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 ( ) G. Berry J. Najdowski sumed natural form of M. sieversii native to central N. Alsmairat R. Beaudry M. G. Nair Department of Horticulture, Pomology Group, Michigan Asia. State University, East Lansing, USA e-mail: [email protected] Keywords Malus Anthocyanin Genetic diversity Fruit Apple Crab MdMYB10 MYB10 R. Michelutti 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

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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 to the skin, and with varying degrees of representative fruit from each accession, on a scale of diffusion into the cortex. This pattern was commonly 1 (faint pinkish tinge), 2 (moderate pink), 3 (pink-red), seen in M. x domestica accessions that showed 4 (moderate red), or 5 (dark red). MdMYB10 allelotype moderately to highly pigmented skin, especially where at the promoter mini/microsatellite region was deter- this was mottled, striped or blushed against a greenish mined by the PCR using oligonucleotide primers or yellowish skin ground color (not shown). In all (50-GGAGGGGAATGAAGAAGAGG-30 and 50-TC cases examined, the strongest subcutaneous pigmen- CACAGAAGCAAACACTGAC-30) that flank this tation within a fruit corresponded to regions of the region, as described by Espley et al. (2009). Source cortex underlying intensely colored skin (Fig. 1a, b tissue for DNA preparation was either bud or leaf. and not shown). Although this trait was seen consis- tently in individual fruit from any single accession, it was inconsistently seen between accessions of the same name among collections (Table 1), suggesting Results strong influence of environment or genetic differences between identically named accessions. The observed Evaluation and documentation of Malus localized pigmentation at the calyx pole of the core accessions exhibiting red fruit flesh was found in a subset of accessions showing intense subcutaneous pigmentation (Fig. 1a; Table 1). Pig- During the period of natural fruit ripening (mid- mentation within the vasculature was observed for a August through early November) in 2009 and/or 2010, small subset of red-skinned accessions, although we evaluated and documented both the internal pattern degree of pigmentation in this pattern was not and the intensity of pigmentation within fruit internal obviously related to intensity of skin color (Fig. 1b tissues in four major Malus live collections. For the and not shown). Relatively large-fruited accessions *2,289 accessions available at the USDA collection, showing the most striking degree of pigmentation in we examined each individual, focusing on a subset of these patterns are listed in Table 1. 120 accessions previously reported by ARS staff to The second general group of accessions showing exhibit pink or red pigmentation in the flesh (http:// internal pigmentation consisted of a diversity of acces- www.ars-grin.gov/cgi-bin/npgs/html/desc.pl?115045), sions classified as wild species forms, M. xdomestica as well as all accessions that exhibited markedly red- cultivars, interspecific hybrids, and other known or tinged or purplish foliage. For the NFC and CCG, each presumed hybrids (Table 2). This group was 123 Euphytica

Fig. 1 Internal patterns of pigmentation among apple acces- throughout the periphery of the cortex, while in M. hybrids sions. M. x domestica ‘Browns Apple’’ (a) represents those ‘Purple Prince’ (e) and ‘Shelley’ (f) it is mainly localized to the with strong subcutaneous and calyx-localized pigmentation. cortex outside the core or within the core, respectively. Many M. hybrid ‘Piotosh’ (b) shows strong subcutaneous and accessions including M. hybrid ‘Sissipuk’ (g) show strong vascular pigmentation. In M. x domestica ‘Pink Pearl’ (c) and pigmentation throughout all internal tissues. In each panel, the M. pumila ‘Niedzwetzkyana’ (d), pigmentation is seen diffusely scale bar is 1 cm distinguished from that described above in that the overall pattern and intensity of pigmentation for acces- pigmentation (1) was distributed more broadly within sions in this group is cataloged in Table 2. internal tissues, (2) was generally much more intense, and (3) was not limited to intensely red-skinned fruit. Allelotypes of MdMYB10 promoter among red- However, both the pattern of pigmentation with respect fleshed Malus genotypes to the internal tissues of the fruit, and the intensity of this pigmentation within those tissues, were highly variable Tandem repeat of a 23-bp element within the apple among accessions in this group (Fig. 1c–g; Table 2). MdMYB10 promoter region was found to be associated For example, accessions showed pigmentation that was with red flesh in several genotypes studied (Espley distributed diffusely throughout the periphery of the et al. 2009). In the cultivar ‘Redfield’, this repeated cortex (Fig. 1c, d), predominant in the cortex outside of promoter element directed ectopic expression of the core (Fig. 1e), limited mainly to the core (Fig. 1f), or MdMYB10 (Espley et al. 2009). To further assess the distributed throughout all internal tissues (Fig. 1g). The specificity of the association between the repeated 123 Euphytica

