Cloning And Functional Analysis Of Genes Controlling Red Leaves In Lettuce

Lettuce leaves have red or green colors.Nowadays,the red leaves of lettuce are appreciated by consumers due to their unique colors and health benefits,and they are attributed to anthocyanin accumulation.The biosynthetic mechanisms of flavonoids have been well studied in petunia,arabidopsis and maize.However,little is known in lettuce.In order to clone the gene controlling red leaves of lettuce,a RIL population derived from a cross between Y37 and S1 was genetic analyzed.Genetic screening showed that four genes which are known involved in anthocyanin biosynthesis?three regulatory genes RLL1-3 and one structural gene RLL4:ANS?can not fully explain the variation of anthocyanins in lettuce leaves,and 114 families were screened.After phenotypic identification,a total of 32 F₄ lines showed segregations of light red and dark red.and one segregating population Q54 that are homozygous for the four genes was further analyzed.A ratio of dark red:light red?3:1 were found in this population,suggesting a novel gene controlling this trait.Bulked segregant analysis in combination with RNA sequencing?BSR-seq?was used to genetically map the causal gene for this trait.25 dark red individuals were chosen as the dark red pool and 25 light red individuals were chosen as the light red pool.Equal amount of leaf tissue from each individual were mixed,The RNAs was extracted and sequenced for each pool.Analysis of the sequencing data reveals considerable different for the allele frequency between the two pools in the63 Mb⁶9 Mb region of chromosome 9.Further,CAPS and SNP markers were designed to screen the populations.Finally,the candidate gene was fine-mapped to the interval of357.5 Kb.Gene annotation showed that three genes associated with anthocyanins were located in this interval.Gene sequence and expression analysis of three genes were compared between two parents.Of them,a gene named RLL5 that encoded the WD40protein were regarded as a candidate gene.The 134 amino acid of RLL5 mutation?V becomes D?occurred in the conserved domain,resulting in loss of function.Differentially expressed gene analysis between the dark red and light red lettuces showed that the expression of anthocyanin related genes in the dark red lettuces were significantly higher than that in the light red lettuces.Howerver,the expression of RLL5gene was not significantly different.The determination of anthocyanin compounds of two colors lettuces showed that there were more than 19 anthocyanin compounds in dark red lettuces,which were higher than the light red lettuces,The higher amount of anthocyanins in dark red lettuces makes them darker red phenotype than the light red lettuces.Besides,we found that the quality of dark red lettuces are superior to the light red lettuces by comparing the nutrient contents.Genetic analysis of M9×S1 F₂ segregating population showed another novel gene controlling the red leaves.Phenotyping show that the ratio of dark red leaves and light red leaves is to 3:1,indicating the trait is controlled by a single gene.Using BSR-seq method,the locus was mapped to 69 Mb²13 Mb region on chromosome 1.Using CAPS and SNP markers,the candidate gene was mapped to 73.58 Mb²13 Mb.Due to lack of molecular marks in this region,the gene was only located in a large candidate region.Cloning genes related to anthocyanin in lettuce will help to cultivate anthocyanin rich lettuce and clarify the molecular mechanism of anthocyanin synthesis and accumulation in lettuce.