R. Velasco

Identification and validation of a QTL influencing bitter pit symptoms in apple (Malus × domestica)

Bitter pit is one of the most economically important physiological disorders affecting apple fruit production, causing soft discrete pitting of the cortical flesh of the apple fruits which renders them unmarketable. The disorder is heritable; however, the environment and cultural practices play a major role in expression of symptoms. Bitter pit has been shown to be controllable to a certain extent using calcium sprays and dips; however, their use does not entirely prevent the incidence of the disorder. Previously, bitter pit has been shown to be controlled by two dominant genes, and markers on linkage group 16 of the apple genome were identified that were significantly associated with the expression of bitter pit symptoms in a genome-wide association study. In this investigation, we identified a major QTL for bitter pit defined by two microsatellite (SSR) markers. The association of the SSRs with the bitter pit locus, and their ability to predict severe symptom expression, was confirmed through screening of individuals with stable phenotypic expression from an additional mapping progeny. The data generated in this current study suggest a two gene model could account for the control of bitter pit symptom expression; however, only one of the loci was detectable, most likely due to dominance of alleles carried by both parents of the mapping progeny used. The SSR markers identified are cost-effective, robust and multi-allelic and thus should prove useful for the identification of seedlings with resistance to bitter pit using marker-assisted selection in apple breeding programs.

Genome Sequencing, Transcriptomics, and Proteomics

This review encompasses the current status of major areas of progress in olive tree genome sequencing, including insights into genome function derived from large-scale gene expressing profiling, and studies on genomic architecture of repetitive sequences, smaller RNA, and proteomics. Olive tree genomics, as well as other omics, is progressing owing to recent developments in next-generation sequencing (NGS) technologies. Biological insights, therefore, are not only resulted from the sequencing initiative, since from genetic mapping, gene expression profiling, gene discovery research, and proteomics over nearly last seven years a large amount of information has been provided by different laboratories. The availability of high-quality genome assembly provides olive biologists with valuable new tools to improve and develop new varieties more efficiently, enabling the implementation of marker-assisted selection and genomic selection, and contributing to the comprehension of the molecular determinants of key traits peculiar to the species of olive tree and giving important clues concerning the evolution of its complex genome.

An integrated approach for increasing breeding efficiency in apple and peach in Europe

Despite the availability of whole genome sequences of apple and peach, there has been a considerable gap between genomics and breeding. To bridge the gap, the European Union funded the FruitBreedomics project (March 2011 to August 2015) involving 28 research institutes and private companies. Three complementary approaches were pursued: (i) tool and software development, (ii) deciphering genetic control of main horticultural traits taking into account allelic diversity and (iii) developing plant materials, tools and methodologies for breeders. Decisive breakthroughs were made including the making available of ready-to-go DNA diagnostic tests for Marker Assisted Breeding, development of new, dense SNP arrays in apple and peach, new phenotypic methods for some complex traits, software for gene/QTL discovery on breeding germplasm via Pedigree Based Analysis (PBA). This resulted in the discovery of highly predictive molecular markers for traits of horticultural interest via PBA and via Genome Wide Association Studies (GWAS) on several European genebank collections. FruitBreedomics also developed pre-breeding plant materials in which multiple sources of resistance were pyramided and software that can support breeders in their selection activities. Through FruitBreedomics, significant progresses were made in the field of apple and peach breeding, genetics, genomics and bioinformatics of which advantage will be made by breeders, germplasm curators and scientists. A major part of the data collected during the project has been stored in the FruitBreedomics database and has been made available to the public. This review covers the scientific discoveries made in this major endeavour, and perspective in the apple and peach breeding and genomics in Europe and beyond.Genomics: New resources for apple and pear breedingA Europe-led effort to bridge the gap between genomics and breeding offers a new resource to support apple and peach production. A team led by François Laurens from the Research Institute in Horticulture and Seeds (IRHS) in Angers, France, review the tools, methodologies and scientific discoveries made through FruitBreedomics, a project that ran between 2011 and 2015 and involved 28 different research and private companies from around the world. These new molecular and bioinformatics tools include DNA arrays for mapping genetic diversity, computer software for analyzing genetic information and novel methods for understanding commercially important traits such as fruit quality and resistance to stresses. The project’s materials should help apple and peach producers better plan their breeding programs so as to meet grower and consumer demand for high-quality fruit with desired characteristics.

