Wheat streak mosaic virus (WSMV), vectored by Wheat curl mite, has been of great economic importa... more Wheat streak mosaic virus (WSMV), vectored by Wheat curl mite, has been of great economic importance in the Great Plains of the United States and Canada. Recently, the virus has been identified in Australia, where it has spread quickly to all major wheat growing areas. The difficulties in finding adequate natural resistance in wheat prompted us to develop transgenic resistance based on RNA interference (RNAi). An
Resistance to both barley yellow dwarf virus (BYDV) and cereal yellow dwarf virus (CYDV) has been... more Resistance to both barley yellow dwarf virus (BYDV) and cereal yellow dwarf virus (CYDV) has been demonstrated in wheat genetic stocks with Thinopyrum intermedium chromatin. A number of resistance-bearing translocations have been reported on chromosome arm 7DL from two independent Th. intermedium sources; one source is the addition line L1 and the other is the spontaneous substitution line P29. Another source of resistance in wheat cytogenetic stocks is available as a 2Ai(2D) substitution line. We used a set of 38 molecular markers and the available deletion stocks to compare the size of the 7DL translocations more comprehensively than has been done previously. We also compared the efficacy of BYDV resistance of the various genetic stocks both before and after transfer to a common genetic background. TC14 was confirmed as carrying the smallest translocation, replacing about 20% of the distal end of 7DL. TC5 and TC10 had 90% of the chromosome arm replaced by Th. intermedium chromatin; the proximal 10% corresponded to wheat chromatin. YW642 appeared to have the whole 7DL replaced by Th. intermedium chromatin, as confirmed by the co-dominant marker cfd68 mapping on the bin nearest the centromere. Translocation line P961341 had bins 3, 7, and 8 replaced by Th. intermedium chromatin, making this the second smallest translocation with BYDV and CYDV resistance. The translocation sizes reported here differ from some of the previous estimates. The translocated Th. intermedium segments appeared to be bigger than the replaced wheat 7DL fragments. All the resistances derived from the L1 and P29 group 7 chromosomes and the 2Ai#2 chromosome were effective in reducing the number of infected plants and the mean virus titre, regardless of the background. Some evidence is discussed suggesting the long arm of the Th. intermedium group 7 chromosome 7Ai#1 carries two resistances, the distal Bdv2 and a proximal second gene.
Rusts and barley yellow dwarf virus (BYDV) are among the main diseases affecting wheat production... more Rusts and barley yellow dwarf virus (BYDV) are among the main diseases affecting wheat production world wide for which wild relatives have been the source of a number of translocations carrying resistance genes. Nevertheless, along with desirable traits, alien translocations often carry deleterious genes. We have generated recombinants in a bread wheat background between two alien translocations: TC5, ex-Thinopyrum (Th) intermedium, carrying BYDV resistance gene Bdv2; and T4m, ex-Th. ponticum, carrying rust resistance genes Lr19 and Sr25. Because both these translocations are on the wheat chromosome arm 7DL, homoeologous recombination was attempted in the double hemizygote (TC5/T4m) in a background homozygous for the ph1b mutation. The identification of recombinants was facilitated by the use of newly developed molecular markers for each of the alien genomes represented in the two translocations and by studying derived F2, F3 and doubled haploid populations. The occurrence of recombination was confirmed with molecular markers and bioassays on families of testcrosses between putative recombinants and bread wheat, and in F2 populations derived from the testcrosses. As a consequence it has been possible to derive a genetic map of markers and resistance genes on these previously fixed alien linkage blocks. We have obtained fertile progeny carrying new tri-genomic recombinant chromosomes. Furthermore we have demonstrated that some of the recombinants carried resistance genes Lr19 and Bdv2 yet lacked the self-elimination trait associated with shortened T4 segments. We have also shown that the recombinant translocations are fixed and stable once removed from the influence of the ph1b. The molecular markers developed in this study will facilitate selection of individuals carrying recombinant Th. intermedium–Th. ponticum translocations (Pontin series) in breeding programs.
