GWAS Screening And Molecular Characteristics Of High-yield Genes In Forsythia Suspensa Fruit

Forsythia suspensa is a deciduous shrub of Oleaceae,which not only has economic and ornamental value,but also has high value in medicinal application.The genetic basis of crop yield is a hot spot in current agricultural research.Identification of genes related to yield traits is the key to improve yield.F.suspensa,as a new medicinal crop,lacks excellent varieties at present.Genes related to fruit yield traits of F.suspensa have not been identified yet.In this study,the plant of F.suspensa in the same field(34.03 N and 111.03 E)in Sanmenxia was used as experimental material.The candidate genes related to fruit yield were screened by Genome-wide association study GWAS and transcriptome data were combined to differentially express genes in four parts of stem,leaf and flower of F.suspensa.Bioinformatics was carried out on four candidate genes affecting the yield of F.suspensa,and Fs WRKY18 was cloned and overexpressed.The specific results are as follows:1.Identification of candidate genes and variant loci for yield-related traits such as seed length,fruit width,fruit thickness and single grain weight of 60 forsythia suspensa plants from the same habitats,13 variable loci related to single grain weight,24 variable loci related to fruit thickness,8 variable loci related to fruit width were found,and four possible genes related to fruit development were screened: WRKY transcription factor 35(WRKY18,EVM0030555),salicylic acid binding protein(SBP2,EVM0012993),auxin response factor6(ARF6,EVM0018222)and alpha-mannosidase(α-MAN,EVM0029771).2.The physical and chemical properties,phylogeny,conserved motifs,chromosome location,interacting proteins and homology of the corresponding proteins were analyzed using bioinformatics.The results showed that the open reading frame of the WRKY18 candidate gene was 768 bp,encoding 255 amino acids,and had a highly conserved full-length sequence.It was a member of the WRKY transcription factor superfamily,and its subcellular location was predicted to be in the nucleus.The sequence expression was similar to that of olive Olea europaea have higher homology.3.According to F.suspensa transcriptome data,it was found that the expression level of auxin response factor(ARF6,EVM0018222)was higher than that of the other three candidate genes,and the expression level of Fs ARF6 in Forsythia flower was higher than that in fruit,stem,and leaf.The expression level was: stem > fruit > leaf.The expression level of salicylic acid binding protein(SBP2,EVM0012993)in the fruit,stem,leaf and flower of F.suspensa was the lowest compared with the other three candidate genes;Compared with Fs SBP2 and Fsα-MAN,Fs SBP2 and Fsα-MAN are highly expressed in the stem,followed by the stem,and the expression level of Fs WRKY18 is higher than that of Fs SBP2 and Fsα-MAN in the fruit of F.suspensa.Related studies have found that Fs WRKY18 has a certain role in the development of the fruit.It is speculated that WRKY18 may be related to F.suspensa fruit size.4.The probability of hydrophilic protein with peptide predicted by WRKY18 protein management property in bioinformatics is 0.065%.The proportions of the four secondary structures predicted by the secondary structure are: Alpha helix accounts for 23.53%,Extended strand accounts for 11.76%,Beta turn accounts for 4.31%,Random coil accounted for 60.39%.Gene expression of the WRKY family found that the gene expression level of Forsythia leaf samples was second in the stem tissue,and the lowest in the flower tissue.The expression level in the whole fruit was higher than that in the flower tissue.The WRKY transcription factor 35(WRKY18,EVM0030555)was cloned and the over-expression vector PC2300-35s-GFP-rbcs was constructed.Agrobacterium tumefaciens solution containing recombinant plasmid was preserved,which provided a certain basis for the subsequent transgenic transformation system of Forsythia suspensa Forsythia suspensa.