Map-based Cloning Of The Genes Controlling Palmately Lobed Leaf In Lettuce And Their Molecular Mechanisms

Cultivated lettuce（Lactuca sativa）,domesticated from prickly lettuce（L.serriola）,is one of the most important green leafy vegetables worldwide.Leaf shape is a vital agronomic trait for leafy vegetables such as lettuce.For leafy vegetables,leaf shape per se is the target of artificial selection.Both cultivated and wild lettuce have the polymorphism of lobed and non-lobed leaves.The polymorphism of lobed leaves and non-lobed leaves in cultivated lettuce was inherited from its progenitor L.serriola,and the causal gene is Ls ZFP4,located on Chromosome 3.Some lettuce cultivars have lobed leaves,varying from pinnately to palmately lobed.Previous studies focused on pinnately lobed leaves in lettuce,but little is known on the genetic and molecular mechanisms underlying the development of palmately lobed leaves in lettuce.In this study,we used bulked segregant analysis（BSA）combined with RNA-seq（BSR-seq）to dissect the genetics underlying the complexity of leaf lobes in lettuce.Two major QTLs controlling lobe complexity were fine mapped and cloned:Ls KN1▽and Ls DRL1.The candidate gene Ls KN1▽was verified using a complementation test.Its molecular mechanism was investigated in detail.The main results are as follows:（1）Genetic analysis of the complexity of leaf lobe in lettuce.We crossed an inbred line（FZ-118）of palmately lobed leaves with a wild lettuce（CGN04971）of pinnately lobed leaves.Individuals from the F₂population showed a continuous distribution of lobe complexity,ranging from pinnately lobed leaves to palmately lobed leaves.Phenotypic variation and BSR-seq analysis suggested three major loci contribute to the complexity of leaf lobes in the F₂segregating population.We defined the loci on chromosomes 2,3 and 7 as Palmately Lobed Leaf 1,2 and 3,（PLL1-3）.（2）The candidate gene for PLL3 is the Ls KN1gene.Using the single gene segregating population with 1,004 individuals,PLL3 was mapped to markers LG7-18.4 and LG7-18.9,a physical interval of 400 kb on chromosome 7 of lettuce.The candidate region contains eight genes,and Ls KN1 is the only gene differentially expressed between pinnately lobed leaves and palmately lobed leaves.While the parent FZ-118with the palmately lobed leaves has the mutated allele Ls KN1▽,which has an insertion of 3,935 bp CACTA-like transposon at+99 bp.The Ls KN1 allele that is upregulated by the insertion of a transposon promotes the development of palmately lobed leaves.The candidate gene Ls KN1▽was verified using a complementation test.Ls KN1 controls palmately lobed leaves through multiple pathways.We showed that Ls KN1 upregulates Ls CUC2 and Ls CUC3 through different mechanisms,and their upregulation is critical for the development of palmately lobed leaves.Ls KN1binds to the promoter of Ls PID to promote auxin biosynthesis,which positively contributes to the development of palmately lobed leaves.In contrast,Ls KN1suppresses GA biosynthesis to promote palmately lobed leaves.Ls KN1 also binds to the promoter of Ls AS1,a dorsiventrality gene,to downregulate its expression.Overexpression of the Ls AS1 gene compromised the effects of the Ls KN1 gene,and changed palmately to pinnately lobed leaves.Our study suggested that the upregulated Ls KN1 gene leads to palmately lobed leaves in lettuce through several pathways,including auxin,gibberellin,and leaf dorsiventrality pathways.（3）Cloning of the PLL1 gene.We used this F₃family to fine map and clone the PLL1gene.We genotyped a total of 1,763 from the F₃family with a series of genetic markers in the candidate region,and consequently delimited the PLL1 gene between markers LG2-203.13 and LG2-203.23,a physical interval of 200 kb on chromosome 2of lettuce.A gene named Ls DRL1 was inserted by a 12.7 kb transposon,leading to palmately lobed leaves.（4）Previous study was shown that one single locus Ls ZFP4 controls lobed and non-lobed polymorphism in Lactuca.Ls ZFP4 is recessive epistatic to PLL1/PLL3 on palmately lobed leaves.We compared the phenotypes of PLL1/PLL3 under the genetic background of Ls ZFP4 and Lszfp4.Interestingly,all the Lszfp4 homozygotes had non-lobed leaves,while all the Ls ZFP4 homozygotes and Ls ZFP4/Lszfp4heterozygotes had palmately lobed leaves.Therefore,the Ls ZFP4 gene is recessive epistatic to PLL1/PLL3 in regulation of palmately lobed leaves in lettuce.Our results provide new insights on the molecular regulation of lobed leaves,and are useful in breeding programs to develop lettuce cultivars with ideal leaf shapes.