Flavonoids play essential roles in human health. Apple (Malus domestica Borkh.), one of the most widely produced and economically important fruit crops in temperate regions, is a significant source of flavonoids in the human diet and is among the top nutritionally rated and most widely consumed fruits worldwide. Epidemiological studies have shown that the consumption of apples, which are rich in a variety of free and easily absorbable flavonoids, is associated with a decreased risk of various diseases. However, apple production is challenged by serious inbreeding problems. The narrowing of the hereditary base has resulted in apples with poor nutritional quality and low flavonoid contents. Recently, there have been advances in our understanding of the roles that Malus sieversii (Ledeb.) M.Roem has played in the process of apple domestication and breeding. In this study, we review the origin of cultivated apples and red-fleshed apples, and discuss the genetic diversity and construction of the core collections of M. sieversii. We also discuss current research progress and breeding programs on red-skinned and red-fleshed apples and summarize the exploitation and utilization of M. sieversii in the breeding of high-flavonoid, and red-fleshed apples. This study highlights a valuable pattern of horticultural crop breeding using wild germplasm resources. The future challenges and directions of research on the molecular mechanisms of flavonoid accumulation and high-flavonoid apple breeding are discussed.
Flavonoids are a major class of polyphenolic compounds produced by secondary metabolic pathways in plants1. They have been extensively studied for their essential roles in human health1,2. Flavonoids from various fruits and vegetables play a key role in reducing disease risk3,4. Apple (Malus domestica Borkh.), one of the most widely produced and economically important fruit crops in temperate regions5, is a significant source of flavonoids in the human diet and is among the top nutritionally rated and most widely consumed fruits worldwide6. There is a traditional American saying, “an apple a day keeps the doctor away.” In the US, 22% of the phenolics in the human diet originate from apples, which makes apples the largest dietary source of phenols7. In Spain, the median and mean total flavonoid intake were reported to be 313.26 and 269.17 mg/day, respectively, and apples are the largest source of dietary flavonoids, accounting for 23% of the total flavonoid intake8. In Finland, apples and onions are the primary sources of dietary flavonoids9.
The free flavonoid content is higher in apples than in other fruits, which results in the availability of more flavonoids for eventual absorption into the human bloodstream10. Apple flavonoid intake has been reported to have positive effects on aging and cognitive decline, cardiovascular health, weight management, asthma, and gastrointestinal health11,12. In addition, numerous epidemiological studies have found that apple consumption is widely associated with a decreased risk of various diseases13,14. However, the genetic diversity and the nutritional quality of modern apple cultivars have decreased during the process of domestication. Therefore, apple breeding with the target of enriching common apple cultivars with beneficial metabolites is a goal of both horticultural research and practice.
The exploration of the origin and evolution of apple and a greater understanding of the mechanisms of development of red-skinned and red-fleshed apples will enable researchers and breeders to cultivate superior and new varieties using different breeding objectives. In this review, we outline the historical domestication process of cultivated apples and red-fleshed apples, discuss recent research on the molecular mechanism of flavonoid synthesis and accumulation in red-skinned and red-fleshed apples, and summarize breeding goals and research progress on red-skinned and red-fleshed apples. We also discuss the exploitation and utilization of Malus sieversii (Ledeb.) M.Roem in the context of breeding for high-flavonoid and red-fleshed apple varieties. This information is significant to introduce wider diversity into apple breeding programs and highlights a valuable pattern of horticultural crop breeding.
Malus sieversii: origin of cultivated apples and red-fleshed apples
Origin of cultivated apples and red-fleshed apples
Apple is the primary fruit grown in temperate regions around the world5. To effectively utilize wild apple resources to breed apple hybrids, it is important to understand the origins of cultivated and red-fleshed apples, the relationship between cultivated apple and its primary wild resources, and the manner in which the key characters of apples have been domesticated.
Many researchers have focused on the origin and evolution of the cultivated apple. As early as 1930, the geneticist Vavilov considered that “the center of diversity is the center of origin” and speculated that the wild apple and its related species in Turkestan (Kazakstan, Kyrgyzstan, Uzbekistan, Turkmenistan, and Tajikistan) were the ancestors of the cultivated apple15. Later, Forsline et al. collected many wild germplasm resources in these regions, and their analyses appeared to confirm the similarity between wild and cultivated apples16,17. Concomitantly in 1996, Janick et al. suggested that “Central Asia” was the area of greatest diversity and the center of origin of the domesticated apple18. Genetic analyses based on random amplified polymorphic DNA (RAPD) data showed that M. sieversii from the Xinjiang Autonomous Region of China was the species most closely related to the cultivated apple M. domestica cv. “Golden Delicious”19. In addition, Rehder suggested that Malus sylvestris Miller could be one of the ancestors of the cultivated apple20. Malus orientalis Uglitz was proposed to be another ancestor of cultivated apple, but M. sieversii was considered to be the most important ancestor21,22.
Until 2010, when the entire apple genome was first sequenced, new advances were developed in the origin and evolution of apples. Comparative analyses of genetic data showed that M. domestica cultivars were more closely related to accessions of the wild species M. sieversii and less closely related to accessions of M. sylvestris, Malus baccata, Malus micromalus, and Malus prunifolia23. This research confirmed that M. sieversii and not M. sylvestris was the ancestor of the cultivated apple. It was also confirmed that M. orientalis and Malus asiatica showed genetic similarity to M. sieversii. Subsequently in 2017, the genome re-sequencing of apple further clarified the evolutionary process of the cultivated apple. A population genetic structure analysis showed that both M. domestica cultivars and M. sylvestris originated from M. sieversii. M. sieversii in Xinjiang was found to be the most primitive and has retained high homology, while M. sieversii in Kazakhstan was found to have a high degree of genetic heterozygosity. M. sieversii migrated westward along the ancient Silk Road and gradually evolved into the cultivated common apple with the hybrid infiltration of M. sylvestris and M. orientalis. The crossing and domestication of M. sieversii and M. baccata along the Silk Road to the East resulted in early Chinese apples (Fig. 1)5.
Aside from cultivated apples, there are a number of high-anthocyanin and red-fleshed apple germplasm resources, and their origin and evolution have rarely been studied. Red-fleshed apples include M. sieversii f. niedzwetzkyana and the cultivated species M. domestica var. niedzwetzkyana24. Analyses of chloroplast and nuclear data showed that M. domestica var. niedzwetzkyana could have originated from the wild apple forests of Central Asia25. Steven, in 2012, identified and classified 3000 red-fleshed apple germplasm resources, including cultivars, wild species, and hybrids. It was inferred that all these red-fleshed apples originated from M. sieversii f. niedzwetzkyana26.
Some studies have shown that there is extremely rich genetic diversity related to polyphenol content, mineral elements, sugar and acid components, and volatile components in M. sieversii in Xinjiang27,28. The total phenolic and flavonoid contents were found to be significantly higher in M. sieversii than in the “Starking” apple variety28. Thus, M. sieversii can be used to breed functional apples with red flesh and high flavonoid contents. High-flavonoid apples will provide more flavonoids to consumers and will benefit human health.
Genetic diversity of Malus sieversii and the construction of core collections
Studies on population genetic structure can provide the basis for in situ conservation and the utilization of germplasm resources29. The genetic structure refers to the non-random distribution of genetic variation in a species or population and indicates the distribution patterns of genetic variation within and between populations30. To elucidate the process, pattern, and mechanism of evolution, it is necessary to first discuss the genetic variation, genetic structure, and their variations within a population, as well as the factors that affect population genetic structure31,32.
