Analysis Of Residual Heterozygosity Variation On Maize Genome And Cloning And Mechanism Dissection Of One Major QTL For Tocopherol In Maize Kernels

Maize（Zea mays L.）is one of the most important crops and the production in China could account for around 22% of the worldwide production.Due to the crossfertilization,maize is also one of the crops that fully have been taken advantage of heterosis.In maize breeding,a serious issue arose that inbreeding can lead to the decrease of fitness in offspring.It was studied that residual heterozygosity（RH）is beneficial to organisms’ survival by improving their fitness.Using 12 biparent populations,we analyzed the patterns of RH across the whole genome and the potential factors affecting them.Meanwhile,RH in advanced inbred lines has been successfully applied into QTL mapping.By analyzing RH of heterogeneous inbred family（HIF）lines,we systematically assessed the potential use of the HIF library for QTL fine mapping.Maize kernels are rich in macronutrients,like starch and lipids,and micronutrients,such as carotenoids and tocopherols,providing food,feed and raw material for humans and animals.There are three main types of tocopherol（α,γ,and δ）enriched in maize kernels and more than 70% of the total tocopherols is stored in the embryo,it was observed the tocopherol content in different individual maize lines varied extensively.Intake of sufficient and various tocopherols is essential for human and animals.It is of great importance to increase the tocopherol content in maize kernels,which would be good for human health.However,only a few genes responsible for the tocopherol accumulation were reported in maize.Here,we used the HIF library to map one major QTL（q VE1）controlling tocopherol content in maize kernels and further analyzed its function.For the two parts,the major results are as follows:1.In total,the HIF library comprises 18,615 unique RH intervals,with an average of 1551 intervals per population and the total length of each line extends approximately 2.8% of the maize genome.Approximately 40% of the RH intervals are shorter than 2 Mb,and within each population,an average of 4 different lines containing RH intervals can be identified for any given genome region.We used 1191 QTLs mapped in the 12 populations,affecting 19 agronomic traits,to calculate the real coverage and resolution.The results demonstrated that 94% of mapped QTLs could be covered in HIF lines,and the mapping resolution approached to1.34 Mb on average using HIF.2.The distribution of RH,including residual heterozygous rate（RHR）and the number of RH intervals（RHN）,was not random across the genome and 7 specific RH hotspot regions were identified.It was inferred that the spurious heterozygous genotype introduced by sequence duplications was insufficient to induce the RH hotspots,and selection may be involved in the formation of RH hotspots during the population construction.3.Only one population B73/BY804 contained significantly more RH in the pericentromeres,and two populations K22/CI7 and KUI3/B77 possessed less RH in the pericentromeres than the remaining chromosome arms.The remaining nine populations didn’t show any RH rate difference between the pericentromeres and the remaining chromosome arms.These findings suggested that the distribution of RH across the chromosomes might be affected by the genetic background.4.In DAN340/K22 RIL population,we detected one major QTL（q VE1）controlling the tocopherol content in maize kernels,which explained 33.5% of phenotypic variations.Choosing appropriate HIFs to develop a larger population,we finally narrowed down the QTL region to a single gene ZmPORB1,which is annotated as a light-dependent NADPH: protochlorophyllide oxidoreductase（POR）playing an important role in the biosynthesis of chlorophyll.ZmPORB1 is a completely dominant gene and controls the γ-and total tocopherol in maize kernels.5.By candidate gene association analysis,it was inferred a 13.7Kb transposon-related presence and absence variation（PAV）in promotor and a nonsynonymous mutation SNP in forth exon were putative causal sites.Compared with NIL^DAN340,it was identified that a 13.7Kb insertion in the allele from NIL^K22 decreased the methylation level at the promotor and repressed the gene expression level in the embryo,which subsequently resulted in the decrease in kernel tocopherol levels.6.ZmPORB1 expression level can be induced by light and hypoxia in leaf and embryo,respectively,inferring that two different responsive elements in the promoter,and the hypoxia-related element could be interrupted by the 13.7Kb insertion and further brought up the dysfunction of ZmPORB1 in maize embryo.7.ZmPORB1 could affect tocopherol by tocopherol regeneration rather than chlorophyll pathway.In embryos of NIL^DAN340 and NIL^K22,no significant difference was observed in the levels of two precursors HGA and PDP;no chlorophyll was detected and no known genes related to chlorophyll and tocopherol pathway showed differential expression.GO analysis showed almost DEGs identified in both 25 days after pollination（DAP）embryo and hypoxia-treated embryo were relevant to stress response,and even,17% of these differentially expressed gene（DEGs）,including ZmPORB1,overlapped between 25 DAP and the hypoxia-treated embryo,which suggested ZmPORB1 would be involved in the hypoxia response of developing kernels.8.There are two paralogous genes of PORB in maize,ZmPORB1 and ZmPORB2.It was studied that significant epistatic interactions occur between these two paralogs.No difference was found between the two alleles of ZmPORB1 which were in the high γ-tocopherol content when ZmPORB2 allele was functional.By contrast,when ZmPORB2 allele was weak,significant difference was found between two alleles of ZmPORB1 and with relative lower tocopherol for both alleles.