QTL Mapping,Cloning And Functional Analysis Of Ear Length In Maize

Maize provides 42%of the worldwide food calories consumed by humans,and it has been one of the most important cereal crops.By 2050,a 50%increase in cereal grain production will be required to meet the need of the estimated human population of～9.7billion.Higher yield has always been a major goal in maize crop breeding.Ear length（EL）is an important component of yield with an increased EL leading to a higher grain yield.Therefore,cloning EL gene and analyzing its molecular mechanism is important for breeding high yield varieties,and ensuring global food security.Through genome wide association study（GWAS）,expression analysis,genetic transformation,an EL gene YIGE1 was cloned.Moreover,an EL QTL cluster,q EL6.2,was also identified.The main results are as follows:1.Using GWAS,an EL QTL was identified and named q EL1.The CRISPR/Cas9technology was used to knock out these four genes.Knockout（KO）lines of the three genes GRMZM2G329040、GRMZM2G703565 and AC208571.4＿FG001 were invariant in EL compared with their respective controls.Notably,GRMZM2G008490 KO lines carrying a 432 bp deletion in coding sequence displayed significant decreased EL(P=3.9×10^-5).In addition,the OE lines had significant increased expression levels(P≤1.6×10^-4)and longer EL(P≤5.1×10^-3)than that of non-transgenic（NT）siblings in four independent transgenic events.And Compared with wild type（WT）plants,mum1 mutants displayed both significant reduced expression levels(P=8.9×10^-4)and shorter EL(P=9.2×10^-8).Taken together,these data indicated that GRMZM2G008490 annotated as an unknown protein,which we referred to as YIGE1 hereafter,is the causal gene for q EL1and positively regulates EL.2.17 SNPs were significantly associated with EL by candidate association study,among which the lead SNP Chr1.S＿50679974（C/T）had the lowest P value.All the significant polymorphisms are located within one LD block（r²>0.50）.YIGE1 expression levels in 2-5mm immature ear of 82 inbred lines were positively correlated with EL(Pearson’s r=0.53,P=1.12×10^-6).Moreover,lines harboring C at Chr1.S＿50679974showed higher expression levels（0.9 vs 1.29,P=0.02）of YIGE1 and longer EL(11.6cm vs 12.4cm,P=7.3×10^-5)than that of lines harboring T.LUC driven by YIGE1 promoter from YIGE1^BK（C allele）had much higher LUC activity than that from YIGE1^YU87-1（T allele）.Next,point mutation of the YIGE1 promoter by substitution of the T allele of YIGE1^YU87-1 with the C allele significantly elevated the promoter activity(P=6.7×10^-3).Conversely,substitution of the C allele of YIGE1^BK with the T allele resulted in lower promoter activity(P=6.5×10^-6).To recapitulate,SNP C/T contributes to variation in promoter activity,expression levels and EL.3.YIGE1 regulates the identity and fate of the inflorescence meristem.Compared to WT plants,mum1 mutants displayed significantly reduced IM size(P=8.5×10^-12),fewer KNPR(P=5.2×10^-7)and shorter EL(P=7.6×10^-3)before pollination.By contrast,the OE lines displayed larger IM size(P=2.5×10^-8),greater KNPR(P=2.8×10^-4)and longer EL(P=4.6×10^-4)before pollination than their NT siblings.RNA-seq was conducted using 0.5mm developing ears from OE and NT lines,and detected 506differentially expressed genes（DEGs）.Gene ontology and KEGG analysis indicated DEGs were significantly enriched in hormone signals,starch and sugar metabolism.Furtherly,significant differences were observed in levels of sucrose,trehalose,D-fructose,glucose and D-sorbitol between NT and OE siblings,which were decreased in OE developing ear.Moreover,developing ears of YIGE1 OE lines accumulated more Indole-3-acetic acid（IAA）and IAA conjugates than NT.It is speculated that YIGE1 may be involved in sugar and auxin signal pathways to regulate ear inflorescence meristem development,thus,affecting floret production and EL in maize.4.The nucleotide diversity（π）in the 1kb regulation region of YIGE1 was much lower in maize than in Z.parviglumis being similar with that found in landraces.The frequency of the favorable C allele increased in order in Z.parvigumis,landrace and maize（0.56 vs 0.62 vs 0.84）.These results suggested that this locus underwent continuous selection and the favorable allele was enriched during maize domestication and improvement.5.Three EL QTL,q EL6.1,q EL6.2 and q EL6.3,which were identified in previous study,were verified in different backgrounds and environments.Furtherly,q EL6.2 was divided into q EL6.2-1 and q EL6.2-2,both of which mapped to 240 kb region and the candidate genes were nominated.In brief,using GWAS,a QTL-YIGE1,which encodes an unknown protein,was identified,and may be involved in sugar and auxin signal pathways to regulate EL and grain yield by affecting pistillate floret number in maize inflorescence morphogenesis.The cloning and dissection of YIGE1,QTL-q EL6.2-1 and q EL6.2-2 not only improve our understanding of the molecular mechanism of inflorescence development,but also provide genetic resource for EL and yield improvement in maize.