Candidate Genes Screening For Growth Period Traits And Function Studies Of BnamiR156 And BnaSPL3 In Brassica Napus

Rapeseed belongs to Brassica,is one of the four major oil crops(soybean,rapeseed,peanut,sunflower)in the world,and also the important economic crops with great production value in China.Brassica napus is widely planted in the world due to its high yield and disease resistance.As a comprehensive marker of plant growth and development,growth period is an important agronomic trait in B.napus,which determines the growing season and planting area.Selecting and breeding early maturing varieties of B.napus with appropriate growth period can avoid high temperature and high humidity at the later stage of growth,which is one of the main objectives of rapeseed breeding in China.In order to explore the genetic basis of the growth period related traits of B.napus and its application in molecular marker-assisted selection,our study used recombinant inbred line(RIL)populations for linkage analysis and natural populations for association analysis to identify QTL for growth period(GP)and related traits(initinal flowering date(DIF),final flowering date(DFF),flowering period(FP)and maturity time(MT)).We further screened the extreme early and late flowering materials from the RIL population and performed transcriptome sequencing of them,combined with the previous mapping data and identified candidate genes related to growth period.At the same time,because the mi R156/SPL module is the regulatory hub for plant growth and development,it plays an important role in a variety of biological processes.Using B.napus “ZS11” as the material,we peformed gene expression profile data and real-time quantitative PCR to analyze the expression patterns of Bnami R156 and Bna SPL3 in different plant tissues,and finally screened out the key members.We first constructed the overexpression vector and expression suppression vector,and then transformed them into Arabidopsis thaliana by inflorescence infection method.Finally,we analyzed their function in the flowering induction of B.napus and their interaction with other flowering genes,which provides theoretical basis for molecular design breeding.The main results are as follows: 1.QTL mapping of growth period and related traitsThe phenotype statistics of five growth periods(DIF,DFF,FP,MT and GP)in the recombinant inbred line population constructed with GH06 as the female parent and P174 as the male parent were carried out for two consecutive years(2017 and 2018),combined with the high density SNP genetic linkage map constructed in our laboratory.83 significant QTLs controlling five growth period-related traits were identified in B.napus by composite interval methods(CIM)with Win QTLCart2.5 software.Among them,15 QTLs were related to at least two growth period traits.Among them,the initial flowering QTL q18 DIF.A01-2,the final flowering QTL q18 DFF.A01 and the maturity time QTL q18 MT.A01-1 detected in 2018 were overlaped with each other,and have the highest additive effect,the contribution rates were 14.99%,15.92% and 18.30%.Therefore,it is speculated that q18 DIF.A01-2 is the main effect growth period QTL.Based on the B.napus genome annotation and the physical location of QTL confidence interval,a total of 73 Arabidopsis flowering homologous genes were identified in QTL analysis.2.Association analysis of growth period and related traitsSelecting 588 oilseed germplasm resources from all over the world,and then we conducted phenotypic statistics of five growth period-related traits for three consecutive years(2017,2018 and 2019).In order to minimize the impact of environmental variation,we calculated the best linear unbiased prediction(BLUP)value for each line of growth period traits.The optimal MLM model was selected,and the 3,856,91 SNP markers covering the whole genome were used to perform the association analysis using 3-years phenotype data and the corresponding BLUP value.We focused on QTLs that were stably detected in at least two environments.Finally,a total of 146 SNP loci significantly related to growth period traits were identified and constituted 19 haplotype blocks.Compared with the previous mapping results of growth period traits,a total of 28 loci are located in or close to the QTL confidence interval identified by previous studies.Using the haplotype block length or 300 kb upstream and downstream of SNPs as the confidence interval,a total of 101 candidate genes homologous to Arabidopsis flowering genes were identified.In addition,5 SNPs were detected to be significantly correlated with two or more growth period traits.3.Transcriptome analysis of early and late flowering materialsEarly-flowering material(18Z134) and late-flowering material(18Z88)were selected from the RIL population,and the leaves in the vegetative and reproductive growth phases were sampled,four c DNA libraries were constructed and sequenced using RNA-Seq.GO enrichment analysis was performed on DEGs shared by the same material at different