Construction Of High-Density Genetic Map And QTL Analysis For Important Agronomic Traits Of Chinese Pink

Dianthus Chinensis（Chinese pink）is one of perennial ornamentals which belongs to Caryophyllaceae.Due to its important ornamental value and resistance to cold and drought,it has been widely used in gardens,park and landscape with large area of land.At present,few researches of molecular biology on D.Chinensis has been reported.It is necessary to develop molecular biology,explore the regulation pathway of important characters and turn traditional breeding to molecular breeding.In this research,We used the double-digest restriction-site-associated DNA sequencing（ddRAD-seq）to identify SNP,and have constructed a high-density genetic map using 140 F₂progenies derived from a cross between‘MH’（single flower）and‘X4’（double flower）parental genotypes.Quantitative trait loci（QTL）mapping analysis was conducted for 12 horticultural traits.Subsequently,the BSR-Seq method was used to excavate the SNP closely linked to candidate genes by using in 30 single and 30 double flower plant pools of F₂generation.The SNP linked to double flower trait,genome of carnation and QTL analysis were used to search candidate genes,and then the candidate genes were cloned and analyzed.Finally,carnation’s genome was used to screen candidate genes for other important agronomic traits.The research are showed as following points:1.Self-crossing 10-generation‘MH’with single flower and self-crossing 10-generation‘X4’with double flower were selected as parents and performed cross.F₁ generation were all presenting intermediate character which indicate that flower type was incomplete dominance in D.Chinensis.The 400 F₂ population came from F₁ self-cross,and 140 of them were randomly selected for constructing genetic map.2.ddRAD-seq was used for sequencing,a total of 381,804,896 reads,about 57.27 Gb raw data were obtained from parents and 140 F₂ progenies.59,841 high quality SNPs were detected.By screening and filtering,2,353 SNPs were selected for constructing genetic map.The first high-density genetic linkage map of D.Chinensis was constructed by Joinmap 4.1 which comprised 15 linkage groups and 2,353 SNPs.The total length of the genetic map was 967.54 cM with an average marker distance of 0.41 cM.The genetic length of LGs ranged from 35.29 cM（LG2）to 95.24 cM（LG11）.3.The statistical analysis for 12 agronomic traits of mapping population was performed.A total of 31 QTLs were detected for 9 phenotype characters.Proportion of phenotypic variance explain（PVE）for these QTLs was varied from 13%to 78.1%.Among these QTLs,qDFT-2 had the highest proportion of PVE with 78.1%.For all three of the color-related traits,the highest LOD scores for associated QTLs were mapped to the same position which corresponded to the 64.86 cM region at the end of LG3.4.In order to accurately screen the candidate genes controlled double flower trait in D.Chinensis.Firstly,we performed scanning electron microscope analysis for flower-bud of two parents.And then,BSR-Seq sequencing analysis was carried out for two parents and two pools（30 single flower plant and 30 double flower plants）in F₂ generation,27.5 G sequencing data were obtained.A total of 126 candidate SNPs were identified to associate with the double flower trait.Through a combination analysis of QTL mapping,BSR-Seq analysis and the reference genome information,we anchored the region control double flower trait and found that the region located at about 2 cM near peak area in the qDFT-2.A total of 327 genes were located in this region,we selected a most likely candidate gene DcAP2L.The full length of DcAP2L was cloned from the total DNA of two parents,single flower pool and double flower pool in F₂ generation.The full length of DcAP2L in single parental and double parental was 3,594 bp and 3,612 bp,respectively.There were21 differences between the two sequences,the double flower parent had a variation of one base at the miR172 binding site.In addition,the full-length sequence of DcAP2L of single flower pool and double flower pool in F₂generation was analyzed to determine the variation of this base at the miR172 binding site was exactly existed in single flower and double flower of D.Chinensis.5.For three of the color-related traits,searching candidate genes were performed together because of their high correlation.For other agronomic traits,searching candidate genes were performed by using genome information of carnation.Candidate genes were screened at 2 cM near the peak location of QTL region.For color related traits,a P450monooxygenase gene CYP90A1 was found to be a possible candidate gene.A candidate gene encodes TRANSDUCIN/WD40-2 protein may be related to stamen number trait,One candidate gene FUS3 may be related to plant height trait,and we screened no candidate genes which could regulate plant width trait,HY5 may be related to the main stem diameter trait,and two genes PLE and TSO1 may be related to the branch number character.In conclusion,the genetic map and QTLs for agronomic trait will serve as a foundation for genomic structure and marker-assisted selection breeding.The candidate genes for double flower trait,stamen number,color-related trait,stem diameter,plant height and branch number character will lay an important foundation for the analysis of genetic mechanism of important horticultural traits,and will facilitate the development of breeding in Dianthus spp..