应用 bulk segregant RNA-Seq（BSR-Seq）和等位基因特异性引物研究大豆白粉病抗性。

Application of bulk segregant RNA-Seq (BSR-Seq) and allele-specific primers to study soybean powdery mildew resistance.

机构信息

Master Program for Plant Medicine, National Taiwan University, Taipei, 106319, Taiwan.

Taoyuan District Agricultural Research and Extension Station. Ministry of Agriculture, Taoyuan, 327005, Taiwan.

出版信息

BMC Plant Biol. 2024 Mar 1;24(1):155. doi: 10.1186/s12870-024-04822-1.

DOI:10.1186/s12870-024-04822-1

PMID:38424508

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC10905810/

Abstract

BACKGROUND

Powdery mildew (PM) is one of the important soybean diseases, and host resistance could practically contribute to soybean PM management. To date, only the Rmd locus on chromosome (Chr) 16 was identified through traditional QTL mapping and GWAS, and it remains unclear if the bulk segregant RNA-Seq (BSR-Seq) methodology is feasible to explore additional PM resistance that might exist in other varieties.

RESULTS

BSR-Seq was applied to contrast genotypes and gene expressions between the resistant bulk (R bulk) and the susceptible bulk (S bulk), as well as the parents. The ∆(SNP-index) and G' value identified several QTL and significant SNPs/Indels on Chr06, Chr15, and Chr16. Differentially expressed genes (DEGs) located within these QTL were identified using HISAT2 and Kallisto, and allele-specific primers (AS-primers) were designed to validate the accuracy of phenotypic prediction. While the AS-primers on Chr06 or Chr15 cannot distinguish the resistant and susceptible phenotypes, AS-primers on Chr16 exhibited 82% accuracy prediction with an additive effect, similar to the SSR marker Satt431.

CONCLUSIONS

Evaluation of additional AS-primers in the linkage disequilibrium (LD) block on Chr16 further confirmed the resistant locus, derived from the resistant parental variety 'Kaohsiung 11' ('KS11'), not only overlaps with the Rmd locus with unique up-regulated LRR genes (Glyma.16G213700 and Glyma.16G215100), but also harbors a down-regulated MLO gene (Glyma.16G145600). Accordingly, this study exemplified the feasibility of BSR-Seq in studying biotrophic disease resistance in soybean, and showed the genetic makeup of soybean variety 'KS11' comprising the Rmd locus and one MLO gene.

摘要

背景

白粉病（PM）是大豆的重要病害之一，利用宿主抗性可有效防治大豆白粉病。迄今为止，仅在第 16 号染色体（Chr）上通过传统的 QTL 作图和 GWAS 鉴定了 Rmd 位点，而利用大量分离群体 RNA 测序（BSR-Seq）方法是否能探索其他品种中可能存在的额外的 PM 抗性尚不清楚。

结果

BSR-Seq 用于对比抗品种和感品种之间的基因表达差异，以及亲本之间的差异。通过 ∆（SNP-index）和 G'值在 Chr06、Chr15 和 Chr16 上确定了几个 QTL 和显著的 SNP/Indels。利用 HISAT2 和 Kallisto 鉴定了这些 QTL 内的差异表达基因（DEGs），并设计了等位基因特异性引物（AS-primers）来验证表型预测的准确性。虽然 Chr06 或 Chr15 上的 AS-primers 不能区分抗品种和感品种，但 Chr16 上的 AS-primers 表现出 82%的准确性预测，且具有加性效应，与 SSR 标记 Satt431 相似。

结论

在 Chr16 的连锁不平衡（LD）块中进一步评估了其他 AS-primers，进一步证实了抗性位点来源于抗性亲本品种“高雄 11 号”（‘KS11’），不仅与 Rmd 位点重叠，且有独特上调的 LRR 基因（Glyma.16G213700 和 Glyma.16G215100），还含有下调的 MLO 基因（Glyma.16G145600）。因此，本研究例证了 BSR-Seq 用于研究大豆生物亲和性病害抗性的可行性，并展示了大豆品种“KS11”的遗传组成，包含 Rmd 位点和一个 MLO 基因。