Determination Of Insertion Site And Analysis Of Adjacent Sequence Features Of Satellite Tandem Repeat PSC119.2 On Wheat Chromosome 7D

In the common wheat genome,repetitive sequences occupy 80% to 90% of the genome.Among them,satellite tandem repeat sequences are an important part of its genome.The mode of evolution and amplification of satellite tandem repeat sequences is most likely associated with rolling loop replication and unequal recombination.Some recent studies suggest that the amplification and evolution of tandem repeat sequences may be associated with certain transposon jumps,but this hypothesis needs to be supported by more experimental data.In this study,we used a mutant material of wheat and rye cross progeny with p Sc119.2 family satellite tandem repeat sequence inserted in chromosome 7D as the experimental subject.Non-denaturing fluorescence in situ hybridization(ND-FISH),single-copy fluorescence in situ hybridization(SC-FISH)and molecular markers were used to study the insertion sites of p Sc119.2 in chromosome 7D.The sequence characteristics of the insertion sites were also analyzed.In order to find the cause of p Sc119.2 repeat sequence jumping between chromosomes.Meanwhile,we examined the methylation status of the sequences adjacent to the insertion site,the condensation degree of the insertion region of the inserted 7D chromosome,and the pairing of the variant 7D meiosis.The specific experimental results are as follows:1.The experimental material was identified using ND-FISH.Strain 17T471-3contained one pair of normal 7D chromosomes and both 7D chromosomes were free of Oligo-p Sc119.2-1 signal.Strain 19T110-5 contained one normal 7D chromosome and one variant 7D chromosome,and one arm of the variant 7D chromosome carried a distinct Oligo-p Sc119.2-1 signal,indicating that the p Sc119.2 tandem repeat sequence was inserted into the 7D chromosome;strain 17T471-10 contained one pair of variant 7D chromosomes,i.e.both 7D chromosomes had Oligo-p Sc119.2-1 signal.2.Twenty single copy sequences of chromosome 7D were obtained,and SC-FISH analysis using these sequences as probes localized the insertion region of the p Sc119.2tandem repeat sequence on chromosome 7D to the short arm of 7D(7DS),within ～0.4 Mb(235,428,953-235,837,690 bp);at meiotic rudimentary prophase,the variant 7D chromosome showed three Oligo-p Sc119.2-1 signal sites on the variant 7D chromosome during meiotic coarse-lineage,indicating that the p Sc119.2 tandem repeat sequence has three insertion sites in this ～0.4 Mb region on the 7DS.Thirty one pairs of 7D-specific primers were also developed and molecularly characterized.One to two pairs of7D-specific primers were designed for each 100 kb interval within the above ～0.4 M flanking and region,but these specific primers did not show differences between normal and variant 7D,and thus the exact locations of the insertion regions were not further determined.3.The sequence characteristics of the insertion region of 0.4 Mb and 0.1 M each upstream and downstream for a total of ～0.6 Mb: 235,428,953-236,096,405 bp were analyzed to clarify the distribution and enrichment characteristics of tandem repeats and transposon arrays of different lengths and types within the insertion region in the genome,and 438 long terminal repeat reverse transcription transposons were screened for a total of9 classes,namely LTR/ Gypsy,DNA/En Spm/CACTA,LTR,LTR/Copia,DNA,DNA/Mariner,DNA/Mu DR,Non LTR/L1,Non LTR/RTE;256 tandem repeats,classified into 6 categories according to the single length,1-10 bp,11-30 bp,31-60 bp The region contains a large number of long terminal repeat reverse transcription transposons,indicating that transposon structural diversity plays an important role in the evolution of satellite tandem repeat sequences,which lays the foundation for studying the evolution and amplification mode of satellite tandem repeats.4.Both heterozygous 7D variant and pure 7D variant materials paired normally during meiotic rudimentary and terminal metaphase;by measuring the relative intermediate lengths between the single copy FISH signals on either side of the p Sc119.2signal in the 7D intermediate chromosomes and comparing the lengths of this interval in normal 7D and variant 7D,the results showed that the relative lengths between the two single copies in the variant chromosomes were significantly higher than those in the 7D normal chromosome;the methylation status near the insertion region was predominantly hemimethylated,with more G↓AATTC methylation sites than C↓CGG methylation sites;the above results provide an experimental basis for analyzing the insertion of tandem repeat sequences by exploring chromosome-related behavior and sequence epigenetics.