Identification And Exploitation Of Genes For Disease Resistance And Grain Protein Content In Hexaploid Triticale

Wheat powdery mildew,caused by Blumeria graminis f.sp.tritici,and wheat leaf rust,caused by Puccinia triticina Eriks,are two important diseases that severely threaten wheat production.Most resistance genes tend to be overcome within several decades due to rapid evolution of physiological races and strong selection for virulent pathogen mutants.Therefore,continuously exploiting naturally occurring resistance genes remains the most effective measure to improve the disease resistance of bread wheat.A hexaploid triticale cultivar Sorento from Poland is highly resistant to wheat powdery mildew isolate E09,leaf rust isolate PHT and stripe rust isolate CYR32.However,the resistance has not been studied and exploited in wheat breeding.In the present study,to incorporate resistance genes against powdery mildew and leaf rust into bread wheat,Xuezao,a wheat line susceptible to powdery mildew and leaf rust,was crossed with the resistant triticale cv.Sorento as the female parent.The F1 hybrids were then backcrossed with Xuezao as the recurrent male parent for several rounds.By means of marker analysis and cytological analysis,it is possible to locate resistance genes on rye chromosomal arms in a wheat background.Increasing grain protein content is another important purpose to improve bread wheat.NAM genes are associated with increased grain protein content(GPC),Zn and Fe content as well as accelerating senescence in wheat.Plant-specific NAM gene is a member of NAC transcriptional factor which not only participates in the formation of apical meristem in plants and response to plant hormone but also is involved in stress tolerance and defensive reaction.Related NAM genes have been reported in wheat,barley and Triticum timopheevii,whereas homologous NAM genes in rye were not investigated by far.Here we aimed to clone the rye NAM-R genes by means of homology-based cloning strategy from the triticale cultivar on our hand and overexpress the genes in Arabidopsis thaliana.All related results are listed as below:1.The powdery mildew and leaf rust resistance have been successfully transferred from hexaploid triticale Sorento into susceptible wheat line Xuezao by backcrossing.By means of phenotype evaluation both at seedling and adult stage,we acquired plants resistant to either of powdery mildew isolate E09 and leaf rust isolate PHT or both in different generation.Transmissions of powdery mildew and leaf rust resistance reached 60%and 50%respectively.2.By marker analysis,we demonstrate that the long arm of the 2R(2RL)chromosome confers resistance to both the leaf rust isolate PHT and powdery mildew isolate E09 at adult-plant and seedling stages,while the long arm of 4R(4RL)confers resistance only to powdery mildew isolate E09 at both stages.Additionally,adult plants containing 2R chromosome in BC2F2 were also resistant to stripe rust isolate CYR32 when this race was applied in field.3.The chromosomal composition of BC2F3 plants containing 2R or 2RL and 4R or 4RL in the form of substitution and translocation were confirmed by GISH(genomic in situ hybridization)and FISH(fluorescence in situ hybridization).Monosomic and disomic substitutions of a wheat chromosome with chromosome 2R or 4R,as well as one 4RS-4DL/4DS-4RL reciprocal translocation homozigote and one 2RL-1DL translocation hemizigote,were recovered.Lines homozygous for 2R and 4R chromosomes were shown to be stable genetically.Such germplasms are of great value in wheat improvement.4.Lines homozygous for 2R were verified using wheat public molecular markers and four rye co-dominant markers.Among the rye co-dominant markers,three and one were located on long and short arm of 2R chromosome,respectively.These co-dominant markers can be applied in MAS(Marker Assisted Selection).5.By inoculating plants homozygous for 2R chromosome with multiple powdery mildew isolates in China,we proved that 2R chromosome of Sorento also conferred resistance to 23 isolates except for one E20 which is also virulent to most of known mildew resistance genes.This suggests that the materials we produced are of great value in improving wheat resistance.6.Here we cloned the rye NAM genes NAM-R1 and NAM-R2 from triticale cultivar Legalo and Sorento using homology-based cloning strategy.By developing specific markers NAMT2 and NAMT3-2,NAM-R1 and NAM-R2 were mapped on short arms of rye chromosomes 6R and 2R respectively using a Sorento x Xuezao backcrossing population developed previously.7.Both of the gDNA and mRNA sequence of NAM-R1 and NAM-R2 shared more than 90%similarities with cloned NAM genes in wheat.By comparing NAM proteins from Triticum turgidum,Aegilops tauschii,Hordeum vulgare,Triticum timopheevii,Oryza sativa,Arabidopsis thaliana,Aegilops speltoides and Haynaldia villosa,we found that the NAM-R1(6RS)of rye,NAM-S1(6SS)of Aegilops speltoides and NAM-V1(6VS)of Haynaldia villosa are more homologous with the sixth homoeologous group of Triticum turgidum(NAM-A1,NAM-B1),Aegilops tauschii(NAM-D1),and Triticum timopheevii(TtiNAM-G1),and correspondingly,the NAM-R2(2RS)shared more similarities with the second homoeologous group of Triticum turgidum(NAM-A2,NAM-B2),Aegilops tauschii(NAM-D2)and Hordeum vulgare(HvNAM-2),These results indicate the homoeologous feature of Poaceae for second and sixth homoeologous group.8.In order to investigate the function of NAM-R1,NAM-R2,NAM-S1 and NAM-V1,they are overexpressed driven by 35S promoter in Arabidopsis thaliana wild type Columbia.By means of Basta screening and PCR identification,positive T2 families were obtained.