# Deciphering the genetic architecture of fruit color in strawberry

Béatrice Denoyes, Alexandre Prohaska, Johann Petit, Christophe Rothan

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Béatrice Denoyes, Alexandre Prohaska, Johann Petit, Christophe Rothan. Deciphering the genetic architecture of fruit color in strawberry. Journal of Experimental Botany, 2023, Advance Access publication, 10.1093/jxb/erad245. hal-04183094

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EXPERT VIEW

# Deciphering the genetic architecture of fruit color in strawberry

Béatrice Denoyes<fnref n="1" />,*, ID, Alexandre Prohaska<fnref n="1" />,<fnref n="2" />, ID, Johann Petit<fnref n="1" />, ID and Christophe Rothan<fnref n="1" />,*, ID

Received 31 March 2023; Editorial decision 26 June 2023; Accepted 28 June 2023

Editor: John Lunn, MPI of Molecular Plant Physiology, Germany

## Abstract

Fruits of *Fragaria* species usually have an appealing bright red color due to the accumulation of anthocyanins, water-soluble flavonoid pigments. Octoploid cultivated strawberry (*Fragaria × ananassa*) is a major horticultural crop for which fruit color and associated nutritional value are main breeding targets. Great diversity in fruit color intensity and pattern is observed not only in cultivated strawberry but also in wild relatives such as its octoploid progenitor *F. chiloensis* or the diploid woodland strawberry *F. vesca*, a model for fruit species in the *Rosaceae*. This review examines our understanding of fruit color formation in strawberry and how ongoing developments will advance it. Natural variations of fruit color as well as color changes during fruit development or in response to several cues have been used to explore the anthocyanin biosynthetic pathway and its regulation. So far, the successful identification of causal genetic variants has been largely driven by the availability of high-throughput genotyping tools and high-quality reference genomes of *F. vesca* and *F. × ananassa*. The current completion of haplotype-resolved genomes of *F. × ananassa* combined with QTL mapping will accelerate the exploitation of the untapped genetic diversity of fruit color and help translate the findings into strawberry improvement.

**Keywords:** Anthocyanins, color, flavonoids, genome, haplotype, MYB, QTL mapping, *Rosaceae*, strawberry.

## Introduction

Octoploid cultivated strawberry (*Fragaria × ananassa*) is a major horticultural crop that issued from a fortuitous cross in the 18th century in France between *F. virginiana* and *F. chiloensis* (Edger et al., 2019). Other species of the *Fragaria* genus cultivated for human consumption include the wild diploid woodland strawberry (*F. vesca*), which serves as a model for fruit crops of the *Rosaceae* family (Gaston et al., 2020). The cultivated and approximately 25 wild strawberry species currently described, which include species ranging from diploids to decaploids, exhibit a high genetic and phenotypic diversity that can be used

to decipher the mechanisms responsible for strawberry fruit color (Castillejo et al., 2020; Qiao et al., 2021).

Considerable effort has been devoted in recent years to breeding cultivated strawberry varieties displaying not only increased resistance to pathogens, high yield, and extended storage period, but also improved fruit sensorial and nutritional quality (Senger et al., 2022). The main sensorial traits include fruit flavor and taste (aroma, sugars, organic acids) and appearance (size and shape, brightness, color). Specialized metabolites responsible for fruit color, mainly anthocyanins, also participate

<footnote n="1">¹ INRAE and Univ. of Bordeaux, UMR 1332 Biologie du Fruit et Pathologie, F-33140 Villenave d'Ornon, France</footnote>

<footnote n="2">² INVENIO, MIN de Brienne, Bordeaux, France</footnote>

<footnote>* Correspondence: christophe.rothan@inrae.fr or beatrice.denoyes@inrae.fr</footnote>
in fruit nutritional value (Mezzetti et al., 2018; Miller et al., 2019). As a result, fruit color, which plays a major role in the attractiveness and nutritional quality of strawberry, has been intensively investigated over the past decade.

Substantial advances have been made in our capacity to identify genetic variations responsible for differences in fruit color, thanks to progress in sequencing and genotyping technologies. Whole genome sequencing (WGS) of *F. × ananassa* led to the release of a reference genome (Edger et al., 2019) and, more recently, of several haplotype-resolved genomes (Hardigan et al., 2021a, Preprint; Lee et al., 2021; Fan et al., 2022; Mao et al., 2023). The implementation of high-throughput genotyping technologies (Bassil et al., 2015; Hardigan et al., 2020) now enables the high-resolution mapping of fruit color quantitative trait loci (QTLs) and the identification of candidate genes (CGs) (Castillejo et al., 2020; Davik et al., 2020; Labadie et al., 2020, 2022; Pott et al., 2020; Manivannan et al., 2021). Excellent reviews (e.g. Whitaker et al., 2020) published recently describe in detail the present state of genetic and genomic studies in strawberry and their application to breeding. In this review, we focus on how our understanding of the genetic architecture of fruit color in strawberry can benefit from recent developments in fruit color phenotyping and in strawberry genomics. We further provide a current and forward-looking perspective on how these findings can be translated to improvement of strawberry fruit color.

## The genus *Fragaria* is genetically diverse

Strawberry belongs to genus *Fragaria*, which contains about 25 species (Liston et al., 2014; Qiao et al., 2021) ranging from diploid (2n=2×=14) to decaploid (2n=10×=70). All species are restricted to single continents or specific areas, except the diploid woodland species *F. vesca*, which is found in both Eurasia and America (Staudt, 1962, 1989). The majority of diploid species and all five tetraploid species are present in China (Liston et al., 2014). The single hexaploid species, *F. moschata*, is present in Europe, including Russia. The two related wild octoploid species, *F. chiloensis* and *F. virginiana*, are present in America with different distributions in South and North America (Staudt, 1962, 1989).

Cultivated strawberry and its wild progenitors, *F. virginiana* and *F. chiloensis*, are allo-octoploids with 2n=8×=56 chromosomes (Box 1). Genetic diversity of *F. × ananassa* has been increased by recurrent interspecific hybridizations with various progenitor accessions to introduce new traits-of-interest (Hardigan et al., 2021b). This species results from a hybridization by chance in the early 1700s in Europe between two related octoploid species, *F. virginiana* and *F. chiloensis* (Darrow, 1966; Hancock, 1999), resulting in a new cultivated fruit species, *F. × ananassa*. Strawberry breeding began in England in the late 1700s, followed by France and Germany. The first selected European cultivars were used as genitors in early American

breeding programs, together with American native cultivars (Darrow, 1966). Today, the genetic diversity in the cultivated strawberry seems to be preserved thanks to the large number of founders and introgression with wild octoploid strawberry germplasm (Hardigan et al., 2021b). Consequently, the genetic structure of this cultivated species displays a large amount of admixture (Bird et al., 2021; Zurn et al., 2022).

Fruit color diversity, ranging from white to dark red, is high in the genus *Fragaria*. For example, white fruits can be found in the diploid species *F. vesca*, *F. nubicola*, *F. pentaphylla*, and *F. nilgerensis* (Qiao et al. 2021) and in the octoploid *F. chiloensis* (Finn et al., 2013). In *F. × ananassa*, the diversity of skin color and flesh color pattern (e.g. white-fleshed genotypes versus red-fleshed genotypes; Fig. 1) is considerable (Aaby et al., 2012; Castillejo et al., 2020). As detailed in Box 1, recent developments in fruit color phenotyping and strawberry genomics now enable the high-resolution mapping and identification of causal genetic variations present amidst the strawberry genetic diversity. In addition, widespread gene editing technologies can simultaneously target the large numbers of homoeoalleles at a given locus found in cultivated strawberry, thus providing a technological means to modify fruit color.

### Recent advances in strawberry genomics provide the key to exploring strawberry diversity

Reconstructing the evolutionary history of the *Fragaria* species was made possible thanks to the increased availability of strawberry genome sequences. Within the *Fragaria* genus, the first genome sequenced was that of the diploid *F. vesca* genotype 'Hawai-4' (Shulaev et al., 2011; Edger et al., 2018; Li et al., 2019), a white fruit-bearing genotype that has been widely used in studies involving stable genetic transformation (Gaston et al. 2020). Since then, high-quality chromosome-level reference genomes of a red-fruited *F. vesca* genotype (Alger et al., 2021) and of various diploid species (J. Zhang et al., 2020; Feng et al., 2021, 2023; Qiao et al., 2021; Sun et al., 2022) have been made available. Recently, the reconstruction of the phylogeny of 10 wild diploid strawberries with whole genome resequencing data revealed complex and widespread introgression across the *Fragaria* genus (Feng et al., 2023).

The allo-octoploid *F. × ananassa* is complex, with four subgenomes derived from diploid species contributing to its genome (Rousseau-Gueutin et al., 2009). The first *F. × ananassa* genome sequence, that of the California variety 'Camarosa', confirmed that the diploid *F. vesca* and *F. iinumae* were ancestors of two of the four subgenomes (Edger et al., 2019). The origin of the remaining two subgenomes, possibly related to *F. viridis* and *F. nipponica* or to a now extinct species related to *F. iinumae*, is still under investigation (Edger et al., 2019, 2020; Liston et al., 2020). Until recently, this complexity has hampered the high-resolution genetic mapping of traits, and the diploid *F. vesca* has been used in parallel as a strawberry model (Gaston et al., 2020). The suitability of *F. vesca* to explore the genetic control
of fruit quality traits, including color, was further reinforced by expression analyses showing the dominance of the *F. vesca* sub-genome for the control of fruit quality traits and, more specifically, anthocyanin biosynthesis (Edger *et al.*, 2019). The recent availability of *F. vesca* and *F. × ananassa* genome sequences triggered the very quick development of genotyping technologies using single nucleotide polymorphism (SNP) arrays based on the *F. vesca* genome (Bassil *et al.*, 2015). Subsequently, an 850K SNP array was developed from the *F. × ananassa* 'Camarosa' genome (Hardigan *et al.*, 2020), from which a widely used (Senger *et al.*, 2022) 50K array with selected chromosome-specific SNPs was designed (Hardigan *et al.*, 2020). SNP array genotyping now allows the high-resolution localization of QTLs on specific chromosomes of *F. × ananassa* homeologous groups (HGs) (Box 1).

