Mechanism Of Double-flower Trait Formation In Roses Based On QTL Mapping And NGS-based BSA

Rose is important plant with high ornamental value in Rosaceae family,and it is one of the most widely used cut flowers and garden flowers in the world.There are 30,000 to 35,000 rose cultivars now,and the number of petals ranges from 5 to more than 200 petals.At present,the molecular mechanism of formation of double flower is still unclear.To analyze the molecular mechanism of double flower formation of rose,in this study,the hybrid offsprings of Rosa chinensis ‘Zhaiye Tengben Yuejihua’×R.chinensis ‘Old Blush’ were used as material,the floral traits were measured and their genetic rules were analyzed.QTL(Quantitative Trait Locus)mapping combined with NGS-based BSA method were used to finely locate the loci of petal number.The candidate genes for regulating the petal number of rose were obtained.The expression pattern analysis and gene function verification were carried out,which provided a reference for the molecular mechanism of the formation of double flower in roses.The main results are as follows:1.Analysis of inheritance of rose flower type characterisricsEight traits,including single/double flower,petal number,pistil number,stamen number,petalized stamen number,flower diameter,petal length and petal width of ‘Old Blush’,‘Zhaiye Tengben Yuejihua’and their 303 hybrid progenies were observed,and found that the number of petals was clearly separated.The number of petals was at least 5 and at most 54,and the ratio of single flower to double flower was about 3∶1.There was a phenomenon of stamen petrification.The variation coefficients ranged from16.28% to 194.77%,and the generalized heritability ranged from 34.96% to 67.19%.The most significant variation was in the number of petrified stamens,while the least was in flower diameter.Genetic models of each trait were determined by major gene + polygene genetic analysis.There was a negative correlation between number of petals and number of stamens,which proved the double flowers stems from stamen petalization in roses.2.QTL mapping of rose flower type traitsBased on the high-density genetic map of diploid roses established in the previous period,QTL analysis of 8 flower-type traits was carried out using CIM compound interval mapping method,and 100 related QTLs were detected.The QTLs with the highest LOD values for double flower,petallized stamen,stamen number and pistil number were in LG3,the QTLs with the highest LOD values for petal number were in LG4,the LOD values for flower diameter and petal width were in LG7,and the LOD values for petal lengths were in LG6.The highest LOD values for QTLs ranged from 3.24 to 12.9,and the explanatory phenotypic variation rates ranged from 3.66% to 13.8%.3.NGS-based BSA was used to accurately locate the QTLs of rose petalsIn hybrid population of Rosa chinensis ‘Zhaiye Tengben Yuejihua’ × R.chinensis ‘Old Blush’,30 single flowers and double flowers with extreme differences in the number of petals were selected to construct a single flower DNA pool and a double flower DNA pool for 50× full Genome re-sequencing.By |Δ(SNP-index)| method,the QTLs of number of petals were located between Chr3:10840001 and Chr3:15320001,and the size of the interval was 4.46 Mb.By ED method,the most significant related loci of petal number were located between Chr3:13200001 and Chr3:13940001,and the size of the interval was 738.45 kb.4.Analysis of expression patterns of candidate genes in different stages of rose flower developmentIntersection of the QTL mapping interval for the number of petals and the NGS-based BSA candidate region,combined with gene function annotation,19 genes were selected as candidate genes for expression pattern analysis.The results showed that Rchi OBHm Chr3g0467361(RGA3),Rchi OBHm Chr3g0467381(RGA2),Rchi OBHm Chr3g0467441,Rchi OBHm Chr3g0468481(Rc AP2L),Rchi OBHm Chr3g0466491(Rc AS1),Rchi OBHm Chr3g0467691(Rc WUS),Rchi OBHm Chr3g0467431(Rc WAT1)and Rchi OBHm Chr3g0467371 in double flower were significantly higher than those in single flower at each developmental stage,so the eight genes were used as candidate genes for functional verification of double petal traits of rose.5.Functional analysis of candidate genes regulating the quantitative traits of rose petalsVIGS technology in roses were constructed,were used in ‘Old Blush’.It was found that the number of petals of roses decreased significantly compared with that of the control and the empty control after Rc AP2 L,Rc AS1 and Rc WUS were silenced.The number of stamens increased with decrease of petal number,and sepal number,pistil number did not change significantly.The candidate genes were cloned and bioinformatics analysis were carried out.In summary,in our study the separation characteristics of flower type traits of rose were clarified.By using QTL mapping and NGS-based BSA,the loci of petal number were positioned in a interval of738.45 kb.By q RT-PCR and VIGS technology,three genes regulating the number of petals of rose were excavated.The results laid a foundation for elucidating the regulation mechanism of the number of petals of rose.