Allelic Variation of MYB10 Is the Major Force Controlling Natural Variation in Skin and Flesh Color in Strawberry (Fragaria spp.) Fruit.

The fruits of diploid and octoploid strawberry (Fragaria spp) show substantial natural variation in color due to distinct anthocyanin accumulation and distribution patterns. Anthocyanin biosynthesis is controlled by a clade of R2R3 MYB transcription factors, among which MYB10 is the main activator in strawberry fruit. Here, we show that mutations in MYB10 cause most of the variation in anthocyanin accumulation and distribution observed in diploid woodland strawberry (F. vesca) and octoploid cultivated strawberry (F ×ananassa). Using a mapping-by-sequencing approach, we identified a gypsy-transposon in MYB10 that truncates the protein and knocks out anthocyanin biosynthesis in a white-fruited F. vesca ecotype. Two additional loss-of-function mutations in MYB10 were identified among geographically diverse white-fruited F. vesca ecotypes. Genetic and transcriptomic analyses of octoploid Fragaria spp revealed that FaMYB10-2, one of three MYB10 homoeologs identified, regulates anthocyanin biosynthesis in developing fruit. Furthermore, independent mutations in MYB10-2 are the underlying cause of natural variation in fruit skin and flesh color in octoploid strawberry. We identified a CACTA-like transposon (FaEnSpm-2) insertion in the MYB10-2 promoter of red-fleshed accessions that was associated with enhanced expression. Our findings suggest that cis-regulatory elements in FaEnSpm-2 are responsible for enhanced MYB10-2 expression and anthocyanin biosynthesis in strawberry fruit flesh.

Methylome, transcriptome, and phenotype changes induced by temperature conditions experienced during sexual reproduction in Fragaria vesca.

Temperature conditions experienced during embryogenesis and seed development may induce epigenetic changes that increase phenotypic variation in plants. Here we investigate if embryogenesis and seed development at two different temperatures (28 vs. 18°C) result in lasting phenotypic effects and DNA methylation changes in woodland strawberry (Fragaria vesca). Using five European ecotypes from Spain (ES12), Iceland (ICE2), Italy (IT4), and Norway (NOR2 and NOR29), we found statistically significant differences between plants from seeds produced at 18 or 28°C in three of four phenotypic features investigated under common garden conditions. This indicates the establishment of a temperature-induced epigenetic memory-like response during embryogenesis and seed development. The memory effect was significant in two ecotypes: in NOR2 flowering time, number of growth points and petiole length were affected, and in ES12 number of growth points was affected. This indicates that genetic differences between ecotypes in their epigenetic machinery, or other allelic differences, impact this type of plasticity. We observed statistically significant differences between ecotypes in DNA methylation marks in repetitive elements, pseudogenes, and genic elements. Leaf transcriptomes were also affected by embryonic temperature in an ecotype-specific manner. Although we observed significant and lasting phenotypic change in at least some ecotypes, there was considerable variation in DNA methylation between individual plants within each temperature treatment. This within-treatment variability in DNA methylation marks in F. vesca progeny may partly be a result of allelic redistribution from recombination during meiosis and subsequent epigenetic reprogramming during embryogenesis.

Signatures of polygenic adaptation align with genome-wide methylation patterns in wild strawberry plants.

Epigenetic inheritance can drive adaptive evolution independently of DNA sequence variation. However, to what extent epigenetic variation represents an autonomous evolutionary force remains largely elusive. Through gene ontology and comparative analyses of genomic and epigenomic variation of wild strawberry plants raised in distinct drought settings, we characterised genome-wide covariation between single nucleotide polymorphisms (SNPs) and differentially methylated cytosines (DMCs). Covariation between SNPs and DMCs was independent of genomic proximity, but instead associated with fitness-related processes such as stress responses, genome regulation and reproduction. We expected this functional SNP-DMC covariation to be driven by adaptive evolution canalising SNP and DMC variation, but instead observed significantly lower covariation with DMCs for adaptive rather than for neutral SNPs. Drought-induced DMCs frequently co-varied with tens of SNPs, suggesting high genomic redundancy as a broad potential basis for polygenic adaptation of gene expression. Our findings suggest that stress-responsive DMCs initially co-vary with many SNPs under increased environmental stress, and that natural selection acting upon several of these SNPs subsequently reduces standing covariation with stress-responsive DMCs. Our study supports DNA methylation profiles that represent complex quantitative traits rather than autonomous evolutionary forces. We provide a conceptual framework for polygenic regulation and adaptation shaping genome-wide methylation patterns in plants.

Evolutionary Genomics of Structural Variation in Asian Rice (Oryza sativa) Domestication.

