Cloning And Functional Analysis Of A Major QTL QEl7 For Ear Length And Kernel Number Per Row In Maize

Maize（Zea mays L.）is the largest crop in our country.Maize yield is critical for ensuring food security and national economic development.Kernel numbers per ear is one of important yield components and is controlled by multiple genes with minor effects in maize.However,understanding the genetic basis and key loci of yield-related traits is still very limited.Dissecting genetic basis and cloning key genes has importantly theoretical and pratical significance for genetic improvement of maize yield traits.Previously,we identified a major QTL qEL7,for maize ear length,kernel number per row and ear weight.In this study,fine mapping of qEL7,transgenic validation,expression analysis,candidate gene association analysis,molecular evolution and favorable allele detection were performed.The major results are as follows:1.Fine mapping and cloning of qEL7:We backcrossed qEL7^SL17（short ear）to qEL7^Ye478（long ear）and identified 7 chromosomal recombination events in approximately 12,000 individual.By progeny test in two years,qEL7 was narrow-downed into a～50.8 kb interval,flanked by marks M4 and M5.Only on gene,Zm00001d020686,which encodes an 1-aminocyclopropane-1-carboxylate oxidase2（ACO2）,was annotated in this region B73 Ref Gen V4,and was referred to as ZmACO2.Sequencing of ZmACO2 from the QTL parents revealed 11 SNPs and 5 In Dels in its5?-untranslated region（UTR）and promoter region,but complete sequence identity in the coding region.These results suggest that sequence variation in the ZmACO2 promoter leads to the differences in ear phenotypes between qEL7^Ye478 and qEL7^SL17.2.Biological function of ZmACO2:To confirm the function of ZmACO2,we created two knockout lines（aco2-cr1 and aco2-cr2）by CRISPR/Cas9 and three overexpression lines（ACO2-OE1,ACO2-OE2 and ACO2-OE3）using maize Ubiquitin promoter-driven ZmACO2 coding region constructs in KN5585 genetic background.aco2-cr1 and aco2-cr2 lines showed an increase in ear length by about 0.86(p=2.37?10^-8)and 0.81 cm(p=1.87?10^-9),in kernel number per row by about 3.49(p=6.82?10^-20)and 1.80 kernels(p=1.44?10^-9),and in ear weight by about 12.31(p=1.41?10^-5)and 11.31 g(p=1.15?10^-4),compared with non-transgenic plants（control-cr）.Overexpressing ZmACO2 lines showed a decrease in ear length by about 1.36(p=5.79?10^-10),1.17(p=1.64?10^-10)and 1.16 cm(p=2.32?10^-9),in kernel number per row by about 2.06(p=3.76?10^-7),3.76(p=1.99?10^-19)and 3.36 kernels(p=42.68?10^-13),and in ear weight by about 19.61(p=5.38?10^-8),17.95(p=1.24?10^-8)and 20.90 g(p=1.54?10^-8),compared with non-transgenic plants（control-OE）.These results confirmed that ZmACO2 negatively controls ear length,kernel number per row and ear weight in maize.3.Expression of ZmACO2 and its biochemical function:ZmACO2 is a constitutively expressed gene,which is mainly expressed in mature leaves and inflorescences.In 2-5mm ears,the relative expression of ZmACO2 in qEL7^SL17 was significantly higher than qEL7^Ye478.These results suggest that ZmACO2 expression is related to ear phenotype difference between qEL7^Ye478 and qEL7^SL17.m RNA in situ hybridization showed that ZmACO2 was specifically enriched in the primordia and adaxial domains of SPMs,the base and adjacent of SMs,FMs,palea and glume in floral organs.These results show that ZmACO2 plays a role in the initiation and development of axillary meristem and the differentiation and development of floral organs.The ZmACO2 enzyme activity results show that ZmACO2 protein can catalyze the substrate ACC conversion into ethylene in vitro.Next,we measured ethylene levels in 2mm and 5 mm developing ears,and found that ethylene levels were significantly lower in the long ear qEL7^Ye478 line than that in short ear qEL7^SL17.Similarly,we further observed decreased ethylene levels in aco2-cr1 line and increased ethylene levels in the ACO2-OE3 line compared with corresponding non-transgenic plants.Together,these