Strawberry Genome Research Articles

Genome-Wide Analysis of the ERF Family and Identification of Potential Genes Involved in Fruit Ripening in Octoploid Strawberry.

Ethylene response factors (ERFs) belonging to the APETALA2/ERF superfamily acted at the end of the ethylene signaling pathway, and they were found to play important roles in plant growth and development. However, the information of ERF genes in strawberry and their involvement in fruit ripening have been limited. Here, a total of 235 ERF members were identified from 426 AP2/ERF genes at octoploid strawberry genome level and classified into six subgroups according to their sequence characteristics and phylogenetic relationship. Conserved motif and gene structure analysis supported the evolutionary conservation of FaERFs. Syntenic analysis showed that four types of duplication events occurred during the expansion of FaERF gene family. Of these, WGD/segmental duplication played a major role. Transcriptomic data of FaERF genes during fruit ripening and in response to abscisic acid screened one activator (FaERF316) and one repressor (FaERF118) that were involved in fruit ripening. Transcriptional regulation analysis showed some transcription factors related to ripening such as ABI4, TCP15, and GLK1 could bind to FaERF316 or FaERF118 promoters, while protein–protein interaction analysis displayed some proteins associated with plant growth and development could interact with FaERF118 or FaERF316. These results suggested that FaERF118 and FaERF316 were potential genes to regulate strawberry ripening. In summary, the present study provides the comprehensive and systematic information on FaERF family evolution and gains insights into FaERF’s potential regulatory mechanism in strawberry ripening.

Genome-Wide Identification, Characterization, and Expression Profile Analysis of CONSTANS-like Genes in Woodland Strawberry (Fragaria vesca).

CONSTANS-like (CO-like) gene is one of the most important regulators in the flowering process of the plant, playing a core role in the photoperiodic flowering induction pathway. In this study, we identified 10 distinct CO-like genes (FveCOs) in woodland strawberry (Fragaria vesca). They were classified into three groups with specific gene structure characteristics or protein domains in each group. The effect of selection pressure on the FveCOs in the woodland strawberry was tested by Ka/Ks, and it was shown that the evolution rate of FveCOs was controlled by purification selection factors. Intraspecific synteny analysis of woodland strawberry FveCOs showed that at least one duplication event existed in the gene family members. Collinearity analysis of woodland strawberry genome with genomes of Arabidopsis, rice (Oryza sativa), and apple (Malus × domestica) showed that CO-like genes of F. vesca and Malus × domestica owned higher similarity for their similar genomes compared with those of other two species. The FveCOs showed different tissue-specific expression patterns. Moreover, real-time quantitative PCR results revealed that the expressions of the most FveCOs followed a 24-h rhythm oscillation under both long-day (LD) and short-day (SD) conditions. Further expression analysis showed that the individual expression changing profile of FveCO3 and FveCO5 was opposite to each other under both LD and SD conditions. Moreover, the expression of FveCO3 and FveCO5 was both negatively correlated with the flowering time variation of the woodland strawberry grown under LD and SD conditions, indicating their potential vital roles in the photoperiodic flowering regulation. Further protein interaction network analysis also showed that most of the candidate interaction proteins of FveCO3 and FveCO5 were predicted to be the flowering regulators. Finally, LUC assay indicated that both FveCO3 and FveCO5 could bind to the promoter of FveFT1, the key regulator of flowering regulation in the woodland strawberry, and thus activate its expression. Taken together, this study laid a foundation for understanding the exact roles of FveCOs in the reproductive development regulation of the woodland strawberry, especially in the photoperiodic flowering process.

Genome-Wide Analysis of Ascorbic Acid Metabolism Related Genes in Fragaria × ananassa and Its Expression Pattern Analysis in Strawberry Fruits.