Once a QTL has been mapped at high-resolution, the next step in deciphering the genetic architecture of a trait is to identify the causal polymorphism. In *F. × ananassa*, up to eight candidate homoealleles carried by eight chromosomes can be found at a given locus (Gaston *et al.*, 2020). A breakthrough advance to analyse the contribution of each homoeallele to a color trait was the development of accurate and cost-effective long-read technologies enabling the assembly and phasing of complex genomes (Dumschott *et al.*, 2020; Hon *et al.*, 2020) (Box 1). So far, haplotype-resolved genomes have been completed for four varieties of *F. × ananassa* ('Royal Royce', 'Wongyo', 'Florida Brillance', and 'Yanli') (Hardigan *et al.*, 2021a, Preprint; Lee *et al.*, 2021; Fan *et al.*, 2022; Mao *et al.*, 2023). Three of them are available at the Genome Database for Rosaceae (Jung *et al.*, 2019). Haplotype-genome sequencing and transcriptome analysis of 'Yanli' has already demonstrated the potential of this approach to investigate mechanisms underlying anthocyanin biosynthesis and therefore to discover hitherto unknown natural genetic variations controlling fruit color.

### *Fruit color is a major visual and nutritional trait in strawberry*

The red color of the strawberry fruit is due to the accumulation during ripening of red-colored anthocyanins. Anthocyanins are flavonoids derived from the phenylpropanoid pathway (Fig. 1). In addition to their role as water-soluble pigments conferring an attractive color to the fruit, anthocyanins are antioxidant molecules with major health benefits (Miller *et al.*, 2019). As with other fruit traits, there is considerable variation between consumer preferences based on geographic region or country. In Asia, for example, white-skinned fruit varieties can be highly valued (Castillejo *et al.*, 2020). Breeders should therefore adapt their objectives to the main market to be reached and consider the intensity, hue, and pattern of the fruit color.

When deciphering the architecture of fruit color, the first step is typically to phenotype large collections of genetic resources (Box 1). Most often, strawberry color is scored by

## Box 1. Key developments in deciphering the genetic architecture of fruit color in strawberry

* Strawberry color phenotyping

(A) Examples of the diversity of fruit color intensity, hue, and pattern found in cultivated strawberry. (B, C) Fruit color intensity and hue can be evaluated by using (B) scoring-based color chart or (C) physical means (CIELAB color space).

(D) Individual phenylpropanoid, flavonoid and anthocyanin compounds can be exhaustively analysed by using MS-based technologies. (E) Automated phenotyping is key to reliably assessing the diversity of fruit color intensity and distribution in large collections (Zingaretti *et al.*, 2022).

* Strawberry genotyping and sequencing

(A) Chromosomes of the allo-octoploid *F. × ananassa* are ranged in seven homoeology groups (HGs), each including eight homoeologous chromosomes with ancestral homology. (B) A haplotype-specific SNP array has been designed thanks to the reference genome of *F. × ananassa* (Hardigan *et al.*, 2020). (C) Haplotype-resolved sequences of *F. × ananassa* were obtained by accurate long-read sequencing (Hardigan *et al.*, 2021a, Preprint; Fan *et al.*, 2022; Mao *et al.*, 2023).

* Natural *MYB10* variations

(A) *MYB10* is a master regulatory gene that targets structural genes of the anthocyanin biosynthetic pathway, resulting in the bright red color of strawberry fruit skin. (B) Various loss-of-function *MYB10* mutations impair anthocyanin biosynthesis in *F. × ananassa*, resulting in white-skinned fruit (Castillejo *et al.*, 2020; Wang *et al.*, 2020; Yuan *et al.*, 2022). (C) Transposon insertion in the *MYB10* promoter induces ectopic expression of *MYB10* in the fruit flesh, resulting in red-skinned and red-fleshed fruit (Castillejo *et al.*, 2020).
* Simultaneous editing of homoeoalleles with the CRISPR/Cas9 system

(A) The CRISPR/Cas9 system targets specific gene sequences where it induces double strand breaks. DNA repair of the breaks can generate mutations. (B) In *F. × ananassa*, multiple homoeoalleles of an anthocyanin transport gene sharing common sequences could be targeted simultaneously (Gao *et al.*, 2020).

## 1. Phenotyping

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## 2. Genotyping - Sequencing

A

Chromosomes ranged in 7
homoeology groups (HGs)

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B

Haplotype-specific
genetic
markers
(SNP array)

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C

Haplotype-resolved
genome
sequence

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## 3. Natural *MYB10* variations

A

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B

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C

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## 4. Simultaneous editing of homoeoalleles with CRISPR/Cas9

A

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B

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<figure><img src="image_26.png" /><figcaption>**Fig. 1.** Synthesis, storage, and regulation of anthocyanins and their contribution to fruit color in cultivated strawberry (*Fragaria × ananassa*). A simplified flavonoid biosynthetic pathway is shown. The various chemical classes are encircled with different colors. All the proteins indicated were studied in *Fragaria × ananassa* with the exception of HY5, bHLH9, and bHLH33, which were only studied in *Fragaria vesca*. Proteins encoded by structural genes: 4CL, 4-coumaroyl:CoA-ligase; ANR, anthocyanidin reductase; ANS, anthocyanidin synthase; C4H, cinnamate-4-hydroxylase; CHS, chalcone synthase; CHI, chalcone isomerase; DFR, dihydroflavanol reductase; F3H/FHT, Flavanone 3 hydrolase; F3'H, flavanone 3' hydrolase; FLS, flavonol synthase; GST, glutathione S-transferase; GT, anthocyanidin glucosyltransferase; LAR, leucoanthocyanidin reductase; PAL, phenylalanine ammonia lyase. Proteins encoded by regulatory genes: bHLH, basic helix-loop-helix transcription factor; HY5, Long Hypocotyl 5; MAPK3, Map Kinase 3; MYB, myeloblastosis transcription factor; RAV1, Related to Abscisic Acid Insensitive 3 (ABI3)/Viviparous 1 (VP1) transcription factor; SCL8, SCARECROW-LIKE 8; TCP11, Teosinte branched, Cycloidea and PCF transcription factor; WD40, WD40-repeat protein.</figcaption></figure>
<table><caption>**Table 1.** Structural genes with roles in biosynthesis and storage of anthocyanins in strawberry fruit</caption><thead><tr><th>Strategy</th><th>Fruit trait studied or affected/main findings</th><th>Pathways or genes</th><th>Species</th><th>References</th></tr></thead><tbody><tr><td colspan="5"><strong>Metabolome analyses and/or gene expression profiling</strong></td></tr><tr><td>Targeted expression profiling<sup>c</sup>; enzyme activities</td><td>Color of developing/ripening fruit</td><td>PP/Flav pathways</td><td>F. x ananassa</td><td>Almeida et al. (2007)</td></tr><tr><td>Targeted expression profiling<sup>c</sup>; enzyme activities</td><td>Color of developing/ripening fruit of various genotypes, environmental effect</td><td>Flav pathway</td><td>F. x ananassa</td><td>Carbone et al. (2009)</td></tr><tr><td>Targeted expression profiling<sup>c</sup>; metabolome</td><td>Various genotypes</td><td>PP/Flav pathways</td><td>F. x ananassa</td><td>Schaart et al. (2013)</td></tr><tr><td>Transcriptome<sup>b,c</sup></td><td>Yellow fruit genotype</td><td>Flav pathway</td><td>F. vesca</td><td>Zhang et al. (2015)</td></tr><tr><td>Transcriptome<sup>b</sup>, metabolome</td><td>Yellow/white fruit genotypes</td><td>PP/Flav pathways-GST</td><td>F. vesca</td><td>Härtl et al. (2017)</td></tr><tr><td>Transcriptome<sup>b</sup></td><td>Yellow fruit genotype, color of developing/ripening fruit, F. vesca eFP browser</td><td>PP/Flav pathways</td><td>F. vesca</td><td>Hawkins et al. (2017)</td></tr><tr><td>Targeted expression profiling<sup>c</sup></td><td>Color of developing/ripening fruit of high anthocyanin cultivars</td><td>PP/Flav pathways</td><td>F. x ananassa</td><td>Hossain et al. (2018)</td></tr><tr><td>Transcriptome<sup>b,c</sup></td><td>White-fleshed and white-skinned genotypes</td><td>Flav pathway</td><td>F. x ananassa</td><td>Zhao et al. (2021)</td></tr><tr><td>Targeted expression profiling<sup>c</sup></td><td>Color of developing/ripening fruit</td><td>PP/Flav pathways</td><td>F. chiloensis</td><td>Mattus-Araya et al. (2022)</td></tr><tr><td colspan="5"><strong>Studies targeted to phenylpropanoid or flavonoid pathway genes</strong></td></tr><tr><td>Transcriptome<sup>b</sup>, metabolome, transient OE</td><td>Pinkish-skinned and white-fleshed genotypes</td><td>C4H</td><td>F. x ananassa</td><td>Jiang et al. (2022)</td></tr><tr><td>Transient RNAi silencing</td><td>Restoration of red flesh color</td><td>CHS</td><td>F. x ananassa</td><td>Hoffmann et al. (2006)</td></tr><tr><td>Transcriptome<sup>a</sup>, metabolome; transient RNAi silencing</td><td>Fruit color reduction and lignin increase</td><td>CHS</td><td>F. x ananassa</td><td>Ring et al. (2013)</td></tr><tr><td>Transient RNAi silencing</td><td>Reduced anthocyanin accumulation</td><td>F3H/FHT</td><td>F. x ananassa</td><td>Jiang et al. (2023)</td></tr><tr><td>Transcriptome<sup>b,c</sup>, metabolome</td><td>Color of developing/ripening fruit, white-fleshed genotype</td><td>PP/Flav pathways-F3'H</td><td>F. x ananassa</td><td>Lin et al. (2018)</td></tr><tr><td>Stable RNAi silencing</td><td>Cyanidin-3-glucoside accumulation</td><td>ANR</td><td>F. x ananassa</td><td>Fischer et al. (2014)</td></tr><tr><td>Transient RNAi silencing</td><td>Premature accumulation of anthocyanins</td><td>GT</td><td>F. x ananassa</td><td>Griesser et al. (2008)</td></tr><tr><td>Transcriptome<sup>b,c</sup>, metabolome, transient OE and RNAi silencing</td><td>Reduced flavan 3-ols content</td><td>GT</td><td>F. x ananassa</td><td>Yuan et al. (2022)</td></tr><tr><td>Mapping-by-sequencing; transient OE and RNAi silencing</td><td>OE: restoration of fruit pigmentation in mutant</td><td>RAP/GST</td><td>F. vesca</td><td>Luo et al. (2018)</td></tr><tr><td>Stable OE; CRISPR/Cas9 gene editing</td><td>Fruit color mutant in F. vesca ENU population</td><td>RAP/GST</td><td>F. x ananassa</td><td>Gao et al. (2020)</td></tr><tr><td></td><td>OE: restoration of fruit pigmentation in mutant</td><td>RAP/GST</td><td>F. vesca</td><td>Gao et al. (2020)</td></tr><tr><td></td><td>CRISPR: reduced fruit coloration</td><td>RAP/GST</td><td>F. x ananassa</td><td>Gao et al. (2020)</td></tr></tbody></table>

Abbreviations: ENU, N-ethyl-N-nitrosourea; Flav, flavonoids; PP, phenylpropanoids. Enzymes encoded by structural genes: ANR, anthocyanidin reductase; C4H, cinnamate-4-hydroxylase; CHS, chalcone synthase; F3H, flavanone 3 hydrolase; F3'H, flavanone 3'-hydrolase; GST, glutathione S-transferase; GT, anthocyanidin glucosyltransferase; RAP, Reduced Anthocyanins in Petioles.

