Genetic Diversity Analysis On Triticum And Aegilops,and Mapping,Utilization And Expression Of Gene(s) Resistant To Disease

Powdery mildew and stripe rust,caused by fungi,are the most devastating diseases of wheat worldwide.Transferring beneficial genes from related plants to common wheat with the help of distant hybridization,is one of the important means to enrich the genetic basis of common wheat increasingly.Excavation and utilization of resistance genes of wheat relatives,screening the molecular markers of resistance genes,analysis of resistance mechanism of resistant germplasm,and transferring and accumulation resistance genes have important theoretical significance and practical value for enriching the disease resistance of wheat genetic resources and clarifying the molecular mechanisms of resistance.In this study,138 synthetic wheat,92 common wheat and its relatives and 1 Aegilops ovata were employed to evaluate genetic diversity,identifify germplasm resistant to powdery mildew and stripe rust,investigate the inheritance of disease resistance and detect adjacent molecular markers,breed new germplasm resistant to disease,constructe and analyze cDNA library of Ae.Ovata leaf inoculated with Blumeria graminis（DC.）E.O.Speer f.sp.tritici（Bgt）.The main results are as follows:1.Genetic diversity of synthetic wheat,common wheat and its relativesHigh-molecular-weight glutenin subunit（HMW-GS）were analyzed by SDS-PAGE of 138 synthetic wheat.Five alleles and one novel type of alleles were identified at the Glu-A1.Thirteen alleles and five novel types of alleles were identified at the Glu-B1 locus and frequencies of subunit 6+8,7+8 and 14+15 were 33.3%,29.7%and 21.7%,respectively.Thirteen alleles and four novel types of alleles identified at the Glu-D1 and frequencies of subunit 1.5+10,2+12,2+12.5 and 5+10 were 21.7%,22.5%,16.7%and 13.0%,respectively.Genetic diversity among 19 Triticum aestivum accessions and 73 accessions of closely related species were analyzed using simple sequence repeat（SSR）markers.Forty-four out of 497 SSR markers were polymorphic.In total 274 alleles were detected（mean 6.32 alleles per locus）.The polymorphic information content（PIC）of the loci ranged from 0.3589 to 0.8854（mean 0.7538）.The D genome had the highest mean number of alleles（6.32）followed by the A and B genomes（6.13 and 5.94,respectively）.The correlation between PIC and allele number was significant in all genome groups（0.7540,0.7361 and 0.7482 for A,B and D genomes,respectively）.Among the seven homologous chromosome groups,genetic diversity was lowest in group 7 and highest in group 5.In cluster and principal component analyses,all accessions grouped according to their genomes were consistent with their taxonomic classification.Accessions with the A and D genomes were clustered into two distinct groups,and AABB accessions showed abundant genetic diversity and a close relationship.T.durum and T.turgidum were clustered together,consistent with their morphological similarity.Cluster analysis indicated emmer is closely related to hexaploid wheat.Compared with common wheat,higher genetic variation was detected in spelt,T.aestivum subsp.yunnanense and subsp.tibetanum.In addition,a close genetic relationship between T.polonicum and T.macha was observed.The results of the clustering and principal component analyses were essentially consistent,but the latter method more explicitly displayed the relationships among wheat and closely related species.2.Excavation,SSR markers and utilization of Disease-resistance genes from Synthetic wheatSynthetic wheat is an important germplasm to transfer benefit gene（s）from alien species to common wheat（Tritium aestivum L.）