Contents lists available at ScienceDirect Molecular and Cellular Probes journal homepage: www.elsevier.com/locate/ymcpr The ribosomal intergenic spacer (IGS) in the potato and tobacco cyst nematodes, Globodera pallida, G. rostochiensis and G. tabacum Mehrdad Madani a,b,* , Len Ward b , Andy Vierstraete c , Solke H. De Boer b , Maurice Moens d,e a Department of Soil Science, University of Manitoba, R3T 2N2, Winnipeg, MB, Canada b Canadian Food Inspection Agency, 93 Mount Edward Road, Charlottetown Laboratory, Charlottetown, PE, Canada c Biology Department, Gent University, K.L. Ledeganckstraat, 35, 9000, Gent, Belgium d Research Institute for Agriculture, Fisheries and Food (ILVO), 9280, Merelbeke, Belgium e Department of Plants and Crops, Ghent University, Coupure Links 653, Ghent, Belgium ARTICLE INFO Keywords: Cyst nematodes Potato IGS PCR 5S gene 28S gene ABSTRACT The potato cyst nematodes Globodera pallida and G. rostochiensis (PCN), and tobacco cyst nematode (TCN), G. tabacum, are the most important parasitic nematodes of potato and tobacco worldwide. Ribosomal DNA provides useful molecular data for diagnostics, the study of polymorphisms and for evolutionary research in eukaryotic organisms including nematodes. Here we present data on the structure and organization of a rarely studied part of the intergenic spacer (IGS) region of the PCN and TCN genome of cyst nematodes. This region has shown potential for diagnostic purposes and population studies in other organisms including nematodes. In nematodes, the ribosomal RNA gene cluster comprises three genes: 5.8S, 18S and 28S rRNA, which are separated by spacer regions: the intergenic spacer (IGS), non-transcribed spacer (NTS), externally transcribed spacer (EST) and the internally transcribed spacer (ITS). The intergenic spacer (IGS) region consists of an external transcribed spacer (ETS) and a non-transcribed spacer (NTS) which is located between the 28S of one repeat and the 18S gene of the next repeat within the rRNA genes cluster. In this study, the first flanking portion of the IGS was amplified, cloned and sequenced from PCN and TCN. Primers were then designed to amplify the whole IGS sequence. PCR amplification of IGS from G. tabacum, G. pallida, and G. rostochiensis yielded respectively: a single amplicon of 3 kb, three amplicons sized 2.5, 2.6 and 2.9 kb, and two amplicons sized 2.8 and 2.9 kb. Results showed that Globodera spp. has more than one variant copy of the IGS, with both long and short repetitive DNA elements. An approximately 400 bp long region without any internal repetitive elements, were identified in a position be- tween the two repetitive regions suggesting that there is a 5S gene in the IGS of these species. 1. Introduction Three cyst nematodes species belonging to the genus Globodera are parasites of Solanaceae plants. The two species of the Potato Cyst Nematodes (PCNs), i.e. G. pallida and G. rostochiensis, primarily infects potato, but also tomato and eggplant and to a lesser extend other Solanum spp. The Tobacco Cyst Nematode (TCN), G. tabacum, is a plant- parasitic nematode that mainly infests tobacco [35]. PCNs have been estimated to cause losses of 9% of total potato production worldwide [51]. Globodera tabacum consists of three subspecies, including G. ta- bacum virginae, G. tabacum solanacearum and G. tabacum tabacum [45]. Globodera tabacum tabacum and G. tabacum solanacearum can reduce crop yield significantly. In Virginia (USA) in 1983, the yield loss of tobacco due to TCN was estimated at 15% [33]. Traditional identification of cyst nematodes is mainly based on the morphology and morphometrics of both females (cysts) and second- stage juveniles (J2) [35]. However, because identification of cyst ne- matodes is time-consuming and difficult, DNA-based identification techniques have been developed for Globodera species [29]. Several molecular markers and genes have been used for the identification of plant-parasitic nematodes of the genera Meloidogyne, Bursaphelenchus, Heterodera and Globodera, and also for the study of their phylogeny and populations [29,30]. The internal transcribed spacers (ITS), the D2-D3 expansion region of the 28S gene, and the 18S gene in ribosomal DNA were the first to be used [40,44]. These were later followed by the study of other genes such as Actin, and more recently the heat shock protein gene (hsp-90) and Cytb of the mitochondrial DNA (mt DNA) [27,30,31,41,50]. In eukaryotes, the 5′ to 3’ structure organization of the multi-gene area of the ribosomal DNA (rDNA) cluster is typically arranged as head https://doi.org/10.1016/j.mcp.2019.101441 Received 17 January 2019; Received in revised form 18 August 2019; Accepted 27 August 2019 * Corresponding author. Department of Soil Science, University of Manitoba, R3T 2N2, Winnipeg, MB, Canada E-mail address: Meh.Madani@yahoo.com (M. Madani). Molecular and Cellular Probes xxx (xxxx) xxxx 0890-8508/ Crown Copyright © 2019 Published by Elsevier Ltd. All rights reserved. Please cite this article as: Mehrdad Madani, et al., Molecular and Cellular Probes, https://doi.org/10.1016/j.mcp.2019.101441