Validation of gene candidate through complementation experiment is crucial and also considered as the gold standard proof of gene identity. Here we report the complementation results through wheat transformation1 of the BED domain containing wheat stripe rust resistance gene candidates Yr5(Yr5a) and YrSP(Yr5b) that were isolated in our previous study2.
A comparative genomic strategy was utilised for extending of the UTRs of Yr5 and YrSP. We used the Yr5 and YrSp sequences to identify haplotypes present in the sequenced genomes of the wheat cultivars Cadenza and Claire. Sequence alignments facilitated the design of conserved primers across the predicted UTR regions. Amplification products were then generated from the wheat isolines (Avocet +Yr5 and Avocet+YrSp wild type genotypes) based on a combination of conserved and Yr5/YrSp candidate gene specific primers. The PCR products amplified from each wildtype genomic DNA were then subcloned into E. coli in order to obtain intact sequence. The final confirmed native 5’ regulatory element (RE) for Yr5 and YrSP candidate genes were 744bp and 671bp, respectively, and the 3’ RE for Yr5 and YrSP were 1,500bp and 2,092bp, respectively.
To validate the candidate gene of Yr5 and to ensure its expression, two constructs were used to generate transgenic wheats in the cultivar Fielder, because the 5’ RE obtained through comparative genomic approach was considered to be suboptimal (744bp). One construct was assembled with the native 5’ and 3’ REs, 744bp and 1,500bp, respectively, and designated as Fielder:Yr5:NativeRE. The second construct, designated as Fielder:Yr5:Sr33RE, was designed by fusing the short native 5’ (225bp) and 3’ Res (47bp) initially obtained via MutRenSeq (Mutagenesis and Resistance gene Enrichment and Sequencing), with the gene regulatory elements from Sr33 (1,531bp for 5’ RE and 996bp for 3’ RE). We obtained 18 and 13 independent primary transgenic T0 plants carrying the Fielder:Yr5:NativeRE and Fielder:Yr5:Sr33RE, respectively. All independent T0 plants with either Fielder:Yr5:NativeRE or Fielder:Yr5:Sr33RE showed resistance to stripe rust pathotype 134 E16 A+17+27+ (PBI rust Culture No. 617), while all the empty vector-transformed Fielder controls were susceptible.
A similar approach was also adopted with two YrSp gene constructs for transformation. One construct was assembled with the native 5’ and 3’ RE, 671bp and 2,092bp, respectively, and designated as Fielder:YrSP:NativeRE. The second construct, designated as Fielder:YrSP:Sr33RE, fused the native 5’ and 3’ REs obtained from MutRenSeq, 225bp and 2,092bp, respectively, together with the gene RE from Sr33 (1,531bp at 5’ UTR and 996bp at 3’ UTR). We obtained 18 and 20 independent T0 plants carrying the Fielder:YrSP:NativeRE and Fielder:YrSP:Sr33RE, respectively. All independent T0 plants with Fielder:YrSP:NativeRE and Fielder:YrSP:Sr33RE showed resistance to stripe rust pathotype 134 E16 A+17+27+, while all the empty vector-transformed Fielder controls were susceptible.
Our transformation results confirmed the candidates for both Yr5 and YrSP, and not only the native full-length regularatory elements, but also the heterologous REs are sufficient to confer resistance.