<sup>a</sup> Microarray transcriptome analysis.

<sup>b</sup> RNAseq transcriptome analysis.

<sup>c</sup> qRT-PCR transcriptome analysis.

visual comparison with a color chart (Labadie et al., 2022). In recent years, a key development was the automatization of the process whereby photographs of fruits taken in standardized conditions are analysed for intensity and pattern of fruit color (Zingaretti et al., 2022). Color hue is usually assessed by colorimeter with the CIELAB color space (Lerceteau-Köhler et al., 2012; Alarfaj et al., 2021). Destructive methods can measure the total anthocyanin content of a fruit extract and tens of different polyphenolic metabolites when using highly sensitive, but more laborious, mass spectrometry (MS)-based technologies (Ring et al., 2013; Urrutia et al., 2016; Crecelius et al. 2017;

Pott et al., 2020; Wang et al., 2020). Detailed knowledge of the composition of anthocyanin-related chemicals in the fruit can further facilitate the identification of structural and regulatory CGs underlying color QTLs (Davik et al., 2020; Labadie et al., 2022).

## Anthocyanin biosynthesis, storage, and regulation

The main classes of flavonoids detected in strawberry (flavanones, anthocyanins, flavonols, flavan-3-ols) are produced through the phenylpropanoid pathway (Fig. 1).
Phenylpropanoids are derived from phenylalanine by successive actions of phenylalanine ammonia-lyase, cinnamate-4-hydroxylase and 4-coumaroyl:CoA-ligase (Fig. 1). Chalcone synthase is the first committed step in the synthesis of flavonoids. Naringenin chalcone (a flavanone) produced by chalcone synthase is converted to naringenin by chalcone isomerase. Naringenin is then converted to pelargonidin, an anthocyanidin, by successive steps catalysed by the flavonoid 3-hydroxylase, dihydroflavonol-4-reductase and anthocyanidin synthase. In a parallel pathway, eriodyctiol produced from naringenin by flavonoid 3'-hydroxylase leads to the production of cyanidin (an anthocyanidin) by flavonoid 3-hydroxylase, dihydroflavonol-4-reductase, and anthocyanidin synthase. Branched pathways produce flavonols (e.g. kaempferol and quercetin) and flavan-3-ols (e.g. epiafzelechin and epicatechin) that produce proanthocyanidins (condensed tannins). Proanthocyanidins accumulate during early fruit development while anthocyanins accumulate during fruit ripening. Pelargonidin and cyanidin, which can be further complexed with flavan-3-ols, are stabilized by anthocyanidin 3-glucosyltransferases, which produce pelargonidin- and cyanidin-glucosides that are stored in the vacuole (Fig. 1). Genes upstream of dihydroflavonols are usually named early biosynthesis genes and downstream genes late biosynthesis genes.

In F × *ananassa*, the main anthocyanin is pelargonidin 3-glucoside while cyanidin 3-glucoside is a minor component; the second most abundant anthocyanin is pelargonidin-3-malonylglucoside, which may represent more than 30% of the anthocyanins, depending on the cultivar (Aaby et al., 2012). Anthocyanin composition can be different in other strawberry species; for example, F. vesca accumulates preferentially cyanidin-glucosides (Urrutia et al., 2016). Leucoanthocyanidins are colorless while anthocyanidins and their derivatives are colored. The relative proportion of the various anthocyanins found in strawberry may have a profound effect on fruit color, with pelargonidin derivatives giving the fruit a bright red appearance and cyanidin derivatives a dark-red appearance (Fischer et al., 2014).

Following their synthesis on the cytoplasmic face of the endoplasmic reticulum, anthocyanins are transported into the vacuole for storage (Fig. 1). The color hue of anthocyanins is sensitive to pH, a process well studied in anthocyanin-colored flowers (Verweij et al., 2008). Therefore, vacuolar localization provides the low pH conditions required to express the intense red coloration of the fruit. Anthocyanin transport may involve several proteins, including glutathione S-transferases that can conjugate glutathione (GSH) to anthocyanins, thus facilitating anthocyanin transfer to the vacuole (Luo et al., 2018; Gao et al., 2020).

The MYB, basic helix-loop-helix (bHLH), and WD40 proteins that form the MBW complex are the main transcriptional regulators of the flavonoid biosynthetic pathway genes (Xu et al., 2015) in strawberry as in other plant species including Rosaceae species such as apple (Espley et al., 2007) (Fig. 1).

In particular, the MYB family play key roles in the positive or negative regulation of proanthocyanidin and anthocyanin genes in developing strawberry (Schaart et al., 2013) and in response to hormonal (abscisic acid, ethylene) treatments (Zhao et al., 2018; Wang et al., 2020; Martínez-Rivas et al., 2023) and abiotic signals such as light and cold, heat, and water stresses (Medina-Puche et al., 2014; Kadomura-Ishikawa et al., 2015; Mao et al., 2022). Various additional transcription factors belonging to different families have been shown to modulate anthocyanin biosynthesis in strawberry (Fig. 1; Table 1).

## Roles of structural anthocyanin-related genes in fruit color formation

Alterations in the expression of enzymes of the phenylpropanoid pathway can lead to changes in anthocyanin content and composition, and thus in the intensity, hue, and/or pattern of color of fruit tissues (Fig. 1). The color diversity found in strawberry genetic resources has been abundantly exploited in diploid and cultivated strawberries to investigate the role of structural and regulatory genes in anthocyanin accumulation, using up-to-date transcriptome and metabolome technologies (Tables 1, 2). An additional source of color diversity that has been exploited is changes in fruit color and associated modifications in the expression of regulatory and structural genes along fruit development and ripening (Tables 1, 2).

