Positional Cloning Of A Novel Flowering Time Gene, QNE1, In Soybean And Analysis Of Its Functional Mechanism In Relation To The E1 Pathway

The time to maturity is one of the major features affecting the variety distribution,also is a prerequisite for a higher yield in soybean.Generally maturity is measured by the days from emergence to flowering and maturity.To date,many loci controlling maturity have been identified by traditional genetic studies,these loci generally was reffered to as E series.As with many researchers'long term endovours,major loci underlying maturity have been successfully cloned.However,some reports have been indicated that there are still some major loci that have not been fine-mapped or cloned.In this study,we developed a F₂ mapping population derived from a cross between Heinong 51?male?and Zhonghuang 13?female?,identified a novel QTL,QNE1?major Qtl near E1?for flowering time.Fine mapping and polymorphism analyis enabled us to identify the candidate gene at a single gene level.Functional conformation was conducted by over-expression of QNE1 in Williams 82.Also the functional correlation with E1 pathway is generally disclosed.1.In this study,a F₂ mapping population,derived a cross between Zhonghuang13?E1/e2/E3-Mi/E4?and Heinong 51?E1/e2/e3-tr/E4?was developed.A set of 96 F₂individuals were genotyped using Illumina SoySNP8k iSelect BeadChip to construct a genetic linkage map.A major QTL for flowering time was mapped onto chromosome6,which was located on a proximal region of the E1 gene.Both parents have been identified to be dominant E1 genotype at the E1 locus.Therefore,this novel QTL for flowering time was termed as to QNE1?major Qtl Near E1?.2.Nine recombinants at QNE1 region identified from 750 individuals of F_2:3populations were used for fine-mapping.The phenotyping of flowering time in the progenies of these recombinants enabled us to narrow down to a 253.22 kb region,in which 12 genes were annotated.Among these genes,the coding regions of two genes,Glyma.06G203600 and Glyma.06G204300,showed polymorphism between Zhonghuang 13 and Heinong 51.Together with our previous report,in which two candidate genes,Glyma.06G204000 and Glyma.06G204300,were identified using another mapping population of Iwaheme×Suinong 10 for QNE1,Glyma.06G204300was regarded as a strong candidate gene for QNE1.3.Based on bioinformatic analysis,we found that:Glyma.06G204300 is a transcription factor which belongs to TCP family.TCP domain of Glyma.06G204300with a b-HLH structure is highly conservative.The evolutionary tree analysis showed QNE1 gene is most closely related to wild soybean.And it belongs to CIN class of TCP family.SNP of Glyma.06G204300 leads to the change in protein secondary structure.4.Subcellular localization of QNE1 candidate gene showed that the gene is mainly distributed in the nucleus.After tests of toxicity to the yeast and self-transcriptional activation,Glyma.06G204300-BD carrier was used as a bait,we screened yeast library and obtained 37 candidate proteins which may interact with Glyma.06G204300.These related proteins merit further confirmation.5.By transformed the overexpression vector of QNE1 candidate gene into the soybean cultivar Williams 82.Eight T₀ transgenic positive strais were obtained,and screening of T₁ transgenic strais are being conducted.6.The results of tissue specific expression showed that QNE1 candidate gene mainly expressed in the leaves and flowers.The transcriptome and expression analysis of gene related to flowering time pathway in different QNE1 genotypes plants were conducted.The results are as follows:1)The transcriptional abundances of GmFT2a and GmFT5a genes and flowering time were significantly associated with genotypes of QNE1,suggesting that QNE1 candidate gene indeed invovled in flowering regulation;2)The transcriptional abundance of E1 gene was not highly correlated to genotypes of QNE1,showing that QNE1 may function differently from the E1 pathway.More accurate experiment design is needed to disclose the relationship with the E1 pathway.In conclusion,we successfully fine mapped QNE1,a novel gene locus for floweing time,to a single gene,Glyma.06G204300.Bioinformatic and functional analysis showed this gene is a novel transcriptional factor,belonging to TCP family.We also discussed functional mechanism of this gene in relation to the E1 pathway.This study enables us to explore the functional mechanism in flowering time regulation,enrich molecular network of the soybean flowering time regulation.