Dissection Of Allelic Variation Underlying Yield-Related Traits In Paeonia Rockii By Using Association Mapping

Tree peony originated in China is a peculiar ornamental,medicinal and oil woody plant.Paeonia rockii,one of the tree peony wild species,is widely distributed in the Qinling Mountains and adjacent areas in central China.Its cultivated germplasm is mainly distributed in northwest China,mainly in Gansu province.In recent years,P.rockii presents an advantage agronomic trait for its seed yield and nutritional value.It has become a valuable emerging woody oil crop and has been widely cultivated in northern China.At present,the effective improvement of seed yield has become an important direction for efficient breeding of P.rockii.However,in this valuable plant,basic research related to its yield is scarce,and the functional loci associated with yield traits has also rarely been identified.Therefore,we first reported the development and application of SSR within transcription factors(TFs)in P.rockii based on the next-generation sequencing(NGS)and single-molecule long-read sequencing(SMLRS).Then,to explore the genetic architecture of 24 yield-related candidate quantitative traits,the association mapping was reported in 420 unrelated cultivated P.rockii individuals based on the developed markers,aiming to identify important allelic loci that regulate yield traits and provide molecular basis for high-yield breeding of oil tree peony.The main results in this study as follows:(1)We screened and constructed a test population consisting of 420 individuals of P.rockii.A total of 24 candidate quantitative traits were investigated and analyzed,which indicated that all these fruit traits showed very significant differences among different accessions(P < 0.01).Through correlation analysis,202 pairs of significantly correlated combinations were obtained(P < 0.05),among which 182 pairs of extremely significantly correlated combinations(P < 0.01)were obtained.Principal component analysis was used to classify 24 traits into six principal components that could better explain the variation of traits in the test population.Through systematic cluster analysis,the test population was divided into eight categories and the 24 quantitative traits were divided into 5 categories.Taken together,the main indicators(4),the secondary indicators(15)and the auxiliary reference indicators(5)of yield formation in P.rockii were determined.(2)We first reported the development and application of expressed sequence tag-simple sequence repeat(EST-SSR)in P.rockii based on NGS and SMLRS.Then 166 EST-SSRs containing six nucleotide repeat types were identified from 959 candidate TFs associated with yield,with an average of one SSR per 5.83 Unigenes.Moreover,102 primer pairs(61.45%)produced successful amplifications in the two transcriptome sequencing(RNA-seq)cultivars(P.rockii ‘Fen Mian Tao Sai'and P.rockii ‘Jing Shun Fen').Among them,58 primer pairs(56.86%),which were from 18 gene families such as AP2,BHLH and HSF,were polymorphic both in the parents of a linkage mapping population(P.suffruticosa ‘Hong Qiao' and P.ostii ‘Feng Dan Bai' M24)and 8 randomly selected accessions of P.rockii.Further,the 58 EST-SSRs indicated a high level of informativeness with the PIC values ranged from 0.32 to 0.91(mean 0.70)when were assessed in 37 tree peony accessions.Transferability studies indicated that amplification ratio of 58 primer pairs ranged from 89.66% to 100%across 7 wild species in Paeonia.Additionally,the main clusters were basically consistent with the known pedigree trees in the genetic relationship study of 62 Paeonia accessions.(3)The genetic diversity of the mapping population was evaluated using the 58 newly developed EST-SSR markers in the 420 P.rockii accessions.Then we detected 483 alleles,and the average value of polymorphic information content(PIC)was 0.514,which proved that the mapping population showed a high level of genetic diversity.In addition,the population structure was divided into four subpopulations(K = 4)through the STRUCTURE and cluster analysis.(4)The results of linkage disequilibrium(LD)test showed that the overall level of LD between 58EST-SSRs was low.Single-marker association analysis using mixture linear models(MLM)showed208 significant associations,involving the 18 quantitative traits and 55 EST-SSRs.These loci were mainly from AP2,MYB and NAC gene families and explained a small proportion of the phenotypic variance.Among them,we identified 13 significant associations including 3 floral quantitative traits and11 EST-SSRs,which explained 4.15% to 25.32%(mean 10.55%)of phenotypic variation.For branch and leaf traits,we identified 129 significant associations including 8 quantitative traits and 50EST-SSRs,which explained 3.25% to 37.45%(mean 10.6%)of phenotypic variation.For fruit traits,we identified 66 significant associations including 7 quantitative traits and 39 EST-SSRs,which explained4.45% to 30.52%(mean 11.86%)of phenotypic variation.(5)The association mapping results were verified in the linkage population,and a total of 2significant associations related to parietal lobule length in the two populations were detected.The 2significant associations showed the same gene effect in both population,respectively(P026 with positive effect and PS17 with negative effect).Taken together,we first identified the 58 EST-SSR loci within TFs associated with yield based on NGS and SMLRS database in P.rockii.Then the association analysis method was used to identify the EST-SSR markers(208)associated with the yield traits of P.rockii.These provide a molecular reference for the selection and genetic improvement of yield traits in P.rockii.