Candidate Genes Association Analysis Of Maize Transcription Factors

Transcription factors are important upstream regulatory factors of plant development.They control the transcription of downstream functional genes by combining them in the promoter regions of downstream genes to exercise regulatory effects on plant phenotypes.Analyzing the regulation of TFs on phenotype can help improve efficiency of molecular breeding by improving the purpose and pertinence of phenotypic observation.Maize growth period is a trait closely related to yield.The heading date,Silking time and pollen shed will allow sufficient time for corn to complete grain filling and maturity in advance,which is beneficial to increase to yield.At the same time,in view of the unique maize-wheat cropping system in the Huanghuaihai area,the shortening of the maize growth period will also ease the pressure on wheat sowing,so that the wheat can develop well before the winter,increase the wheat yield,and then increase the annual grain yield.In order to analyze the regulation of TFs on the phenotypes,including growth period,of key maize traits,in this study,508 inbred lines were served as the natural GWAS population.Based on 21 kinds of maize major agronomic and quality traits,GWAS was performed on 2034 maize TFs S.The main conclusions obtained are as follows:1)Under the Q+K model,the GWAS was performed on 2034 TFs,and a total of 3578 SNPs were identified as significantly associated with 21 phenotypes(P<10-3).The phenotypic variation explained by these SNPs ranged from 2.37% to 68.15%.Among them,the significant TFs associated with axis color are 105,which is the most.Among the transcription factor families with more than 10 family members,the G2-like family has the highest proportion of transcription factor significant members,and the FHA family contains the largest number of SNPs per gene on average.Significant SNPs are located in the intron region,exon region,5’UTR region and 3’UTR,accounting for 47.19%,28.20%,12.23% and 12.39%,respectively.2)Indepth analysis of TFs related to traits in corn growth period(heading date,silking time and pollen shed).Correlation analysis of the three phenotypes showed that the three phenotypes were significantly correlated.There are 75,68 and 58 TFs associated with treading date,Silking time and pollen shed,respectively.About half of these TFs are associated with more than one phenotype,and 24 TFs are associated with three phenotypes.There are 551 SNPs that are significantly related to growth period,of which 206 are SNPs that are significantly related to traits at heading date,177 are SNPs that are significantly related to traits at pollen shed,and 168 are SNPs that are significantly related to traits at Silking time.There were 75 significant SNPs simultaneously associated with the phenotypes at the Silking time and heading date,as well as the phenotypes at the Silking time and heading date;128 significant SNPs were simultaneously associated with the phenotype at the heading date and pollen shed.3)Forty transcription factor genes containing 3 or more significant SNPs related to growth stages were selected.Through DAP-seq,key cis sites and downstream genes regulated by these TFs were obtained.Twenty-two TFs obtained DAP binding site information,and the cis sites where these TFs bound are consistent with the reports on the model plant Arabidopsis.Downstream genes bound by TFs are significantly enriched in organic nitrogen compound anabolism,meiosis,RNA binding,DNA binding activity,serine/threonine protein kinase activity,etc.Different TFs had coregulated downstream genes.For example,Zm00001d017660 is co-regulated by TFs of 9 transcription factor families,which encodes a photoperiod-independent early flowering gene;the downstream binding gene Zm00001d020364 common to the NAC and MYB TFs and the downstream gene combined with NAC,MYB and ARF(Zm00001d047269).Regulate the expression of early floral protein 4.These results show that using candidate gene association analysis combined with DAP-seq,the key regulatory transcription factors upstream of growth stage-related traits are NAC,MYB and ARF transcription factors,which directly bind to the photoperiod-independent early flowering genes in the downstream key genes.Hezaohua gene 4 and other downstream key functional gene promoters regulate the transcription of these genes and ultimately lead to the phenotype of changes in the growth period.The correlation(LD)analysis of NAC,MYB and ARF transcription factors shows that the significant sites related to the phenotype are highly correlated with nearby SNPs.4)An overexpression vector and a CRISPR/Cas9 knockout vector were constructed for one of the TF related to the growth period of maize,Zm IAA10,and the maize genetic transformation was carried out.Zm IAA10 T2 overexpression positive plants(3 independent transformation events)showed the heading stage,the powdering stage and the silk stage were earlier than the control(KN5585);while the Zm IAA10 knock-out positive plants(3 independent editing events)had no early growth period Phenotype.The results indicate that the use of candidate gene association analysis can obtain reliable trait-related upstream regulatory TFs.This study explored a highly maneuverable method of establishing a link between regulatory genes and phenotypic traits,which can provide key genetic resources and molecular markers for molecular breeding of maize,and help improve the purpose and efficiency of molecular breeding.