An isozyme molecular variation analysis was conducted for 259 M. sieversii seedlings in Central Asia. The results showed that 85% of the isozyme variation was caused by differences among wild individual plants within the population, and the other 15% of variation was explained by differences among the sampling regions. There was no isozyme variation among populations in the same region22. Volk et al. analyzed the genetic diversity of 591 individual M. sieversii plants and found that most of the genetic differentiation was caused by differences among individual plants within the population33. In China, the Xinjiang wild fruit forest located east of the Tianshan Mountains is a rare “oceanic” broad-leaved forest type in the desert region. It is a remnant community of broad-leaved forest in this tertiary warm temperate zone, and it is distinct from the wild fruit forests in the west Tianshan Mountains of Central Asia34. M. sieversii in Xinjiang is a major component of the wild fruit forest in the Tianshan Mountains, and its genetic diversity is extremely rich27,35. Analyses of the peroxidase isozyme spectrum of M. sieversii in Xinjiang showed that there were clear differences among different M. sieversii apple populations but not within the same population36. When four M. sieversii populations in the Yili and Tacheng areas of Xinjiang were analyzed using simple sequence repeat and sequence-related amplified polymorphic markers, most of the genetic differentiation was within the population, and the genetic distance among the populations was significantly correlated with the geographic distance. The Gongliu population was the most genetically diverse of the populations tested and therefore had priority for protection37,38.
Although M. sieversii populations in Xinjiang have been severely damaged, they are still distributed across 1800 h m2, and there are nearly 1 million copies of the genetic resource34. The huge quantity of germplasm resources makes it very difficult to carry out ex-situ or in vitro conservation. In 1984, the concept of a core collection was proposed39. The construction of a core collection provides opportunities for the in-depth evaluation of the germplasm resources and the effective protection and utilization of those resources40. Marker-assisted sampling methods based on RAPD data were proposed41. Marshall and Brown suggested that the most important index to evaluate genetic diversity was the number of alleles42. Volk et al. surveyed and collected M. sieversii from Kazakhstan. Thirty-five of the 124 lines obtained were selected to construct the core collection33. Zhang et al. proposed a method to construct the core collection of M. sieversii in Xinjiang based on molecular markers43. Liu et al. suggested that the core collection of M. sieversii should be constructed based on the results of stepwise clustering, Mahalanobis distance, and single linkage analyses combined with preferred sampling (20% sampling proportion)44. The construction of the core collection of M. sieversii laid the foundation for its ex-situ conservation. To establish a multi-level conservation system for M. sieversii germplasm resources, research should focus on its ex-situ conservation, including the protection of its original habitat, and its preservation in vitro, either as plantlets or organs. These measures will be of great significance for the scientific protection and sustainable and efficient utilization of M. sieversii germplasm resources in apple breeding.
Mechanisms regulating flavonoids in red-skinned and red-fleshed apples
The complexity of the regulation mechanism of flavonoid biosynthesis is the embodiment of the diversity of apple germplasm resources. Therefore, in addition to understanding the origin and evolution of apple germplasm resources, it is highly significant to explore the metabolic mechanism of flavonoid production in apple. The pathways of flavonoid synthesis have been elucidated in many plant species45,46,47,48. Phenylalanine is a direct precursor for the synthesis of anthocyanins and other flavonoids, and it is converted by phenylalanine ammonia lyase (PAL) into 4-coumaroyl-CoA and malonyl-CoA46,49. The next key reaction is the conversion from 4-coumaroyl-CoA into dihydroflavonol, which can be converted into anthocyanins, flavonols and other flavonoids via the activities of different enzymes (Fig. 2).
Flavonoid biosynthesis in red-skinned apple
The coloring of red-skinned apples is primarily determined by anthocyanin47. Anthocyanin biosynthesis involves the coordinated expression of structural genes and transcription factors47, and is influenced by other internal factors (endogenous hormones) and external factors (external environment, light, and temperature)50. Transcription factors determine the temporal and spatial patterns of gene expression and the expression level of structural genes, thus regulating the intensity and pattern of coloration in red-skinned apples. Members of three transcription factor families (MYB, bHLH, and WD40) function together in a ternary MYB–bHLH–WD40 (MBW) protein complex to participate in anthocyanidin pathways, a role that is widely conserved among plant species51,52,53. In the red-skinned apple, MdMYB1 and MdMYBA, which regulate the biosynthesis of anthocyanin in fruit skin, were the first transcription factors to be isolated and characterized (Fig. 2)47,54. Since then, research on anthocyanidin synthesis in apple has progressed considerably. Ubiquitin ligase MdCOP1 was suggested to interact with MdMYB1 to regulate light-induced anthocyanin synthesis and apple skin coloration55. Subsequently, MdMYB3 was also reported to promote anthocyanin accumulation in the apple skin56. MdbHLH3 can bind to the promoters of MdDFR and MdUFGT and promote anthocyanin synthesis57. However, the transcription factor MdTTG1 in the WD40 class does not bind to the promoters of MdDFR and MdUFGT or interact with MdMYB1. The regulation of anthocyanin by MdTTG1 may be achieved by the interaction with MdbHLH3 and MdbHLH3358.
In addition to gene regulation, anthocyanin synthesis in red-skinned apples is affected by light, temperature, and endogenous hormones. The expression of MdMYBA in the apple skin was induced by low temperature, and MdMYBA specifically combined with the promoter of MdANS to promote anthocyanin synthesis54. Low temperature induced the phosphorylation of MdbHLH3 and enhanced its transcriptional activation activity on the promoter of the anthocyanin structural genes, resulting in the large-scale accumulation of anthocyanin57. However, high temperature was found to change the activity of the BMW complex and reduce anthocyanin accumulation, leading to lighter-colored apple skin59. MdSnRK1.1 can interact with MdJAZ18 to regulate the biosynthesis of anthocyanin induced by sucrose60. Jasmonic acid can increase the binding of MdMYB9 and MdMYB11 to the promoter of downstream structural genes in the anthocyanin metabolic pathway and promote anthocyanin accumulation61. In addition, studies have shown that plant nutrients also regulated the synthesis of anthocyanin62. For example, the redness of the apple skin can be influenced by the foliar application of CaCl2 and/or the nitrogen status63,64.
In conclusion, various transcription factors regulate anthocyanin synthesis via different mechanisms in red-skinned apple, and anthocyanin synthesis is also affected by the stage of growth and development and by environmental factors. Consumers prefer the appearance of red-skinned apples, and red apples have a higher commodity value65. However, apples are often consumed without the skin, and thus, the healthy flavonoids and anthocyanins are lost66. In addition, serious inbreeding problems have narrowed the hereditary base of cultivated apple varieties, resulting in fruit with poor nutritional quality and low flavonoid contents67,68. Tsao et al. analyzed eight apple cultivars and found that the flavonoid contents were five times higher in the peels than in the flesh69. Similarly, McGhie et al. found that, on average, 46% of the polyphenolics in whole apples were located in the skin, and the skin contained essentially all of the flavonols and anthocyanin70. However, consumers eat much smaller amounts of apple peels than flesh. Thus, the breeding of red-fleshed apples with high flavonoid content is a target of apple breeders worldwide. Although a few red-fleshed apple varieties have been bred, no high-quality commoditized red-fleshed apple varieties are available on the market at present. To produce new red-fleshed apple lines, wild red-fleshed apples and/or other apple resources are being used to carry out distant hybridization breeding. The cultivation of functional red-fleshed apple varieties is of great significance to expand the genetic basis of cultivated apple varieties and to increase the human intake of flavonoids to maintain health.
