Group Crop Health, Faculty of Agricultural and Environmental Sciences, University of Rostock, Satower Str. 48, 18059, Rostock, Germany.
Saatzucht Steinach GmbH & Co KG, Wittelsbacherstrasse 15, 94377, Steinach, Germany.
Theor Appl Genet. 2016 Oct;129(10):1915-32. doi: 10.1007/s00122-016-2749-4. Epub 2016 Jul 19.
Molecular markers including a potential resistance gene co-segregating with the LpPg1 stem rust resistance locus in perennial ryegrass were identified by massive analysis of cDNA ends (MACE) transcriptome profiling. Stem rust caused by Puccinia graminis subsp. graminicola is a severe fungal disease in the forage crop perennial ryegrass and other grasses. The previously identified LpPg1 locus confers efficient resistance against the pathogen. The aim of this study was to identify candidate genes involved in rust resistance and to use them as a resource for the development of molecular markers for LpPg1. To identify such candidates, bulked segregant analysis was combined with NGS-based massive analysis of cDNA ends (MACE) transcriptome profiling. Total RNA was isolated from bulks of infected and non-infected leaf segments from susceptible and resistant genotypes of a full-sibling mapping population and their respective parental lines and MACE was performed. Bioinformatic analysis detected 330 resistance-specific SNPs in 178 transcripts and 341 transcripts that were exclusively expressed in the resistant bulk. The sequences of many of these transcripts were homologous to genes in distinct regions of chromosomes one and four of the model grass Brachypodium distachyon. Of these, 30 were genetically mapped to a 50.8 cM spanning region surrounding the LpPg1 locus. One candidate NBS-LRR gene co-segregated with the resistance locus. Quantitative analysis of gene expression suggests that LpPg1 mediates an efficient resistance mechanism characterized by early recognition of the pathogen, fast defense signaling and rapid induction of antifungal proteins. We demonstrate here that MACE is a cost-efficient, fast and reliable tool that detects polymorphisms for genetic mapping of candidate resistance genes and simultaneously reveals deep insight into the molecular and genetic base of resistance.
通过大规模 cDNA 末端分析 (MACE) 转录组分析，鉴定了与多年生黑麦草 LpPg1 抗秆锈病基因共分离的分子标记。禾柄锈菌 (Puccinia graminis subsp. graminicola) 引起的秆锈病是饲料作物多年生黑麦草和其他禾本科植物的一种严重真菌病害。先前鉴定的 LpPg1 基因座赋予了对病原体的有效抗性。本研究旨在鉴定参与锈病抗性的候选基因，并将其用作 LpPg1 分子标记开发的资源。为了鉴定这些候选基因，结合了混池分离分析和基于 NGS 的大规模 cDNA 末端分析 (MACE) 转录组分析。从易感和抗性全同胞图谱群体的感病和抗性基因型的受感染和未感染叶段的混池分离物中分离总 RNA，并进行 MACE。生物信息学分析在 178 个转录本和 341 个仅在抗性混池中表达的转录本中检测到 330 个抗性特异性 SNP。这些转录本的许多序列与模式禾本科植物短柄草染色体 1 和 4 上不同区域的基因同源。其中 30 个被遗传定位到围绕 LpPg1 基因座的 50.8 cM 跨度区域。一个候选 NBS-LRR 基因与抗性基因座共分离。基因表达的定量分析表明，LpPg1 介导一种有效的抗性机制，其特征在于对病原体的早期识别、快速防御信号和快速诱导抗真菌蛋白。我们在这里证明，MACE 是一种具有成本效益、快速可靠的工具，可用于候选抗性基因的遗传作图中的多态性检测，同时深入了解抗性的分子和遗传基础。
