Efficient Induction And Identification Of Translocation With Small Segments From Short Arm Of Chromosome 6V Of Haynaldia Villosa

Wheat production and quality are affected by biotic and abiotic stresses sereiously,and the improvement of the resistance to diseases and pests,and the adaptability to various environments are the main objectives of modern wheat breeding.H.villosa(syn. Dasypyrum villosum L.,2n=14,VV) have been proved to be highly resistant to wheat diseases and pests,tolerant to coldness and dryness,and is considered as an important genetic resource for wheat improvement.The translocation lines with small alien fragment, especially the interstitial translocation lines are best for use of alien useful genes in wheat breeding.However,up to now,few Triticum aestivum-H.villosa translocation lines with small alien segment have been reported.Therefore,it is necessary to establish a high efficient strategy for the induction and identification of alien translocation lines.In the present study,the mature female gametes of wheat-H.villosa 6VS/6AL translocation line were irradiated by ⁶⁰Co-γray with 160Rad/M dosage rate and three dosages(1600,1920,2240Rad) before flowering.These irradiated florets were emasculated the same day,and then covered with paper bags.After 2-3 days,these florets were pollinated with normal fresh pollens from T.aestivum cv.Chinese Spring.Genomic in situ hybridization(GISH) at mitosis metaphase of root-tip cell of M₁ plants was used to detect the chromosomes structural changes involving 6VS of H.villosa.Among the 534 M₁ plants screened,97 plants contained chromosome structural changes with small segment of 6VS,including 80 interstitial translocated chromosomes,57 terminal translocated chromosomes and 55 deleted chromosomes.For the 2240 Rad dosage treatment,the induction frequencies of interstitial translocation,terminal translocation and deletion were 21.02%,14.01%,and 14.65%respectively,which were much higher than that previously reported.The M₂ seeds were obtained by backcrossing of 74 M₁ plants involving 146 chromosomes with structural changes of 6VS,and it was found that the structure aberrations in the M₁ plants could be transmitted to their progenies.Irradiating mature female gametes of whole arm translocation may be a new and highly efficient approach for creation of small segment chromosome structural changes,especially for interstitial translocations.These small fragments of 6VS obtained in the present research are different in both length of fragment and breakage location,and are useful for fine location of Pm21 gene,physical mapping of 6VS and development of translocation lines with small fragment.Using a software PRIMER5.0PCR,primers were designed according to the sequences of ESTs located in the different bins of the short arm of homeologous group 6 chromosomes of wheat.Three molecular markers specific for 6VS(6BS28-386,6DS38-730,6AS6-740) were developed.Twelve deletion lines involved in the short arm of homeologous group 6 chromosomes of wheat and H.villosa were used to physically locate 8 specific markers of 6VS.The markers CINAU17-1086,6VS19-381 were located in the chromosome bin of FL (fragment length) value 0.58-1.00 and 0.58-0.99,the marker CINAU15-902 was located in the chromosome bin of FL value 0.45-0.58,the markers CINAU16-1650,6DS38-730, 6BS28-386 were located in the chromosome bin of FL value 0.35-0.45,and the markers CINAU18-723 and 6AS6-740 were located in the bin of FL value 0.00-0.45.These markers are usefull both for identification of structural changes with 6VS and fine mapping of Pm21.Through the back-crosses between some M₁ plants with chromosome changes(female parent) and common wheat(male parent),8 M₂ plants with single small interstitial translocation chromosomes of 6VS have been obtained.The above 8 6VS specific molecular markers were used to define the fagment of 6VS.The fragment of 6VS in translocation line NJ9-30,NJ2-2(or NJ2-3) had FL value 0.00-0.58,0.35-0.58,respectively. The fragment of 6VS in the other lines(NJ6-23,NJ6-14 etc) was involved in the segment of FL value 0.00-0.35.Two heterozygous insterstitial translocation lines with small segment of 6VS,which showed high resistance to powdery mildew,were selected,and homozygous interstitial translocation lines with Pm21 will be obtained.The Pm21 gene was further located in the region of 6VS with FL value 0.35-0.58 by using these genetic stocks of 6VS and powdery mildew resistance identification.