| Matury(especially flowering time)is a major ecological trait that relates to distribution and yield of soybean varieties.The mapping and analyze of maturity genes is very important to breed excellent cultivars with wide ecological adaptability.So far,a number of major E genes that regulate maturity period in soybean have been cloned.Their application has improved the efficiency and yield of breeding,but it has not yet completely solved breeding problem.Therefore,the aim of this study is to clone and functional analysis the new maturity gene to provide theoretical basis for improving the adaptability and yield of soybean.The detailed studies are as follows:1.MA recombinant inbred line derived from a cross between Minsoy(Ele2E3E4)and Archer(elase2E3E4)for detecting flowering time QTL in LD conditions.Though parents resequencing and SLAF-seq of MA population,a high-density genetic map with total length of 3,588.94 cM was used for QTL mapping,comprising of 5074 SLAF markers.Using this genetic map and flowering time of F6 and Fs MA population,three stable QTLs(TOF5,TOF6.1,and TOF7)were identified for the flowering time,which were mapped to chromosomes 5,6,and 7,respectively.Among them,TOF 6.1 closely linked with E1.And the El genotype between parents is different,these results further indicates that TOF 6.1 is the El gene.The contribution of TOF7 to flowering time is second only to TOF 6.1,and its coding gene has not been reported.Therefore,TOF7 is a new locus for flowering time.2.Used genic map on chromosome 7 and flowering time of MA population over two years,TOF7 were detected between Chr7:3904858 and Chr7:4936114,about 1.03Mb.Additionally,residual heterozygous lines(RHLs)#13 and#131 were selected which might segregate at the TOF7 locus.For flowering time and maturity of RHLs#13 and#131,the observed frequencies of F2 progenies all fit a 3:1 ratio,and segregation ratios of F3 progenies all fit a 1:2:1 ratio.These results revealed that a single dominant gene encode TOF7 locus.The phenotypes detected from two near-isogenic lines(NILs)revealed that NIL-TOF7 plants significantly promoted flowering time and maturity than NIL-tof7,which strongly suggest that TOF7 controls early-flowering time and maturity under natural LD environments.Moreover,TOF7 gene was further fine-mapped to an～138 kb interval.The TOF7 was identified as the best BLAST hit to critical gene of photoperiod regulated flowering in Arabidopsis.3.Though sequence analysis,a nonsynonymous base of the TOF7 gene CDS is different between M and A,which mutates the 9th amino acid located in the N-terminal signal region of the encoding protein.Furthermore,TOF7 protein in parent M mainly located in nucleus,while tof7 protein in parent A located in the cytoplasm and nucleus.Functional complementarity experiments show that TOF7 gene in parent M can restore the precocious phenotype of Arabidopsis homologous gene mutants,but the tof7 gene in parent A can not restore.Finally,TOF7 gene was mutated by the base deletion through CRISPR-cas9 technology.In LD conditions,the mutants significantly flowered on average 5-6 days later than WT plants,and the maturity date delayed about 15 days compared to WT plants.These results fully prove that the non-synonymous mutation of TOF7 leads to changes of TOF7 protein subcellular location,which delay flowering time and maturity.Additionally,NIL-tof7 and TOF7 cas9-mutants increased yield of plant compared to NIL-TOF7 and WT plants,respectively,indicating the importance of TOF7 gene for breeding.4.TOF7 gene exhibits the expression pattern of 24h clock rhythm,and mainly expressed in triple leaves.During the signal pathway of photoperiod regulation flowering,TOF7 gene promotes flowering by inhibiting the expression of El,then up-regulating the expression of GmFT2a and GmFT5a gene.These results will complement the research of biological clocks genes on the regulation network of flowering in soybeanIn conclusion,TOF7 was fine-mapped as a novel and dominant QTL for flowering time according on QTL location.TOF7 is proved to promote flowering and maturity in soybean by various methods such as subcellular localization,functional complementarity and mutants,and TOF7 participates in the flowering regulatory network composed of soybean E1-E4.These findings will not only add more knowledge on photoperiod regulated maturity but also provide powerful theoretical basis for soybean adaptability and molecular breeding. |
