Construction Of Oligo-FISH Based Karyotype Of Haynaldia Villosa Andcharacterization Of Wheat-H. Villosa 6VL Chromosome Aberrations

Wheat(Triticum aestivum L.)is one of the most important grain crops in the world.Haynaldia villosa(L.)Schur,a wild relative of wheat,posseses many traits that which are useful for wheat improvement,such asresistances to diseases and tolerances to various stresses.The development of alien chromosome lines such as addition/substitution,whole arm translocation and especially small fragment translocation will greatly facilitate the study and utilization of alien genes.In previous studies by chromosome engineering,such alien lines involving different Haynaldia villasa chromosomes have been developed and genes conferring resistances powdery mildew,yellow mosaic virus and good grain quality have been transferred into wheat and allocated on specific H.villasa chromosomes or chromosome fragments.Among them,the whole arm translocation T6VS·6AL having the powdery mildew resistance gene Pm21 are now widely used in wheat breeding for disease resistance in China.To localize,transfer and utilize more genes of interest from H.villosa,in this study,oligo-FISH based karyotype of H.villosawas constructed and the diversification of five H.villosa accessions were analyzed based on their FISH signal polymorphism.The 6VL has been reported to confer resistances to stem rust,nematode,and etc.A whole arm translocation T6AS·6VL has been developed in previous study.To develop and characterize new alien translocations involving 6VL,in this study,chromosome specificmolecular markers were developed.The homoeologous chromosome pairing system and irradiationwere used to induce chromosome rearrangements and used new chromosome aberrations involving chromosome 6VL were characterized.Combining with FISH karyotype and molecular marker analysis,a physical map of 6VL chromosome was constructed.All these have laid a solid foundation for mapping and utilization of alien genes.The main results obtainedin thisresearch are as follows:1.Construction of oligo-FISH based karyotype of H.villosaIn the present research,a microsatellite Afa was clonedfrom H.villosaand an oligo probe oligo-pDvAfa was synthesized.This new probe together with four previously reported oligo probes,oligo-pSc119.2,oligo-pAsl,(GAA)10,and(AAC)10 were used for FISH analysis of H.villosa accession 91C43.It was found that the FISH signal distributionon H.villosa chromosomes were differentfor different probes.The FISH signals of probes oligo-pAsl and oligo-pDvAfa were distributed along the full length of all H.villosa chromosomes,those of probe oligo-pSc 119.2 were predominantly located at the telomeric or subtelomeric regions of both arms,whearas those of probes(GAA)10 and(AAC)10 were predominantly at the centromeric and pericentromeric regions.Thus the combination of probes located at different chromosome regions could create rich signals for accurate identification of each of the H.villosa chromosomes.Further FISH of these probes in five different H.villosa accessions showed that the signal pattern for the five accessions are similar.However,we observed that the FISH signals for all probes were less intensivein accession VP20 than other four accessions.There are signal polymorphism for some chromosomes among different accessions,such as chromosomes 3 V and 7V.This indicated that there was diversification in species H.villosa.2.Development of specific EST-PCR markers for chromosome 6VL of H.villosaA total of 297 primer pairs were designed according to the EST sequences previously allocated in different bins of wheat chromosome 6AL,6BL and 6DL.These primer pairs were used for amplification using the genomic DNA of Chinese spring(CS),CS nulli-tetrasomic lines(N6AT6B,N6BT6A and N6DT6A),H.villosa,T.durum,T.durum-H.villosa amphiploidy(AABBVV),T.aestivum-H.villosa disomic addition lines(DA1V-DA7V),translocation lines(T6VS·6AL,T6AS·6VL).Among them,32 primer pairs could amplify specific bands for chromosome 6V.Thirty-two were specific markers for 6VL,and the remained 1 was specific marker for 6VS based on the PCR in the two translocation lines.3.Induction of 6VL chromosome aberrations using the CS phlb mutantThe translocation line T6AS·6VL which is homozygous for ph1b were used to cross with the CS ph1b mutant.The derived 1028 F2 plants were characterized by Genomic in situ hybridization(GISH).Among them,a total of 70 6VL chromosome aberrations were identified,including 55 terminal translocations and 15 interstitial translocations.These potential 6VL translocations were self-fertilized and by GISH of the F4 population,22 homozygous 6VL translocations involving were identified,including 19 terminal translocations,3 interstitial translocations.In addition,3 were identified to be 6VL ditelosomic lines.4.Induction of 6VL chromosome aberrations using irradiation of the T6AS·6VLThe pollen of translocation line T6AS·6VL were irradiated by 60Co-y ray(1,200 rad)before flowering,and then pollinated to emasculated spikes of common wheat variety Yangmai 158 or Yangmai 15.The M1 plants were screened by GISH for 6VL chromosome aberrations.In the 115 M1 plants derived from Yangmail58×T6AS·6VL,the 10 6VL chromosome aberrations were identified,including 4 terminal translocations,2 interstitial translocations and 4 other unclassified types.In the 165 M1 plants derived from Yangmai15×T6AS·6VL,the 28 6VL chromosome aberrations were identified including 14 terminal translocations,5 interstitial translocations,6 deletion chromosomes and 3 other unclassified types.The mature female gametes of the esmaculated spikes of T6AS·6VL were irradiated by 60Co-γ ray(1,200 rad)before flowering,and then pollinated using normal pollens of common wheat variety Nannong 06Y86.The derived 96 Mi plants were screened by GISH for 6VL chromosome aberrations.Eleven 6VL chromosome aberrations were identified,including 5 terminal translocations,2 interstitial translocations,2 deletion chromosomes and 3 other unclassified types.The above identified 6VL chromosome aberrations were further self-fertilized or backcrossed to common wheat.GISH of the derived M3 or BC1F2 progenies showed that 40 were 6VL chromosome aberrations,including 13 homozygous translocations[the 2 is from(Yangmai158×T6AS·6VL)M3,the 7 is from(Yangmai 15 ×T6AS·6VL)M3,the 1 is from(Yangmai 15 ×T6AS·6VL)×Yangmai 15 BC1F2,the 2 is from(T6AS·6VL×Nannong 06Y86)M3,the 1 is from(T6AS·6VL×Nannong 06Y86)×Nannong 06Y86 BC1F2],the 14 heterozygous translocation and 13 unclassified types.5.Cytological physical map of 6VLBased on the amplification results of 31 6VL-specific EST-PCR markers and 716VL-specific IT markers in the chromosome aberrations by CS ph1b,102 molecular markers specific for the short arm of chromosome 6VL were further allocated to 9 different regions.