Ascorbic acid (AsA) is an important antioxidant for scavenging reactive oxygen species and it is essential for human health. Strawberry (Fragaria × ananassa) fruits are rich in AsA. In recent years, strawberry has been regarded as a model for non-climacteric fruit ripening. However, in contrast to climacteric fruits, such as tomato, the regulatory mechanism of AsA accumulation in strawberry fruits remains largely unknown. In this study, we first identified 125 AsA metabolism-related genes from the cultivated strawberry “Camarosa” genome. The expression pattern analysis using an available RNA-seq data showed that the AsA biosynthetic-related genes in the D-mannose/L-galactose pathway were downregulated remarkably during fruit ripening which was opposite to the increasing AsA content in fruits. The D-galacturonate reductase gene (GalUR) in the D-Galacturonic acid pathway was extremely upregulated in strawberry receptacles during fruit ripening. The FaGalUR gene above belongs to the aldo-keto reductases (AKR) superfamily and has been proposed to participate in AsA biosynthesis in strawberry fruits. To explore whether there are other genes in the AKR superfamily involved in regulating AsA accumulation during strawberry fruit ripening, we further implemented a genome-wide analysis of the AKR superfamily using the octoploid strawberry genome. A total of 80 FaAKR genes were identified from the genome and divided into 20 subgroups based on phylogenetic analysis. These FaAKR genes were unevenly distributed on 23 chromosomes. Among them, nine genes showed increased expression in receptacles as the fruit ripened, and notably, FaAKR23 was the most dramatically upregulated FaAKR gene in receptacles. Compared with fruits at green stage, its expression level increased by 142-fold at red stage. The qRT-PCR results supported that the expression of FaAKR23 was increased significantly during fruit ripening. In particular, the FaAKR23 was the only FaAKR gene that was significantly upregulated by abscisic acid (ABA) and suppressed by nordihydroguaiaretic acid (NDGA, an ABA biosynthesis blocker), indicating FaAKR23 might play important roles in ABA-mediated strawberry fruit ripening. In a word, our study provides useful information on the AsA metabolism during strawberry fruit ripening and will help understand the mechanism of AsA accumulation in strawberry fruits.

Genome-wide identification and characterization of the abiotic-stress-responsive lipoxygenase gene family in diploid woodland strawberry (Fragaria vesca)

Lipoxygenase (LOXs) is a kind of dioxygenase without heme and iron, which plays an important role in the development and adaptation of many plants to the environment. However, the study of strawberry LOX gene family has not been reported. In this study, 14 LOX genes were identified from the diploid woodland strawberry genome. The phylogenetic tree divides the FvLOX gene into two subfamilies: 9-LOX and 13-LOX. Gene duplication event analysis showed that whole-genome duplication (WGD)/segmental duplication and dispersed duplication effectively promoted the expansion of strawberry LOX family. QRT-PCR analysis showed that FvLOX genes were expressed in different tissues. Expression profile analysis showed that FvLOX1 and FvLOX8 were up-regulated under low temperature stress, FvLOX3 and FvLOX7 were up-regulated under drought stress, FvLOX6 and FvLOX9 were up-regulated under salt stress, FvLOX2, FvLOX3 and FvLOX6 were up-regulated under salicylic acid (SA) treatment, FvLOX3, FvLOX11 and FvLOX14 were up-regulated under methyl jasmonate (MeJA) treatment, FvLOX4 and FvLOX14 were up-regulated under abscisic acid (ABA) treatment. Promoter analysis showed that FvLOX genes were involved in plant growth and development and stress response. We analyzed and identified the whole genome of strawberry FvLOX family and characterized a variety of FvLOX candidate genes involved in abiotic stress response. This study laid a theoretical and empirical foundation for the response mechanism of strawberry to abiotic stress.

Genome-Wide Identification of Strawberry Metal Tolerance Proteins and Their Expression under Cadmium Toxicity

Metal tolerance proteins (MTPs) are divalent cation transporters, known to upkeep the mineral nutrition of plants and heavy metal transport at cell, tissue, or whole plant levels. However, information related to evolutionary relationships and biological functions of MTP genes in strawberry (Fragaria vesca L.) remain elusive. Herein, we identified 12 MTP genes from the strawberry genome and divided them into three main groups (i.e., Zn-MTP, Fe/Zn MTP, and Mn-MTP), which is similar to MTP grouping in Arabidopsis and rice. The strawberry MTPs (FvMTPs) are predicted to be localized in the vacuole, while open reading frame (ORF) length ranged from 1113 to 2589 bp with 370 to 862 amino acids, and possess 4 to 6 transmembrane domains (TMDs), except for FvMTP12 that possessed 16 TMDs. All the FvMTP genes had putative cation efflux and cation diffusion facilitator domains along with a zinc dimerization (ZT-dimer) domain in Mn-MTPs. The collinear analysis suggested their conservation between strawberry and Arabidopsis MTPs. Promoter analysis also demonstrated that some of them might possibly be regulated by hormones and abiotic stress factors. Moreover, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis proposed that FvMTP genes are involved in cation transport and homeostasis. The expression analysis showed that FvMTP1, FvMTP1.1, and FvMTP4 were significantly induced in leaf samples, while FvMTP1.1 and FvMTP4 were significantly regulated in roots of cadmium (Cd)-treated strawberry plants during progressive stress duration. The findings of Cd accumulation depicted that Cd contents were significantly higher in root tissues than that of leaf tissues of strawberry. These results are indicative of their response during the specific duration in Cd detoxification, while further functional studies can accurately verify their specific role.

