Positional Cloning And Characterization Reveal The Role Of FvePHP In The Regulation Of Narrow Leaflets And Early Flowering In Strawberry

Strawberry fruit is favored by consumers because of its unique flavor and rich nutrition.Leaves are the main organ of plants photosynthesis.Flowering time is a key agronomic trait that has a significant impact on plant yield and commodity value.Therefore,it is of great significance to explore the genes of strawberry leaf development and flowering.In this study,a mutant with narrow leaflet and early flowering（nlef）phenotype was identified from ethyl methanesulfonate mutagenesis Fragaria vesca’Yellow Wonder’（YW）population.F₁ plants were obtained by crossing nlef mutant with woodland strawberry’Ruegen’（RG）.BSA sequencing was performed on F₂ population after F₁ generation self-crossing,and then the candidate genes were located and identified by molecular markers.The main results are as follows:1.The plant height,crown diameter,leaflet length,leaflet width,serrate number,and leaflet area of nlef plants were significantly smaller than that of wild type plants.Scanning electron microscopy showed that the mutant leaf cells were significantly smaller than the wild type.Paraffin section observation showed that the leaf thickness of the mutant was significantly larger than that of the wild type.The results of flowering time investigation showed that the flowering rate of the mutant was 5.71%when the wild-type plants did not flower 93 days after sowing,and the flowering rate of the mutant nlef reached 100%at 127 days after sowing,while only 42%of the mutant plants bloomed,indicating that the mutant plants bloomed earlier than the wild-type plants.2.The F₁ plants was obtained by crossing mutant nlef and RG,and F₁ showed the same phenotypic characteristics as the wild-type.The ratio of wild-type to mutant plants in F₂population was approximately 3:1（277:97）,indicating that the mutant type was regulated by a recessive gene.3.The results of BSA-seq showed that a total of 676,004 SNPs were identified in two pooled sequencing data and two parents.The Euclidean distance algorithm and the SNP-index were used to analyze SNPs and In Dels,respectively.The results of the two algorithms show that the candidate interval is located on the Fvb1 chromosome.Through the In Del molecular marker,the candidate interval was located in Indel-8054 and Indel-9652 and size was 1.94 Mb,that is from 16,302,018 to 18,245,390 on the reference genome.The results of manual screening SNP of homozygous mutation in the parental mutant and F₂ mutant pool indicating two variants occurred in the upstream and downstream of the gene,two SNPs occurred in the intron,one was located in the gene,two SNPs occurred in the alternative splicing site,and three In Dels occurred in the upstream of the gene.one occurs in the intron.Upstream and downstream,intronic and intergenic mutations do not change the sequence of the coding region of the gene.Alternative splice site mutations may cause incorrect splicing patterns and lead to loss of gene function.Therefore,we further verified the genes with alternative splice site.Genotyping analysis was performed in F₂ mutant plants by competitive allele-specific PCR.The results showed that the SNPs of the two alternative splicing site mutations were co-segregated with the mutants.RT-PCR was used to verify in mutant and wild-type parents showed that only Fve PHP had alternative splicing in the mutant.Validation of six exchange individual plants using the d CAPS marker developed based on the SNP of this alternative splice site mutation showed that the marker co-segregated with the mutant phenotype.Therefore,Fve PHP may be a candidate gene4.To confirm the mutation of splicing site of the Fve PHP gene,the DNA of YW and nlef were used as templates for PCR amplification and sequencing.The results showed that in the mutant nlef,the splice donor site at the 5’end of the second intron of the Fve PHP gene was mutated（C-T）.Meanwhile,RT-PCR were performed in YW and nlef,and the amplified products were introduced in T vector for sequencing and analysis.The results showed that there are two new transcripts in the mutant nlef,one is retained an intron（Fve PHP-IR）another is deleted part of the exon（Fve PHP-PED）.There was a stop codon in the intron retained by the Fve PHP-IR transcript,resulting in premature termination of the protein translation.The 54 bp exon sequence of Fve PHP-PED was deleted and translated into a complete truncated protein,resulting in the destruction of the integrity of the conserved domain of the protein.5.The virus-induced gene silencing in wild-type YW showed that the height of the plant was reduced,the new leaves were deformed,leaflet was smaller.Over-expression of Fve PHP in nlef showed that the leaf morphology of transgenic plants was similar to that of the wild type in the tissue culture period,Phenotypic investigation after transplanting showed that overexpression of Fve PHP largely restored flowering time,plant height,leaf size,margin serration and other phenotypes.6.Fve PHP localized in the nucleus by subcellular localization.Analysis of the expression characteristics of Fve PHP showed that the gene was highly expressed in strawberry leaves,flowers and fruits.Promoter analysis of the Fve PHP gene revealed multiple elements including auxin response elements,ABA response elements and stress related elements.7.q RT-PCR analysis of several key flowering genes showed that the expression levels of Fve AP1,Fve SEP1,Fve SEP3,and Fve FUL were significantly increased in the shoot apical meristems（SAM）of mutant.Meanwhile,Fve FT was also significantly increased in the mutant leaves.In addition,there was no significant difference in the expression of Fve AP1 and Fve SOC1 between the transgenic plants and the wild-type plants.These results indicated that the early flowering of the mutant was related to the high expression of flowering regulatory genes.8.A number DEGs involved in cell wall metabolism,secondary metabolism,and plant hormones were identified by transcriptome sequencing of YW and nlef leaves.For example,auxin related genes Fv LAX,Fv IAA13,Fv IAA26-like,and Fv ARF5.In addition,the promoter regulators of differential genes were predicted,and the results showed that the transcription factors were mainly enriched in the process of cell division and floral meristem development.Proteomic analysis of YW and nlef leaves showed that the differential proteins were enriched in the macromolecule catalytic pathway,cell wall composition,DNA metabolic pathway.KEGG results showed that the differential proteins were significantly enriched in the DNA damage repair pathway.9.Expression analysis of DNA damage repair genes showed that the expression of DNA damage repair genes was significantly higher in the mutant SAM than in the wild-type.It is speculated that DNA damage affects the cell cycle and cell enlargement leads to plant organ abnormalities.