Genetic Diversity of Red-Fleshed Apples (Malus)
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Euphytica DOI 10.1007/s10681-011-0579-7 Genetic diversity of red-fleshed apples (Malus) Steven van Nocker • Garrett Berry • James Najdowski • Roberto Michelutti • Margie Luffman • Philip Forsline • Nihad Alsmairat • Randy Beaudry • Muraleedharan G. Nair • Matthew Ordidge Received: 31 May 2011 / Accepted: 3 November 2011 Ó Springer Science+Business Media B.V. 2011 Abstract Anthocyanins are flavonoid pigments including the core and cortex (flesh). Red-fleshed imparting red, blue, or purple pigmentation to fruits, apple genotypes are an attractive starting point for flowers and foliage. These compounds are powerful development of novel varieties for consumption and antioxidants in vitro, and are widely believed to nutraceutical use through traditional breeding and contribute to human health. The fruit of the domestic biotechnology. However, cultivar development is apple (Malus x domestica) is a popular and important limited by lack of characterization of the diversity source of nutrients, and is considered one of the top of genetic backgrounds showing this trait. We iden- ‘functional foods’—those foods that have inherent tified and cataloged red-fleshed apple genotypes from health-promoting benefits beyond basic nutritional four Malus diversity collections representing over value. The pigmentation of typical red apple fruits 3,000 accessions including domestic cultivars, wild results from accumulation of anthocyanin in the skin. species, and named hybrids. We found a striking However, numerous genotypes of Malus are known range of flesh color intensity and pattern among that synthesize anthocyanin in additional fruit tissues accessions, including those carrying the MYB10 R6 allele conferring ectopic expression of a key tran- scriptional regulator of anthocyanin biosynthesis. Although MYB10 R6 was strongly associated with red-fleshed fruit among genotypes, this allele was Electronic supplementary material The online version of this article (doi:10.1007/s10681-011-0579-7) contains neither sufficient nor required for this trait in all supplementary material, which is available to authorized users. genotypes. Nearly all red-fleshed accessions tested could be traced back to ‘Niedzwetzkyana’, a pre- & S. van Nocker ( ) Á G. Berry Á J. Najdowski Á sumed natural form of M. sieversii native to central N. Alsmairat Á R. Beaudry Á M. G. Nair Department of Horticulture, Pomology Group, Michigan Asia. State University, East Lansing, USA e-mail: [email protected] Keywords Malus Á Anthocyanin Á Genetic diversity Á Fruit Á Apple Á Crab Á MdMYB10 Á MYB10 R. Michelutti Á M. Luffman Agriculture and AgriFood Canada, Harrow, ON, Canada Abbreviations P. Forsline USDA United States Department of Agriculture USDA-ARS, Geneva, NY, USA CCG Canadian Clonal Genebank M. Ordidge MSU Michigan State University Brogdale National Fruit Collection, Kent, UK NFC National Fruit Collection 123 Euphytica Introduction levels of phenylalanine ammonia-lyase (PAL), a key enzyme in the anthocyanin biosynthetic pathway (Faragher 1983). Anthocyanins comprise a class of flavonoid pigment In the typical red apple fruit, anthocyanin accumu- that is widely used in nature to impart pigmentation to lation is limited to the skin (Steyn 2009), comprised of fruits, flowers, and foliage, and for photoprotection, only a few epidermal and hypodermal cell layers, especially under conditions of stress (Davies 2009; whereas the internal tissues generally lack anthocya- Hatier and Gould 2009; Steyn 2009). These com- nin and are non-pigmented. In these tissues, collec- pounds may also function in free-radical scavenging tively referred to as the fruit ‘flesh’, anthocyanin during normal growth and development (Hatier and production may be limited by levels of MdMYB10, a Gould 2009). Anthocyanins are widely distributed, presumed transcription factor that promotes synthesis occurring in all higher land plants with the exception of of multiple key enzymes involved in anthocyanin the Caryophyllales (Harborne 1996). Anthocyanins are biosynthesis (Espley et al. 2007; Lin-Wang et al. also chemically diverse, representing various stable 2010), because ectopic expression of the MdMYB10 glucosides of an array of anthocyanidins, and more gene in transgenic apple was sufficient to drive than 500 distinct natural anthocyanins have so far been anthocyanin production in several organs and tissues reported (reviewed by Andersen and Jordheim 2005). normally lacking anthocyanin (Espley et al. 2007). In Anthocyanins accumulate in the vacuole of the cell, addition, enhanced MdMYB10 RNA levels in cortical and depending on the specific anthocyanin and vacu- tissues were associated with the red pigmentation of olar pH, can impart red, blue, or purple pigmentation to the