Genome-Wide Investigation of G6PDH Gene in Strawberry: Evolution and Expression Analysis during Development and Stress.

As one of the key enzymes in the pentose phosphate pathway (PPP), glucose-6-phosphate dehydrogenase (G6PDH) provides NADPH and plays an important role in plant development and stress responses. However, little information was available about the G6PDH genes in strawberry (Fragaria × ananassa). The recent release of the whole-genome sequence of strawberry allowed us to perform a genome-wide investigation into the organization and expression profiling of strawberry G6PDH genes. In the present study, 19 strawberry G6PDH genes (FaG6PDHs) were identified from the strawberry genome database. They were designated as FaG6PDH1 to FaG6PDH19, respectively, according to the conserved domain of each subfamily and multiple sequence alignment with Arabidopsis. According to their structural and phylogenetic features, the 19 FaG6PDHs were further classified into five types: Cy, P1, P1.1, P2 and PO. The number and location of exons and introns are similar, suggesting that genes of the same type are very similar and are alleles. A cis-element analysis inferred that FaG6PDHs possessed at least one stress-responsive cis-acting element. Expression profiles derived from transcriptome data analysis exhibited distinct expression patterns of FaG6PDHs genes in different developmental stages. Real-time quantitative PCR was used to detect the expression level of five types FaG6PDHs genes and demonstrated that the genes were expressed and responded to multiple abiotic stress and hormonal treatments.

FaPKc2.2 negatively modulates strawberry fruit ripening by reprograming the carbon metabolic pathway

Although the main contribution of pyruvate kinases in the energy providing and regulation of plant growth or seed development have been discovered, the potential roles of these enzymes in the fruit ripening process has not been well elucidated so far. In this study, a total of 53 pyruvate kinases (PKs) were identified in the genome of octoploid strawberry. Expression specificity in the fruit led to the identification of a cytosolic PK (FxaC_15g00080, namely FaPKc2.2), which was inhibited by both ABA and sucrose, either alone or synergistically. It was expressed in both vegetative and reproductive organs. Subcellular localization analysis confirmed that the FaPKc2.2 was a cytosolic PK. Transient overexpression of the FaPKc2.2 obviously inhibited the ripening of strawberry fruits. Biochemical and transcriptomic data further revealed that the upregulation of the FaPKc2.2 systematically reprogrammed metabolisms, mainly by channeling the main flux from carbon to amino-acid related nitrogen biomolecules. These results demonstrated the regulatory role of the FaPKc2.2 in the fruit ripening and highlighted the probability of utilizing the gene in strawberry fruit quality improvements.

Genome-wide characterization and expression of the TLP gene family associated with Colletotrichum gloeosporioides inoculation in Fragaria × ananassa.

BackgroundColletotrichum gloeosporioides, a soil-borne fungal pathogen, causes significant yield losses in many plants, including cultivated strawberry (Fragaria × ananassa, 2n = 8x = 56). Thaumatin-like proteins (TLPs) are a large and complex family of proteins that play a vital role in plant host defense and other physiological processes.MethodsTo enhance our understanding of the antifungal activity of F. × ananassa TLPs (FaTLP), we investigated the genome-wide identification of FaTLP gene families and their expression patterns in F. × ananassa plants upon pathogen infection. Moreover, we used RNA sequencing (RNA-seq) to detect the differences in the expression patterns of TLP genes between different resistant strawberry cultivars in response to C. gloeosporioides infection.ResultsIn total, 76 TLP genes were identified from the octoploid cultivated strawberry genome with a mean length of 1,439 bp. They were distributed on 24 F. × ananassa chromosomes. The FaTLP family was then divided into ten groups (Group I–X) according to the comparative phylogenetic results. Group VIII contained the highest number of TLP family genes. qRT-PCR analysis results indicated that FaTLP40, FaTLP41, FaTLP43, FaTLP68, and FaTLP75 were upregulated following C. gloeosporioides infection in the resistant octoploid strawberry.ConclusionsThe data showed some differences in TLP gene expression patterns across different resistant strawberry cultivars, as well as faster TLP defense responses to pathogenic fungi in resistant cultivars. This study will aid in the characterization of TLP gene family members found in octoploid strawberries and their potential biological functions in plants’ defenses against pathogenic fungi.

