Transcription analysis of apple fruit development using cDNA microarrays

The knowledge of the molecular mechanisms underlying fruit quality traits is fundamental to devise efficient marker-assisted selection strategies and to improve apple breeding. In this study, cDNA microarray technology was used to identify genes whose expression changes during fruit development and maturation thus potentially involved in fruit quality traits. The expression profile of 1,536 transcripts was analysed by microarray hybridisation. A total of 177 genes resulted to be differentially expressed in at least one of the developmental stages considered. Gene ontology annotation was employed to univocally describe gene function, while cluster analysis allowed grouping genes according to their expression profile. An overview of the transcriptional changes and of the metabolic pathways involved in fruit development was obtained. As expected, August and September are the two months where the largest number of differentially expressed genes was observed. In particular, 85 genes resulted to be up-regulated in September. Even though most of the differentially expressed genes are involved in primary metabolism, several other interesting functions were detected and will be presented

Microsatellite markers spanning the apple ( Malus x domestica Borkh.) genome

A new set of 148 apple microsatellite markers has been developed and mapped on the apple reference linkage map Fiesta x Discovery. One-hundred and seventeen markers were developed from genomic libraries enriched with the repeats GA, GT, AAG, AAC and ATC; 31 were developed from EST sequences. Markers derived from sequences containing dinucleotide repeats were generally more polymorphic than sequences containing trinucleotide repeats. Additional eight SSRs from published apple, pear, and Sorbus torminalis SSRs, whose position on the apple genome was unknown, have also been mapped. The transferability of SSRs across Maloideae species resulted in being efficient with 41% of the markers successfully transferred. For all 156 SSRs, the primer sequences, repeat type, map position, and quality of the amplification products are reported. Also presented are allele sizes, ranges, and number of SSRs found in a set of nine cultivars. All this information and those of the previous CH-SSR series can be searched at the apple SSR database ( http://www.hidras.unimi.it ) to which updates and comments can be added. A large number of apple ESTs containing SSR repeats are available and should be used for the development of new apple SSRs. The apple SSR database is also meant to become an international platform for coordinating this effort. The increased coverage of the apple genome with SSRs allowed the selection of a set of 86 reliable, highly polymorphic, and overall the apple genome well-scattered SSRs. These SSRs cover about 85% of the genome with an average distance of one marker per 15c

Diversity arrays technology (DArT) markers in apple for genetic linkage maps

Diversity Arrays Technology (DArT) provides a high-throughput whole-genome genotyping platform for the detection and scoring of hundreds of polymorphic loci without any need for prior sequence information. The work presented here details the development and performance of a DArT genotyping array for apple. This is the first paper on DArT in horticultural trees. Genetic mapping of DArT markers in two mapping populations and their integration with other marker types showed that DArT is a powerful high-throughput method for obtaining accurate and reproducible marker data, despite the low cost per data point. This method appears to be suitable for aligning the genetic maps of different segregating populations. The standard complexity reduction method, based on the methylation-sensitive PstI restriction enzyme, resulted in a high frequency of markers, although there was 52–54% redundancy due to the repeated sampling of highly similar sequences. Sequencing of the marker clones showed that they are significantly enriched for low-copy, genic regions. The genome coverage using the standard method was 55–76%. For improved genome coverage, an alternative complexity reduction method was examined, which resulted in less redundancy and additional segregating markers. The DArT markers proved to be of high quality and were very suitable for genetic mapping at low cost for the apple, providing moderate genome coverage

Combinations of Plant Water-Stress and Neonicotinoids Can Lead to Secondary Outbreaks of Banks Grass Mite (Oligonychus Pratensis Banks)

