A rapid genotyping method for an obligate fungal pathogen, Puccinia striiformis f.sp. tritici, based on DNA extraction from infected leaf and Multiplex PCR genotyping
© Ali et al; licensee BioMed Central Ltd. 2011
Received: 21 February 2011
Accepted: 20 July 2011
Published: 20 July 2011
Puccinia striiformis f.sp. tritici (PST), an obligate fungal pathogen causing wheat yellow/stripe rust, a serious disease, has been used to understand the evolution of crop pathogen using molecular markers. However, numerous questions regarding its evolutionary history and recent migration routes still remains to be addressed, which need the genotyping of a large number of isolates, a process that is limited by both DNA extraction and genotyping methods. To address the two issues, we developed here a method for direct DNA extraction from infected leaves combined with optimized SSR multiplexing.
We report here an efficient protocol for direct fungal DNA extraction from infected leaves, avoiding the costly and time consuming step of spore multiplication. The genotyping strategy we propose, amplified a total of 20 SSRs in three Multiplex PCR reactions, which were highly polymorphic and were able to differentiate different PST populations with high efficiency and accuracy.
These two developments enabled a genotyping strategy that could contribute to the development of molecular epidemiology of yellow rust disease, both at a regional or worldwide scale.
Puccinia striiformis f.sp. tritici (PST), an obligate basidiomycete that causes wheat yellow/stripe rust, a serious disease in all major wheat growing regions [1–5]. The development of different molecular markers has aided the description of possible PST migration patterns , the emergence of high temperature-adapted strains [7, 8] and the existence of recombination [9, 10]. Despite these recent developments, numerous questions still need to be addressed, e.g. the evolutionary history of PST, its centre of origin, its historic migration pathways or more recent migrations causing new epidemics. These studies necessitate the genotyping of a large number of isolates, a process that is limited by both DNA extraction and genotyping methods.
Two or three cycles of PST spore multiplication on plants are usually necessary after sampling before DNA extraction. Because of its obligate nature, PST cannot be cultured on routine media to obtain sufficient biomass for DNA extraction . Spore production may be further complicated when dealing with exotic isolates, which involve the mandatory use of expensive, time-consuming and wholly-contained facilities. In addition, using a given set of susceptible varieties to increase the spores of exotic isolates may give rise to bias. We had previously observed very low levels of infection or even resistance reactions in previously considered fully susceptible varieties such as cv. Victo , Michigan Amber and Cartago when inoculated with Pakistani isolates. This can result in the loss of isolates having avirulence factors recognized by unknown resistance genes in varieties used to increase spores. One alternative is to extract DNA from one or few spores, and then increase it through a whole genome multiple displacement amplification  before performing genotyping. However, the sophistication required for this method, as well as the need to prevent any contamination from other organisms, limits its use. We therefore tested here a third procedure, i.e. the direct extraction of fungal and plant DNA from single spore-infected leaf.
Another issue was the availability of a set of molecular markers sufficient to describe the population structure of a pathogen. The use of microsatellites/simple sequence repeat (SSR) markers with co-dominance and high polymorphism is of considerable value to the study of dikaryotic fungi such as PST . When SSR detection and allele sizing are performed using an automated DNA fragment analyzer based on the separation of fluorescently-labeled amplicons, accurate and efficient genotyping can be achieved . A good way to further enhance the efficiency of SSR genotyping is to multiplex SSR amplifications. Multiplex PCR refers to the simultaneous amplification of several markers in a single reaction, thereby saving the time and money required to manage each PCR reaction separately . This method has been reported as achieving the same specificity as single conventional PCR reactions . We report here a protocol that enabled the amplification of a set of highly informative SSR markers for PST studies, by means of three multiplex PCR reactions for PST.
