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Fragaria nilgerrensis is a wild diploid strawberry species endemic to east and southeast region in Asia and provides a rich source of genetic variations for strawberry improvement. Here, we present a chromosome‐scale assembly of F. nilgerrensis using single‐molecule real‐time (SMRT) Pacific Biosciences sequencing and chromosome conformation capture (Hi‐C) genome scaffolding. The genome assembly size was 270.3 Mb, with a contig N50 of ∼8.5 Mb. A total of 28 780 genes and 117.2 Mb of transposable elements were annotated for this genome. Next, detailed comparative genomics with the high‐quality F. vesca reference genome was conducted to obtain the difference among transposable elements, SNPs, Indels, and so on. The genome size of F. nilgerrensis was enhanced by around 50 Mb relatively to F. vesca, which is mainly due to expansion of transposable elements. In comparison with the F. vesca genome, we identified 4 561 825 SNPs, 846 301 Indels, 4243 inversions, 35 498 translocations and 10 099 relocations. We also found a marked expansion of genes involved in phenylpropanoid biosynthesis, starch and sucrose metabolism, cyanoamino acid metabolism, plant–pathogen interaction, brassinosteroid biosynthesis and plant hormone signal transduction in F. nilgerrensis, which may account for its specific phenotypes and considerable environmental adaptability. Interestingly, we found sequence variations in the upstream regulatory region of FnMYB10, a core transcriptional activator of anthocyanin biosynthesis, resulted in the low expression level of the FnMYB10 gene, which is likely responsible for white fruit phenotype of F. nilgerrensis. The high‐quality F. nilgerrensis genome will be a valuable resource for biological research and comparative genomics research. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression by base pairing to mRNA target sequences, and play crucial roles in plant development and stress responses. The knowledge on post-transcriptional regulation by miRNAs in strawberry is rather limited so far. In order to understand the role of miRNA in the molecular control during strawberry fruit development, small RNA libraries were constructed from fruits at the turning stage of strawberry cultivar 'Sachinoka' and its white-flesh mutant by using the Solexa platform. One hundred and twenty conserved miRNAs belonging to 27 miRNA families and 33 putative novel strawberry miRNAs were identified in both libraries. Their target genes were predicted using the Fragaria vesca genome. Nine of all miRNAs showed significant expression differences between two types of samples. Four miRNAs were up-regulated and five were down-regulated in white-flesh mutant. The sequencing results were partially validated by quantitative RT-PCR. Among them, the expression of miR399a shows the biggest change between the two samples. The prediction of its target gene showed that miR399 may play an important role in phosphate homeostasis of strawberry fruits. Furthermore, we deduce that the expression of miR399 has negative correlation with the content of sugars. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are 20-24 nucleotide (nt) non-coding regulatory RNAs which play critical roles in plant growth and development. miRNA-encoding genes, which are transcribed by RNA polymerase II, are involved in a variety of processes, including developmental and morphogenesis systems, and hormone and stress responses. To investigate miRNA responses to tissue culture conditions, quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to detect differences in miRNA expression between in vitro micropropagated strawberry plants and transplanted micropropagated strawberry plants. Four miRNAs were differentially expressed between them, including one up-regulated gene (miR156) and three down-regulated genes (miR164, miR172 and miR390). The ratios of miRNA expression levels in in vitro micropropagated strawberry plants to micropropagated plants transplanted into soil in greenhouse for 4 months for miR156, miR164, miR172 and miR390 were 6.757, 0.046, 0.035 and 0.050, respectively. The ratio of miR156 expression levels in micropropagated plants transplanted into soil for 5 months to levels in the conventionally propagating runner plants was 3.785. miR156 was expressed highly and was strikingly inversely proportional to the expressions of its target gene SPL9 and miR172 in in vitro micropropagated strawberry plants. We speculate that high expression of miR156 is the main reason for rejuvenation in micropropagated plants. [ABSTRACT FROM AUTHOR]

