Construction of a subtracted gDNA microarray for rapid discovery of molecular markers associated with day-neutrality in strawberry (Fragaria x ananassa)

Breeding day-neutral strawberry (Fragaria x ananassa Duchesne) is pivotal to extend fruit-bearing season and increase the efficiency of production. However, genetic improvement of day-neutrality by the means of molecular marker technologies remains slow due to genome complexity of octoploid strawberry. This study employs an innovative approach by integrating the Subtracted Diversity Array (SDA) technology and Bulked Segregant Analysis (BSA) to facilitate the identification of molecular markers associated with day-neutrality in octoploid strawberry. A Fragaria Discovery Panel (FDP) containing 287 features specific to strawberry genome was constructed as a platform for rapid screening of DNA polymorphism between one short day (SD) strawberry DNA bulk and three day-neutral (DN) bulks varrying in flowering strength. Differential array hybridisation patterns between the DN and SD bulks revealed a novel molecular marker, FaP2E11, closely linked to CYTOKININ OXIDASE 1 (CKX1) gene possibly involved in promoting flowering under non-inductive condition. Interestingly, a 12 bp deletion was observed within the FaP2E11 sequence cloned from SD genotypes but not DN genotypes. As cytokinin is required to induce flowering, this result indicates that full sequence of FaP2E11 and the sequence with deletion are allelic variants linked to the low enzyme activity CKX1 and the wild type alleles, respectively. To selectively capture flowering associated sequences specific to strawberry, genomic DNA subtraction was performed between a pool of five strawberry genotypes and nine non-angiosperm species using Suppression Subtractive Hybridisation (SSH) technique. A total of 287 subtracted fragments were used to construct a strawberry-specific Subtracted Diversity Array (SDA). Validation of the array revealed a high subtraction efficiency (99%), indicating that the subtracted fragments are strawberry-specific. To investigate the ability of SDA for marker-trait association, three segregating populations: (1) DN ‘01-061-311’ x SD ‘Juliette’, (2) DN ‘01-061-311’ x DN ‘05-069-63’ and (3) DN ‘01-061-311’ x DN ‘05-069-194’ were chosen for BSA based on flowering habits of parental genotypes. The DNA of the individuals derived from the F1 populations were pooled into four bulks: strong day-neutral (DN1), intermediate day-neutral (DN2), weak day-neutral (DN3) and short day (SD) according to flowering strength. Each DNA bulk was hybridised separately onto the SDA. All hybridisations were performed with six technical replicates and two biological replicates to ensure microarray reproducibility, resulting in 12 data points per feature.