In this study, we developed and optimized a multiplex polymerase chain reaction (PCR) for simultaneous detection of two important leaf rust resistance genes Lr24 and Lr68. Seedling resistance gene Lr24 and slow rusting gene Lr68 encoding resistance to leaf rust were identified in forty-one advanced salt-tolerant wheat genotypes using molecular markers viz. Sr24#50 and csGS, respectively. For re-validation of PCR reaction results, a seedling resistance test (SRT) was performed in controlled glasshouse conditions. Genotypes were evaluated under epiphytotic conditions for SRT by artificial inoculation with a spectrum of Puccinia triticina pathotypes. Results of SRT were supplemented by PCR results, indicated that the gene Lr24/Sr24 conferred leaf rust resistance in two wheat genotypes namely HD 2851 and KRL2029. In advanced line KRL2029, along with Lr24/Sr24 other rust resistance genes (Lr26/Sr31/ Yr9) were also present. Molecular marker csGS for a known slow rusting gene Lr68 was amplified in fifteen salt-tolerant wheat genotypes indicated the presence of Lr68 in these lines. These lines could also be utilized as donors for slow rusting characteristics. Combination of both the genes could provide resistance to leaf rust and stem rust. Presences of respective genes in salt-tolerant lines were confirmed by SRT as well as molecular markers separately and multiplex PCR assay was designed. Multiplex PCR method allowed to reduce the cost and time of analyses as well as reducing the workload.

在本研究中，我们开发并优化了多重聚合酶链反应 (PCR)，用于同时检测两个重要的叶锈病抗性基因Lr24和Lr68。使用分子标记，在41个高级耐盐小麦基因型中鉴定了编码叶锈病抗性的幼苗抗性基因Lr24和慢锈病基因Lr68。分别为Sr24#50和csGS。为了重新验证 PCR 反应结果，在受控温室条件下进行了幼苗抗性测试 (SRT)。在 SRT 的附生条件下，通过人工接种一系列小麦锈菌 (Puccinia triticina) 来评估基因型病理类型。SRT结果与PCR结果相辅相成，表明基因Lr24/Sr24赋予HD 2851和KRL2029两种小麦基因型叶锈病抗性。在高级品系 KRL2029 中，除了Lr24/Sr24 外，还存在其他锈病抗性基因 ( Lr26/Sr31/Yr9 )。已知慢锈基因Lr68 的分子标记csGS在 15 个耐盐小麦基因型中扩增，表明这些品系中存在Lr68 。这些线也可以用作缓慢生锈特性的供体。这两个基因的组合可以提供对叶锈病和茎锈病的抗性。通过SRT和分子标记分别确认耐盐品系中各个基因的存在，并设计了多重PCR测定。多重 PCR 方法可以降低分析的成本和时间，并减少工作量。