Final Report Summary - IOF FLAVOR (CONTROL OF AROMA COMPOUNDS IN STRAWBERRY) Intensive breeding for the improvement of agronomic traits, such as yield, disease resistance, fruit firmness and size has contributed to a progressive decline of strawberry flavor (taste and aroma). Furthermore, the complexity of volatile composition and their quantitative genetics has discouraged breeders of the genetic improvement of this trait. The overall objective of the IOF FLAVOR project is the identification and characterization of strawberry genes controlling natural variation in key flavor compounds and the elucidation of molecular mechanisms controlling their concentration. The final key goal of this proposal is the translation of genomic and phenotypic data into markers suitable for breeding tastier strawberries.One of the first objectives of this project was to develop a genetic map of strawberry genome with high density of molecular markers to enable efficient identification of QTLs and candidate genes. To achieve this objective we have optimized DArT and DArT-seq platforms for high-throughput genotyping in strawberry, a crop with a genetically complex octoploid genome (Sánchez-Sevilla et al., 2015).The second objective was the Identification of genes controlling the content of essential volatiles in strawberry fruits. The IOF Flavor Project has used information generated in previous genetic studies of strawberry flavor to elucidate the molecular mechanisms controlling major QTLs for key volatiles. Identification of the genes regulating these important compounds would be a major step for strawberry improvement. Our attention has been focused on major QTLs that are stable, and therefore suitable for marker-assisted breeding. During the first period of the project, that took place in the laboratory of Dr. Kevin Folta in the University of Florida, we have identified the key gene FaFAD1 necessary for the accumulation of γ-decalactone, a volatile that contributes to strawberry flavor with peachy and sweet notes. The results have been published in two companion manuscripts in the journal BMC genomics (Sánchez-Sevilla et al., 2014; Chambers et al., 2014). The variation in the content of another important lactone, γ-dodecalactone, has been mapped to linkage group VII-1 in strawberry. Using a combination of genetic and next-generation sequencing analysis we have identified two candidate genes for the variation in this volatile. Functional analyses such as expression studies and over-expression and silencing of candidate genes are being done in order to functionally validate them.Esters, formed by esterification of alcohols and acyl-CoA, constitute the largest and one of the most important volatiles contributing to the aroma of strawberry fruit. A common QTL controlling the content of 10 important esters, such as butyl and octyl hexanoates and butanoates, and 2 alcohols, 1-octanol and 1-decanol, was detected in LG VI-1 in a previous work. We have identified 20 contrasting lines among the 95 progeny lines of the ‘232’ x ‘1392’ population and analyzed differential expression using RNA-seq between the two pools of 10 lines. This analysis has resulted in the identification of two candidate genes, both located in LG VI at the position were the closest marker to the QTL for esters maps in the reference F. vesca genome. The expression of both candidate genes was correlated with the variation in esters content. To investigate the role of these two genes in ester biosynthesis, we are currently analyzing the effect of over- and down-regulation of both genes by transient expression studies in fruits of different strawberry cultivars. These ongoing experiments will hopefully identify if any of these candidate genes are able to affect volatile levels in strawberry fruits and clarify the molecular mechanisms controlling their concentration.The key goal of this proposal is the translation of genomic and phenotypic data into markers suitable for strawberry breeding. An important activity of the IOF FLAVOR project is the development and validation of closely linked markers to the identified genes for future implementation in marker assisted breeding programs. Selected markers are been assessed in a wide collection of cultivars to determine whether they predict the corresponding traits accurately. As a result of this project we have analyzed the ability of 2 markers in the genes FaOMT and FaFAD1 to predict the presence of mesifurane and γ-decalactone among strawberry cultivars (manuscript in preparation). Both markers were able to predict the traits with reliability higher than 91%. A simple PCR test for these volatiles in fruits has been developed and disseminated to strawberry breeders. We will continue marker development and validation with additional volatile traits once other candidate genes under study are functionally evaluated.In contrast to other important traits easier to quantify, strawberry aroma results from a complex interaction of hundreds of volatile compounds resulting from the activity of many genes from different biosynthetic pathways. Usually, breeders select for flavor based in sugar and acid measurements, and panel tests. Aroma evaluation using instrumental analysis, such as GC-MS, during breeding programs is expensive due to the high number of selections that need to be tested. The implementation of markers such as the ones developed in the IOF-FLAVOR Project for the selection of cultivars with improved flavor will surely accelerate breeding programs for the selection of flavorful strawberries. Therefore, the availability of DNA markers able to predict high concentration of particular volatiles will have a high socio-economic impact on the strawberry breeding industry. Furthermore, consumers will also benefit from buying and enjoying tastier fruit.
