Functional Analysis Of Maize Ehd1 Mutant And Hetetosis Loci Identification Of Maize Kernel Related Traits

Maize is the first important cereal crop and plays a critical role on national food security in China.Genes controlling maize kernel traits should be cloned to reveal the molecular mechanism of kernel development,which is helpful for maize yield improvement.Maize kernel traits are controlled by quantitive trait loci and exhibit heterosis.Dissection of QTL and heterotic loci of maize kernel traits are useful for increasing grain quality and yield,as well as predicting hybrid performance.ehd1 is a natural mutant identified during two-cycle inbred line selection.It has defects in kernel development and vegetative growth,resulting in no more than 33% 100-kernel weight of the wild type.Genetic analysis showed that this trait was controlled by a single recessive gene,named Zm EHD1.This gene was cloned and its function was validated in this study.The QTL and heterotic loci for kernel traits were detected using two test populations with two year two location phenotype.The two test populations were derived from crossing a set of 184 chromosome segment substitution lines(CSSLs)to two inbred lines(Zheng58 and Xun9058).The detailed results are as follows:1.The vegetative growth and kernel development were both seriously retarded in ehd1 mutant.Compared with the wild type,ehd1 mutant has lower germination rate,shrunken endosperm and embryo,1/3 of the 100-kernel weight of the WT,less cytoplasm and fewer starch granules in endosperm cell,ehd1 had shorter and fewer seedling primary roots.2.The Zm EHD1 gene was fine mapped to a ~6-kb region on chr4 using a F3 mapping population containing ~53,000 individuals(EHD1/EHD1 and EHD1/ehd1).There are two protein coding genes,one is GRMZM2G052740,the other is GRMZM2G052740.GRMZM2G052740 gene has three nonsynonymous SNPs between mutant and wild type.Then,this gene was validated as the target gene ofehd1 mutant by genetic transformation of GRMZM2G052740CRISPR-Cas9 vector and the allelism test.3.The Zm EHD1 encodes a typical EH domain protein.Subcellular localization showed that the Zm EHD1 protein in WT mainly expressed on the PM.However,the Zm EHD1 protein in mutant expressed not only on the PM but also in the cytoplasm.Zm EHD1 is a constitutive gene and its m RNA has high abundance.Importantly,Zm EHD1 was upregulated at all sampling times in the ehd1 mutant compared with in the WT.4.Uptake of FM4-64 was conducted to test the endocytosis process in seedling of the ehd1 mutant and WT,the positive transgenic individual and the control.The results showed that mutation of Zm EHD1 delayed internalization of FM4-64 in ehd1 mutant and Zm EHD1 knock-out mutants.Additionally,the results of inhibition of FM-64 endocytic recycling by fungal toxin BFA suggested that Zm EHD1 was important in endocytic transport.The endocytosis was proved to be regulated by physical interaction of Zm EHD1 and Zm AP2 ? subunit by Bi FC,split-ubiquitin yeast two-hybrid and pull-down assays.5.RNA-seqanalysis was performed using endosperm of the ehd1 mutant and the WT at 15 DAP.The result showed that the mutation of Zm EHD1 affected the expression of auxin related genes.Experiments related to auxin were performed to detect its influence on phenotype.The response of horizontally placed mesocotyl-coleoptiles to gravity is delayed in ehd1 mutant and Zm EHD1 knock-out mutants.The free IAA content in the kernels of the ehd1 mutant was ~30% lower than that in the WT.The DR5::RFP signals were significantly reduced in the root caps in ehd1 mutant and the PIN1a::YFP signals in the roots of ehd1 mutant showed more diffuse localization than that in WT line,and could be detected in root cortex.The germination rate phenotypic defects of ehd1 mutant and Zm EHD1 knock-out mutants can be rescued by exogenous low concentrations of 1-NAA.6.56 QTL and 120 HL for kernel traits were identified using a CSSL population and its two test populations.Out of them,only four QTL and HL were detected at the same or overlapping introgression region.57 and 63 different HL were identified for four kernel traits in the CSSLs ×Xun9058and CSSLs ×Zheng58 populations.There were 9 and 6 HL that could be identified at the two locations for CSSLs × Zheng58 and CSSLs × Xun9058 populations,respectively.And only 21 HL were found in both of the two test populations.These results showed that most HL for the four kernel traits were different for these two test populations.The common HL might be the important loci for the Reid × Tangsipingtou heterotic mode,and could be used to predict hybrid performance in maize breeding.