Map-based Cloning Of Three Genes Controlling Anthocyanin Accumulation In Lettuce Leaves And Their Molecular Mechanisms

Lettuce?Lactuca sativa?is one of the most important vegetables in the world.Red lettuce is increasingly appreciated by consumers because of its color and its health benefits.The red color of lettuce leaves is mainly caused by anthocyanin accumulation in leaf epidermal cells.Leaf color is one of the most important agronomic traits in lettuce,and the metabolites?anthocyanins?underlying the color are effective components of anticancer and anti-aging due to its strong antioxidation.However,there are still few studies on the color of lettuce leaves.In this study,a red lettuce was crossed with a green lettuce,and an F₂ population segregating leaf color was constructed.The color trait was genetically analyzed using bulked segregant analysis?BSA?in combination with the second-generation sequencing technology.Three genes?RLL1-3?controlling red leaves of lettuce were cloned using map-based cloning.The molecular mechanism of these three genes and their evolution were studied in detail.The main results are as follows:1.Genetic analysis of leaf color of lettuce.A F₂ segregating population was constructed through a cross between a red and a green lettuce cultivars.Phenotypic variation and BSA+RNA-seq analysis suggested multiple loci controlling leaf color in this population,including both qualitative and quantitative loci.Molecular markers were designed at the candidate regions and used to screen the F₂ population.Consequently,two major loci,named RLL1 and RLL2 were identified on chromosome 5.2.Construction of single gene segregating population.Molecular markers were designed at the RLL1 and RLL2 candidate regions and were used to screen the F₂population.Individuals that were homozygous at one locus but heterozygous at the other locus were selected for further genetic analysis.Selfing seeds were collected from those individuals,and segregating sub-populations with ideal phenotypic variation were chosen to fine map and clone the two genes,respectively.3.Cloning of the RLL1 gene and its role in anthocyanin biosynthesis.Using the single gene segregating population with 1,751 individuals,RLL1 was mapped to404.3-407.2 Mb on chromosome 5.In the candidate region,there is a gene encoding a bHLH transcription factor.Genetic transformation showed that this gene is the RLL1 gene.In the green parent,the RLL1 gene had a 5-bp frame-shift deletion,leading to its loss of function.RLL1 regulates multiple genes in the anthocyanin biosynthesis pathway,and Yeast-one-Hybrid analysis showed that RLL1 bands to the promoter of some anthocyanin pathway structure genes.4.Cloning of the RLL2 gene and its role in anthocyanin biosynthesis.Using the single gene segregating populations of RLL2,the RLL2 gene was mapped to a gap on chromosome 5 of lettuce.We then screened the unmapped scaffolds,searching for SNPs that showed significant frequency difference between the two pools.A unmapped scaffold was identified and markers on this scaffold co-segregated with color in the single gene segregating population.On the scaffold,a gene encoding a R2R3-MYB transcription factor is present in the red parent and highly expressed,but absent in the green parent,exhibiting presence/absence polymorphism.Transformation of this gene into a green genotype successfully changed its leaves to red,confirming the function of the RLL2 gene.The RLL2 protein binds to the promoter of multiple genes in the anthocyanin biosynthesis pathway,consistent with upregulation of several genes in the red individuals in the single gene segregating population.5.The cloning of the RLL3 gene and its functional analysis.A subpopulation which was homozygous of RLL1 and rll2 showed color variation.BSA+RNA-seq analysis of this subpopulation discovered the third locus?RLL3?controlling leaf color in this population.RLL3 is a negative regulatory factor for anthocyanin synthesis.Map-based cloning of the RLL3 gene showed that it encodes an R3-MYB transcription factor.Our data suggested that RLL3 inhibits the synthesis of anthocyanin by competing with R2R3-MYB for binding to the RLL1?bHLH?transcription factor.6.Evolutionary analysis of the RLL1-RLL3 genes.PCR amplification of the sequences of the RLL1 and RLL3 genes showed that all functional change mutations occurred after domestication.The loss of function mutation in the RLL1 gene led to pure green leaves,while the mutations in RLL3 gene resulted in red leaves even under non-stress conditions.Therefore,the selections for leaf color in lettuce went to two opposite directions,showing typical of disruptive selection.The cloning of three genes controlling leaf color of lettuce can be applied directly to lettuce breeding programs.The functional analysis of these three genes generated insight on the regulation of anthocyanin biosynthesis in plants.The evolution study provided a typical example for disruptive selection.