Development of a species-specific PCR to detect the cereal cyst nematode, Heterodera latipons

Abstract

Several Heterodera species can reduce the yield of wheat and barley, among which H. avenae, H. filipjevi and H. latipons are economically the most important. Their identification, based on morphological characteristics, is not straightforward but can be made easier using molecular techniques. In this study, we developed species-specific primers for the detection of H. latipons. The actin gene of eight Heterodera species was partially sequenced and, after purifying and sequencing the PCR products, all sequences were aligned to find unique sites. The alignment showed moderate to very high similarities between the species. However, a small fragment of the actin gene was suitable for the construction of a potentially useful species-specific primer for H. latipons. The optimised PCR was subsequently tested with several populations of 14 Heterodera species and a single population of Punctodera punctata. Heterodera latipons was represented by 16 populations originating from six different countries. The primer set (Hlat-act), designed using AlleleID 7.73, was shown to be very specific. To test its sensitivity further, the PCR was conducted on DNA extracted from five second-stage juveniles (J2) of H. latipons mixed with five or 100 J2 belonging to H. avenae. The PCR was able to detect up to 1:10 dilution of the DNA obtained from five J2. The results showed that a specific and sensitive H. latipons species-specific PCR was constructed.

Characterization of cereal cyst nematodes (Heterodera spp.) in Morocco based on morphology, morphometrics and rDNA-ITS sequence analysis

“…All cysts of H. avenae had prominent bullae, but no underbridge; H. latipons, Unauthenticated Download Date | 5/10/18 8:59 PM however, had a strong underbridge and lacked distinct bullae in the vulval cone. Previous studies (Wouts and Sturhan 1995;Subbotin et al 2003) Species-specific primers for PCR have been developed to complement the traditional species identification of H. avenae (Toumi et al 2013a;Yan et al 2013) and H. latipons (Toumi et al 2013b). Several genes were successfully used to identify many species of Heterodera (Subbotin et al 1999;Yan et al 2013).…”

“…forward primer 5'-CGT AAC AAG GTA GCT GTA G-3' and the reverse primer 5'-TCC TCC GCT AAA TGA TAT G-3' , were used to detect H. avenae in the DNA extracts of 11 populations. Extracts that were not identified as belonging to H. avenae were used in a PCR with the species-specific primers set Hla-acti-F (5'-ACT TCA TGA TCG AGT TGT AGG TGG ACT CG-3') and Hla-acti-F (5'-ACC TCA CTG ACT ACC GAT GAA GAT TC-3') (Toumi et al 2013b) along Unauthenticated Download Date | 5/10/18 8:59 PM with the universal reverse primers (Ferris et al 1993) to eventually characterise H. latipons.…”

“…The ther mal cycler programme consisted of 5 min at 95°C, 30 cycles of 30 s at 94°C, 30 s at 58°C and 45 s at 72°C, followed by a final elongation step of 8 min at 72°C. For the detection of H. latipons, 2 µl of the DNA extract was added to the PCR reaction mixture containing 21 µl ddH 2 O, 25 µl 2× DreamTaq PCR Master Mix (Fermentas Life Sciences, Germany), and 1 µM of each of the primers Hla-acti (Toumi et al 2013b) and Ferris et al (1993). The programme of the thermal cycler consisted of 5 min at 95°C; 50 cycles of 30 s at 94°C, 45 s at 50°C and 45 s at 72°C, followed by a final elongation step of 8 min at 72°C.…”

Parasitism effects on white clover by root-knot and cyst nematodes and molecular separation of Heterodera daverti from H. trifolii

“…The new sequences of ITS rRNA and coxI of H. daverti were aligned using ClustalX 1.83 (Thompson et al 1997) with default parameters with corresponding published gene sequences of cyst nematodes the Schachtii group species (Subbotin et al 2001;Toumi et al 2013). Outgroup taxa for each dataset were chosen according to the results of a previous study (Subbotin et al 2001).…”

Development of qPCR assays for quantitative detection of Heterodera avenae and H. latipons

“…Therefore, development of molecular tools will allow the identification and discrimination between species, but also their precise quantification. Most recently, it was demonstrated that a speciesspecific PCR assay provides an efficient tool for an accurate, rapid and sensitive detection of three species of CCN (Yan and Smiley 2009;Qi et al 2012;Peng et al 2013;Toumi et al 2013a;Yan et al 2014). However, none of the species-specific primers were selected for quantification purposes; yet, quantification is very essential in breeding programs and extension activities.…”