Cloning And Functional Analysis Of The Lateral Suppressor Gene (CLS)in Cucumber

Commercial cucumber (Cucumis sativus L.; 2n=2x=14) has been of culinary importance to humans for millennia. With the increased requirement in cucumber-like vegetables, yield has been a focus of cucumber breeders for over 50 years. The lateral branch number is an important agronomic trait for cucumber production, to which, though, pickling (processing) and fresh-eaten cucumber cultivars have different requirements. Therefore, studies on the genetic and molecular mechanisms of cucumber lateral branching will be of help to cucumber breeders to create special cucumber plant architectures. In the past years, however, the studies on cucumber branching have been restricted on the processing cucumber types, whereas, these studies were only focused on the number, locations and effects of the detectable QTLs for possible maker-assisted selection (MAS) of an appropriate branch trait in cucumber breeding and not involved in the branching mechanisms of cucumber.Based on this situation, to explore the possible branching mechanisms in cucumber materials in our laboratory, which could be readily classified into three types in terms of branch numbers, i.e. multiple branching (multi-branching), fewer branching and non-branching types. The grafting and decaption experiments, bud examination and genetic analyses on these cucumber lines suggested that the non-branching phenotype of S61 is caused by a defect in lateral bud formation and controlled by a single recessive gene. In order to isolate a Lateral Suppressor (LS)-homologous gene, Cucumber Lateral Suppressor (CLS), we used the homo-based cloning strategy according to the conserved structures of the lateral suppressor genes, LS, LAS and MOC1. Homology analysis showed that the cDNA encoded protein is a member of the plant-specific GRAS family proteins which contains the characteristic sequence motifs of the GRAS family. The phylogenetic analysis showed that CLS together with the known LATERAL SUPPRESSORs in tomato, rice, Arabidopsis, carrot, forms a distinct cluster, implying that CLS may have a function similar to LS/LAS/MOC1. Because no conventional nuclear localization signal (NLS) domain was predicted in CLS by in silico analysis with the predication program, the instantaneous expression was permored. Nuclear targeting of CLS is consistent with its predicted transcription regulatory role. Southern blotting experiment showed that only one copy of CLS exists in cucumber genome. Interestingly, the CLS could LAS. Next, semi-quantitative RT-PCR analysis showed that CLS could be detected in all tested tissues except the internode. Moreover, RNA in situ hybridization revealed that the pattern of CLS transcript accumulation in axillary meristems was similar to that of LAS in Arabidopsis, suggesting that CLS plays an important role in cucumber lateral meristem initiation. Remarkably, the CLS transcript signal could not be detected in the axils of leaf primordia in the non-branching line, S61, indicating that the CLS expression defect is responsible to its non-branching phenotype. However, no nucleotide changes was found in the ORF region of the CLS from S61 except for the three nucleotide changes in 3'UTR and some other alterations in the intron region away from the splicing boundaries. Based on these changes, a CAPS marker was designed. But the recessive phenotype wasn't co-segration with the CAPS marker in the 06/61 F2 population.All the results suggest that the lateral branching development in cucumber can also be divided into two major processes. One is the lateral bud inination and the other is the outgrowth of the lateral buds. CLS plays a key role in cucumber axillary meristem initiation. The CLS expression defect is responsible to the branchless phenotype of S61.