As shown in Table 1, since the early identification of phenylpropanoid and flavonoid pathway genes by Almeida et al. (2007), a large number of studies have investigated structural genes involved in anthocyanin biosynthesis and storage and branched pathways in strawberry. Various omics technologies including metabolomics (e.g. LC-electrospray ionization-MS or ultraperformance LC-MS/MS) and transcriptomics (microarrays, RNAseq) used alone or in combination have been used to investigate the contribution of the anthocyanin pathway and, more broadly, of the phenylpropanoid pathway to fruit color in F. vesca and F × *ananassa*. Sources of color variability were fruit developmental stages (green versus ripening stages), response to various signals (abscisic acid, light, temperature) and natural or artificially induced variations in skin and/or flesh color. Most findings have been functionally validated by transient expression assay in the fruit (Hoffmann et al., 2006), a technique well-suited for the visual assessment of the function of anthocyanin-related CGs by overexpression or RNAi silencing. Stable genetic transformation, which is much more time-consuming and tedious, has also been used in F. vesca and F × *ananassa* to evaluate the function of various GCs and their natural variants, including by CRISPR/Cas9 gene editing (Gao et al., 2020) (Box 1). These studies demonstrated that the modulation of the expression of enzymes involved in phenylpropanoid and anthocyanin synthesis or transport effectively affects the quantity and/or composition of fruit anthocyanins. They further pinpointed the important impact on fruit color of the trade-off between the formation of colored
<table><caption>**Table 2.** Regulatory genes with roles in anthocyanin accumulation in strawberry fruit</caption><thead><tr><th>Main strategies</th><th>Trait studied or affected/main findings</th><th>Main regulatory genes</th><th>Species</th><th>References</th></tr></thead><tbody><tr><td>MYB10</td><td>Stable OE and RNAi silencing (MYB10); stable RNAi silencing (bHLH33)</td><td>Color of developing/ripening fruit<br>Increased (OE) or reduced (RNAi) Anth accumulation (MYB10)<br>No effect (bHLH33)</td><td>MYB10<br>bHLH33</td><td>F. vesca</td><td>Lin-Wang et al. (2014)</td></tr><tr><td>Transient RNAi silencing (MYB10), transcriptome<sup>a,c</sup></td><td>Transient OE and RNAi silencing (MYB10)</td><td>Color of developing/ripening fruit, water stress treatment<br>MYB10 gene targets in PP/Flav pathway</td><td>MYB10</td><td>F. x ananassa</td><td>Medina-Puche et al. (2014)</td></tr><tr><td>Transient OE and RNAi silencing (MYB10)</td><td>DNA variant analysis, transient OE of WT and mutant MYB10</td><td>Light and ABA induction of MYB10<br>Reduced (RNAi) Anth accumulation (MYB10)<br>MYB10 gene targets in Flav pathway</td><td>MYB10</td><td>F. x ananassa</td><td>Kadomura-Ishikawa et al. (2015)</td></tr><tr><td>Genetic mapping, candidate gene analysis</td><td>Transcriptome<sup>b,c</sup>, metabolome, transient OE of WT and mutant MYB10 in strawberry and tobacco</td><td>Yellow fruit genotypes<br>MYB10 mutation, restoration of red coloration with OE of WT MYB10 but not mutant MYB10</td><td>MYB10</td><td>F. vesca</td><td>Hawkins et al. (2016)</td></tr><tr><td>Transient OE in fruit and promoter binding activity of WT and mutant MYB10</td><td>QTLseq analysis, transient OE or RNAi of MYB10 in fruit, metabolome, transcriptome<sup>b</sup>, GWAS, Design of genetic marker</td><td>Yellow fruit genotypes<br>MYB10 mutation<br>White-fruited genotype, color in developing fruit, ABA level<br>Restoration of red coloration with OE of MYB10</td><td>MYB10<br>MYB family<br>MYB1<br>MYB10</td><td>F. vesca<br>F. x ananassa</td><td>Zhang et al. (2017)<br>Wang et al. (2020)</td></tr><tr><td>WGS of F. nilgerrensis and comparison with F. vesca, transient OE in tobacco of F. nilgerrensis and F. vesca MYB10</td><td>Transcriptome<sup>b,c</sup>, mapping of color QTLs, candidate gene analysis</td><td>Yellow fruit genotype<br>Restoration of red coloration with OE of WT but not mutant MYB10<br>Biparental populations derived from white-fruited F. vesca and white or red fleshed or skinned F. x ananassa or F. chiloensis genotypes<br>MYB10 loss-of-function or gain-of-function alleles in all three Fragaria spp., functional validation, MYB10 gene targets in PP/Flav pathways</td><td>MYB10</td><td>F. vesca<br>F. x ananassa<br>F. chiloensis</td><td>Chen et al. (2020)<br>Castillejo et al. (2020)</td></tr><tr><td>WGS analysis of diploid Fragaria spp.</td><td>Transcriptome<sup>b,c</sup>, mapping of color QTLs, candidate gene analysis</td><td>White-fruited species<br>Reduced expression of MYB10 in F. nilgerrensis</td><td>MYB10</td><td>F. nilgerrensis<br>F. vesca</td><td>Zhang J et al. (2020)</td></tr><tr><td>WGS</td><td>Other regulatory genes</td><td>Genotypes with skin, flesh and achene color variations, biparental population<br>Co-localization of MYB10 with main color QTL</td><td>MYB10</td><td>F. x ananassa</td><td>Manivannan et al. (2021)</td></tr><tr><td>Stable ectopic expression of Arabidopsis MBW complex genes in strawberry</td><td>Transient RNAi silencing of MYB1</td><td>White/pink/yellow fruit species and accessions<br>Allelic variations of MYB10 in various species linked to fruit color</td><td>MYB10</td><td>F. vesca, F. nubicola, F. pentaphylla, F. nilgerrensis</td><td>Qiao et al. (2021)</td></tr><tr><td>Transient OE of MYB5, promoter binding and protein/protein interaction experiments, transcriptome<sup>b</sup>, metabolome</td><td>Other regulatory genes</td><td>White-fleshed fruit genotype<br>Frameshift mutation in MYB10</td><td>MYB10</td><td>F. x ananassa</td><td>Yuan et al. (2022)</td></tr><tr><td>Ectopic expression in tobacco of strawberry MYB1</td><td>Tobacco flower color</td><td>Tobacco flower color</td><td>MYB1</td><td>F. x ananassa</td><td>Aharoni et al. (2001)</td></tr><tr><td>Transient RNAi silencing of MYB1</td><td>Increased Anth accumulation and decreased flavan-3-ols<br>MYB1 gene targets in Flav pathway</td><td>Increased PA accumulation, decreased Anth accumulation<br>MYB1 gene targets in Flav pathway</td><td>MYB1</td><td>F. chiloensis</td><td>Salvatierra et al. (2013)</td></tr><tr><td>Stable ectopic expression of Arabidopsis MBW complex genes in strawberry</td><td>Increased PA accumulation, decreased Anth accumulation<br>MYB1 gene targets in Flav pathway</td><td>Increased PA accumulation, decreased Anth accumulation<br>MYB1 gene targets in Flav pathway</td><td>MYB9/MYB11, bHLH3, TTG1</td><td>F. x ananassa</td><td>Schaart et al. (2013)</td></tr><tr><td>Transient OE of MYB5, promoter binding and protein/protein interaction experiments, transcriptome<sup>b</sup>, metabolome</td><td>Positive regulation of Anth and PA biosynthesis<br>MYB5 gene targets in Flav pathway</td><td>Positive regulation of Anth and PA biosynthesis<br>MYB5 gene targets in Flav pathway</td><td>MYB5</td><td>F. x ananassa</td><td>Jiang et al. (2023)</td></tr></tbody></table>
<table><caption>**Table 2. Continued**</caption><thead><tr><th>Main strategies</th><th>Trait studied or affected/main findings</th><th>Main regulatory genes</th><th>Species</th><th>References</th></tr></thead><tbody><tr><td>Transient OE of MBW complex genes, promoter-TFs interactions</td><td>MYB5/10 promotion of accumulation of Anth and PA MBW gene targets in Flav pathway</td><td>MYB5–MYB10<br>MYB9–MYB11<br>EGL3–LWD1</td><td>F. × ananassa</td><td>Yue et al. (2023)</td></tr><tr><td>Transcriptome, stable RNAi silencing</td><td>Color of developing/ripening fruit, ABA treatment<br>Regulation of pelargonidin and cyanidin malonyl derivatives</td><td>MYB123<br>bHLH3</td><td>F. × ananassa</td><td>Martinez-Rivas et al. (2023)</td></tr><tr><td>Expression profiling of regulatory genes<sup>c</sup></td><td>Color in developing fruit of high Anth cultivars</td><td>MYB and bHLH families</td><td>F. × ananassa</td><td>Hossain et al. (2018)</td></tr><tr><td>Transcription profiling of bHLH<sup>c</sup></td><td>White-flesh genotypes, color in developing fruit, ABA and ethylene treatments<br>bHLH candidates for Anth accumulation</td><td>bHLH family</td><td>F. × ananassa</td><td>Zhao et al. (2018)</td></tr><tr><td>Ectopic expression in Arabidopsis, transient OE and VIGS silencing in F. vesca</td><td>Light induction of bHLH9<br>Promotion of Anth accumulation by bHLH9 OE and reduction of Anth by VIGS of bHLH9–bHLH9 interaction with HY5 and binding to promoters of Anth-related genes</td><td>bHLH9<br>HY5</td><td>F. vesca</td><td>Li et al. (2020)</td></tr><tr><td>Transient OE and RNAi silencing of MYB10 activating RAV1 (AP2/ERF)</td><td>Promotion of Anth accumulation by RAV1 OE and reduction of Anth by RAV1 RNAi, RAV1 activation of promoters of Flav pathway genes</td><td>RAV1</td><td>F. × ananassa</td><td>Zhang Z et al. (2020)</td></tr><tr><td>Transient OE and RNAi silencing in F. × ananassa, stable OE and CRISPR gene editing in F. vesca</td><td>Low temperature treatment<br>Reduction by MAPK3 of MYB10 transcriptional activity and CHS degradation</td><td>MAPK3</td><td>F. × ananassa<br>F. vesca</td><td>Mao et al. (2022)</td></tr><tr><td>Transcriptome<sup>b,c</sup>, transient RNAi silencing and OE of candidate TFs</td><td>Biparental population<br>TF gene targets in Flav pathway</td><td>SCL8<br>TCP11</td><td>F. × ananassa</td><td>Pillet et al. (2015)</td></tr></tbody></table>

Abbreviations: Anth, anthocyanins; Flav, flavonoids; OE, overexpression; PA, proathocyanidins; PP, phenylpropanoids; RNAi, RNA interference; TF, transcription factor; VIGS, virus induced gene silencing; WGS, whole genome sequencing. Proteins encoded by regulatory genes: bHLH, basic helix-loop-helix transcription factor; HY5, Long Hypocotyl 5; MAPK3, Map Kinase 3; MBW, MYB–bHLH–WD40 complex; MYB, myeloblastosis transcription factor; RAV1, Related to Abscisic Acid Insensitive 3 (ABI3)/Viviparous 1 (VP1) transcription factor; SCL8, SCARECROW-LIKE 8; TCP11, Teosinte branched, Cycloidea, and PCF transcription factor; WD40, WD40-repeat protein.

anthocyanins and that of colorless compounds synthesized via branched pathways, an example of which is the synthesis by anthocyanidin reductase (ANR) of flavan-3-ols at the expense of anthocyanins (Fischer et al., 2014).

## MYB10 emerges as a master regulator of anthocyanin content and pattern in the fruit

Similar genetic resources and strategies have been used to identify regulatory genes involved in the control of fruit color (Table 2). Their possible roles in the regulation of fruit anthocyanins have been further validated in planta by transgenic or transient gene expression assays and for transcription factors by mechanistic studies (e.g. protein-protein interactions and promoter-transcription factor binding assays). Not surprisingly, given the well-established role of the MBW ternary complex in phenylpropanoid regulation (Xu et al., 2015), various members of the MYB family have been shown to play a prominent role in anthocyanin accumulation in strawberry. In most

cases, their gene targets could be determined (Fig. 1; Table 2). MYB10 regulates the expression of early biosynthesis and late biosynthesis genes and therefore emerged as a master regulator of fruit color in strawberry (Medina-Puche et al., 2014). Explorations of diverse accessions of natural white/yellow-fruited genotypes of F. vesca, F. nilgerrensis, and F. × ananassa led to the discovery of various loss-of-function MYB10 mutations that impaired anthocyanin accumulation in the fruit (Hawkins et al., 2016; Zhang et al., 2017; Chen et al., 2020; Wang et al., 2020; Yuan et al., 2022). In addition, genome sequencing of numerous accessions of five diploid Fragaria species uncovered multiple independent single base mutations of the MYB10 gene associated with white fruit in several diploid species (Qiao et al., 2021). A key development was the systematic analysis of MYB10 alleles in F. vesca, F. chiloensis, and F. × ananassa (Castillejo et al., 2020) (Table 2; Box 1). By studying various F. chiloensis accessions and biparental populations issued from either white-fruited F. vesca or from white- and red-fleshed or skinned F. × ananassa, there were uncovered not only a range of

<footnote><sup>a</sup> Microarray transcriptome analysis.</footnote>

<footnote><sup>b</sup> RNAseq transcriptome analysis.</footnote>

<footnote><sup>c</sup> qRT-PCR transcriptome analysis.</footnote>
different loss-of-function alleles but also a gain-of-function allele responsible for the red-color of the fruit flesh, from which a genetic marker was derived (Box 1). The gain-of-function is due to the insertion of a transposon in the *MYB10* promoter that triggers anthocyanin biosynthesis. Haplotype sequencing of ‘Yanli’ coupled with transcriptome analysis of the ripe fruit provided further insight into the structural diversity and expression complexity of anthocyanin-related homoeoalleles (Mao *et al.*, 2023). It showed, for example, that homoeoalleles of several anthocyanin structural genes were absent from one or several chromosomes while a dominant *MYB10* homoeoallele located in a single chromosome from the *F. iinumae* subgenome was responsible for ~75% of *MYB10* expression (Mao *et al.*, 2023). Upstream in the regulatory network, a MAP kinase (MAPK3) was shown to reduce the *MYB10* transcriptional activity in response to cold (Mao *et al.*, 2022).