Thinopyrum intermedium translocations derived from the wheat (Triticum aestivum L.) substitution ... more Thinopyrum intermedium translocations derived from the wheat (Triticum aestivum L.) substitution line P-29 were previously characterized by RFLP. We have further analyzed these lines and additional related germplasm with publicly available STS and SSRs. Primers which showed a polymorphism between wheat and P-29, were tested in all recombinant and nulli-tetrasomic lines confirming their position on chromosome 7D. The resulting 7D/7E chromosome maps appeared as a mosaic of wheat and Th. intermedium chromatin sections. To verify the composition of the translocation lines suggested by the RFLP-PCR map, F2 progeny of two crosses (CS/216-1 and CS/260-1) were analyzed with molecular markers. Both populations gave an unexpectedly diverse number of recombinant individuals, suggesting that interstitial translocations occur more frequently than previously thought. This analysis also showed that there is a wide range in the number and position of the interstitial translocations within a given line such as the mosaic chromosome in recombinant line 260-1/CS-26, which has four Th. intermedium chromosome segments. Phenotypic data of the two populations suggested the presence of one gene which we have called Bdv3 to differentiate it from the previously reported orthologous gene Bdv2. Using the PCR-based molecular markers identified in this study, 5 out of 12 elite lines that showed good yields and no YDV symptoms contained Th. intermedium chromatin. Due to the multiple components involved in the YDV disease complex, combining selection for YDV resistance with the molecular markers and maps identified in this study will increase the efficiency of introgressing Th. intermedium chromatin containing YDV resistance or other beneficial traits into elite wheat germplasm.
Wheat streak mosaic virus (WSMV), vectored by Wheat curl mite, has been of great economic importa... more Wheat streak mosaic virus (WSMV), vectored by Wheat curl mite, has been of great economic importance in the Great Plains of the United States and Canada. Recently, the virus has been identified in Australia, where it has spread quickly to all major wheat growing areas. The difficulties in finding adequate natural resistance in wheat prompted us to develop transgenic resistance based on RNA interference (RNAi). An RNAi construct was designed to target the nuclear inclusion protein ‘a’ (NIa) gene of WSMV. Wheat was stably cotransformed with two plasmids: pStargate-NIa expressing hairpin RNA (hpRNA) including WSMV sequence and pCMneoSTLS2 with the nptII selectable marker. When T1 progeny were assayed against WSMV, ten of sixteen families showed complete resistance in transgenic segregants. The resistance was classified as immunity by four criteria: no disease symptoms were produced; ELISA readings were as in uninoculated plants; viral sequences could not be detected by RT-PCR from leaf extracts; and leaf extracts failed to give infections in susceptible plants when used in test-inoculation experiments. Southern blot hybridization analysis indicated hpRNA transgene integrated into the wheat genome. Moreover, accumulation of small RNAs derived from the hpRNA transgene sequence positively correlated with immunity. We also showed that the selectable marker gene nptII segregated independently of the hpRNA transgene in some transgenics, and therefore demonstrated that it is possible using these techniques, to produce marker-free WSMV immune transgenic plants. This is the first report of immunity in wheat to WSMV using a spliceable intron hpRNA strategy.
We searched for QTLs involved in tolerance to barley yellow dwarf (BYD), a serious viral disease ... more We searched for QTLs involved in tolerance to barley yellow dwarf (BYD), a serious viral disease of small grain cereals in two wheat populations, Opata × Synthetic (ITMI)and Frontana × INIA66 (F × I), for which marker data had previously been generated. The populations were evaluated in replicated field trials under artificial inoculation with a BYDV-PAV-Mex isolate and under disease-free conditions. Disease symptoms (yellowing, dwarfism and biomass reduction) were visually recorded and agronomic traits (number of tillers,height, biomass, yield and thousand-kernel weight) were measured on five plants per plot. Phenotypic data on all evaluated traits showed normal distribution with high correlation between visual estimates and measured values. Heritabilities were mostly moderate to high in the 114 lines of the ITMI population, and from low to moderate in the 117 lines of the F × I population. QTL analyses were based on genetic maps containing 443 loci for the ITMI population and 317 loci for the F × I population. Using composite interval mapping, 22 QTLs in the ITMI population and seven in the F × I population were detected, explaining9.8–43.3% of total phenotypic variation (σ2P)per agronomic trait in the first population, and 4.1–13.7% in the second. Individual QTLs explained less than 15.8%of σ2P. In the F × I population a minor QTL explaining 7% of σ2P for yellowing was detected on the short arm of 7D, linked to leaf tip necrosis, a morphological marker for linked genes Bdv1, Yr18 andLr34. A QTL consistently detected for several traits on 2D in the ITMI population and on the short arm of group 6 chromosome(6S) in F × I explained 10–15% of σ2P. The large number of QTLs having mostly small effects and the continuous distribution of all evaluated traits confirmed the polygenic nature and complexity of BYD tolerance in wheat.
