| Retrotransposons which are ubiquitous and abundant in plant genomes mightcontribute to agronomic variation (maturity date, flesh color) by increasing allelicdiversity or by changing the regulation of gene expression. Somatic mutation, a mainbiological phenomenon of citrus and other fruits, provide the useful materials not only forthe formation of bud sport but also for retrotransposon activity. The present researchisolated RT (reverse transcriptase) sequences from copia-like retrotransposons for theinvestigation of their heterogeneity, activity and genomic distribution in citrus; undertissue-culture and colchicine-treated conditions, the citrus callus were used to study themechanism about DNA methylation of pol domain structure from gypsy-likeretrotransposons. With the experimental procedures modified and optimized, three novelmarker methods of IRAP, REMAP, SSAP, were developed for independent innovation inCitrus. The main results were as follows:1. RT (reverse transcriptase) sequences from copia-like retrotransposons were clonedand charazterized. Results showed:1) Copia-like sequences were amplified from three citrus genomic DNA (Cara Calanavel, shatian pummelo and Murcott tangor) by PCR with degenerateoligonucleotide primers corresponding to highly conserved RT domains in thecopia-like retrotransposons. PCR fragments of roughly 240bp were isolated and70 cloned were sequenced, 40 different RT sequences were obtained. Thenucleotide sequence similarity shown by computer analysis between inter- andintra-group was in a wide range, which indicates high level of sequenceheterogeneity among these clones and groups. Among these sequences, 16 cloneshad unique nucleotide sequences (intact sequences), and the others had aframeshift, a stop codon, or both. According to alignment and phylogeneticanalysis, all sequences were divided into seven groups.2) Southern analysis revealed the distribution of RT sequence (Too10) in differentcitrus genomes. Methylation analysis showed there was high level of methylationin citrus genomes.3) The total copy number of the copia-like retrotransposons in citrus species genomewas estimated by dot-blot hybridization. The results showed the copia-like retrotransposons (Tcc10) covered nearly 17% of citrus genome, and its copynumber in Cgrandis (cv. Shatian pummel) lower than in Creticulata (cv.Bendiguangju mandarin) and C. sinensis (cv. Cara Cara navels orange).2. A potential site, closely related with gypsy-like retrotransposons (Designated asMckre) was obtained by RAPD analysis from the tetraploid callus of Murcott tangorunder colchicines treatment. Results showed both Mckre had an uninterrupted openreading frame (ORF) encoding 232 amino acids which contained conservedretrotransposon pol motifs with the invariant DD motif, the highly conserved SKCEF ofthe reverse transcriptase active site block and approximately 40 residues in itsdownstream. Southern blot revealed that Mckre was of multiply copy. DNAdemethylation was observed in the callus that had been cultured for three mouths andtreated with 0.1% colchicine for 48 h and 72 h, by Mspâ… /Hpaâ…¡digestions and Southernblot. And the altered methylation patterns were related with gypsy-like retrotransposons(Mckre), nonetheless, none of copia-like retrotransposons (Tcc10) and pigment gene(Lcyb) showed apparent methylation activation.3. Seven IRAP primers were designed according to LTR sequences of copia-like andgypsy-like retrotransposons in citrus. The IRAP and REMAP band patterns between thegenotypes exhibited high level of polymorphism. In total, combined IRAP and REMAPanalysis, 196 scorable amplification products ranging from 200 bp to 2000 bp weregenerated, 147 of which were polymorphic, with an average of 5 polymorphic bands perprimer combination. The level of polymorphism was 75 %. Tweenty-four representativeaccessions of Citrus, Fortunella, Poncirus were selected, and amplified by the IRAP andREMAP primers. Loci among the 24 genotypes ranged from 2 to 8 with an average of3.54, and the mean PIC value was 0.537. Pairwise comparison was conducted between allthe genotypes included in this study. Jaccard's similarity coefficients calculated fromIRAP and REMAP data varied from 0.19 between Bendiguangju mandarin and 'HB'pummelo, to 0.99 between 'Washington' navel orange and 'Cara Cara' navel orange, witha mean of 0.59. Neighbor-joining cluster results based upon the IRAP and REMAP datashowed that all the samples were divided into 7 clusters, namely orange-mandarin (C.sinensis-C.reticulata), Calamonidn (C. reticulata), pummelo (C. grandiss), citron-lime (C.medica-C.aurantifolia), ichang papeda (C.ichangensis), kumquat (Fortunella spp), andtrifoliate orange (Poncirus spp) clusters.4. RAPD, SSR, AFLP, SSAP and ISTR analyses were used to detect the variationsamong 14 bud mutant cultivars from navel orange, sweet orange and mandarin. No polymorphism was found by AFLP, SSR and ISTR. RAPD marker was ineffective, out of50 primers, only one revealed polymorphisms, showing one missing band in 'Cara Cara'navel orange. In SSAP, fragments were amplified using 91 pairs of LTR/MseI selectiveprimers, and 9 primers showed DNA polymorphisms. These results also showed SSAPtechnique was an effective method for identification of citrus bud mutant cultivars. Tenspecific bands derived from SSAP analysis were sequenced. Sequence analysis showedthat those amino acids were identified with corresponding genes involved mainly insignal transmission, anti-adversity and ribosome protein, and might be closely relatedwith citrus fruit ripening.
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