More *MYB* genes regulating the flavonoid pathway in strawberry fruit have been characterized recently, including *MYB5*, *MYB9*, *MYB11*, *MYB123*, and the negative regulator *MYB1* (Table 2). Other members of the MBW complex that were studied include *bHLH* genes (*bHLH3*, *bHLH9*, *bHLH33*) and WD40 genes (Zhao *et al.*, 2018; Yue *et al.* 2023). Additional transcription factors of various families including TCP11 and SLC8 (Pillet *et al.*, 2015), HY5 (Li *et al.*, 2020) and an AP2/ERF (RAV1) (Z. Zhang *et al.*, 2020) were also shown to be involved in the regulation of anthocyanins in strawberry. These results further demonstrate that, in addition to *MYB10*, many actors may participate in the regulation of the natural variations in fruit color observed in genetic resources.

## Beyond *MYB10*: using untapped genetic diversity to discover new natural variations in fruit color

Thanks to the exploration of anthocyanin-related pathways and genes in *Fragaria*, the number of color-related CGs has greatly increased in recent years. These findings already provide a solid basis for understanding anthocyanin accumulation in the fruit. However, the demonstration by transient or stable genetic transformation that a CG affects fruit color is usually not sufficient to ascertain its value in the genetic control of color. Moreover, the octoploid status of *F. × ananassa* raises specific issues regarding loss-of-function mutations. As the inactivation of one or several homoeoalleles can be compensated by the expression of other homoeoalleles, loss-of-function mutations may have an impact on fruit color only if at most one or a few homoeoalleles are active in *F. × ananassa*. This is indeed the case for the various loss-of-function mutations found in the *F. × ananassa* *MYB10* gene (Table 2). They likely produce white/yellow fruits in *F. × ananassa* (Table 2) only because at most two *MYB10* homoeoalleles of the same subgenome are expressed in the fruit (Castillejo *et al.*, 2020; Mao *et al.*, 2023). In contrast, genetic variations resulting in the overexpression or enhanced enzymatic activity of a single homoeoallele have likely a dominant effect on fruit color, whatever the number

of active homoeoalleles in the genome. The few examples of CG-based genetic markers for color selection by marker-assisted selection, e.g. *MYB10* (Castillejo *et al.*, 2020) or *ANR* (Labadie *et al.*, 2022), were designed after natural genetic variations present in regulatory regions of the CGs. Information on the number and genomic localization of expressed CG homoeoalleles is therefore precious for the design of genetic markers, or the simultaneous edition of all the homoeoalleles (Gao *et al.*, 2020). These findings also underscore the importance of using the large untapped diversity found in cultivated strawberry for color improvement.

A way to combine a CG catalog with the diversity of genetic resources is to analyse expression QTLs, with the aim to link natural variations of CG transcript level to color variations and thereby develop reliable markers for selection in *F. × ananassa* (Barbey *et al.*, 2020) (Table 3). However, this approach limits the focus to known expressional CGs and does not allow the discovery of new genetic variants with original functions. To further explore the color diversity, biparental or multiparental (Wada *et al.*, 2017) populations can be used to map chromosome regions responsible for quantitative variations of fruit color. Since the early studies (Zorrilla-Fontanesi *et al.*, 2011; Lerceteau-Köhler *et al.*, 2012) (Table 3), the mapping resolution of QTL analyses has been considerably increased and biochemical technologies now permit the identification and quantification of tens of secondary metabolites related to fruit color and nutritional value (e.g. in Pott *et al.*, 2020) (Box 1).

The development of high-throughput genotyping arrays (Hardigan *et al.*, 2020) now permits the high-resolution mapping of a color QTL to a narrow genomic region on a single subgenome (Davik *et al.*, 2020; Pott *et al.*, 2020; Manivannan *et al.*, 2021; Labadie *et al.*, 2022) (Table 3; Box 1). Once a CG has been identified in the QTL region, by mining literature or combining transcriptome and metabolome analyses with genetic data, WGS data obtained from the parents can be analysed to identify GC polymorphisms, design genetic markers and validate them for breeding. This strategy has been, for example, used to design an *ANR*-specific homeoallelic marker for a major color (pelargonidin-3-glucoside content) QTL (Labadie *et al.*, 2022). High-resolution color QTLs can also be detected by screening genetic resources by genome-wide association studies (GWAS). A GWAS scans the genome for significant associations between genetic markers and the trait studied and is thus an alternative to the genetic mapping of QTLs. It can efficiently link color variations to the genetic information provided by genotyping arrays, as done for strawberry aroma (Fan *et al.*, 2022), or by genotyping by sequencing (Vining *et al.*, 2017). The recent completion of haplotype-resolved genomes for several *F. × ananassa* varieties (Hardigan *et al.*, 2021a, Preprint; Lee *et al.*, 2021; Fan *et al.*, 2022; Mao *et al.*, 2023) (Box 1) opens the way to accurately determining the chromosomal location and polymorphisms of the homeoallelic variants responsible for color variation. However, care
<table><caption>**Table 3.** Genetic analysis of quantitative variations of fruit color in strawberry</caption><thead><tr><th>Main strategies: QTL and GWAS analyses</th><th>Fruit trait studied or affected/main findings</th><th>Pathways or candidate genes</th><th>Species</th><th>References</th></tr></thead><tbody><tr><td>Mapping of fruit color QTLs</td><td>Diversity of fruit color and Anth content in biparental population</td><td>—</td><td>F. × ananassa</td><td>Zorrilla-Fontanesi et al. (2011)</td></tr><tr><td>Mapping of fruit color QTLs</td><td>Diversity of fruit color and Anth content in biparental population</td><td>—</td><td>F. × ananassa</td><td>Lerceteau-Köhler et al. (2012)</td></tr><tr><td>Metabolome, transcriptome<sup>a</sup>, mapping of color QTLs and mQTLs of polyphenolic compounds, transient RNAi silencing</td><td>Diversity of fruit color and PP/Flav compounds in biparental populations</td><td><strong>POD</strong></td><td>F. × ananassa</td><td>Ring et al. (2013)</td></tr><tr><td>Mapping of fruit color QTLs</td><td>Diversity of fruit color in biparental population</td><td>—</td><td>F. × ananassa</td><td>Castro and Lewers (2016)</td></tr><tr><td>Metabolome, mapping of mQTLs of polyphenolic compounds</td><td>Diversity of PP/Flav compounds in near isogenic lines population</td><td>PP/Flav candidate genes</td><td>F. vesca</td><td>Urrutia et al. (2016)</td></tr><tr><td>eQTLs of PP/Flav candidate genes</td><td>Diversity of fruit color in biparental populations</td><td>PP/Flav candidate genes</td><td>F. × ananassa</td><td>Barbey et al. (2020)</td></tr><tr><td>GWAS and mapping of mQTLs of polyphenolic compounds</td><td>Diversity of PP/Flav compounds in biparental populations</td><td>PP/Flav candidate genes</td><td>F. × ananassa</td><td>Davik et al. (2020)</td></tr><tr><td>Metabolome, mapping of mQTLs of polyphenolic compounds</td><td>Diversity of PP/Flav compounds in biparental populations</td><td><strong>MT</strong></td><td>F. × ananassa</td><td>Pott et al. (2020)</td></tr><tr><td>Mapping of fruit color QTLs</td><td>Diversity of fruit color and PP/Flav compounds in biparental population</td><td><strong>FaF3'H</strong></td><td>F. × ananassa</td><td>Alarfaj et al. (2021)</td></tr><tr><td>Transcriptome<sup>b,c</sup>, mapping of fruit color QTLs</td><td>Diversity of fruit skin, flesh and achene color in biparental population, parental line with pink skin and white flesh and core color</td><td>PP/Flav candidate genes</td><td>F. × ananassa</td><td>Manivannan et al. (2021)</td></tr><tr><td>Metabolome, transcriptome<sup>a</sup>, mapping of fruit color QTLs and mQTLs of polyphenolic compounds, WGS, design of genetic marker</td><td>Diversity of fruit color and PP/Flav compounds content in biparental population</td><td>PP/Flav candidate genes</td><td>F. × ananassa</td><td>Labadie et al. (2022)</td></tr></tbody></table>

Proteins encoded by main candidate genes in bold are: ANR, F3'H, POD.

Abbreviations: ANR, anthocyanidin reductase; Anth, anthocyanins; eQTL, expression QTL; F3'H, Flavanone 3'-hydrolase; Flav, flavonoids; GWAS, genome wide association studies; MT, malonyltransferase; POD, Peroxidase; PP, phenylpropanoids; QTL, quantitative trait locus; WGS, whole genome sequencing.

must be taken in the interpretation of GWAS data, as indirect association may produce statistically significant results at loci unrelated to the trait (Platt et al., 2010).

## Towards the selection of strawberry varieties with improved fruit color

In addition to its potential value in understanding the mechanisms involved in fruit color, information on the genetic architecture of fruit color can be used to select strawberry varieties with desirable traits. Once identified and validated, genetic variations underlying a major color trait can be translated to strawberry color improvement through marker-assisted selection (Castillejo et al., 2020; Labadie et al., 2022). Because multiple homoeoalleles can be simultaneously edited by CRISPR/Cas9 (Gao et al., 2020), gene editing can also be used to validate the findings or even engineer new strawberry varieties (Box 1).

Of course, to select superior strawberry varieties, breeders must take into account additional traits linked to the quality of the fruit (sweetness, acidity, aroma, shape, etc.) and the plant (resistance to pathogens, flowering pattern, yield, etc.) (Labadie et al., 2020; Whitaker et al., 2020; Senger et al., 2022). Combining several markers linked to various traits is an effective way of accelerating marker-assisted selection or selection by means of the genomic prediction models (genomic selection, GS) recently developed. GS links the traits studied, including fruit color, to genome-wide molecular markers to select the most suitable individuals from the population to breed new strawberry varieties with the desired traits. GS has been explored for strawberry resistance to Botritis cinerea (Petrasch et al., 2022) and Phytophtora cactorum (Jiménez et al., 2022) and fruit quality and yield (Gezan et al., 2017; Osorio et al., 2021). GS can use SNP markers and machine learning approaches to determine the genetic value of unseen lines for fruit quality traits, e.g. fruit color and shape in Solanaceae (Tong et al., 2022). As previous

<footnote><sup>a</sup> Microarray transcriptome analysis.</footnote>

<footnote><sup>b</sup> RNAseq transcriptome analysis.</footnote>

<footnote><sup>c</sup> qRT-PCR transcriptome analysis.</footnote>
knowledge of causal genetic variations is not required, GS can be well adapted to the selection of color traits in strawberry where, except for major QTLs such as *MYB10*, color intensity is mostly governed by numerous small QTLs (Davik *et al.*, 2020; Pott *et al.*, 2020; Labadie *et al.*, 2022). A recent example is the application of GS to the selection of strawberry F₁ hybrids with high fruit firmness or high pericarp color (Yamamoto *et al.*, 2021).