Table 1 Large-fruited accessions showing enhanced degree of Table 1 continued common internal pigmentation patterns Name Accessiona Pattern Name Accessiona Pattern Stoke Red NFC1989-110 SC Aldwick Beauty NFC1970-001 C/SC PI589697 SC Alton PI589138 N Violette NFC1945-002 SC NFC1966-035 SC Vista Bella PI588819 N Atlas MAL0923 SC/V NFC1971-048 C/SC NFC1950-127 SC Wealthy PI588788 SC Barry PI589428 SC NFC2000-098 N NFC1958-018 SC C calyx, SC subcutaneous, V vascular, N no pigmentation Beauty of Bath PI589664 N a Accession source is indicated by prefix: NFC-, NFC; PI- or NFC1966-146 SC GMAL-, USDA collection; MAL-, CCG; other accession Ben’s Red NFC1957-208 C/SC names, MSU collection Bloody Ploughman NFC1962-042 SC Brown’s Apple PI589662 N element and this trait in the breadth of apple MAL0939 C/SC genotypes, we determined MdMYB10 promoter allel- NFC1989-067 SC otype for 110 diverse, red- or non-red-fleshed acces- Burgundy PI588835 SC sions from the MSU collection, as well as most red- NFC1975-035 SC fleshed accessions identified in the USDA collection Crimson Beauty of Bath NFC1975-303 SC and CCG. This analysis employed PCR to amplify a Devon Crimson Queen NFC1953-081 SC promoter segment that includes the position of the Discovery PI589900 SC element, and distinguished the non-repeated element

NFC1973-189 SC form (R1) from the hexarepeat form found in ‘Red- Duchess’ Favourite PI126873 SC field’ (R6) based on product size (Espley et al. 2009). NFC2000-031 SC/V Analysis of most of these accessions resulted in either Edgar NFC1967-068 C/SC or both products, which we interpreted as homozygous

Feltham Beauty NFC1921-083 C/SC (R1/R1 or R6/R6) or heterozygous (R1/R6), respec- Folkestone NFC1964-069 SC tively. An exception was an accession of M. coronaria, Julyred PI588942 N a non-red-fruited species, which produced a product NFC1974-061 C/SC that was intermediate in length between the R1 and R6 Laxton’s Herald PI131463 N products. Direct sequencing revealed that this pro- NFC1938-007 SC moter contained only a single R element (R1), with the Marriage-Maker NFC1955-064 C/SC observed increase in size due to expansion of a Minjon NFC1951-060 SC polyadenine stretch at position *660 relative to the Morden 363 PI589234 SC MdMYB10 start codon (not shown). For several Pfirsichroter Summerapfel PI589595 N accessions, this PCR did not generate any product, NFC1945-071 SC even after repeated attempts on biological replicates Pioneer 20 MAL0933 SC (Table 2 and not shown), potentially due to sequence Piotash MAL0926 SC/V variation disrupting the oligonucleotide primer bind- Red Devil NFC1999-070 SC ing sites. For the red- or non-red-fleshed accessions analyzed Red Sauce PI589087 N from the MSU collection, pigmented flesh was asso- NFC1967-077 C/SC/V ciated with presence of the R allele, extending Rubens NFC1955-008 SC 6 previous observations (Espley et al. 2009) (Table S1). Scarlet Pippin MAL0348 SC/V Of the 82 non-red accessions, 76 contained only the R St. Lawrence MAL0263 SC 1 allele. The remaining six were either R /R or R NFC1950-305 N 1 6 6 (Table S1). We confirmed the presence of the R6 allele

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Table 2 Pigmentation pattern, intensity, and MdMYB10 allelotype of intensely red-fleshed apples t Classification Accession Pattern Intensitya MdMYB10 allelotype