Barley yellow dwarf (BYD) is the most important viral disease of small cereal grains. True resist... more Barley yellow dwarf (BYD) is the most important viral disease of small cereal grains. True resistance to the disease is not found in wheat (Triticum aestivum L.), but it has been introgressed from Thinopyrum intermedium (Ti) on chromosome 7DL of recombinant wheat lines designated TC. The objectives of our study were to identify a high through-put scoring tool for the presence of the translocated Th. intermedium fragment and to assess its suitability for evaluating resistance to BYDV in segregating populations. Segregation of the Ti fragment was followed in the F2 population of an Anza (bread wheat) by TC14/2*Spear (TC14) cross. Resistance to BYDV isolates PAV-Mex and MAV-Mex in F3, F4, and F5 populations was evaluated under field and/or greenhouse conditions by measuring the virus titers of infected plants using ELISA, and the number of infected plants per plot. The SSR marker gwm37 was polymorphic for the translocation. In F4 lines it was associated with the physical presence of an intact translocation on chromosome 7DL and with low virus titers of BYDV-PAV. Reductions in virus titer of 27% and 55% in the F3 and 18% and 45% in the F5 populations were observed when the fragment was present in the heterozygous and homozygous states, respectively, confirming a dosage effect of the resistance allele. A lower proportion of infected individuals in the field was associated with the presence of the fragment, indicating a mechanism that may interfere with aphid feeding or virus translocation within infected plants. Despite significant differences between groups with and without the fragment, the OD values of infected lines overlapped, and it was not possible to definitively detect the fragment based solely on ELISA. We conclude that gwm37 is a reliable marker for the Ti translocation that will allow efficient detection of the translocation in breeding populations and greatly assist in selecting BYDV-resistant wheats in the absence of the disease.
Ayala, L., van Ginkel, M., Khairallah, M., Keller, B., and Henry, M. 2001. Expression of Thinopyr... more Ayala, L., van Ginkel, M., Khairallah, M., Keller, B., and Henry, M. 2001. Expression of Thinopyrum intermedium-derived Barley yellow dwarf virus resistance in elite bread wheat backgrounds. Phytopathology 91:55-62.
Wheat streak mosaic virus (WSMV), vectored by Wheat curl mite, has been of great economic importa... more Wheat streak mosaic virus (WSMV), vectored by Wheat curl mite, has been of great economic importance in the Great Plains of the United States and Canada. Recently, the virus has been identified in Australia, where it has spread quickly to all major wheat growing areas. The difficulties in finding adequate natural resistance in wheat prompted us to develop transgenic resistance based on RNA interference (RNAi). An
Resistance to both barley yellow dwarf virus (BYDV) and cereal yellow dwarf virus (CYDV) has been... more Resistance to both barley yellow dwarf virus (BYDV) and cereal yellow dwarf virus (CYDV) has been demonstrated in wheat genetic stocks with Thinopyrum intermedium chromatin. A number of resistance-bearing translocations have been reported on chromosome arm 7DL from two independent Th. intermedium sources; one source is the addition line L1 and the other is the spontaneous substitution line P29. Another source of resistance in wheat cytogenetic stocks is available as a 2Ai(2D) substitution line. We used a set of 38 molecular markers and the available deletion stocks to compare the size of the 7DL translocations more comprehensively than has been done previously. We also compared the efficacy of BYDV resistance of the various genetic stocks both before and after transfer to a common genetic background. TC14 was confirmed as carrying the smallest translocation, replacing about 20% of the distal end of 7DL. TC5 and TC10 had 90% of the chromosome arm replaced by Th. intermedium chromatin; the proximal 10% corresponded to wheat chromatin. YW642 appeared to have the whole 7DL replaced by Th. intermedium chromatin, as confirmed by the co-dominant marker cfd68 mapping on the bin nearest the centromere. Translocation line P961341 had bins 3, 7, and 8 replaced by Th. intermedium chromatin, making this the second smallest translocation with BYDV and CYDV resistance. The translocation sizes reported here differ from some of the previous estimates. The translocated Th. intermedium segments appeared to be bigger than the replaced wheat 7DL fragments. All the resistances derived from the L1 and P29 group 7 chromosomes and the 2Ai#2 chromosome were effective in reducing the number of infected plants and the mean virus titre, regardless of the background. Some evidence is discussed suggesting the long arm of the Th. intermedium group 7 chromosome 7Ai#1 carries two resistances, the distal Bdv2 and a proximal second gene.