Improvements to PST DNA extraction
Development of an efficient and multiplexed set of SSR for the PST population study
Description of three PCR multiplexes enabling the genotyping of 20 Puccinia striiformis f.sp. tritici SSR
Allele size range
Provided by X. Chen
Provided by X. Chen
The PCR reactions were performed using a QIAGEN kit containing a single mix of Taq-polymerase, MgCl, dNTPs and buffer, referred to as the Type-it microsatellite kit specially designed for Multiplex PCR reactions. Each reaction contained 2 μL water, 1 μL Q-solution, 1 μL of the primer mix (containing 2 μM of each SSR primer), 5 μL of the Type-it mix and 1 μL (15 ng) of DNA. The amount of DNA was increased to 3 μL and no water was added for infected plant leaves, as the PST DNA was diluted with plant DNA. An optimization step was performed to identify the optimum melting temperature for all the SSRs in a given Multiplex. The optimum PCR conditions thus determined were the same for all three multiplexes, with preheating at 95°C for 5 min followed by 30 cycles of 95°C for 30 s, 57°C for 90 s and 72°C for 30 s, with a final extension step at 60°C for 30 minutes, using an iCycler (Biorad) thermocycler. The PCR products were run on a 2% agarose gel to reveal the amplification products. The amounts of PCR product added to 35 μL of the Sample Loading Solution, containing 0.4 μL Beckman Coulter 400-bp size standard, varied as follows:1.5 μL for Multiplex-1 and Multiplex-3 and 2.2 for Multiplex-2 in the case of spore DNA; 3 μL for Multiplex-1 and Multiplex-2 and 4 μL for Multiplex-2 in the case of infected leaf DNA. Amplicon fragments were separated using a Beckman Coulter CEQ-8000 DNA Analyzer with the default FRAG-3 run method. The fragments were read using CEQ-8000 Genetic Analysis System Software (Beckman Coulter) to record alleles manually for each locus according to amplicon fragment lengths. All the alleles were readable and there was no difference between allele lengths whether the PCR was performed for each SSR separately or in a multiplex reaction. Allele sizes were within the range of previously reported alleles for previously developed SSR markers [13, 16], while for two new SSRs, the allele sizes were within the range of 211-213 bp for WU-6 and 325-334 bp for WU-12. The technique was then used to genotype more than a thousand isolates representing the worldwide PST population and we found no ambiguity in terms of allele reading, together with an efficient discriminating power for this set of SSRs. The data on these worldwide set of isolates would be used to infer about the PST phylogeny and evolutionary biology. The development of this multiplex-based amplification technique, together with the reading of allele length through a sequencer, achieved gains in time and accuracy, as well as regarding the reproducibility of the results.
The extraction of DNA from infected leaves, together with a Multiplex-based PCR reaction and the reading of fluorescently labeled alleles through a sequencer thus provides a ready-to-use method for the efficient genotyping of PST, and enables clear gains in terms of time, money, reproducibility and accuracy. Because of the high level of polymorphism of the SSR markers selected, the proposed SSR set could also constitute a genotyping reference at worldwide level, enabling the rapid comparison of genetic analyses. Such easily comparable sets of markers constitute an essential tool for molecular epidemiology and to trace emerging races in a fungus that is known for its highly efficient long distance migration . Furthermore, the genotyping of large number of isolates from different geographical regions coupled with recent population genetics analyses would assist to address ancestral relationship between different geographically spaced populations, describe the ancient migration routes and the role of host and geography on pathogen population structuring. This will help us to understand overall evolution of pathogens and to consequently orientate disease management strategies.
We would like to thank Laurent Gérard and Nathalie Galet for their technical assistance during the multiplication of yellow rust isolates. We thank X. Chen for kindly sharing the list of 13 sequences for candidate SSRs. This work was supported by the European Integrated Project Bioexploit, FOOD-CT-2005-513959 and the ANR (Agence Nationale de la Recherche) through the EMERFUNDIS project (ANR 07-BDIV-003). Sajid Ali was supported by the Higher Education Commission, the Government of Pakistan.
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