Promoter analysis of the SPATULA (FvSPT) and SPIRAL (FvSPR) genes in the woodland diploid strawberry (Fragaria vesca L.)

The aim of this study was to identify transcription factor (TF) binding sites and cis-regulatory elements (CREs) on the promoters of FvSPR1-like2 (SPIRAL) and FvSPT (SPATULA) genes in the woodland diploid strawberry (Fragaria vesca L.). We identified: (1) MYB59, WRKY25 and WRKY8 TFs which play a role in ethylene signaling; (2) ARF family of TFs which play a role in ARF-mediated auxin signaling on the promoter of FvSPR1-like2 gene; (3) ARR family of TFs which play a role in cytokinin signaling; (4) ERF family of TFs which play a role in ethylene signaling on the promoter of FvSPT. This bioinformatic analysis of TFs and CREs may provide a better understanding of the function of genes involved in, and the mechanism underlying, non-climateric ripening during strawberry fruit maturation.

Establishment of A Rapid and Stable Infected System by Agrobacterium-Mediated Transformation of Germination Seeds in Diploid Strawberry.

Background Strawberry (Fragaria) is regarded as a model plant for both Rosaceae and non-climacteric fruit ripening. Although much progress has been made in identification of gene function using stable and transient genetic transformation systems in strawberry, the limitation is, more or less, are present. To this end, development of a rapid, efficient, and stable transformation system is required for strawberry research and breeding. Results Here, using diploid Hawaii-4 (Fragaria vesca) seeds and a reporter gene of CHLH (the H subunit of magnesium chelatase magnesium chelatase) key to chlorophyll synthesis, we first develop a rapid, efficient, and stable infected system by the Agrobacterium-mediated seed infection to silence the reporter gene, reaching an infection frequency with 28.3% through a series of optimization elements, including seed full imbibition and initial germination, shaking infection for 24 h, dark cultivation on MS medium for 3 d at 24 ℃, light culture on MS-Tim medium for 1 week at 24 ℃, and vector construction tagged with fluorescence label. Taken together, radicle-emergence germination seeds, appropriate Agrobacterium concentration and infection time are critical for successful infection, finally obtaining the infected kanamycin-resistant seedlings of T1 generation by infected wild seeds within 1 month and T2 generation-infected plants within 4 months. Conclusions The Agrobacterium-mediated infection of germinating seeds (AMTGS) in diploid strawberry (F. vesca) is first established, providing a useful tool for gene function identification and improved agronomic traits in strawberry.

Establishment of a Rapid and Stable Transgenic System by Agrobacterium-Mediated Transformation of the Germination Seeds in Diploid Strawberry.

BackgroundStrawberry (Fragaria) is regarded as a model plant for both Rosaceae and non-climacteric fruit ripening. Although much progress has been made in the identification of gene function using traditional, stable and transient genetic transformation systems in strawberry, the limitation is, more or less, present. Thus, development of a rapid, efficient, and stable transformation system is required for strawberry research and breeding.ResultsHere, using diploid Hawaii-4 (Fragaria vesca) seeds and a reporter gene of CHLH (the H subunit of magnesium chelatase magnesium chelatase) , we first develop a new, rapid, efficient, and stable transgenic system by the Agrobacterium-mediated seed transformation to silence the reporter gene, obtaining a transformation frequency with 10 % through a series of optimization conditions, including full imbibition and initial germination, shaking infection for 24 h, dark cultivation on MS medium for 3 d at 24 ℃, light culture on MS-Tim medium for 1 week at 24 ℃, and vector construction carried fluorescence label. Taken together, radicle-emergence germination seeds, appropriate Agrobacterium concentration and infection time are critical for successful transformation, obtaining transgenic kanamycin-resistant seedlings within 1 month and T2 generation transgenic plants within 4 months. ConclusionsWe first have successfully established Agrobacterium-mediated transformation of germinating seeds (AMTGS) in diploid strawberry (F. vesca), providing a useful tool for studying non-climacteric fruit ripening and strawberry breeding.

Reinvigoration of diploid strawberry (Fragaria vesca) during adventitious shoot regeneration

Diploid strawberry (Fragaria vesca ‘Baiguo’) is a model plant for studying functional genomics in Rosaceae. Adventitious shoot regeneration is essential for functional genomics by Agrobacterium tumefaciens-mediated transformation. An efficient shoot regeneration method using diploid strawberry leaf explants was conducted on 1/2MS + 1/2B5 medium that contained 2.0 mg L−1 TDZ over 14 days of dark culture; this induced the maximum percentage of shoot regeneration (96.44 ± 1.60%) and the highest number of shoots per explant (23.46 ± 2.14) after 11 weeks of culture. The explants considerably enlarged after 12 days; then, turned greenish brown after 30 days, yellowish brown after 36 days, and completely brown and necrotic after 48 days. Large numbers of adventitious shoots were produced from 48 to 66 days, and the shoots elongated from 66 to 78 days; this represents a critical period of reinvigoration, which included 30 days for leaf explant chlorosis, 36 days for adventitious shoot appearance, and 48 days for generation of numerous shoots. During the reinvigoration process, higher expressions of the hormone synthesis-related genes Ciszog1, CKX2, CKX3, CKX7, YUC2, YUC6, YUC10, YUC9, and GA2ox were detected from 30 to 48 days. Our results indicate that these genes may regulate reinvigoration of shoot regeneration.

Identification and Expression Analysis of Fragaria Vesca MLO Genes Involved in Interaction with Powdery Mildew (Podosphaera Aphanis)

Strawberry powdery mildew, caused by Podosphaeraaphanis is a major fungal disease that affects strawberry yield and quality. In the model plant species Arabidopsis and the crop plants barley, tomato and pea, the Mildew resistance locus O (MLO) proteins have been found to be required for powdery mildew susceptibility. The present study, based on the sequence of a wild plum (Prunus americana) MLO protein, identified 16 MLO genes within the genome of woodland strawberry, Fragaria vesca and examined their expression pattern in response to powdery mildew infection in three diploid strawberry cultivars. Phylogenetic analysis showed that the FvMLO genes can be classified into six clades. Four FvMLO genes were grouped into clade III, which comprises MLO genes from Arabidopsis, tomato and grapevine that mediate powdery mildew susceptibility. A RNA-seq analysis of two diploid strawberry cultivars, F. vescassp. vesca accession Hawaii 4 (HW) and F. vesca f. semperflorens line “Yellow Wonder 5AF7” (YW) at 1 d (1 DAI) and 8 d (8 DAI) after infection showed the expression of 12 out of the 16 FvMLO genes. The comparison of Fragments Per Kilobase of transcript per Million mapped reads (FPKM values) detected by RNA-seq and expression values of qRT-PCR for FvMLO genes showed substantial agreement. The FvMLO3 gene, which was grouped in clade III and orthologous to the Arabidopsis,tomato and grapevine genes, was highly expressed in YW compared to other FvMLO genes across varieties. The results showed that FvMLO genes can be used as potential candidates to engineer powdery mildew resistance in strawberry based on MLO suppression or genome editing.