Develpoment Of EST-SSR Marker And Construction Of Genetic Map In 'Guizimai No.1'

Common wheat?2n=6x=42?is a heterologous hexaploid crop,which has a larger genome and also contains a number of repeat sequences,so the development of new molecular markers can not only provide a new approach to study of wheat genome,but also help for genetic diversity analysis,gene mapping and molecular-assisted selection in wheat.Based on the transcriptome sequencing results of‘Guizimai No.1',the total Unigenes sequence was exploited for EST-SSR markers,the developement of markers were screened for polymorphism,mapped on chromosome and analyzed genetic diversity in wheat.Through GBS?genotype-by-sequencing?technology and the screened markers,110 individual plants of F₂ generation obtained from the hybridization between‘Guizimai No.1'and‘Zhongyan Extra Large'were used to construct the genetic linkage map,and carry out chromosome localization of the powdery mildew resistance gene derived from‘Guizimai No.1'.The results were as followed:1.119,572 total Unigenes sequences were obtained based on the RNA-Seq analysis of‘Guizimai No.1',and 8,749 EST sequences?accounting for 7.3%of all Unigenes sequences?containing microsatellite sites were obtained by using MISA?version 1.0?software to detect and analyze the SSR loci,and one SSR per 8.04 kb on average.Single nucleotide repeat types^Hexanucleotide repeat types has 1,906sequences?21.8%of total SSRs?,1,838 sequences?21%of total SSRs?,4,706sequences?53.8%of total SSRs?,264 sequences?3%of total SSRs?,26 sequences?0.3%of total SSRs?and 9 sequences?0.1%of total SSRs?,respectively.The six types of nucleotide repeats contained 258 different repeat motif,while dinucleotide repeats occurring most frequently with AG/CT and AC/GT,and trinucleotide repeats with CCG/CGG most frequently.2.The primers of 8,749 EST sequences with SSR loci were designed by using System Primer 5.0,and 5,503 primers were successfully designed,accounting for 62.9%of the total EST-SSRs sequences.702 pairs EST-SSRs primers from them were randomly selected and Chinese spring,‘Guizimai No.1'and‘Zhongyan Extra Large'were selected as materials to identify the effectiveness of these primers,the results showed that 341 pairs of EST-SSR primers could amplify stable and clear bands in the three materials,53 of them showed polymorphism between their parents.Chinese Spring Nulli-tetrasomes were selected as materials to mapped 153 pairs of effective primers on 16 chromosomes except 3A,5A,3B,4B and 1D.In additon,162 EST-SSR primer pairs were integrated into the genetic map of‘Guizimai No.1×Zhongyan Extra Large'.341 pairs of EST-SSR markers are effective and can be used as molecular breeding research in wheat and related species in future.3.Genetic diversity analysis was performed in 52 wheat materials by using 53pairs EST-SSR primers that polymorphism exsit in two parents.18 pairs of EST-SSR primers showed polymorphism in 52 wheat materials,and the polymorphic rate was34%.A total of 100 alleles were detected by 18 pairs of primers,with a variation of3¹0 alleles and an average of 5.6 alleles per marker.The primer polymorphism information index?PIC value?varied from 0.303 to 0.791,and the average of PIC value was 0.568.52 wheat varieties?lines?were successfully distinguished by the 18 pairs of EST-SSR markers,which indicated the developed EST-SSR markers are effective and that can be used to genetic diversity analysis.4.Based on Genotyping-by-Sequencing,we constructed a genetic linkage map with a total length of 5,402.12 cM,and a total of 23,134 SNPs covering 21chromosomes.The variation of chromosome length was 77.84 cM?6D?⁷63.53cM?3B?,and the variation of chromosome coverage SNP number was 134?6D?⁶,288?3B?.The average distance between markers of the map was 0.23 cM,in which the marker density on chromosome 6A was the highest,and the average distance between markers was only 0.10 cM,while the marker density on chromosome 5D was the lowest,and the average distance between markers was 1.11 cM.5.A LOD value of 34.8 was detected on chromosome 6A by Composite Interval Mapping?CIM?,indicating that the powdery mildew resistance gene from‘Guizimai No.1'was located on chromosome 6A.Using the constructed high-density linkage map,the powdery mildew resistance gene was mapped in the 2.3 cM region of GBS-SNP marker chr6A-307802221 and chr6A-309885836.