Map-based Cloning And Characterization Of The Key Genes Controlling Trichome Formation In Tomato

Trichomes exist in the epidermis of nearly all terrestrial plants,including unicellular and multicellular trichomes,glandular and non-glandular trichomes.The molecular mechanism of unicellular trichomes in Arabidopsis is well characterized.However,knowledge about the regulatory pathway of multicellular trichomes in tomato(Solanum lycopersicum)is limited.1.Tomato germplasm resources are very rich.179 accessions(163 normal trichome accessions and 18 no trichome accessions)were used to perform GWAS for trichome formation in tomato.A single locus on chromosome 10 was detected.At the same time,only introgression line(IL)10-2 showed trichome absent through screening trichome-related traits in the 50 ILs,which cover the entire genome of S.pennellii in the background of the cultivated species M82.This result showed that there is a key gene on chromosome 10.h mutant LA3172 is the same as IL10-2 and showed trichome absent.So we constructed two F2 population:Ailsa Craig x IL10-2 and LA3172×LA0411(S.pimpinellifolium).Genetic analysis indicated that the two hair absent phenotype are regulated by a single recessive gene.Finally,this gene was delimited in the region between MP-31 and MP-26.Sequence analysis showed four predicted genes in this region.There is a nucleotide length polymorphism between LA3724 and AC in ORF3.This result showed that ORF3 may be the garget gene.2.We also collected many hair absent materials from all over the world.Sequence analysis showed that there were three type mutations in those hair absent materials:a deletion of 1440bp such as LA3172 and LA0085;an early stop codon resulting from reading frame shift mutations such as LA04432 and 3-605;amino acid substitution such as LA1589 and IL10-2.3.The hair absent phenotype was rescued when ORF3 was expressed in h mutant LA3172.On the contrary,silencing of ORF3 in tomato Ailsa Craig plants by RNA interference(RNAi)resulted in trichome disappearance in all aerial parts.The result showed ORF3 is the right candidate for H gene.4.Trichomes on LA3172 and IL10-2 were observed by SEM showed that type I trichomes completely disappeared on stems of LA3172 and IL10-2.This result showed that H gene plays an important role in the formation of this type of trichomes.5.The spatial expression pattern of Hwas examined by quantitative RT-PCR including roots,young stems,young leaves,function leaves,shoot apexes,flowers,mature-green fruits showed that H was constitutively expressed in all tissues and being higher in the flowers and fruits.This result indicated that H gene not only plays an important role in trichomes formation but also flowers and fruits development.6.Over expression of H gene in tobacco can induced much more trichomes formation.ZFP8(Niben101Scf00749g00003.1),the homologue of H in tobacco,and CA10g21340,the homologue of H gene in pepper also can regulated trichomes formation when over expression in tomato.Those results showed that H gene and its homologs in Solanaceae species are functionally conserved through evolution.7.The disappearance of type I trichomes in both W_ohh and wowohh plants suggested that H gene may be a recessive epistatic factor to the Wo gene.The result showed that H expression was up-regulated in Wo allelic mutants(LA3560,LA3186,and LA0258),whereas down-regulated in the Wo-RNAi plants,indicating that H gene may be positively regulated by Wo.H directly interacts with Wo in vivo and in vitro by yeast two hybrid and GST-pull down.This result indicated that H gene regulated type I trichomes formation by interated with Wo.8.LA3724 was found to have less and shorter trichomes compared with Alisa Craig and its stamens do not form a typically closed anther cone.Trichomes on Ailsa Craig are strings of cells perpendicular to the epidermis,but most of trichomes on LA3724 are forked,some of which are not strings of cells perpendicular to the epidermis but with two parallel cells on the stalk of trichomes by SEM.Based on the high-density molecular linkage map,it was suggested that dl is located in the long arm of chromosome 8.To clone and characterize d/,we constructed two F2 populations derived from the two crosses of dl mutant:LA3724 ×IL8-1 and LA3724 × LA1 589(Solarium pimpinellifolium)and developed 12 molecular markers.Finally,dl was delimited in the 109kb region between 52.81Mb and 52.92Mb.Sequence analysis predicted the presence of seven putative open reading frames.There is no nucleotide polymorphism between LA3724 and AC in seven putative open reading frames.Surprisingly,the aquaporin-like protein gene was evidently upregulated in dl mutant.Thus,we inferred that this gene is a potential candidate for the phenotypes.