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

in Nematology
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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.

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

in Nematology



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    PCR amplification of DNA from eight different Heterodera species with the universal actin gene primers (Hs-actF and Hs-actR) revealing a 420-bp fragment. Lanes: L = 100 bp DNA ladder (Fermentas Life Sciences); 1, 2 = H. latipons (Fa3); 3, 4 = H. avenae (Fa1); 5 = H. hordecalis (E69); 6, 7 = H. filipjevi (Did23b); 8 = H. glycines (HGRiggs); 9 = H. schachtii (HSPol); 10 = H. betae (DCP1248); 11 = H. ciceri (FaC3); 12 = Negative control. (See Table 1 for codes.)

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    A graphical map of the Actin 1 gene showing the positions of the designed species-specific primers. The shaded and white boxes represent the exons (Ex) and introns (In), respectively. The sizes of the boxes correspond with the lengths of the exons and introns.

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    Gradient PCR with the Heterodera latipons-specific PCR (Hlat-act primers) using DNA of H. latipons (Fa3). Temperature range from 43 to 60°C. L = 100 bp DNA ladder (Fermentas Life Sciences).

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    Selected results of the Heterodera latipons-specific PCR (Hlat-act primers) using DNA from all samples (see Table 1 for codes). Lanes: L = 100 bp DNA ladder (Fermentas Life Sciences); 1 = Heterodera pratensis (DCP1041A); 2 = Punctodera punctata (DCP1041B); 3 = H. avenae (Fa1); 4 = H. hordecalis (E69); 5 = H. glycines (HGRiggs); 6 = H. schachtii (HSPol); 7 = H. betae (DCP1248); 8 = H. filipjevi (Fa125); 9 = H. goettingiana (MP1); 10 = H. humuli (MP5); 11 = H. ciceri (FaC3); 12 = H. trifolii (HT9); 13 = H. carotae (DCP1734); 14 = H. daverti (HD11); 15-22 = different populations of H. latipons (15 = HL5; 16 = HLCyp; 17 = Fa7A1; 18 = Fa3; 19 = HLMorc; 20 = Mus1; 21 = HLIran; 22 = HL50); 23 = Negative control.

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    Result of the sensitivity test of the Hlat-act primers in a PCR with 1 μl undiluted or diluted DNA of Heterodera latipons (Hl) mixed with 1 μl DNA from H. avenae (Ha). Lanes: 1, 2 = 1:2 dilution of 5 second-stage juveniles (J2) of Hl; 3, 4 = 5 J2 of Ha; 5, 6 = 100 J2 of Ha; 7, 8 = 5 J2 of Hl; 9, 10 = 1:5 dilution of 5 J2 of Hl; 11, 12 = 1:10 dilution of 5 J2 of Hl; 13, 14 = 1:50 dilution of 5 J2 of Hl; 15, 16 = 5 J2 of Hl and 100 J2 of Ha; 17, 18 = 1:2 dilution of 5 J2 of Hl and 100 J2 of Ha; 19, 20 = 5 J2 of Hl and 5 J2 of Ha; 21, 22 = 1:5 dilution of 5 J2 of Hl and 100 J2 of Ha; 23 = negative control; L = 100-bp DNA ladder (Fermentas Life Sciences).


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