Rusts and barley yellow dwarf virus (BYDV) are among the main diseases affecting wheat production... more Rusts and barley yellow dwarf virus (BYDV) are among the main diseases affecting wheat production world wide for which wild relatives have been the source of a number of translocations carrying resistance genes. Nevertheless, along with desirable traits, alien translocations often carry deleterious genes. We have generated recombinants in a bread wheat background between two alien translocations: TC5, ex-Thinopyrum (Th) intermedium, carrying BYDV resistance gene Bdv2; and T4m, ex-Th. ponticum, carrying rust resistance genes Lr19 and Sr25. Because both these translocations are on the wheat chromosome arm 7DL, homoeologous recombination was attempted in the double hemizygote (TC5/T4m) in a background homozygous for the ph1b mutation. The identification of recombinants was facilitated by the use of newly developed molecular markers for each of the alien genomes represented in the two translocations and by studying derived F2, F3 and doubled haploid populations. The occurrence of recombination was confirmed with molecular markers and bioassays on families of testcrosses between putative recombinants and bread wheat, and in F2 populations derived from the testcrosses. As a consequence it has been possible to derive a genetic map of markers and resistance genes on these previously fixed alien linkage blocks. We have obtained fertile progeny carrying new tri-genomic recombinant chromosomes. Furthermore we have demonstrated that some of the recombinants carried resistance genes Lr19 and Bdv2 yet lacked the self-elimination trait associated with shortened T4 segments. We have also shown that the recombinant translocations are fixed and stable once removed from the influence of the ph1b. The molecular markers developed in this study will facilitate selection of individuals carrying recombinant Th. intermedium–Th. ponticum translocations (Pontin series) in breeding programs.
Thinopyrum intermedium translocations derived from the wheat (Triticum aestivum L.) substitution ... more Thinopyrum intermedium translocations derived from the wheat (Triticum aestivum L.) substitution line P-29 were previously characterized by RFLP. We have further analyzed these lines and additional related germplasm with publicly available STS and SSRs. Primers which showed a polymorphism between wheat and P-29, were tested in all recombinant and nulli-tetrasomic lines confirming their position on chromosome 7D. The resulting 7D/7E chromosome maps appeared as a mosaic of wheat and Th. intermedium chromatin sections. To verify the composition of the translocation lines suggested by the RFLP-PCR map, F2 progeny of two crosses (CS/216-1 and CS/260-1) were analyzed with molecular markers. Both populations gave an unexpectedly diverse number of recombinant individuals, suggesting that interstitial translocations occur more frequently than previously thought. This analysis also showed that there is a wide range in the number and position of the interstitial translocations within a given line such as the mosaic chromosome in recombinant line 260-1/CS-26, which has four Th. intermedium chromosome segments. Phenotypic data of the two populations suggested the presence of one gene which we have called Bdv3 to differentiate it from the previously reported orthologous gene Bdv2. Using the PCR-based molecular markers identified in this study, 5 out of 12 elite lines that showed good yields and no YDV symptoms contained Th. intermedium chromatin. Due to the multiple components involved in the YDV disease complex, combining selection for YDV resistance with the molecular markers and maps identified in this study will increase the efficiency of introgressing Th. intermedium chromatin containing YDV resistance or other beneficial traits into elite wheat germplasm.
Wheat streak mosaic virus (WSMV), vectored by Wheat curl mite, has been of great economic importa... more Wheat streak mosaic virus (WSMV), vectored by Wheat curl mite, has been of great economic importance in the Great Plains of the United States and Canada. Recently, the virus has been identified in Australia, where it has spread quickly to all major wheat growing areas. The difficulties in finding adequate natural resistance in wheat prompted us to develop transgenic resistance based on RNA interference (RNAi). An RNAi construct was designed to target the nuclear inclusion protein ‘a’ (NIa) gene of WSMV. Wheat was stably cotransformed with two plasmids: pStargate-NIa expressing hairpin RNA (hpRNA) including WSMV sequence and pCMneoSTLS2 with the nptII selectable marker. When T1 progeny were assayed against WSMV, ten of sixteen families showed complete resistance in transgenic segregants. The resistance was classified as immunity by four criteria: no disease symptoms were produced; ELISA readings were as in uninoculated plants; viral sequences could not be detected by RT-PCR from leaf extracts; and leaf extracts failed to give infections in susceptible plants when used in test-inoculation experiments. Southern blot hybridization analysis indicated hpRNA transgene integrated into the wheat genome. Moreover, accumulation of small RNAs derived from the hpRNA transgene sequence positively correlated with immunity. We also showed that the selectable marker gene nptII segregated independently of the hpRNA transgene in some transgenics, and therefore demonstrated that it is possible using these techniques, to produce marker-free WSMV immune transgenic plants. This is the first report of immunity in wheat to WSMV using a spliceable intron hpRNA strategy.