Flavonoid biosynthesis in red-fleshed apple
The regulation of flavonoid synthesis in red-fleshed apples is more complex than that in red-skinned apples. Red-fleshed apples can be divided into Type 1 (red coloration in the fruit flesh, skin, leaves, and other vegetative tissues) and Type 2 (red coloration only in the fruit flesh). MdMYB10, an allele of MdMYB1/MYBA responsible for only the apple skin color, determines the red pigmentation of the Type 1 red-fleshed apple71. The Type 1 red-fleshed apple has a minisatellite-like structure comprising six tandem repeats in the promoter of MdMYB10 (R6:MdMYB10), while white-fleshed apple has only one (R1:MdMYB10). The R6 repeat sequences are self-binding sites of the MdMYB10 protein and are positively correlated with the self-activating activity of its promoter (Fig. 2)72. The overexpression of MdMYB10 in transgenic “Royal Gala” conferred a red-fleshed phenotype73. Although MdMYB10 is highly expressed in Type 1 red-fleshed apples, it is not expressed in Type 2 red-fleshed apples. Instead, another MYB transcription factor close to MdMYB10, designated MdMYB110a, is correlated with the red pigmentation in Type 2 red-fleshed apples. MdMYB110a is not expressed in Type 1 red-fleshed apples74. These findings were confirmed in a study of the Type 2 red-fleshed apple “JPP35”75. Type 2 red-fleshed apples show more variation in the coverage and intensity of the red coloration of the cortex, indicating a more complex genetic control mechanism than that found in Type 1 red-fleshed apples76.
In addition to MdMYB10, other studies have also identified some positive and negative regulatory factors in the red-fleshed apple. For example, silencing ANS in a red apple cultivar almost completely blocked the biosynthesis of anthocyanin in the transgenic plants77. Wang et al. characterized a PA1-type MYB transcription factor, MdMYBPA1, from the red-fleshed apple and found that MdMYBPA1 responded to low temperature by redirecting the flavonoid biosynthetic pathway from proanthocyanidin to anthocyanin production78. In addition, a transcriptomic analysis of the red-fleshed apples reveals the role of MdWRKY11 in flavonoid and anthocyanin biosynthesis79. Cytokinin was suggested to promote the accumulation of anthocyanin by inhibiting the expression of MdMYB308 in red-fleshed apples80. MdARF3 was induced by auxin to inhibit anthocyanin synthesis in red-fleshed apple calluses81. Xu et al. found that an anthocyanin negative regulatory gene, MdMYB16, may interact with MdbHLH33 to control red pigmentation82. Another gene, MdHB1, has also been shown to negatively regulate anthocyanin synthesis. Its overexpression reduced the flesh content of anthocyanin in the red-fleshed apple “Ballerina”83. In addition to anthocyanin synthesis, there are few studies on the synthesis of proanthocyanidins and flavonols in red-fleshed apples. Recently, two novel R2R3-MYB transcription factors related to flavonoid synthesis, designated MYB12 and MYB22, were cloned and identified from a red-fleshed apple. These transcription factors were demonstrated to play important roles in regulating the synthesis of proanthocyanidin and flavonols in red-fleshed apples (Fig. 2)84.
Breeding of red-skinned and red-fleshed apples
Breeding of red-skinned apples
With the increasing demand for higher quality apples, variety improvement has become an important goal for breeders85. Apple quality includes appearance, flavor, texture, storage and transportation properties, fresh food characteristics, suitability for processing, and genetic make-up85. In recent years, due to the rapid development of molecular biology technologies, there has been much progress in research on the mechanism of apple quality formation. To breed apples with improved flavor, Brown et al. and Visser et al. studied the classification standard of apple sugar and acid content, and the relationship between sugar and acid content and fruit flavor86,87. A recent study on the effect of SWEET genes on sugar accumulation in apple fruit provided the basis for the genetic improvement of flavor quality88. For texture quality breeding, researchers have targeted alleles related to ethylene, which plays an important role in climacteric fruits, such as tomato, apple, peach, and banana89. Ethylene production was found to be lower in the MdACS1-2/-2 homozygous variety than in the MdACS1-1/-2 heterozygous and MdACS1-1/-1 homozygous varieties90. To improve the processing qualities of apple, Huang et al. phenotyped hybrids derived from five biparental crosses of M. asiatica and M. domestica and demonstrated that relatively high acidity is an important breeding objective for fresh juice-specific apple cultivars91.
The red color of the skin is an important appearance trait of apple and largely determines its market value. Consumers always associate red skin with good taste, ripeness, and flavor; therefore, red-skinned apple varieties are preferred65. To breed red-skinned apples, identifying the genetic characteristics and regulation mechanism of red-skinned apples is the core issue. In early studies on the genetics and breeding of red-skinned apples, most breeders believed that the red color of the apple skin was a quality trait controlled by a dominant single gene. At the beginning of the last century, Crane et al. proposed for the first time that the synthesis of anthocyanin in the apple skin was controlled by the dominant single gene Rf, which was subsequently confirmed in other studies92,93. With the progress of biotechnology, the development of molecular markers has provided a theoretical basis for red-skinned apple breeding. Studies using molecular markers showed that yellow-skinned varieties were homozygous recessive, while red-skinned varieties were heterozygous or homozygous dominant94,95. Takos et al. obtained a MYB gene and a derived cleaved amplified polymorphic sequence marker in a study on the progenies of “Lady Williams” (red-skinned) and “Golden Delicious” (yellow-skinned)47. They found that MdMYB1-1 linked fragments could be amplified from most of the red cultivars, but none of the non-red cultivars.
In later studies, an increasing number of breeders suggested that the red-skin trait was controlled by multiple genes96,97. Lespinasse et al. analyzed the hybrid combinations of “Richared” and “Reinette di Landsberg” and found that the yellow-skinned trait was dominant to the red-skinned trait and was controlled by two dominant complementary genes. When there were two dominant genes, the fruit was yellow; otherwise, the fruit was red96. Other researchers suggested that the red-skinned trait was a quantitative trait controlled by many genes24,98. Sheng et al. proposed that the red-skinned trait was not only controlled by a dominant gene but also by the growth environment99. Ju et al. suggested that the red-skinned trait was controlled by a transcription factor and a structural gene, which participated in anthocyanin synthesis, and that environmental conditions affected skin color by regulating these genes100. Therefore, studies on the regulation of anthocyanin synthesis are highly significant to improve the appearance, quality, and commodity value of red-skinned apples.
It cannot be ignored that red sports selection is an important technique in red-skinned apple breeding. Approximately 30% of the apple varieties have been selected from apple sports, and their yields account for approximately half of the total apple production worldwide. Most of them are red sports101,102. Lines with changes in epigenetic modifications are known as sports, and sport selection can improve quality traits, such as the coloring, growth habit, and the timing of maturity in fruit trees. Epigenetic DNA methylation greatly affects anthocyanin synthesis and the coloration of the apple skin. Several studies have described the mechanisms of coloration in red sports. The increased anthocyanin synthesis in the red bud of “Ralls” was due to higher enzyme activities of CHI and DFR103. Additional research on the red bud of “Ralls” showed that the structural genes in anthocyanin biosynthesis were upregulated because of the increased expression of MdMYB1, and the low methylation level of the MdMYB1 promoter was associated with its increased expression104. El-Sharkawy et al. suggested that differential methylation levels in the MR3 and MR7 promoter regions of MdMYB10 were the epigenetic factors causing the color mutation105. Overall, the methylation level of the MdMYB1 promoter determines apple color in color bud mutants. The formation of apple red buds is caused by the demethylation of the MdMYB1 promoter. Additional research is required to identify the gene(s) involved in the demethylation process and how to recognize such loci.
