Microsatellite allele dose and configuration establishment (MADCE): an integrated approach for genetic studies in allopolyploids.

BACKGROUND: Genetic studies in allopolyploid plants are challenging because of the presence of similar sub-genomes, which leads to multiple alleles and complex segregation ratios. In this study, we describe a novel method for establishing the exact dose and configuration of microsatellite alleles for any accession of an allopolyploid plant species. The method, named Microsatellite Allele Dose and Configuration Establishment (MADCE), can be applied to mapping populations and pedigreed (breeding) germplasm in allopolyploids. RESULTS: Two case studies are presented to demonstrate the power and robustness of the MADCE method. In the mapping case, five microsatellites were analysed. These microsatellites amplified 35 different alleles based on size. Using MADCE, we uncovered 30 highly informative segregating alleles. A conventional approach would have yielded only 19 fully informative and six partially informative alleles. Of the ten alleles that were present in all progeny (and thereby ignored or considered homozygous when using conventional approaches), six were found to segregate by dosage when analysed with MADCE. Moreover, the full allelic configuration of the mapping parents could be established, including null alleles, homozygous loci, and alleles that were present on multiple homoeologues. In the second case, 21 pedigreed cultivars were analysed using MADCE, resulting in the establishment of the full allelic configuration for all 21 cultivars and a tracing of allele flow over multiple generations. CONCLUSIONS: The procedure described in this study (MADCE) enhances the efficiency and information content of mapping studies in allopolyploids. More importantly, it is the first technique to allow the determination of the full allelic configuration in pedigreed breeding germplasm from allopolyploid plants. This enables pedigree-based marker-trait association studies the use of algorithms developed for diploid crops, and it may increase the effectiveness of LD-based association studies. The MADCE method therefore enables researchers to tackle many of the genotyping problems that arise when performing mapping, pedigree, and association studies in allopolyploids. We discuss the merits of MADCE in comparison to other marker systems in polyploids, including SNPs, and how MADCE could aid in the development of SNP markers in allopolyploids.

Measurement of lipid transfer protein in 88 apple cultivars.

BACKGROUND: Fruits are a major cause of food allergy in adults. Lipid transfer proteins (LTP) are implicated in severe allergic reactions to fruits, but little is known about LTP content in different cultivars. OBJECTIVE: Determination of the levels of LTP in a wide range of apple cultivars. METHODS: LTP was measured in apples from 53 cultivars grown in Italy and 35 grown in The Netherlands, using three different immunoassays: a competitive ELISA (cELISA), a sandwich ELISA (sELISA) and a RAST inhibition (RI). Selected cultivars were evaluated using the basophil histamine release test (BHR), skin prick test (SPT) and double-blind, placebo-controlled food challenge (DBPCFC). RESULTS: LTP levels measured with the three immunoassays were significantly correlated, as judged by Pearson's correlation (0.61 < Rp < 0.65; p < 0.0001), but differed with respect to the actual quantities: 3.4-253.2 (sELISA), 2.7-120.2 (cELISA) and 0.4-47.3 microg/g tissue (RI). Between cultivars, LTP titers varied over about a two-log range. Pilot in vitro and in vivo biological testing (BHR, SPT and DBPCFC) with selected cultivars supported the observed differences in LTP levels. CONCLUSIONS: Around 100-fold differences in LTP levels exist between apple cultivars. Whether the lowest observed levels of LTP warrant designation as hypo-allergenic requires more extensive confirmation by oral challenges. Determination of cultivar variation in LTP levels provides important information for growers and consumers. Comparison to earlier reported Mal d 1 levels in the same cultivars reveals that a designation as low allergenic does not always coincide for both allergens.