High-quality de novo assembly of the apple genome and methylome dynamics of early fruit development

Using the latest sequencing and optical mapping technologies, we have produced a high-quality de novo assembly of the apple (Malus domestica Borkh.) genome. Repeat sequences, which represented over half of the assembly, provided an unprecedented opportunity to investigate the uncharacterized regions of a tree genome; we identified a new hyper-repetitive retrotransposon sequence that was over-represented in heterochromatic regions and estimated that a major burst of different transposable elements (TEs) occurred 21 million years ago. Notably, the timing of this TE burst coincided with the uplift of the Tian Shan mountains, which is thought to be the center of the location where the apple originated, suggesting that TEs and associated processes may have contributed to the diversification of the apple ancestor and possibly to its divergence from pear. Finally, genome-wide DNA methylation data suggest that epigenetic marks may contribute to agronomically relevant aspects, such as apple fruit development

The improved assembly of the European Pear

Apple and Pear diverged from each other between 5.4 and 21.5 MYA and are believed to share a common genome duplication event between 35 and 50 MYA (Velasco et al. 2010, Wu et al. 2012). Size differences have been observed between the Apple and Pear genomes which are estimated at 527Mb (Pyrus x Bretschneideri Rehd) and 700Mb (Malus x Domestica Borkh) respectively (Wu et al. 2013, Li et al. 2016). The difference in genome size has been accounted for primarily by the proliferation of transposable elements, with the gene space thought to be fairly similar between the two species (Wu et al. 2012). Comparative genomics of the lineage has however, been hampered by the fragmented nature of the reference assemblies. A new chromosome scale assembly was recently produced (Daccord et al. 2017) and now also a chromosome scale assmble of the European Pear (this study), which shows strong collinearity with Apple, greatly facilitating the comparative study of these genomes

Identification of a leucine-rich repeat receptor-like serine/threonine-protein kinase as a candidate gene for Rvi12 (Vb)-based apple scab resistance

Apple scab caused by Venturia inaequalis is the most important fungal disease of apples (Malus × domestica). Currently, the disease is controlled by up to 15 fungicide applications to the crop per year. Resistant apple cultivars will help promote the sustainable control of scab in commercial orchards. The breakdown of the Rvi6 (Vf) major-gene based resistance, the most used resistance gene in apple breeding, prompted the identification and characterization of new scab resistance genes. By using a large segregating population, the Rvi12 scab resistance gene was previously mapped to a genetic location flanked by molecular markers SNP_23.599 and SNP_24.482. Starting from these markers, utilizing chromosome walking of a Hansen’s baccata #2 (HB2) BAC-library; a single BAC clone spanning the Rvi12 interval was identified. Following Pacific Biosciences (PacBio) RS II sequencing and the use of the hierarchical genome assembly process (HGAP) assembly of the BAC clone sequence, the Rvi12 resistance locus was localized to a 62.3-kb genomic region. Gene prediction and in silico characterization identified a single candidate resistance gene. The gene, named here as Rvi12_Cd5, belongs to the LRR receptor-like serine/threonine-protein kinase family. In silico comparison of the resistance allele from HB2 and the susceptible allele from Golden Delicious (GD) identified the presence of an additional intron in the HB2 allele. Conserved domain analysis identified the presence of four additional LRR motifs in the susceptible allele compared to the resistance allele. The constitutive expression of Rvi12_Cd5 in HB2, together with its structural similarity to known resistance genes, makes it the most likely candidate for Rvi12 scab resistance in appl

Development of the Axiom® Apple480K SNP genotyping array and its application for genome wide association study in apple

