稻熱病抗性相關分子標誌之開發與臺灣水稻品種Pik基因座單倍型分析

Molecular marker development for rice blast resistance genes and the analysis of Pik locus haplotypes in Taiwan rice cultivars

許龍欣(Hsu, L. H) ； 王聖善(S. S. Wang) ； 陳榮坤(R. K. Chen)

《臺南區農業改良場研究彙報》 70期 (2017/12) Pp. 11-23

摘要

稻熱病為水稻重要病害，而病害防治手段中較經濟安全的方式為育成抗病品種。以往臺灣稻作育種利用之抗病親本來源較少，對特定病原菌篩選壓力大，抗病品種往往在大面積推廣栽培數年內即失去抗性，因此擴大抗病基因來源及育成廣抗幅品種為育種者持續努力之目標，利用分子標誌輔助回交選種可提高抗病育種之效率。本試驗設計之7組TaqManSNP分子標誌於水稻品種臺南11號及抗病基因供給親LTH單基因系間確認具有多型性後，搭配即時定量PCR平臺快速篩檢31個LTH抗病單基因系及39個栽培品種，結果顯示4個稉稻栽培品種具有Pita，9個秈稻品種具有Pib，這13個品種可應用於臺灣品種抗病性改良。而依據Pik基因座上5個對偶基因Pik、Pik-h、Pik-m、Pi1、Pik-s序列之設計5組SNP分子標記，接著依參試水稻之Pik基因座上5個SNP位點構成之單倍型（haplotype）差異，將70個參試品種區分為16種單倍型。結果顯示，21個稉型栽培品種與Pik-s有相同之單倍型；6個稉型栽培品種與Pik有相同單倍型；而4個秈型栽培品種（TNS22、TCSW2、1003、IRBB66）則在5個位點皆無法判別基因型。本試驗之SNP分子標誌可做為抗病育種快速篩選回交子代之工具，而不同水稻品種之Pik對偶基因單倍型分析結果，可提供抗病育種材料基因型之評估資訊。現有技術：以分子標誌輔助作物回交選種，不受外在環境之限制，可精準選拔目標基因、加速輪迴親背景回復效率而提高育種效率。創新內容：本研究採螢光標定探針系統之SNP分子標記，可配合即時定量PCR快速偵測目標SNP位點基因型。對產業影響：藉由抗病基因分子標誌篩檢臺灣水稻品種後，分子標誌資訊與抗病基因檢測結果皆可提供抗病育種親本選擇之參考資訊。

關鍵字

LTH單基因系 ； Pik基因座 ； 單倍型 ； 單一核苷酸多型性 ； 稻熱病抗病基因

並列摘要

Rice blast is one of the most damaging diseases of rice cultivated in Taiwan. The most economical and eco-friendly strategy for decreasing the loss caused by disease is to develop disease resistant cultivars. Many of the old ＂blast-resistant＂ cultivars in Taiwan have lost their resistance few years after their release when the cultivation area of one single popular cultivar increased quickly in several years. The blast fungus races can evolve so fast that they can overcome the major resistance genes in short time. Therefore, the target which rice breeder engage in is to develop broad-spectrum and durable resistant cultivars. In this study, we developed 7 TaqMan probe-based SNP genotyping markers target to 3 loci (6 resistant alleles) to distinguish TN11 and R genes donor cultivars. The screening of 39 Taiwan rice cultivars with Pita and Pib markers show that 4 Taiwan japonica cultivars have Pita gene and 9 indica cultivars have Pib gene. Five markers based on Pik, Pik-h, Pik-m and Pi1, the 4 alleles which locate in Pik locus, were used to analyze the total 70 cultivars. The 5 SNPs site consist 16 composite genotypes, or haplotype, of Pik locus. No cultivar has the same haplotype with Pi1 or Pik-m, while 6 japonica cultivars have the same haplotype with Pik, 21 japonica cultivars have the same haplotype with Pik-s, and haplotypes of 6 LTH lines are the same with Pik. Four cultivars seem no Pik locus exists. The results of this study, both of SNP marker for R genes and the analysis of 70 rice cultivars, can provide useful information to improve rice blast resistance. What is already known on this subject? Marker-assisted backcross selection can increase the breeding efficiency through precise tracking target genes and accelerating the recovery of recurrent parent genome without environment limitation. What are the new findings? We use fluorescence-labeled probe SNP markers instead of traditional way relying for large amount of gel-electrophoresis to analyze our target alleles. We can detect the target region SNP rapidly by using this kind markers together with real-time PCR. What is the expected impact on this field? Those resistant gene specific markers we developed and the results in this study about the R gene resource in Taiwan rice cultivars can provide useful information to improve rice blast resistance.

並列關鍵字

Haplotype ； LTH lines ； Pik locus ； Rice blast resistant gene ； Single nucleotide polymorphism (SNP)