Genetic Cloning And Functional Analysis Of The Genes Controlling Plant Architecture Of Butterhead Lettuce

Lettuce,which belongs to Asteraceae,is a popular vegetable around the world.The morphology of lettuce is highly diverse,and it can be classified into several horticultural types including butterhead lettuce,looseleaf lettuce,romaine lettuce,crisphead lettuce and stem lettuce.Butterhead lettuce is a unique horticultural type,which has round leaves,compact plant architecture,and loose leafy head.The genetic analysis of butterhead plant architecture and the cloning of the associated genes may enrich our understanding of plant architecture,and contribute to the future genetic improvement of lettuce.In this study,we cloned the genes controlling butterhead plant architecture,and verfied their functions.The main research results are as follows:（1）In this study,we crossed a butterhead lettuce（W6-29885）with a stem lettuce（Ws1168）.The phenotype of F₁hybrid was similar to the parent of stem lettuce.We constructed an F₃ subfamily,which showed segregation of looseleaf plant architecture and butterhead plant architecture,with a Mendelian ratio of 3:1.Bulk Segregant Analysis+RNA-seq（BSR）analysis followed by fine mapping delimited the gene controlling butterhead plant architecture to the region of 55,769,129 bp─60,189,439 bp on chromosome 1.This candidate region coincided with the locus controlling compact plant architecture in a previous study in our lab.The candidate region contains a member of the AGCVIIIa family LG114959（LsKIPK）,which is homologous to the At KIPK（AT3G52890）gene in Arabidopsis.A nonsense mutation was found in the first exon of the Lskipk gene in the butterhead parent,leading to lack of the kinase domain.We verified that the LsKIPK gene was a key gene controlling compact and butterhead plant architecture of lettuce through complementation test and knockout assay.LsKIPK protein was located in cell membrane,and highly expressed in mesophyll and midvein of lettuce.Lskipk mutation reduced the length of midvein epidermal cells,and led to shortening leaves.The loss-of-function of Lskipk also enhanced the mechanical strength of the leaf lamina joint and midvein,resuling in reduced leaf angle.The LsKIPK genotypes were investigated in cultivated and wild lettuce.There were two loss-of-function alleles of LsKIPK.Besides the allele with the nonsense mutation,there is another loss-of-function allele,which has a nonsynonymous mutation in the conserved kinase domain.The two nonfunctional alleles were only found in cultivated lettuce,and therefore likely occurred after domestication of lettuce.（2）We constructed an F₂ population through crossing crisphead lettuce PI536734and butterhead lettuce PI577118.In the F₂ population,there was segregation of non-butterhead plant architecture and butterhead plant architecture,with a Mendelian ratio of 3:1.BSR analysis indicated that the locus controlling butterhead plant architecture in this population was located around at 70 Mb on chromosome 4.Fine mapping revealed the candidate interval of 70,046,962─70,539,919 bp on chromosome 4.Map-based cloning identified the gene LG4403145 as the candidate gene controlling butterhead plant architecture,which encodes an AAA-ATPase.This candidate gene has a 19 bp insertion in the first exon,resulting in frameshift mutation in the butterhead parent.We verified the function of LsATPase on controlling compact and butterhead plant architecture by complementation test and knockout assay.LsATPase protein was located in cell membrane.Lsatpase reduced the length of epidermal cells in the leaf midvein,leading to smaller leaf length.And Lsatpase also enhanced the mechanical strength of the leaf lamina joint and midvein,leading to reducing the leaf angle.Screening the LsATPase gene in lettuce accessions identified two nonfunctional alleles.The second loos-of-function allele had insertion of a nucleotide A in the first exon,mainly found in stem lettuce.Both loss-of-function mutations occurred after domestication.Lsatpase and Lskipk were equally important for the development of butterhead lettuce.（3）LsKIPK and LsATPase complementation plants were crossed with commercial butterhead cultivars.The F₁ phenotype confirmed that Lskipk Lsatpase double mutants are necessary and sufficient for the plant architecture of butterhead lettuce.Moreover,genetic analysis suggested that both Lskipk Lsatpase inhibited the effect of Lssaw1 and LsKN1▽that promote leafy head phenotype.（4）In order to further analyze the regulatory mechanism of LsKIPK and LsATPase on plant architecture development,we identified a recombinant inbred line,which was heterozygous at both loci（LsKIPK Lskipk/LsATPase Lsatpase）,from the population between stem lettuce Y37 and looseleaf lettuce S1.Four genotypes of LsKIPK LsATPase,LsKIPK Lsatpase,Lskipk LsATPase and Lskipk Lsatpase were selected for RNA sequencing.Results showed that Lskipk and Lsatpase affected the expression of genes related to cell wall formation and cytoskeleton.Complementation plants had thicker cell wall of xylem cell and parenchyma cell of leaf midvein than recipient.In summary,we identified two key genes LsKIPK and LsATPase,controlling butterhead plant architecture,and revealed Lskipk Lsatpase was sufficient and necessary for the development of butterhead plant architecture.It was found that the two genes regulated the expression of genes associated with cell development,changed the cell wall thickness and cell size,and affected the growth state of leaf veins,resulting in controlling the development of lettuce plant architecture.The results provided new gene resources and breeding strategies for the future lettuce programs.