New polymorphic EST-Based molecular markers in three Malus X domestica (Borkh.) cultivars (Fiesta, Prima, Discovery)

Molecular markers derived from fruit expressed genes and from other candidate genes controlling fruit traits are a useful tool to map genes involved in fruit development and quality. Functional markers for fruit quality traits were developed for three apple cultivars ('Fiesta', 'Prima', 'Discovery') following three main approaches. Initially, the public sequence databases were screened for transcription factors (TFs), leading to the identification of 425 Malus EST (Expressed Sequence Tags); among which 28 SSR containing sequences were selected and tested for polymorphism. A second set of genes, from which molecular markers were developed, was obtained from microarray studies, performed in our lab, aimed at identifying genes involved in fruit development and maturation. Three hundred genes resulted differentially expressed during different fruit developmental stages. The corresponding ESTs provided a second source of functional markers. To reduce redundancy, the 300 EST sequences were assembled and used to identify corresponding unique transcripts: 153 sequences resulted to be unique while 147 redundant sequences were assembled in 36 contigs. In total, 189 sequences were screened for the presence of SSR (Simple Sequence Repeat), while the sequences not containing repeated elements were BLAST compared against PlantGDB-assembled Unique Transcript database (PUT) and the resulting PUTs were analyzed to identify repeated motifs. Furthermore, INDEL (INsertion/DELetion) and SNP (Single Nucleotide Polymorphism) were searched in the assembled sequences. Finally, the third approach, to identify ESTs, involved in fruit development and maturation, exploited the information gathered in other species to identify homologous sequences potentially involved in fruit formation and in determining its physiological composition. In total, more than 100 PCR primer pairs were designed, flanking SSR, INDEL, or SNP containing regions or including most of the differentially expressed sequences while sequence reactions for the three cultivars were performed when polymorphism were not detected. SNPs detection was performed digesting PCR products with different restriction enzymes, by means of SSCP (Single Strand Conformation Polymorphism) or presence/absence of band while SSRs and INDELs were scored as length polymorphism. The forty-seven polymorphic markers developed in this study will be useful to increase mapping resolution in 'Fiesta' x 'Discovery' and/or 'Prima' x 'Fiesta' reference maps. The work is part of the European project named HiDRAS (High-quality Disease Resistant Apples for a Sustainable Agriculture).