The power of chromosome-scale, haplotype-resolved genomes

The assembly of chromosome-scale and haplotype-resolved reference genomes is now more easily attainable, largely due to various improvements in both assembly algorithms and long-read sequencing technologies (see recent review by Michael and VanBuren, 2020 Michael T.P. VanBuren R. Building near-complete plant genomes. Curr. Opin. Plant Biol. 2020; 54: 26-33 Google Scholar ). Due to these technological advancements, there has been a shift away from using highly inbred accessions, in some instances double haploids, to instead assembling the genomes of important reference genotypes. For example, a haplotype-resolved genome was recently assembled for an important cultivated garden strawberry (Fragaria x ananassa) cultivar Royal Royce, which is not only highly heterozygous but also octoploid (8×) ( Hardigan et al., 2021 Hardigan M.A. Feldmann M.J. Pincot D.D.A. Famula R.A. Vachev M.V. Madera M.A. Zerbe P. Mars K. Peluso P. Rank D. et al. Blueprint for phasing and assembling the genomes of heterozygous polyploids: application to the octoploid genome of strawberry. Preprint at bioRxiv. 2021; https://doi.org/10.1101/2021.11.03.467115 Google Scholar ). Similarly, haplotype-phased genomes have been assembled for other important crops, including cassava (Manihot esculenta) ( Hu et al., 2021 Hu W. Ji C. Shi H. Liang Z. Ding Z. Ye J. Ou W. Zhou G. Tie W. Yan Y. et al. Allele-defined genome reveals biallelic differentiation during cassava evolution. Mol. Plant. 2021; 14: 851-854 Google Scholar ) and potato (Solanum tuberosum) ( Hoopes et al., 2022 Hoopes G. Meng X. Hamilton J.P. Achakkagari S.R. de Alves Freitas Guesdes F. Bolger M.E. Coombs J.J. Esselink D. Kaiser N.R. Kodde L. et al. Phased, chromosome-scale genome assemblies of tetraploid potato reveals a complex genome, transcriptome, and predicted proteome landscape underpinning genetic diversity. Mol. Plant. 2022; (S1674-2052, 00003-X)https://doi.org/10.1016/j.molp.2022.01.003 Google Scholar ). This has resulted in the development of new genomic resources that can be directly used in guiding molecular breeding efforts, without worrying about the gene presence-absence variation that exists within a crop or species ( Golicz et al., 2016 Golicz A.A. Batley J. Edwards D. Towards plant pangenomics. Plant Biotechnol. J. 2016; 14: 1099-1105 Google Scholar ; Bayer et al., 2021 Bayer P.E. Scheben A. Golicz A.A. Yuan Y. Faure S. Lee H. Chawla H.S. Anderson R. Bancroft I. Raman H. et al. Modelling of gene loss propensity in the pangenomes of three Brassica species suggests different mechanisms between polyploids and diploids. Plant Biotechnol. J. 2021; 19: 2488-2500 Google Scholar ). An additional benefit of having a reference genome of an important cultivar and/or genotype, versus from a highly inbred line, is that valuable insights that are potentially gained from the analysis of genetic variants present among haplotypes are not lost, allowing for additional insights.

Strawberry, a New Natural Host of Brassica Yellows Virus in China.