We searched for QTLs involved in tolerance to barley yellow dwarf (BYD), a serious viral disease ... more We searched for QTLs involved in tolerance to barley yellow dwarf (BYD), a serious viral disease of small grain cereals in two wheat populations, Opata × Synthetic (ITMI)and Frontana × INIA66 (F × I), for which marker data had previously been generated. The populations were evaluated in replicated field trials under artificial inoculation with a BYDV-PAV-Mex isolate and under disease-free conditions. Disease symptoms (yellowing, dwarfism and biomass reduction) were visually recorded and agronomic traits (number of tillers,height, biomass, yield and thousand-kernel weight) were measured on five plants per plot. Phenotypic data on all evaluated traits showed normal distribution with high correlation between visual estimates and measured values. Heritabilities were mostly moderate to high in the 114 lines of the ITMI population, and from low to moderate in the 117 lines of the F × I population. QTL analyses were based on genetic maps containing 443 loci for the ITMI population and 317 loci for the F × I population. Using composite interval mapping, 22 QTLs in the ITMI population and seven in the F × I population were detected, explaining9.8–43.3% of total phenotypic variation (σ2P)per agronomic trait in the first population, and 4.1–13.7% in the second. Individual QTLs explained less than 15.8%of σ2P. In the F × I population a minor QTL explaining 7% of σ2P for yellowing was detected on the short arm of 7D, linked to leaf tip necrosis, a morphological marker for linked genes Bdv1, Yr18 andLr34. A QTL consistently detected for several traits on 2D in the ITMI population and on the short arm of group 6 chromosome(6S) in F × I explained 10–15% of σ2P. The large number of QTLs having mostly small effects and the continuous distribution of all evaluated traits confirmed the polygenic nature and complexity of BYD tolerance in wheat.
Barley yellow dwarf (BYD) is the most important viral disease of small cereal grains. True resist... more Barley yellow dwarf (BYD) is the most important viral disease of small cereal grains. True resistance to the disease is not found in wheat (Triticum aestivum L.), but it has been introgressed from Thinopyrum intermedium (Ti) on chromosome 7DL of recombinant wheat lines designated TC. The objectives of our study were to identify a high through-put scoring tool for the presence of the translocated Th. intermedium fragment and to assess its suitability for evaluating resistance to BYDV in segregating populations. Segregation of the Ti fragment was followed in the F2 population of an Anza (bread wheat) by TC14/2*Spear (TC14) cross. Resistance to BYDV isolates PAV-Mex and MAV-Mex in F3, F4, and F5 populations was evaluated under field and/or greenhouse conditions by measuring the virus titers of infected plants using ELISA, and the number of infected plants per plot. The SSR marker gwm37 was polymorphic for the translocation. In F4 lines it was associated with the physical presence of an intact translocation on chromosome 7DL and with low virus titers of BYDV-PAV. Reductions in virus titer of 27% and 55% in the F3 and 18% and 45% in the F5 populations were observed when the fragment was present in the heterozygous and homozygous states, respectively, confirming a dosage effect of the resistance allele. A lower proportion of infected individuals in the field was associated with the presence of the fragment, indicating a mechanism that may interfere with aphid feeding or virus translocation within infected plants. Despite significant differences between groups with and without the fragment, the OD values of infected lines overlapped, and it was not possible to definitively detect the fragment based solely on ELISA. We conclude that gwm37 is a reliable marker for the Ti translocation that will allow efficient detection of the translocation in breeding populations and greatly assist in selecting BYDV-resistant wheats in the absence of the disease.
Ayala, L., van Ginkel, M., Khairallah, M., Keller, B., and Henry, M. 2001. Expression of Thinopyr... more Ayala, L., van Ginkel, M., Khairallah, M., Keller, B., and Henry, M. 2001. Expression of Thinopyrum intermedium-derived Barley yellow dwarf virus resistance in elite bread wheat backgrounds. Phytopathology 91:55-62.
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Papers by Ligia Ayala