During the last decade, high-throughput genotyping has facilitated the dissection of complex traits in species with large/complex genomes and high level of genetic diversity. Array-based marker systems have been increasingly adopted for high-throughput genotyping, not only in model organisms, but also in many non-model plant species for which genomic resources are now available. A new high-density Affymetrix Axiom® SNP array has been built for the domesticated apple (Malus x domestica). It gathers more than 487K SNPs that are evenly distributed over the 17 chromosomes. The array has been built from the high-depth resequencing (~10-20x) of 63 different varieties covering most of the genetic diversity in cultivated apple. SNPs have been chosen by applying a focal points approach to enrich genic regions, but also to reach a uniform coverage of non-genic regions as to support SNP haplotype approaches. A total of 1324 apple accessions, including the 92 progenies of two mapping populations, have been genotyped with the Axiom®Apple480K to assess the effectiveness of the array. The majority of SNPs (359,994; 74%) fell in the most interesting class of Poly High Resolution polymorphisms. A novel filtering procedure was also devised to identify a subset of 275K robust markers that can be safely used for germplasm surveys in apple. A first application to genome wide association (GWA) study of two phenology traits (flowering time and maturity date) in six European germplasm collections is also presented. The Axiom®Apple480K has been publicly released and will likely be a reference tool for GWA studies in apple

Development and validation of the Axiom®Apple480K SNP genotyping array

Cultivated apple (Malus × domestica Borkh.) is one of the most important fruit crops in temperate regions, and has great economic and cultural value. The apple genome is highly heterozygous and has undergone a recent duplication which, combined with a rapid linkage disequilibrium decay, makes it difficult to perform genome-wide association (GWA) studies. Single nucleotide polymorphism arrays offer highly multiplexed assays at a relatively low cost per data point and can be a valid tool for the identification of the markers associated with traits of interest. Here, we describe the development and validation of a 487K SNP Affymetrix Axiom® genotyping array for apple and discuss its potential applications. The array has been built from the high-depth resequencing of 63 different cultivars covering most of the genetic diversity in cultivated apple. The SNPs were chosen by applying a focal points approach to enrich genic regions, but also to reach a uniform coverage of non-genic regions. A total of 1324 apple accessions, including the 92 progenies of two mapping populations, have been genotyped with the Axiom®Apple480K to assess the effectiveness of the array. A large majority of SNPs (359 994 or 74%) fell in the stringent class of poly high resolution polymorphisms. We also devised a filtering procedure to identify a subset of 275K very robust markers that can be safely used for germplasm surveys in apple. The Axiom®Apple480K has now been commercially released both for public and proprietary use and will likely be a reference tool for GWA studies in apple

High-quality de novo assembly of the apple genome and methylome dynamics of early fruit development

Using the latest sequencing and optical mapping technologies, we have produced a high-quality de novo assembly of the apple (Malus domestica Borkh.) genome. Repeat sequences, which represented over half of the assembly, provided an unprecedented opportunity to investigate the uncharacterized regions of a tree genome; we identified a new hyper-repetitive retrotransposon sequence that was over-represented in heterochromatic regions and estimated that a major burst of different transposable elements (TEs) occurred 21 million years ago. Notably, the timing of this TE burst coincided with the uplift of the Tian Shan mountains, which is thought to be the center of the location where the apple originated, suggesting that TEs and associated processes may have contributed to the diversification of the apple ancestor and possibly to its divergence from pear. Finally, genome-wide DNA methylation data suggest that epigenetic marks may contribute to agronomically relevant aspects, such as apple fruit development

Novel and emerging biotechnological crop protection approaches

Traditional breeding or genetically modified organisms (GMOs) have for a long time been the sole approaches to effectively cope with biotic and abiotic stresses and implement the quality traits of crops. However, emerging diseases as well as unpredictable climate changes affecting agriculture over the entire globe force scientists to find alternative solutions required to quickly overcome seasonal crises. In this review, we first focus on cisgenesis and genome editing as challenging biotechnological approaches for breeding crops more tolerant to biotic and abiotic stresses. In addition, we take into consideration a toolbox of new techniques based on applications of RNA interference and epigenome modifications, which can be adopted for improving plant resilience. Recent advances in these biotechnological applications are mainly reported for non‐model plants and woody crops in particular. Indeed, the characterization of RNAi machinery in plants is fundamental to transform available information into biologically or biotechnologically applicable knowledge. Finally, here we discuss how these innovative and environmentally friendly techniques combined with traditional breeding can sustain a modern agriculture and be of potential contribution to climate change mitigation