Functional Analysis Of Flowering Regulator BnaA3.FRI And CSSL-based Flowering QTL Mapping In Brassica Napus L.

Flowering is a quantitative trait that controlled by a plenty of genes,timely flowering will keep plants from disadvantage environments and benefits the sexual reproductive,and also,it is a basis for higher yield for crops.Oilseed rape(Brassica napus L.,B.napus)is one of the most important oil crops in the world with significant economic value.There are three growth types,spring-type,semi-winter type and winter-type,of B.napus according to their flowering time and vernalization requirement.A number of flowering QTLs and flowering candidate genes had been identified in B.napus through QTL mapping combined with comparative genome mapping with Arabidopsis.However,the mechanism of flowering control in B.napus is still rarely known: 1)few studies on molecular mechanism analysis of major flowering genes,2)micro-effect QTL is rarely mapping and lacking functional analysis,3)the interations between flowering genes are rarely studied.To further understand the flowering control in B.napus,we investigated the association between Bna A3.FRI and growth types and local adaption of B.napus,and the molecular mechanism of Bna A3.FRI on flowering time control.Inaddition,we mapped a micro-effect flowering QTL on A2 chromosome by using chromosome segment substitution lines(CSSL)generated from backcross of No.2127(donor patent)with ZY821(resipient parent).The main results were as follows:1.Functional analysis of flowering gene Bna A3.FRI in B.napusFRI is a major determinant of flowering time and ecotype in Arabidopsis,it represses flowering by activating the expression of flowering repressor FLC.There are four FRI orthologous,Bna A3.FRI,Bna A10.FRI,Bna C3.FRI and Bna C9.FRI,in B.napus.Linkage and association studies showed Bna A3.FRI was contributed to the growth type differentiation and flowering time variation in B.napus.We amplified and sequenced the four Bna FRI copied from five winter type and five spring type B.napus,and identified 30 polymorphic sites in the regions of 200 bp upstream of ATG and 2.2 Kb coding area of Bna A3.FRI,and 30 and 50 polymorphic sites in the coding area of Bna A10.FRI and Bna C3.FRI,respectively.However,none polymorphic sites was observed in Bna C9.FRI.Further haplotype analysis of Bna A3.FRI,Bna A10.FRI,and Bna C3.FRI using INDEL markers in 17 winter type rapseed showed that the orthologus Bna A3.FRI was highly conserved in winter type of rapeseed.Extensive polymorphsm analysis of Bna A3.FRI by sequencing in additional 20 B.napus accessions revealed a total of 33 polymorphic sites in the regions of 200 bp upstream of ATG and 2.2 Kb coding area,including 5 In Dels(two located at the 200 bp upstream of ATG)and 28 SNPs(all located at coding region and 16 of them caused amino acid deletion/substitution).These 33 polymorphic sites form nine haplotypes,including three major haplotypes(hap1,hap2 and hap8,which presented 10,8,and 5 times,respectively).Further haplotype analysis of Bna A3.FRI based on In Del markers in a panel of 174 rapeseed accessions inferred three major haplotypes,m HAP1(22.4%),m HAP2(60.3%)and m HAP3(15.5%).Haplotype conponent analysis of Bna A3.FRI in different growth types of rapeseed showed that,all of the winter-type rapeseed(17/17,100%)contain m HAP1 and this was significentlly higher than the m HAP1 ratio in semi-winter(8/118,6.8%)and spring type(15/39,38.5%,respectively)of rapeseed.Further analysis of Bna A3.FRI haplotype in rapeseed from different regions indicated that,the ratio of m HAP1(5/108,4.6%)and m HAP3(11/108,10.2%)in rapeseed accessions from Asia and were significantly lower than the ratio of m HAP2(90/108,83.3%).In contrust,the m HAP1 ratio in rapeseed from Europe is as high as 58.5%(24/41).These results suggest that variation of Bna A3.FRI associates with the growth type differentiation and local adaption of B.napus.Flowering time comparison of semi-winter type or spring B.napus with different Bna A3.FRI m HAPs showed that the flowering time of accessions with different Bna A3.FRI m HAPs were significantly different.Further functional analysis of Bna A3.FRI hap1 and hap2 in Arabidopsis Col(FLCfri)showed both hap1 and hap2 activated the expression of At FLC and delayed flowering.However,the function of hap1 was significantly stronger than hap2.To further analysis the influence of sequence variations on expression level and protein function of Bna A3.FRI,we expressed the hap2,hap3,and hap8 under the control of pormoter from hap1,and the same time,expressed hap1 under the control of promoter from hap2.Transgenic analysis in Col showed that all of the four Bna A3.FRI haplotypes actived the expression of At FLC and delayed flowering.However,the function of Bna A3.FRI genes from hap2,hap3,and hap8 was significantly weaker than the one from hap1.These results suggest that variations in the gene sequence of Bna A3.FRI resulted weaker FRI protein and finally causing early flowering.2.CSSL-based flowering QTL mapping in B.napusPrevious work in our lab on whole genome chromosome segment substitution lines(CSSL)contrustion identified many early flowering lines,in which three A2 chromosome segment substitution lines,3W066,3W071 and 3W081 were obtained through foreground and background analysis on BC4F1 plants.The average flowering days of the three BC4F2 populations were 145.7 ± 18.3 d,84.3 ± 34.14 d and 136.8 ± 29.27 d,respectively,all of which were significantly early than the late flowering parent ZY821(161.7 ± 4.34 d)in the same environment.The CSSL 3W066 which contained the least background introgression segment(showed 94.75% genome identity to ZY821)was then used for flowering QTL mapping.A linkage map that contains 16 In Del or SSR markers of the introgression segment on A2 chromosome was constructed.The flowering time and genotype for each plant in BC4F3 population in WH2011(2011/2012,semi-winter environment)and BC4F4 population in GS2012(2012,spring environment)were investigated.Two flowering QTLs,q FTA2.1a and q FTA2.1b,were detected on the A2 chromosome introgression segment at WH2011 environment,which explained 25.4% and 49.1% flowering time variations,respectively.In addition,two flowering QTLs,q FTA2.2a and q FTA2.2b,were detected at GS2012 environment,which explained 13.1% and 3.7% flowering time variations,respectively.The genotype and flowering time were investigated for each plant in the BC4F3?BC4F4 and BC4F5 populations that grown in semi-winter environments of WH2011,WH2012 and WH2013,respectively.The QTL q FTA2.1a was finally delimited in the region between marker KH77 and KH78,with a physic distance of 245 Kb through aligning with the B.napus reference genome,in which an important flowering gene Bna FT.A2 was identifed.Moreover,three NILs(NIL-1,NIL-2 and NIL-3)with different flowering times and lengths of introgression segments were generated through self-crossing(the NIL-1 contains the longest introgression segment and covers the introgression segments in NIL-2 and NIL-3,NIL-2 contains both the q FTA2.1a and q FTA2.1b loci,NIL-3 only contains the q FTA2.1a locus).The flowering time of the three NILs and ZY821 in WH2013 were 66.5 ± 11 d?140 ± 8.94 d?156.3 ± 7.18 d and163.1±2.03 d,respectively.This resoult indicates that the A2 chromosome introgession segment of 3W066 contains more than two flowering QTLs that with additive effect,and the q FTA2.1a locus we mapped is a micro-effect QTL.