Breeding of red-fleshed apples
In the global apple industry, most of the significant commercial apple cultivars have white or off-white flesh, such as “Golden Delicious”, “Red Delicious”, “Fuji”, “Gala”, “Granny Smith”, and “Jonathan”. The use of a limited number of parents has greatly narrowed the genetic base of the existing cultivated apple varieties, which makes it difficult to obtain important breakthroughs in characters and phenotypes67. Thus, red-fleshed apples have attracted much attention from breeders because of their appealing flesh color.
The breeding of red-fleshed apple can be traced back to 1897 when Hansen encountered an unusual apple species in the wild fruit forest of Turkestan. This apple species had reddish-purple fruit skin, flesh, blossoms, and juvenile foliage. It was named M. pumila var. niedzwetzkyana, after its discoverer, the Russian botanist Niedzwetzky. He crossed M. pumila var. niedzwetzkyana with established varieties to create some new red-fleshed apples, the best known of which is “Almata”106. There is another pink-fleshed apple with a dull pale-green or whitish-yellow skin that is native to Siberia and the Caucasus. This apple, which is called “Surprise”, is widely thought to be a descendant of M. pumila var. niedzwetskyana107. After many years of apple breeding until 1944, Albert Etter produced approximately 30 high-quality red-fleshed descendants of “Surprise”, but only “Pink Pearl” was commercially released108. The Hansen apple shows deep red pigmentation not only in the apple flesh but also in the fruit skin, blossom, and juvenile foliage. This was later defined as the Type 1 phenotype76. The Etter apple only has pink flesh in the cortex, and the red pigmentation is not found in the leaves, stems, or other vegetative tissues. This was later defined as the Type 2 phenotype109. The pioneering work of Hansen and Etter in creating the two types of red-fleshed apple laid the foundation for the breeding of red-fleshed apple worldwide. In England, Fishman gave new names to Etter’s pink-fleshed apples; they were marketed under the “Rosetta” series title from 1973 and included “Pink Pearmain”, “Blush Rosette”, “Thornberry”, “Rubaiyat”, “Christmas Pink”, “Grenadine”, and “Pink Parfait”108. In Japan, Sekido et al. have been breeding Type 2 red-fleshed apples since 1989. The red-fleshed apple cultivar “Pink Pearl” and its progeny “JPP35” (“Jonathan” × “Pink Pearl”) were used as paternal parents to produce new red-fleshed cultivars109. In Germany, the Type 2 red-fleshed apple “Weirouge” was bred in Weihenstephan and first registered in 1997110. “Baya Marisa” was then bred using “Weirouge” as a parent. In New Zealand, the HortResearch apple breeding program, which was established in 1998, used the Type 1 red-fleshed cultivated apple “Redfield”, derived from “Wolf River” × M. pumila var. niedzwetzkyana111, for Type 1 breeding. For Type 2 breeding, descendants of “Sangrado” were used as the primary red-fleshed parents75. In Switzerland, a series of several red-fleshed apple cultivars, “Redlove”, have been bred and released by Markus Kobelt over the past decade, including “Redlove Sirena”, “Redlove Calypso”, “Redlove Circe”, “Redlove Era”, and “Redlove Odysso”112.
The German breeders Neumüller and Dittrich investigated apple seedlings originating from more than 40 cross-combinations to breed Type 1 red-fleshed apples. They found that a greater proportion of the red-fleshed progeny was obtained when the female parent was a Type 1 red-fleshed apple than when the male parent was a Type 1 red-fleshed apple113. In Japan, the breeding of the red-fleshed apple has primarily concentrated on the Type 2 red-fleshed apple, and the linkage between red-flesh traits and S-genotypes have been investigated. The S3-RNase allele in the red-fleshed apple cultivar “Pink Pearl” and its progeny, “JPP35”, were found to be linked with their red-flesh trait, which provided suitable cultivar combinations for efficient red-fleshed apple breeding114,115. In addition, to breed new red-fleshed apples containing both the MdMYB10 and MdMYB110a genes, “Geneva” (Type 1) and “Pink Pearl” (Type 2) were crossed as paternal parents116. More than 3000 red-fleshed apple germplasm resources have been identified worldwide, and almost all the red types were found to the contain the R6:MdMYB10 locus with the exception of “Pink Pearl”. Of the 82 non-red types, 76 contained only the R1:MdMYB10 locus26. These findings provide additional evidence that the genetic background of red-fleshed apple is complex, and the mutation of any structural gene or transcription factor may affect the red-flesh phenotype.
Utilization of Malus sieversii in red-fleshed apple breeding
M. sieversii belongs to the Tertiary relict species34. There are many variations in its fruit morphology, color, and flavor, plant height, tree shape, and other phenotypic characters. Thus, it is a critical genetic resource for apple breeding117. In addition, M. sieversii populations have developed resistance to drought, cold, pests, and barren soils under natural selection118, and therefore, this species has been widely used in breeding programs to improve the stress resistance of cultivated apples. In a study on the drought tolerance of six apple species, including M. sieversii, Malus toringoides was the most drought-resistant species followed by M. sieversii and Malus transitoria119. Twelve putative M. sieversii Hsp20 genes were identified from RNA-Seq data, indicating that the heat tolerance of M. sieversii may be associated with Hsp20120. In a study on biotic stress resistance, M. sieversii was found to be resistant to scab, and therefore, it has been used as a source of scab resistance in cultivar breeding121,122. Luby et al. evaluated the resistance of different provenances to fire blight, and found that provenances of M. sieversii had better resistance to fire blight and were suitable materials to breed for disease resistance123.
While M. sieversii has been used widely to breed for disease resistance, it has been less commonly used to breed for red-fleshed apples in recent years. Instead, some cultivated apples with good flavor quality have been used more widely for red-fleshed apple breeding. Although the early breeding of red-fleshed apples used M. sieversii and its descendants, the recent objectives of more colorful red flesh, better crispness, and better flavor have led to other lines being favored for red-fleshed apple breeding. Consequently, the other favorable characteristics of M. sieversii, such as its high-flavonoid content, short juvenile phase, and other excellent wild characteristics, have been neglected in the recent history of red-fleshed apple breeding.
Red-fleshed apples are not necessarily rich in flavonoids. In addition to anthocyanin, the primary pigment in red flesh, other flavonoid components are highly significant to the value of apples to human health. When selecting red-fleshed apples, breeders should monitor the changes in other flavonoids during the selection process124. Balancing the red-flesh phenotype with a high flavonoid content is the key to the effective utilization of M. sieversii f. niedzwetzkyana. Chen et al. directly used M. sieversii f. niedzwetzkyana as parents to breed “high-flavonoid functional apples”, which are defined as “apples that are rich in flavonoids in their red-fleshed fruits, that have a good quality of taste and flavor, and have a good quality of appearance and storage”67. In 2006, the F1 hybrid population of M. sieversii f. niedzwetzkyana was constructed in China125. Xu et al. determined the flavonoid composition and contents of four red-fleshed apple strains in the F1 hybrid population and suggested that the differential expression of MdMYB10, MdbHLH3, MdMYB12, MdMYB16, and MdMYB111 at different stages of development could explain the differences in flavonoid contents126. The phenolic compounds that accumulated in five Type 2 red-fleshed apples with marked differences in the coverage and intensity of the red coloration of the cortex were also characterized. The results suggested that MdMYB110a could regulate the synthesis of other phenolic compounds, as well as anthocyanin127. Wang et al. conducted a comparative transcriptome analysis between red- and white-fleshed apples in the F1 population using RNA-Seq and found that 22 upregulated genes in red-fleshed apples were associated with flavonoid biosynthesis128.
