水稻臺農82號疊氮化鈉誘變系WM1370之抗稻熱病基因座分析

Analysis of rice blast resistance loci in WM1370, a sodium azide induced mutant line derived from 'Tainung 82'

水稻是重要糧食作物，由Pyricularia oryzae引起的稻熱病嚴重影響水稻生產，而使用抗病品種為最有效且環保的防治方法。水稻臺農82號為對稻熱病感病之稉稻，經過連續五代疊氮化鈉誘變及病圃篩選後，獲得44個抗稻熱病誘變系。本研究針對抗稻熱病及紋枯病的誘變系WM1370，進行9株本土稻熱病菌菌株之接種，發現其具有廣幅抗性；另一方面，透過PCR增幅定序臺農82號及誘變系WM1370之已知抗稻熱病基因座Pi2/9、Pik、Pita及Ptr後，發現臺農82號帶有Pik-s，但在Pi2/9、Pita及Ptr為感病等位基因，WM1370則帶有Pik-s及Pita抗病等位基因，而在Pi2/9及Ptr呈現不同變異。為探討WM1370之抗性遺傳，將其與感病秈稻CO39雜交後之636個F2子代，於2018年稻熱病旱田病圃分析抗病表現，並以84組簡單重複序列（simple sequence repeat, SSR）分子標誌進行集群分離分析（bulked segregant analysis），發現抗性與第12號染色體上3 Mb及17.8 Mb兩處分子標誌連鎖；再透過F_(2:3)至F_(2:5)子代於2019-2021年稻熱病旱田病圃進行抗性評估，同時使用5組SSR、8組競爭性等位基因特異性（KBioscience competitive allele-specific PCR, KASP）及10組插入／缺失（insertion/deletion, Indel）分子標誌進行基因型鑑定，連鎖分析之結果將WM1370之抗性區段縮小至約10.7-14.6Mb區間，候選基因包括Ptr（與抗病品系Katy之等位蛋白具有一個胺基酸差異L763F）。WM1370於第12號染色體上橫跨Pita（10.6 Mb）及10.7-14.6 Mb抗病基因座之區段，以及本研究新開發與抗病區段緊密連鎖之分子標誌，皆提供未來WM1370在育J. Plant Med.種應用上的重要基礎，希望藉此擴充臺灣水稻抗稻熱病基因庫，促進抗病新品種育成。

Rice is an important staple crop. Rice blast caused by Pyricularia oryzae can seriously affect rice production, and the use of genetic resistance is the most effective and eco-friendly method to control this disease. Tainung 82 (TNG82) is a japonica rice susceptible to rice blast. Forty-four rice blast resistance lines were generated after five generations of sodium azide mutagenesis and disease screening in the blast nursery. In this study, WM1370, the mutant line resistant to rice blast and sheath blight, was inoculated with nine local P. oryzae isolates. WM1370 showed broad-spectrum resistance to all nine isolates. Additionally, TNG82 and WM1370 were sequenced for known rice blast resistance loci Pi2/9, Pik, Pita, and Ptr. TNG82 carries Pik-s and susceptibility alleles at Pi2/9, Pita, and Ptr. WM1370 carries resistance alleles at Pik-s and Pita, and have different mutations at Pi2/9 and Ptr. To understand the genetics of resistance in WM1370, 636 F_2 progenies of WM1370 crossed with the susceptible indica cultivar CO39 were evaluated for disease severity in Chiayi upland blast nursery in 2018. Bulked segregant analysis using 84 simple sequence repeat (SSR) markers suggested that the resistance was associated with two markers located at 3 Mb and 17.8 Mb on chromosome 12. Linkage analysis was conducted using CO39 x WM1370 F_(2:3) to F_(2:5) progenies, by evaluating their resistance in Chiayi upland blast nursery in 2019-2021 and their genotypes at five SSR, eight KBioscience competitive allele-specific PCR (KASP), and ten insertion-deletion (Indel) markers. The resistance segment of WM1370 was fine-mapped to approximately 10.7 to 14.6 Mb, which contains Ptr [with predicted one amino acid difference (L763F) from the allelic protein in the resistance cultivar, Katy]. The resistance segment across Pita (10.6 Mb) and the resistance locus at 10.7-14.6 Mb on chromosome 12 in WM1370, and the newly developed markers tightly linked to the resistance segment, can serve as an important basis for the application of WM1370. It is expected to expand the pool of rice blast resistance genes in Taiwan, and promote the breeding of new resistant varieties in the future.