developmental stages.The significantly enriched GO terms include response to cold,negative regulation of floral organ shedding,intracellular hormone transport,and long-day flowering.The results of KEGG enrichment analysis showed that the circadian rhythm-plant and plant hormone signal transduction were significantly enriched,which may be involved in the plant period transition and flowering process.The GO and KEGG enrichment analysis of DEG from different materials in the same development stage showed that DEGs involved in red light,far-red light response and circadian rhythm may be the main reason for the difference in flowering.Based on the BLASTP analysis,a total of 125 DEGs involved in flowering induction were detected,which are involved in the five major flowering pathways,including circadian rhythm/photoperiod,vernalization,gibberellin signaling and age pathways,other genes encode flowering time integrator genes or floral meristems identify genes,these DEGs form a complex flowering regulatory network in B.napus.4.Screening candidate genes of growth period and related traits in B.napusThe DEGs identified by RNA-seq were integrated with the significant QTLs identified by association analysis and linkage analysis.A total of 12 DEGs were further identified as candidate genes for growth period traits.The sequence variation of 12 candidate genes in 588 cultivated rapeseeds was further analyzed.SNP polymorphism sites were found in 7 candidate genes,and formed different haplotypes.Among them,the haplotypes Bna SOC1.A05-Haplb and Bna LNK2.C06-Hapla show more favorable early maturity traits,which can be used for molecular marker assisted breeding of growth period.5.Identification of mi R156 target genes in B.napusAccording to the amino acid sequence of SPL3 in Arabidopsis,five SPL3 homologous genes were identified in B.napus using BLASTP methods.Pfam and SMART analysis confirmed that the five SPL3 genes have complete SBP domain.Using TAPIR and ps RNATarget,it is predicted that the five Bna SPL3 are potential target genes of mi R156 in B.napus.Consistent with the predicted results,the multiple sequence alignment showed that the 3'-UTR regions of the five SPL3 genes all contained the mi R156 recognize sites(MRE).6.Analysis the expression pattern of mi R156 and SPL3According to the ZS11 transcriptome results of different tissues,the five Bna SPL3 are expressed specifically in the pistil.q RT-PCR results showed that: Bna SPL3.Cnng was mainly expressed in petals and pistils,Bna SPL3.C04 was expressed specifically in roots,flowers,buds,and pistils,Bna SPL3.A05 was highly expressed in buds and pistils;mature Bnami R156 b was mainly expressed in stems and sepals.The expression pattern of Bna SPL3.C04 is opposite to that of mi R156 b,and it is speculated that it may be negatively regulated by Bnami R156 b.Therefore,Bna SPL3.C04 is used as a candidate gene for next functional verification.7.Function study of mi R156 and SPL3.C04 in flowering induction in B.napusThe miR156b and miR156 g precursor gene sequence in B.napus were cloned to construct the mi R156 overexpression vector,the Arabidopsis IPS1 was cloned and the mimicry156 vector that inhibited the expression of mi R156 was constructed using IPS1 as the backbone.They were transformed into the Arabidopsis thaliana,and then homozygous T3 generation transgenic plants were screened by PPT(glyphosate)and PCR detection methods.Compared with the wild-type,transgenic Arabidopsis overexpressing mi R156 b and mi R156 g showed increased number of leaves,late flowering,and delayed transition from vegetative to reproductive stage;MIM156 transgenic Arabidopsis showed early flowering,reduced number of leaves,and earlier stage transition compared with the wild-type.In addition to the flowering,mi R156 can also regulate plant architecture.Compared with the control,mi R156 overexpression plants showed reduced plant height and increased branches,while the MIM156 transgenic plants showed no significant difference compared with the control.The pEarly Gate101-Bna SPL3.C04 overexpression vector was also constructed and introduced into A.thaliana by inflorescence dip methods.After PPT screening and PCR detection,homozygous T3 generation transgenic plants were obtained.Compared with the wild control,the transgenic line showed earlier flowering and increased plant height.It indicates that mi R156 and SPL3 are important regulators of flowering and plant growth and development in B.napus.8.Bna SPL3.C04 participate in the flowering pathwayWe performed transcriptome analysis of transgenic Arabidopsis(35S::MIM156,35S::mi R156 b and 35S::SPL3.C04),KEGG enrichment analysis showed that DEGs were mainly enriched in plant hormone signal transduction and circadian rhythm pathways.The results of q RT-PCR analysis further indicated that Bna SPL3.CO4 integrates multiple flowering signal pathways including age,photoperiod and GA pathway through flowering time integrator SOC1 and FUL to induce Arabidopsis phase transition and early flowering.