Spider mites, a cosmopolitan pest of agricultural and landscape plants, thrive under hot and dry conditions, which could become more frequent and extreme due to climate change. Recent work has shown that neonicotinoids, a widely used class of systemic insecticides that have come under scrutiny for non-target effects, can elevate spider mite populations. Both water-stress and neonicotinoids independently alter plant resistance against herbivores. Yet, the interaction between these two factors on spider mites is unclear, particularly for Banks grass mite (Oligonychus pratensis; BGM). We conducted a field study to examine the effects of water-stress (optimal irrigation = 100% estimated evapotranspiration (ET) replacement, water stress = 25% of the water provided to optimally irrigated plants) and neonicotinoid seed treatments (control, clothianidin, thiamethoxam) on resident mite populations in corn (Zea mays, hybrid KSC7112). Our field study was followed by a manipulative field cage study and a parallel greenhouse study, where we tested the effects of water-stress and neonicotinoids on BGM and plant responses. We found that water-stress and clothianidin consistently increased BGM densities, while thiamethoxam-treated plants only had this effect when plants were mature. Water-stress and BGM herbivory had a greater effect on plant defenses than neonicotinoids alone, and the combination of BGM herbivory with the two abiotic factors increased the concentration of total soluble proteins. These results suggest that spider mite outbreaks by combinations of changes in plant defenses and protein concentration are triggered by water-stress and neonicotinoids, but the severity of the infestations varies depending on the insecticide active ingredient

The European project HiDRAS : innovative multidisciplinary approaches to breed high- quality disease resistant apples

The European project HiDRAS (High-quality Disease Resistant Apples for a Sustainable Agriculture) is a collaborative effort between 11 European groups (University of Milano and University of Bologna, Italy; Plant Research International, The Netherlands; INRA Centre d'Angers, France; Swiss Federal Institute of Technology and Swiss Federal Research Station, Switzerland; Horticultural Research International, U.K.; Federal Centre for Breeding Research on Cultivated Plants, Germany; Centre de Recherches Agronomiques de Gembloux, Belgium; Warsaw Agricultural University and Research Institute of Pomology and Floriculture, Poland) aimed at the identification of genetic factors controlling fruit quality. Breeders will be provided with new tools, such as molecular markers linked to fruit quality and pathogen resistance, to improve "marker assisted selection". An innovative approach based on the phenotypic and molecular characterisation of a large number of related genotypes will be adopted. New software will be developed to fully exploit genotypic, phenotypic and pedigree data, with the aim of identifying fruit quality QTLs and to follow the transmission of their alleles along the pedigrees. A large number of highly informative markers, including markers from fruit-quality-related genes will be generated and an apple genetic map with evenly distributed markers will be constructed to increase the efficiency of the QTL analysis. Consumer's needs will be tested to study the quality parameters that determine the success of a new apple variety in different European countries. In those occasions our scientific research will be presented increasing the trust of European citizens towards disease resistant apples, therefore favouring their diffusion. An apple data repository will be established to collect and share data among the participants. Initially database access will be limited but later it could be opened to the scientific community. The original and well concerted multidisciplinary approach, including apple breeding, genetics, molecular biology, statistics and bioinformatics, will ensure the achievement of the project objectives

Apple fruit : towards the identification of genes showing a modulated expression during development

Fruit development is a fundamental process determining fruit quality; therefore the knowledge of the genes involved allows the deployment of more efficient selection methods. In order to detect these genes, cDNA microarrays were constructed using two subtractive cDNA libraries. About 1600 clones, derived from the two libraries have been printed in duplicate on the slides. Hybridizations were always performed comparing mRNA from fruits of the cultivar 'Prima', collected in May, with different mRNA samples isolated from the same plant at different developmental stages (June, July, August, and September). The analysis of the expression profiles suggests that about 10% of the genes present on the slides show a modulated expression during fruit development. 75% of sequences have known functions and the most represented categories are: primary metabolism (17%), stress and defense (10%), aminoacid and protein metabolism (9%). A high proportion (24%) corresponds to genes encoding proteins with unknown function. Gene Ontology annotation was employed to univocally describe the differentially expressed genes, while clustering analysis allowed grouping genes according to their expression profiles. In June and July, genes have similar expression patterns and their changes in expression level are not significant. On the contrary, August and September are characterized by a high number of genes up or down regulated. Microarray data were experimentally validated by an independent method, Quantitative RT-PCR, and about 70% of them were confirmed. Though not all the data were confirmed, microarray results are a good tool to identify candidate genes potentially correlated with fruit quality QTLs. Molecular markers are going to be generated from the most interesting transcripts and their position is going to be determined on the apple linkage map. Subsequent QTL analysis will allow associating and correlating those genes with fruit quality traits