Molecular Evolution And Association Analysis Of ZmFKF1, A Clock-Controlled Gene In Maize

Broadening the genetic diversity is the foundation to achieve higher breeding objectives in corn, such as high-yield, quality and resistance. However, the photoperiod sensitive of maize introduced from tropical and subtropical seriously restricted the use of superior germplasm. Recently, researchers had fully excavated superior gene resources, and constructed gene regulatory networks, and development molecular markers for molecular breeding by using the comparative genomics methods in different species. In this study, we performed a comprehensive computational analysis of FKF1 and the LOV-F-box gene family, and cloned FKF1 in several important Poaceae species for phylogenetic analysis. And try to excavate excellent allelic variations in 89 lines of association panel by candidate gene association analysis. RT-PCR was used to detect the expression profile of ZmFKF1b in shoot tips and leaves of maize inbred lines under 7/17 hours light treatments. We measured several physiological indicators of maize leaves under two photoperiodic, which may help to excavate new evaluation index for photoperiod sensitivity. The main results were as follows:(1) Through bioinformatics approach,115 candidate LOV-F-box genes were identified in 47 species, and all of them were existed in vascular land lant, those mosses and aquatic plants were not identified. All identified candidate genes can be clustered into FKF1 and ZTL/LKP2 two groups, each group can be then divided into monocots and dicots. Indicate that the differentiation of monocots and dicots is later than the division of FKF1 and ZTL/LKP2, but earlier than ZTL/LKP2 or ZTLs. All FKF1 in 32 species also clustered into monocots and dicots, much more, it can be consistent with classification of species as well.(2) Two ZmFKF1 were identified in maize B73 genome database, named ZmFKF1a and ZmFKF1b. In this study, we cloned full-length sequence of ZmFKF1b, OsFKFl, AtFKF1 contained UTR and TaFKF1 without UTR. Phylogenetic analysis show that all maize and teosinte clustered together. Whats more, all maize germplasm divided into temperate, subtropical and tropical/subtropical three groups was well. Besides, several evidences, including nucleotide polymorphism analysis, selection tests and phylogenetic analysis, support that ZmFKF1b has been under strong positive selection, especially in the coding region of gene, these results imply that the protein of ZmFKFlb has functional differentiation in different maize.(3) Nucleotide polymorphism of ZmFKF1b was evaluated using the 89 inbred lines of the association panel. Totally 62 polymorphic sites were identified in thefull length genesequences obtained, including 49 SNPs (one per 64 bp) and 13 InDels (one per 242 bp).12 out of 18 SNPs in coding region is synonymous mutations, and two extron each has an InDel. Linkage disequilibrium analysis of ZmFKF1b indicate that, there were several LD blocks in ZmFKF1b, the LD decayed rapidly below to R2=0.1 within 3300 bp. Using the general linear model to association analysis between the polymorphic sites in ZmFKF1b and phenotypic traits.11 sites of identified significant loci affect the variability of maize flowing time of male and female in Beijing, significance level (P<0.001).They can explain 15.91-22.05% phenotypic variation(R2) of flowing time.8 sites were associated with anthesis and silking stage under two different observation points at P<0.01 level.12 sites were significantly associated with the tassel length (P<0.01), no significant loci were identified associated with anthesis-silking interval (ASI) and plant height.(4) In this study, RT-PCR was used to detect the expression profile of ZmFKF1b in shoots tips and leaves of maize inbred lines under 17/7h light treatments. Under 7h light treatment, the expression level of ZmFKF1b in shoots tips and leaves of Huangzao4 were all increased, and downregulated under 17h. While in CMT-L106, the expression level tends to be stable, and also downregulated under 17h. The result imply that two different photoperiod uninduced the expression of sensitive materials, and do a great influence to the floral reversion and flowingting time, while inhibiting its expression of insensitives materials under long day treatment, and delayed flowering time.(5) Use the choosed extreme materials to measure various physiological indicators under two light treatments. It shows that maize undered 17h have higher chlorophyll content than 7h, and the difference between photoperiod-sensitive lines was biger. The free amino acid content was higher under short-day treatment, and it was increased under two treatments. Huangzao4 and 622016-ZCN-2 are two photoperiod insensitive lines, which in short-day has very high content of free amino acids. The starch content and soluble protein content were increased with period of treatment. Soluble sugar content reduced in early treatment and then increased at the end of treatment. More interesting, the lowest value period is consistent with the flower bud differentiation of male, it indicate that the soluble sugar content can do as an important physiological indicator to evaluat photoperiod sensitivity. The photosynthetic rate (Pn), stomatal conductance (Gs), intercellular CO2 concentration (Ci) and transpiration rate (Tr) four Photosynthetictraits were investigated at different growth stages. We found that the more sensitive the material is the higher photosynthetic efficiency it well, and with the increase of processing time each measurement tend to decrease. Indicate that these physiological traits can also evaluat photoperiod sensitivity.