| Ty1-copia retrotransposon are mobile genetic elements,which transpose replicativelythrough RNA intermediates.They are ubiquitous, present in high copy numbers as highlyheterogeneous populations and show insertional polymorphism both with in and betweenspecies in plants and generate stable mutations. In order to use these characteristics, molecularmarkers based on retrotransposon were frequently used to study genetic diversity, toestablish phylogenies, to generate genetic linkage maps and to identify markers linked toagronomically importantly genes because of covering whole genomes and highlier level ofpolymorphism.In these paper,we studied the characteristics of Ty1-copia retrotransposon ingenome of ‘Zaosu’pear and its mutant and compared the differences between them to discusswhether transpose of Ty1-copia retrotransposon induced mutant,which was significantlyuseful for breeding.The main results as follows:1. Using degenerate oligonucleotide primers corresponding to the conserved domains of theRT of Ty1-copia retrotransposon, a polymerase chain reaction（PCR）system was establishedand modified to amplify it. Twenty-nine fragments of260bp approximately in size wereamplified from genomic DNA of ‘Zaosu’pear, and thirty-one from its red mutant. Comparedwith ‘Zaosu’pear, the amino acid sequences of RT in red skin mutant displayed much moremutations,including termination, deletion and substitution accounted for the heterogeneity ofRT sequences. In dendogram analysis,the sequence of red mutant from the forth family had acloser relationship with Malus x domestica and Prunus mume,suggesting they may have thesame origin.2. We successfully amplified RT sequences of260bp approximately in size from10different species in pear, meaning Ty1-copia group retrotransposon spread extensively.Therewere no insertion in these sequences results and neither‘Zaosu’nor its red mutant detectedtranscriptional activities of Ty1-copia retrotransposon,even if ‘Zaosu’propagated on MS solidmedium supplemented with1.0mg/L6-BA and0.3mg/L IBA for three month.3. Real-time PCR was creatively used to estimate the copy number of Ty1-copia groupretrotransposon in ‘Zaosu’and its red mutant. There were more copies of Ty1-copia groupretrotransposon in red mutant than ‘Zaosu’. 4. According to the homologous sequences,9363bp and9632bp fully length were obtainedfrom‘Zaosu’,which named PbTYZS1and PbTYZS2,respectively. Another sequence9652bp insize from red mutant,named as PbTYRM1.DNA fragment Cluster analysis of these sequencesshowed a great heterogeneity among each domain isolated from the same genotype,whichwere totally belong to Ty1-copia group retrotransposon. The average amino acid sequenceheterogeneous between pears and other individual species were ranged from27%to40%. Thelength of LTR sequences of PbTYZS1, PbTYZS2and PbTYRM1were1801bp-1815bp,noneof which had the same sequence with the similarity of93.79%~94.39%.Cis-element analysisshowed that LTR sequences of PbTYZS1, PbTYZS2and PbTYRM1contained manystress-responsive elements and auxin-responsive elements, which included the singleresponsive elements such as gibberellins, salicylic acid, low temperature anddehydration-responsive, cold-responsive element and integrity elements to response tomultiple environment challenges and auxin induces.5. Based on the sequence of PbTYZS1,we established and optimized IRAP molecularmarker and developed5pairs of IRAP marker primers all of which revealed multiplepolymorphism.‘Zaosu’and its two kinds of mutants, red mutant and extremely precocious mutantwere distinguished by IRAP molecular marker based on5primers.There were totally286polymorphic,clear bands,in which29bands revealed polymorphism,and could differentiate budmutants from ‘Zaosu’.The polymorphism of each marker primer was10.20%,12.90%,7.35%,14.58%,11.11%.After analysis and calculation,the average of geneticdistance between ‘Zaosu’and red mutant was0.104,and0.115with extremely precociousmutant.The polymorphism between species was18.89%and20.41%. |
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