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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(q RT-PCR). A bottleneck for the analysis is the acquisition of a good yield of high quality RNA suitable for q RT-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(Li Cl, SDS and Na Cl) to solve the problems and selected the best to obtain high quality and quantity RNA for use in q RT-PCR. In the three improved methods, the Na Cl method was proven to be the best for extracting RNA from urediniospores of and wheat leaves infected by P. striiformis f. sp. tritici, although the modified Li Cl and SDS methods also increased yield of RNA compared to the previous methods. The improved Na Cl 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 q RT-PCR analysis; 2) the use of low-cost Na Cl 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 Na Cl 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 μg 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 Li Cl, SDS, Na Cl and traditional Trizol methods, respectively. The method could be used to study the overwintering rates of P. 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.
Stripe rust, caused by Puccinia striiformis f. Sp. Tritici, is an important wheat disease in China, Critical 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 (q RT-PCR). A bottleneck for the analysis is the acquisition of a good yield of high quality RNA suitable for q RT-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 (LiCl, SDS and NaCl) to solve t he problems and selected the best to obtain high quality and quantity RNA for use in q RT-PCR. The improved Na Cl 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 q RT-PCR analysis; 2) the use of low-cost Na Cl to replace more expensive Trizol can reduce the cost; 3) the yield and quality of RNA can increased the amount of RNA was increased three times from urediniospores of P. striiformis f. sp. tritici compared to from the extraction kit. Approximately, 10.11 μg 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 amount obtained from the previous LiCl, SDS, NaCl and traditional Trizol methods, respectively. method could be used to study the overwintering rates of P. 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.