| 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.Anemone-type chrysanthemum is famous for its oddly shaped,colorful petals,with higher ornamental value,anemone-type is also one of the important goals of chrysanthemum breeding.At present,the genetic mechanisms of anemone-type chrysanthemum’s inflorescence-related traits remained unclear.In view of this,67 representative anemone-type chrysanthemum varieties were analyzed for phenotypic assessment and genetic diversity analysis;the genetic rules of anemone-related traits were analyzed using cross combinations of multiple different genetic backgrounds;the special molecular marker loci was associated anemone-type via BSA,then developing a SCAR for further verification;the significant QTLs was detected in floral and epistatic interaction effects were analyzed in different floral traits based on linkage mapping;the significant SNPs and candidate genes were associated with floral trait in chrysanthemum via genome-wide association analysis（GWAS）.The findings of the current study give insights into the complex genetic architecture of anemone-related traits in chrysanthemum from the perspective of both classical and molecular quantitative genetics,and provide important theoretical basis and valuable breeding materials for traditional crossbreeding and modern molecular breeding of chrysanthemum.The mains results are as follows:1.A panel of 67 accessions was characterized with respect to both floral morphology,assessed using a set of 12 inflorescence-associated traits,and to genotype,using sequence-related amplified polymorphism（SRAP）fingerprinting.Abundant variation and a high level of broad-sense heritability（>88%）were revealed for all traits except number of tubular floret lobes.Of the possible 66 pairwise inter-trait combinations,37 proved to be significantly correlated.A principal component analysis of the trait data showed that the first four principal components explained～78%of the variance;meanwhile a hierarchical clustering analysis revealed a number of discrete groups reflecting the various cultivated types,but was not predictive of provenance.The SRAP genotyping employed 22 primer combinations and generated 238 informative fragments.A moderately high level of the Jaccard’s similarity coefficient（mean 0.67）was calculated for the examined panel.A hierarchical clustering analysis based on the genotypic data revealed four groups,which were clearly reflective of cultivated type;and moreover,a comparison of the within-cluster means for each of the two key anemone-related traits-tubular floret length and tubular floret length/style length-showed that the genotype-based clusterings were associated with the anemone levels,albeit imperfectively.A principal coordinate analysis showed that the first two coordinates accounted for about 14%of the total variation.2.Nine cross combinations from seven parents were conducted to investigate flower patterns,maternal effect,and the relationships between the heterosis of six tubular floret traits and parental genetic distance.The results showed that highly significant（P<0.05）differences among the parents and cross combinations in various traits.Abundant variation was revealed for almost floral traits,thus indicating great potential for the selection of lines with the desirable traits.The segregation ratios of anemone to nonanemone in the F1 hybrids were 1:3（nonanemone × anemone type or anemone × nonanemone type）and 3:1（anemone × anemone type）except two populations from parents ’Nannong Xuefeng’ and’QX096’.The hybrids’ performance were more inclined to vulnerable parent,and involved less influence of maternal or paternal effects.Moreover,floral traits in current cross combinations displayed some extend decline and exhibited negative mid parent heterosis（MPH）values.Phenotypic distance was significantly negative correlated with MPH for floral traits except style length and disk flower diameter.Unfortunately,no significant correlations were observed between molecular marker-based genetic distance and MPH for all floral traits.3.The genetic variation in anemone traits was investigated in a segregating intercultivar F1 population derived from the anemone type ’Nannong Xuefeng’and the nonanemone type ’QX096’,and anemone-specific markers were developed via the bulked segregant analysis（BSA）method.A high variation coefficient（>30%）was obtained for most tubular floret traits relevant to the anemone type.The anemone morphology of the hybrids was intermediate between that of their parents,and some transgressive individuals were observed,indicating great potential for the selection of lines with the desirable anemone type.In the BSA,4 of the 500 SRAP primer combinations were identified as informative between the bulks of the anemone and nonanemone F1 individuals.After cloning and sequencing,only one SRAP marker,i.e.,M11E1272,was successfully converted to the SCAR168.The SCAR marker was validation in ’Nannong Xuefeng’ ×’QX096’（80 lines）and ’Nannong Xuefeng’ × ’Monalisa’（144 lines）populations demonstrated that the marker SCAR168 could truly discriminate anemone and nonanemone types with a high coincidence ratio 87.86%～92.50%.In addition,a general linear model（GLM）based association analysis（P<0.01）revealed that the SCAR marker exhibited a positive effect on the anemone traits and explained 20.83%～33.66%phenotypic variation.4.The inheritance of a set of traits associated with floral form was investigated using a quantitative trait locus（QTL）mapping approach applied to a segregating population bred from a cross between the two varieties ’Nannong Xuefeng’ and ’Monalisa’.The coefficient of variation of the various traits ranged from 12.79%to 59.23%,with an average of 28.0%,and there was notable transgressive segregation in both directions.A set of 26 QTL was uncovered,explaining a portion of the variation for eight of the eleven traits;individually,the loci accounted for between 6.34%and 11.86%of the phenotypic variation.A further 16 pairs of epistatically acting QTL was identified,associated with a contribution of between 3.69%and 13.63%of the phenotypic variance;the majority of these interactions involved background loci.The conclusion was that both additive and epistatic effects were involved in the genetic determination of floral form in chrysanthemum.5.A genome-wide association study was performed using filtered 66,323 high quality SNPs in the panel of 102 chrysanthemum accessions.The phenotype descriptive statistics showed no significant in the two years,the variation coefficient range is 11.60%to 88.62%,and the broad sense heritability was between 47.20%and 90.40%,and most of the traits were non-continuous and skewed distribution trends.22 SNPs were associated with nine traits via mrMLM software,and the highly favorable SNP alleles that explained 6.67%～27.24%of the phenotypic variation were subsequently identified according to the phenotypic effects.Finally,7 putative candidate genes for tubular floret related traits were identified and validated by quantitative real-time PCR. |
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