The Genetic Basis Of Carotenoid Content In Maize Kernel

Carotenoids are natural pigments which play an important role in human health as antioxidants and vitamin A precursors.Among the main food stable crops,maize is the one of the most stable food crop that has high quality of nutrients.The carotenoid contents of maize kernel are much higher than that of other staple crops.Thus,improving the carotenoid contents in maize kernel is a priority choice for breeders to realize nutrition biofortification.Many efforts have been undertaken to improve carotenoid contents and to understand the genetic mechanism of carotenoid biosynthesis in maize kernels.In this study,three researches were performed:1)The genetic architecture of carotenoids in maize kernel dissected by using high-density single nucleotide polymorphism markers and simple sequence repeat(SSR)-based linkage map in By804×B73 recombinant inbred line population;2)The genetic basis of maize carotenoid biosynthesis and accumulation in maize kernels was dissected using Mo17×X26-4 introgression population;3)Association between ZmTIM15 and carotenoid content in maize kernel was investigated by candidate gene association analysis,expression analysis and functional investigation.The major results are listed as follows:1)A total of 81 QTLs were detected by using two linkage maps,high-density bin map and simple sequence repeat(SSR)-based linkage map,with one to seven QTLs for each trait,explain 4.21%-47.53%of the phenotypic variation in the By804×B73 RIL population.A comparison of the QTL mapping efficiency between the two linkage maps revealed the high-density bin map had higher resolution,the bin map enhanced the resolution of QTL mapping from an average of 9.45 cM to an average of 4.57 cM.In the current study 46 additional QTLs were identified,with 16 being common with previous studies and 14 newly identified.Among the results,29.6%(24/81)of QTLs explained>10%of phenotypic variation,and 70.4%(57/81)of≤10%.These results suggest that a few large-effects QTLs,together with a variable number of minor-effect QTLs,mainly contribute to most of the genetic components of carotenoids in maize kernel in By804xB73 RIL population,which represents the complexity of carotenoid biosynthesis and accumulation in maize.2)Carotenoid contents in Mo17xX26-4 introgression population showed abundant phenotypic variation with fold change ranging from 2.33(zeaxanthin/total carotenoids)to 12.59(lutein).ANOVA analysis indicated that genotype and environment had a highly significant effect on each carotenoid trait and the broad-sense heritability ranged from 0.54-0.98.After 1000 permutation tests by the composite interval mapping method presented in WinQTLCart 2.5,a total of 52 individual QTLs were identified for sixteen carotenoid related traits in 19 genomic regions across nine chromosomes.Each individual QTL explained 3.63-38.50%of the phenotypic variation.Among of these QTLs,three major QTLs each accounted for over 15%of the phenotypic variations,and mainly distributed on chromosome 7,8 and 10.Additionally,five pairs of epistatic interaction among two loci with additive effect were detected,and explained 2.98-7.78%of carotenoid variation.These results suggest that a few major QTLs with large additive effect,together with a number of minor-effect QTLs and a certain number of epitasis QTLs with additive effects,contribute to the genetic components of carotenoid contents in maize kernels in Mo17×X26-4 introgression population.3)A previous genome-wide association study(GWAS)identified ZmTIM15 significantly associated with carotenoid related traits.ZmTIM15 encode protein TIM15 with unknown function in maize.To investigate the association between ZmTIM15 and carotenoid content in maize kernel,ZmTIM15 was re-sequenced in a maize association panel of 508 inbred lines.The candidate gene association analysis identified 37 polymorphic sites with minor allele frequency of ≥0.05 in ZmTIM15 significantly associated with P-carotene,lutein,lutein/total carotenoids,zeaxanthin and RATIO at P≤0.01.The most significant polymorphic site was SNP64 and associated with RATIO trait at P of 2.01×10-8.SNP64 is a nonsynomynous SNP,which is located in the first exon containing C-to-T transition and leads to proline to serine change.For further function investigation,two maize alleles,ZmTIM15B73(C in SNP64)and ZmTIM15MB73(T in SNP64)were transferred into Arabidopsis mutants for ZmTIM15 orthologous gene in Arabidopsis.As expected,both ZmTIM15B73 and ZmTIM15MB73 can complete the morphology deficient of carotenoid contents in Arabidopsis mutants.In addition,ZmTIM15MB73 transgenic plants significantly increased 14%anteraxanthin,11%violaxanthin,33%lutein,11%zeaxanthin,and 30%of total carotenoid content compared with ZmTIM15B73 transgenic plants.These results suggest that ZmTIM15 was associated with carotenoid contents and SNP64 was the causal variant in ZmTIM15 to alter the natural variation of carotenoids.