Studies On LTR Retrotranspon And Its IRAP, REMAP Molecular Markers In Narcissus Tazetta L.var. Chinensis Roem

Chinese narcissus (Narcissus tazetta L.var. chinensis Roem) is a kind of triploidplant. The genome is about15-22.5G. The plant blooms but has no seeds; it onlyreproduces by vegetative propagation. Chinese narcissus has almost1,000yearscultural history, but the varieties are not rich and the flower colors are not abundant.The development of new varieties can only be found from individual variationin cultivated plants; but the formation of new species caused by tiny gene mutations isdifficult to distinguish by traditional molecular marker techniques. In this paper, thefirst BAC library of Chinese narcissus was constructed and partial BAC ends weresequenced. The primers of conserved region for narcissus reverse transcriptase weredeveloped depending on the end sequences information which was obtained; the geneof narcissus retrotransposons reverse transcriptase was cloned as well. The IRAP,REMAP markers of narcissus retrotransposons were developed and some narcissusvarieties were identified by these markers. The main results are as follows:1. Construction of Chinese Narcissus BAC libraryThe extraction method of Chinese narcissus high molecular weight DNA wasestablished; and a partial Chinese narcissus BAC library was constructed. There are69,120clones in this library; and the average insert size was about87kb. No emptyclone was found. Southern hybridization results showed that there was no chloroplastDNA contamination.2. Research of Narcissus retrotransposons RT geneThe conserved region primers of narcissus reverse transcriptase were developed;and the43independent retrotransposons RT gene sequences were isolated fromChinese 'Zhangzhou narcissus',' Nanri Island narcissus',' Ziva ', and ' Tahiti' varieties.Blast results showed that the43amino acid sequence homologous rates were between64%and95%. Nineteen of these gene sequences have continuous ORF that showedthey should have potential transcriptional activity. Neighbor-Jioning phylogenetic treewas obtained and43amino acid sequences were divided into7groups based on theanalysis by MEGA5. The RT sequences in Family4were all from the ploidy species.Only2RT sequences were in Family5and were all from 'Tahiti' and ' Ziva 'variety.Family2, Family3and Family5groups can not be translated into amino acidssequence, there is no transcriptional activity;5sequences of Family7have continuoustranslation and could have the potential transcriptional activity of function.3. Research and development of Narcissus retrotransposons IRAP and REMAPmarkersAccording to the sequences of narcissus retrotransposons3'-LTR,11specificIRAP positive and negative primers were designed by Primer Premier5.0. Single-primed PCR was done using11primers. Good polymorphisms was found inPCR with primer IRAP4, IRAP7, IRAP8, IRAP9, and IRAP11.16SSR primers weredeveloped from3′-LTR sequence using3bases repeat set by GRAMENE Ssrtool.176primer combinations were formed by mixing11IRAP primers and16SSR primersrespectively. Good polymorphisms was found by PCR with randomly selectedprimers.4. Application of IRAP and REMAP markers of narcissus reverse transcriptiontransposonSix varieties of Chinese narcissus were selected to distinguish including ChineseZhangzhou narcissus single flowered form ' Jinzhan Yintai ', Zhangzhou narcissusdouble flowered form' Yu Linglong ', wild type ' Nanri Island narcissus','PingTannarcissus 'and the new breeding varieties' Golden Triangle ',' Yunxiang narcissus'.The results of RAPD and ISSR PCR were compared with the results of PCR usingprimers with IRAP or REMAP. The results showed that it can not be distinguished the5varieties except ' Yunxiang narcissus' by using RAPD and ISSR PCR, but PCRusing IRAP primers or REMAP primers can distinguish all the6varieties very clearly.