Generation Of GmFT1a And GmFT4 Mutantsusing A Widely-adapted Soybean Variety Of Zhonghuang 39

Soybean is a typical photoperiod sensitive short-day crop,and its yield,quality,resistance and ecological adaptability are closely related to photoperiod sensitivity and growth traits.With the development of modern molecular biology,several photoperiod related genes have been identified,and a molecular regulatory network for photoperiod regulation of soybean reproductive development has been preliminarily constructed,among which the FT family genes are the integration nodes of multiple flowering pathways.Ten FT homologous genes have been isolated from soybean,among which Gm FT2a and Gm FT5a both promote plant flowering function.However,the expression pattern of Gm FT1a under different photoperiod treatments was opposite to that of Gm FT2a/Gm FT5a,and the vegetative growth period,reproductive growth period and whole growth period of over-expressed Gm FT1a were significantly prolonged,indicating that Gm FT1a was a soybean flowering suppressor gene.Gm FT4 is one of the homologous genes of FT gene family in soybean,and its expression is regulated by E1,an important growth period regulation gene unique to soybean.Previous studies have found that over-expression of soybean Gm FT4 in model plants can delay flowering time in Arabidopsis thaliana,but the effects of over-expression of soybean Gm FT4 on flowering induction and growth period after flowering have not been reported.In this study,agrobacterium tumefaciens mediated genetic transformation of soybean cotyledon nodes was used to create Gm FT1a and Gm FT4 gene editing materials with the transgenic soybean cultivar Zhonghuang 39 as the transformation receptor,and CRISPR/Cas9 technology was used to observe the phenotypic changes of flowering stage and evaluate the breeding value of Gm FT1a and Gm FT4.The main research results are as follows:（1）Gm FT1a and Gm FT4 were knocked out by CRISPR/Cas9 genome editing technology using Zhonghuang 39,the main soybean variety,as the receptor material,and mutant materials were successfully created.Sequencing results showed that CRISPR/Cas9 had a mutation efficiency of 28.6%in the T₀generation Gm FT1a target,and a mutation efficiency of 48.0%in the T₀generation Gm FT4 target was screened out in the T₁generation.Two types of editing types were screened out in the T₁generation.The first is caused by 1-bp insertion and the second by 5-bp deletion.The homozygous mutants of Gm FT1a and Gm FT4 were successfully obtained by screening and identification of progenies.（2）Flowering time of Gmft1a and Gmft4 homozygous mutants was identified under artificially controlled long day conditions（LD,16 h light/8 h dark）and short day conditions（SD,12 h light/12 h dark）.The identification results showed that the Gmft1a knockout line with Zhonghuang 39 as the receptor had no obvious difference in flowering time under the condition of long and short days compared with the wild type.The Gmft4 knockout line with zhonghuang 39 as the receptor showed early flowering compared with the wild-type material in the long day condition,but there was no obvious difference in flowering time between the Gmft4 knockout line and the wild-type material in the short day condition.（3）In this study,Gm FT1a and Gm FT4 gene editing materials were successfully created using the widely adapted cultivar Zhonghuang 39 as the transformation receptor,and there was no significant difference between the transformation efficiency and that of model cultivar Jack as the receptor.The early flowering mutant material obtained in this study effectively shortened the flowering period of the plant,which is expected to play a huge breeding potential in higher latitudes,but also provides a basis for the genetic breeding of soybean adapted to different ecological regions.