Genetic Mechanism Underlying Red Spots On Lettuce Leaves

Lettuce is one of the most important vegetables and widely grown all over the world.Due to its short cultivation period and suitable for hydroponic growing methods,lettuce are widely used in new agricultural systems such as plant factory.Lettuce is a rich source of vitamin A and vitamin K.However,the majority of lettuce is water,resulting in deficiency of nutrition.In lettuce,the red leaves were a result of anthocyanin accumulation.Based on the high antioxidant activity of anthocyanin,high content of anthocyanin in lettuce is a main goal for breeding.However,the anthocyanin accumulation in lettuce leaves depends on light intensity,limiting its application.We found a few accessions displaying irregular red spots on green leaves,and the formation of spots was not affected by light.Elucidating the genetic mechanism underlying the formation of spots has a great significance in lettuce improvement to breed a red lettuce which can accumulate anthocyanins even in low light conditions such as in plant factory.In this study,we dissected the genetic mechanism underlying the red spots on lettuce leaves.Finally we cloned two genes responsible for the formation of spots and fine mapped one loci controlling density of spots.Combined with the transcriptome sequencing,the genes involved in the formation of spots were identified and a model was proposed.The main results were listed below:1.Cytological observation and metabolite analysis showed that the red spots are a result of random distribution of cyaniding-3-O-β-glucopyranoside in the epidermal and subepidermal of adaxial side and epidermal of abaxial side.2.Two genes responsible for the formation of spots were cloned by forward genetic approach.And they encode ANS and R2R3-MYB transcription factor,respectively.A nonsense mutation was occurred in green lettuce,and transformation of ANS into green lettuce recovered red leaves,suggesting ANS regulates the biosynthesis of anthocyanin.Besides,the loss of LSL2 A also led to green leaves.Overexpression of LSL2 A in green lettuce,the leaves turned red.However,complementation of LSL2 A into same green lettuce,leaves displayed spotted,indicating LSL2 A was responsible for the formation of spots.Moreover,transgenic experiment showed that the promoter of LSL2 A had spatio-temporal expression pattern,indicating the causal gene for red spots is the regulatory gene of LSL2 A.3.124 cultivars and 145 wild genotypes of lettuce were used to investigate the evolution of LSL2 gene family in Lactuca.Consequently,a total of 34 distinct LSL2 genes were obtained and a phylogenetic tree was constructed.These LSL2 genes can be divided into three groups according to phylogenetic tree.Our data suggest that there are three homologs in the ancestral wild lettuce L.virosa,and genotypes with varied number of homologs are a result of deletion or duplication.The LSL2 haplotype in spotted lettuce is selected from the L.serriola with three homologs.4.LSL3 was assumed to encode C2H2 type zinc finger protein.Candidate region in dense spotted lettuce was assembled by Nanopore sequencing technique and annotated manually.Comparison between several genotypes showed that the point mutation in ZFP was unique in dense spotted lettuce.Phylogenetic tree revealed that ZFP belonged to subset C3 in Arabidopsis,indicating ZFP may regulate the transposon activity by small RNA.5.Differentially expressed gene(DEG)analysis between red-spotted region and green region on the same leaves identified 141 differentially expressed genes,in which 5genes(CHS,DFR,ANS,LSL2 and b HLH42)were involved in biosynthesis of anthocyanin.Further analysis showed that LSL2 A and LSL2 B specifically expressed in spotted region.Knocking out LSL2 A led to disappearance of the spots.In contrast,no phenotypic change was found when the LSL2 B was knocked out,suggesting that the LSL2 A was responsible for red spots.RT-PCR showed that LSL2 B was not expressed when knocking out LSL2 A,indicating LSL2 A regulates the expression of LSL2 B.6.By constructing a segregating population through crossing a red cultivar with a red-spotted cultivar,the gene controlling red leaf was an allel of LSL2 with two In Del in the promoter.q PCR showed that LSL2 A was highly expressed in red lettuce,and promoter activity analysis revealed the weak activation ability of pro LSL2 A.Transformation of the RLL2 A from the red parent into a green genotype recovered red color.Therefore,the mutations in the promoter region affect its transcription and caused leaf color variation.Furthermore,all spotted lettuce harbored the LSL2 haplotype,and all red lettuce harbored RLL2 haplotype in GWAS population,and q PCR revealed that the expression of LSL2 A was dramatically reduced in spotted lettuce,suggesting all spotted lettuce were caused by the insertions in the promoter of LSL2 A.In this study,we successfully cloned two genes that are indispensible for the redspotted leaves in lettuce and fine mapped the locus controlling dense of spots.Based on above results,the mechanism for the formation of spots was propsed.There is a transcription factor and this transcription factor regulates LSL2 A.The spatio-temporally expression of this transcription factor resulted in irregular red spots by activating LSL2 A.Red spots exists on leaves of L.sativa and L.serriola,even on the leaves of L.virosa,indicating this is an ancient event.Hundreds and thousands of seeds harvesting from spotted lettuce were germinated,but no revertant with full red leaves was found,indicating high expression of this transcription factor might have side effect to lettuce.In future,full red revertant can be obtained by tissue culture and applied for breeding lettuce with high content anthocyanins and independent to high intensity light.