Development Of Simple Sequence Repeat(SSR) Markers From Functional Genes And Establishment Of Molecular Identification Card For Tree Peony

Tree peony(Paeonia suffruticosa Andr.),is native to China which has a long history of cultivation and abundant germplasm resources.It is widely cultivated all over the world.As a "plant available" plant,the breeding objectives have become more specific and diverse.Therefore,it is particularly important to use tree peony germplasm resources to breed new varieties of tree peony suitable for modern market demand and to improve the efficiency of peony breeding.Based on important functional gene sequence of tree peony,the development of DNA molecular markers and the construction of DNA molecular identity cards have important guiding significance for the utilization,protection and identification of tree peony germplasm resources.SSR markers are considered to be ideal molecular markers in molecular marker-assisted breeding because of their high polymorphism,co-dominant inheritance,simple operation,reproducibility and stability.Therefore,based on the SSR marker,this study developed molecular markers of peony functional genes and constructed molecular identifiers for190 tree peony varieties.The main findings are as follows:1.The 42 functional gene sequences that have been developed by the predecessors were analyzed,and 132 SSR loci were found from 35 cDNA sequences.Among them,the trinucleotide repeat is the most abundant type,totaling 58,accounting for 43.9% of the total number of SSR markers,followed by hexa-,di-,hepta-,penta,octa and tetra-nucleotide repeats.Through primer design,121 pairs of SSR primers were synthesized,among which 49 pairs of primers(40.5%)were successfully amplified,and 8pairs of primers(16.3%)were polymorphic.The polymorphism of 8 pairs of polymorphic SSR primers among 16 peony cultivars and their versatility among 8 peony samples were detected.The polymorphism information ranged from 0.26 to 0.85 with a mean value of 0.54.The versatility test found that 8 pairs of markers can be effectively amplified and the amplification rate reaches 100%.The results showed that the newly developed functional gene SSR molecular markers provided important genetic resources for the development of peony and related species breeding.2.Using the eight pairs of polymorphic SSR primers successfully developed in this study,combined with the 12 pairs of highly polymorphic SSR primer primers developed by the predecessors,the molecular identification of 190 peony varieties was constructed by capillary electrophoresis.The number of alleles of 20 pairs of primers in 190 peony varieties ranged from 2-13,with an average of 7.3;The number of bands is in the range of 3-36,with an average of 16.9 bands per pair of primers.Twenty pairs of primers were arranged in ascending order according to the band type,and each sample was coded by numbers or letters and serially connected into a DNA molecular identity card.The results showed that 20 pairs of primers constituted DNA molecular identification cards to completely distinguish 190 peony samples.53 peony cultivars have specific alleles.Six pairs of primers were found with the least primers to distinguish the most diverse quality(PS193+PSC3+PS273+PSC4+PS033+PS095,PS193+PSC3+PS273+PSC4+PS095+PS033 or PS193+PSC3+PS273+PSC4+PS095+PS158).The 190 peony varieties can be completely distinguished,providing a fast and effective means for identifying and protecting peony germplasm resources.