Fine Mapping And Cloning Of The Yield Trait Related Genes Emp10 And Ter1 In Maize

The development of kernel and inflorescence play crucial a role in determining the kernel weight and kernel number per ear,while the kernel weight and kernel number per ear are the most important elements for the yield of maize ear.Therefore,cloning the key QTL/genes related to maize kernel and inflorescence development,discovering their functions and regulatory mechanisms have important theoretical and practical significance.Here,we cloned the Empty pericarp10?Emp10?which played a role in maize kernel development,and clarified the mechanism of Emp10 affecting kernel development.Besides,using the introgressed line of teosinte in maize inbred line,we fine-mapped a major QTL derived from teosinte affecting kernel row number,named as teosinte ear rank1?ter1?.The results obtained were as follows:1.Phenotypic characterization of emp10.At maturity,the emp10 seeds exhibit empty pericarp and are unviable,which resulted from the developmental arrest of the embryo and endosperm.Cytological observations revealed that the emp10 embryos were blocked in the proembryo stage,the endosperm central cells were filled with few small starch granules in the emp10 mutant,and the basal endosperm transfer layer cells in the emp10 kernels were small and lacked cell wall ingrowths.2.Positional cloning of Emp10.Genetic characterization indicated that the emp kernel phenotype resulted from a monogenic recessive variation.Using 6912 individuals,we mapped Emp10 into a 64.5 kb physical interval,and found that in the emp10 mutant,GM16 contained a 431 bp deletion encompassing 5?UTR and exon 1 and the GM16transcript was nearly undetectable.These results indicated that GM16 is possible causal gene for emp10.Emp10 encodes a P-type PPR protein forming 10 PPR motifs.Furthermore,we found that EMP10 is highly homologous with Sobic.001g092300 in sorghum.3.Expression and localization of Emp10.Spatiotemporal expression analysis showed that Emp10 was expressed in all of the tissues tested and was highly accumulated in ear and kernel.Subcellular localization analysis indicated that EMP10 targeted the mitochondria.4.The mechanism of Emp10 affecting kernel development.By compared the transcripts of mitochondrial genes,we found that intron 1 remained in nad2 transcripts in emp10.Both BN-PAGE and NADH dehydrogenase activity staining failed to detect any trace of complex I activity in emp10.Furthermore,we examined the endosperm cells with transmission electron microscopy and found that the emp10 mitochondria showed a poorly developed membrane system with large internal spaces and dissociated cristae structures.Additionally,the expression levels of AOX2 and AOX3 were significantly higher in emp10 than that in WT,which might contribute to energy supply for seed development briefly.These results indicated that the loss of Emp10 function disturbed the oxidative phosphorylation of electron transport chain,which led to the shortage of energy supply for kernel development,such that,the seed arrested and displayed empty pericarp at maturity.5.Fine-mapping of ter1.Ter1,semidominant mutation,was isolated from Zong3 x Teosinte BC₂F₇ population previously,and accounted for the aborted pedicellate spikelets,thus decreased kernel row number.In this study,we crossed Ter1 to maize inbred line C01to obtain the F₂ segregating population which contained 3822 individuals and 16 different recombinant types were discovered.Using recombinant-derived progeny test,we narrowed ter1 into a³00 kb interval flanked by markers C4 and M4 according to the B73 reference genome?RefGen V4?.6.Positive teosinte BAC clones selection and sequencing.We identified 10 positive clones from 70565 BAC clones by PCR,and a0085P23/a0131J09 were selected for sequencing.The sequencing result showed that the interval flanked by C4 and M4 was about 250 kb in teosinte,the difference between teosinte and maize focused on the intergenic region.Both teosinte and maize contained two candidate genes,Zm674 and Zm675,in this interval.7.Gene structure and expression level of ter1 candidate genes.Zm674 and Zm675encoded transmembrane protein which contained Tmemb₁85A,RhaT transmembrane domain,respectively.The C-terminus of ZM674 included a C3HC4 zinc finger domain.Additionally,we analyzed the expression level of Zm674 and Zm675,Zm674 didn't have any change,while Zm675 was increased in Ter1 significantly.8.Analysis of ter1 candidate genes.We designed specific primer on Zm674 and Zm675to amplify and analyze the sequence variation between Zong3 and Ter1.The coding region of Zm674 contained one SNP but didn't changed encoded amino acid.While Zm675 had 12 SNPs on the coding region and six of them could change five amino acid,furthermore,the SNP644 was almost fixed in maize and teosinte.88.6%?163/181?of teosinte material were fixed as A,while 97.3%?496/510?of maize lines were fixed as G.9.Vector construction and genetic transformation.To clone ter1,we constructed gene editing vector based on CRISPR-Cas9 system and transformed them into maize already.