High-quality haplotype-resolved genome assembly of cultivated octoploid strawberry

Cultivated strawberry(Fragaria×ananassa),a perennial herb belonging to the family Rosaceae,is a complex octoploid with high heterozygosity at most loci.However,there is no research on the haplotype of the octoploid strawberry genome.Here we aimed to obtain a high-quality genome of the cultivated strawberry cultivar,“Yanli”,using single molecule real-time sequencing and high-throughput chromosome conformation capture technology.The“Yanli”genome was 823 Mb in size,with a long terminal repeat assembly index of 14.99.The genome was phased into two haplotypes,Hap1(825 Mb with contig N50 of 26.70 Mb)and Hap2(808 Mb with contig N50 of 27.51 Mb).Using the combination of Hap1 and Hap2,we obtained for the first time a haplotype-resolved genome with 56 chromosomes for the cultivated octoploid strawberry.We identified a∼10 Mb inversion and translocation on chromosome 2-1.104957 and 102356 protein-coding genes were annotated in Hap1 and Hap2,respectively.Analysis of the genes related to the anthocyanin biosynthesis pathway revealed the structural diversity and complexity in the expression of the alleles in the octoploid F.×ananassa genome.In summary,we obtained a high-quality haplotype-resolved genome assembly of F.×ananassa,which will provide the foundation for investigating gene function and evolution of the genome of cultivated octoploid strawberry.

Genome-wide identification and expression analysis of auxin response factor(ARF) gene family in strawberry(Fragaria vesca)

Auxin signaling plays a significant role in the whole process of plant growth and development from embryogenesis to senescence.Auxin response factors(ARFs) are reported to regulate the expression of auxin response genes by binding to auxin response elements.ARF is the most critical transcription factor family which has been released in most species,but few reports in strawberry.In this study,the structure characterization of 12 FvARF genes in strawberry,their expression patterns at different development stages,different organizations,and different indole-3-acetic acid(IAA) treatments were analyzed.The expression of 12 FvARFs was found in all experiment tissues and showed almost the same trend during fruit development.All FvARFs respond to the treatment of IAA.Our study provides comprehensive information on ARF family in strawberry,including gene structures,chromosome locations,phylogenetic relationships and expression patterns.The information on FvARF genes paves the way for future research on strawberry ARF genes.

A splice site mutation in the FvePHP gene is associated with leaf development and f lowering time in woodland strawberry

Leaves and f lowers are crucial for the growth and development of higher plants.In this study we identified a mutant with narrow leaf lets and early f lowering(nlef)in an ethyl methanesulfonate-mutagenized population of woodland strawberry(Fragaria vesca)and aimed to identify the candidate gene.Genetic analysis revealed that a single recessive gene,nlef,controlled the mutant phenotype.We found that FvH4_1g25470,which encodes a putative DNA polymeraseαwith a polymerase and histidinol phosphatase domain(PHP),might be the candidate gene,using bulked segregant analysis with whole-genome sequencing,molecular markers,and cloning analyses.A splice donor site mutation(C to T)at the 5-end of the second intron led to an erroneous splice event that reduced the expression level of the full-length transcript of FvePHP in mutant plants.FvePHP was localized in the nucleus and was highly expressed in leaves.Silencing of FvePHP using the virus-induced gene silencing method resulted in partial developmental defects in strawberry leaves.Overexpression of the FvePHP gene can largely restore the mutant phenotype.The expression levels of FveSEP1,FveSEP3,FveAP1,FveFUL,and FveFT were higher in the mutants than those in‘Yellow Wonder’plants,probably contributing to the early f lowering phenotype in mutant plants.Our results indicate that mutation in FvePHP is associated with multiple developmental pathways.These results aid in understanding the role of DNA polymerase in strawberry development.

Woodland strawberry WRKY71 acts as a promoter of flowering via a transcriptional regulatory cascade

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.

Auxin-induced AUXIN RESPONSE FACTOR4 activates APETALA1 and FRUITFULL to promote flowering in woodland strawberry

Flowering time is known to be regulated by numerous pathways,such as the autonomous,gibberellin,aging,photoperiod-mediated,and vernalization pathways.These regulatory mechanisms involve both environmental triggers and endogenous hormonal cues.Additional flowering control mechanisms mediated by other phytohormones,such as auxin,are less well understood.We found that in cultivated strawberry(Fragaria×ananassa),the expression of auxin response factor4(FaARF4)was higher in the flowering stage than in the vegetative stage.Overexpression of FaARF4 in Arabidopsis thaliana and woodland strawberry(Fragaria vesca)resulted in transgenic plants flowering earlier than control plants.In addition,FveARF4-silenced strawberry plants showed delayed flowering compared to control plants,indicating that FaARF4 and FveARF4 function similarly in regulating flowering.Further studies showed that ARF4 can bind to the promoters of the floral meristem identity genes APETALA1(AP1)and FRUITFULL(FUL),inducing their expression and,consequently,flowering in woodland strawberry.Our studies reveal an auxin-mediated flowering pathway in strawberry involving the induction of ARF4 expression.