1764 x domestica PI392302 T 3.5 (not named) x atrosanguinea CC2605*01 OC 2 R1/R6 (not named) 90P048*01 T 4 R1/R6 Adams Hybrid PI588885 OC 3.5 R1/R6 MAL0481 OC 3 R1/R6 90P046-47 OC 3 R1/R6 Alamata Hybrid PI588932 CO, OC 2.5 R1/R6 Aldenhamensis x purpurea PI589016 CO 1 MAL0733 CO, P 1.5 R1/R6 CC1352*04 CO, P 2 R1/R6 Almey Hybrid PI588824 CO 1 R1/R6 Amisk MAL0554 T 3.5 Arrow Hybrid PI148703 T 3 R1/R6 Athabasca Hybrid PI588899 CO 1 R6 CC2736*01 R1/R6 Babine Hybrid PI148490 CO 2 R1/R6 Barbara Ann Hybrid PI589314 T 4 R1/R6 CC3048*02 R1/R6 Baskatong NFC1980-098 T 4.5 MAL1150 R6 Bedford CC1877*01 R1/R6 Bellefleur Krasny x domestica NFC1975-342 P 2 Bellefleur Rekord x domestica PI259447 P 2 Budagovsky 54-118 x domestica PI589482 P 1 R6 Budagovsky 57-233 x domestica PI437039 P 1 R1/R6 Cameron PI589500 OC 4 R6 MAL0442 T 4 R6 Candied Apple MAL0482 OC 2.5 R1/R6 Caravel x domestica NFC1965-008 P 1.5 Cheal’s Weeping x domestica PI589354 T 2 R1/R6 Cowichan Hybrid PI589057 CO, P 2 R1/R6 NFC1976-167 T 2.5 MAL0919 P 2.5 R1/R6 Crab1 MAL0698 T 1.5 R6 Crab3 MAL0699 T 3 R6 Crab4 MAL0780 T 1 Crab6 MAL0701 T 3 R1/R6 Crab7 MAL0756 T 3 R6 Cranberry Hybrid PI589180 T 5 R6 Crimson Brilliant Hybrid PI588767 CC1879*01 R1/R6 Dainty Hybrid PI644149 R6 Edith Hybrid PI307503 T 4 R1/R6

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Table 2 continued t Classification Accession Pattern Intensitya MdMYB10 allelotype

Eleyi x purpurea PI589364 T 2.5 R1/R6 GMAL542 Erie CC1547*01 R6 Ferrill’s Crimson MAL0550 T 4 R1/R6 FORM 181 (35-01) Hybrid PI613969 P 2.5 R1/R6 Freeman Hybrid Hybrid PI589737 T 3 R1/R6 Garry Hybrid PI589510 2.5 MAL0446 P 1 Geneva x domestica PI589079 CO, OC 3 MAL0932 CO, OC 2.5 Geneva Crab MAL0563 CO, OC 2.5 Gloriosa Hybrid PI589273 CO, OC 2 R1/R6 GMAL 220 x adstringens PI588898 T 2.5 GMAL 2614 PI589858 OC 1.5 R1/R6 GMAL 2617 PI589861 CO 1 R1/R6 GMAL 33 x moerlandsii PI437056 T 4 Hedwigiae x domestica PI307501 T 1 R6 Henrietta Crosby Hybrid PI589251 T 4 R6 CC1929*01 T 3.5 R6 Henry F. Dupont Hybrid PI589732 OC 3 R1/R6 Hopa Hybrid PI588990 T 2.5 R1/R6 CC1611*01 R1/R6 Hoser Hybrid PI589506 T 3 R6 Indian Magic Hybrid PI588912 CO 1.5 R1/R6 Indian Summer Hybrid PI589733 OC 1.5 R1/R6 Irene Hybrid PI589862 T 3.5 R1/R6 Jay Darling Hybrid PI589245 OC 3.5 R1/R6 Kamsomolez x domestica PI589169 P 2 Kelsey Hybrid PI590173 CO, P 2 R1/R6 MAL0385 CO 2 Kingsmere Hybrid PI589388 CO 2.5 R1/R6 Kizil alma 826 x domestica PI30327 CO 0.5 Kobendza x purpurea PI588888 OC 2 R1/R6 Kobenza Hybrid PI589508 T 3 R1/R6 Kornicensis x purpurea PI588911 T 4 R1/R6 CC2741*01 T 2.5 R1/R6 KSC 28 MAL0963 CO 2 Kuldzhinka Krupnoplodnaya x domestica NFC1975-343 CO, P 2 Lemoine x purpurea PI78034 CC2744*01 R1/R6 Leslie MAL0553 T 3.5 R6 Liset Hybrid PI588864 T 2.5 R6 M. marjorensis ‘Formosa’ PI589411 CO, P 2 R1/R6 Magnus MAL0186 CO 1.5