Conventional hybrid breeding has remained the primary breeding method throughout the breeding process of the red-fleshed apple. Although hybrid breeding is an effective method to create new apple varieties, the long juvenile phase restricts its development. Therefore, shortening the juvenile phase has become an important research topic in apple breeding, and there have been some breakthroughs in recent years129,130,131. Flachowsky et al. transferred the flower-forming gene of Betula pendula BpMADS4 into the apple cultivar “Pinova” and obtained an early flowering phenotype101. The first rapid and efficient breeding system for apple disease resistance was established using the BpMADS4 transgenic line “T1190”, which shortened the breeding period from 30–40 years to several years102. Yamagishi et al. inoculated virus vectors containing the Arabidopsis flower-forming gene AtFT under the control of a strong promoter and the knock-down apple gene MdTFL1-1 into the cotyledons of apple seedlings. Most of the seedlings bloomed, bore fruit, and produced seeds normally that year103. Nocker et al. suggested that grafting high-node buds onto dwarf rootstocks could shorten the juvenile phase132. However, such methods are labor-intensive and not suitable for large numbers of seedlings in a selection plot. The length of the juvenile phase of the hybrid offspring differs significantly among different apple varieties133. Chen et al. observed that the first filial generation has a short juvenile phase67. The juvenile phase of the five cross-combinations with M. sieversii f. niedzwetzkyana as parents was only 2.33–4.33 years, while that of the control hybrid combination of “Golden Delicious” and “Hanfu” was 3.33–5.33 years. This indicated that the utilization of M. sieversii f. niedzwetzkyana was useful to shorten the juvenile phase and improve the breeding efficiency of red-fleshed apples68.
The genome resequencing of M. sieversii has been completed. In addition, three complementary approaches to bridge the gap between genomics and breeding have been proposed134. It is expected that more excellent traits of M. sieversii f. niedzwetzkyana germplasm resources will be explored and utilized for red-fleshed apple breeding.
Conclusions and perspectives
The complexity of the regulation of flavonoid biosynthesis is the embodiment of the diversity of red-fleshed and high-flavonoid apple germplasm resources. Therefore, research on the formation of the unique flavonoid profiles of red-fleshed apples is of great significance. Flavonoid metabolism in red-fleshed apples is affected by their genetic background and by environmental factors. Many genes in the flavonoid biosynthetic pathway have been cloned, identified, and partially functionally verified. However, due to the complexity of the metabolic pathway, additional research is required to explore the roles of other regulation mechanisms, such as protein modifications and small RNAs, in anthocyanin and flavonoid synthesis in red-fleshed apples.
The breeding of high-flavonoid red-fleshed apples involves the effective integration and balancing of multiple quality traits. For example, higher flavonoid contents in fruit can decrease fruit quality by creating a more astringent taste. Similarly, an increase in the anthocyanin content can lead to the accumulation of malic acid. Improving high-flavonoid apples by crossing them with existing cultivars with high sweetness, excellent flavor, high crispness, and other extreme phenotypes is the key focus in the next step of breeding. In addition, the more excellent traits of the M. sieversii f. niedzwetzkyana germplasm resources need to be explored in more detail and utilized to breed red-fleshed apples and early maturing or late-ripening red-fleshed high-flavonoid apple lines with excellent storage and transportation qualities.
Winkel-Shirley, B. It takes a garden. How work on diverse plant species has contributed to an understanding of flavonoid metabolism. Plant Physiol. 127, 1399–1404 (2001).
Krisetherton, P. M. & Keen, C. L. Evidence that the antioxidant flavonoids in tea and cocoa are beneficial for cardiovascular health. Curr. Opin. Lipidol. 13, 41 (2002).
Hertog, M. G., Hollman, P. C. & Katan, M. B. Content of potentially anticarcinogenic flavonoids of 28 vegetables and 9 fruits commonly consumed in The Netherlands. J. Agric. Food Chem. 40, 2379–2383 (1992).
Knekt, P. et al. Flavonoid intake and risk of chronic diseases. Am. J. Clin. Nutr. 76, 560 (2002).
Duan, N. et al. Genome re-sequencing reveals the history of apple and supports a two-stage model for fruit enlargement. Nat. Commun. 8, 249 (2017).
Boyer, J. & Liu, R. H. Apple phytochemicals and their health benefits. Nutr. J. 3, 5 (2004).
Vinson, J. A., Su, X., Zubik, L. & Bose, P. Phenol antioxidant quantity and quality in foods: fruits. J. Agric. Food Chem. 49, 5315 (2001).
Zamora-Ros, R. et al. Estimation of dietary sources and flavonoid intake in a Spanish adult population (EPIC-Spain). J. Am. Diet. Assoc. 110, 390 (2010).
Hertog, M. G., Feskens, E. J. & Kromhout, D. Antioxidant flavonols and coronary heart disease risk. Lancet 349, 699 (1997).
Chu, Y. F., Sun, J., Wu, X. Z. & Liu, H. R. Antioxidant and antiproliferative activities of common vegetables. J. Agric. Food Chem. 50, 7449–7454 (2002).
Hyson, D. A. A comprehensive review of apples and apple components and their relationship to human health. Adv. Nutr. 2, 408 (2011).
Bondonnoa, C. P. et al. Flavonoid-rich apples and nitrate-rich spinach augment nitric oxide status and improve endothelial function in healthy men and women: a randomized controlled trial. Free Radic. Biol. Med. 52, 95 (2012).
Eberhardt, M. V., Lee, C. Y. & Liu, R. H. Antioxidant activity of fresh apples. Nature 405, 903 (2000).
Williamson, G. & Manach, C. Bioavailability and bioefficacy of polyphenols in humans. II. Review of 93 intervention studies. Am. J. Clin. Nutr. 81, 243S (2005).
Vavilov, N. I. Wild progenitors of the fruit trees of Turkistan and the Caucasus and the problem of the origin of fruit trees. 9th International Horticultural Congress, Reports and Proceedings 271–286 (1930).
Forsline, P. L., Dickson, E. E. & Djangaliev, A. D. Collection of wild Malus, Vitis and other fruit species genetic resources in Kazakstan and neighboring republics. Hortic. Sci. 29, 433 (1994).
Forsline, P. L. Adding diversity to the national apple germplasm collection: collecting wild apples in Kazakstan. N. Y. Fruit Quart. 3, 3–6 (1995).
Janick, J. & Moore, J. N. Apples. In Fruit Breeding: Tree and Tropical Fruits 1–77 (John Wiley & Sons, New Jersey, 1996).
Zhou, Z. Q. & Li, Y. N. The RAPD evidence for the phylogenetic relationship of the closely related species of cultivated apple. Genet. Resour. Crop Evol. 47, 353–357 (2000).
Rehder, A. Manual of Cultivated Trees and Shrubs Hardy in North America 7-7 (The Macmillan Company, London, 1927).
Korban, S. S. & Skirvin, R. M. Nomenclature of the cultivated apple. HortScience 19, 177–180 (1984).
