Mapping,Cloning And Functional Aanlysis Of Few Spines 1 In Cucucmber

Fruit spine density is an important quality trait for marketing in cucumber(Cucumis sativus L.).However,none of genes related to cucumber spine density has been cloned,so,the molecular mechanism of cucumber spine density is still unclear.The mapping and cloning of fs1 could contribute to study on the molecular mechanism of cucumber spine density,which will provide theoretical basis and technical support for the molecular breeding of spine density.In our study,we separated a mutant,few spines 1(fs1),from a North-China type cucumber inbred line,CNS2(wild tye,WT).Compared with WT,the fs1 mutant has fruit with a much lower density of spines,but there are no significant differences in fruit size,leaf,stem and other organs between fs1 and WT.Therefore,fs1 is an ideal material to study the mechanism of fruit spine density in cucumber.Two F2 populations for mapping fs1 were constructed.One cross was between fs1 and WT,and the other between fs1 and North China-type‘Chinese long'9930(9930).To mapping fs1,BSA(Bulk Segregant Analysis)combined with genome resequencing and map-based cloning was used.Transcriptomic analysis of tender fruits at anthesis stage from mutant and non-mutant individuals from the above F2 population were performed and some genes affected by fs1 were found.To confirm the candidate gene,we performed genetic transformation of cucumber.The results are as follows: 1.We constructed a F2 population with fs1 and WT.Leaf samples from mutant and non-mutant individuals from the above F2 population were combined into two separate pools and subjected to genome resequencing.Combined with SNVs and the phenotypes,we located fs1 on the end of chromosome 6.2.It was difficult to develop polymorphic markers in this region between fs1 and WT because of their highly similar genetic background.We constructed another F2 population with fs1 and North China-type 9930.Based on the genome resequencing data,4 polymorphic CAPS markers and 1 polymorphic SNP marker were identified between fs1 mutant and 9930.Using CAPS1 and MM2 markers,130 recombinant lines were chosen out from 5,400 F2 plants derived from the cross between fs1 and 9930.Three more markers(two CAPS and one SNP)were developed to analyze the 32 recombinant plants with mutant phenotype,and fs1 locus was narrowed down to an approximately 110.4-kb region between markers MM1 and MM2.There were a total of 25 predicted genes in this region.3.All 25 genes in 110.4-kb region,including their promoter region,were sequenced and compared between fs1 and WT.A fragment substitution in the promoter of Csa6G514870 was discovered.We found that a 10-bp fragment from –833 to –823 bp before the start codon of Csa6G514870 was replaced with 812 bp in fs1 compared with WT.Csa6G514870 encodes a PROTODERMAL FACTOR 2(PDF2)protein that belongs to the HD-Zip IV subfamily.The expression level of Csa6G514870 in fruits at anthesis stage was higher in fs1 compared to WT.This indicated that Csa6G514870 might be the best candidate gene.4.To further study the functions of fs1,we performed genetic transformation of cucumber.We got 20 regenerated plants,they need to further be validated.5.We got four genes(Csa5M606310,Csa6M501990,Csa3M10181 and Csa3M824990)from fs1 transcriptome compared with cucumber trichome related transcriptome or expression profile,which may be involved in cucumber spine density control network.6.Based on the substitution in the promoter of Csa6G514870,a molecular marker,MM3,was developed,it was a co-segregating maker with the trait of spine density.The 812 fragment wich is in fs1 is also present in wild cucumber(C.sativus var.hardwickii)indicating that the fragment substitution was a key factor determining fruit spine density,and was the key reason that caused spine density changed during domestication.In our study,the best candidate gene for fs1 locus was isolated though map-based cloning,and the key factor determining fruit spine density was found.This work provides a solid foundation not only for understanding the molecular mechanism of fruit spine density,but also for molecular breeding in cucumber.