| The Triticeae genus contains more than 400 wild species.Mining and transferring of favorable genes from these important resources can broaden the genetic diversity of wheat for breeding.Roegneria ciliaris(Trin.)Nevski(2n=4x=28,ScScYcYc),a perennial tetraploid wild relative of wheat,possess many useful traits for wheat improvement,such as resistances to Fusarium head blight,wheat streak mosaic virus,and barley yellow dwarf virus,tolerances to drought and cold,etc.In order to make more effective utilization of the excellent gene of R.ciliaris and elucidate the phylogenetic relationships between R.ciliaris and other different species,a complete set of wheat-R.ciliaris addition lines have been constructed by the chromosome engineering.In the present work,a FISH-based karyotype of R.ciliaris was established using a complete set of wheat-R.ciliaris addition lines.In order to identify the individual chromosome of R.ciliaris,molecular markers specific to the individual chromosome of R.ciliaris were developed.To evaluate the potential resistance of R.ciliaris to FHB,the Inayama komugi-R.ciliaris amphiploid and a complete set of DALs were evaluated for reaction to FHB over seven successive growth seasons.In order to locate the FHB resistance gene of R.ciliaris,chromosome aberrations involving chromosome 1Yc were induced by irradiation.Combining the analysis by FISH and molecular marker,a physical map of 1Yc chromosome was constructed.All these results have laid a solid foundation for mapping and utilization of alien genes of R.ciliaris.The main results obtained in this research are as follows:1.Establishment of a FISH-based karyotype of R.ciliaris using a complete set of wheatR.ciliaris addition linesFour repetitive DNA sequences[5S rDNA,45S rDNA,RcAfa and(GAA)10]were used as probes for FISH analysis.Based on the FISH patterns of the alien chromosomes in 14 wheat-R.ciliaris DALs.we established a FISH-based karyotype of R.ciliaris.FISH using two repetitive DNA probes showed that RcAfa signaling is much more abundant than(GAA)10 on R.ciliaris chromosomes.RcAfa signals were distributed on 11 R.ciliaris chromosomes,except 3 Yc,5Yc and 6Yc.Most signals were located at the telometric regions on both arms of 1Sc,4Sc,6Sc,1Yc,2Yc and 7Yc,on the short arms of 4Yc and 5Sc,and on the long arms of 3Sc and 7Sc.The signals of 2Sc were located at the pericentric region of its long arm.In general,FISH signals of RcAfa were more intense in genome Sc than in Yc.However,we found that 2Yc had more RcAfa signals than 2Sc,and 7Yc had more RcAfa signals than 7Sc.(GAA)10 signals were distributed on nine R.ciliaris chromosomes.The signals were mainly located at the centromeres or pericentromeric regions.Signals on 2Yc and 7Sc were located at the telomeres of their long arms;signals of 2Sc,3 Yc,5Sc and 5Yc were located at centromeric regions of their long arms;signals of 4Ycand 6Yc were located at centromeric regions of their short arms,and signals of 4Sc were located at its centromeric region.FISH using 45S rDNA as probe showed that there are two SAT chromosomes in R.ciliaris.The NOR of 1 Sc was at the middle of the short arm and the NOR of 5Sc was in the telomeric region of this short arm.These two chromosomes could be distinguished from other R.ciliaris chromosomes based on 45S rDNA probe.The presence of 5S rDNA FISH signals on the short arm was a unique character for 5Yc.To summarize,12 pairs of R.ciliaris chromosome could be unambiguously distinguished from each other by sequential GISH/FISH based on combining these four probes.However,1 Yc and 7Yc showed similar FISH patterns and could not distinguish using the current probes.2.Development and assignment of molecular markers specific for R.ciliaris chromosomesMolecular markers provide a rapid approach for accurate identification of R.ciliaris chromosomes transferred into wheat background.In the present study,a total of 1917 primer pairs previously mapped to the seven homoeologous groups of wheat were used and amplified in CS,Inayama komugi(Ik),Ik-R.ciliaris amphiploid and R.ciliaris.We assigned the 774 IT markers to 14 R.ciliaris chromosomes using wheat-R.ciliaris DALs.Based on the type of amplification product,we divided 774 IT molecular markers into six types.The first type could specifically identify the Sc genome chromosomes.This type had 260 markers.which accounts for 33.59%of the total markers.We assigned 73,28,22,75,41,21 and 0 IT markers to the 1Sc-7Sc chromosome,respectively.The second type of IT molecular marker could specifically identify the Yc genome chromosome.This type had 204 markers,accounting for 26.36%of the total markers.We assigned 5,17,18,41,54,34 and 35 IT markers to chromosomes 1Yc-7Yc,respectively.The third