Cloning And Functional Characterization Of A Major QTL KERNEL LENGTH9 (KL9) In Zea Mays

Kernel length,kernel width and kernel thickness,three related criteria closely related to the maize kernel size,directly affect the kernel weight and ultimately influence maize yield.Hence,studying the genetic and molecular basis of kernel size by using the method of forward genetics will be beneficial for the improvement of maize yield.In this study,we cloned a major QTL KERNEL LENGTH 9（KL9）,named ZmKL9,using the constructed near-isogenic lines of kernel-related traits(Mc^qKL9 and Mc),which could control kernel length and hundred kernel weight.Furthermore,The function of ZmKL9was confirmed by genetic transformation.The major results are as follows:1.The construction and phenotypic identification of KL9’s near isogenic lines.The Mc^qKL9,contained the KL9 in the Mc background,screened to obtain from the BC₄F₂population constructed by using V671（large grain）and Mc（small grain）.By comparing the differences in kernel size and other agronomic traits between Mc^qKL9 and Mc,it was shown that the KL9 could increase a single kernel length by 0.39 mm(p=3.15×10^-12)and hundred kernel weight by 1.48 g（p=0.0002）without affecting the kernel width（p=0.1）.Meanwhile,the KL9 could affect increase maize yield by increasing ear length and ear weight without affecting the growth period.Phenotypic statistics and observation of kernel length were performed on early developed kernels of Mc^qKL9 and Mc at 6,9 and 14 DAPs.Phenotypic identification results showed that the kernels of Mc^qKL9 were significantly longer(p=4.52×10^-12,p=2.28×10^-31,p=1.57×10^-11)than that of Mc at the three period.Cytological sections revealed that KL9 may regulate kernel length by affecting the number of endosperm cells.2.The positional cloning and identification of key candidate genes of KL9.To identify KL9,we backcrossed Mc^qKL9to Mc and constructed a selfed-backcross population of～34,000 individuals.We screened recombinants and performed progeny tests for KL9 through multi-year and multi-environment,and finally mapped KL9 to a736 kb region of chromosome 9.By combining the expression pattern and RNA-Seq analysis of the twenty annotated genes within the interval showed that only seven of them had the expression level,among which the expression level of Zm00001d046718 was highest and differentially expressed in 6 DAP seeds of Mc^qKL9and Mc.The candidate regional association mapping was conducted on KL9 candidate interval by using the kernal-related traits of the 513 association inbred lines,and Zm00001d046718 was found to be significantly associated with 100-kernel weight.Comparing the nucleotides sequence of Zm00001d046718 in the Mc^qKL9 and Mc,we found that there were multiple variations in the promoter region of the gene.Meanwhile,three SNP variations in the coding region of the gene resulted in the changes of three amino acids.Importantly,we found that a 288 bp（TE1）and a 557 bp（TE2）TE variations in 5’UTR and the first intron of Zm00001d046718,respectively.Therefore,this gene was selected as a key candidate gene for KL9,named ZmKL9,encoding a b ZIP functional domain transcription factor.3.Expression pattern analysis and transcriptional activation verification of ZmKL9.RT-qPCR revealed that ZmKL9 was expressed in many tissues of the B73 maize inbred line,indicating that it is a constitutively expressed gene.Meanwhile,the expression level of ZmKL9 was differentially expressed in 6 DAP seeds of the Mc^qKL9 and Mc.Luciferase activity test showed that the insertion of 288 bp transposon in 5’UTR region of ZmKL9 in Mc may affect the expression of ZmKL9.Subcellular localization analysis that ZmKL9 was mainly located on the nucleus.Transcriptional activation experiments in yeast showed that the b ZIP functional domain of ZmKL9 was necessary for transcriptional activation while the TMD functional domain had no effect on transcriptional activation.4.Function characterization of ZmKL9.We generated transgenic maize plants that over-expressed it（KL9-OE）in inbred line B104.Two over-expression lines（OE12 and OE13）were analyzed.Compared with the self-crossing ears and kernels of the negative plants,the ear length increased by 0.98 cm(p=2.94×10^-7)and 1.09 cm(p=3.19×10^-6),respectively,the ear weight increased by19.59 g(p=3.64×10^-5)and 15.72 g（p=0.00039）,respectively.The average kernel length of the single kernel on self-crossbred ear was increased by 0.53 mm(p=7.79×10^-6)and0.3 mm（p=0.0027）,and the average grain weight was increased by 2.09 g(p=4.66×10^-5)and 0.74 g（p=0.05）,respectively.These results indicated that ZmKL9 had a vital function of regulating kernel length and hundred kernel weight,and also significantly affected two yield traits,ear length and ear weight.5.The genetic effects of ZmKL9^TE+allele in natural populations and the domestication selection of ZmKL9.The PCR amplification analysis of 513 modern maize inbred lines showed that 4lines only contained TE1 insertion,and 18 lines both contained TE1 and TE2 insertion.Further comparison of the kernel length of the three haplotypes showed that the kernel length which contained the TE insertion was 8.73±0.36 mm,which was significantly lower(9.30±0.73 mm,p=1.05×10^-6)than that of the lines without TE insertion.This indicated that TE-PAV was an important variation site affecting kernel length in natural population.The candidate gene association analysis indicated that the TE1 transposon located in the 5’UTR region of the ZmKL9 was significantly correlated with the kernel length(p=1.18×10^-5).The TE-PAV was amplificated from 183 teosintes,276 landraces and 538 maize inbred lines,indicating that it was a rare allele and not selected during maize domestication.In summary,we cloned a QTL by map-based cloning for KL9（qKL9）as a gene that encodes a b ZIP transcription factor which functions as a regulator of kernel length（KL）.The role of ZmKL9 in regulating kernel development was confirmed by the methods of breeding effect evaluation of the target intervel,the candidate regional association analysis,gene expression analysis and genetic transformation.Further study found that the presence or absence of variation in the insertion of the ZmKL9 5’-UTR 288 bp transposon element was an important natural variation,and as a rare mutation locus repressing the expression of ZmKL9,thus affecting kernel length and maize grain yield.