The WRKY proteins are a large family of transcription factors that play important roles in stress responses and plant development. However, the roles of most WRKYs in strawberry are not well known. In this study, FvWRKY71 was isolated from the woodland strawberry 'Ruegen'. FvWRKY71 was highly expressed in the shoot apex and red fruit. Subcellular localization analysis showed that FvWRKY71 was located in the nucleus. Transactivation analysis showed that FvWRKY71 presented transcriptional activation activity in yeast. Overexpression of FvWRKY71 in Arabidopsis and woodland strawberry revealed early flowering in the transgenic plants compared with the wild-type control. Gene expression analysis indicated that the transcript levels of the flowering time and development integrator genes AP1, LFY, FT, AGL42, FUL, FPF1, SEP1, SEP2, and SEP3 were increased in FvWRKY71-overexpressing Arabidopsis and strawberry plants compared with the wild-type controls, which may result in accelerated flowering in transgenic plants. Furthermore, FvWRKY71 was proven to directly bind to the W-boxes (TTGACT/C) of the FvFUL, FvSEP1, FvAGL42, FvLFY, and FvFPF1 promoters in vitro and in vivo. Taken together, our results reveal a transcriptional regulatory cascade of FvWRKY71 involved in promoting flowering in woodland strawberry. [ABSTRACT FROM AUTHOR]

Abstract: The most popular retrotransposon-based molecular marker system at the present time is the sequence-specific amplification polymorphism (S-SAP) system, which is widely used for various applications including phylogenetic analysis. In this study, we successfully developed a new S-SAP marker system based on FaRE1 retrotransposon in strawberry genome. Among 34 primer combinations used to develop the S-SAP marker system, seven primer combinations produced high quality and reliability banding patterns. A total of 288 polymorphic bands from 722 amplified bands were detected in 13 strawberry accessions, with a polymorphic ratio of 39.8%. Phylogenetic analysis showed that genetic coefficient ranged from 0.67 to 1.00 for 13 strawberry accessions. An 187bp S-SAP band was detected from 50 strawberry plantlets regenerated from leaves which were cultured on MS media supplemented with 1.0mg/l thidiazuron (TDZ) and 0.1mg/l 2,4-dichlorophenoxyacetic acid (2,4-D). The available evidences proved S-SAP marker had high level of polymorphism in strawberry accessions. [Copyright &y& Elsevier]

• FvPHO2 gene-edited plants of woodland strawberry were obtained by CRISPR/cas9 technology. • Knockout of the FvPHO2 gene can increase the phosphorus content in the leaves and fruits of woodland strawberry. • Knockout of the FvPHO2 gene can improve the anthocyanin content and soluble solids content in the mature fruits of woodland strawberry. Phosphorus (P) plays a vital role in plant growth and reproduction. Our previous study found that sugar content positively correlated with phosphorus content in strawberry fruits. PHO2, a ubiquitin-conjugating E2 enzyme, is a key negative regulator in phosphorus uptake and transport. Thus, PHO2 may be an ideal candidate gene for the breeding of P-efficiency and high-quality cultivars based on the method of gene editing. In this study, the FvPHO2 gene of woodland strawberry (Fragaria vesca) was edited with CRISPR/Cas9 technology and we obtained T 0 generation transgenic mutants, through identification, we selected pho2–1 # 19 and pho2–2 # 9 as representatives of Fvpho2 mutants and analyzed them. Then we obtained T 1 generation by selfing. Phosphorus (P) content in the leaves of Fvpho2 mutants was significantly increased by 11%-89% compared with the wild-type (WT). Similarly, the P content in the mature fruits of Fvpho2 mutants was increased by 40%-64% compared with the WT. However, the plant height of Fvpho2 mutants was significantly decreased by 7%-18% compared with the WT. Interestingly, the anthocyanin content and soluble solids content has been increased by 22%-38% and 26%-32% respectively. This study will lay the foundation for the quality breeding of strawberries by editing the FvPHO2 gene. [ABSTRACT FROM AUTHOR]