Beschreibung:
Anthocyanins are secondary metabolites found in higher plants that contribute to the colors of flowers and fruits. In apples (Malus domestica Borkh.), several steps of the anthocyanin pathway are coordinately regulated, suggesting control by common transcription factors. A gene encoding an R2R3 MYB transcription factor was isolated from apple (cv Cripps' Pink) and designated MdMYB1. Analysis of the deduced amino acid sequence suggests that this gene encodes an ortholog of anthocyanin regulators in other plants. The expression of MdMYB1 in both Arabidopsis (Arabidopsis thaliana) plants and cultured grape cells induced the ectopic synthesis of anthocyanin. In the grape (Vitis vinifera) cells MdMYB1 stimulated transcription from the promoters of two apple genes encoding anthocyanin biosynthetic enzymes. In ripening apple fruit the transcription of MdMYB1 was correlated with anthocyanin synthesis in red skin sectors of fruit. When dark-grown fruit were exposed to sunlight, MdMYB1 transcript levels increased over several days, correlating with anthocyanin synthesis in the skin. MdMYB1 gene transcripts were more abundant in red skin apple cultivars compared to non-red skin cultivars. Several polymorphisms were identified in the promoter of MdMYB1. A derived cleaved amplified polymorphic sequence marker designed to one of these polymorphisms segregated with the inheritance of skin color in progeny from a cross of an unnamed red skin selection (a sibling of Cripps' Pink) and the non-red skin cultivar Golden Delicious. We conclude that MdMYB1 coordinately regulates genes in the anthocyanin pathway and the expression level of this regulator is the genetic basis for apple skin color. Next-generation DNA sequencing (NGS) produces vast amounts of DNA sequence data, but it is not specifically designed to generate data suitable for genetic mapping. Recently developed DNA library preparation methods for NGS have helped solve this problem, however, by combining the use of reduced representation libraries with DNA sample barcoding to generate genome-wide genotype data from a common set of genetic markers across a large number of samples. Here we use such a method, called genotyping-by-sequencing (GBS), to produce a data set for genetic mapping in an F1 population of apples (Malus × domestica) segregating for skin color. We show that GBS produces a relatively large, but extremely sparse, genotype matrix: over 270,000 SNPs were discovered but most SNPs have too much missing data across samples to be useful for genetic mapping. After filtering for genotype quality and missing data, only 6% of the 85 million DNA sequence reads contributed to useful genotype calls. Despite this limitation, using existing software and a set of simple heuristics, we generated a final genotype matrix containing 3967 SNPs from 89 DNA samples from a single lane of Illumina HiSeq and used it to create a saturated genetic linkage map and to identify a known QTL underlying apple skin color. We therefore demonstrate that GBS is a cost-effective method for generating genome-wide SNP data suitable for genetic mapping in a highly diverse and heterozygous agricultural species. We anticipate future improvements to the GBS analysis pipeline presented here that will enhance the utility of next-generation DNA sequence data for the purposes of genetic mapping across diverse species.