Fine Mapping Of QHS4-a Major QTL For Starch Content In Maize Kernel And Analyze Candidate Gene For Starch Content In Maize Kernel Using Recombinant Inbred Lines

As the world's largest crop,corn is not only the human food and animal feed,but also the main source is an important industrial raw material.Starch,protein,oil constitute the three major components of maize grain,wherein the starch content of the highest,accounting for about 65%of maize grain.Maize starch content can not only increase corn yields,but also increase the production of corn ethanol as an industrial raw materials and other specialty produce added value.Corn starch content of the control major QTL fine mapping and cloning for the study of metabolic pathways and analytical corn starch corn starch genetic improvement provides an important basis and foundation.In this study,and DuPont-Pioneer company,in the early detection of the three major QTL on chromosome 4 in maize detected by a control corn starch content of the main effect QTL-qHS4 for fine positioning.First,by building on qHS4 near-isogenic lines were fine mapping,and then in the large-scale sequencing RNA-seq analysis obtained by expressing a large number of corn starch content related genes and QTL within the section for a more detailed analysis,Finally linkage analysis and expression analysis of the response analysis and selection of candidate genes for functional analysis and identification of functional sites.In addition,our group is still preliminary work in our laboratory,K22 and CI7 cross deriving an recombinant inbred lines population.We had three environments phenotype results and used high-density maps of maize starch content related QTL mapping.We were detceting six main QTLs and then candidate genes function were predicted an analyzed in the mapping regions.The main results are as follows:1.In the near-isogenic lines constructed BC5 generations effect on qHS4 value assessments carried out,the detected value and the QTL effect of recombinant inbred consistent verify the authenticity of the qHS4 effect value.Use the filters in the BC5 population with 736 recombinant individuls,qHS4 was mapped between marker MZA15842-4 and MZA11894-8 between with a 10.56cM interval,thus narrowing the QTL interval.In order to further reduce the section on qHS4 loci for fine positioning of in 2011 Hainan and 2012 Beijing,we use molecular markers between MZA15842-4 and MZA11894-8 screening the new recombinant events,screened 2355 recombinant individuls in 2012 Hainan,Beijing and Xinjiang progeny tessting.The qHS4 limited between mark MZA12884-19 and MZA8887-4 with genetic distance 1.95cM,and physical distance is 3.68MB.After we had limited the qHS4 between mark MZA12884-19 and MZA8887-4,we developed five SNP markers,in 2013 Xinjiang progeny test,qHS4 was mapped between mark IDP6613 and PZE-104117199,and the physical distance is 0.31MB.This region includes overlop four BACs,with 7 genes.2.In order to detect QTL target gene expression levels within the section on the impact of the laboratory and DuPont-Pioneer companies to apply two near-isogenic lines 14 days after pollination carried out 21 days after pollination,RNA-seq analysis in qHS4 fine positioning.In this study,a total of 41083 gene can be detected,within the section in which the gene QTL 4467 genes.For different periods of grain starch content of detected 14 days after pollination starch content in two near-isogenic lines the difference between the maximum.For 14 days after pollination,pollination biology repeated twice after 21 correlation analysis,analysis of chromosome 4 within the section 14DAP and 21DAP candidate in near-isogenic lines and control family co-expression of 517 genes,of which 60 4 groups of genes common in the expression of the material reached a significant level(P-value<0.05).The expression of these genes in 21 DAP amount is higher than 14DAP,the 60 expressed genes were clustered annotation features found in the formation of starch synthesis and carbohydrate-related genes that may be involved in starch metabolism in biological processes,as candidate genes can be considered starch content?3.Regional association analysis within qHS4 interval identified 12 independent SNPs which were significantly associated with kernel oil concentration(P<0.05).Though gene annotation and fuctional prediction,two candidate genes involved in the same fine mapping locus were identified.GRMZM2G016805 and GRMZM2G320723 encode Zinc finger,DHHC-type,and palmitoyltransferase Pentatricopeptide repeat,respectively.No significant expression difference of GRMZM2G016805 was detected correlated with starch content,and GRMZM2G320723 was positively correlated with starch content.This suggesting that GRMZM2G016805 maybe not cause the observed differences between the expression levels and the target traits,and GRMZM2G320723 maybe cause the observed differences between the expression levels and the target traits in the association panel.4.Using recombinant inbred lines starch content of positioning results show a total of 4 and its component traits associated genomic regions,the use of two maps.Meanwhile,compared with the low-density SSR map,more QTL detection for high-density maps,important sites of the total phenotypic variance than those of low-density linkage map.In the high density map has 30 annotated genes support the accuracy of the high density of the map data set BLUP construction.Among of these,GRMZM2G068701,GRMZM2G077316,GRMZM2G032280 and GRMZM2G050080 encode protein function mybe related to lipid-starch metabolic activity according to the public data,5.We genotyped the two RIL populations with Illumina Illumina MaizeSNP50 and construct high-density genetic maps.Based on the genetic linkage map constructed using 2,386 bins as markers,six quantitative trait loci(QTLs)for starch content in maize kernels were detected in the CI7/K22 RIL population.Each QTL accounted for 4.7%(qSTA9-1)to 10.6%(qSTA4-1)of the starch variation.6 QTL which collectively explained 48.6%of the total phenotypic variation were detected.The QTL interval was further reduced using the bin-mapmethod,with the physical distance of a single bin at the QTL peak ranging from 81.7 kb to 2.2 Mb.Based on thefunctional annotations and prior knowledge of the genes in the top bin,seven genes were considered as potentialcandidate genes for the identified QTLs.Three of the genes encode enzymes in non-starch metabolism but may indirectly affect starch biosynthesis,and four genes may act as regulators of starch biosynthesis.