DNA marker development and validation for the level of methyl butanoate in strawberries

Strawberry (Fragaria x ananassa Dutch.) breeding with intense flavour and better quality has always been a priority of the Australian breeding program. One of the main reasons is that foreign sourced commercial varieties do not always produce consistence flavour across the season to satisfy customer perception under Australian growing conditions. Previous analysis of genotype-by-environment interactions of flavour compounds suggested that the inheritance of some compounds is highly influenced by the genetic composition of the genotype. Therefore, our current research has focused on screening polymorphic markers between two bulks of plants possessing high and low levels of key flavour compounds with high genetic influence. The aroma profile of the parental genotypes, Juliette and 07-102-41 breeding line and 50 F₁ progeny plants were analysed using solid phase micro-extraction (SPME) and gas chromatography coupled with mass spectrometry (GC-MS). The results showed that no methyl butanoate was detected in Juliette whilst significantly high levels of the compound were identified in 07-102-41. Frequency distribution was generated to categorise progeny plants into two extremes bulks according to high and low levels of methyl butanoate detected in each plant. A strawberry-specific Subtracted Diversity Array (SDA) was then used as a platform for screening polymorphic markers between the two phenotypic extreme bulks. The resulting microarray data set was subjected to Discriminant function analysis, Fisher’s ratio and Independent samples t-test for the identification of polymorphic markers associated with the biosynthesis of methyl butanoate in Australian-grown strawberries. DNA sequence analysis of a polymorphic marker, FLP1D7 revealed a putative C/T Single Nucleotide Polymorphism (SNP) between Juliette and 07-102-41. For the validation of the marker, a primer set was designed to amplify the region flanking SNP. The putative marker was validated on the F₁ progeny plants derived from the two phenotypic extreme bulks and a wider range of germplasm including the Australian commercial varieties Melba, San Andreas, Portola, Palomar, Camino Real and Albion. DNA sequencing of the putative marker confirmed the association between the C/T SNP and the levels of methyl butanoate in the strawberry genotypes tested.