EHA105. The pBICAL1, a binary plasmid for the expression of RNA1 of ALSV in plants, was also introduced into EHA105. To amplify and produce recombinant ALSV particles, leaves of Nicotiana benthamiana were infected with pBICAL1/EHA105 and pBICAL2-ParPDS/EHA105 at the same time. The amplified ParPDS-ALSV in N. benthamiana was isolated and infected to the cotyledons of Prunus seeds right after germination by a particle bombardment. Uniform discoloration of the upper leaves, a typical knock-down phenotype of PDS, was observed several weeks after inoculation in a certain range of fruit tree species in Prunus.

Genetic variation takes many forms, from large chromosomal anomalies to individual base pair changes. Copy number variants (CNVs) are generally considered to be gains or losses of DNA segments larger than 1 kb. In humans CNVs are a factor in determining phenotype, as they provide rapid genetic response to a changing environment and have been associated with risk of disease. Preliminary analyses of plant genomes have indicated that CNVs are important to the evolution of plant genomes which have been under strong selection. Surprisingly, CNVs have been an untapped source of genomic variation, compared with SNPs, for understanding the genetic control of key traits in plants and as tools to improve selection efficiency of new varieties. Domesticated apples first appeared in the Near East around 4,000 years ago and it seems pertinent to investigate the CNV of apple varieties, as many CNVs may have emerged since its domestication. We have performed a genome-wide analysis of CNV using low coverage genome re-sequencing of 34 apple varieties using the R package CNVrd2. CNVs were detected across the apple genome. CNVs associated with candidate genes for aroma, and pest and disease resistance were identified.

Apple Replant Disease (ARD) causes a serious economic loss for the apple industry. Although it has been known that a complex of necrotrophic fungi and oomycetes are the primary causal agents of ARD, the molecular response in apple to infection by these pathogens has not previously been examined. In this study, we identified apple genes that are activated by Pythium ultimum, an oomycete that is a significant component of the ARD pathogen complex. The apple genes examined in this study include those involved in ethylene (ET) biosynthesis and jasmonic acid (JA) biosynthesis, a transcription factor gene that functions as an ET/JA signal integrator (MdERF: ethylene response factor) and a pathogenesis-related (PR) gene that is a target of ERF (MdCHIB: CHITINASEB or beta-chitinase). Using real-time quantitative reverse transcription PCR, target gene expression was shown to a 10-60 fold up-regulation in apple root stock seedlings 1-2 days post P. ultimum inoculation. Transcriptional regulation of these target genes by exogenous application of ethylene and jasmonate was also examined. MdERF was not only up-regulated by either ethylene or JA, but the enhanced expression of MdERF was significantly greater when exposed to the combination of these hormones. For MdACS and MdAOS, ethylene and JA serve as a positive or negative regulator depending upon gene isoform and the plant tissues in which expression is monitored. Our data demonstrate that ET/JA cross-talk plays an important role in the apple rootstock response to infection by P. ultimum.

Bud dormancy release in Rosaceae depends on a minimum accumulation of chilling hours. DORMANCY ASSOCIATED MADS-BOX (DAM) genes are negative flowering regulators and strong candidates for the regulation of bud growth cessation and dormancy maintenance. Epigenetic mechanisms appear to control the silencing of DAM genes in peach in a similar way to its ortologous in Arabidopsis during vernalization. To better understand the molecular mechanism of dormancy release in sweet cherry (Prunus avium var. Bing); we isolated the promoter and the first intron of DAM6. Histone modifications and DNA methylation pattern kinetic of these regions, in response to chilling hours (CH) accumulation, was studied in floral buds sampled weekly during the dormancy period. Using Chromatin Immunoprecipitation (ChIP) essay, we observed that the presence of H3K27me3 and histone variant H2A.Z increased reaching a maximum at 1038 CH in the promoter. This histone modification and histone variant are related to silencing of gene expression. On the other hand it was observed that the presence of H3K4me3, a modification related to gene activation, decreases with CH accumulation in the promoter. In parallel, the results obtained from bisulfite sequencing show an increase of DNA methylation in the first intron of DAM6, and suggest a dynamic change of methylated cytosines, as the chilling requirement is being fulfilled indicating silencing of DAM6. Our results strength the role of epigenetic mechanisms in chilling requirement for dormancy release and blooming in Rosaceous species as sweet cherry. This work is supported by FONDEF G09I1008; FONDAP CRG 15070009 and Basal PFB-16.

The genus Potentilla (Rosaceae) is large and diverse and is closely-related to the economically important Rubus and Fragaria. Potentilla species exhibit many morphological similarities to Fragaria, but the majority do not form fleshy accessory ‘berries’ characteristic of all members of the Fragaria. The close relatedness, morphological similarities, small genome size and high homozygosity of a number of Potentilla species make them an ideal comparative organism to study characteristic features of Fragaria such as ‘berry’ formation. To develop an experimental system to study such traits, the genome of P. micrantha was sequenced using the Illumina HiSeq and MiSeq and the PacBio RS sequencing platforms. Data were generated from overlapping paired end libraries, large insert mate-pair libraries PacBio SMRT-bell libraries. The sequence data was assembled and a genome with an approximate size of 315 Mb was recovered, and confirmed with flow-cytometry against a F. vesca ‘Hawaii 4’ standard. RNA-seq was performed on reproductive tissue samples from five stages of fruit development in P. micrantha, corresponding to the stages of development defined for F. vesca. Additionally RNA-seq was performed on young leaf samples. Comparisons of the P. micrantha genome and transcriptome sequence to that of F. vesca ‘Hawaii 4’ revealed a high degree of expected synteny, but also clear patterns of global and localized divergence. The details and implications of these differences with respect to fruit development will be discussed.