Characterization Of Tyl-copia Retrotransposons From Apple Genome And Establishment Of S-SAP Molecular Markers

Retrotransposons are powerful tools for studies of gene cloning,biodiversity and phylogeny due to their ubiquitous distribution,high copy numbers,a considerable degree of sequence heterogeneity and irreversible insertion in plant kingdom.Apples are important temperate fruit and widely distributed throughout the world.In this study,based on isolating of RT sequence by degenerate primers aimed at RT conserved motifs,the different methods were explored to identify LTR sequences and complete sequences of Ty1-copia retrotransposon from apple genome,and the gene orders and conserved motifs were analyzed with bioinformitics.In addition,based on the isolated LTR sequences in 3' end,the S-SAP technique of apple was established,and further 24 Fuji bud sport clones and 11 Delicious bud sport clones were distinguished by S-SAP.The main results are as followed:Adaptor-mediated nested PCR and modified sitefinding-PCR succeeded in isolating the LTR sequences of Ty1-copia retrotransposons,while adaptor-mediated,semi-nested degenerative PCR and specific PCR were failure to isolate LTR sequences.Based on known sequences of approximate 260 bp,five 3' end LTR sequences of Ty1-copia retrotransposons were isolated using adaptor-mediated nested PCR,and one 3' end LTR sequences was characterized by modified sitefinding-PCR.Based on known sequences of approximate 260 bp,four Ty1-copia retrotransposons complete sequences,named as Mdtcr1,Mdtcr2,Mdtcr3 and Mdtcr4,were isolated by combination of Adaptor-mediated nested PCR and modified sitefinding-PCR methods.The lengths were 5147 bp,5280 bp,4428 bp and 4884 bp for Mdtcr1,Mdtcr2,Mdtcr3 and Mdtcr4 respectively.The elements presented typical structure of Ty1-copia retrotransposons in which POL gene order was PR-INT-RT-RNaseH,and the homology and phylogeny relationship between isolated retrotransposons and other Ty1-copia retrotransposons in INT,RT and RNaseH region further proved that the four elements belonged to Ty1-copia retrotransposons. They contained character motifs of different regions of Ty1-copia retrotransposons,such as C-C-H-C motif in GAG,D(T/S)G sites in PR,H-H-C-C motif in INT,motifâ… (TAFLHG), motifâ…¡(YGLKQ)and motifâ…¢(YVDDML)in RT,motifâ… [KHI(F)D(E)]and motifâ…¡[A(S)DX₂TK]in RNaseH region,characteristics sequences TGGT in PBS,conserved sequences AGGGG in PPT and LTR boundary motif 5'-TG-CA-3',etc.The four TEs presented different in integrity of ORF,size and organization of LTR sequences.Cis-element analysis showed that the 3' end LTR sequences of Mdtcr1-Mdtcr4 contained many stress-responsive elements and auxin-responsive elements,which included the single responsive elements such as gibberellins,salicylic acid,low temperature and dehydration-responsive,cold-responsive element and integrity elements to response to multiple environment challenges and auxin induces.The S-SAP molecular system was established based on the isolated 3' end LTR sequences of Ty1-copia retrotransposons.The ten primer combinations were selected from eighty primer combinations,which were high polymorphic,clear bands,even distribution and qualified for apple S-SAP analysis.Twenty four Fuji bud sport clones,and 11 Delicious bud sport clones were distinguished by S-SAP molecular marker and the results showed that there were four primer combinations(9L/Mtcc,9L/Paat,LTRP1/Mtcc and YLDLTR/Mggt)and three primer combinations(YLDLTR/Paat,9LTR/Mtcc and LTRP1/Mtcc)that were able to differentiate the Fuji bud sport clones and Delicious bud sport clones.The phylogenetic relationship among 24 Fuji bud sport clones and 11 Delicious bud sport clones base on SAHN analysis showed that Fuji bud sport clones could be grouped into five distinct families based on similarity 0.88.The first group contained 18 bud sport clones,which were Naga-fu No.1 Fuji, spur Fuji,Naga-fu No.3 Fuji,Huimin spur Fuji,Early Fuji,Iwate line 1 Fuji,Naga-fu No.2 Fuji,Naga-fu No.6 Fuji,Naga-fu No.7 Fuji,Yanfu 3 No.3 Fuji,Giant Fuji,Aki-Fu 1 Fuji, Iwa-Fu 10 Fuji,Kudo Fuji,Line 1 Fuji,Naga-fu No.4 Fuji,Changhong Fuji and Miyazaki spur Fuji.The second group contained Naga-fu No.1 Fuji and 2001 Fuji.The third group was comprised of Aki-Fu Fuji and Mori-ho-fu No.1 Fuji,and Elite spur Fuji and Fuji were as a single group respectively.Eleven Delicious bud sport clones could be grouped into four distinct families based on similarity 0.95.The first group contained Starking Delicious,Stark spur Ultra Red Delicious,Houjiadian Red Spur Delicious,Aizhuang Delicious,Xinyuanshuai Delicious,Tangshanfengchan Delicious,Wellspur Delicious,Miller's spur Delicious.New Starkspur Golden Delicious,Red Spur Delicious and Stark Spur Supreme Red Delicious were as a single group respectively.Fuji and Delicious bud sport clones were as a single group based on similarity 0.75,which showed that the developed S-SAP technology in this study could effectively distinguish cultivars and bud sport clones investigated.This indicated that the apple bud sport clones might related to Ty1-copia retrotransposons.