## Conclusion and perspectives

The wide diversity of fruit color intensity and pattern found in cultivated strawberry and its wild relatives can help decipher the genetic architecture of fruit color. Gaining insight into the genetic factors affecting natural variations in external (skin) and internal (flesh) fruit color is crucial for the efficient modification of this trait in new cultivars. Considerable work has been already done to identify structural and regulatory genes involved in the accumulation of red anthocyanins in the fruit. Among these is the *MYB10* gene whose natural variations play key roles in clear-cut color variations of *Fragaria* species.

A recent breakthrough advance for the allo-octoploid F × *ananassa* is the development of accurate long-read sequencing technologies that, when coupled with high-throughput genotyping, enable tackling of the untapped color diversity of strawberry genetic resources. Given the plummeting cost of these technologies (Eisenstein, 2023), we may envisage in the near future that haplotype-resolved genome sequences can be obtained for several of the genotypes under investigation, or even all of them. Providing that robust phenotyping technologies can be implemented, this should considerably accelerate the pace at which we decipher the genetic architecture of fruit color in strawberry. High-resolution mapping of QTLs to a distinct chromosome allows mining candidate genes and polymorphisms in a narrow genomic region and combining this information with expression analysis of homoeoalleles. A recent strawberry flavor study that combined multi-omics (transcriptome, metabolome), haplotype sequencing, and GWAS could successfully uncover fruit flavor genes and their regulatory elements (Fan *et al.*, 2022), thus providing a roadmap for exploring the color of strawberry. In the near future, we may expect that, in addition to known color-related genes, genes of previously unknown function are isolated. While this should deepen our understanding of color control in strawberry, it can also be very challenging.

## Author contributions

CR wrote the manuscript with contributions of BD. AP commented on GWAS and GS. JP commented on biochemical pathways and performed the artwork. All authors including BD, AP, JP, and CR edited the manuscript.

## Conflict of interest

The authors declare they have no conflicts of interest.

## Funding

The authors gratefully acknowledge support from Région Nouvelle-Aquitaine (AgirClim project No. 2018-1R20202) and European Union's Horizon 2020 research and innovation program (BreedingValue project No. 101000747). We apologize to our colleagues whose work could not be cited due to space limitations and constraints to cite mainly publications from the last 3 years.

## References

Aaby K, Mazur S, Nes A, Skrede G. 2012. Phenolic compounds in strawberry (*Fragaria* × *ananassa* Duch.) fruits: Composition in 27 cultivars and changes during ripening. Food Chemistry 132, 86–97.

Aharoni A, De Vos CH, Wein M, Sun Z, Greco R, Kroon A, Mol JN, O'Connell AP. 2001. The strawberry FaMYB1 transcription factor suppresses anthocyanin and flavonol accumulation in transgenic tobacco. The Plant Journal 28, 319–332.

Alarfaj R, El-Soda M, Antanaviciute L, Vickerstaff R, Hand P, Harrison RJ, Wagstaff C. 2021. Mapping QTL underlying fruit quality traits in an F₁ strawberry population. The Journal of Horticultural Science and Biotechnology 96, 634–645.

Alger EI, Platts AE, Deb SK, et al. 2021. Chromosome-scale genome for a red-fruited, perpetual flowering and runnerless woodland strawberry (*Fragaria vesca*). Frontiers in Genetics 12, 671371. doi:10.3389/fgene.2021.671371

Almeida JR, D'Amico E, Preuss A, et al. 2007. Characterization of major enzymes and genes involved in flavonoid and proanthocyanidin biosynthesis during fruit development in strawberry (*Fragaria × nananassa*). Archives of Biochemistry and Biophysics 465, 61–71. doi:10.1016/j.abb.2007.04.040.

Barbey C, Hogshead M, Schwartz AE, Mourad N, Verma S, Lee S, Whitaker VM, Folta KM. 2020. The genetics of differential gene expression related to fruit traits in strawberry (*Fragaria × nananassa*). Frontiers in Genetics 10, 1317. doi:10.3389/fgene.2019.01317.

Bassil NV, Davis TM, Zhang H, et al. 2015. Development and preliminary evaluation of a 90 K Axiom® SNP array for the allo-octoploid cultivated strawberry *Fragaria × nananassa*. BMC Genomics 16, 155. doi:10.1186/s12864-015-1310-1

Bird KA, Hardigan MA, Ragsdale AP, Knapp SJ, VanBuren R, Edger PP. 2021. Diversification, spread, and admixture of octoploid strawberry in the Western Hemisphere. American Journal of Botany 108, 2269–2281. doi:10.1002/ajb2.1776

Carbone F, Preuss A, De Vos RC, D'Amico E, Perrotta G, Bovy AG, Martens S, Rosati C. 2009. Developmental, genetic and environmental factors affect the expression of flavonoid genes, enzymes and metabolites in strawberry fruits. Plant, Cell and Environment 32, 1117–1131. doi:10.1111/j.1365-3040.2009.01994.x.

Castillejo C, Waurich V, Wagner H, et al. 2020. Allelic variation of MYB10 is the major force controlling natural variation in skin and flesh color in strawberry (*Fragaria* spp.) fruit. The Plant Cell 32, 3723–3749. doi:10.1105/tpc.20.00474.

Castro P, Lewers KS. 2016. Identification of quantitative trait loci (QTL) for fruit-quality traits and number of weeks of flowering in the cultivated strawberry. Molecular Breeding 36, 138.

Chen G, Xu P, Pan J, Li Y, Zhou J, Kuang H, Lian H. 2020. Inhibition of FvMYB10 transcriptional activity promotes color loss in strawberry fruit. Plant Science 298, 110578. doi:10.1016/j.plantsci.2020.110578

Crecelius AC, Hölscher D, Hoffmann T, Schneider B, Fischer TC, Hanke MV, Flachowsky H, Schwab W, Schubert US. 2017. Spatial and
temporal localization of flavonoid metabolites in strawberry fruit (*Fragaria × ananassa*). Journal of Agricultural and Food Chemistry **65**, 3559–3568. doi:10.1021/acs.jafc.7b00584.

Darrow G. 1966. The Strawberry: History Breeding and Physiology. New York: Holt, Rinehart and Winston.

Davik J, Aaby K, Buti M, Alsheikh M, Šurbanovski N, Martens S, Røen D, Sargent DJ. 2020. Major-effect candidate genes identified in cultivated strawberry (*Fragaria × ananassa* Duch.) for ellagic acid deoxyhexoside and pelargonidin-3-O-malonylglucoside biosynthesis, key polyphenolic compounds. Horticulture Research **7**, 125. doi:10.1038/s41438-020-00347-4.

Dumschott K, Schmidt MH, Chawla HS, Snowdon R, Usadel B. 2020. Oxford Nanopore sequencing: new opportunities for plant genomics? Journal of Experimental Botany **71**, 5313–5322. doi:10.1093/jxb/eraa263.

Edger PP, McKain MR, Yocca AE, Knapp SJ, Qiao Q, Zhang T. 2020. Reply to: Revisiting the origin of octoploid strawberry. Nature Genetics **52**, 5–7. doi:10.1038/s41588-019-0544-2.

Edger PP, Poorten TJ, VanBuren R, et al. 2019. Origin and evolution of the octoploid strawberry genome. Nature Genetics **51**, 541–547. doi:10.1038/s41588-019-0356-4.

Edger PP, VanBuren R, Colle M, et al. 2018. Single-molecule sequencing and optical mapping yields an improved genome of woodland strawberry (*Fragaria vesca*) with chromosome-scale contiguity. GigaScience **7**, gix124. doi:10.1093/gigascience/gix124.

Eisenstein M. 2023. Illumina faces short-read rivals. Nature Biotechnology **41**, 3–5. doi:10.1038/s41587-022-01632-4.

Espley RV, Hellens RP, Putterill J, Stevenson DE, Kutty-Amma S, Allan AC. 2007. Red colouration in apple fruit is due to the activity of the MYB transcription factor, MdMYB10. The Plant Journal **49**, 414–427. doi:10.1111/j.1365-313X.2006.02964.x.

Fan Z, Tieman DM, Knapp SJ, et al. 2022. A multi-omics framework reveals strawberry flavor genes and their regulatory elements. New Phytologist **236**, 1089–1107. doi:10.1111/nph.18416.

Feng C, Wang J, Harris AJ, Folta KM, Zhao M, Kang M. 2021. Tracing the diploid ancestry of the cultivated octoploid strawberry. Molecular Biology and Evolution **38**, 478–485. doi:10.1093/molbev/msaa238.

Feng C, Wang J, Liston A, Kang M. 2023. Recombination variation shapes phylogeny and introgression in wild diploid strawberries. Molecular Biology and Evolution **40**, msad049. doi:10.1093/molbev/msad049.

Finn CE, Retamales JB, Lobos GA, Hancock JH. 2013. The Chilean strawberry (*Fragaria chiloensis*): over 1000 years of domestication. HortScience **48**, 418–421.

Fischer TC, Mirbeth B, Rentsch J, Sutter C, Ring L, Flachowsky H, Habegger R, Hoffmann T, Hanke MV, Schwab W. 2014. Premature and ectopic anthocyanin formation by silencing of anthocyanidin reductase in strawberry (*Fragaria × ananassa*). New Phytologist **201**, 440–451. doi:10.1111/nph.12528.

Gao Q, Luo H, Li Y, Liu Z, Kang C. 2020. Genetic modulation of RAP alters fruit coloration in both wild and cultivated strawberry. Plant Biotechnology Journal **18**, 1550–1561. doi:10.1111/pbi.13317.

Gaston A, Osorio S, Denoyes B, Rothan C. 2020. Applying the Solanaceae strategies to strawberry crop improvement. Trends in Plant Science **25**, 130–140. doi:10.1016/j.tplants.2019.10.003.