Lamboy, W. F. et al. Partitioning of Allozyme Diversity in Wild Populations of Malus sieversii L. and Implications for Germplasm Collection 619 (American Society for Horticultural Science, Lexington, 1996).
Velasco, R. et al. The genome of the domesticated apple (Malus x domestica Borkh.). Nat. Genet. 42, 833 (2010).
Shu, H.R. Apple Science 29–60 (China Agricultural Press, Beijing, 1999).
Harris, S. A., Robinson, J. P. & Juniper, B. E. Genetic clues to the origin of the apple. Trends Genet. 18, 426–430 (2002).
Van, N. S. et al. Genetic diversity of red-fleshed apples (Malus). Euphytica 185, 281–293 (2012).
Chen, X. et al. Genetic diversity of volatile components in Xinjiang wild apple (Malus sieversii). J. Genet. Genom. 34, 171 (2007).
Zhang, X. Y. et al. Genetic diversity of mineral elements, sugar and acid components in Malus sieversii(Ldb.)Roem. Acta Hortic. Sin. 35, 277–280 (2008).
Song, G. Conservation Biology 11–19 (Zhejiang Science and Technology Press, Zhejiang, 1997).
Hamrick, J. L. Plant population genetics and evolution. Am. J. Bot. 69, 1685–1693 (1982).
Song, G. & Yuan, H. D. Principle and Methodologies of Biodiversity Studies 123–140 (Chinese Science and Technology Press, Beijing, 1994).
Hamrick, J. L. & Loveless, M. D. Associations Between the Breeding System and the Genetic Structure of Tropical Tree Populations (Westview Press, Boulder, 1989).
Volk, G. M. et al. Ex situ conservation of vegetatively propagated species: development of a seed-based core collection for Malus sieversii. J. Am. Soc. Hortic. Sci. 130, 203–210 (2005).
Zhang, X. S. On the eco-geographical characters and the problems of classification of the wild fruit-tree forest in the Ili Valley of Sinkiang. Acta Bot. Sin. 15, 239–253 (1973).
Tao, F. et al. Genetic diversity of fruit morphological traits and content of mineral element in Malus sieversii(Ldb.) Roem. and its elite seedlings. J. Plant Genet. Resour. 7, 270–276 (2006).
Li, T. J., Hu, Z. H. & Wang, L. Primary studies on genetic diversity of the pox isoenzyme banding patterns of Xinjiang wild apple [Malus sieversii (Lebed.) Roem.]. Agric. Sci. Tianjin 9, 27–29 (2003).
Zhang, C. et al. Genetic structure of Malus sieversii population from Xinjiang, China, revealed by SSR markers. J. Genet. Genom. 34, 947–955 (2007).
Zhang, C. Y. et al. SRAP markers for population genetic structure and genetic diversity in Malus sieversii from Xinjiang, China. Acta Hortic. Sin. 36, 7–14 (2009).
Frankel, O. H. & Brown, A. H. D. Plant genetic resources today: a critical appraisal. Proceedings International Conference of Genetics 249–257 (1984).
Wang, Y. K. et al. Advances in core collection of fruit germplasm. J. Plant Genet. Resour. 11(6), 380–385 (2010).
Ghislain, M., Zhang, D., Fajardo, D., Huaman, Z. & Hijmans, R. J. Marker-assisted sampling of the cultivated Andean potato Solanum phureja collection using RAPD markers. Genet. Resour. Crop Evol. 46, 547–555 (1999).
Marshall, D. R. & Brown, A. H. D. Optimum Sampling Strategies in Genetic Conservation, Cambridge University Press, Cambridge, 53–80 (1975).
Zhang, C. Y. et al. A method for constructing core collection of Malus sieversii using molecular markers. Sci. Agric. Sin. 42, 597–604 (2009).
Liu, Z. C. et al. Study on method of constructing core collection of Malus sieversii based on quantitative traits. Sci. Agric. Sin. 43, 358–370 (2010).
Wienand, U., Weydemann, U., Niesbachklösgen, U., Peterson, P. A. & Saedler, H. Molecular cloning of the c2 locus of Zea mays, the gene coding for chalcone synthase. Mol. Gen. Genet. 203, 202–207 (1986).
Boss, P. K., Davies, C. & Robinson, S. P. Analysis of the expression of anthocyanin pathway genes in developing Vitis vinifera L. cv Shiraz Grape Berries and the implications for pathway regulation. Plant Physiol. 111, 1059–1066 (1996).
Takos, A. M. et al. Light-induced expression of a MYB gene regulates anthocyanin biosynthesis in red apples. Plant Physiol. 142, 1216 (2006).
Almeida, J. et al. Characterization of major enzymes and genes involved in flavonoid and proanthocyanidin biosynthesis during fruit development in strawberry (Fragaria x ananassa). Arch. Biochem. Biophys. 465, 61–71 (2007).
Jaakola, L. et al. Expression of genes involved in anthocyanin biosynthesis in relation to anthocyanin, proanthocyanidin, and flavonol levels during bilberry fruit development. Plant Physiol. 130, 729–739 (2002).
Ubi, B. E. et al. Expression analysis of anthocyanin biosynthetic genes in apple skin: effect of UV-B and temperature. Plant Sci. 170, 571–578 (2006).
Nesi, N., Jond, C., Debeaujon, I., Caboche, M. & Lepiniec, L. The Arabidopsis TT2 gene encodes an R2R3 MYB domain protein that acts as a key determinant for proanthocyanidin accumulation in developing seed. Plant Cell 13, 2099–2114 (2001).
Gonzalez, A., Zhao, M., Leavitt, J. M. & Lloyd, A. M. Regulation of the anthocyanin biosynthetic pathway by the TTG1/bHLH/Myb transcriptional complex in Arabidopsis seedlings. Plant J. 53, 814–827 (2008).
Schaart, J. G. et al. Identification and characterization of MYB-bHLH-WD40 regulatory complexes controlling proanthocyanidin biosynthesis in strawberry (Fragaria×ananassa) fruits. New Phytol. 197, 454–467 (2013).
Ban, Y. et al. Isolation and functional analysis of a MYB transcription factor gene that is a key regulator for the development of red coloration in apple skin. Plant Cell Physiol. 48, 958 (2007).
Li, Y. Y. et al. MdCOP1 ubiquitin E3 ligases interact with MdMYB1 to regulate light-induced anthocyanin biosynthesis and red fruit coloration in apple. Plant Physiol. 160, 1011–1022 (2012).
Vimolmangkang, S., Han, Y., Wei, G. & Korban, S. S. An apple MYB transcription factor, MdMYB3, is involved in regulation of anthocyanin biosynthesis and flower development. BMC Plant Biol. 13, 176 (2013).
Xie, X. B. et al. The bHLH transcription factor MdbHLH3 promotes anthocyanin accumulation and fruit colouration in response to low temperature in apples. Plant Cell Environ. 35, 1884–1897 (2012).
An, X. H., Tian, Y., Chen, K. Q., Wang, X. F. & Hao, Y. J. The apple WD40 protein MdTTG1 interacts with bHLH but not MYB proteins to regulate anthocyanin accumulation. J. Plant Physiol. 169, 710–717 (2012).
Lin-Wang, K. et al. High temperature reduces apple fruit colour via modulation of the anthocyanin regulatory complex. Plant Cell Environ. 34, 1176 (2011).
Liu, X. J. et al. MdSnRK1.1 interacts with MdJAZ18 to regulate sucrose-induced anthocyanin and proanthocyanidin accumulation in apple. J. Exp. Bot. 68, 2977 (2017).