Structural variants (SVs) are a largely unstudied feature of plant genome evolution, despite the fact that SVs contribute substantially to phenotypes. In this study, we discovered SVs across a population sample of 347 high-coverage, resequenced genomes of Asian rice (Oryza sativa) and its wild ancestor (O. rufipogon). In addition to this short-read data set, we also inferred SVs from whole-genome assemblies and long-read data. Comparisons among data sets revealed different features of genome variability. For example, genome alignment identified a large (∼4.3 Mb) inversion in indica rice varieties relative to japonica varieties, and long-read analyses suggest that ∼9% of genes from the outgroup (O. longistaminata) are hemizygous. We focused, however, on the resequencing sample to investigate the population genomics of SVs. Clustering analyses with SVs recapitulated the rice cultivar groups that were also inferred from SNPs. However, the site-frequency spectrum of each SV type-which included inversions, duplications, deletions, translocations, and mobile element insertions-was skewed toward lower frequency variants than synonymous SNPs, suggesting that SVs may be predominantly deleterious. Among transposable elements, SINE and mariner insertions were found at especially low frequency. We also used SVs to study domestication by contrasting between rice and O. rufipogon. Cultivated genomes contained ∼25% more derived SVs and mobile element insertions than O. rufipogon, indicating that SVs contribute to the cost of domestication in rice. Peaks of SV divergence were enriched for known domestication genes, but we also detected hundreds of genes gained and lost during domestication, some of which were enriched for traits of agronomic interest.

Altered regulation of TERMINAL FLOWER 1 causes the unique vernalisation response in an arctic woodland strawberry accession.

Vernalisation requirement is an agriculturally important trait that postpones the development of cold-sensitive floral organs until the spring. The family Rosaceae includes many agriculturally important fruit and berry crops that suffer from crop losses caused by frost injury to overwintering flower buds. Recently, a vernalisation-requiring accession of the Rosaceae model woodland strawberry (Fragaria vesca) has been identified in northern Norway. Understanding the molecular basis of the vernalisation requirement in this accession would advance the development of strawberry cultivars better adapted to temperate climate. We use gene silencing, gene expression analysis, genetic mapping and population genomics to study the genetic basis of the vernalisation requirement in woodland strawberry. Our results indicate that the woodland strawberry vernalisation requirement is endemic to northern Norwegian population, and mapping data suggest the orthologue of TERMINAL FLOWER1 (FvTFL1) as the causal floral repressor. We demonstrate that exceptionally low temperatures are needed to downregulate FvTFL1 and to make these plants competent to induce flowering at low postvernalisation temperatures in the spring. We show that altered regulation of FvTFL1 in the northern Norwegian woodland strawberry accession postpones flower induction until the spring, allowing plants to avoid winter injuries of flower buds that commonly occur in temperate regions.

Selection for population-specific adaptation shaped patterns of variation in the photoperiod pathway genes in Arabidopsis lyrata during post-glacial colonization.

Spatially varying selection can lead to population-specific adaptation, which is often recognized at the phenotypic level; however, the genetic evidence is weaker in many groups of organisms. In plants, environmental shifts that occur due to colonization of a novel environment may require adaptive changes in the timing of growth and flowering, which are often governed by location-specific environmental cues such as day length. We studied locally varying selection in 19 flowering time loci in nine populations of the perennial herb Arabidopsis lyrata, which has a wide but patchy distribution in temperate and boreal regions of the northern hemisphere. The populations differ in their recent population demographic and colonization histories and current environmental conditions, especially in the growing season length. We searched for population-specific molecular signatures of directional selection by comparing a set of candidate flowering time loci with a genomic reference set within each population using multiple approaches and contrasted the patterns of different populations. The candidate loci possessed approximately 20% of the diversity of the reference loci. On average the flowering time loci had more rare alleles (a smaller Tajima's D) and an excess of highly differentiated sites relative to the reference, suggesting positive selection. The strongest signal of selection was detected in photoperiodic pathway loci in the colonizing populations of Northwestern Europe, whereas no evidence of positive selection was detected in the Central European populations. These findings emphasized the population-specific nature of selection and suggested that photoperiodic adaptation was important during postglacial colonization of the species.

A recent local sweep at the PHYA locus in the Northern European Spiterstulen population of Arabidopsis lyrata.

Northern and central European Arabidopsis lyrata ssp. petraea populations are locally adapted to prevailing climatic conditions through differences in timing of life history events. The timing of flowering and, in perennials, the timing of growth cessation influence fitness. Phytochrome A may have an important role in regulating these life history traits as it perceives changes in daylength. We asked whether PHYA has contributed to local adaptation to the northern conditions in A. l. petraea. To search for signals of directional selection at the PHYA locus, we resequenced PHYA and 9 short fragments around PHYA from a 57-kb region from a German (Plech) and a Norwegian (Spiterstulen) population and compared patterns of differentiation and diversity to a set of 19 reference loci around the genome. First, we found that the populations were highly differentiated: there were three nonsynonymous fixed differences at the PHYA locus, which was in stark contrast with the total four fixed differences in the 19 reference loci. Compatible with a sweep hypothesis, variation was almost completely removed from the 9.4-kb region around PHYA in the northern Spiterstulen population. The overall level of linkage disequilibrium (LD) was higher in Spiterstulen, but there was no LD across the PHYA locus in the population, which is also a known consequence of a selective sweep. The sweep has likely occurred after the last glacial maximum, which suggests that it has contributed to adaptation to the northern conditions.

