Molecular characterization of the Pratylenchus vulnus populations on cereals in Turkey
Published Online: Aug 25, 2020
Page range: 1 - 4
DOI: https://doi.org/10.21307/jofnem-2020-084
Keywords
© 2020 Mehmet Sait Karaca et al., published by Sciendo
This work is licensed under the Creative Commons Attribution 4.0 International License.
Root lesion nematodes of the genus
The genus
In Turkey,
A total of 17 nematode populations obtained from wheat and barley cultivated fields in Konya and Karaman provinces in Central Anatolian Plateau in Turkey on April in 2016 and 2017 were investigated. Two populations were from Center (lat: 37.256700, lon: 33.402159, barley soil) and Ayrancı (lat: 37. 461495, lon: 33. 899667, barley soil) districts in Karaman province and 15 populations were from Çumra (lat: 37. 652138, lon: 32. 810197, wheat soil), Güneysınır (lat: 37. 267285, lon: 32. 703158, wheat plant), Bozkır (lat: 37.215825, lon: 32. 566730, wheat plant), Yalıhöyük (lat: 37.334460, lon: 32.094510, wheat plant), Beyşehir (lat: 37. 708799, lon: 31. 711387, barley plant), Yunak (lat: 38.813822, lon: 31.753218, wheat plant), Kulu (lat: 39.083393, lon: 32.985203, wheat plant; lat: 39.065963, lon: 33.054807, wheat plant; lat: 39.039809, lon: 33.040935, wheat soil; lat: 38.965648, lon: 33.008739, barley plant), Cihanbeyli (lat: 38.627013, lon: 32.920812, wheat plant; lat: 38.468969, lon: 32.833310, wheat plant), Karatay (lat: 37.877460, lon: 32.914944, wheat plant; lat: 37.978114, lon: 32.710529, barley plant), and Kadınhanı (lat: 38.573150, lon: 32.276917, wheat plant) districts in Konya province.
Nematodes were previously identified with PCR fragments at 287 bp using species-specific D3b-R/Pvul-F primer for
Total genomic DNA was extracted from five individual nematodes in 30 µl extraction buffer as described by Yavuzaslanoglu et al. (2018). The sample was placed at −20°C for 1 hr and then incubated at 65°C for 1 hr. The proteinase was deactivated at 95°C for 10 min. DNA template was re-suspended in 20 µl TE (10 mM Tris-HCl, 1 mM EDTA, pH: 8.0) (Al-Banna et al., 1997). Prepared DNA suspension was preserved at −20°C until use.
Real-time PCR experiment was set up with DNAs of 17 nematode populations. Negative amplification control (NAC) included distilled water and positive amplification control (PAC) included
The PCR mixture including the nematode specific primer set was vortexed for 2 sec and transferred 15 µL into each well. A 5 µL of each DNA sample were added into their designated well. Real-Time PCR was run including the following steps; initial template denaturation for 3 min at 95°C, 35 cycles of amplification were DNA denaturation for 10 sec at 95°C, annealing for 60 sec at 63°C, and extension for 30 sec at 72°C. DNA melting curve analysis of the amplicon was performed by increasing the temperature from 72 to 95°C at 0.2 to 0.5°C/sec (ramp). The fluorescent signal was measured using the FAM or SYBR/FAM channel, after every cycle and after every temperature increment of the PCR melting curve.
Sequence alignments of one of the
Nematode DNAs was purified using ExoSAP-ITTM PCR Product Cleanup Reagent (Thermo Fisher Scientific, USA) prior to sequence alignment.
Purified DNA samples were sequenced using ABI3730XL Sanger sequencing device and BigDye Terminator v3.1 Cycle sequencing kit (Applied Biosystems, Foster City, CA) in Macrogen laboratory in the Netherlands.
The sequences were deposited into the GenBank database and compared with those of the other
The 17
Figure 1:
Melting curve of specific amplicons for
The sequences of D2 to D3 expansion region of 28S rDNA for the one population of
Data blast revealed a sequence similarity of 98.66% with
The real-time PCR was used to estimate the accuracy and sensitivity of molecular identification of
In many research works, D3 expansion region of 26S rDNA has been using for evaluation and identification among species and genera of nematodes (Huang and Yan, 2017) as well as 28S rRNA (Subbotin et al., 2008; Lopez-Nicora et al., 2012; Janssen et al., 2017).
High similarity rate between the sequence alignments of
Molecular identification is specific and reliable in distinguishing nematode species (Nguyen et al., 2017). Nematode species difficult to distinguish morphologically from other species can be successfully identified using molecular characterization.
Detection of