Isolation And Characterization Of LTR Retrotransposon Sequences From Strawberry Genome

Strawberry(Fragaria spp.)is a kind of rosaceous herbaceous plant that propagates vegetatively. During the past years,strawberry production had been developed fast.Strawberry micro-propagation technique had been applied widely on the strawberry industry.The phenomenon of somaclone variation had been discovered in tissue culture plants,when they were transplanted in the filed.One of the important facts of somaoclone is the retrotransposon's transposition activity.The major research of this article:The conserved domains of reverse transcriptase(RT)genes of Ty1-copia group retrotransposons were isolated from the octoploid cultivated strawberry(Fragaria ananassa Duch),the diploids wild strawberry(Fragaria vesca Duch,Fragaria viridis Duch,Fragaria madscurica Staudt)and the tetraploids wild strawberry (Fragaria orientalis Lozinsk);Phylogenetic analysis of RT sequences of Ty1-copia groups retrotransposons from different ploidy strawberries was made;The conserved domains of reverse transcriptase RT genes of Ty3-gypsy group retrotransposons were isolated from the octoploid cultivated strawberry(Fragaria ananassa);The copy number and the transcription activity of Ty1-copia and Ty3-gypsy retrotransposons were detected.1.Nineteen unique RT gene sequences of Ty1-copia group retrotransposons were isolated from the octoploid cultivated strawberry(F.ananassa)using degenerate PCR amplification.The sequences were similar with the RT gene sequences of Ty1-copia group retrotransposons in other species which have been submitted on NCBI.The average amino acid sequence heterogeneous ranged from 1 to 70%.Among the 19 unique sequences,5 contained premature stop codons and/or frame shift,while the remaining 14 sequences had potentially function.These 19 Ty1-copia group RT sequences were designated as 7 groups by using phylogenetic analysis.The sequences with stop codons fell in the same clade of group 1;34 unique RT gene sequences of Ty1-copia group retrotransposons were isolated from the diploids wild strawberry(F. vesca,F.viridis,F.madscurica).The sequences were similar with the RT gene sequences of Ty1-copia group retrotransposons in other species which have been submitted on NCBI.The average amino acid sequence heterogeneous between individual sequences ranged from 2.3 to 55.06%.Among the 34 unique sequences,12 contained premature stop codons,while the remaining 22 sequences had potentially function. These 34 Ty1-copia group RT sequences were designated as 7 groups by using phylogenetic analysis.The sequences with stop codons fell in the same clade of group 7 unique RT gene sequences of Ty1-copia group retrotransposons were isolated from the tetraploid wild strawberry(F.orientalis).The sequences were similar with the RT gene sequences of Ty1-copia group retrotransposons of other species which have been submitted on NCBI.The average amino acid sequence heterogeneous between individual sequences ranged from 1.12 to 55.06%.Among the 13 unique sequences,2 contained premature stop codons,while the remaining 11 sequences had potentially function.These 13 RT sequences of Ty1-copia group were designated as 4 groups by using phylogenetic analysis.The sequences with stop codons fell in the same clade of group 4.2.Ten unique RT gene sequences of Ty3-gypsy group retrotransposons were isolated from the octoploid cultivated strawberry(F.ananassa)using degenerate PCR amplification.The sequences were similar with the RT gene sequences of Ty3-gypsy group retrotransposons in other species which have been submitted on NCBI.The average amino acid sequence heterogeneous between individual sequences ranged from 1 to 30%.Among the 10 unique sequences,2 contained premature stop codons and 2 sequences were not completed,while the remaining 6 sequences had potentially function.These 10 RT sequences of Ty3-gypsy group from octoploid cultivated strawberry(F.ananassa)were designated as 2 groups using phylogenetic tree analysis.3.The phylogenetic analysis of Ty1-copia group retrotransposons RT gene sequences,which from octoploid cultivated strawberry(F.ananassa),diploids wild strawberry(F.vesca,F.viridis,F.madscurica) and tetraploids wild strawberry(F.orientalis),was made.The kindred of F.viridi and F.orientalis were more closed than the others,wd3-1,which came from F.vesca,was the most eldest RT gene sequences of Ty1-copia group retrotransposons from strawberry.4.Southeron dot blotting hybridization analysis was used to estimate the copy numbers of Ty1-copia and Ty3-gypsy gruops retrotransposons in F.ananassa genome.The copy number of Ty1-copia and Ty3-gypsy group retrotransposons was about 2,875 molecules per genome and about 348 molecules per genome,respectively.Both types of LTR retrotransposons contributed to 15.8%in the F.ananassa genome and the proportion of Ty1-copia retrotransposons was higher(13.5%)than that of Ty3-gypsy retrotransposons(2.3%).5.RT-PCR technique was used to detect the transcription activity of both Ty1-copia and Ty3-gypsy group retrotransposons,in the octoploid cultivated strawberry.The results shew that both of the filed plants and the tissue culture plants haven't detected transcription activity sequences of any LTR retrotransposon groups.The transcription activity sequences of Ty1-copia group were found in filed plants,which were treated with 2 mM salicylic acid,50 mM methl jamonate,50 mM ABA and 50 mM 2,4-D for 16 h,and the callus which was induced by two different regeneration culture medium.One was MS supplemented with 6-BA 0.5mg/L,GA₃ 0.5 mg/L,TDZ 2.0 mg/L and IBA 0.02 mg/L,the other was MS supplemented with 6-BA 0.5mg/L,GA₃ 0.5 mg/L,TDZ 1.0 mg/L and 2,4-D 0.1 mg/L.6.ys11-11,ys11-27 and xp4-2 were different from the other19 unique transcription activity RT gene sequences of Ty1-copia group retrotransposons.Average amino acid sequence heterogeneous of the remaining 19 sequences ranged from 1 to 6.51%,which was lower than the sequences which isolated from strawberry genome DNA.