results suggest that ZmACO2 catalyzes ACC conversion into ethylene in vitro and in vivo.4.Association mapping of ZmACO2:We sequenced the 3,539 bp genomic region,covering the promoter and gene body of ZmACO2,in 214 diverse inbred lines.A total of56 variants with MAF>0.05 were detected.Of these,five variants were significantly associated with ear length at p=1.73?10^-4,including one 7 bp In Del and four SNPs in the ZmACO2 promoter region.The five associated sites,which were in complete linkage disequilibrium,formed two haplotypes:Hap^Ye478 and Hap^SL17.Those inbred lines carrying Hap^Ye478 exhibited a longer ear(p=6.94?10^-5)with more kernels per row（p=0.004）than inbred lines carrying Hap^SL17,and expression of ZmACO2 in lines with the Hap^Ye478 was significantly lower than in the Hap^SL17 lines(p=1.30?10^-4).These results confirm that the variants in the ZmACO2 promoter affect ZmACO2 expression,and it is an important causal site that affects the variation of ear length and kernel numbers in natural populations.5.Evaluation of potential breeding value of qEL7 and ZmACO2:We generated 26F₁ hybrids by crossing both qEL7^Ye478 and qEL7^SL17 with 13 diverse inbred lines,respectively.Under different genetic backgrounds,the hybrids crossed from qEL7^Ye478showed an increase in ear length by 0.69-1.72 cm,in kernel number per row by 1.82-3.73kernels,and in ear weight by 12.12-36.25 g,compared with the hybrids crossed from qEL7^SL17.Similarly,we generated 12 F₁hybrids by crossing both aco2-cr1 line and control with 6 diverse inbred lines,respectively.The results showed that the hybrids crossed from aco2-cr1 showed an increase in ear length by 0.56-1.21 cm,in kernel number per row by 1.91-2.43 kernels,in ear weight by 16.25-28.36 g,and in grain yield per ear by 9.05-29.60 g,compared with the hybrids crossed from control-cr.In summary,both the natural qEL7 allele and newly created aco2-cr1 allele by CRISPR/Cas9 increase grain yield of hybrids under diverse genetic backgrounds.6.The potential pathway of ZmACO2 regulates ear-related traits:RNAs isolated from～2 mm ears of qEL7^Ye478 and qEL7^SL17 were used for transcriptome analysis,and found that 7 IAA,8 JA,17 CK,6 BR and 2 GA biosynthesis-related genes are differentially expressed.We further measured the IAA and JA content in the developing ear,and showed that the content of IAA and JA in qEL7^SL17 was significantly higher than that in qEL7^Ye478.In addition,inflorescence development-related genes were also detected,including FEA4,BIF4 and VT2 in the auxin pathway and TS1,TS2 in the JA pathway.Therefore,ethylene might affect ear development by phytohormone cross-talk and regulating inflorescence development-related genes,and the underlying mechanisms still need to be revealed by further studies.7.Molecular domestication of ZmACO2:We calculated nucleotide diversity around ZmACO2 within a 100 kb interval on chromosome 7 in 198 teosinte,354 landrace and513 inbred lines.We found that the ZmACO2 region within a 40 kb interval exhibited obvious reduction of nucleotide diversity in landrace maize as compared with teosinte,indicating that this region may have undergone selection.Next,we resequenced ZmACO2 promoter and gene body in 53 teosinte and 44 landrace lines to examine the selection pressure of this region during maize domestication and improvement.Nucleotide diversity,Tajima’s D and Hudson-Kreitman-Aguade（HKA）test results indicate that ZmACO2 locus has a selective role during the maize domestication,and the favorable haplotype Hap^Ye478 is enriched in modern maize inbreds.The above research results confirmed the biological function and breeding value of ZmACO2.And it was discovered that ethylene plays an important role in regulating inflorescence meristem activity and floret fertility,thereby regulating the formation of kernel number in maize.This is to clarify the genetic basis of kernel number formation in maize.It also provides gene resources for the genetic improvement of yield-related traits in maize.