Brassica yellows virus (BrYV; genus Polerovirus, family Solemoviridae) has an icosahedral spherical virion with a positive-sense single-stranded RNA genome and it is distinguished from turnip yellows virus (TuYV) based on differences in ORF0 and ORF5 (Xiang et al., 2011). To investigate the occurrence and distribution of viruses infecting strawberry (Fragaria ananassa) in the main production areas in China, a survey of nine greenhouses (667 m2 each) was conducted in the cities of Yantai and Beijing, China in August 2020. About 1% of strawberry plants in each greenhouse showed virus-like symptoms of chlorotic spots; 89 symptomatic leaf samples were randomly collected for virus testing. Total RNA was extracted from a pool of eight samples of four different cultivars (Hokowase: 2, Mibao: 2, Sagahonoka: 2, Monterey: 2) from Yantai using RNAprep Pure Plant Plus Kit (TianGen, China). A cDNA library was constructed by NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina® (NEB, USA) after ribosomal RNA-depletion using an Epicentre Ribo-Zero™ rRNA Removal Kit (Epicentre, USA). High-throughput sequencing was done on Illumina Hiseq 4000, generating 70,931,850 high-quality 150 bp paired-end reads. Clean reads were de novo assembled by Trinity (v2.2.0) and the resulting contigs were screened by BLASTn and BLASTx against GenBank database as described previously (Grabherr et al., 2013). A total of 1,432,164 high-quality reads unmapped to the strawberry genome were obtained and assembled into 93 contigs (ranging from 33 to 8,031 nt). Seven of these contigs (277 to 1,254 nt) shared 98.2 to 100% nt identities with BrYV-A (accession no. HQ388348) and covered 89.5% of the genome of BrYV-A. Subsequent analyses indicated the presence of Strawberry pallidosis-associated virus and Strawberry mottle virus in the analyzed sample, both have been reported in strawberry in China (Shi et al., 2018; Fan et al., 2021). To confirm BrYV infection, total RNA was isolated from the eight samples used for HTS and reverse transcription polymerase chain reaction (RT-PCR) was conducted with two pairs of specific primers (CP and rtp, Supplementary Table 1) designed based on the assembled contigs. PCR products with expected sizes (587 and 609 bp) were observed in one sample (cv. Mibao). BLASTn analysis indicated that the amplicons (accession no. MW548437 and MW548438) shared 98.6% and 99.3% nt identity with BrYV-A, respectively. To obtain the complete sequence of the putative BrYV isolate, the gaps were bridged and the terminal sequences were determined using 5' and 3' RACE kits (Clontech, China) based on the assembled contigs. The complete genome sequence of the putative BrYV isolate has a length of 5,666 nt (accession no. MZ666129) and shares more than 94.3% nt identities with other BrYV isolates. Phylogenetic analysis indicated that the isolate grouped closely with BrYV and further from TuYV (Figure S1). In addition, 11 samples (cv. Benihoppe) of the remaining 81 symptomatic strawberry samples tested positive for BrYV by RT-PCR with the two pairs of primers mentioned above. The sequences (accession no. MZ407232 and MZ407233) revealed 99.5% and 99.3% nt identities with MW548437 and MW548438. To the best of our knowledge, this is the first report of natural infection of BrYV in strawberry plants. Our findings expand the host range of BrYV, but disease association is difficult to establish due to presence of mixed infection and non-fulfillment of Koch's postulates.

Characterization and regulation mechanism analysis of ubiquitin-conjugating family genes in strawberry reveals a potential role in fruit ripening

BackgroundE2 ubiquitin-conjugating (UBC) enzymes are an integral component of the ubiquitin proteasome system that play an important role in plant development, growth, and external stress responses. Several UBC genes have been identified in various plants. However, no studies exploring the functions of UBC genes in regulating fruit of strawberry have been reported. In the present study, a systematic analysis of the entire UBC family members were conducted in the genome of strawberry (Fragaria ×ananassa) based on bioinformatics method, and the gene functioning in strawberry ripening was explored.ResultsA total of 191 UBC genes were identified in the genome of cultivated strawberry. These genes were unevenly distributed across the 28 chromosomes from the 4 subgenomes of cultivated strawberry, ranging from 3 to 11 genes per chromosome. Moreover, the expansion of FaUBC genes in strawberry was mainly driven by WGD. All the FaUBC genes were clarified into 13 groups and most of them were included in the group VI. The gene structure analysis showed that the number of exons varied from 1 to 23, and the structure of genes had few differences within the same groups but a distinction in different groups. Identification of the cis-acting elements of the promoter revealed multiple regulatory elements that responded to plant growth and development, phytohormone responsive, and abiotic and biotic stress. Data from functional annotation indicated that FaUBC genes play a role in a variety of biological processes. The RNA-seq data showed that FaUBC genes displayed different expression pattern during the fruit ripening process and clarified into 6 clusters. In particular, cluster 3 exhibiting a sudden expression increase in the turning red stage were speculated to be involved in fruit ripening. Hence, two FaUBC genes (FaUBC76 and FaUBC78) were selected for gene function analysis by transient over-expression method. The results indicated that FaUBC76 has a positive effect on the fruit development and ripening in strawberry by up-regulating accumulation of anthocyanins. Moreover, expression of some maturity-related genes were also significantly increased, further supporting a role for FaUBC76 in the regulation of fruit ripening or softening. On the contrary, the overexpression of FaUBC78 significantly increased the firmness of strawberry fruit, indicating that FaUBC78 had a positive role in inhibiting the decrease of strawberry fruit firmness.ConclusionOur study not only provide comprehensive information on system evolution and function on UBC genes, but also give a new insight into explore the roles of FaUBC genes in the regulation of strawberry ripening.

