Distribution Of Tandem Repeats In Genomes Of Wheat And Related Species And Its Application For Chromosome Identification

Repetitive DNA sequences account for a high proportion(> 70%)of the genome size in Triticeae species.As the most widely cultivated Triticeae crop,wheat(Triticum aestivum L.)has a genome containing more than 85% repetitive DNA.However,few studies have been performed on the structure and function of repetitive sequences in the genomes of Triticeae species.The repeated sequences can be broadly classified into tandem repeats(TRs)and interspersed repeats(IRs),only a few TRs have been utilized in molecular markers development,evolutionary research and cytogenetic analysis.Recently,the genomic sequencing have been completed for a number of Triticeae species with the improvement of assembly technology,and the decrease of cost for plant whole-genome sequencing.It allowed us to perform genome-wide analysis of the structure and function of TRs in wheat and its related species,and develop more TR probes for chromosome identification.In the present study,by combining genomic and molecular cytogenetic studies,the distribution of TRs in the reference genomes of wheat and its related species were identified.A series of new oligo probes which could produce clear and stable fluorescence in situ hybridization(FISH)signals on wheat chromosomes were obtained.Which resulted in precise identification of the representative wheat-alien translocation chromosomes.The main results are as follows:1.The establishment of a web service for visualization of genome-wide TRs distribution.On the basis of the physical characteristics of TR families in the genome,based on NCBI-BLAST+,the B2DSC(http://mcgb.uestc.edu.cn/b2dsc)web server was developed.It can be widely used for visualizing the physical distribution and enrichment of repeats in a genome.Moreover,B2 DSC can also be used to design TR based FISH probes and predict their physical location of FISH signals.Meanwhile,the FISH hybridization patterns of available probes can be used to evaluate or further improve the assembly quality involved the target repeats in specific regions of the genome.2.Genome-wide investigation of the TRs in genomes of wheat and its related species.A database(http://mcgb.uestc.edu.cn/tr)containing the non-redundant TRs(NR-TRs)in the genomes of wheat(T.aestivum,AABBDD),Triticum urartu(AA),Aegilops tauschii(DD),Triticum dicoccoides(AABB)and cultivated barley(Hordeum vulgare,HH)was constructed.It was found that the distribution of TRs occupied 2-5%of the wheat chromosomes.TR arrays are not random dispersal across the A,B and D sub-genomes,and TRs showed a higher density on D than A or B sub-genome chromosomes.The distribution and enrichment characteristics of TR arrays of different lengths in wheat genome were clarified.The results indicated that the structural diversity of repeats played an important role in wheat genome evolution.3.The association analysis between TRs and the regulation of gene expression in the wheat genome.For the high confidence(HC)genes,the abundance of 1-10 bp TR arrays within the 100 bp region around transcription start sites(TSS)is much higher than that in other genomic regions,and 31-60 bp TR arrays are most abundant in the regions approximately 500 bp downstream of transcription terminal sites(TTS).In the wheat genome,TR arrays with more than 50 copies of repeat units were observed in the1 Kb region upstream TSS or the 300 bp region downstream of TTS of the HC genes,and they were found to be either silenced or in low expression.The Germin-like protein genes with TRs inserted into their CDS regions were not expressed at all.It was found that 93 low-copy TR arrays in wheat may contain potential pre-microRNA sequences,which will shed light on the future studies on genome-wide expression regulation network by TRs.4.Development of new oligo probes based on the highly accumulated TRs.On the basis of the genome-wide analysis of the clustered TR arrays,a total of 44 highly accumulated TRs in wheat were obtained,and 16 new oligo probes were designed.These new probes could produce clear and stable non-denaturing FISH(ND-FISH)hybridization signals on wheat chromosomes.By combining the ND-FISH results for the present and previously published TR oligo probes(TR-Oligos),an integrated chromosome physical map for TR-Oligos was constructed,which may greatly improve the resolution of FISH for the identification of specific chromosome segments in Triticeae species.5.The research on the distribution and evolution of a highly accumulated minisatellite family in wheat.As the most abundant minisatellite with a length of 44 bp in wheat,Ta-3A1 has predicted with no less than 69,135 copies in the wheat genome.The total length of the Ta-3A1 repeats is about 3.02 Mb.Ta-3A1 repeats are mainly concentrated in short intervals of wheat chromosome arms 3AL,5AL,7AS,7AL,5BLand 5DS.Combining the phylogenetic analysis and the comparative physical distribution analysis,we found that rapid changes occurred in copy number and chromosomal locations of Ta-3A1 among the different species in the tribe Triticeae,which may have been associated with chromosomal rearrangements during speciation and polyploidization.6.The produced TR-Oligos allowed to precisely identify the chromosome structure rearrangements based on the newly developed oligo probes,we investigated the chromosomal constructions of the wheat-Thinopyrum intermedium introgression line Z4,wheat-rye-Thinopyrum ponticum translocation line Amigo,and wheat cultivar Chuanmai 62(CM62)by multiple ND-FISH analysis.The breakpoints of the translocated chromosome in Z4(Tr-I)were accurately identified,of them,the breakpoint of 3A was located at about 532.13 Mb of 3AL,and 47 Mb fragment of 3DS was inserted in the terminal of Tr-I.The FISH analysis confirmed that Amigo contains wheat-rye translocation chromosome 1RS.1AL,and the translocation of 7BS.7AS and7 BL.7AL.Besides,it was confirmed that the chromosome fragment length of Th.ponticum in the satellite region of 1B is about 120 Mb.Using multiple TR-Oligos,the breakpoints of CM62 were located at 99-151 Mb of 5B and 310-379 Mb of 7B.These results confirm that multiple ND-FISH can identify the chromosome segments rearrangements of wheat and its relative species with largely increased resolution.In summary,our studies focused on the TRs of wheat and its related species,developed a web server for displaying the genome-wide distribution of TR copies,which has been a sharing platform for TR analysis.The new TR oligo probes and the integrated physical map for TR-Oligos,have been successfully used to accurately identify the translocation of wheat-alien chromosome segments,which was comprehensively combined the new approaches of molecular cytogenetics and genomics.The results of this paper have important scientific significance not only for the theoretical analysis of wheat genome organization,function and evolution,but also for benefit future wheat breeding activities based on chromosome manipulation and engineering.