Development And Analysis Of A Genome-wide Suite Of Specific SSR Markers In Aegilops Tauschii And Chinese Spring Wheat(Triticum Aestivum L.)

Bread wheat(Triticum aestivum L.)is one of the most important grain crops in the world.According to the Food and Agriculture Organization of the United Nations,wheat is a staple food for about 40% of the world's population(http://faostat.fao.org),and is also the main source of vegetable protein in the human food.However,domestication and cultivation have reduced the genetic variation in bread wheat,which threaten wheat breeding process in the future.There is an urgent need to broaden the genetic basis of wheat through breeding,specifically by identifying,incorporating,and using key genes from wild relatives.Aegilops tauschii is the diploid D-genome donor of hexaploid wheat,and it possesses many useful genes for high yield,disease and insect resistances,and grain quality.So,Aegilops tauschii is an important genetic resources for the genetic improvement of common wheat.In recent years,molecular markers have become available for Ae.Tauschii and common wheat.Simple sequence repeat(SSR)markers,also known as microsatellites,are a class of highly polymorphic molecular markers that have good reproducibility and stability.The work draft of the Chinese spring wheat and Ae.Tauschii whole genome sequence have been plotted,which lay a foundation for the large-scale development of genome-wide SSR markers.In the present study,we developed genome-wide SSR markers for Aegilops tauschii on a large scale using bioinformatics methods.Then analyzed the diversity of wheat species and constructed genetic linkage map using developed markers.In the end,we developed the specific markers of wheat and Aegilops tauschii to improve the working efficiency of molecular breeding.The experiment results are as follows:(1)The SSRs present in the Ae.tauschii genome were identified and located using MIcroSAtellite.A total of 93,204 SSRs were identified at a density of 22.48 per Mb.Of these,3,060 were compound SSRs,and the90,144 ‘pure' SSRs(containing a single repeat motif)could be divided into486 types.Among these types,the percentage of dinucleotide repeats was thehighest,followed by hexanucleotides and trinucleotides,while the percentage of pentanucleotide repeats was the lowest.Among all the repeats,highest proportion were AG/TC,followed by AC/TG,AAG/TTC,AT/TA,AAC/TTG,whose proportion were 18.93%,18.93%,8.94%,7.81% and 4.18%respectively.(2)PCR primers were designed with Primer 3.0.All of the SSRs identified were used for primer design.Primer pairs for a total of 86,616 SSR markers are designed successfully.We analyzed the polymorphism of the markers in the genome of Ae.tauschii using re-PCR.As a result,8,400 markers were predicted to be single locus and 78,216 markers were predicted to be multiple locus.The percentage of single locus markers was 9.70%.Among the 8,400 single locus markers,the largest proportion(3,063;36.46%)were dinucleotide repeats,followed by hexanucleotides and trinucleotides,and 243(2.89%)were composed of compound repeats.Based on the scaffold information anchored to the integated genetic map of Jia,we located 1,555 single locus SSRs on the genome of Ae.Tauschii.The map spanned 1,044 cM with an average distance between marker loci of 0.67 cM.The percentage of these SSR loci on each chromosome is as follows: 1D to 7D: 20.19%,17.36%,11.64%,7.91%,17.17%,10.10%,and 15.63%.(3)A total of 35 single locus markers were randomly selected for validation in the genome of Ae.Tauschii.We found that 15(42.9%)amplified a single locus,eight(22.9%)amplified multiple identical loci,and 12(34.2%)gave no amplification products.This result showed that the consistency with re-PCR was 42.9%.The phylogenetic tree was constructed using UPGMA cluster analysis in NTSYS.The genetic similarity coefficients ranged from0.6-1,and averaged 0.8.This results showed that the unit point markers can be used for system analysis of wheat varieties.We selected 19 loci which showed differences among the accessions to build a binary matrix to calculate the PIC.The PIC varied from 0.083 to 0.5844 with a mean of 0.2957.(4)We screened specific SSR markers from the Ae.Tauschii and Chinese spring wheat's SSR markers which were predicted to be single locus.We got 80,374 specific markers of wheat and 1,525 specific markers of Ae.Tauschii.The using of these specific markers will improve the efficiency of breeding work in the further.