Institute of Plant Protection, Henan Academy of Agricultural Sciences; Key Laboratory of Crop Integrated Pest Management of the Southern of North China, Ministry of Agriculture of the People's Republic of China, Zhengzhou, 450002, China.
School of Life Sciences, Henan University, Kaifeng, 475001, China.
BMC Plant Biol. 2022 Feb 23;22(1):83. doi: 10.1186/s12870-022-03448-5.
Wheat powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is an important disease affecting wheat production. Planting resistant cultivars is an effective, safe, and economical method to control the disease. Map construction using next-generation sequencing facilitates gene cloning based on genetic maps and high-throughput gene expression studies. In this study, specific-locus amplified fragment sequencing (SLAF) was used to analyze Huixianhong (female parent), Hongyoumai (male parent) and two bulks (50 homozygous resistant and 50 susceptible F segregating population derived from Huixianhong × Hongyoumai to determine a candidate gene region for resistance to powdery mildew on the long arm of chromosome 7B in wheat landrace Hongyoumai. Gene expressions of candidate regions were obtained using bulked segregant RNA-seq in 10 homozygous resistant and 10 susceptible progeny inoculated by Bgt.. Candidate genes were obtained using homology-based cloning in two parents.
A 12.95 Mb long candidate region in chromosome 7BL was identified, and five blocks in SLAF matched the scaffold of the existing co-segregation marker Xmp1207. In the candidate region, 39 differentially expressed genes were identified using RNA-seq, including RGA4 (Wheat_Chr_Trans_newGene_16173)-a disease resistance protein whose expression was upregulated in the resistant pool at 16 h post inoculation with Bgt. Quantitative reverse transcription (qRT)-PCR was used to further verify the expression patterns in Wheat_Chr_Trans_newGene_16173 that were significantly different in the two parents Hongyoumai and Huixianhong. Two RGA4 genes were cloned based on the sequence of Wheat_Chr_Trans_newGene_16173, respectively from two parent and there was one amino acid mutation: S to G in Huixianhong on 510 loci.
The combination of SLAF and BSR-seq methods identified a candidate region of pmHYM in the chromosome 7BL of wheat landrace cultivar Hongyoumai. Comparative analysis between the scaffold of co-segregating marker Xmp1207 and SLAF-seq showed five matching blocks. qRT-PCR showed that only the resistant gene Wheat_Chr_Trans_newGene_16173 was significantly upregulated in the resistant parent Hongyoumai after inoculation with Bgt, and gene cloning revealed a difference in one amino acid between the two parent genes, indicating it was involved in the resistance response and may be the candidate resistance gene pmHYM.
小麦白粉病由禾布氏白粉菌（Bgt）引起，是影响小麦生产的重要病害。种植抗性品种是控制病害的有效、安全、经济的方法。基于遗传图谱和高通量基因表达研究，利用下一代测序进行图谱构建有助于基因克隆。在这项研究中，利用特异位点扩增片段测序（SLAF）分析了小麦地方品种红花麦（母本）、红优麦（父本）和两个群体（50 个纯合抗白粉病和 50 个感病的 F 分离群体），以确定红花麦染色体 7B 长臂上抗白粉病的候选基因区域。利用 Bgt 接种的 10 个纯合抗性和 10 个纯合感病后代的混池 RNA-seq 获得候选区域的基因表达。在两个亲本中，利用同源克隆获得候选基因。
在染色体 7BL 上鉴定到一个 12.95 Mb 长的候选区域，SLAF 中有 5 个块与现有共分离标记 Xmp1207 的支架匹配。在候选区域中，通过 RNA-seq 鉴定到 39 个差异表达基因，包括 RGA4（小麦_染色体_新基因_16173）-一种抗病蛋白，其在接种 Bgt 后 16 小时的抗性池中转录水平上调。利用定量反转录（qRT）-PCR 进一步验证了在两个亲本红花麦和灰红米中表达模式显著不同的 Wheat_Chr_Trans_newGene_16173。根据 Wheat_Chr_Trans_newGene_16173 的序列，分别从两个亲本中克隆了两个 RGA4 基因，在灰红米的 510 位上有一个氨基酸突变：S 到 G。
SLAF 和 BSR-seq 方法的结合鉴定了小麦地方品种红花麦 7BL 上的 pmHYM 候选区域。共分离标记 Xmp1207 支架与 SLAF-seq 的比较分析显示有 5 个匹配块。qRT-PCR 显示，接种 Bgt 后，抗性亲本红花麦中仅 Wheat_Chr_Trans_newGene_16173 基因显著上调，基因克隆显示两个亲本基因在一个氨基酸上存在差异，表明该基因可能参与了抗性反应，可能是候选抗性基因 pmHYM。
