Development Of Wheat New Germplasm With Rust Resistance And Discovery Of New Genes

Rust pathogens are the most widespread wheat disease in the world.There are three wheat rust diseases:stem,leaf,and stripe(or yellow)rust,caused by Puccinia graminis f.sp.tritici(Pgt),P.triticina(Ptr),and P.striformis f.sp.tritici(Pst),respectively,which cause significant and widespread crop losses once these rust diseases became epidemics.Worse no seeds you can harvest.There are two ways to control rust in cereals,chemical control and genetic control.Chemical control is effective in disease emergency prevention and control,but it costs and also brings environmental pollution problems.Genetic control,or the planting of resistant cultivars or varieties,is the most effective,economically safe and environmentally friendly approach.Since the 1970s,the plant breeders and pathologists worldwide have focused on identification of new or effective resistance genes.However,wheat cultivars can be easily overcomed by pathogen evolution and single resistance gene widely used in wheat breeding only a few years later.The identification and application of new disease-resistance genes is the most effective means of control the rust losses.So,identification and characterisation of the new resistance genes in wheat related species,Triticum monococcum ssp.monococcum,Triticum urartu,durum wheat,and bread wheat will enrich the gene pool and enhance rust resistance in wheat cultivars.This research main focus on(1)Molecular cytogenetic identification of new synthetic T.turgidum ssp.turgidum-T.urartu amphiploids;(2)Development and identification of new synthetic T.turgidum-T.monococcum amphiploids;(3)Molecular mapping and seedling stripe rust resistance gene in bread wheat WGH54;(4)Molecular mapping of adult plant stripe rust resistance gene in durum wheat stweart and fine mapping of adult plant stripe rust resistance in wheat accession Aus27284;(5)Introgression of Lr34 genes from bread wheat enhances the rust resistance of durum wheat.(1)Molecular cytogenetic identification of new synthetic T.turgidum ssp.turgidum-T.urartu amphiploidsIn order to transfer the useful gene of T.urartu to common wheat,four synthetic T.turgidum ssp.turgidum-T.urartu amphiploids(AABBAuAu)were produced by the colchicine treatment of the F1 hybrids between the Chinese tetraploid landraces of T.turgidum ssp.turgidum and T.urartu.In this study,the chromosome constitutions,agronomic character and high molecular weight subunits of these amphiploids were investigated.It was showed that the plant height was ranged from 108.45 cm to 139.43 cm,the tiller number ranged from 7.3 to 7.5,ear length ranged from 10.23 cm to 12.17 cm,the spikelets ranged from 16.26 to 22.06,the selfed seedset ratios ranged from 37.77%to 70.46%.Individual chromosomes of the T.turgidum ssp.turgidum-T.urartu amphiploids could be identified by multicolor fluorescence in situ hybridization using the combination of oligonucleotides probes of Oligo-pSc119.2-1,Oligo-pTa535-1,Oligo-pTa71-2,pTa-713 and(AAC)5.The 42 chromosomes of the amphiploids usually paired with bivalents,along with a small number of univalent,trivalent and tetravalent.All four amphiploids exhibited high resistance to predominant stripe rust races CYR31,CYR32,CYR33,CYR34,and SY11-4 at the adult stage.1Ay subunits from two different T.urartu accessions were expressed in the corresponding amphiploids.These T.turgidum ssp.turgidum-T.urartu amphiploids are new resources for bread wheat improvement(2)Development and identification of new synthetic T.turgidum-T.monococcum amphiploidsTriticum monococcum ssp.monococcum has useful traits for bread wheat improvement.The synthesis of Triticum turgidum-T.monococcum amphiploids is an essential step for transferring genes from T.monococcum into bread wheat.In this study,264 wide hybridization combinations were done by crossing 60 T.turgidum lines belonging to five subspecies with 83 T.monococcum accessions.Without embryo rescue and hormone treatment,from the 10,810 florets pollinated,1983 seeds were obtained,with a mean crossability of 18.34%(range 0-89.29%).Many hybrid seeds(90.73%,923/1017)could germinate and produce plants.A total of 56 new amphiploids(AABBAmAm)were produced by colchicine treatment of T.turgidum × T.monococcum F1 hybrids.The chromosome constitution of amphiploids was characterized by fluorescence in situ hybridization using oligonucleotides probes with different chromosome and subchromosome specificities.Sodium dodecyl sulphate polyacrylamide gel electrophoresis analysis indicated that the Glu-A1m-b,Glu-A1m-c,Glu-A1m-d and Glu-A1m-h proteins of T.monococcum were expressed in some amphiploids.Despite resistance reduction in several cases,45 out of 56 amphiploids exhibited resistance to the current predominant Chinese stripe rust races at both the seedling and adult plant stage.These novel amphiploids provide new germplasm for the potential improvement of bread wheat quality and stripe rust resistance.