| Lettuce（Lactuca sativa）is an important worldwide vegetable.It has rich diversity of leaf shapes,which provides ideal research materials for studying leaf development.Heading is a unique phenotype in vegetables.This trait exists in Chinese cabbage,endive,cabbage,and lettuce.However,only little is known on the genetic and molecular mechanisms of heading leaves.In this study,we genetically analyzed the heading trait in lettuce and identified two candidate genes associated with heading.The details are as follows:Genetic analysis of heading in lettuce.In this study,we used an F2segregating population derived from a cross between a crisphead and a looseleaf lettuce to identify genes regulating heading leaves.From the F2 population,20 heading and 20 non-heading individuals were mixed into two pools,respectively.RNAs were extracted from the young leaves of the two pools and sequenced using Illumina platform.Allele frequencies of the two pools were calculated,and three regions on chromosmes showed obvious difference on allele frequencies between the two pools suggesting three QTLs controlling lettuce heading in this population.We screened F2populations using CAPS markers and selected individuals that are heterozygous at one QTL but homozygous for others.These selected individuals were selfed to generate F2:3populations.As a result,two populations for two different loci,which showed Mendian ratio,were obtained.These two loci were named as Lettuce Heading Locus 4（LHL4）and Lettuce Heading Locus 5（LHL5）,respectively.Fine mapping and cloning of LHL4.LHL4 was fine mapped to a 230 Kb candidate region on chromosome 2 using 1,008 individuals from an F2:3population.Five genes were expressed in the candidate region.Among them,Ls DRL1,which is a homolog of Arabidopsis At DRL1 and may be associated with brassinosteroid（BRs）metabolism,showed significant differential expression between the two pools.Furthermore,compared with non-heading parent,a 12 Kb sequence was inserted in the coding region of the Ls DRL1 gene from the heading parent.The remaining 4 genes were not involved in leaf development,and they did not exhibit structural variation or differential expression pattern between the two parents.Therefore,the Ls DRL1 gene was considered as a candidate gene for LHL4.We overexpressed and CRISPR knockout Ls DRL1 in heading plant and looseleaf plant,resepectively.Unfortunately,no obvious phenotypic changes were observed between the positive T0plants and controls.Fine mapping and cloning of the LHL5 gene.Approximately 1,000 plants were genotyped and phenotyped,and the LHL5 gene was mapped to in a 1.3 Mb interval on chromosome 5.A total of 30 genes were present in the candidate interval.Among them,8genes exhibited SNPs or small In Dels in the coding region between the two parents,while no differential expressions were observed between the two parents for any of the 30 genes.One of these 8 genes with polymorphsim encoded a transcription factor of the TCP family.The TCP gene from the looseleaf parent had 6-base insertion in its coding region compared with heading parent.The remaining genes showed no known function associated with leaf development.Therefore,the TCP gene was the most likely candidate gene of LHL5.CRISPR vector was constructed to knockout Ls TCP in both loose leaf plant and heading lettuce and one positive Ls TCP8 edited plant was obtained.The effects of the Ls TCP on heading will be evaluated in future.This study dissected the genetic mechanisms of heading in lettuce;the candidate genes identified in this study provide excellent resources for future studies on heading mechanism;our results are also helpful for breeding lettuce varieties with different leaf shapes in the future. |
