Fine Mapping And Identification A Candidate Gene For The Gene ZmLW1 Which Related To Leaf Width In Maize

Leaf area is one of the key influencing factors of yield in maize,and leaf width,as a core component of leaf area,plays a vital role in the light capture of photosynthesis.Therefore,studying and cloning the genetic mechanism of genes controlling leaf width through forward genetics is of great value to the improvement of core inbred line in future breeding and can help increase corn yield.In this study,CSSLs populations constructed with Ye478 and Qi319,were used to map QTL on chromosome 1 and 7 related to leaf shape,which is used to go through mutual verification with preliminary positioning results.Segregating populations were constructed with Ye478 and CSSL TY24.Genetic analysis and fine mapping of genes on long arm of chromosome 1 that simultaneously control three-ear leaves’width were performed,combined with gene sequencing and quantitative PCR technology to identify candidate genes,to clarify gene function sites and temporal and spatial expression,which provides a theoretical basis for the selection and breeding of Ye478 improved line.The main findings are as follows:1.Traits such as leaf width,leaf area and leaf angle all show obvious super-parental advantages,there is an obvious positive correlation between leaf width and leaf area（P<0.01）,and between leaf above ear and leaf under ear of three-ear leaves（P<0.05 in Shenyang,P<0.01in Sanya）,and an obvious negative correlation between leaf angle and leaf area,and no obvious correlation between leaf width and leaf angle.2.The genetic background detection of CSSLs populations showed that families in the CSSLs population had a high background recovery rate（93.9%-99.65%）.Through comparing differences between the genetic background and leaf traits of the families and that of Ye478,QTL that steadily control width of the three-ear leaves was found on chromosome 1（between287.75～290.45Mb）and chromosome 7（between 126.35～126.6Mb）,QTL that control area of the three-ear leaves was found on chromosome 7（between 135.2～137.05Mb）,QTL that control area of leaf above ear and ear leaf was found on chromosome 1（between 75.7～78.1Mb）,QTL that steadily control leaf angle under ear was respectively found on chromosome 1（between249.15～249.65Mb）and chromosome 7（between 52.9～55.65M and between 159.35～159.45M b）.3.CSSL TY24 with background recovery rate of 98.55%was screened as near-isogenic lines of Ye478 for future fine mapping by CSSLs population genetic background detection and phenotypic research,and it showed no obvious difference to Ye478 in agronomic traits except leaf width.4.The width of the three-ear leaves of the three segregating populations constructed with Ye478 and TY24 as parents all had high genetic diversity.The analysis results of the genetic model showed that the width of the three-ear leaves conformed to a pair of major gene+polygene mixed inheritance model,which had a high major gene heritability of over 40%and that increased as the number of self-generation increases.5.Genotyping and phenotypic measure of 178 individual plants in the BC₁F₂ population found the QTL,with the phenotypic contribution rate were between 16.51%～19.31%,controlling width of three-ear leaves was detected between markers m90 and umc1118 on chromosome 1 with the physical length of 295Kb,which was named as LEAF WIDTH1（Zm LW1）.Twelve recombinant haplotypes were detected in BC₁F₄ through five polymorphic markers.According to the genetic background and phenotypic values of type 2,type 7 and type10,Zm LW1 was anchored between markers n55 and n47 which located at295,910,645～295,951,903bp,with physical length of 41,258bp,where contained five genes encoding proteins according to the B73＿v4 reference genome sequence.6.The CDS sequence analysis of the five genes showed that only the two genes（LOC103645700 and LOC100279743）had meaningful mutations.Analysis of the expression levels of the five genes in three-ear leaves of Ye478 and TY24 found that only gene LOC100279743 was significantly different between the three-ear leaves in two materials.The relative expression of TY24 in three-ear leaves were respectively 1.72,14.91 and 4.35 times of that of Ye478 and the other four genes had no significant difference.It was preliminarily determined that LOC100279743 was a candidate gene for controlling the width of three-ear-leaves of maize.7.The analysis of the exon sequence of LOC100279743 in materials with different leaf widths showed that the leaf width was obviously related to a 9bp indel site,the average leaf width of the inbred lines containing the 9bp fragment was 6.89cm,while the average leaf width of the inbred lines not containing the fragment was 12.06cm,but had no correlation to other sites.Genotype detection of the 9bp In Del locus in 462 individual plants which leaf widths was significantly smaller than that of Ye478 in the BC₁F₄ population,and it was found that this locus was co-segregated with the three-ear leaves width.Therefore,it was determined that LOC100279743 was gene Zm LW1 controlling leaf width in maize,and the 9bp In Del site was the function site of it.8.Analysis of the Zm LW1 genome sequence revealed that the gene contains two exons:the first was the signal peptide coding sequence,and the second was the protein encoding sequence.Zm LW1 encoded a protein containing 161 amino acids,which was found to belong to the Phytocyanin gene family by functional domain analysis.The analysis of the expression level of Zm LW1 in different organs of the two materials showed that Zm LW1 had a higher expression level in mature leaves,and had the highest expression level in the ear leaf of TY24（16.22）,while in Ye478,it had the highest in the first leaf under ear（5.98）,and TY24showed higher expression over Ye478 at the same leaves position（P<0.01）.9.The study on the leaf tissue structure of Ye478 and TY24 found that Ye478 was significantly higher than TY24 in terms of primary vein spacing and primary and secondary vein number,but there was no significant difference in the secondary vein spacing and leaf cell size of the two inbred lines.Therefore,the difference in the distance between the primary veins of Ye478 and TY24 was caused by the change of the number of secondary veins,and that affected the leaf width of the two materials together with the number of primary veins.