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Table 2 continued t Classification Accession Pattern Intensitya MdMYB10 allelotype

Makamik Hybrid PI588876 T 3.5 Manito x domestica PI589155 CO 1 MAL0871 CO 1 Maypole Hybrid NFC1987-043 CO, OC 3.5 NA 40298 Hybrid PI589009 OC 1 Nipissing Hybrid PI148496 CO,P 1.5 R1/R6 MAL0933 P 2 Northern Lights x domestica PI590171 P 1.5 Oekonomierat Echtermeyer x domestica PI78170 OC 2 R1/R6 CC2556*01 P 2 R1/R6 Okanagan Hybrid PI148708 CO,P 2 Ottawa 7 Hybrid PI588882 T 2.5 R6 MAL0525 T 2.5 R6 Otterson Hybrid PI590178 CO,OC 4 R1/R6 Paradisiaca Atrosanguinea PI136488 T 3 R1/R6 Pink Beauty Hybrid PI589213 CO,P 2 R6 MAL0846 Pink Pearl x domestica PI588980 P 1 R1 MAL0977 P 2 R1 Pioneer Scarlet Hybrid PI589247 T,CO 3 R6 MAL0928 T 2.5 R6 PK.14 x domestica PI274840 CO,P 2 R1/R6 Prairie Fire Hybrid PI589820 T 2.5 R1/R6 Prince Charming x domestica PI588834 T 3.5 Profusion x moerlandsii PI589449 T 2 R1/R6 PI588887 T 4 R1/R6 MAL0735 OC 3.5 R6 MAL0455 CO, OC 2.5 R1/R6 CC3058*01 OC 2.5 R1/R6 Purple Prince 20030960*04 OC 4 R1/R6 Purple Wave Hybrid PI589499 T 5 R1/R6 NFC1980-104 T 4 Pyramidal Hybrid PI588763 T 2.5 R6 Radiant Hybrid MAL0509 T 1 PI588947 CO 1 R6 Red Barron 88N051*01 OC 3 R1/R6 Red Jacket Hybrid PI589250 CO, OC 2 R1/R6 Red Silver Hybrid PI589321 T 4 R6 Red Splendor Hybrid PI588822 T 3.5 R1/R6 MAL0450 OC 3 R1/R6 20060298*04 OC 2 R1/R6 Redfield x domestica PI589211 CO, P 1.5 R1/R6 Redflesh Hybrid PI589010 CO, P 2.5 R1/R6 Roberts Crab Hybrid PI437057 T 5 R6

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Table 2 continued t Classification Accession Pattern Intensitya MdMYB10 allelotype

