The Strawberry DNA Testing Handbook was recently developed to assist breeders in identifying published DNA tests and understanding how to apply them. This community resource is available at the Genome Database for Rosaceae and will be continually updated as existing tests are improved and new tests are published . While locus-specific DNA tests are highly useful in parent and seedling selection for traits with simple genetics, genome-wide prediction has become the strategy of choice for improving genetically complex traits in crop species. The goal is predictive, and the utility of this strategy has been demonstrated in strawberry for parent selection for yield and quality traits where it was shownthat: markers are more effective than pedigrees for estimating breeding values, even when phenotypic information is present; phenotyping effort can be reduced by using trials of advanced selections as training populations; and individuals with high predicted performance can be used as parents one year early in the breeding cycle. The future of strawberry breeding research is rife with opportunities in the genomics era. In particular, candidate-gene approaches will be dramatically enhanced by the “Camarosa” reference and other octoploid genomic resources, given the ability to pinpoint sequence variations among subgenomes and thus distinguish among homoeologous alleles. Genes involved in fruit volatile compound biosynthesis are particularly attractive targets given the importance of aroma to flavor and sweetness perception. Gene identification will, in turn, fuel the development of new DNA tests and enhance existing tests.
Questions for the future include the following: What level of genetic gains can be achieved simply by developing markers in causal genes, round nursery pots eliminating problems of recombination between marker and gene? Will functional characterization of genes lead to gene edited strawberries in the commercial realm? A tool that may help to uncover gene/trait associations in strawberry and help identify “missing heritability” is expression QTL . In essence, eQTL are segregating genomic regions influencing differential gene expression. With RNAseq alone, it is often difficult to discern whether changes in transcript accumulation are due to genetics, environment or stochastic effects. A recent eQTL analysis identified a subset of strawberry fruit genes whose differential expression is determined by genotype, the extent of that genetic influence, and markers that can be used for selection of desired gene-expression ranges. Thus, eQTL analyses may reveal marker/trait associations in cases where strawberry phenotypes are influenced by transcript abundance. In other cases, eQTL controlling transcripts of undetermined function can support candidate-gene discovery and trait-based gene cloning. In a recent example, simple cross-referencing of trait-QTL and eQTL markers identified a causal aroma biosynthesis gene in melon. For complex traits controlled by many loci, the area of genome-wide prediction presents a number of practically important research questions for the future. Will the newest SNP array, with its whole-genome coverage and wealth of subgenome-specific markers, help increase prediction accuracies? How large should training populations be to achieve maximum predictive power, and how many breeding cycles can be included? Given that the vast majority of octoploid strawberry cultivars are asexually propagated, can non-additive effects be modeled to enhance predictions of clonal performance? When will low-density genotyping be affordable enough to select for complex traits in seedling populations, as opposed to selection only among parents?
They have been answered in other crop species, and we expect that they will soon be answered in strawberry as well.Strawberries are perennial rosette plants that form a determinate inflorescence from the apical meristem of the crown. Their axillary meristems can differentiate into either branch crowns, that are able to bear additional inflorescences, or runners. Because of these alternative fates of axillary meristems, there is a strong trade-off between flowering and runnering. Strawberries can be divided into two main groups according to their flowering habits. Seasonal flowering strawberries produce flower initials under day lengths below a critical limit , whereas perpetualflowering strawberries produce new inflorescences continuously once induced to flower. In the diploid woodland strawberry F. vesca, the dominant progenitor of the octoploid cultivated strawberry, two classical mutants affecting flowering and runnering are known. Recessive mutations in the SF Locus and Runnering locus cause PF and runnerless phenotypes, respectively. The F. vesca homolog of TERMINAL FLOWER1 was found as a candidate gene for SFL independently by two groups, and Koskela et al. demonstrated the function of FvTFL1 as a major floral repressor that causes the seasonal flowering habit. The R locus was also recently mapped, and a mutation in a gene encoding gibberellin biosynthetic enzyme GA20-oxidase was found. This gene is highly expressed in axillary buds, and the mutated enzyme is not able to convert GA12 to GA20, a precursor of bio-active GA1. Studies in cultivated strawberry indicate at least partial conservation of the genetic pathway in woodland strawberry. Based on available data in woodland strawberry, a genetic model can be proposed . In SF genotypes, FvTFL1 integrates environmental signals to control flowering, and flower induction only occurs after the down regulation of this gene by cool temperatures below 13 °C or by short days at temperatures of 13–20 °C, whereas higher temperatures prevent flower induction by activating FvTFL1. Genes involved in the temperature regulation of FvTFL1 await elucidation, but the photoperiodic pathway is quite well understood. Under long days, the woodland strawberry homolog of CONSTANS activates FLOWERING LOCUS T1 in leaves, which leads to the upregulation of SUPPRESSOR OF THE OVEREXPRESSION OF CONSTANS1 in the shoot apex.