Gezan SA, Osorio LF, Verma S, Whitaker VM. 2017. An experimental validation of genomic selection in octoploid strawberry. Horticulture Research **4**, 16070. doi:10.1038/hortres.2016.70.

Griesser M, Hoffmann T, Bellido ML, Rosati C, Fink B, Kurtzer R, Aharoni A, Muñoz-Blanco J, Schwab W. 2008. Redirection of flavonoid biosynthesis through the down-regulation of an anthocyanidin glucosyltransferase in ripening strawberry fruit. Plant Physiology **146**, 1528–1539. doi:10.1104/pp.107.114280.

Hancock JF. 1999. Strawberries. Wallingford, UK: CABI Publishing.

Hardigan MA, Feldmann MJ, Lorant A, Bird KA, Famula R, Acharya C, Cole G, Edger PP, Knapp SJ. 2020. Genome synteny has been conserved among the octoploid progenitors of cultivated strawberry over millions of years of evolution. Frontiers in Plant Science **10**, 1789. doi:10.3389/fpls.2019.01789.

Hardigan MA, Feldmann MJ, Pincot DDA, et al. 2021a. Blueprint for phasing and assembling the genomes of heterozygous polyploids: application to the octoploid genome of strawberry. bioRxiv, 467115. doi:10.1101/2021.11.03.467115. [Preprint].

Hardigan MA, Lorant A, Pincot DDA, et al. 2021b. Unraveling the complex hybrid ancestry and domestication history of cultivated strawberry. Molecular Biology and Evolution **38**, 2285–2305. doi:10.1093/molbev/msab024.

Härtl K, Denton A, Franz-Oberdorf K, Hoffmann T, Spornraft M, Usadel B, Schwab W. 2017. Early metabolic and transcriptional variations in fruit of natural white-fruited *Fragaria vesca* genotypes. Scientific Reports **7**, 45113. doi:10.1038/srep45113.

Hawkins C, Caruana J, Li J, Zawora C, Darwish O, Wu J, Alkharouf N, Liu Z. 2017. An eFP browser for visualizing strawberry fruit and flower transcriptsomes. Horticulture Research **4**, 17029. doi:10.1038/hortres.2017.29.

Hawkins C, Caruana J, Schiksnis E, Liu Z. 2016. Genome-scale DNA variant analysis and functional validation of a SNP underlying yellow fruit color in wild strawberry. Scientific Reports **6**, 29017. doi:10.1038/srep29017.

Hoffmann T, Kalinowski G, Schwab W. 2006. RNAi-induced silencing of gene expression in strawberry fruit (*Fragaria × ananassa*) by agroinfiltration: a rapid assay for gene function analysis. The Plant Journal **48**, 818–826. doi:10.1111/j.1365-313X.2006.02913.x.

Hon T, Mars K, Young G, et al. 2020. Highly accurate long-read HiFi sequencing data for five complex genomes. Scientific Data **7**, 399. doi:10.1038/s41597-020-00743-4.

Hossain MR, Kim H-T, Shanmugam A, Nath UK, Goswami G, Song J-Y, Park J-I, Nou I-S. 2018. Expression profiling of regulatory and biosynthetic genes in contrastingly anthocyanin rich strawberry (*Fragaria × ananassa*) cultivars reveals key genetic determinants of fruit color. International Journal of Molecular Sciences **19**, 656. doi:10.3390/ijms19030656.

Jiang L, Yue M, Liu Y, Ye Y, Zhang Y, Lin Y, Wang X, Chen Q, Tang H. 2022. Alterations of phenylpropanoid biosynthesis lead to the natural formation of pinkish-skinned and white-fleshed strawberry (*Fragaria × ananassa*). International Journal of Molecular Sciences **23**, 7375. doi:10.3390/ijms23137375.

Jiang L, Yue M, Liu Y, et al. 2023. A novel R2R3-MYB transcription factor FaMYB5 positively regulates anthocyanin and proanthocyanidin biosynthesis in cultivated strawberries (*Fragaria × ananassa*). Plant Biotechnology Journal **21**, 1140–1158. doi:10.1111/pbi.14024.

Jiménez NP, Feldmann MJ, Famula RA, Pincot DDA, Bjornson M, Cole GS, Knapp SJ. 2022. Harnessing underutilized gene bank diversity and genomic prediction of cross usefulness to enhance resistance to *Phytophthora cactorum* in strawberry. The Plant Genome **16**, e20275. doi:10.1002/tpg2.20275.

Jung S, Lee T, Cheng CH, et al. 2019. 15 years of GDR: New data and functionality in the Genome Database for Rosaceae. Nucleic Acids Research **47**, D1137–D1145. doi:10.1093/nar/gky1000.

Kadomura-Ishikawa Y, Miyawaki K, Takahashi A, Masuda T, Noji S. 2015. Light and abscisic acid independently regulated FaMYB10 in *Fragaria × ananassa* fruit. Planta **241**, 953–965. doi:10.1007/s00425-014-2228-6.

Labadie M, Vallin G, Petit A, Ring L, Hoffmann T, Gaston A, Potier A, Schwab W, Rothan C, Denoyes B. 2020. Quantitative trait loci for flavonoids provide new insights into the genetic architecture of strawberry (*Fragaria × ananassa*) fruit quality. Journal of Agricultural and Food Chemistry **68**, 6927–6939. doi:10.1021/acs.jafc.0c01855.

Labadie M, Vallin G, Potier A, et al. 2022. High resolution quantitative trait locus mapping and whole genome sequencing enable the design of an anthocyanidin reductase-specific homoeo-allelic marker for fruit colour improvement in octoploid strawberry (*Fragaria × ananassa*). Frontiers in Plant Science **13**, 869655. doi:10.3389/fpls.2022.869655.

Lee HE, Manivannan A, Lee SY, et al. 2021. Chromosome level assembly of homozygous inbred line 'Wongyo 3115' facilitates the construction of a high-density linkage map and identification of QTLs associated with fruit firmness in octoploid strawberry (*Fragaria × ananassa*). Frontiers in Plant Science **12**, 696229. doi:10.3389/fpls.2021.696229.

Lerceteau-Köhler E, Moing A, Guérin G, Renaud C, Petit A, Rothan C, Denoyes B. 2012. Genetic dissection of fruit quality traits in the octoploid cultivated strawberry highlights the role of homoeo-QTL in their
control. Theoretical and Applied Genetics **124**, 1059–1077. doi:10.1007/s00122-011-1769-3.

Li Y, Pi M, Gao Q, Liu Z, Kang C. 2019. Updated annotation of the wild strawberry *Fragaria vesca* V4 genome. Horticulture Research **6**, 61. doi:10.1038/s41438-019-0142-6.

Li Y, Xu P, Chen G, Wu J, Liu Z, Lian H. 2020. FvbHLH9 functions as a positive regulator of anthocyanin biosynthesis by forming a HY5-bHLH9 transcription complex in strawberry fruits. Plant and Cell Physiology **61**, 826–837. doi:10.1093/pcp/pcaa010.

Lin Y, Jiang L, Chen Q, Li Y, Zhang Y, Luo Y, Zhang Y, Sun B, Wang X, Tang H. 2018. Comparative transcriptome profiling analysis of red- and white-fleshed strawberry (*Fragaria × ananassa*) provides new insight into the regulation of the anthocyanin pathway. Plant and Cell Physiology **59**, 1844–1859. doi:10.1093/pcp/pcy098.

Lin-Wang K, McGhie TK, Wang M, Liu Y, Warren B, Storey R, Espley RV, Allan AC. 2014. Engineering the anthocyanin regulatory complex of strawberry (*Fragaria vesca*). Frontiers in Plant Science **5**, 651. doi:10.3389/fpls.2014.00651.

Liston A, Cronn R, Ashman T-L. 2014. *Fragaria*: a genus with deep historical roots and ripe for evolutionary and ecological insights. American Journal of Botany **101**, 1686–1699.

Liston A, Wei N, Tennessen JA, Li J, Dong M, Ashman TL. 2020. Revisiting the origin of octoploid strawberry. Nature Genetics **52**, 2–4. doi:10.1038/s41588-019-0543-3.

Luo H, Dai C, Li Y, Feng J, Liu Z, Kang C. 2018. *Reduced Anthocyanins in Petioles* codes for a GST anthocyanin transporter that is essential for the foliage and fruit coloration in strawberry. Journal of Experimental Botany **69**, 2595–2608. doi:10.1093/jxb/ery096.

Manivannan A, Han K, Lee SY, Lee HE, Hong JP, Kim J, Lee YR, Lee ES, Kim DS. 2021. Genome-wide analysis of MYB10 transcription factor in *Fragaria* and identification of QTLs associated with fruit color in octoploid strawberry. International Journal of Molecular Sciences **22**, 12587. doi:10.3390/ijms222212587.

Mao W, Han Y, Chen Y, et al. 2022. Low temperature inhibits anthocyanin accumulation in strawberry fruit by activating FvMAPK3-induced phosphorylation of FvMYB10 and degradation of Chalcone Synthase 1. The Plant Cell **34**, 1226–1249. doi:10.1093/plcell/koac006.

Mao J, Wang Y, Wang B, et al. 2023. High-quality haplotype-resolved genome assembly of cultivated octoploid strawberry. Horticulture Research **10**, uhad002. doi:10.1093/hr/uhad002.

Martínez-Rivas FJ, Blanco-Portales R, Pérez-Serratosa M, et al. 2023. FaMYB123 interacts with FabHLH3 to regulate the late steps of anthocyanin and flavonoid accumulation during ripening. The Plant Journal **114**, 683–698. doi:10.1111/tpj.16166.

Mattus-Araya E, Guajardo J, Herrera R, Moya-León MA. 2022. ABA speeds up the progress of color in developing *F. chiloensis* fruit through the activation of *PAL*, *CHS* and *ANS*, key genes of the phenylpropanoid/flavonoid and anthocyanin pathways. International Journal of Molecular Sciences **23**, 3854. doi:10.3390/ijms23073854.

Medina-Puche L, Cumplido-Laso G, Amil-Ruiz F, Hoffmann T, Ring L, Rodríguez-Franco A, Caballero JL, Schwab W, Muñoz-Blanco J, Blanco-Portales R. 2014. MYB10 plays a major role in the regulation of flavonoid/phenylpropanoid metabolism during ripening of *Fragaria × ananassa* fruits. Journal of Experimental Botany **65**, 401–417. doi:10.1093/jxb/ert377.