(3)Molecular mapping of adult plant stripe rust resistance in durum wheat cultivar Stewart and its relationship with Yr80 gene in bread wheat Aus27284Adult plant resistance to stripe rust disease was detected in durum wheat cultivar Stewart.To genetically characterise this resistance,Stewart was crossed with the highly susceptible durum cultivar Bansi and an F2 genetic map consisting of 7718 polymorphic markers was generated using a 90K wheat Infinium iSelect SNP array.Scoring of adult plant disease resistance(APR)to stripe rust in 260 F3 families from this population coupled with QTL analysis identified two resistance QTL segregating in this family,both derived from the Stewart parent.One locus was mapped in the Lr46/Yr29/Pm39 region and was confirmed as Lr46 using a near perfect marker.The second locus(Yr3BL)was located on the distal region of chromosome 3BL.F3,F5,and F7 RILs from the Stewart × Bansi family were developed and field trialled for stripe rust disease resistance in 2017,2018 and 2019,respectively,and genotyped with targeted genotyping-by-sequencing assays.The Lr46 locus was detected in all trials whereas the weaker Yr3BL QTL was only detected in F3 and F5 field trials.Field data in 2019 showed that the overall resistance is lower than that in 2017-2018 crop growing seasons.Therefore,at the serious rust outbreaks growing season,the weaker Yr3BL QTL resistance was not satisfactory.These analyses positioned Yr3BL on an interval equivalent to the 610 Mb-619 Mb region of the Chinese Spring chromosome 3BL sequence.Evidence for additivity of Yr3BL and Lr46 was observed.Comparative mapping indicated that the Yr3BL locus co-locates with the recently described Yr80 adult plant stripe rust resistance locus identified in bread wheat land race Aus27284 in a 55 MB interval on chromosome 3BL.To further compare Yr3BL with Yr80 map locations,nineteen F6 recombinant lines from a Aus27284 × Avocet cross were obtained from the University of Sydney and an additional 4 F7 recombinants identified by flanking marker(KASP65624 and IWA6720)screening of 1500 seed derived from two F6 families that segregate for Yr80 resistance.These recombinants were field trialed for stripe rust resistance in 2019 and genotyped with STS,KASP and RNASeq markers thereby reducing the bread wheat Yr80 interval to a 2.1 Mb region that encodes 15 genes in the Chinese Spring reference genome sequence.This fine mapping also indicated that Yr3BL and Yr80 are in fact separate loci with Yr3BL located 55 Mb distal to Yr80.Closely linked markers identified in this study will be useful for marker assisted selection of these loci in both durum and bread wheat breeding programs.(4)Molecular mapping and seedling stripe rust resistance gene in bread wheat WGH54Previously WGH54 exhibited a high level of stripe rust resistance against Puccinia striiformis f.sp.tritici(Pst)pathotypes both at seedling and adult stage,indicating that WGH54 contains all stage resistance genes.To identify the genes in WGH54,WGH54 was crossed with the susceptible parent Avocet S(AvS),and F2 and F3 generations were raised.Monogenic segregation observed among 50 WGH54/AvS F2 families.Equal amounts of genomic DNA from 10 resistant and 10 susceptible F2 RILs were separately pooled for the iSelect wheat 90 K Infinium SNP array.The resistance to susceptible ration is 3:1 in F2 plants,suggesting that a single dominant gene may confer resistance in WGH54,temporarily named YrWGH54.YrWGH54 was identified and mapped on the long arm of chromosome 2A,combined with 90k SNP data,a number of KASP and SSR markers.Four EMS mutants were obtained,among them,one of them is deletion and YrWGH54 was fine mapped to 0.3Mb region using the F2,F3 and deletion,which have 5 encodes resistanceassociated protein genes in Chinese Spring,two durum wheats(Zavitan and Kronos)reference genome sequence.Closely linked markers identified in this study will be useful for marker assisted selection of the loci in bread wheat breeding programs.This study also will contribute to the cloning of the candidate genes.(5)Introgression of the D genome encoded Lr34/Yr18/Sr57/Pm38/Ltn1 adult plant resistance gene into Triticum turgidum(durum wheat)Lr34/Yr18/Sr57/Pm38/Ltn1(hereafter referred to as Lr34)is a multi-pathogen adult plant resistance gene that provides resistance to wheat leaf rust,stripe rust,stem rust and powdery mildew diseases.It is located on chromosome 7D of bread wheat(ABD genomes)and is not readily available for durum wheat(AB genomes)improvement.Here we have introgressed this gene into chromosome 7A of durum wheat by using a phlc mutant to induce heterologous recombination.A durum wheat chromosome disomic substitution line Langdon 7D/7A,which contains the 7D chromosome from bread wheat cultivar Chinese Spring,was crossed with a homozygous phlc mutant line(cv.Cappelli).F1 plants were then backcrossed to Cappelli phlc and BC1 plants identified that were homozygous for phlc and also contained chromosome 7D.Progeny from these lines were sought that contained recombinant 7D/7A chromosomes using PCR to identify plants containing Lr34 but that also had reduced levels of a D-genome specific repeat sequence(Dgas)compared with Langdon 7D/7A homozygous and artificial heterozygous controls.Lines containing Lr34-encoding small D genome introgressions on chromosome 7A were characterised using PCR markers,90K wheat Infinium iSelect SNP arrays and FISH/GISH cytogenetics.Two lines containing the smallest D-genome introgressions were then crossed for three generations using the susceptible durum cultivar Bansi as a recurrent parent.It was shown that the 7D introgressions in the backcrossed Bansi lines provided characteristic Lr34mediated adult plant resistance to wheat stripe rust disease caused by Puccinia striiformis f.sp.tritici.These 7D chromosome introgressions are currently being backcrossed into several elite durum cultivars for global improvement of durum disease resistance.