In PF woodland strawberries that are lacking a functional FvTFL1, this FvCOFvFT1-FvSOC1 pathway promotes flowering, whereas in SF genotypes upregulation of FvTFL1 by FvSOC1 reverses the outcome of the pathway. Actual flower induction is poorly understood, but the role of FvFT3, APETALA1 , and FRUITFULL genes that are activated in the shoot apex after the down regulation of FvTFL1 by short days or cool temperature should be further explored. Another important challenge is to understand the flowering process in the context of the yearly growth cycle and to identify allelic variation that can be used for breeding new cultivars better adapted to diverse climates. Open questions include, for example, how is flower initiation and differentiation regulated? How is floral development connected to dormancy? PF cultivars are commercially quite important, but the genetic control of the trait clearly differs from PF in woodland strawberry. A major locus controlling PF was identified, named perpetual flowering and runnering because the PF allele also reduced runnering. Several additional studies in different crossing populations have confirmed PFRU and narrowed the chromosome region. The causal gene is not known, but several candidate genes have been suggested. Interestingly, a QTL controlling flowering time in woodland strawberry was mapped to the same region of chromosome four. These data suggest the presence of either two important flowering genes in this region or different alleles of the same gene that control PF and flowering time. Identification of the PF gene or genes in cultivated strawberry is obviously a research question of high importance both scientifically and commercially. Better understanding the trade-off between flowering and runnering is also an important area of inquiry, plastic flower pots because it might assist plant breeders and growers in controlling the balance between vegetative and sexual reproduction. Several lines of evidence suggest that GA controls the fate of axillary meristems in strawberries. Guttridge and Thompson found that runnerless woodland strawberry mutants began to form runners after GA treatment, and similar reversion was observed in a recent mutant screen that led to the identification of suppressor of runnerless, a gene that encodes a DELLA growth repressor of the GA signaling pathway. Inhibitors of GA biosynthesis, in contrast, enhance crown branching and yield in cultivated strawberry.
Furthermore, FvSOC1 was found to control runner formation and regulate the expression of several GA bio-synthetic genes, including the recently identified FvGA20ox4, which likely encodes a rate limiting enzyme of the GA bio-synthetic pathway in axillary buds. These data suggest a model in which FvSOC1 activates FvGA20ox4 and possibly other GA bio-synthetic genes in axillary buds, leading to high bio-active GA1 levels, degradation of SLR proteins, and runner formation .In contrast to most fruits, the fleshy tissue of Fragaria is a modified stem tip called the receptacle. The true fruits of Fragaria are dried ovaries called achenes, each of which contains a single seed. The receptacle together with attached achenes are what is typically refered to as the “fruit”. Although connected by fibrovascular strands, molecular analysis using microarrays and RNA-seq show that the achenes and receptacle exhibit asynchronous transcriptional programs that reflect differences in timing of maturation of the two tissues. Fruit set requires a sufficient percentage of fertilized achenes due to their production of auxin. Early studies showed that strawberry is non-climacteric, not appearing to respond to exogenous ethylene. However, the role of ethylene in strawberry maturation is reassessed later in this section in light of more recent data. The control of fruit set and development by plant hormones that interact and synchronize signals between the developing seed and surrounding tissues is graphically described in McAtee et al., and a comprehensive discussion of hormonal regulation of fruit ripening in nonclimacteric as compared to climacteric fruit can be found in Cherian et al.. Although a complete picture of how hormonal regulation and crosstalk underlie the molecular mechanisms of development and ripening has not yet emerged in either diploid or octoploid strawberry, considerable progress has been achieved due to gene expression analyses using microarrays, RNAseq, agroinfiltration for transient gene silencing, stable transformation with reporter genes or altered expression, and virus-induced gene silencing. The high-quality diploid and octoploid strawberry genomes and associated resources now available will greatly accelerate discovery. There is still much to learn from the diploid model, and fruit development in the octoploid is likely to involve a more complex interplay of homoeologous genes. In addition, due to potential for interactions among products of homoeologs in the octoploid, careful holistic analysis of octoploid fruit development in achenes and during development remains to be accomplished by combining highly sensitive and accurate transcriptomic, proteomic, and metabolomic methods. That auxin and GAs are primary hormonal players during early development of both the receptacle and achenes is well supported by hormonal analyses of F. × ananassa and transcriptome data from F. vesca. F. vesca transcriptome data from fertilization to the large green fruit stage , were analyzed for evidence of biosynthesis and activity of most of the major hormones, as well as for IAA transport. Genes in the IAA biosynthesis pathway are actively expressed by the endosperm, and perhaps integuments, of the newly fertilized ovary, closely followed by expression of GA biosynthesis genes. This transcriptome foundation now needs to be expanded with detailed hormone metabolic and transport studies, perhaps more easily accomplished by feeding studies with the correct reference compounds and mass spectrometric analyses using larger octoploid fruit. As illustrated in Fig. 3, there are many steps in the process of fruit set and early development that still require direct study. We know that IAA levels in intact F. × ananassa fruit rise rapidly following fertilization and peak at the small green fruit stage, thereafter decreasing to low levels at the white stage and to very low, but homeostatically regulated, levels in red ripe fruit. Interestingly, separate analysis of white fruits and achenes showed that almost all the IAA measured in the intact fruit is in the achenes, with barely detectable levels in the receptacle. It has been suggested that low receptacle IAA levels are required for ABA biosynthesis to start and ripening to commence. GA1, likely the only bio-active GA in fruit, also increased after fertilization, reaching a peak in the large green stage. Like IAA, GA1 levels are low during ripening stages. Analysis of spatial expression in F. vesca showed that genes encoding AUX/IAA transcriptional coregulators and auxin response factors , are highly expressed in the expanding receptacle, as expected if IAA is being released from the achenes to stimulate receptacle growth prior to ripening. How, and in what form, auxin travels from the fertilized achenes to the receptacle is still unknown, as are the signals that turn on auxin biosynthesis in precise tissues in response to fertilization. Auxin transport might be most easily addressed in the larger, octoploid organs. As indicated by the diploid transcriptome studies, such analyses should include measurements of conjugated IAA biosynthesis and possible movement using mass labeled compounds. GA receptor protein genes are also highly expressed in expanding F. vesca fruit, as are DELLA repressor genes.