Map-based Cloning And Functional Analysis Of Round Leaf Gene Csrl In Cucumber (Cucumis Sativus L.)

Leaf is the most important source organ of cucumber?Cucumis sativus L.?due to its vital functions in photosynthesis,respiration,and photoperception.Leaf morphology is an important part of plant morphogenesis.Leaf shape is an important part of?ideal plant type?,which is very important for plant growth and development and improvement of agricultural productivity.Excavation and cloning of leaf-shaped control genes is of great significance for revealing the regulation mechanism of plant leaf shape and accelerating the plant type breeding process of crops.A number of leaf-shaped control genes have been cloned on other plants,but in the cucurbit crops,the mining of leaf-shaped control genes is rarely reported.The research team identified two round leaf mutants C356 and C949 from the EMS mutant library of cucumber?Cucumis sativus L.?inbred line CCMC.In this study,we used the map-based cloning and MutMap method to clone the cucumber round leaf genes Csrl-1 and Csrl-2,and carried out preliminary functional analysis of the candidate genes.The research results laid the foundation for further study on the regulation mechanism of cucumber leaf shape.The main results for this study are as follows:1.Phenotype and genetic analysis of cucumber round leaf mutantThe phenotypes of mutants C356 and C949 were observed.Compared with the wild type,both mutants showed a rounded leaf shape,a relatively smooth leaf margin,and a missing or reduced flowering calyx.The petal top of the female flower closed in a rod shape.The mutant C949 plants were relatively low compared to the mutant C356 plants.A field survey of the round leaf traits of the mutants showed that the F₁ of both mutants showed normal leaf shape.In the F_2:3 populations,the segregation ratio of normal leaf-shaped plants and round-leaf plants was consistent with the 3:1 trait segregation ratio by chi-square??²?test.The results showed that the round leaf trait of the mutant was qualitative trait controlled by the recessive single genes,Csrl-1 and Csrl-2,respectively.2.Map-based Cloning of Cucumber Round Leaf Mutant Gene CsrlIn this study,we conducted fine mapping for the Csrl-1 and Csrl-2 mutation in C356and C949,respectively.We delimited the Csrl-1 gene to a 188.9 kb region in cucumber Chr1by genetic mapping using 1196 plants of F₂ populations and 18 pairs of polymorphic SSR markers and newly developed dCAPS markers.The results of MutMap analysis showed that only one non-synonymous SNP1G19389180 located in the CsPID1 gene met the filtering criteria in the Csrl-1 localization interval.Combined with the gene in the Csrl-1 localization interval and the analysis result of MutMap,the CsPID1 gene was the most possible candidate gene for Csrl-1.The Csrl-1 candidate gene CsPID1 was cloned.The sequence alignment showed that a C to T base mutation occurred at the 1000 bp position of the CsPID1 gene CDS region of C356,resulting in the amino acid being mutated from Pro³³⁴ in wild-type to Ser³³⁴ in C356.The dCAPS marker NWdCAPS360 developed by this non-synonymous SNP was verified by linkage analysis in a large population,and the results showed that the marker was co-segregated with exchanged individuals.Meanwhile in order to detect whether the SNP locus exists in natural germplasm,the SNP was also verified by uniqueness assay among 100 cucumber lines in natural germplasms.The results showed that the SNP site was only present in the mutant.Using the same SSR molecular marker as Csrl-1,the Csrl-2 gene was also mapped to the same 188.9 kb localization region and was the same candidate gene CsPID1.Using the MutMap method,it was also found that only one non-synonymous SNP1G19389177 located in the CsPID1 gene met its filtration criteria.Combined with the gene in the Csrl-2localization interval and the analysis result of MutMap,the CsPID1 gene was the most possible candidate gene for Csrl-2.The Csrl-2 candidate gene CsPID1 of mutant C949 and wild type CCMC was cloned.The sequence alignment showed that which has a G to A transition at the 1003 bp position of the CsPID1 gene CDS region of C949,resulting in the amino acid being mutated from Glu³³⁵ in wild-type to Lys³³⁵ in C949.The candidate genes for C356 and C949 are both CsPID1,but the mutation sites of the gene are different in the two mutants.The allelicity of these two mutants was tested.The results showed that the segregation ratios of normal leaf plants and round leaf plants in the hybrid progeny were consistent with the ratio of 3:1 trait segregation.Csrl-2 was allelic to Csrl-1.3.Functional Analysis of CsPID1 geneThe expression of CsPID1 in different tissues and organs of CCMC and C356 was detected by qPCR.The results showed that the expression level of CsPID1 gene in roots,leaves and female flowers of C356 was significantly lower than that of wild type,which may result in the less developed roots,round leaves and abnormal female flowers,respectively in the rl-1 mutant.CsPID1 encodes a Ser/Thr protein kinase,which is involved in the polar transport of auxin.Bioinformatics analysis showed that there are three homologous genes of PID in the cucumber genome:CsPID1,CsPID2 and CSPID2L?CsPID2-like?,while the expression of the CsPID1 gene was significantly different in the leaves of wild type and C356 suggesting the CsPID1 gene may plays a major role in the formation of round leaf.At the same time,the 35S-CsPID1 overexpression vector was transformed into Arabidopsis thaliana.The positive transgenic plants showed a phenotype similar to that of the overexpressed AtPID gene.This result also supports CsPID1 as a candidate gene.4.Phenomenon of female infertility in mutantThrough a series of comprehensive analysis,such as anatomical observation,pollen vigor identification,cross-test,fluorescence electron microscopy,and qPCR analysis,it was found that the fertility of the pollen grains of the mutant plants was normal,and the male flowers were used as the male parent to pollinate and produce seeds normally.It is indicated that the mutant fruit without seed may be caused by female infertility.Further studies have shown that the main reason for female infertility is due to fertilization cannot be completed due to the absence of ovules in the mutant.Loss of CsPID1 function may cause interference with auxin efflux,resulting in failure or delay in the formation of the maximum auxin required for the initial formation of the placenta in the cucumber ovary and the growth of the ovule primordia,ultimately resulting in the inability to form ovules.