Identification Of Maize Small Wrinkled Leaf Mutant Swl1 And Cloning Of The Encoding Gene

As the source,maize leaves are the material basis of yield formation.Photosynthesis has a very important impact on dry matter accumulation and yield.The development of maize leaves is regulated by a series of genes.Any mutation may cause changes in leaf morphology or leaf composition and content.Leaf type mutation is a valuable material for the breeders to pursue"ideal plant type".Therefore,it is of great theoretical significance and application value to clone the gene related to the leaf development of maize and to analyze its function.Maize EMS（methanesulfonate）mutant library is a"treasure house"for functional genomics research in maize.We used EMS to mutagen the pollen of Maize Backbone Inbred Line RP125 to obtain a phenotypic stable mutant swl1（small wrinkled leaf 1）.This study investigated the agronomic characters of the mutant,the mutant leaves were observed by histology,the F₂ and BC₁F₁ segregation population was constructed with the maize inbred line B73.The mutation gene was located and cloned by BSR-Seq analysis and Map-based cloning.and the results were as follows:1.swl1 mutant is a maize wrinkle leaf mutant.The leaf of the mutant is shrunken,and the leaf area is reduced.The plant height,leaf area,grain length and grain width of the mutant are different from wild type.The contents of fatty acids,primary alcohols and alkanes in mutant leaves were higher than those in wild type,but the results in flower silk were just the opposite.It shows that the mutant gene maybe plays different roles in the waxy synthesis pathway in different tissues.Cytological observation of leaf tissue shows that the epidermis of the mutant leaf epidermis is malformed and the arrangement is irregular.It is presumed to cause the shrinkage of the leaf surface.2.Using swl1 and B73 to construct BC₁F₁ and F₂ segregation population,the parental positive and negative cross test excludes the possibility of the influence of cytoplasmic genetic material,and the genetic analysis of two genetic populations indicates that the mutation is controlled by the recessive single gene.The F₂were genotyped by BSA（Bulked Segregant Analysis）strategy and more than 200 polymorphism SSR markers.It was found that the SSR markers mmc0481,umc1680,umc1941 on chromosome 5 were co-separated with mutant phenotypes.It was preliminarily determined that the gene was located on chromosome 5.Then,a number of Indel markers nearby the three SSR makers were developed by using maize B73 reference genome information and RP125 sequencing.The polymorphic markers were used to detect the genotypes of 1276 recessive single strains,and the target genes were finely located between indel19 and indel14,and the physical distance was 134 KB.The results of BSR-seq coincide with the results of the map.Combining the B73 reference genome annotation information,5 candidate genes were found in the segment,and the specific primers of all the genes were designed and cloned in the mutant and wild type.The sequencing comparison found a T to A mutation on the last exon of the Zm00001d017251gene,The valine converted into aspartic acid in its encoded protein sequence,which resulted in the change of function of SWL1 protein.Sequencing of mixed pools of 3 recessive individuals and 100 recessive DNA of F2 population revealed that the mutation site was real.3.The search for maize genome database found that the total length of SWL1（Zm00001d017251）genome was 6594 bp,and the total length of c DNA was 4696 bp,including 7 exons and 6 introns,and 318 amino acids were encoded.In the NCBI site,the SWL1 protein was found to be BLAST.In Arabidopsis and sorghum,the function of SWL1 is related to the wax composition.The protein encoded by the gene is related to the wax composition of the epidermis.The protein domain site analysis found that the SWL1 protein C has a WAX2 domain,and the domain and the epidermal wax are in the domain.Qualitative synthesis is related.After mutation,this domain changes its normal function,which affects the regulation of wax synthesis.The phylogenetic tree analysis showed that SWL1 protein had high homology with the sorghum homologous protein from the same family as gramineous plants,indicating that the gene was highly conserved in different species.4.Based on the phenotype of the swl1 mutant,we screened a mutant similar to the swl1phenotype in the EMS mutant library and screened an allelic mutant,named swl2,which had a base G mutation on the last exon of the Zm00001d017251 gene,which led to its encoded protein Glutamic acid is changed into lysine.The change of amino acid results in the change of function of SWL2 protein.The hybridization between swl1 and swl2 showed that the F1phenotype was consistent with the parents,indicating that the phenotype of swl1 and swl2 was controlled by the same gene.