Stripe rust, caused by Puccinia striiformis f. sp. tritici, is an important wheat disease in China, seriously threatening wheat production. Understanding the winter survival of the fungus is a key for predicting the s...Stripe rust, caused by Puccinia striiformis f. sp. tritici, is an important wheat disease in China, seriously threatening wheat production. Understanding the winter survival of the fungus is a key for predicting the spring epidemics of the disease, which determines the crop loss. Estimation of P. striiformis f. sp. tritici winter survival requires processing a large number of samples for sensitive detection of the pathogen in wheat leaf tissue using real-time quantitative reverse transcription PCR （qRT-PCR）. A bottleneck for the analysis is the acquisition of a good yield of high quality RNA suitable for qRT-PCR to distinguish dead and alive fungal hyphae inside leaves. Although several methods have been described in the literatures and commercial kits are available for RNA extraction, these methods are mostly too complicated, expensive and inefficient. Thus, we modified three previously reported RNA extraction methods with common and low-cost reagents （LiCI, SDS and NaCI） to solve the problems and selected the best to obtain high quality and quantity RNA for use in qRT-PCR. In the three improved methods, the NaCI method was proven to be the best for extracting RNAfrom urediniospores of and wheat leaves infected by P. striiformis f. sp. tritici, although the modified LiCI and SDS methods also increased yield of RNA compared to the previous methods. The improved NaCI method has the following advantages： 1） Complete transfer of urediniospores of P. striiformis f. sp. tritici from the mortar and pestle can ensure the initial amount of RNA for the qRT-PCR analysis; 2） the use of low-cost NaCI to replace more expensive Trizol can reduce the cost; 3） the yield and quality of RNA can be increased; 4） the improved method is more suitable for a large number and high quantity of samples from fields. Using the improved NaCI method, the amount of RNA was increased three times from urediniospores of P. striiformis f. sp. tritici compared from the extraction kit. Approximately, 10.11 IJg total RNA of high quality was obtained from 100 mg of infected leaves, which was 8.8, 6.5, 3.4 and 2.1 folds of the amounts obtained from the previous LiCI, SDS, NaCI and traditional Trizol methods, respectively. The method could be used to study the overwintering rates of R striiformis f. sp. tritici over a large region of wheat production for predicting epidemic levels by determining pathogen survival levels after winter. The method can alsobe used in any studies which need a large number of high quality RNA samples.展开更多
基金supported by the National Key Basic Research Program of China (2013CB127700)the Na-tional Natural Science Foundation of China (31071640 and 31271985)partially supported by the 111 Project from Education Ministry of China (B07049)
文摘Stripe rust, caused by Puccinia striiformis f. sp. tritici, is an important wheat disease in China, seriously threatening wheat production. Understanding the winter survival of the fungus is a key for predicting the spring epidemics of the disease, which determines the crop loss. Estimation of P. striiformis f. sp. tritici winter survival requires processing a large number of samples for sensitive detection of the pathogen in wheat leaf tissue using real-time quantitative reverse transcription PCR （qRT-PCR）. A bottleneck for the analysis is the acquisition of a good yield of high quality RNA suitable for qRT-PCR to distinguish dead and alive fungal hyphae inside leaves. Although several methods have been described in the literatures and commercial kits are available for RNA extraction, these methods are mostly too complicated, expensive and inefficient. Thus, we modified three previously reported RNA extraction methods with common and low-cost reagents （LiCI, SDS and NaCI） to solve the problems and selected the best to obtain high quality and quantity RNA for use in qRT-PCR. In the three improved methods, the NaCI method was proven to be the best for extracting RNAfrom urediniospores of and wheat leaves infected by P. striiformis f. sp. tritici, although the modified LiCI and SDS methods also increased yield of RNA compared to the previous methods. The improved NaCI method has the following advantages： 1） Complete transfer of urediniospores of P. striiformis f. sp. tritici from the mortar and pestle can ensure the initial amount of RNA for the qRT-PCR analysis; 2） the use of low-cost NaCI to replace more expensive Trizol can reduce the cost; 3） the yield and quality of RNA can be increased; 4） the improved method is more suitable for a large number and high quantity of samples from fields. Using the improved NaCI method, the amount of RNA was increased three times from urediniospores of P. striiformis f. sp. tritici compared from the extraction kit. Approximately, 10.11 IJg total RNA of high quality was obtained from 100 mg of infected leaves, which was 8.8, 6.5, 3.4 and 2.1 folds of the amounts obtained from the previous LiCI, SDS, NaCI and traditional Trizol methods, respectively. The method could be used to study the overwintering rates of R striiformis f. sp. tritici over a large region of wheat production for predicting epidemic levels by determining pathogen survival levels after winter. The method can alsobe used in any studies which need a large number of high quality RNA samples.