.Agronomic characters,powdery mildew resistance,and stripe rust resistance of 138 synthetic wheat were identified and evaluated.The resistant ance identification results to powedery mildew showed that 27（19.5%）synthetic wheat accessions were resistant to powdery mildew and 21,2 and 4 out of 27 accessions were immune,highly resistant and moderately resistant to powdery mildew,respectively.The resistantance identification results to stripe rust showed that 55（39.9%）synthetic wheat accessions were resistant to stripe rust and 28,12 and 15 out of 55 accessions were immune,highly resistant and moderately resistant to stripe rust,respectively.10 synthetic wheat accessions were resistant to both powdery mildew and stripe rust.Synthetic wheat SE5785 resistance to powdery and SE5756 resistance were selected for next research and utilization.We constructed a segregating population of 126 F2 plants derived from SE5785/Xiaoyan22.Genetic analysis revealed that a single recessive gene,PmSE5785,was responsible for seedling stage powdery mildew resistance in SE5785,temporialy.A genetic map comprising of Xbarc59,Xgwm539,Xcfd62,Xwmc817 and PmSE5785,spanned 18.9 cM on chromosome 2D.Xbarc59 was the closest markers that are linked to SE5785 at genetic distances of 7.3 cM.Analysis indicated that PmSE5785 may be a new gene.In the offspring from the cross or backcross SE5785 and excellent common wheat cultivar Xiaoyan 22 suscetiple to powdery mildew,we devoleped new germplasm N07228-1 and N07228-2 with large seed,resistance to powdery mildew and better agronomic traits.We constructed a segregating population of 116 F2 plants derived from SE5756/Xinong 979.Genetic analysis revealed that a single dominant gene,YrSE5756,was responsible for seedling stage stripe rust resistance in SE5756.A genetic map comprising of Xwmc626,Xwmc269,Xgwm1l,Xbarx137,Xwmc419,Xwmc85,Xgpw5237,Xwmcl34,WE173,Xwmc631 and YrSE5756,spanned 70.1 cM on chromosome IBS.Xwmc419 and Xwmc85 were flanking markers that are tightly linked to YrSE5756 at genetic distances of 2.3 and 1.8 cM.In the offspring from the cross or backcross SE5756 and excellent common wheat cultivar Xinong 979 suscetiple to stripe rust,we devoleped new germplasm N07178-1,N07178-2,N08256-1 and N08256-2 with large seed,resistance to stripe rust and better agronomic traits.3.Transferring of powdery mildew resistance gene（s）from Aegilops to common and devolepment of new germplasmDevolepment of amphidiploid between tetraploid wheat and Ae.Tauschii:F1 seed setting rate of 57 combinations between 23 tetraploid and 8 Ae.Tauschii resistance to powdery mildew were analyzed.Tetraploid as female parent,seed setting rate ranged from 0.74%to 7.7%and MY3478 was highest（7.70%）.Ae.Tauschii as male parent,seed setting rate ranged from 1.7%to 5.25%and SY41 was highest（5.25%）.MY3478 and SY41 may contain fructification genes.The morphological and cytological of the combination（MY3478/SY41）with the highest seed setting rate were investigated.The morphological traits was similar to common wheat,but the hard and difficult threshing shell similar Ae.Tauschii.Chromosome number of F2 generation is mainly 41 and 42.A synthetic wheat NA0928-1-1-1,resistant to powdery mildew and stable morphologically and cytologically,was bred.Transferring of powdery mildew resistance gene from Ae.Ovata to common wheat:F₁ generation between common wheat Chinese Spring and Ae.Ovata was identified using morphology,HMW-GS,cytology and molecular marker and it was true hybrid.With the increasing generation of selfing and backcross in derivatives,in higher generations of selfing and backcross,the chromosome number gradually decreased and eventually tended to common wheat.Backcrossing was better to fasten the process than selfing.Based on SSR markers identification resulits in its parent and derivatives,23 pairs of SSR primers had the specificity bands of Ae.ovata in six derivatives.We got some Chinese Spring-Ae.Ovata derivatives resistant to powdery mildew.4.Constrution and analyse of cDNA library of Ae.Ovata leaf inoculated with BgtTotal RNA was extracted from Ae.Ovata leaves inoculated with Bgt at 12 h,24 h,36 h,48 h and 96 h,respectively,equal amount of which then were mixed as reverse transcription template.The cDNA library was constructed.After screening repeated and redundancy sequences,409 unigenes were obtained.BlastX alignment in nr-protein database revealed that 365（89.44%）unigenes predicted genes were highly similarity to known proteins.Using Pathway software 307 unigenes with known function involved in cellular processes（8.31%）,environmental information processing（13.45%）,genetic information processing（11.25%）,defense resistance gene（8.07%）,metabolism（31.05%）,organismal sy stems（2.93%）,hypothetical protein（14.18%）,no significant similarity found（10.76%）.These EST lay the foundation for later resistance related gene identification.