Development Of Wheat-h.villosa 3V Chromosome Structural Aberrantions And Physical Localization Of Gliadin Subunits On Wheat-h.villosa 4VL Chromosome

Haynaldia villosa(2n=2x=14)is one of the diploid wild relatives of wheat.It has many good characteristics needed for wheat cultivation,such as disease resistance,cold and drought resistance,strong tillering ability,high protein content,etc.The development of alien chromosome lines such as addition/substitution and especially small fragment translocation will greatly facilitate the study and utilization of alien genes.There are many potential excellent genes.on the 3V,4VL chromosomes of wheat,such as resistance to eye spot,total erosion,fusarium mosaic,salt tolerance and the presence of a site encoding α-gliadin.In order to better explore and utilize the excellent genes of 3V and 4VL chromosomes in H.villosa,a complete set of alien addition lines of wheat-H.villosa were identified for resistance to stripe rust.The resistance genes of H.villosa were excavated;In order to locate the excellent genes on the 3V and 4VL chromosomes of H.villosa,the structural variants involving different fragment sizes of 3V and 4VL chromosomes of H.villosa were induced by the the Chinese Spring ph1b mutants and pollen irradiation;the structural variants involving different fragment sizes of 3V and 4VL chromosomes of H.villosa were identified by GISH/FISH;these structural variants were further analyzed by molecular markers,and physical map of 3V and 4VL chromosomes of H.villosa were constructed,respectively.The main results are as follows:1.Chromosome localization of stripe rust resistance in a set of alien addition lines of wheat-H.villosaIn 2019 year and 2020 year,a set of alien addition lines of wheat-H.villosa were identified for resistance to stripe rust at seedling stage and adult stage.The results indicated that DA3V showed stable resistance to stripe rust.It is suggested that the genes related to stripe rust resistance may be located on the 3 V chromosome of Haynaldia villosa.2.Development of the structural aberrantions involving the wheat-H.villosa 3V chromosomeIn order to precisely locate the genes related to stripe rust resistance on the 3V chromosome of Haynaldia villosa,F1 was obtained by crossing the Chinese Spring ph1b mutant with wheat-H.villosa alien addition line DA3V and then F1 was selfed or backcrossed.Molecular markers were used to screen the 3V chromosome and ph1bph1b in the selfed and backcrossed generations,respectively.32 and 3 plants with a single 3V chromosome and ph1bph1b were obtained from the selfed and backcrossed generations,respectively.During 2018-2019.the progenies of the 35 plants identified by the previous generation were germinated and transplanted,and 506 single plants were obtained.In order to speed up the efficiency of identification of the structural variants of the 3V chromosome,five molecular markers(CINAU1242,CINAU1246,CINAU1239,CINAU1173,CINAU1156)located in different segments of the 3V chromosome were used to analyze the 506 plants,and 65 potential recombinant exchange plants were screened.In order to obtain stable 3V chromosomal structural variants,GISH/FISH analysis was carried out on the recombinant exchange strains selected from the previous generation from 2019 to 2020.Among the 337 F4 and BC1F3 single plants of(DA3V×CS ph1bph1b),111 chromosomal structural variants were obtained,including 18 different types:4 large fragment translocation lines,4 terminal translocation lines,2 homozygous whole arm translocation lines,2 telosomics,and 6 complex chromosome structural variation types.3.Cytological physical map of 3V chromosome of H.villosaBased on the amplification results of specific markers in the structural variants,211 IT markers specific for the 3V chromosome were further allocated to 9 different regions,and the physical map of 3V was constructed.4.Induction of 4VL chromosomal structural variants of Triticum aestivum by CS ph1bph1b mutant and pollen radiationFrom 2018 to 2019,CS ph1bph1b mutant was used to cross with the T4VL·4DS homozygous arm translocation line(having ph1b recessive homozygous background)and in the progenies of backcrossing of the pollen radiation of T.durum-H.villosa by 60Co-γ ray,the results of GISH/FISH analysis showed that 14 chromosome structural variants were obtained,including 1 terminal translocations,1 whole arm translocations,2 insertion translocations,4 deletion lines and 6 other materials.5.Cytological physical map of 4VL chromosome of H.villosaBased on the amplification results of specific markers in the structural variants,73 IT markers specific for the 4VL chromosome were further allocated to 6 different regions,and the physical map of 4VL was constructed.6.Physical location of α-gliadin in the 4VL chromosome of H.villosaIn order to further locate the gliadin subunit on the 4VL chromosome of H.villosa,we used SDS-PAGE gel electrophoresis to analyze 14 different types of structural variants,and found that T4VL·4DS homozygous whole arm translocation line(18SJJ3-14)and two heterozygous terminal translocation lines(18SJJ59-7 and 18SJJ59-2)could amplify the specific bands of α-gliadin on the 4VL chromosome of H.villosa,while the other structural variants(two homozygous deletion and interstitial translocation lines)had no specific bands of gliadin.Combined with SDS-PAGE,cytology and molecular markers,α-gliadin was finally located at the terminal region of 4VL between CINAU1981 and CINAU1232.According to the reference genomic data(unpublished),α-gliadin in the 4VL chromosome of H.villosa was located between 448Mb-483Mb of 4VL physical segment.