An, X. H. et al. MdMYB9 and MdMYB11 are involved in the regulation of the JA-induced biosynthesis of anthocyanin and proanthocyanidin in apples. Plant Cell Physiol. 56, 650 (2015).
Jezek, M., Zörb, C., Merkt, N. & Geilfus, C. M. Anthocyanin management in fruits by fertilization. J. Agric. Food Chem. 66, 753–764 (2018).
Kadir, S. A. Fruit quality at harvest of “Jonathan” apple treated with foliarly-applied calcium chloride. J. Plant Nutr. 27, 1991–2006 (2005).
Thomasraese, J. & Drake, S. R. Nitrogen fertilization and elemental composition affects fruit quality of â ˜Fujiâ™ apples. J. Plant Nutr. 20, 1797–1809 (1997).
King, M. C. & Cliff, M. A. Development of a model for prediction of consumer liking from visual attributes of new and established apple cultivars. J. Am. Pomol. Soc. 4, 223–229 (2002).
Giomaro, G. et al. Polyphenols profile and antioxidant activity of skin and pulp of a rare apple from Marche region (Italy). Chem. Cent. J. 8, 45 (2014).
Chen, X. S. et al. Discussion on today’s world apple industry trends and the suggestions on sustainable and efficient development of apple industry in China. J. Fruit Sci. 27, 598–604 (2010).
Chen, X. S. et al. Genetic variation of F1 population between Malus sieversii f. neidzwetzkyana and apple varieties and evaluation on fruit characters of functional apple excellent strains. Sci. Agric. Sin. 47, 2193–2204 (2014).
Tsao, R., Yang, R., Young, J. C. & Zhu, H. Polyphenolic profiles in eight apple cultivars using High-Performance Liquid Chromatography (HPLC). J. Agric. Food Chem. 51, 6347–6353 (2003).
Mcghie, T. K., Martin Hunt, A. & Barnett, L. E. Cultivar and growing region determine the antioxidant polyphenolic concentration and composition of apples grown in New Zealand. J. Agric. Food Chem. 53, 3065–3070 (2005).
Espley, R. V. et al. Red colouration in apple fruit is due to the activity of the MYB transcription factor, MdMYB10. Plant J. 49, 414 (2007).
Espley, R. V. et al. Multiple repeats of a promoter segment causes transcription factor autoregulation in red apples. Plant Cell 21, 168 (2009).
Espley, R. V. et al. Analysis of genetically modified red-fleshed apples reveals effects on growth and consumer attributes. Plant Biotechnol. J. 11, 408–419 (2013).
Chagné, D. et al. An ancient duplication of apple MYB transcription factors is responsible for novel red fruit-flesh phenotypes. Plant Physiol. 161, 225 (2013).
Umemura, H., Otagaki, S., Wada, M., Kondo, S. & Matsumoto, S. Expression and functional analysis of a novel MYB gene, MdMYB110a_JP, responsible for red flesh, not skin color in apple fruit. Planta 238, 65–76 (2013).
Volz, R. K. et al. Breeding for red flesh colour in apple: progress and challenges. Acta Hortic. 814, 337–342 (2009).
Szankowski, I. et al. Shift in polyphenol profile and sublethal phenotype caused by silencing of anthocyanidin synthase in apple (Malus sp.). Planta 229, 681 (2009).
Wang, N. et al. The proanthocyanidin-specific transcription factor MdMYBPA1 initiates anthocyanin synthesis under low temperature conditions in red-fleshed apple. Plant J. https://doi.org/10.1111/tpj.14013 (2018).
Wang, N. et al. Transcriptomic analysis of red-fleshed apples reveals the novel role of MdWRKY11 in flavonoid and anthocyanin biosynthesis. J. Agric. Food Chem. 66, 7076–7086 (2018).
Wang, Y. C. et al. Molecular cloning and expression analysis of cytokinins responsive gene MdMYB308 in red flesh apple. Sci. Agric. Sin. 50, 4178–4185 (2017).
Wang, Y. et al. Molecular cloning and expression analysis of an auxin signaling related gene MdARF3 in red flesh apple. Acta Hortic. Sin. 44, 633–643 (2017).
Xu, H. et al. The molecular mechanism underlying anthocyanin metabolism in apple using the MdMYB16 and MdbHLH33 genes. Plant Mol. Biol. 94, 149–165 (2017).
Jiang, Y. et al. MdHB1 down-regulation activates anthocyanin biosynthesis in the white-fleshed apple cultivar ‘Granny Smith’. J. Exp. Bot. 68, 1055 (2017).
Wang, N. et al. MYB12 and MYB22 play essential roles in proanthocyanidin and flavonol synthesis in red-fleshed apple (Malus sieversii f.niedzwetzkyana). Plant J. 90, 276–292 (2017).
Chen, X. S. et al. Genetic improvement and promotion of fruit quality of main fruit trees. Sci. Agric. Sin. 48, 3524–3540 (2015).
Visser, T., Schaap, A. A. & Vries, D. P. D. Acidity and sweetness in apple and pear. Euphytica 17, 153–167 (1968).
Brown, A. G. & Harvey, D. M. The nature and inheritance of sweetness and acidity in the cultivated apple. Euphytica 20, 68–80 (1971).
Zhen, Q. et al. Developing gene-tagged molecular markers for evaluation of genetic association of apple SWEET genes with fruit sugar accumulation. Hortic. Res. 5, 14 (2018).
Tacken, E. et al. The role of ethylene and cold temperature in the regulation of the apple POLYGALACTURONASE1 gene and fruit softening. Plant Physiol. 153, 294–305 (2010).
Harada, T. et al. An allele of the 1-aminocyclopropane-1-carboxylate synthase gene (Md-ACS1) accounts for the low level of ethylene production in climacteric fruits of some apple cultivars. Theor. Appl. Genet. 101, 742–746 (2000).
Huang, Z. et al. Relatively high acidity is an important breeding objective for fresh juice-specific apple cultivars. Sci. Hortic. 233, 29–37 (2018).
Crane, M. B. & Lawrence, W. J. C. Genetical studies in cultivated apples. J. Genet. 28, 265–296 (1933).
Klein, L. G. The inheritance of certain fruit characters in the apple. Proc. Am. Soc. Hortic. Sci. 72, 1–14 (1958).
Cheng, F. S., Weeden, N. F. & Brown, S. K. Identification of co-dominant RAPD markers tightly linked to fruit skin color in apple. Theor. Appl. Genet. 93, 222–227 (1996).
Zhao, J., Tian, Y. K., Wang, C. H., Dai, H. Y. & Wang, D. Identification of RAPD marker linked to the red skin traits of apples. J. Fruit Sci. 23, 165–168 (2006).
Lespinasse, Y., Lespinasse, J. M. & Ganne, B. Inheritance of two agronomical characters in the apple tree (Malus Pumila MILL.): compact type habit and fruit colour. Acta Hortic. 159, 35–48 (1985).
Lespinasse, Y., Fouillet, A., Flick, J. D., Lespinasse, J. M. & Delort, F. Contribution to genetic studies in apple. Acta Hortic. 224, 99–108 (1988).
Brown, S. K. Genetics of apple. Plant Breed. Rev. 9, 342–343 (1992).
Sheng, B. C. & Yu, M. L. Genetic studies on color of apple peel. Fruit. Sci. 4, 191–194 (1993).