The Gene FvTST1 From Strawberry Modulates Endogenous Sugars Enhancing Plant Growth and Fruit Ripening.

Sugar is an important carbon source and contributes significantly to the improvement of plant growth and fruit flavor quality. Sugar transport through the tonoplast is important for intracellular homeostasis and metabolic balance in plant cells. There are four tonoplast sugar transporters (FvTST1-4) in strawberry genome. The qRT-PCR results indicated that FvTST1 has a differential expression pattern in different tissues and developmental stages, and exhibited highest expression level in mature fruits. The yeast complementation assay showed that FvTST1 can mediate the uptake of different sugars, such as fructose, glucose, sucrose, and mannose. Subcellular localization analyses revealed that FvTST1 was mainly targeted to the tonoplast. Transient expression of FvTST1 in strawberry fruits enhanced both fruit ripening and sugar accumulation. Furthermore, FvTST1-transformed tomato plants exhibited higher sucrose and auxin content, enhanced seed germination and vegetative growth, higher photosynthetic rate, early flowering, and bore fruit; fructose and glucose levels were higher in transgenic fruits than those in the control. Transcriptomic analysis indicated that the auxin signaling pathway was highly enriched pathway in up-regulated Gene-ontology terms. In transgenic plants, genes encoding transcription factors, such as phytochrome-interacting factors PIF1, -3, and -4, as well as their potential target genes, were also induced. Collectively, the results show that FvTST1 enhances plant growth and fruit ripening by modulating endogenous sugars, and highlight the biological significance of this gene for future breeding purposes.

GDS: A Genomic Database for Strawberries (Fragaria spp.)

Strawberry species (Fragaria spp.) are known as the “queen of fruits” and are cultivated around the world. Over the past few years, eight strawberry genome sequences have been released. The reuse of these large amount of genomic data, and the more large-scale comparative analyses are very challenging to both plant biologists and strawberry breeders. To promote the reuse and exploration of strawberry genomic data and enable extensive analyses using various bioinformatics tools, we have developed the Genome Database for Strawberry (GDS). This platform integrates the genome collection, storage, integration, analysis, and dissemination of large amounts of data for researchers engaged in the study of strawberry. We collected and formatted the eight published strawberry genomes. We constructed the GDS based on Linux, Apache, PHP and MySQL. Different bioinformatic software were integrated. The GDS contains data from eight strawberry species, as well as multiple tools such as BLAST, JBrowse, synteny analysis, and gene search. It has a designed interface and user-friendly tools that perform a variety of query tasks with a few simple operations. In the future, we hope that the GDS will serve as a community resource for the study of strawberries.

QTL Analysis for Chlorophyll Content in Strawberry (Fragaria × ananassa Duch.) Leaves

Chlorophyll is an important factor facilitating plants to capture, allocate and transforms light energy and plays a major role in yield formation. Strawberry is one of the most important fruit crops worldwide. Breeding strawberry for better light utilization by improving photosynthetic efficiency can improve the yield potential. In strawberry, genetic studies have been done for several traits, but no reports on the genetic mapping of chlorophyll content in leaves. In the present study, we used two independent F2 mapping populations (BS-F2 and BC-F2) and, Axiom 35 K strawberry chip and genotyping-by-sequencing derived single nucleotide polymorphisms based linkage maps to identify the quantitative trait loci (QTLs) controlling leaf chlorophyll content. SPAD values were used to estimate the leaf chlorophyll content of parental lines and F2 populations. A total of seven QTLs, including major and minor effects, common and specific to populations, were identified across the strawberry genome explaining phenotypic variation (R2) ranging from 1.4 to 26.4%. Candidate genes associated with the photosynthesis and chlorophyll content were inferred in commonly detected QTLs. This work thus provides not only information for novel loci controlling chlorophyll content in strawberry leaves but also forms the basis for future marker assisted breeding in strawberry to select the plants for required chlorophyll content.

