Positional Cloning And Identification Of Runner Producing Gene In Strawberry

The propagation of strawberry is mainly based on strawberry runners,while the genes controlling runner production have not been well characterized.To identify genes responsible for runner production,we characterized a mutant that exhibited runners in mutant population obtained from the non-runnering woodland strawberry ‘Yellow Wonder'(YW)and named as strawberry runner production(srp).Genetic analyses indicated that srp gene was controlled by a single recessive gene.Whole genome sequencing combines with bulked segregant analysis of the two parents srp mutant and a non-runnering woodland strawberry ‘Ruegen'(RG)and bulked F2 individuals were conducted and analyzed for detecting the causal mutation.At the same time,we used traditional molecular markers to locate,found a candidate gene and verified it.The main results are as follows:1.Mutant srp can produce runner continuously,internode elongation,leaf number decreased compared with wild type YW,plant height,crown diameter and leaf size all increased significantly,and crown diameter decreased slightly.The hybridization between the mutant and wild type RG is the same as that of the wild type.F1 group has basically the same phenotype as the wild type.Whether the F2 group has the separation ratio of runner or non-runner,and the expected value of 1:3,which indicates that the character of runner is controlled by a recessive single gene.2.Whole genome re-sequencing BSA was used to locate the mutant,and 480106 SNP were detected in total.The SNP index of runner pool was calculated,and 1583 SNP were identified on 7 chromosomes.The peak of the frequency of SNP appeared on Fvb4,indicating that this chromosome is most likely to be the gene interval regulating srp.The candidate SNP was analyzed.A SNP mutation(W601*,TGG?TGA)was found at the C terminus of DELLA gene Fve RGA1 of the gibberellin signal pathway,leading to the early termination of the coding.We think Fve RGA1 is the most likely candidate gene to control the srp mutant trait.3.Indel markers were used to identify the traditional genetic mapping.On chromosome4(Fvb4),Indel markers and CAPS/d CAPS primers were designed according to the difference of sequencing data.06210-Hinf I was isolated from the F2 population obtained from the mutant srp and RG hybrid,and was developed from the single base mutation produced byFve RGA1 gene.Therefore,these data have further demonstrated that Fve RGA1 is the best candidate gene for the srp.4.We found conversion from G to A in the adenine guanine 1803 rd base position(1803G-A)of Fve RGA1 by gene cloning and Sanger sequencing,this result was consistent with that of re-sequencing.5.The RNAi vector of Fve RGA1 gene was constructed,and then the vector was transformed into the wild-type woodland strawberry YW and RG with two different genetic backgrounds.The transcriptional level of Fve RGA1 gene in all transgenic lines was significantly downregulated,and the transgenic plants could produce runners which was consistent with the phenotype of mutant,indicating that Fve RGA1 is the gene controlling runner production.6.Fve RGA1 was cloned from wild-type YW and fused with GUS gene to construct Fve RGA1 overexpression vector p RI101-Fve RGA1-GUS and transformed mutant srp.The internodes of transgenic plants overexpressing the Fve RGA1-GUS fusion gene returned to normal,but runners still produced.We speculated that the fusion of Fve RGA1 and GUS affected the gene's function.Therefore,the Fve RGA1 overexpression vector p RI101-Fve RGA1 without GUS gene was constructed and transformed four times,but no resistant shoot has been obtained so far.7.Subcellular localization analysis indicated that Fve RGA1 was localized in the nucleus.A transcription analysis suggested that Fve RGA1 was expressed ubiquitously in all examined strawberry organs,especially in young leaves,petioles and stem apices.The promoter of Fve RGA1 was analyzed,and many cis acting elements were found,including the gibberellin response element.8.The stem tip of YW and srp were selected for transcriptome sequencing,and 6different expression genes were selected and verified.