Genome-Wide Association Studies And Function Characterization Of Candidate Genes For Main Horticultural Traits In Chrysanthemum

Chrysanthemum(Chrysanthemum morifolium Ramat.),as one of the ten most famous flowers in China and one of the four most popular cut flowers over the world,is of high ornamental and economic value,which occupies an important position in flower industry.As a species,chrysanthemum has rich germplasm resources and wide genetic variation.During the past 1,600 years of breeding activity,chrysanthemums have developed some major cultivated types,i.e.cut-flower,garden,traditional types,potplant,and gound-cover types,diverse in plant architecture and inflorescence traits.However,the phylogenetic relationships harbored in various cultivated types and the wild progenitors remain obscure as yet.Most of the horticultural traits of interest in breeding of chrysanthemum are quantitative traits controlled by complex genetic mechanisms.Despite an enormous amount of research effect into genetic basis of some key horticultural traits in chrysanthemum,it still not very clear.In this study,a large number of SNPs in chrysanthemum were developed via the specific-locus amplified fragment sequencing(SLAF-seq)approach.Phylogenetic relationship,population structure and population differentiation levels of five different chrysanthemum types were characterized by the acquisition of SNP makers.GWAS was performed to dissect the genetic control of some horticultural traits(flower color,flower shape,ray floret type,cultivated type,capitulum diameter,number of ray florets and flowering time),and some significant associated SNP loci and candidate genes were identified.Moreover,two derived cleaved amplified polymorphic sequence(dCAPS)markers were developed to facilitate molecular marker-assisted selection in chrysanthemum.Furtherly,the candidate genes deteted by GWAS were used for functional analysis.The main contents and conclusions are as follows:1.In this study,199 chrysanthemum entries representing each of the five common types were sequenced using specific-locus amplified fragment sequencing(SLAF-seq),and a set of 92,830 high-quality single nucleotide polymorphisms(SNPs)with a minor allele frequency of at least 5%was defined.The result of phylogenetic analysis corresponded well with the phenotypic classification.Based on the analysis of phylogenetic relationship,population structure and population differentiation levels(as measured by FST)of the five different types,we found that the small-flowered types,spray cut chrysanthemum(SCC)and potted and ground chrysanthemum(PGC),are more closely related to the wild progenitor species(WC)than are the large-flowered ones,disbud cut chrysanthemum(DCC)and traditional chrysanthemum(TC);and the PGC type was closest.571 genetic regions appeared to have experienced selection in the separation of PGC from DCC,and that between PGC from TC.A genome-wide association analysis revealed that seven SNPs lying within six genes were predictive of three key horticultural traits(ray floret type,cultivated type,flower shape),but no association with flower color was detected.2.We conducted a GWAS based on two years of phenotype data and a set of 92,617 SNPs using a panel of 107 diverse cut chrysanthemums to dissect the genetic control of three floral traits.A total of 81 SNPs were significantly associated with the three floral traits(capitulum diameter,number of ray florets and flowering time)in at least one environment,with an individual allele explaining 22.72%to 38.67%of the phenotypic variation.In addition,44 of these associations were detected repeatedly across both years of field trialing.Fifteen highly favorable alleles were identified for the three target traits by computing the phenotypic effect values for the stable associations.Furthermore,dosage pyramiding effects of the favorable SNP alleles were observed,and there was a significant(P<0.01)linear correlation between highly favorable allele numbers with the phenotypic values of capitulum diameter,number of ray florets and flowering time,with correlation coefficients of 0.39,0.42 and 0.28,respectively.SNP locus Marker7840-50 related to flowering time and another SNP locus Marker7810-165 related to capitulum diameter were converted to two dCAPS markers that co-segregated with corresponding phenotypic performance with an average efficiency of 82.7%and 87.2%,respectively.Finally,six putative candidate genes were identified that contribute to flowering time and capitulum diameter.These results provide valuable insights into molecular marker-assisted selection(MAS)in chrysanthemum breeding programs.3.The molecular functions of candidate CmTPL1-1 gene was further study.CmTPLl-1 was cloned from cut chrysanthemum ’Jinba’ according to our transcriptome data.CmTPL1-1 had the highest expression level in stem,and the following were leaf and flower.CmTPL1-1 was located in the nucleus by subcellular localization analysis and had no transcription activation.CmTPL1-1 was transformed into Arabidopsis(Col-0)with Agrobacterium EHA105 containing the genetic transformation expression vector pMDC43-CmTPL1-1.In Arabidopsis thaliana,over-expression CmTPL1-1 plants had smaller plants and leaves,more rosette leaves,more meristems,changed flower shape and the number of stamen were decreasesd.In order to clarify the molecular network mechanism of CmTPL1-1 involved in plant growth and development,especially for floral development.CmWOX4,CmLBD3 and CmLBD36 interaction protein were screened from the yeast cDNA library of chrysanthemum ’Jinba’ by CmTPL1-1.CmWOX4 and CmLBD38 both had no transcriptional activation activity,while CmLBD36 had obvious transactivation activity.Yeast two-hybrid and bimolecular fluorescence complementation proved that CmWOX4,CmLBD38 and CmLBD36 interacted with CmTPL1-1 respectively.Taken together,these results revealed that CmTPL1-1 regulates plant growth and development by interacting with Cm WOX4,CmLBD38 and CmLBD36.4.The molecular functions of candidate CmPUB43 gene were initial determined.A gene encoding a U-box ubiquitin ligase was isolated from our transcriptome data and designated as CmPUB43.CmPUB43 belonged to Class II U-box gene,and contained a U-box domain and three ARM repeats at the C-terminus.The phylogenetic tree clustere’d CmPUB43 with AaPUB43 of Artemisia annua.Subcellular localization predictions indicated that CmPUB43 encoded products were located in the cytoplasm and nucleus.We constructed expression vector pMDC43-CmPUB43,and introduced the pMDC43-CmPUB43 into Arabidopsis(Col-0)with Agrobacterium EHA105.The transgenic Arabidopsis lines displayed smaller plants and leaves,more rosette leaves,and asymmetry distribution of petals.The result indicated that CmPUB43 gene is involved in the regulation of plant growth and development,and laid the foundation for the further study on the mechanism of CmPUB43 gene.