Robinson Hybrid PI589455 T 3 R1/R6 Rose Bud x domestica PI589130 P 1.5 Rosedale Hybrid PI589252 CO, P 1.5 R1/R6 Rosseau MAL0587 T 3.5 R1/R6 Royal Raindrops 20080097*06 T 4.5 R1/R6 Royalty Hybrid CC3066*01 R6 PI588823 T 5 R6 Rudolph Hybrid PI589514 P 1 R1/R6 Scugog Hybrid MAL0591 CO, P 2 R1/R6 PI589628 CO, P 3.5 R1/R6 Selkirk Hybrid MAL0447 CO, OC 2.5 R1/R6 PI589460 T 3 R1/R6 Severnii Bujon x domestica PI107245 CO, P 1 R1/R6 Shelley Hybrid MAL0875 T 2 R1/R6 PI437048 T 2 R1/R6 Simcoe x adstringens PI148709 CO 1.5 R6 Simpson (10-35) Hybrid PI589244 OC 3 Sissipuk Hybrid MAL0588 T 3.5 R6 PI148500 T 3 R6 Slocan x domestica PI148710 CO, P 2 R1/R6 Sparkler Hybrid PI588809 T 4 R1/R6 Strathmore Hybrid PI588774 T 1.5 R6 MAL0876 CO, P 2 R6 Striped Beauty Hybrid PI588766 OC 2 Sundog Hybrid PI230685 P 3 R1/R6 MAL0766 T 1 R1/R6 Timiskaming Hybrid PI151644 T 4.5 R6 Tomiko MAL0589 T 4 Totem Hybrid NFC1996-072 T 4 Vanguard CC1564*01 CO 1 R6 Wabiskaw x domestica PI148503 CO, P 2.5 R1/R6 Wierdakii x purpurea PI588894 CO, P 1.5 R1/R6 Williams MAL0863 P 1.5 T throughout, CO core, OC outer cortex, P peripheral a 1 (faint pinkish tinge), 2 (moderate pink), 3 (pink-red), 4 (moderate red), or 5 (dark red)

and lack of internal pigmentation in these six acces- the R1 form, yet exhibited strong internal pigmentation sions in multiple individuals and in two successive (Fig. 1). years. For the remaining, R6-containing accessions in the Analysis of red-fleshed accessions identified from USDA collection and CCG, we noted that the intensity the USDA collection and CCG revealed that almost all of pigmentation was not associated with presumed that could be genotyped contained an R6 allele heterozygosity or homozygosity of the R6 allele 2 (Table 2). The single exception was ‘Pink Pearl’, (v P [ 0.05). For example, several R1/R6 accessions which showed only a single product corresponding to including the cultivar ‘Purple Wave’ produced 123 Euphytica

Table 3 Pigmentation pattern, intensity, and MdMYB10 allelotype of identified species forms Name Classification Accession Pattern Intensitya MdMYB10 allelotype

Niedzwetzkyana M. pumila PI589857 P 2 R1/R6 Niedzwetzkyana M. pumila PI589225 P 2 R1/R6 Niedzwetzkyana M. pumila PI589287 P 1.5 R1/R6 Form 35 (33-01) M. sieversii PI613967 P 1 R1/R6 Kaz 95 05-01P-22 M. sieversii PI633918 P 2 R6 USSR-89-35-01 M. sieversii PI629318 P 1 R1/R6 Gloriosa Mandshurica PI588754 T 2 R1/R6 (no name) Tschonoskii CC1895*01 T 4 R1/R6 P peripheral, T throughout a 1 (faint pinkish tinge), 2 (moderate pink), 3 (pink-red), 4 (moderate red), or 5 (dark red)

‘Exzellenz Thiel’ ‘Oekonomierat Echtermeyer’ x scheideckeri ‘Gloriosa’ ‘Wolf River’ ‘Bluebeard’ ‘Redfield’ sieboldii ‘Purple Prince’ ‘Liset’ x atrosanguinea ‘Profusion’ x purpurea ‘Lemoinei’ x purpurea ‘Kornicensis’ x purpurea ‘Kobendza’ x purpurea ‘Aldehamensis’ x purpurea ‘Eleyi’ ‘Selkirk’ ‘Tomiko’ ‘Pink Beauty’ ‘Sundog’ ‘Almey’ ‘Athabasca’ ‘Timiskaming’ ‘Jay Darling’ Ottawa 521 ‘Babine’ ‘Crimson Brilliant’ pumila ‘Niedzwetzkyana’ ‘Wabiscaw’ ‘Irene’ sieboldii ‘zumi’ ‘Athabasca’ ‘Purple Wave’ op ‘Totem’ ‘Nipissing’ Wijik McIntosh ‘Sissipuk’ ‘Maypole’ ‘Simcoe’ baccata ‘Baskatong’ ‘Meach’ ‘Kingsmere’ sieboldii ‘Makamik’ ‘Garry’ ‘Geneva’ ‘Amisk’ ‘Hopa’ ‘Radiant’ ‘Erie’ ‘Vanguard’ ‘Cowichan’ ‘Sparkler’ ‘Scugog’ op ‘Rosseau’ ‘Arrow’ op ‘Cameron’ ‘Katherine’ ‘Redflesh’ coronaria ‘Elk River’ ‘Cranberry’ ‘Dolgo’ ‘Henrietta Crosby’ x arnoldiana ‘Dorothea’ ‘Barbara Ann’ unnamed halliana ‘Parkmanii’ op x purpurea ‘Eleyi’ ‘Henry DuPont’ unknown ‘Cluster’ ‘Surprise’ ‘Royalty’ ‘Bedford’ ‘Pink Pearl’ ‘Dainty’ op op op