Role of vernalization and of duplicated FLOWERING LOCUS C in the perennial Arabidopsis lyrata.

FLOWERING LOCUS C (FLC) is one of the main genes influencing the vernalization requirement and natural flowering time variation in the annual Arabidopsis thaliana. Here we studied the effects of vernalization on flowering and its genetic basis in the perennial Arabidopsis lyrata. Two tandemly duplicated FLC genes (FLC1 and FLC2) were compared with respect to expression and DNA sequence. The effect of vernalization on flowering and on the expression of FLC1 was studied in three European populations. The genetic basis of the FLC1 expression difference between two of the populations was further studied by expression quantitative trait locus (eQTL) mapping and sequence analysis. FLC1 was shown to have a likely role in the vernalization requirement for flowering in A. lyrata. Vernalization decreased its expression and the northern study populations showed higher FLC1 expression than the southern one. eQTL mapping between two of the populations revealed one eQTL affecting FLC1 expression in the genomic region containing the FLC genes. Most FLC1 sequence differences between the study populations were found in the promoter region and in the first intron. Variation in the FLC1 sequence may cause differences in FLC1 expression between late- and early-flowering A. lyrata populations.

Warmer temperature during asexual reproduction induce methylome, transcriptomic, and lasting phenotypic changes in Fragaria vesca ecotypes.

Plants must adapt with increasing speed to global warming to maintain their fitness. One rapid adaptation mechanism is epigenetic memory, which may provide organisms sufficient time to adapt to climate change. We studied how the perennial Fragaria vesca adapted to warmer temperatures (28°C vs. 18°C) over three asexual generations. Differences in flowering time, stolon number, and petiole length were induced by warmer temperature in one or more ecotypes after three asexual generations and persisted in a common garden environment. Induced methylome changes differed between the four ecotypes from Norway, Iceland, Italy, and Spain, but shared methylome responses were also identified. Most differentially methylated regions (DMRs) occurred in the CHG context, and most CHG and CHH DMRs were hypermethylated at the warmer temperature. In eight CHG DMR peaks, a highly similar methylation pattern could be observed between ecotypes. On average, 13% of the differentially methylated genes between ecotypes also showed a temperature-induced change in gene expression. We observed ecotype-specific methylation and expression patterns for genes related to gibberellin metabolism, flowering time, and epigenetic mechanisms. Furthermore, we observed a negative correlation with gene expression when repetitive elements were found near (±2 kb) or inside genes. In conclusion, lasting phenotypic changes indicative of an epigenetic memory were induced by warmer temperature and were accompanied by changes in DNA methylation patterns. Both shared methylation patterns and transcriptome differences between F. vesca accessions were observed, indicating that DNA methylation may be involved in both general and ecotype-specific phenotypic variation.

Population genetic analysis of a global collection of Fragaria vesca using microsatellite markers.

The woodland strawberry, Fragaria vesca, holds great promise as a model organism. It not only represents the important Rosaceae family that includes economically important species such as apples, pears, peaches and roses, but it also complements the well-known model organism Arabidopsis thaliana in key areas such as perennial life cycle and the development of fleshy fruit. Analysis of wild populations of A. thaliana has shed light on several important developmental pathways controlling, for example, flowering time and plant growth, suggesting that a similar approach using F. vesca might add to our understanding on the development of rosaceous species and perennials in general. As a first step, 298 F. vesca plants were analyzed using microsatellite markers with the primary aim of analyzing population structure and distribution of genetic diversity. Of the 68 markers tested, 56 were polymorphic, with an average of 4.46 alleles per locus. Our analysis partly confirms previous classification of F. vesca subspecies in North America and suggests two groups within the subsp. bracteata. In addition, F. vesca subsp. vesca forms a single global population with evidence that the Icelandic group is a separate cluster from the main Eurasian population.

Additive QTLs on three chromosomes control flowering time in woodland strawberry (Fragaria vesca L.).

Flowering time is an important trait that affects survival, reproduction and yield in both wild and cultivated plants. Therefore, many studies have focused on the identification of flowering time quantitative trait locus (QTLs) in different crops, and molecular control of this trait has been extensively investigated in model species. Here we report the mapping of QTLs for flowering time and vegetative traits in a large woodland strawberry mapping population that was phenotyped both under field conditions and in a greenhouse after flower induction in the field. The greenhouse experiment revealed additive QTLs in three linkage groups (LG), two on both LG4 and LG7, and one on LG6 that explain about half of the flowering time variance in the population. Three of the QTLs were newly identified in this study, and one co-localized with the previously characterized FvTFL1 gene. An additional strong QTL corresponding to previously mapped PFRU was detected in both field and greenhouse experiments indicating that gene(s) in this locus can control the timing of flowering in different environments in addition to the duration of flowering and axillary bud differentiation to runners and branch crowns. Several putative flowering time genes were identified in these QTL regions that await functional validation. Our results indicate that a few major QTLs may control flowering time and axillary bud differentiation in strawberries. We suggest that the identification of causal genes in the diploid strawberry may enable fine tuning of flowering time and vegetative growth in the closely related octoploid cultivated strawberry.