Genetic Mapping Of The Nulliplex-branch Gene In Cotton And Adaptive Evolutionary Analysis Of Pebp Gene Family

OBJECTIVE:Plant architecture is an important comprehensive character affecting crop yield and quality.Flowering is the final response of flowering plants to internal signals and external stimuli,marking the transition of plants from vegetative growth stage to reproductive growth stage,and also profoundly affecting the growth characteristics and the type of plants architecture.Cotton is a plant with indeterminate growth and continuously produce lateral branches and inflorescences from the shoot apical meristem?SAM?of the main stem under natural conditions,forming an indeterminate plant architecture.The branch of cotton includes vegetative branch and fruit branch,which including determinate and indeterminate branch.Nulliplex branch is a typical of deteminate fruit branch,which directly produces one or more flowers clustering from the main stem,thus forming a very compact plant architecture.Focus on nulliplex fruit branch of cotton,and based on fine mapping and preliminary validation of its genetic control genes,the analysis of bioinformation and adaptive evolution of PEBP gene families was carried out for the purpose of reconstructing the evolutionary patterns and history of PEBP gene family,uncovering the dynamics and pivotal molecular information of evolution.The dissertation can lay the foundation of analyzing the multiple functions of PEBP gene families in cotton,illuminating the molecular mechanism of nulliplex branch and guiding breeding for ideal plant structure.METHODS:?1?In this study,using the method of Bulked Segregant Analysis?BSA?and combined with Next Generation Sequencing technologies?NGS?,F2 generation hybrid was produced by crossing between Xinhai18?G.barbadense genus,nulliplex fruit branch?and TM-1?G.hirsutum genus,normal branch?for constructing group and fine mapping of controlling gene?s?of cotton nulliplex fruit branch;?2?The controlling gene of nulliplex_branch?gb_nb1?was preliminarily determined through the complementary test of cotton GhNB;?3?Based on the preliminarily validation of gb_nb1,PEBP genes in the representative genomes of Gossypium?including 10 diploid species and 5 tetraploid species?were identified and cloned by blasting the annotation dataset of Gossypium genomes and homologous cloning,and bioinformatics analysis was carried out;?4?The adaptive evolutionary analysis of the PEBP gene family of cotton and rosids was conducted using the Branch model,Site model and Branch-Site model.?5?Combined with Whole-Genome-Duplication?WGD?events occurring in plant evolution,the evolutionary pattern and characteristic of the expansion and deletion of the cotton PEBP gene family were analyzed,and the evolutionary history of cotton PEBP gene family was reconstructed by identifying of the orthologous grouping,phylogenetic analysis,the identification of ancestral genes,divergence time analysis.RESULTS and CONCLUSION:?1?Genetic analysis showed that the nulliplex branch trait was found to be controlled by a single recessive gene gb_nb1.The single nucleotide polymorphisms?SNPs?between the parents were obtained by using the method of Reduced-Representation Sequencing,and the distribution characteristics of SNP between the two populations of nulliplex branch and normal branch in the F₂ generation were identified.Based on bulk segregation analysis,allelic single-nucleotide polymorphisms?SNPs?were discovered by reduced-representation sequencing between the parents,and their profiles were also characterized in the nulliplex branch and normal branch bulks constructed using the F2 plants.A candidate⁹.0 Mb-long region comprising 42 SNP markers was found to be associated with gb_nb1,which helped localize it at the⁶00-kb interval on Chr16 by segregation analysis in the F₂ population.?2?The nulliplex_branch genes gb_nb1 were preliminarily verified by complementary test of cotton GhNB gene in A.thaliana.The results showed that the arabidopsis ATC homologous GhNB?Gh_D07G1075?was the controlling gene of nulliplex branch trait in cotton,which belonged to PEBP gene family.?3?A total of 160 PEBP genes from 15 species?10 Gossypium diploid species and 5tetraploid species?were identified and cloned by blasting and homologous cloning.Phylogenetic analysis showed that the PEBP family in Gossypium was divided into 3subfamilies,namely MFT_like Subfamily,FT_like Subfamily and TFL1_like Subfamily,including 8 orthologous groups?OG?,termed as MFT-L1,MFT-L2,FT,BFT-L1,BFT-L2,TFL1-L1,TFL1-L2 and CEN.The genome of diploid species contained 8 single-copy PEBP genes belonging to different orthologous groups,while the genome of tetraploid species contained 16double-copy PEBP genes.?4?Bioinformatics analysis showed that,in terms of gene length,gene structure,DNA,CDS and protein sequence,there were significant differences among the different orthologous groups,while PEBP genes from different Gossypium species in the same orthologous group were highly similar or even completely identical.The Gossypium PEBP gene has a typical of gene structure,which including 4 exons and 3 introns.The analysis of spatial and temporal expression patterns of PEBP genes in G.hirsutum showed that the spatial and temporal expression patterns of different PEBP genes were significantly different.Bioinformatics analysis of PEBP gene promoters showed that the number,type and distribution characteristics of cis-regulatory elements were significantly different.The number of cis-regulatory elements in the promoter region of the PEBP genes of C.subellipsoidea and P.patens was significantly less than that of the PEBP genes in Gossypium.The cis-regulatory elements in the promoter of the PEBP genes in Gossypium were unevenly distributed,and there were significant differences in the type and number of cis-regulatory elements contained in promoters of the different orthologous groups.?5?The adaptive evolution analysis of the PEBP gene family in Gossypium showed that the mean selective pressure???of the cotton PEBP gene family was only 0.12396,indicating that the cotton PEBP gene family was an extremely conservative gene family with highly negative?purifying?selection.The analysis using the Branching Model showed that the selective pressure of MFT-L1,MFT-L2 and FT branches?orthologous group?in the Gossypium PEBP family was significantly different from that of the background branches.Based on the Branch-Site Model,7 and 2 positive selective sites were identified in MFT-L2 and FT branches of Gossypium PEBP family,respectively,while none in MFT-L1 branches.?6?The mean selective pressure value?of the PEBP gene family in rosids plants was0.09675,which indicated the evolutionary conservation of the PEBP gene family.The results of Branch Model analysis showed that there was significant heterogeneity in selective pressure between MFT or FT branches and the background branches.13 and 6 positive selective sites were detected in MFT and FT branches by using the Branch-Site Model,respectively.3D modeling of protein molecules showed that,except for 69V in VvFT protein molecules,which corresponding to 65T in VvTFL1,all positive selection sites detected were distributed on the surface of PEBP protein molecules.?7?At least 2 copies of the PEBP gene family presented in the algae ancestor of plants.The differentiation of MFT-L1 and MFT-L2 branches corresponds to the WGD events in the ancestors of angiosperm,while TFL1-L1 and TFL1-L2,BFT-L1 and BFT-L2 are derived from the WGD events in Gossypium ancestor.With the WGD events in evolutionary of plants,the gene duplication,deletion and differentiation in the PEBP gene family also occurred.The extant PEBP genes in Gossypium were the results of previous WGD events,gene deletion and gene differentiation.An evolutionary pattern of cotton PEBP family were reconstructed in the dissertation.