Fig. 2 Documented ancestry of red-fleshed apples. Individual documentation is referenced in Table S3

intensely colored fruit, whereas several R6/R6 culti- representatives of M. sieversii (syn. M. pumila) col- vars including ‘Radiant’ produced fruit with only a lected from Kazakhstan, and three accessions classi- slightly pink tinge (Tables 2 and S2). Together with fied as M. pumila Niedzwetzkyana, a presumed natural the identification of the several non-pigmented, form thought to be originally sourced from central

R6-containing accessions (above), this suggests that Asia (Reviewed in Fiala Fr 1994). Two red-fleshed the activity of the R6 allele of MdMYB10 is extensively species accessions, classified as M. mandshurica and modified by genotype. M. tschonoskii, were presumed heterozygous (R1/R6). All of the red-fleshed accessions classified as wild These accessions might not be typical of the species, species that were analyzed also contained at least one as two additional accessions of M. mandshurica

R6 allele (Table 3). These accessions included three and three additional accessions of M. tschonoskii 123 Euphytica maintained at USDA collection and originally sourced We identified many, generally unrecognized geno- from wild material neither exhibited red-fleshed fruit types that, in contrast, exhibited internal pigmentation nor contained an R6 allele (not shown). that was typically more intense and broadly distributed Parentage of many of the red-fleshed cultivars within the flesh. These include ‘Redfield’, previously identified in this study has been previously docu- characterized as exhibiting ectopic expression of the mented (Fiala Fr 1994; Jefferson 1970; USDA ARS MdMYB10 gene, resulting from the R6 promoter National Genetic Resources Program 2011). We element (Espley et al. 2009). We identified the R6 integrated this information to reconstruct pedigrees element within nearly all red-fleshed accessions (Fig. 2; Table S3). This revealed that nearly all tested, strongly suggesting that this trait in these varieties could be traced back to ‘Niedzwetzkyana’. accessions is dependent on R6. However, it is clear that The exceptions were ‘Bedford’, ‘Royalty’, ‘Dainty’, the presence of the R6 allele alone was insufficient to and the aforementioned ‘Pink Pearl’. ‘Bedford’ was drive development of pigmented fruit flesh under our introduced by the Canadian Department of Agricul- observation conditions, as we identified numerous ture in Brandon, Manitoba where several other accessions that contained the R6 element yet did not ‘Niedzwetzkyana’-derived cultivars were introduced develop significant pigmentation. This could be due to and maintained (Jefferson 1970) and thus is possibly a loss of function mutation within MdMYB10 itself, hybrid with ‘Niedzwetzkyana’ (Table S3). Anecdotal could be effected upstream of MdMYB10 through the information provided by Jefferson (1970) suggest that activity of other factors required for promoting ‘Royalty’ and ‘Dainty’ are also derived from ‘Nied- MdMYB10 expression, or downstream, by the avail- zwetzkyana’. Thus, with the exception of ‘Pink Pearl’, ability of MdMYB10 protein partners or anthocyanin both internal pigmentation and presence of the R6 biosynthetic substrates. These and other similar vari- allele in this subset of accessions can be simply eties may define genetic mutations in the anthocyanin explained by derivation from a common ancestor, biosynthetic pathway that could lead to further insight ‘Niedzwetzkyana’. into its activity or regulation. In addition, many

R6-containing accessions produced fruit exhibiting various internal coloration patterns and intensities, and

Discussion this was not obviously related to whether R6 was apparently heterozygous or homozygous. Collec- In this study we undertook a concerted effort to identify tively, these observations suggest that the effect of and characterize Malus species, forms and cultivars R6 is extensively modified in a genotype-specific that exhibit red-fleshed fruit. This effort was greatly manner. There are numerous potential mechanisms facilitated by both the maintenance of large numbers of that might drive differences in coloration patterns and