Characterization of 25 full-length S-RNase alleles, including flanking regions, from a pool of resequenced apple cultivars

Key messageData obtained from Illumina resequencing of 63 apple cultivars were used to obtain full-length S-RNase sequences using a strategy based on both alignment and de novo assembly of reads.AbstractThe reproductive biology of apple is regulated by the S-RNase-based gametophytic self-incompatibility system, that is genetically controlled by the single, multi-genic and multi-allelic S locus. Resequencing of apple cultivars provided a huge amount of genetic data, that can be aligned to the reference genome in order to characterize variation to a genome-wide level. However, this approach is not immediately adaptable to the S-locus, due to some peculiar features such as the high degree of polymorphism, lack of colinearity between haplotypes and extensive presence of repetitive elements. In this study we describe a dedicated procedure aimed at characterizing S-RNase alleles from resequenced cultivars. The S-genotype of 63 apple accessions is reported; the full length coding sequence was determined for the 25xa0S-RNase alleles present in the 63 resequenced cultivars; these included 10 previously incomplete sequences (S5, S6a, S6b, S8, S11, S23, S39, S46, S50 and S58). Moreover, sequence divergence clearly suggests that alleles S6a and S6b, proposed to be neutral variants of the same alleles, should be instead considered different specificities. The promoter sequences have also been analyzed, highlighting regions of homology conserved among all the alleles.

A study of gene expression changes at the Bp-2 locus associated with bitter pit symptom expression in apples (Malus pumila)

Bitter pit is a physiological disorder of apples that develops in the latter stages of fruit development and during storage. It is characterized by localized necrotic cells that collapse and form pits in the epidermis and outer cortex of fruit. The disorder has been associated with low calcium concentrations, and poor calcium distribution within the fruit. To date, the mechanism that leads to individual cell necrosis, while surrounding cells remain healthy, is not fully understood. In order to ascertain the underlying process of bitter pit incidence in apple fruit, a large mapping population of “Braeburn” (susceptible to bitter pit)u2009×u2009“Cameo” (resistant to bitter pit) was used to map the trait over two growing seasons. A subset of 94 genotypes from the mapping population representing the full range of phenotypes in the same ratio as the full population were selected for genotyping and functional characterization. RNA-Seq analysis on fruit samples of three resistant and three susceptible lines at seven developmental stages (21, 42, 63, 84, 105, 126, and 147xa0days post fertilization) identified a number of candidate genes displaying differential gene expression. A subset of candidate genes selected based on their position within the identified QTL interval on linkage group 16 were validated by RT-qPCR, and two candidate genes displaying differential gene expression were highlighted as strong candidates for the control of bitter pit symptom expression at the Bp-2 locus.

A joint Laimburg – FEM molecular markers project for apple fruit quality traits using the Pedigree Based Analysis strategy

In apple breeding fruit quality is a crucial aspect to guarantee commercial success of new varieties. Among the several quality attributes, individual sugars and acid components, as well as texture, are considered important features due to their significant contribution to sensory quality and consumers acceptance. In traditional breeding, however, selection for these particular traits is a time consuming process, due to the long unproductive juvenile phase of apple seedlings. This step can be assisted to date by the use of molecular markers, an important diagnostic tool useful to anticipate and assist the identification of novel apple varieties characterized by superior fruit quality behaviour. However, the routinely implementation of molecular markers in breeding programs is still limited. In this study a new approach named Pedigree Based Analysis will be used on six full-sib progenies and their pedigree (composed by 15 varieties, represented by founders and ancestors), to target the main set of quantitative trait loci (QTLs). This strategy will allow the identification of the most valuable alleles present in the breeding activities of both Italian institutes. The ultimate goal of the Laimburg - FEM joint project is the identification and validation of molecular markers as a suitable tool for Marker Assisted Breeding in the two on-going apple breeding programs.

Assessing the genetic variability of grape clones

In this work, to understand clonal genetic variability within six wine grape cultivars, we adopted three different methods. Firstly, we carried out a Transposon Display analysis on 141 grape genotypes, which correspond to 47 clones (both registered and biotypes) belonging to the ?Pinot Noir?, ?Pinot Gris?, ?Pinot Blanc?, ?Meunier?, ?Teroldego? and ?Gewurztraminer? cultivars in 3 biological replicates (plants). We tested them using 17 primers targeting specific regions of 6 different Transposable Element families. Secondly, by using 6 triallelic SSR markers we have analysed four tissues (leaf, berry skin, flesh and root) of the 19 registered clones. Finally, we have genotyped the same set of samples by the SNPlexTM Genotyping System, testing 430 electronic SNPs identified in coding and non-coding regions of the ?Pinot Noir? ENTAV 115 grape genome. Based on SNPlex results, we are currently resequencing the regions with a high level of polymorphism to confirm the presence of clonal mutations. Here we report the results of the polymorphisms identified with the different approaches and putatively enabling molecular characterization of clones within international and local grape varieties.