Map Based Cloning And Functional Analyisis Of The Major QTL Affecting Kernel Tocopherol Content In Mazie

A continuous intake from common and novel dietary sources of tocopherols is advantageous for human's health.Unlike rice and wheat,maize can accumulate tocopherols with the highest levels in kernel.Although the tocopherols biosynthesis pathway in Arabidopsis is well-known,the related study in maize remains poor.In this study,a major QTL(qVE5)affecting tocopherol content in the maize kernel was identified by linkage analysis in three independent bi-parental mapping populations.This QTL was cloned with detailed functional analysis.The major achievements are as following:1.Fine mapping of a major QTL:Three RIL populations,including ZONG3/YU8701:ZY,K22/CI7:KC and B73/BY804:BB,were genotyped with high-density SNPs and phenotyped in three environments.A major QTL,qVE5,affecting yT and total tocopherol content of kernels was mapped on chromosome 5 in all three populations in multiple environments.qVE5 accounted for a large proportion of phenotypic variation in each RIL population ranged between 9.75%to 42.47%for y-tocopherol content.Using a residual inbred family line(HIF-1171)derived from KC population,q VE5 was narrowed down to a 170-kb region based on B73(AGPv3)genome.Only one predicted gene,GRMZM2G073351,encoding a protochlorophyllide oxidoreductase(ZmPORB2)was identified in the target region.2 Overexpression of ZmPORB2 increased tocopherol content both in leaves and kernels:Three independent positive transgenic Ti families showed 5-to 8-fold increase of the level of ZmPORB2 mRNA expression at the six-leaf stage leaves compared with the negative controls.?T,?T and total tocopherol content in leaves were significantly increased from 28.11%to 50.59%in six-leaf stage leaves and from 8.12%to 19.24%in mature kernels compared with the negative controls.3 ZmPORB is conserved in higher plants:Protochlorophyllide reductases(PORs)genes are conserved among higher plants.Subcellular localization of ZmPORB2 was localized in the chloroplast.ZmPORB2 was highly expressed in the light and completely suppressed in the dark based on the greenhouse experiment.These results indicate that the function of ZmPORB2 may be similar with the light-dependent protochlorophyllide oxidoreductase B in other higher plants.4.InDe1058 likely affects kernel tocopherol content by regulating ZmPORB2 expression:ZmPORB2 was re-sequenced in 508 inbred lines.Candidate gene association mapping shows a 0/5/8 bp InDel(InDe1058)was located at 59 bp upstream of ATG within the 5' untranslated region was the polymorphic site showing strongest association with the y-tocopherol(P=3.67E-13)and total-tocopherol content(P=1.81E-13).It was also significant association with ZmPORB2 mRNA level in kernel of 15DAP based on 368 inbred liens which implied ZmPORB2 may affect the phenotype by regulating the expression level.5.Tocopherol accumulation in kernel associates with the photosynthetic tissue:The NILs of ZmPORB2 with two different genotypes as "K22" and "CI7" were selfed and crossed each other.Mature kernels from selfed and crossed ears were measured for three tocopherol content traits(?T,?T and total tocopherol).Significant differences in kernel tocopherol content were observed between NILCI7 and NILK22 as well as between NILK22CI7(NILCI7 ×NILK22)and NILCI7K22(NILK22 ×NILCT7).There were no differences between the selfed homozygous kernels of NILCI7 and hybrid kernels of NILCI7K22 for all of the measured traits.Similar results were observed between NILK22 and NILK22C17.In the progeny test based on the NIL family,the yT,total tocopherol content in the kernel of heterozygous genotype was very close to the mid-parent value.These results implied that ZmPORB2 could modulate tocopherol content in maize kernels,and that tocopherol accumulation in kernels associates with the maternal photosynthetic tissue genotype.6.Dramatic change in tocopherol content and ZmPORB2 expression in flag leaves was observed after pollination:A controlled experiment was designed in which half of NILK22 and NILCI7 plants were self-pollinated(P-Leaf),and the rest were non-pollinated by bagging the ears before flowering(NP-Leaf).ZmPORB2 expression was very low in the kernels or NP-Leaf and exhibited no fluctuations across the experimental time period.The expression level of ZmPORB2 in P-Leaf rapidly increased from 0 DAP to 8DAP and decreased from 8DAP reaching the lowest level at 16DAP.In NP-Leaf yT,aT and total tocopherol contents remained stable but with a low level,consistent with the pattern of ZmPORB2 expression in this tissue.Dramatic changes are evident for tocopherol content in the P-Leaf,its peak abundance at 16 DAP.7.ZmPORB2 participates in chlorophyll metabolism and tocopherol biosynthetic pathways:Chlorophyll a+b contents were at steady state and no distinctions were observed between the leaves of pollinated and non-pollinated NILs within 20 DAP.There was also no difference in Pchilde content between NILK22 and NILCI7 in either P-or NP-Leaves at 12 DAP.A difference in Childe a content was,however,identified between NILK22 and NILCI7 at 12 DAP in both P-and NP-Leaves.Pchilde and Childe a content was significantly increased in P-Leaves compared with NP-Leaves at 12 DAP.Pchilde and Childe a content was significantly increased in P-Leaves compared with NP-Leaves at 12 DAP.By contrast,the content of Pheophorbide a remained no change at 12 DAP compared with NP-Leaves,and there was no difference in the levels of this metabolite between NILCI7 and NILk22.Phytol content was elevated in P-leaves compared with NP-Leaves at 12 DAP and a significant difference was observed between the levels of this metabolite between NILK22 and NILCI7 in P-Leaves but not in NP-Leaves.The CHLG expression pattern was very similar to that of ZmPORB2 in P-Leaves within 20 DAP.These data collectively indicate that ZmPORB2 participates both in chlorophyll metabolism and tocopherol biosynthetic pathways in maize leaf.8.Phytol released from chlorophyll after pollination is involved in tocopherol synthesis,both increasing tocopherol content in leaves,and also affecting tocopherol accumulation in kernels.