Genetic Variation Analysis Of Mutagenic Grafted Plum Lines Based On SRAP And IRAP Markers

There is a long cultivation history of plum in China,and it has become an important stone and deciduous fruit tree all over the world.But genetic resources of plum germplasm are increasingly dried up and some genes controlling excellent characters are becoming more and more poor with the in-depth development of breeding.An old 'Jidan' plum tree that possesses a series of excellent characters such as extra-large fruit?succulent crisp and strong resistance were found in Shimian,Sichuan when we investigated fruit germplasm resources in recent years.Although its fruit setting rete is low and the fruit quality is not high enough,it is urgent and necessary to make full use of its germplasm resources and create rich variation to promote genetic improvement and germplasm innovation through effective breeding methods.Sport selection is an important source of breeding new varieties of fruit trees,but due to accidental mutation and low mutation rate,mutation breeding process of fruit germplasm is relatively slow.As the most extensive and effective chemical mutagens,EMS not only can improve the cell mutation rate greatly but also modify a particular trait of germplasm easily.Therefore,the combination of EM S mutation breeding,sport selection and molecular identification were used in this study,in which the branches of an old 'Jidan,plum tree(about 50 years old)were used as experimental materials,the mutant population were produced by single bud grafting after EMS treatment,setting the grafted seedlings without scion mutagenic treatment as control at the same time,the phenotypic characters of the mutant population were observed,and the genetic variation and identification were analyzed by SRAP and IRAP molecular markers.The main results of the study are as follows:1.The single bud grafting was used to creat the mutant population after plum branches were mutagenized with 0.5%EMS for 30 min.Then the variations of biological characteristics such as plant height?branch number?leaf color and shape were found,including dwarfing,more branches,red stems,irregular leaf shapes,softening leaves and branches,leaf curl and shrinkage,etiolated plants,the total mutation rate was 37.9%.2.15 SRAP primers were screened out from 224 pairs of primers for PCR amplification and genetic variation analysis of the 227 grafting strains whose scions were mutagenized with EMS and 1 grafting strain without mutagenic treatment(CK).A total of 65 samples were detected with different bands,from which 136 bands were amplified,the polymorphic bands were 74,the polymorphism rate was about 54.4%and the genetic similarity coefficients were between 0.691-1,among which 47 variants had minor variation and a close genetic relationship with CK while the other 18 materials were clustered into one class,and the genetic composition of the population was same or similar,but the genetic relationship was far from CK,having a larger variation.3.12 IRAP primers were designed for PCR amplification and genetic variation analysis of the CK and 227 grafting strains.The polymorphism was amplificated in 5 primers,a total of 31 samples were detected with different bands,from which 39 bands were amplified,12 bands were polymorphic,the polymorphism rate was about 30.8%and the genetic similarity coefficients were between 0.769-1,among which 13 variants(11 variants were detected by SRAP)had minor variation and a close genetic relationship with CK while the other 18 materials(all variants were detected by SRAP,having a larger variation from CK)were clustered into one class,couldn't be distinguished by the used primers,and the genetic composition of the population was same,but the genetic relationship was far from CK,having a larger variation.4.Different amplification regions of different molecular markers and different markers led to the differences of amplification and identification results,however,the cluster results based on PCR amplification and primer discemibility of SRAP and IRAP were consistent.The results of phenotypic variation and molecular identification showeed that,there were many mutation sites and a number of variation types in the plum germplasm,most of the variant trains had a small difference and a high genetic similarity between each other while 18 trains were the same or similar variation types,having a large variation at DNA level.The correspondence between phenotypic variation and DNA molecular variation needs further study.5.Although the IRAP primers used in this study were less than SRAP,the overall polymorphism and average amplified variation sites and identified variant materials by per primer of SRAP were higher,the amplification and analysis results of the two molecular markers showed that SRAP was more suitable for mutation identification and genetic analysis in plum than IRAP.Polymorphism of IRAP illustrated that retrotransposons may be involved in the formation of plum variation,and the addition or deletion of differential bands indicated that transposon insertion and retrotransposons recombination was one of the important mechanisms of plum variation.