Cloning And Functional Analysis Of A Maize Flowering Time Gene,ZmCCT9

Maize(Zea mays ssp.mays)was domesticated in southwestern Mexico～9,000 y ago from its wild progenitor,teosinte(Zea mays ssp.parviglumis).Although teosinte is distributed only in the narrow Balsas River Basin in southwestern Mexico,modern maize is distributed from latitude 58° north to 40°south and is one of the most widely planted crops in the world.Maize is a short-day plant with photoperiod sensitivity.Most tropical maize is sensitive to photoperiod,which can only flower and mature under short-day conditions(SDs),while temperate maize is insensitive to photoperiod,which has fully adapted to the temperate long-day conditions(LDs).Therefore,the adaptive changes of photoperiod sensitivity played a key role in the adaptation process of maize to different ecological environments.Further analysis of the genetic basis of maize photoperiod is of great significance to understand the molecular mechanisms of maize adaptation to various ecological environments and to employ elit tropical germplasm.In this study,using a large population of 866 maize-teosinte BC2S3 recombinant inbred lines derived from a cross between W22 and teosinte(CIMMYT accession 8759),we successfully cloned an important maize photoperiod gene ZmCCT9 with map-based cloning method.Through candidate gene association analysis,molecular biology analysis,genetic transformation and population genetics analysis,we demonstrated the biological function and selective features of ZmCCT9.The major results are as follows:1.Based on the 19,838 SNP markers that were evenly distributed across maize genome,we performed quantitative trait locus(QTL)mapping for days to anthesis(DTA)under LDs and detected a large-effect QTL(qDTA9)on chromosome 9.Using a large of fine mapping population(n=5,394),qDTA9 was delimited to a 2.4kb noncoding region between markers M115705 and M115707 following a recombinant-derived progeny testing strategy.2.We performed an association analysis by resequencing the 2.4kb noncoding region in an association panel containing 513 diverse maize inbred lines that was scored for DTA under LDs.Interestingly,we found that a Harbinger-like transposable element,located 57kb upstream of a CCT transcription factor(ZmCCT9),exhibited the strongest association with DTA(P=5.26×10-7).We also tested the association of the Harbinger-like element with growing degree days(GDD)collected in six locations at diverse latitudes.Interestingly,the Harbinger-like element exhibited significant associations with GDD only at higher latitudes,potentially indicating a role for the Harbinger-like element in latitudinal adaptation.3.Expression analysis of ZmCCT9 showed that ZmCCT9 predominately accumulates in mature leaves at floral transition and is diurnally regulated.RNA in situ hybridization analysis showed that ZmCCT9 primarily accumulates in leaf vascular bundles and sclerenchyma fiber cells.Phenotypic analysis of the near-isogenic lines(NILs)for qDTA9 under LDs and SDs showed that ZmCCT9 is regulated by photoperiod and confers long-day-dependent flowering repression.4.To validate the function of ZmCCT9,we obtained three independent transgenic plants using CRISPR/Cas9 technology.Homozygous progenies of these three null mutants were planted under LDs to investigate their flowering times.Notably,all three null mutants flowered significantly earlier than wild-type plants,indicating that ZmCCT9 is involved in the regulation of flowering time in maize.5.Through(a)congruence analysis between the Harbinger-like genotypes and the QTL segregation patterns in the maize NAM population,(b)maize protoplast transient expression assay,and(c)allele-specific expression assays in F1 crosses,we demonstrate that the Harbinger-like element acts in cis to repress ZmCCT9 expression and thereby promote flowering under LDs.6.To determine the regulatory relationship of ZmCCT9 in the maize photoperiod pathway,we examined the expression patterns of several known photoperiod-related genes in NILs and transgenic plants.ZmCCT9 represses flowering by negatively regulating expression of the florigen ZCN8.7.We genotyped the Harbinger-like locus in 73 diverse teosinte accessions.Interestingly,none of the investigated teosintes carried the Harbinger-like element,indicating that the Harbinger-like transposon insertion is most likely a de novo mutation that occurred after initial domestication.We analyzed the nucleotide diversity in a panel of 27 diverse maize inbred lines and 19 teosinte accessions.Interestingly,we detected significant selection signals in maize lines carrying the Harbinger-like element,however,no significant selection signals were detected in maize lines lacking the Harbinger-like element.These results showed that the Harbinger-like element was selected during maize domestication.8.We investigated the genotypes of the Harbinger-like transposon insertion at ZmCCT9 and the CACTA-like transposon insertion at ZmCCT10 in 1,008 maize landrace accessions in the Americas.Both transposons showed strong associations with latitude,with both predominantly accumlating at higher latitudes.Closer inspection showed that the CACTA-like element has a relatively higher frequency than the Harbinger-like element at low latitudes,suggesting that the CACTA-like element may arise earlier than the Harbinger-like element.9.Molecular dating analysis and transposon insertion frequency analysis in the association population showed that the CACTA-like element arose～2,624 y earlier than the Harbinger-like element and might have played distinct roles as maize spread geographically from its tropical origin to the temperate zones.The CACTA-like element is likely to have played a crucial role in reducing the photoperiod response of maize just after the initial domestication,whereas the Harbinger-like element arose more recently and mainly contributed to accelerating the spread of maize to higher latitudes.In summary,this work uncovered a distant Harbinger-like transposon acting in cis to alter ZmCCT9 expression to regulate maize flowering time.ZmCCT9 confers long-day-dependent flowering repression by negatively regulating the expression of the florigen ZCN8.The two transposon insertions at ZmCCT10 and ZmCCT9 arose sequentially after the initial domestication of maize and were targeted by selection as maize spread from its tropical origin to higher latitudes.Therefore,the cloning of ZmCCT9 not only further enhances the understanding of the process of maize domestication and adaptation but also provides new gene resource for selective breeding.