水稻稻熱病抗性基因全基因體關聯定位

Abstract

稻熱病 (Rice blast) 由子囊真菌Magnaporthe oryzae 引起，為水稻產區每年發生的重要病害，抗病育種為已知最有效及環境友善的防治方法，然而病原菌演化快速，抗病品種在大面積種植後，常於3-5年內失去抗性。為有效防治此病害，必須持續發掘新的抗性基因及了解現有抗性品種的抗性來源。本研究目標是以關聯定位的方法，尋找312多樣性種原及21個商業品種之稻熱病抗性基因(座)，應用於抗病育種。在表現型分析中，我們以D41-2和12YL-DL3-2兩株具代表性的本土稻熱病菌株，於生長箱內進行接種試驗並記錄各品種病斑面積百分比、病斑型態及抗感病特性，為減少不同次接種試驗造成的誤差，病斑面積百分比的資料以最佳線性無偏估法 (best linear unbiased predictor, BLUP) 進行資料校正，並選用重複試驗中表現一致的品種資料進行關聯定位分析。在基因型分析部分，則是針對商業品種進行genotyping by sequencing (GBS) 分析，並和多樣性種原的44K SNPs資料以及臺農84號、臺南11號之全基因體次世代定序 (next-generation sequencing, NGS) 資料進行合併。GBS 和 44K SNPs 資料的重疊性小於0.05%，NGS和44K SNPs 資料則是可以正常合併。用D41-2接種資料進行關聯定位分析，共得到57個與抗病顯著相關的區域，其中有26個區域和已知的稻熱病R 基因十分接近，17個區域經過Gramene資料庫比對得到636個候選基因，其餘14個區域並未比對到任何已知R 基因或候選基因。以12YL-DL-3-2接種資料進行分析，共得到60個顯著相關的區域，其中35個區域接近已知R 基因，25個區域經過資料庫比對得到472個候選基因，剩下的區域則沒有比對到任何已知基因。兩組資料共同最高的位點皆落在第12條染色體，接近Pita 抗性基因座的位置。經過單套體 (haplotype) 分析，評估各個抗性品種可能帶有抗性基因座之位置，相關資訊可做為未來抗性基因驗證分析及稻熱病抗病育種之基礎。

Rice blast, caused by Magnaporthe oryzae, is an important rice disease annually occurring in rice-growing regions. Resistance breeding is the most effective and environmental-friendly method to manage blast disease. However, the resistance can easily be overcome in 3-5 years due to the fast evolution of M. oryzae. Discovering novel R genes and revealing the resistance genes in commercial varieties can help develop more effective breeding and disease management strategies. This study aimed to use genome-wide association mapping to identify the blast resistance genes in 312 diverse accessions and 21 commercial rice varieties. In phenotyping experiments, two representative M. oryzae isolates, D41-2 and 12YL-DL-3-2, were chosen to inoculate plant materials in the growth chamber. Diseased leaf area, lesion type, and resistance/susceptible reaction were recorded. To eliminate the bias among different inoculation experiments, best linear unbiased predictor (BLUP) values were calculated for the diseased leaf area data. Only the consistent data among different repetitions were chosen for further association analysis. In genotyping experiments, we conducted Illumina next-generation sequencing (NGS) for Tainan 11 (TN11) and Tainung 84 (TNG84) and used genotyping by sequencing (GBS) to discover single nucleotide polymorphisms (SNPs) in commercial rice varieties. After integration of the cross-platform genotype data, including NGS, GBS, and the 44K SNPs data from rice diversity panel, we found that the overlapped SNPs between GBS and 44K data were less than 5%, while NGS data can be successfully combined with 44K SNPs data. In the association analysis, 57 regions were found significantly associated with resistance to the isolate D41-2, with 26 regions colocalized with known R genes and/or quantitative trait loci (QTLs), 17 regions containing 636 defense-related candidate genes, and 14 regions that did not match any known R genes/QTLs or defense-related candidate genes. Sixty regions were significantly associated with resistance to the 12YL-DL-3-2 isolate, with 35 regions colocalized with known R genes/QTLs, and 25 regions containing 472 defense-related candidate genes. The highest peak from the datasets of both isolates was located at the Pita locus on chromosome 12. Analysis of haplotypes revealed the loci conditioning resistance in each resistant accessions. The results will be useful for validation of identified resistance genes/QTLs and donor selection for future resistance breeding.