Isolation Of Thinopyrum Ponticum Genome Specific Repetitive Sequences For Effective Detection Of Alien Segments In Wheat

Thinopyrum genus as a tertiary gene pool of wheat,has many useful traits,such as tolerance to both abiotic and biotic stresses.DNA repetitive sequence is one major component for genomes of most eukaryotes.Isolation of genome specific repeats is very helpful for understanding sub-genome component of polyploid species,their evolution and utilization in crop improvement.In this thesis,Th.ponticum genome specific repetitive sequences were isolated by screening Th.ponticum plasmid library via dot-blot hybridization,assisted by fluorescence in situ hybridization(FISH)analysis.A set of PCR primers was designed for detecting Th.ponticum chromosomal fragments.Major results and conclusions were summarized below.1.Isolation of Th.ponticum genome specific repetitive sequences.Nineteen specific repeats were obtained after screening Th.ponticum plasmid library via dot-blot hybridization,using Th.ponticum genomic DNA as probe and CS genomic DNA as block.2.Seven Th.ponticum genome specific repeats were obtained.FISH analysis showed they dispersedly distributed on all chromosomes of Th.ponticum and Th.intermedium.Furthermore,they could discriminate chromosomes of either Th.ponticum or Th.intermedium from wheat chromosomes without wheat DNA block by FISH.3.They were LTR-retrotransposons.The size of Th.ponticum genome specific repeats varied from 2 to 5 Kb,with GC content higher than 50%.Results of alignment in Triticeae Repeat Sequence Database and NCBI indicated the repeats belonged to Gypsy retrotransposon family.4.The specificity of these repeats was further proved in wheat-Th.ponticum substitution lines and translocation line.Wheat-Th.intermedium 4St(4D)substitution,wheat-Th.ponticum 4E(4D)substitution and wheat-Th.ponticum 4DS/4St translocation line GDR4 were probed by A05,B08,B11,D05,E04,F05 and F08 respectively.No wheat blocking DNA was used in FISH hybridization.5.Ten pairs of primers were developed for efficient amplification of Th.ponticum segments in wheat.Their detection efficiency ranged from 73.3%to 95%in 109 wheat-Th.ponticum derivatives.