type was co-dominant markers that can distinguish homoeologous chromosomes from Sc and Yc genomes.This type had 111 markers,accounting for 14.34%of the total number of markers.We assigned 12,16,22,44,21 and 18 IT markers to homoeologous group 2 to 6,respectively.IT markers which amplified the same specific band in homoeologous chromosomes from the Sc and the Yc genomes were classified as the fourth type.This type had 101 markers,accounting for 13.05%.The fifth type was that a specific band could be amplified on the R.ciliaris,but there were no specific bands in the 14 wheat-R.ciliaris DALs.There were 93 markers of this type,and these markers accounted for 12.66%of the total number of markers.26,14,8,10,15,7 and 13 IT markers were mapped to the homoeologous group 1 to 7,respectively.The sixth type was that a specific band could be amplified on the R.ciliaris,but there were no specific bands on the homoeologous chromosomes and there were specific bands in other homoeologous chromosomes.There were 5 markers of this type,and these markers accounted for 0.65%of the total number of markers.2,2 and 1 IT markers were mapped to the homoeologous group 5 to 7,respectively.3.Chromosome localization of FHB resistance in R.ciliarisTo evaluate the potential resistance of R.ciliaris to FHB,the IK-R.ciliaris amphiploid and a complete set of DALs were evaluated for reaction to FHB over seven successive growth seasons.Compared with the background varieties(IK and CS)and susceptible control(Mianyang 8545),the amphiploid and seven DALs(DA1Sc,DA1 Yc,DA2Yc,DA3Sc,DA4Sc,DA5 Yc and DA6Sc)showed stable FHB resistance.We presumed that these six chromosomes of R.ciliaris may confer resistance gene to FHB.In order to prove the resistance of DALs to FHB coming from the alien R.ciliaris chromosome,we used the disease resistance material DA1Yc and the susceptible wheat variety Alondra’s to create the F2 population,and analyzed the transmission and the FHB resistance of alien chromosome 1 Yc.The results showed that the transmission frequency of 1Yc chromosome with 32.56%had a significant difference to the theoretical value(χ2c=123.93>χ20.05,1=3.84)after the chi-square test,which indicated that the 1 Yc chromosome was easily lost during meiosis in common wheat background.In the F2 population,we separated the population into two groups according to molecular markers specific for alien chromosome of R.ciliaris,group one with 1Yc and group two without 1 Yc.We found that there were large differences in the degree of disease between with 1Yc and without 1Yc groups.The average disease spikelet rate of the population with 1Yc was 21%,and the average disease spikelet rate of the population without 1 Yc was 43%.The results of significant difference analysis showed that the average disease spikelet rate of these two populations was significant,indicating that the disease resistance gene may be derived from alien chromosome 1 Yc and the 1 Yc chromosome can effectively improve the resistance of FHB.4.Induction of 1Yc chromosome aberrations using irradiation of the DA1YcThe pollen of translocation line DA1 Yc were irradiated by 60Co-γ ray(1,200 rad)before flowering,and then pollinated to emasculated spikes of common wheat variety Alondra’s.The M1 plants were screened by GISH for 1Yc chromosome aberrations.In the 261 M1 plants derived from Alondra’s×DA1Yc,the 32 chromosome aberrations involving 1Yc were identified,including 2 terminal translocations,2 whole-arm translocations,8 large fragment translocations,2 telomere,1 deletion chromosome and 17 other unclassified types.The mature female gametes of the emasculated spikes of DA1 Yc,were irradiated by 60Co-γ ray(1,400 rad)before flowering,and then pollinated using normal pollens of common wheat variety Alondra’s.The derived 112 M1 plants were screened by GISH for 1Yc chromosome aberrations.23 chromosome aberrations involving 1Yc were identified,including 4 terminal translocations,2 interstitial translocations,4 whole-arm translocations,3 large fragment translocations,1 deletion chromosome and 8 other unclassified types.The above identified 1Yc chromosome aberrations were further self-fertilized or backcrossed to common wheat.GISH of the derived M3 or BC1F2 progenies identified 33 chromosome aberrations involving 1Yc,including 7 terminal translocations,13 large fragment translocations,2 whole-arm translocations,1 deletion chromosome and 10 other unclassified types.5.Cytological physical map of 1 YcBased on the amplification results of 45 1Yc-specific IT markers in the chromosome aberrations by irradiation.45 molecular markers specific for the 1Yc chromosome were further allocated to 16 different regions. |
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