Mezzetti B, Giampieri F, Zhang YT, Zhong CF. 2018. Status of strawberry breeding programs and cultivation systems in Europe and the rest of the world. Journal of Berry Research **8**, 205–221. doi:10.3233/JBR-180314.

Miller K, Feucht W, Schmid M. 2019. Bioactive compounds of strawberry and blueberry and their potential health effects based on human intervention studies: a brief overview. *Nutrients* **11**, 1510. doi:10.3390/nu11071510.

Osorio LF, Gezan SA, Verma S, Whitaker VM. 2021. Independent validation of genomic prediction in strawberry over multiple cycles. Frontiers in Genetics **11**, 596258. doi:10.3389/fgene.2020.596258.

Petrasch S, Mesquida-Pesci SD, Pincot DDA, et al. 2022. Genomic prediction of strawberry resistance to postharvest fruit decay caused by

the fungal pathogen *Botrytis cinerea*. G3: Genes, Genomes, Genetics **12**, jkab378. doi:10.1093/g3journal/jkab378.

Pillet J, Yu HW, Chambers AH, Whitaker VM, Folta KM. 2015. Identification of candidate flavonoid pathway genes using transcriptome correlation network analysis in ripe strawberry (*Fragaria × ananassa*) fruits. Journal of Experimental Botany **66**, 4455–4467. doi:10.1093/jxb/erv205.

Platt A, Vilhjálmsson BJ, Nordborg M. 2010. Conditions under which genome-wide association studies will be positively misleading. Genetics **186**, 1045–1052. doi:10.1534/genetics.110.121665.

Pott DM, Vallarino JG, Cruz-Rus E, Willmitzer L, Sánchez-Sevilla JF, Amaya I, Osorio S. 2020. Genetic analysis of phenylpropanoids and antioxidant capacity in strawberry fruit reveals mQTL hotspots and candidate genes. Scientific Reports **10**, 20197. doi:10.1038/s41598-020-76946-x.

Qiao Q, Edger PP, Xue L, et al. 2021. Evolutionary history and pan-genome dynamics of strawberry (*Fragaria* spp.). Proceedings of the National Academy of Sciences, USA **118**, e2105431118. doi:10.1073/pnas.2105431118.

Ring L, Yeh SY, Hücherig S, et al. 2013. Metabolic interaction between anthocyanin and lignin biosynthesis is associated with peroxidase FaPRX27 in strawberry fruit. Plant Physiology **163**, 43–60. doi:10.1104/pp.113.222778.

Rousseau-Gueutin M, Gaston A, Aïnouche A, Aïnouche ML, Olbricht K, Staudt G, Richard L, Denoyes-Rothan B. 2009. Tracking the evolutionary history of polyploidy in *Fragaria* L. (strawberry): new insights from phylogenetic analyses of low-copy nuclear genes. Molecular Phylogenetics and Evolution **51**, 515–530. doi:10.1016/j.ympev.2008.12.024.

Salvatierra A, Pimentel P, Moya-León MA, Herrera R. 2013. Increased accumulation of anthocyanins in *Fragaria chiloensis* fruits by transient suppression of FcMYB1 gene. Phytochemistry **90**, 25–36. doi:10.1016/j.phytochem.2013.02.016.

Schaart JG, Dubos C, Romero De La Fuente I, van Houwelingen AMML, de Vos RCH, Jonker HH, Xu W, Routaboul JM, Lepiniec L, Bovy AG. 2013. Identification and characterization of MYB-bHLH-WD40 regulatory complexes controlling proanthocyanidin biosynthesis in strawberry (*Fragaria × ananassa*) fruits. New Phytologist **197**, 454–467. doi:10.1111/nph.12017.

Senger E, Osorio S, Olbricht K, et al. 2022. Towards smart and sustainable development of modern berry cultivars in Europe. The Plant Journal **111**, 1238–1251. doi:10.1111/tpj.15876.

Shulaev V, Sargent DJ, Crowhurst RN, et al. 2011. The genome of woodland strawberry (*Fragaria vesca*). Nature Genetics **43**, 109–116. doi:10.1038/ng.740.

Staudt G. 1962. Taxonomic studies in the genus *Fragaria*. Typification of *Fragaria* species known at the time of Linnaeus. Canadian Journal of Botany **40**, 869–886.

Staudt G. 1989. The species of *Fragaria*, their taxonomy and geographical distribution. Acta Horticulturae **265**, 23–34.

Sun R, Li S, Chang L, et al. 2022. Chromosome-level genome assembly of *Fragaria pentaphylla* using PacBio and Hi-C technologies. Frontiers in Genetics **13**, 873711. doi:10.3389/fgene.2022.873711.

Tong H, Nankar AN, Liu J, et al. 2022. Genomic prediction of morphometric and colorimetric traits in Solanaceous fruits. Horticulture Research **9**, uha072. doi:10.1093/hr/uhac072.

Urrutia M, Schwab W, Hoffmann T, Monfort A. 2016. Genetic dissection of the (poly)phenol profile of diploid strawberry (*Fragaria vesca*) fruits using a NIL collection. Plant Science **242**, 151–168. doi:10.1016/j.plantsci.2015.07.019.

Verweij W, Spelt C, Di Sansebastiano GP, Vermeer J, Reale L, Ferranti F, Koes R, Quattrochcio F. 2008. An H⁺ P-ATPase on the tonoplast determines vacuolar pH and flower colour. Nature Cell Biology **10**, 1456–1462. doi:10.1038/ncb1805.

Vining KJ, Salinas N, Tennessen JA, Zurn JD, Sargent DJ, Hancock J, Bassil NV. 2017. Genotyping-by-sequencing enables linkage mapping in three octoploid cultivated strawberry families. PeerJ **5**, e3731. doi:10.7717/peerj.3731.

Wada T, Oku K, Nagano S, et al. 2017. Development and characterization of a strawberry MAGIC population derived from crosses with six strawberry cultivars. Breeding Science **67**, 370–381. doi:10.1270/jsbbs.17009.
Wang H, Zhang H, Yang Y, et al. 2020. The control of red colour by a family of MYB transcription factors in octoploid strawberry (*Fragaria* × *ananassa*) fruits. Plant Biotechnology Journal **18**, 1169–1184. doi:10.1111/pbi.13282.

Whitaker VM, Knapp SJ, Hardigan MA, et al. 2020. A roadmap for research in octoploid strawberry. Horticulture Research **7**, 33. doi:10.1038/s41438-020-0252-1.

Xu W, Dubos C, Lepiniec L. 2015. Transcriptional control of flavonoid biosynthesis by MYB-bHLH-WDR complexes. Trends in Plant Science **20**, 176–185.

Yamamoto E, Kataoka S, Shirasawa K, Noguchi Y, Isobe S. 2021. Genomic selection for F₁ hybrid breeding in strawberry (*Fragaria × ananassa*). Frontiers in Plant Science **12**, 645111. doi:10.3389/fpls.2021.645111.

Yuan H, Cai W, Chen X, Pang F, Wang J, Zhao M. 2022. Heterozygous frameshift mutation in FaMYB10 is responsible for the natural formation of red and white-fleshed strawberry (*Fragaria × ananassa* Duch). Frontiers in Plant Science **13**, 1027567. doi:10.3389/fpls.2022.1027567.

Yue M, Jiang L, Zhang N, et al. 2023. Regulation of flavonoids in strawberry fruits by FaMYB5/FaMYB10 dominated MYB-bHLH-WD40 ternary complexes. Frontiers in Plant Science **14**, 1145670. doi:10.3389/fpls.2023.1145670.

Zhang J, Lei Y, Wang B, et al. 2020. The high-quality genome of diploid strawberry (*Fragaria nilgerrensis*) provides new insights into anthocyanin accumulation. Plant Biotechnology Journal **18**, 1908–1924. doi:10.1111/pbi.13351.

Zhang J, Zhang Y, Dou Y, Li W, Wang S, Shi W, Sun Y, Zhang Z. 2017. Single nucleotide mutation in *FvMYB10* may lead to the yellow fruit in *Fragaria* vesca. Molecular Breeding **37**, 35. doi:10.1007/s11032-017-0625-9.

Zhang Y, Li W, Dou Y, Zhang J, Jiang G, Miao L, Han G, Liu Y, Li H, Zhang Z. 2015. Transcript quantification by RNA-Seq reveals differentially expressed genes in the red and yellow fruits of *Fragaria* vesca. PLoS One **10**, e0144356. doi:10.1371/journal.pone.0144356.

Zhang Z, Shi Y, Ma Y, et al. 2020. The strawberry transcription factor FaRAV1 positively regulates anthocyanin accumulation by activation of FaMYB10 and anthocyanin pathway genes. Plant Biotechnology Journal **18**, 2267–2279. doi:10.1111/pbi.13382.

Zhao F, Li G, Hu P, Zhao X, Li L, Wei W, Feng J, Zhou H. 2018. Identification of basic/helix-loop-helix transcription factors reveals candidate genes involved in anthocyanin biosynthesis from the strawberry white-flesh mutant. Scientific Reports **8**, 2721. doi:10.1038/s41598-018-21136-z.

Zhao F, Song P, Zhang X, Li G, Hu P, Aslam A, Zhao X, Zhou H. 2021. Identification of candidate genes influencing anthocyanin biosynthesis during the development and ripening of red and white strawberry fruits via comparative transcriptome analysis. PeerJ **9**, e10739. doi:10.7717/peerj.10739.

Zingaretti LM, Monfort A, Pérez-Enciso M. 2022. Automatic fruit morphology phenotype and genetic analysis: an application in the octoploid strawberry. Plant Phenomics **2021**, 9812910. doi:10.34133/2021/9812910. Erratum in: Plant Phenomics 2022, 9873618.

Zorrilla-Fontanesi Y, Cabeza A, Domínguez P, Medina JJ, Valpuesta V, Denoyes-Rothan B, Sánchez-Sevilla JF, Amaya I. 2011. Quantitative trait loci and underlying candidate genes controlling agronomical and fruit quality traits in octoploid strawberry (*Fragaria × ananassa*). Theoretical and Applied Genetics **123**, 755–778. doi:10.1007/s00122-011-1624-6.

Zurn JD, Hummer KE, Bassil NV. 2022. Exploring the diversity and genetic structure of the U.S. National Cultivated Strawberry Collection. Horticulture Research **9**, uhac125. doi:10.1093/hr/uhac125.