Identification of molecular markers associated with fruit flavour and day-neutrality in strawberry (Fragaria x ananassa) using a microarray-based approach
Creators: name: Gor, Mian Chee
Publisher: name: RMIT University
Year: 2024
Subjects: classificationCode: 310505
subject: Gene expression (incl. microarray and other genome-wide approaches)
classificationCode: 340109
subject: Separation science

description: Breeding strawberry (Fragaria x ananassa) with enhanced fruit flavour and day-neutrality is one of the top priorities of the Australian Strawberry Breeding Program. Although several genes involved in the biosynthetic pathways of key volatile compounds and molecular control of flowering response in strawberry have been identified, the development and application of molecular markers associated with these traits remain limited. This study aims to identify molecular markers closely linked to genes controlling strawberry flavour and day-neutrality by combining the Subtracted Diversity Array (SDA) and the Bulked Segregant Analysis (BSA) approaches. To develop a platform for molecular marker identification, a SDA was constructed using a broad subtraction approach between five strawberry genotypes and nine non-angiosperm species, resulting in 287 features specific to the strawberry genome.<br><br> Hierarchical clustering demonstrated that the strawberry-specific SDA was able to correctly cluster 15 strawberry genotypes based on known pedigree information. The aroma profiles and the SDA data of five selected genotypes produced highly similar hierarchical dendrograms. Three of the branch point markers, FLP1C6, FLP1E7 and FLP2E1 showed good correlation with ethyl hexanoate, methyl esters and linalool, respectively. These results indicate that the strawberry-specific SDA may be capable of detecting markers associated with aroma compound production. Subsequently, 50 F1 progeny derived from 07-102-41 x Juliette cross were selected for BSA based on the extreme levels of key volatile compounds assessed to identify molecular markers associated with fruit flavour. For the discovery of molecular markers associated with day-neutrality, the F1 progeny derived from a day-neutral (DN) x short day (SD) and two DN x DN crosses were selected for BSA according to the extremes of flowering response. Differential hybridisation patterns between the extreme DNA bulks were analysed using Discriminant Function Analysis (DFA), Fisher’s ratio and Independent Samples t-Test. Data analysis successfully identified a set of the most predictive features that best discriminated between the extreme DNA bulks. DNA sequence analyses showed that FLP1D7, FLP1A7 and FLP3E12 features were linked to genes possibly involved in the biosynthesis of methyl butanoate, γ-dodecalactone and linalool, respectively.<br><br> Moreover, a putative C/T SNP was observed for FLP1D7 feature, suggesting the presence of allelic variants in this locus. Interestingly, FLP2E11 was found to be closely linked to a CYTOKININ OXIDASE (CKX1) gene possibly involved in promoting flowering under non-inductive condition. A 12 bp deletion was observed in the FLP2E11 sequence cloned from the SD but not the DN genotypes. These results indicate that allelic variants are present in this locus. It is hypothesised that these FLP2E11 allelic variants are linked to the wild type and the low activity CKX1 alleles. A hypothetical model is proposed to demonstrate the mechanism of flowering control regulated by CKX1 based on the hypothesis that flowering strength may be regulated by the copy number of CKX1 alleles. This is the first report on the application of SDA-BSA for the identification of molecular markers potentially associated with important genes controlling fruit flavour and day-neutrality in strawberry. This robust platform should prove useful for future marker-trait association studies in strawberry.