Malus accessions in nationally-funded germplasm intensities among R6-containing accessions. For collections, which intend to exemplify genetic diver- example, at the molecular level, the ability of sity, and by the widespread cultivation of a subset of MdMYB10 itself to act as a transcriptional regulator those genotypes with superior ornamental value. could be influenced both by its peptide sequence and Our work revealed several aspects of such geno- on the number, arrangement and sequence of respec- types that should be of interest for breeding. First, tive cis-acting elements in genes that it directly targets. many domestic cultivars with highly pigmented skin Biochemically, anthocyanin levels are expected to be exhibit internal pigmentation, but this was typically influenced by availability of any of numerous inter- subtle and most often limited to the cell layers mediate compounds as well as intracellular (vacuolar) subtending the epidermis. Such an effect could be pH. At the developmental level, apparent pigmenta- trivially explained by intercellular diffusion or traf- tion could be influenced by the number of anthocy- ficking of anthocyanin biosynthetic intermediates, anin-producing cells/cell layers. Explanations also enzymes, or regulatory factors such as MdMYB10 include variation in sensitivity/response to influences that may normally be constrained to the skin. A few that normally promote anthocyanin levels in apple, cultivars also exhibited pigmentation within the vas- including light and cool temperatures. Such modifiers culature, a feature that may also result from ectopic may be uncovered through genetic experiments expression of such factors. utilizing selected genotypes identified in this study. 123 Euphytica

We also identified an apparent R6 allele in acces- and M. sieboldii) and interspecific hybrids [M. x sions classified as species forms of M. mandshurica scheideckeri (=M. floribunda 9 M. prunifolia), and M. tschnoskii. The identification of R6 in forms of M. atrosanguinea (=M. halliana 9 M. toringo), and several Malus species led Espley et al. (2009)to M. x arnoldiana (=M. baccata 9 M. floribunda)]. conclude that the R6 allele was evolutionarily ancient. This genetic diversity should be exploited to create Given our finding that other, well documented, informed crosses generating new cultivars with supe- representatives of M. mandshurica and M. tschonoskii rior qualities for industrial nutraceutical production did not contain this allele, however, it is probably more from fruit, including disease resistance, yield, ease of likely that these R6-containing accessions are recent mechanical harvesting, and degree of anthocyanin hybrids and simply misclassified. Accurate identifica- accumulation. tion and propagation of apple is challenging for many reasons, including chimaeric composition of many Acknowledgments We thank Jeff Wilson (MSU Campus plants (often with rootstock, interstock and scion of Planning and Administration) and Dr. Frank Telewski (MSU Department of Plant Biology and MSU Campus Planning and distinct genotypes), open pollination, tendency of Administration) for assistance with locating Malus accessions nurseries to propagate plants during dormancy when within the MSU campus. We acknowledge the assistance of few phenotypic markers are apparent, tolerance of Tyler Harris, MSU Multicultural Apprentice Program, with fruit apple to polyploidy, possibility of periclinal chimae- collection and processing and Mary Pennell (NFC Horticultural Curator). This work was supported by the Michigan Apple rism, and accumulation of somatic mutation during Commission, the Michigan Department of Agriculture (Grant repeated vegetative propagation. No. 791N0200091), and the Education Foundation of the An exceptional accession identified in this study is American Society of Plant Biologists. ‘Pink Pearl’, which showed only a single product corresponding to the MdMYB10 R1 allele, yet was References strongly colored internally. We confirmed this pre- sumed allelotype and pigmentation in independent Andersen OM, Jordheim M (2005) The anthocyanins. In: accessions of this variety from both the USDA Andersen OM, Markham KR (eds) Flavonoids: chemistry collection and CCG. In addition, the absence of the biochemistry and applications. CRC Press, Boca Raton, MdMYB10 R allele in ‘Pink Pearl’ was recently noted pp 471–552 6 Arthur JM (1936) Radiation and anthocyanin pigments. In: by Sekido et al. (2010), who additionally determined Duggar BM (ed) Biological Effects of Radiation Vol I. that red flesh in this variety resulted from a dominant McGraw-Hill, New York, pp 471–552 allele linked to the self-incompatibility S-locus. ‘Pink Davies KM (2009) Modifying anthocyanin production in flow- Pearl’ was selected prior to 1944 as an open-pollinated ers. In: Gould K, Davies K, Winefield C (eds) Anthocya- nins: biosynthesis functions and applications. Springer, seedling of ‘Surprise’ (USDA ARS National Genetic New York, pp 471–552 Resources Program 2011). The accession of ‘Surprise’ Espley RV, Hellens RP, Putterill J et al (2007) Red colouration evaluated in this study (USDA PI 134672) did not in apple fruit is due to the activity of the MYB transcription show pigmented flesh, even under environmental factor, MdMYB10. Plant J 49:414–427 Espley RV, Brendolise C, Chagne´ D et al (2009) Multiple conditions sufficient for strong internal color devel- repeats of a promoter segment causes transcription factor opment in Pink Pearl. Further genetic and molecular autoregulation in red apples. Plant Cell 21:168–183 characterization of ‘Pink Pearl’ should define a novel, Faragher JD (1983) Temperature regulation of anthocyanin gain of function modification of the anthocyanin accumulation in apple skin. J Exp Bot 34:1291–1298 Fiala Fr JL (1994) Flowering crabapples: the genus Malus. biosynthetic pathway that could be used in breeding as Timber Press, Portland an alternative to, or enhancer of, MdMYB10 R6. Harborne JB (1996) The evolution of flavonoid pigments in Many of the most popularly planted ornamental plants. In: Swain T (ed) Comparative photochemistry. crabapples are derived from open-pollinated progeny Academic Press, London, pp 271–295 Hatier J-HB, Gould KS (2009) Anthocyanin function in vege- of ‘Niedzwetzkyana’, or from ‘Niedzwetzkya- tative organs. In: Gould K, Davies K, Winefield C (eds) na’ 9 M. baccata (Fig. 2). These varieties are noto- Anthocyanins: biosynthesis functions and applications. riously susceptible to diseases including apple scab Springer, New York, pp 1–20 and fire blight (Jefferson 1970). However, included in Jefferson RM (1970) History, progeny and locations of crab- apples of documented authentic origin. National Arbore- the parentage of other red-fleshed varieties are a tum contribution, No. 2. Agricultural Research Service, US diverse range of species (M. coronaria, M. halliana, Dept of Agriculture, Washington DC 123 Euphytica