cortex in the variety ‘Redfield’ (also called ‘Red the cell and plant tissue. Like other flavonoids, Field’) (Espley et al. 2007). anthocyanins act as powerful antioxidants, at least in Besides ‘Redfield’, several additional known apple vitro, and have attracted a great deal of interest from the genotypes, both domestic and ornamental, exhibit red- public and research community for their potential to fleshed fruit (Espley et al. 2009; Mulabagal et al. 2007). positively impact human health. Although health- We previously showed that anthocyanin accumulation promotive effects of dietary flavonoids have yet to be in fruit of such genotypes can range to 1 mg/g fresh generally substantiated (Lotito and Frei 2006), their weight or more (Mulabagal et al. 2007). This germ- association with human health, especially related to plasm is an attractive starting point for development of their capacity for pigmentation, has led to consumer novel varieties for consumption and nutraceutical use increase in demand for anthocyanin-containing prod- through traditional breeding and biotechnology. The ucts, as well as efforts to identify those natural foods, genetic alteration resulting in ectopic expression of the especially fruits, that are richest in anthocyanins. MdMYB10 gene in ‘Redfield’ was also associated with The fruit of the domestic apple (Malus x domestica) red flesh in at least 15 additional genotypes (Espley is a ubiquitous and important source of nutrients, and et al. 2009). However, our preliminary observations is considered as one of the top functional foods—those revealed a large degree of variation in the degree and foods that have inherent health-promoting benefits pattern of pigmentation within the cortex among some beyond basic nutritional value. Much of this apparent of these genotypes (not shown), suggesting that this value is probably due to the obvious accumulations of allele is subject to extensive genetic modification. anthocyanins in the skin of most cultivated apple In this study, we identified and cataloged red- varieties. Several studies have characterized the fleshed apple genotypes from four major Malus biochemistry, physiology, and genetics of anthocyanin diversity collections collectively representing over production in apples (Reviewed in Telias et al. 2010). 3,000 known apple genotypes including M. x domes- The major anthocyanin in typical apple fruit skin is tica cultivars, wild species, and named hybrids. cyanidin-3-galactoside (Sando 1937). Anthocyanin production in apple fruit skin is associated with fruit maturity and ripening, and has been found to be Materials and methods stimulated by light (Arthur 1936; Siegelman and Hendricks 1952) and cool temperatures (Faragher The United States Department of Agriculture (USDA) 1983). This latter effect was associated with enhanced Malus Germplasm Collection is maintained at the 123 Euphytica USDA-Agricultural Research Service (ARS) Plant including *2,200 accessions, we utilized collection Genetic Resources Unit in Geneva, NY. Plants were records to target those accessions that had been pre- 5–10 years old and were grafted on BUD 9, Malling 7 viously noted or anecdotally known as exhibiting or EMLA 7 rootstocks. The National Fruit Collection internal pink or red pigmentation. In addition, we (NFC) in Brogdale, Kent, UK is owned by the UK examined each individual of the MSU collection, Department for Environment, Food and Rural Affairs which consists of 1,263 individuals representing 154 and maintained and curated by the University of known species and hybrids and *252 unknown Reading, Reading, UK. Trees were 18–35 years old genotypes. Many accessions in these collections did and grafted on Malling 9 rootstocks. The Canadian not produce fruit in one or both years of the study and Clonal Genebank (CCG) is managed by Agriculture thus were not considered. and AgriFood Canada and is located in Harrow, We classified accessions into two general groups. In Ontario, Canada. The Michigan State University the first group, which was composed of relatively (MSU) Malus collection is maintained on the main large-fruited ([4 cm diameter) accessions mostly campus of MSU, East Lansing, MI, USA. All of these classified as M. x domestica, pigmentation was collections were managed in accordance with com- typically subtle and highly localized to the subepider- mercial practice for insect or microbial pests. Only mis, the cortex near the calyx pole of the core, and/or healthy and vigorous trees were selected for evalua- the vasculature within the cortex (Fig. 1a, b). The most tion. Species, hybrid and cultivar nomenclature common pattern that we encountered was subepider- exactly followed collection assignments. Intensity of mal (subcutaneous), where pigmentation was stron- flesh color was designated visually from at least five gest closest