Maize Pan-Transcriptome Construction And Functional Analysis Of Maize Flowering Repressor ZmCOL3

Maize was domesticated from teosinte(Zea mays ssp.parviglumis)about 10,000 years ago in tropical region of Mexico.As one of the most important crops,maize shows an amazing degree of genetic variation.With the accomplishment of B73 reference genome,it offers the foundation for genetic analysis of maize complex traits.However,not all maize genes were elucidated in the current reference genome and it is much more valuable to dig up novel genes in other maize genomes.As the rapid development of next-generation sequencing technology,the cost of genome sequencing has been increasingly reduced.While,compared to genome sequencing,RNA-seq is a more economical and efficient approach to study novel genes at different genetic backgrounds.The first part in this study was about maize pan-transcriptome construction by de novo assembly with the help of deep RNA-seq of kernels at 15 days after pollen in a diverse panel with 368 inbred lines.Genome-wide association study(GWAS)was performed to explore the contribution of maize pan-transcriptome to phenotypes,such as agronomic traits,metabolic traits,gene expression level and so on.The main results are as follows: 1.Among 38,032 reference genes,13,382 were identified as ePAV(expression Presence and Absence Variation),which expressed in some lines while not expressed in other lines.These ePAV genes were enriched in regulation-related processes.And genotype data from array showed that about 1% of them were real PAV(Presence and Absence Variation)at genomic level.2.2,355 novel sequences with a total length of 1.9 Mb were obtained by de novo assembly.1,318 of them were annotated by comparing to the Poaceae and other model species.The remained unannotated ones included: 145 had coding potential,248 may be smRNA(small RNA)precursors and 644 were considered as lncRNA(long non-coding RNA).3.There were 2,023 ePAV genes and 814 novel transcripts significantly associated with agronomic traits and metabolic traits.And the complementary of ePAV and novel transcripts between parents in a test-cross population may result in trait heterosis.4.Maize pan-transcriptome is closed type and the number of expression genes in maize kernels is about 63,000.Under the simple assumption that around 70% ~ 80% of the total genes were expressed in maize kernels,it was predicted that the number of expression genes within the whole maize pan-genome is close to 78,000 ~ 84,000.It's probably to identify much more novel genes since a diverse maize panel was used in this study.And GWAS was performed to explore the genetic basis of maize complex traits at transcript level.The second part in this paper was about functional validation of maize flowering repressor ZmCOL3.Flowering time is an important adaptation trait.Understanding the molecular mechanism and regulation pathway of flowering time in maize is in favor of the utilization of germplasm resources.Previous studies showed that CCT gene family plays an important role in plant flowering time.In this research,we identified the CCT genes in the maize genome and studied their functions by comparing their locations in the genome with the flowering time related QTLs(Quantitative Trait Locus),evaluating the associations among natural variations of each CCT gene with flowering time by using candidate gene association studies and homolog analysis.Moreover,the function of a strong candidate gene,ZmCOL3,was validated by gene transformation in maize.Exploration of functional sites in ZmCOL3 and construction of its regulatory network were also done.The main results are as follows: 1.There were 53 CCT genes in maize genome and these genes could be divide into four subfamilies: 27 COL genes,15 CMF genes,8 PRR genes and 3 TIFY genes.2.Among 53 maize CCT genes,28 of them were located at the flowering time QTL regions via linkage analysis in a previous study,15 genes were significantly associated with flowering time and 13 genes were homolog to rice heading genes.3 genes were constantly identified by all three ways.3.ZmCOL3 belongs to COL subfamily.It expressed higher in leaves,internodes,and embryo.And its expression reached highest levels at 3.5 h after sunrise.Protein ZmCOL3 was located in nuclear by subcellular localization.As a flowering repressor,overexpression of ZmCOL3 delayed flowering time about 3~4 days under both long day and short day conditions.4.The deletion of one cytosine in ZmCOL3's 3'UTR and the insertion of a 551 bp fragment in promoter region were the potential causal polymorphisms,which may account for the maize adaptation from tropical to temperate regions.ZmCOL3 may interfere with the circadian clock or bind to the promoter of ZmCCT to regulate the gene transcription,which might delay flowering.By means of linkage analysis,candidate gene association studies and homolog analysis,it was identified a maize flowering repressor ZmCOL3 in this study and meanwhile,ZmCOL3 was added into the photoperiod pathway in maize,which provide insights into the underlying molecular mechanism of maize flowering time and even facilitate maize genetic improvement in the future.