Cloning And Functional Study Of Soybean Flowering Gene Tof21

Soybean（Glycine max[L.]Merr.）is one of the important grain crops in China,and as a typical short-day plant,photoperiod regulation of soybean flowering is the key factor for soybean to adapt to different ecological regions.Soybean varieties in middle and high latitudes will delay flowering when planted in high latitude,with prolong growing period and unable to receive seeds,whereas they are planted in low latitude,they will bloom early,mature early and low yields.Therefore,it is of great significance to clone the important genes that regulate soybean flowering and improve the regulatory network of soybean flowering to ensure the stable yield of soybean in different latitudes.Based on 424 soybean germplasm resources collected from all over China and combined with soybean flowering and resequencing data,the laboratory identified a flowering site on chromosome 11 and 12,respectively,named Tof11 and Tof12.The loss-of-function alleles of tof11 and tof12 could significantly promote soybean flowering and improve the adaptation of soybean to high latitudes.The tof11 and tof12 have been subjected to strong artificial selection in the process of soybean domestication,result in tof11/tof12 genotypes become fixed in cultivated soybeans.Therefore,tof11/tof12 genotypes cannot be used to adapt soybeans to higher latitudes.However,higher latitude regions（such as Far East of Russia,Heihe of China,etc.）tend to have earlier flowering and maturity,making them only suitable for planting soybeans.Therefore,it is very important to further explore other key genes of flowering period on this basis.In this study,we identified a novel locus regulating flowering stage in soybean,through forward genetic approach;The coding gene was identified by CRISPR/Cas9 and stable transgenic technology.The molecular mechanism was analyzed by transient transformation of tobacco leaves,population genetic analysis and quantitative real-time PCR.This study will further improve and enrich the molecular regulatory network of soybean flowering.The specific research results are as follows:1.424 soybean varieties were divided into two subgroups under the background of Tof11/Tof12 and tof11/tof12.GWAS analysis showed that in the subgroup under tof11/tof12background,a new site for controlling flowering stage of soybean was identified on chromosome 18 and named Tof21（Time of flowering 21）;2.A F₂population derived from the cross between W82（early flowering soybean varieties）and GDL49（late flowering soybean varieties）was uesd to identify QTL,a QTL for controlling the flowering trait was detected,also at a similar position on chromosome 18;3.Using linkage disequilibrium analysis and fine mapping analysis,Tof21 was located in the 120 kb interval,which contains a MYB-like transcription factor that may be involved in the regulation flowering time of soybean,and was presumed to be a candidate gene for this site;4.The flowering time of the near-isogenic lines NIL-Tof21 and NIL-tof21 was observed under long sunshine conditions,and it was found that compared with NIL-Tof21,NIL-tof21bloomed later and could not mature normally,indicating that Tof21 was an important flowering site;5.Two Tof21 overexpressing soybean transgenic lines Tof21-OE17 and Tof21-OE18under W82 background were obtained by overstable transgenic method.Both Tof21overexpressing transgenic lines showed early flowering and early maturity compared with W82 under long sunshine condition.tof21^CRunder the background of W82-E1 was created by CRISPR/Cas9 and stable transgenic technology.Under long sunshine conditions,tof21^CRsignificantly delayed flowering compared with W82-E1,suggesting that Tof21 site was encoded by this transcription factor;6.Transient transformation of tobacco leaves,population genetic analysis and quantitative real-time PCR showed that Tof11/Tof12 was located upstream of Tof21 and inhibited the expression of Tof21,and the tof11/tof12/Tof21 genotypes combination flowered earliest,thus enabling soybean to adapt to higher latitudes.7.Through transient transformation of tobacco leaves and population genetic analysis,it was demonstrated that Tof21 was located upstream of E1,a key gene for soybean flowering,and was able to repress the transcription of E1,thus promoting soybean flowering.In summary,this study identified Tof21,a new site regulating flowering in soybean,through forward genetic approach,cloned the coding gene of Tof21,and analyzed the molecular regulatory network of Tof21 that regulates flowering stage of soybean.This study has important application value in providing a new molecular module for breeding high-yield soybean varieties that adapt to higher latitudes.