Genetic Diversity And The Evolution Of The Microsatellite Segment Among Triticeae Species As Revealed By Wheat Microsatellite Markers

Microsatellite DNA is considered as one of the excellent apprpoaches in studing species evolution and genetic diversity.Tandem repeat sequences play ansignificant role in plant genome,at present,the three functions is widely admitted:first,they are composition of open reading frame,second,they participate in genome adjustment activity.Third,they compose the brittleness locus in chromosome.Genome is that a complicated information deposited system in genome system structure.Repeat sequences influence important life activities such as DNA duplication,chromosome separation,gene expression.Though several functions has been regarded, it is still a long way in the function study on tandem repeat sequences.so it is necessary to perform further study on SSR evolution and biology function.Large amount of studies suggest microsatellite has advantage in the field of detecting polymorphic in species,and it has been already widely used in genetic diversity,phylogenetic relationships,variety identification,building fingerprint map,linkage genetic map,the locations of quantitative trait loci,pedigree analysis and molecular marker-assisted Breeding.Though the application foreground of microsatellites was very wider, exploit SSR marker from species is a very dearly and time-consuming job,because it need people to exploit appropriately polymorphic locus from known sequences,this point is differ from other molecular markers. From 1990s,people had taken an idea whether SSR marker could be applied to wheat species.The research found that the flanking sequence of microsatellite DNA is comparatively stably.The microsatellite marker from a certain species could be applied to nearly species,it can reducing workload of getting microsatellite.Many wheat microsatellite primer sets were successfully used for amplication of DNA from several related species, such as rye,Dasypyrum,Aegilops.It is a very easy and effective measure that enhance the quantity of relative species and get more special molecular markers.Thus,microsatellite DNA seemed to be very useful for clarifying the evolutionary relationships of closely related populations.Consequently,the aim of this study was to examine the transferability and phylogenetic relationships of SSR markers,getting more specific ssr markers,along with studying the variation and evolution of the microsatellite from closely related populations,this research shows the results as follows:1.Genetic diversity and phylogenetic relationships among triticeae species as revealed by wheat microsatellite markersUsing 218 genomic wheat(Triticum aestivum) microsatellite markers to evaluate the genetic diversity and phylogenetic relationships of 22 triticeae species by PCR ampification.39 wheat SSR primers could generate polymorphism bands and the repetitiveness is fine were selected.These primers generated 213 polymorphic alleles in 22 accessions,with an average of 5.4 alleles per primer.The polymorphism information content (PIC) values of the loci ranged from 0.38～0.89,with an average PIC of 0.57.The value of genetic similarity(GS) was varied from 0.46 to 0.94, with an average GS of 0.67.The cluster result showed that all species were classified into 4 species,wheat,Lophopyrum elongatum,Haynaldia villosa,Secale cereale were each belong to one group.The result of cluster is consistent with the results of the traditional taxonomy,which indicate that microsatellites are a useful way to analyze genetic relationship between wheat and its related species.Ampliated polymorphic bands in every species evaluating wheat microsatellite markers can be identify triticeae species.2.Development of Dasypyrum Genome Specific Marker by Using Wheat Microsatellites One hundred and twenty-six SSR primer pairs,distributed in chromosome 1A to 7A,1B to 7B,1D to 7D of Triticum aestivum,were investigated on common wheat,Dasypyrum,Haynaldia villosa,Lophopyrum elongatum,rye.12 SSR markers can amplify rye-specific bands in rye,8 markers can amplify specific bands in Dasypyrum,4 markers can ampify specific bands in Aegilops,9 markers can amplify specific bands in Haynaldia villosa,10 markers can amplify specific bands in Lophopyrum elongatum,and most of these bands are about 500bp,600bp,and 800bp in size.In this experiment,2 genome specific repetitive DNA sequences were separated and identified,(1) A specific polymorphic DNA fragment of about 1000bp amplified by primer pair Xgwm260 was obtained in all lines containing rye chromosomes,but there were not the case in the tested materials.Furthermore,PCR analysis was performed on a set of Chinese Spring×Imperial addition,the result showed that the fourth pair of rye chromosomes contain Xgwm260.The PCR product was recovered,cloned and sequenced,the length of the fragment is 988bp,named PMD260-1000,at the 941 location CACTA combining with SP1(transcription factors),GC-BOX existing sp1,the sequence is inversion repeated,open reading frame was found in the sequence,deduce that fragment structure may be that retrotransposon.Therefore,Xgwm260 is a genome-specific polymorphic DNA segment for genera of rye,and it could be used as a molecular marker for detection of chromosomes of rye in wheat.(2) Xgwm232 also could amplified rye-specific bands and the band is 491bp in size.Take the same measure as above to indicate the fragment is a genome-specific polymorphic DNA segment for genera of rye,and locate on the sixth pair of rye chromosomes.The NCBI Blast revealed that two fragments all had no high homology.Therefore,they could be 2 new DNA sequences.3.Study Variation and Evolution of wheat microsatellite by Sequencing Analysis This experiment cloned some special repeated segments,after sequencing,Put the sequencing result into GENEBANK in NCBI,comparing similarity,and analysis the sequence structure by the software DNAMAN, to study the mutation of these fragments during its long process of evolution.The research found this repeated sequence has stably segment,there were also several conserved motifs such as CAAT-box and GAGA-box,which are related to regulation of transcription,and the sequence similar with nucleotide sequence transcription factor binding sites.(1) The similarity degree of sequence compare between PMD120-500 and the sequence in gamma-gliadin gene reached 97%.The TATA-BOX in this sequence had the mutation in the process of evolution.The difference from this genome sequence existing in the flank of repeated sequence,so it cause mutation in the binding aera,and failed to amplicate tetraploid wheat.The similarity degree of sequence compare between PMD120-500 and a section of Triticum monococcum actin geneand Triticum aestivum cultivar Renan clone BAC 930H14 reached 100%,the location between 100 and 110 has a TATA box,also relative to Amino acid sequence,this sequence may relative with the initial of transcription.(2)The PMD120-500 is inverted repeat sequence,TATAA frame were exited in up-downstream site respectively,and a TATA frame,all belong to promoter characteristic sequence.And the similarity degree of sequence compare between PMD120-500 and the sequence in gamma-gliadin gene G2656 reached 91%,two AATAAA frames have changed,the terminator mutation in the process of evolution could be caused by mutation,Insertion and deletion on base.It could affect the space structure between regulatory elements,the similarity degree of sequence compare between PMD120-500 and the sequence in Hordeum vulgare subsp,vulgare eIF4E gene locus reached 91%,and have mutation in AATAAA frame,insert a G in the AATAAA frame,conduced the Hordeum vulgare lack this regulatory element.