Genetic Diversity Analysis Of Intraspecific Hybridization Between Plumbago Auriculata And Plumbago Auriculata F.alba

Plumbago auriculata Lam.,a woody flower with extremely high ornamental and medicinal value,is introduced and cultivated worldwide.The introduction and cultivation in our country is mainly used for urban garden viewing.However,the single variety of P.auriculata restricts its commercialization development.Also,the cultivated varieties are too single to meet the needs of various climatic regions and multi-color varieties.Cross-breeding is a traditional breeding method,which can produce new phenotypes by gene recombination,and then select plants with excellent characters from offspring.At present,there are only polyploid induction of P.auriculata in the breeding field,and there is no report on hybrid breeding.Thus,the progenies were generated by the reciprocal cross between P.auriculata and P.auriculata f.alba（a variant species of P.auriculata with a white corolla）.After sowing,26 horticultural traits were tested at flowering stage to analysis the genetic variation of hybridization.At the same time,the ISSR-PCR and SRAP-PCR reaction system of P.auriculata were established and optimized,and the ISSR and SRAP primers were selected to carry out molecular labeling of hybridization,and the genetic diversity of progenies was analyzed on the basis of molecular markers.The results can judge whether the hybrids has the value of breeding,and lay an important foundation for the breeding of new varieties of P.auriculata with abundant germplasm resources.The results are as follows:（1）The variation of 19 quantitative characters of hybridization was tested.The results showed that the coefficient of variation of the quantitative characters of flowers was between 5.95%and 61.22%,the coefficient of variation of the beginning of the flowers was the largest,and the coefficient of variation of the sepal length was the smallest.The variation coefficient of leaf character was between 9.26%and 15.92%,and the coefficient of variation of the blade length was the largest,and the leaf shape index was the smallest,and the leaf shape index showed that the shape of the F₁generation blade was stable and the shape of the blade was oval.In that quantitative trait,the effective inflorescences of the plant,the length of the total flower stem,the number of branches,the coefficient of variation of the degree of dispersion of the upper part of the plant are more than 30%,which means those traits have breeding potential and value.（2）The distribution proportion of seven qualitative traits was calculated.The results showed that the"flat"pattern was the main pattern in the apex shape of F₁ petals,and the proportion of"convex tip"with higher ornamental value was 9.41%in F₁generation,but one plant of the same length of pistil and stamen appeared.The flower color of hybrid progenies was in the middle tone of their parents.The shape of leaf apex was mainly"acute tip",while the proportion of"semi-circular"and"oblong"of the two phenotypes of bracket was basically 1:1,and the ratio of"divergence"and"extreme divergence"in the upper part of the plant was about the same as that of the parents,indicating that the branches of F₁ generation were not upright,and the whole plant type was mainly scattered,which was consistent with that of their parents.（3）The Pearson correlation coefficients among phenotypic traits were calculated by SPSS 22.0.The results showed that there was a significant positive correlation between quantitative traits in flowers and leaves.The number of effective inflorescences was negatively correlated with the initial flowering stage and positively correlated with the plant width,which indicated that the earlier the flowering,the greater the number of effective inflorescences.There was a significant positive correlation between leaf length and leaf width and single flower length,indicating that the larger the leaf was,the larger the petal diameter was.The degree of divergence at the upper end of the plant was negatively correlated with the number of branches,which meant that the more branches there were,the more compact the plant type was and the more upright it grew.A total of seven principal components were extracted by principal component analysis,which explained 65.171%of F₁ generation variation.Flower traits and leaf traits were the main factors causing F₁ representative type differences.（4）The ISSR-PCR and SRAP-PCR reaction system were established and optimized by orthogonal test and single factor test.The results showed that the primary factor that affected the ISSR-PCR reaction was Mg²⁺,the established and optimized 25μL ISSR-PCR reaction system was Taq polymerase 1 U,template DNA 100 ng,d NTPs0.15 mmol/L,primer 0.4μmol/L,Mg²⁺0.5 mmol/L.The primary factor that affected the SRAP-PCR reaction was the primer,and the 25μL SRAP-PCR reaction system established and optimized was Taq polymerase 1.25 U,template DNA 100 ng,d NTPs0.15 mmol/L,primer 1.0μmol/L,Mg²⁺2.5 mmol/L.（5）In this study,8 ISSR primers and 7 pairs of SRAP primers were screened.A total of 61 loci were amplified by 15 primers in F₁ generation,of which 58 loci were polymorphic,with an average of 3.86 loci per primer,and the proportion of polymorphism loci was 95.08%.The results showed that the selected primers had high ability to reveal the polymorphism of F₁ generation.（6）The selected primers were used to mark F₁generation.The results showed that the genetic diversity of progenies produced by combination W_LB_S was the highest and that of B_SW_L was the lowest,that is,when P.auriculata f.alba was used as female parent,the genetic diversity of hybrid progenies was higher than that of progenies when P.auriculata was used as female parent.The results of AMOVA analysis showed that the variation of 85 F₁generations mainly came from within the population.Genetic similarity analysis showed that the genetic similarity of B_SW_L combination and B_LW_Scombination was the lowest,while that of W_LB_S and W_SB_L was the highest.In conclusion,the offspring produced by P.auriculata and P.auriculata f.alba have high genetic diversity at both phenotypic and molecular levels.These results imply that we obtained a group of genetically different progenies through intraspecific hybridization of P.auriculata,which enriched the resource bank for breeding varieties of P.auriculata.