LTR Sequences Isolation Of Ty1-copia Group Retrotransposon And Germplasm Resources Evaluation Of Tree Peony

Paeonia suffruticosa Andrews is ligneous plants which belong to the deciduousshrub in Paeoniaceae, Paeonia L. and Sect. Mouton DC. It has an ancient origin, is oneof the primitive angiosperms which was accepted by the scientific community. And ithas high economic value. After thousands of years of natural selection and artificialintroduction and cultivation in China, peony has accumulated a wealth of geneticdiversity resources, but currently who uses the retrotransposon research on peonygermplasm resources evaluation has not been reported. This test isolated Ty1-copiagroup retrotransposon LTR sequences from peony genome for the first time and carrieson the systematic analysis, finally the test designed primers for peony SSAP markersexperiment according to the LTR sequences to evaluate the germplasm resources. Themain results were as follows:1. The nested PCR method was used to isolate the tree peony Ty1-copia groupretrotransposon LTR sequence. The test which combined the high-efficiency thermalasymmetric interlaced PCR (hiTAIL-PCR), annealing control primer (ACP) systemand suppression PCR with the degenerate RNase H nested and PPT primer, PCRproducts from the third round of PCR were cloned into the PMD-18T vector using theTA Cloning Kit (TaKaRa, Dalian, China). The ligation products were transformed intoDH5α competent cell and positive clones were sequenced by Sun-Biotech Co.(Beijing,China). A total of22independent clones were randomly selected and sequenced.20sequences possessed expected PPT primer and an UP primer. The sequence wascompared by DNAstar, and we could find that a continuous GA nucleotide motif,GGGGGAAGA, a polypurine tract, highly conserved in PPT motif, was observed in all20sequences in5region, and it was followed by the TG/TA sequence0-3bp after PPT.In3region, all the sequence existed UP primer The sequences has been deposited in GenBank, and the accession numbers are from KC519444to KC519464. Afterexclusion of repeated sequences.2. The test was based on iPBS-PCR method, amplified the objective sequencefrom northwest Plain varieties ’Red Hydrangea’ and Central Plains varieties’Luoyanghong’, after the polymorphic fragments were cloned, sequenced and analysedby DNAMAN、DNAstar and Vector NTI et al. which is the relevant bioinformaticssoftware,12LTR sequences were got from peony LTR retrotransposons. Thenucleotide sequence has the high heterogeneity, deletion mutation is the main behavior,the length of the nucleotide sequences varied from313to894bp, and homologousranged from31.1%to65.8%. Cluster Analysis was made between the amino a MCIDsequence and the listed LTR retrotransposon from different plant LTR amino a MCIDsequence. It turned out that there was a high homology between some other plants.There may be a transverse transfer between the LTR retrotransposons of them.3. Sequence-specific amplification polymorphism (SSAP) retrotransposon basedmolecular marker for germplasm identification and genetic relationship analysis of treepeony were investigated for the first time, and performance of each SSAP primer pairwas also studied in detail.8retrotransposon primers were designed based on LTRsequence which isolated from Ty1-copia group retrotransposon in tree peony in test1.After screened with7adapter primer Mse I or EcoR I which have3selective basescombinations, the test utilized12primer pairs to precede SSAP molecular marker test,A total of415robust fragments among151types tree peony genotypes which containwild species、 Peony variety abroad and the domestic varieties of peony from differentregions examined, an average of35polymorphic bands were produced per primer pair.Indicating that SSAP is a high multiplex ratio and high polymorphic marker system,furthermore it has high application value for tree peony study. After using the UPGMAcluster analysis method, varieties, producing area and flower color were analysis. Inaddition, A cultivar identification diagram (manual cultivar identification diagram,MCID) was made manually to discriminate the151tree peony cultivars using fourpolymorphic SSAP primer combinations.151tree peony varieties could bedistinguished using4times PCR at the molecular level. Compared to the clustering tree,the manual MCID could be easy to read and have more practical use. In other words,the primers for distinguishing between any two varieties could be found according tothe MCID chart. This method has a wide range of versatility in the other species. Furthermore, it will have a positive meaning in the identification of tree peonygermplasm resources and promote sustainable development of peony industry. Thestudy of SSAP molecular marker in tree peony not only developed the new method todiscuss peony germplasm resources research but also for germplasm resources researchof peony and cross breeding lays the experimental basis and theoretical basis.