Ju, Z., Liu, C., Yuan, Y., Wang, Y. & Liu, G. Coloration potential, anthocyanin accumulation, and enzyme activity in fruit of commercial apple cultivars and their F1 progeny. Sci. Hortic. 79, 39–50 (1999).
Yi, K. et al. Review on identification and utilization of apple sport selection. J. Fruit Sci. 23, 745–749 (2006).
Wang, C. Z. et al. Analysis of aroma components and related enzymes of fatty acid metabolism of red bud sports. Acta Hortic. Sin. 39, 2447–2456 (2012).
Liu, X. J. et al. Studies on anthocyanin biosynthesis and activities of related enzymes of ‘Ralls’ and its bud mutation. Acta Hortic. Sin. 36, 1249–1254 (2009).
Xu, Y. et al. Comparison of MdMYB1 sequences and expression of anthocyanin biosynthetic and regulatory genes between Malus domestica Borkh. cultivar ‘Ralls’ and its blushed sport. Euphytica 185, 157–170 (2012).
Elsharkawy, I., Dong, L. & Xu, K. Transcriptome analysis of an apple (Malus×domestica) yellow fruit somatic mutation identifies a gene network module highly associated with anthocyanin and epigenetic regulation. J. Exp. Bot. 66, 7359–7376 (2015).
Deacon, N. The Diversity of Red Fleshed Apples, Vol. 2017. http://www.suttonelms.org.uk/apple104.html.
Greenmantle, N. The Rosetta Apples, Vol. 2017. http://www.greenmantlenursery.com/fruit/rosetta-apples.htm (2009).
Fishman, R. Albert Etter and the Pink-Fleshed Daughters of Surprise, Vol. 2017. http://www.appleluscious.com/albert_etter_article/albert_etter_article_page_1.html (1995).
Sekido, K. et al. Efficient breeding system for red-fleshed apple based on linkage with S3-RNase allele in ‘Pink Pearl’. Infect. Immun. 45, 534–537 (2010).
Sadilova, E., Stintzing, F. C. & Carle, R. Chemical quality parameters and anthocyanin pattern of red-fleshed Weirouge apples. J. Appl. Bot. Food Qual. Angew. Bot. 80, 82–87 (2006).
Brooks, R. M. & Olmo, H. P. Register of Fruit and Nut Varieties, 707 (University of California Press, Berkeley, 1997).
Red Fleshed Apple Trees, Vol. 2017 http://www.lubera.co.uk/plants/fruit-trees/apple-trees/red-fleshed-apple-trees-redloves/ (2016).
Neumüller, M. & Dittrich, F. Breeding for type 1 red-fleshed apple varieties: using the red-fleshed apple variety as female parent yields a higher percentage of red-leafed seedlings. Acta Hortic. 1172, 241–244 (2017).
Sekido, K. et al. Efficient breeding system for red-fleshed apple based on linkage with S3-RNase allele in ‘Pink Pearl’. Infect. Immun. 45, 534–537 (2010).
Umemura, H., Shiratake, K., Matsumoto, S., Maejima, T. & Komatsu, H. Practical breeding of red-fleshed apple: cultivar combination for efficient red-fleshed progeny production. HortScience 46, 1098–1101 (2011).
Hamada, Y. et al. Breeding depression of red flesh apple progeny containing both functional MdMYB10 and MYB110a_JP genes. Plant Breed. 134, 239–246 (2015).
Forsline, P. L. & Aldwinckle, H. S. Evaluation of Malus sieversii seedling populations for disease resistance and horticultural traits. Acta Hortic. 663, 529–534 (2004).
Zhang, Y. M. et al. Advances in research of the Malus sieversii (Lebed.) Roem.Acta Hortic. Sin. 36, 447–452 (2009).
Yang, J., Yang, E. & Yang, H. A study on drought resistance of genus Malus seedling. Acta Agric. Boreall—Sin. 11, 81–86 (1996).
Yang, M. et al. Identification of MsHsp20 gene family in Malus sieversii and functional characterization of MsHsp16.9 in heat tolerance. Front. Plant Sci. 8, 1761 (2017).
Aldwinckle, H. S., Forsline, P. L., Gustafson, H. L. & Hokanson, S. C. Evaluation of apple scab resistance of Malus sieversii populations from Central Asia. HortScience 32, 440 (1997).
Bus, V. G. et al. The Vh8 locus of a new gene-for-gene interaction between Venturia inaequalis and the wild apple Malus sieversii is closely linked to the Vh2 locus in Malus pumila R12740-7A. New Phytol. 166, 1035–1049 (2005).
Luby, J. J., Alspach, P. A., Bus, V. G. M. & Oraguzie, N. C. Field resistance to fire blight in a diverse apple (Malus sp.) germplasm collection. J. Am. Soc. Hortic. Sci. 127, 245–253 (2002).
Volz, R. K., Mcghie, T. K. & Kumar, S. Variation and genetic parameters of fruit colour and polyphenol composition in an apple seedling population segregating for red leaf. Tree Genet. Genomes 10, 953–964 (2014).
Liu, Z. C., Miao, W. D., Liu, D. L. & Chen, X. S. Construction and evaluation of the segregation population in Malus sieversii. J. Fruit Sci. 29, 722–728 (2012).
Hai-Feng, X. U. et al. Content and analysis of biosynthesis-related genes of flavonoid among four strains of Malus sieversii f. neidzwetzkyana F1 population. Sci. Agric. Sin. 49, 3174–3187 (2016).
Sato, H. et al. Varietal differences in phenolic compounds metabolism of type 2 red-fleshed apples. Sci. Hortic. 219, 1–9 (2017).
Wang, N. et al. Comparative transcriptomes analysis of red- and white-fleshed apples in an F1 population of Malus sieversii f. niedzwetzkyana crossed with M. domestica ‘Fuji’. PLoS One 10, e0133468 (2015).
Flachowsky, H., Peil, A., Sopanen, T., Elo, A. & Hanke, V. Overexpression of BpMADS4 from silver birch (Betula pendula Roth.) induces early‐flowering in apple (Malus × domestica Borkh.). Plant Breed. 126, 137–145 (2010).
Flachowsky, H. et al. Application of a high‐speed breeding technology to apple (Malus × domestica) based on transgenic early flowering plants and marker‐assisted selection. New Phytol. 192, 364–377 (2011).
Yamagishi, N., Kishigami, R. & Yoshikawa, N. Reduced generation time of apple seedlings to within a year by means of a plant virus vector: a new plant-breeding technique with no transmission of genetic modification to the next generation. Plant Biotechnol. J. 12, 60–68 (2014).
Van, N. S. & Gardiner, S. E. Breeding better cultivars, faster: applications of new technologies for the rapid deployment of superior horticultural tree crops. Hortic. Res. 1, 14022 (2014).
Visser, T. Juvenile phase and growth of apple and pear seedlings. Euphytica 13, 119–129 (1964).
Laurens, F. et al. An integrated approach for increasing breeding efficiency in apple and peach in Europe. Hortic. Res. 5, 11 (2018).
This work was supported by the National Key Research Project of China (2016YFC0501505) and the National Natural Science Foundation of China (CN) (31572091, 31730080). We thank Jennifer Smith, Ph.D., from Liwen Bianji, Edanz Group China, for editing the English text of a draft of this manuscript.
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Wang, N., Jiang, S., Zhang, Z. et al. Malus sieversii: the origin, flavonoid synthesis mechanism, and breeding of red-skinned and red-fleshed apples. Hortic Res 5, 70 (2018). https://doi.org/10.1038/s41438-018-0084-4
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