Studies On The Lettuce Genomic Evolution And Genetic Cloning Of A Major Gene Responsible For Leaf Color Variation In Lettuce

Lettuce(Lactuca sativa)is one of the most important vegetables worldwide.Different horticultural types of lettuce exhibit tremendous morphological variation.However,the molecular basis for domestication and divergence among different horticultural types of lettuce remains unknown.In this study,a set of germplasm includes wild and cultivated lettuce accessions were RNA sequenced and studied.The lettuce population structure,genetic diversity,domestication and diversification were comprehensively analyzed.Expression quantitative trait loci(e QTL)were investigated at the whole-genome level and the regulatory networks for flavonoid biosynthesis were constructed.The results of genome-wide association studies(GWAS)were then combined with the e QTL and regulatory network analyses to identify candidate genes controlling anthocyanin accumulation in lettuce.Furthermore,a major gene responsible for leaf green color variation was cloned based on association mapping and linkage mapping.The main results are as follows:(1)With RNA sequencing of 240 Lactuca spp.genotypes which represented most of the phenotypic diversity in our collection,we constructed the lettuce genetic variation map and transcription map,which consisted of 1,133,865 high-quality SNPs and 24,042 gene expression profiles,respectively.(2)Population genetic analyses of wild and cultivated lettuce using the variation map suggests that cultivated lettuce has a strong population structure,with cultivars of the same horticultural type grouped together.Demographic modeling showed that lettuce had a single domestication event,and subsequent diversification led to phenotypically distinct horticultural types.Leafy lettuce types were brought to China most likely via the Silk Road,and continuous selection resulted in a new horticultural type,stem lettuce.(3)A list of regions were identified as putative selective sweeps that occurred during domestication and divergence,respectively.We found genes involved in stress,organ development and metabolic processes are selected during domestication,and genes associated with leaf-heading and secondary stem growth are selected in crisphead and stem types,respectively.Besides,we also found that most of the introgressed regions were contributed by L.serriola through introgression analysis.(4)A total of 5,311 e QTLs regulating the expression of 4,105 genes were identified using GWAS,which are the molecular basis of the rich phenotypic variation in lettuce.Six candidate genes responsible for the variation of anthocyanins in lettuce leaves were identified by a combination of the regulatory networks for flavonoid biosynthesis and the GWAS results,including genes that are known to be involved in flavonoid biosynthesis,such as MYB113,b HLH42 and ANS,etc.(5)One significant locus associated with leaf green color variation was identified using association mapping,and this locus was then fine-mapped to 5,376 bp interval on LG4 using linkage mapping.Ls GLK was located in this interval and was identified as the causal gene.The gene function of Ls GLK was confirmed using complementation test.Sequence analysis revealed that an autonomous CACTA transposon(7,434 bp)inserted into 10 bp downstream from stop codon,and then followed by the duplication of exons 3-6.Moreover,the transposon insertion resulted in disordered alternative splicing of Lsglk.Evolutionary analysis revealed that the insertion of CACTA transposon occurred after lettuce domestication and then spread among butterhead and looseleaf lettuce during lettuce diversification.Our results not only provide lettuce genomic resources,but also lead to deeper insights into domestication,accumulation of anthocyanins and leaf green color variation in lettuce.Data obtained in this study will facilitate biological research and breeding of lettuce cultivars with improved nutritional value.