Evolutionary history and pan-genome dynamics of strawberry (Fragaria spp.)

Strawberry (Fragaria spp.) has emerged as a model system for various fundamental and applied research in recent years. In total, the genomes of five different species have been sequenced over the past 10 y. Here, we report chromosome-scale reference genomes for five strawberry species, including three newly sequenced species' genomes, and genome resequencing data for 128 additional accessions to estimate the genetic diversity, structure, and demographic history of key Fragaria species. Our analyses obtained fully resolved and strongly supported phylogenies and divergence times for most diploid strawberry species. These analyses also uncovered a new diploid species (Fragaria emeiensis Jia J. Lei). Finally, we constructed a pan-genome for Fragaria and examined the evolutionary dynamics of gene families. Notably, we identified multiple independent single base mutations of the MYB10 gene associated with white pigmented fruit shared by different strawberry species. These reference genomes and datasets, combined with our phylogenetic estimates, should serve as a powerful comparative genomic platform and resource for future studies in strawberry.

A Strategy for the Production and Molecular Validation of Agrobacterium-Mediated Intragenic Octoploid Strawberry.

Intragenesis is an all-native engineering technology for crop improvement. Using an intragenic strategy to bring genes from wild species to cultivated strawberry could expand the genetic variability. A robust regeneration protocol was developed for the strawberry cv. ‘Shanghai Angel’ by optimizing the dose of Thidiazuron and identifying the most suitable explants. The expression cassette was assembled with all DNA fragments from F. vesca, harboring a sugar transporter gene FvSTP8 driven by a fruit-specific FvKnox promoter. Transformed strawberry was developed through an Agrobacterium-mediated strategy without any selectable markers. Other than PCR selection, probe-based duplex droplet digital PCR (ddPCR) was performed to determine the T-DNA insert. Four independent transformed shoots were obtained with a maximum of 5.3% efficiency. Two lines were confirmed to be chimeras, while the other two were complete transformants with six and 11 copies of the intragene, respectively. The presence of a vector backbone beyond the T-DNA in these transformants indicated that intragenic strawberries were not obtained. The current work optimized the procedures for producing transformed strawberry without antibiotic selection, and accurately determined the insertion copies by ddPCR in the strawberry genome for the first time. These strategies might be promising for the engineering of ‘Shanghai Angel’ and other cultivars to improve agronomic traits.

Genome-Wide Identification and Characterization of the Abiotic-Stress-Responsive GRF Gene Family in Diploid Woodland Strawberry (Fragaria vesca).

Growth regulatory factors (GRF) are plant-specific transcription factors that play an important role in plant resistance to stress. This gene family in strawberry has not been investigated previously. In this study, 10 GRF genes were identified in the genome of the diploid woodland strawberry (Fragaria vesca). Chromosome analysis showed that the 10 FvGRF genes were unevenly distributed on five chromosomes. Phylogenetic analysis resolved the FvGRF proteins into five groups. Genes of similar structure were placed in the same group, which was indicative of functional redundance. Whole-genome duplication/segmental duplication and dispersed duplication events effectively promoted expansion of the strawberry GRF gene family. Quantitative reverse transcription-PCR analysis suggested that FvGRF genes played potential roles in the growth and development of vegetative organs. Expression profile analysis revealed that FvGRF3, FvGRF5, and FvGRF7 were up-regulated under low-temperature stress, FvGRF4 and FvGRF9 were up-regulated under high-temperature stress, FvGRF6 and FvGRF8 were up-regulated under drought stress, FvGRF3, FvGRF6, and FvGRF8 were up-regulated under salt stress, FvGRF2, FvGRF7, and FvGRF9 were up-regulated under salicylic acid treatment, and FvGRF3, FvGRF7, FvGRF9, and FvGRF10 were up-regulated under abscisic acid treatment. Promoter analysis indicated that FvGRF genes were involved in plant growth and development and stress response. These results provide a theoretical and empirical foundation for the elucidation of the mechanisms of abiotic stress responses in strawberry.

