National Key Facility for Crop Gene Resources and Genetic Improvement / Key Laboratory of Biology and Genetic Improvement of Triticeae Crops, Ministry of Agriculture / Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, 100081, China.
J Appl Genet. 2013 Nov;54(4):417-26. doi: 10.1007/s13353-013-0172-y. Epub 2013 Oct 1.
Wheat-Dasypyrum villosum translocated chromosomes T6V#2S•6AL and T6V#4S•6DL are known to confer excellent resistance to wheat powdery mildew (PM). However, it is difficult to distinguish the two sources of PM resistance genes through multi-pathotype testing because to date no virulence for them has been found. To reveal the relationship between the PM resistance genes from the two translocations, the sequence of the Stpk-V gene, a key member of powdery mildew resistance locus Pm21, was used as a reference to isolate homologous genes from a D. villosum accession No.1026 and its derivatives 6V#4(6D) disomic substitution (DS) line RW15 and T6V#4S•6DL translocation line Pm97033. Two genes Stpk-V2 and Stpk-V3 were cloned from No.1026. Sequence alignment showed that Stpk-V2 and Stpk-V3 shared 98.2 % and 96.2 % of their DNA and 99.3 % and 100 % of their amino acids in identity with Stpk-V. Compared with Stpk-V, a 22-bp direct sequence repeat and a miniature inverted-repeat transposable element (MITE) were found in the intron 4 of Stpk-V2 and Stpk-V3, respectively. However, Stpk-V2 was not present in DS line RW15 and translocation line Pm97033 based on the PCR result, indicating that Stpk-V2 did not contribute to the PM resistance of RW15 and Pm97033. In the promoter region, a 78-bp insertion was found not only in Stpk-V2 and Stpk-V3, but also in its orthologous gene Stpk-A of wheat. In addition, there was a 17 bp/8 bp deletion/insertion in the putative promoter of Stpk-V3 in comparison with that of Stpk-V/Stpk-V2. Real-time quantitative RT-PCR analysis indicated that the expression levels of Stpk-V and Stpk-V3 genes in the translocation lines were induced by the pathogen, but Stpk-V had a higher expression level than Stpk-V3 at 12 h after inoculation with Bgt. The diversity of Stpk-V gene will help to explore new resistance genes to PM in D. villosum for wheat breeding.
已知小麦-长穗偃麦草易位染色体 T6V#2S•6AL 和 T6V#4S•6DL 可赋予小麦对白粉病（PM）的优异抗性。然而，由于迄今为止尚未发现针对它们的毒力，因此通过多病原体测试很难区分这两个 PM 抗性基因来源。为了揭示来自这两个易位的 PM 抗性基因之间的关系，使用 Pm21 抗病基因座的关键成员 Stpk-V 基因的序列作为参考，从长穗偃麦草 No.1026 及其衍生的 6V#4(6D) 二体代换（DS）系 RW15 和 T6V#4S•6DL 易位系 Pm97033 中分离同源基因。从 No.1026 克隆了两个基因 Stpk-V2 和 Stpk-V3。序列比对表明，Stpk-V2 和 Stpk-V3 在 DNA 上的同源性为 98.2%，在氨基酸上的同源性为 99.3%，与 Stpk-V 相同。与 Stpk-V 相比，Stpk-V2 和 Stpk-V3 的内含子 4 中分别发现了一个 22-bp 的直接序列重复和一个微型反向重复转座元件（MITE）。然而，根据 PCR 结果，Stpk-V2 不存在于 DS 系 RW15 和易位系 Pm97033 中，表明 Stpk-V2 并未导致 RW15 和 Pm97033 对 PM 的抗性。在启动子区域，不仅在 Stpk-V2 和 Stpk-V3 中，而且在其同源基因小麦中的 Stpk-A 中发现了一个 78-bp 的插入。此外，与 Stpk-V/Stpk-V2 相比，Stpk-V3 的假定启动子中存在一个 17bp/8bp 的缺失/插入。实时定量 RT-PCR 分析表明，在病原菌接种后 12 小时，转座系中 Stpk-V 和 Stpk-V3 基因的表达水平被诱导，但接种 Bgt 后 Stpk-V 的表达水平高于 Stpk-V3。Stpk-V 基因的多样性将有助于探索小麦长穗偃麦草中 PM 的新抗性基因。