Lin-Wang K, Bolitho K, Grafton K et al (2010) An R2R3 MYB Siegelman HW, Hendricks SB (1952) Photocontrol of antho- transcription factor associated with regulation of the cyanin synthesis in apple skin. Plant Physiol 33:185–190 anthocyanin biosynthetic pathway in Rosaceae. BMC Plant Steyn WJ (2009) Prevalence and functions of anthocyanins in Biol 10:50 fruits. In: Gould K, Davies K, Winefield C (eds) Antho- Lotito SB, Frei B (2006) Consumption of flavonoid-rich foods cyanins: biosynthesis functions and applications. Springer, and increased plasma antioxidant capacity in humans: New York, pp 85–105 Cause, consequence or epiphenomenon? Res Radical Biol Telias A, Bradeen JM, Luby JJ et al (2010) Regulation of Med 41:1727–1746 anthocyanin accumulation in apple peel. In: Janick J (ed) Mulabagal V, van Nocker S, Dewitt D, Nair M (2007) Cultivars Horticultural Reviews, vol 38. John Wiley & Sons Inc, of apple fruits that are not marketed with potential for Hoboken, pp 357–391 anthocyanin production. J Agric Food Chem 55:8165– USDA, ARS, National Genetic Resources Program (2011) 8169 Germplasm Resources Information Network (GRIN), Sando CE (1937) Coloring matters of Grimes Golden, Jonathan, (Online Database). National Germplasm Resources Labo- Stayman and Winesap apples. J Biol Chem 117:45–56 ratory, Beltsville. Available: http://www.ars-grin.gov/ Sekido K, Hayashi Y, Yamada K et al (2010) Efficient breeding cgi-bin/npgs/html/crop.pl?115 (17 February 2011) system for red-fleshed apple based on linkage with S3- RNase allele in ‘Pink Pearl’. HortSci 45:534–537

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