The Molecular Mechanism Of The Retrotransposon Atr1Inducing Spur Mutation In Applt Delicious Clones

Partial sequences of Ty1-copial-like retrotransposon atr1in apple genome, whichcould be activated under stress conditions, were isolated using the long terminal repeatsequence in atr1(atr1-LTRs) by IPCR technique. The special fragments were isolated inDelicious clones spur sports by IRAP-PCR, and The partial flanking sequences of whichwere amplified in Delicious clones spur sports. The alignment result indicated that it issame for the special fragments in the four spur sports. The research laid the foundation onthe mechanism that retrotransponsons induced the mutation of spur type.1. The isolation of partial flanking sequences of retrotransponsons atr1-LTRs. Theflanking sequences of atr1-LTRs were isolated using the primers which were designedbased on a500bp fragment of atr1-LTRs by IPCR technique. After enzyme digestionadequately by restriction enzymes HindⅢ in ‘Meiguihong’ genome DNA, self-looped andligated were made with T4ligase, then the reaction products were used as the templates forPCR. A1896bp fragment was amplified after two circles of nested PCR. Finally, a2124bpfragment was obtained and cloned from ‘Meiguihong’ genome DNA using the primersdesigned based on the1896bp fragment and atr1-LTRs. The sequences analysis indicated itcontained “the partial fragment of reverse transcriptase(RT)、RNsesH、the partial of3’-LTRand its flanking sequences”.2. The special fragments of the Delicious clones spur sports were isolated accordingto atr1-LTRs. The ‘Delicious’ and its spur sports ‘Huashuai1’,‘Aihong’,‘Oregon SpurDelicious’ and ‘Meiguihong’ genome DNA were used as templates for PCR. The specialfragments was amplified by optimal IRAP-PCR using the primers designed according toatr1-LTRs. The sequencing results showed that two special fragments named Hs1and Hs1’with the size of809bp and758bp were obtained in ‘Huashuai1’, and other specialfragments named Ah1、Alg1and Mgh1with the size of809bp were obtained in“Aihong”、‘Oregon Spur Delicious’、“Meiguihong”. The long primers designed based on the specialfragments were utilized in PCR technique using the templates of ‘Delicious’ and its spursports genome DNA. The IRAP marker was successfully converted into SCAR marker.3. Improved TAIL-PCR technique was used to amplify the flanking sequences of thespecial fragments isolated in Delicious clones spur sports. It utilized three nested specialprimers designed in the end region of Mgh1with shorter arbitrary degenerated primerrespectively. The sequencing results were obtained after three circles of optimal PCR. The special fragments Hs2、Ah2、Alg2and Mgh2were amplified using the primers designedbased on the sequencing results and Mgh1in Delicious clones spur sports. Then threenested special primers designed in the end region of Hs2with shorter arbitrary degeneratedprimer were boned respectively. The sequencing results were obtained after three circles ofPCR. The special fragments Hs3、Ah3、Alg3and Mgh3were amplified using the primersdesigned based on the sequencing results and Hs2in Delicious clones spur sports.