Identifying strawberry Whirly family transcription factors and their expressions in response to crown rot

Crown rot is one of the most destructive diseases of cultivated strawberry. The correlation between Whirly family transcription factors, the one class of known resistance genes, and strawberry crown rot resistance has not been studied. In this study, the Whirlys of Fragaria × ananassa, F. iinumae, F. vesca, F. viridis and F. nilgerrensis were characterized by searching the strawberry genome database and analyzing the presence of Whirly domains. Five FaWHYs, two FiWHYs, three FnWHYs, two FviWHYs and four FvWHYs were identified from their respective genome. Two gene clusters with segmental duplications were obtained from the gene cluster analysis with two and three FaWHYs, and three FaWHYs showed syntenic relationships with AtWHYs of Arabidopsis thaliana. FiWHY1, FvWHY2 and FviWHY1 showed syntenic relationships with FaWHY1 and FaWHY2. At the same time, FiWHY2, FvWHY3, FviWHY2 and FnWHY3 exhibited similar syntenic relationships with FaWHY4 and FaWHY5. In addition, FnWHY1 and FnWHY2 corresponded to both FaWHY1 and FaWHY2. Gene expression analysis revealed that five FaWHYs were expressed in crowns, and the regulation of FaWHYs was always consistent with the cis-elements in their promoters. All of them were downregulated by crown rot infected. Together, these results provided a basis for further functional studies of the FaWHYs proteins and their responses to crown rot.

Genomic Characterization of the Fruity Aroma Gene, FaFAD1, Reveals a Gene Dosage Effect on γ-Decalactone Production in Strawberry (Fragaria × ananassa).

Strawberries produce numerous volatile compounds that contribute to the unique flavors of fruits. Among the many volatiles, γ-decalactone (γ-D) has the greatest contribution to the characteristic fruity aroma in strawberry fruit. The presence or absence of γ-D is controlled by a single locus, FaFAD1. However, this locus has not yet been systematically characterized in the octoploid strawberry genome. It has also been reported that the volatile content greatly varies among the strawberry varieties possessing FaFAD1, suggesting that another genetic factor could be responsible for the different levels of γ-D in fruit. In this study, we explored the genomic structure of FaFAD1 and determined the allele dosage of FaFAD1 that regulates variations of γ-D production in cultivated octoploid strawberry. The genome-wide association studies confirmed the major locus FaFAD1 that regulates the γ-D production in cultivated strawberry. With the hybrid capture-based next-generation sequencing analysis, a major presence–absence variation of FaFAD1 was discovered among γ-D producers and non-producers. To explore the genomic structure of FaFAD1 in the octoploid strawberry, three bacterial artificial chromosome (BAC) libraries were developed. A deletion of 8,262 bp was consistently found in the FaFAD1 region of γ-D non-producing varieties. With the newly developed InDel-based codominant marker genotyping, along with γ-D metabolite profiling data, we revealed the impact of gene dosage effect for the production of γ-D in the octoploid strawberry varieties. Altogether, this study provides systematic information of the prominent role of FaFAD1 presence and absence polymorphism in producing γ-D and proposes that both alleles of FaFAD1 are required to produce the highest content of fruity aroma in strawberry fruit.

Reannotation of the cultivated strawberry genome and establishment of a strawberry genome database

Cultivated strawberry (Fragaria × ananassa) is an important fruit crop species whose fruits are enjoyed by many worldwide. An octoploid of hybrid origin, the complex genome of this species was recently sequenced, serving as a key reference genome for cultivated strawberry and related species of the Rosaceae family. The current annotation of the F. ananassa genome mainly relies on ab initio predictions and, to a lesser extent, transcriptome data. Here, we present the structure and functional reannotation of the F. ananassa genome based on one PacBio full-length RNA library and ninety-two Illumina RNA-Seq libraries. This improved annotation of the F. ananassa genome, v1.0.a2, comprises a total of 108,447 gene models, with 97.85% complete BUSCOs. The models of 19,174 genes were modified, 360 new genes were identified, and 11,044 genes were found to have alternatively spliced isoforms. Additionally, we constructed a strawberry genome database (SGD) for strawberry gene homolog searching and annotation downloading. Finally, the transcriptome of the receptacles and achenes of F. ananassa at four developmental stages were reanalyzed and qualified, and the expression profiles of all the genes in this annotation are also provided. Together, this study provides an updated annotation of the F. ananassa genome, which will facilitate genomic analyses across the Rosaceae family and gene functional studies in cultivated strawberry.