MOLECULAR METHODS OF CHARACTERIZATION AND IDENTIFICATION OF GLOBODERA ROSTOCHIENSIS AND GLOBODERA PALLIDA POPULATIONS

The cyst nematodes belonging to the genus Globodera are big worldwide problem in countries were Solanaceaous plants growing. Knowledge of species-composition in populations of Globodera rostochiensis and Globodera pallida is very important for selection of appropriate measure of nematode regulations occurrence. Interand intraspecific variability among species of Globodera rostochiensis and Globodera pallida were studied intensively with the use of molecular analyses of DNA methods. This review summarize and compare of methods chosen to distinguishing between Globodera, both pathotypes and species.


INTRODUCTION
Globodera rostochiensis (Wollenweber) Behrens and G. pallida (Stone) Behrens belongs to the most economically important pests of potato causing up to 50% of yield damages (Nicol et al., 2011). They were introduced into Europe from South America in the 1600s (Evans et al., 1975;Baldwin and Mundo-Ocampo, 1991) and knowledge of the genetic characteristics of each separate introduction might form the basis for classifying the various populations and virulence groups. Pathotype scheme proposed by Kort in 1977 differentiates some pathotypes on the basis of qualitative differences in relation to major resistance genes and others on the basis of quantitative differences in reproduction of populations on differential hosts with polygenic resistance (Kort et al., 1977). DOI: 10.37317/ pbss-2019-0008 Nowadays, many molecular techniques based on DNA analysis is used to searching for variations among nematode populations comes from different geographical regions. Molecular data could be a powerful source of information for the systematics of nematodes and conduct to the determine the range of genetic diversity in relation to virulence of potato cyst nematode (PCN) populations (Hyman and Powers, 1991;Ferris et al., 1991;Baldwin, 1992;Ferris and Ferris, 1992;Bossis and Mugniery, 1993;Powers and Adams, 1993;Blok et al., 1998). Reliable identification and characterization of nematode populations is a prerequisite to studying their genetic variation, interaction with host plants, virulence and to many aspects of PCN control and management. The choice of molecular technique used to the estimation of genetic diversity of populations depends on research question.

rDNA
Region of rDNA in eukaryotic species consists three ribosomal RNA genes -18S, 28S, 5.8, internal transcribed regions -ITS 1 and ITS2 and an external non -transcribed spacer region. In nematodes three ribosomal genes are the best characterized gene regions (Powers, 2004). They are highly conserve but the comparative analysis of differences in coding and non-coding regions of ribosomal DNA (rDNA) can be a popular method for inter-and intraspecies identification of many organisms. DNA sequencing of ITS regions within rDNA allows revealing single base-pair substitution and analysis of this regions has been used to examine phylogenetic relationships between population of the same species. Phylogenetic analysis of 41 ITS region sequences of Globodera parasitizing solanaceous plants obtained by Subbotin and co-authors (2000) suggest that the rDNA in genome of G. rostochiensis and G. pallida populations is present as a mixture of haplotypes with different sequences. Ferris et al., (1993) compared sequence data from internal transcribed spacer (ITS) of the Globodera spp. and found that they have characteristic inter-and intraspecific variations and that the choice of region in the rDNA can influence the amount of variability detected between isolates.
In 1998 Blok studied intraspecific variation of 18 population of G. pallida from Europe and South America in relation to their original introduction into Europe amplifying the repeated region of ITS1/5,8/ITS2 together with 3' end of 18S and 5' end of 28S gene. They found one population of G. pallida from South America more distinct then the others, in different molecular method: RFLP and sequence analysis (Blok et al., 1998) and earlier in SSR and RAPD method (Blok et al., 1997) in comparison of ITS2 region of all tested populations. Identical approaches carry out on 16 Ukrainian population of G. pallida and G. rostochiensis by Pypylenko et al. (2008) shows similarity of Globodera pallida population in 97,8% based on sequences 938bp in comparison to published sequences of European population of PCN described by Blok et al. (1998) and Subbotin et al. (2000). Polymorphism of sequence of the first inter-nal transcribed spacer (ITS1) has been widely used to identifying and assessing genetic variability of three Russian population of Globodera rostochiensis comes from different geographical localities (Chrisanfova et al., 2008). Low variation of the ITS-1 sequences and high conservativeness of this genome region failed to distinguish the isolates groups within the species with the used of this particular method. Comparative analysis of rDNA (fragments ITS1/5.8S/ ITS2) sequences of 16 polish Globodera populations show 97-98% identify in G. rostochiensis and 94-95% identity in G. pallida isolates and allow for splitting populations into three phylogenetic groups originate from Central and Eastern part of the country (Nowaczyk et al., 2011). Molecular characterization of Serbian Globodera isolates reported by Oro et al. (2012) based on comparison of variation occurred in ITS-1 region assumed that possible ancestors of PCN population originated from Peru but presence of G. pallida is not the result of direct import of infected potatoes from Latin America but from England where the population from York has the same sequence like Serbian ones.

mtDNA
Animal mitochondrial genomes (mtDNA) are often used in phylogenetic and population studies. Due to maternal mode of inheritance and lack of recombination mtDNA seems to be the best marker for searching of evolutionary connection between populations (Avise, 1994;Harrison, 1989;Birky, 2008). At present there are available a complete nucleotide sequences for 76 species which revealed differences in size, structure and gene content (Armstrong et al., 2000). The mtDNA encodes the 22 transfer RNAs, two ribosomal RNAs and 12 or 13 proteins involved in transport of electrons and oxidative phosphorylation (Pont-Kingdon et al., 1995). In metazoan mtDNA can occur as a single circular molecules, a linear chromosome or two 8-kb linear molecules (Bridge et al., 1992).
In 2000 Armstrong provided evidence about multipartite structure of mtDNA of Globodera pallida. Molecular approaches carry out on British population of G. pallida reveal at least 6 small, circular mitochondrial DNAs (scmtDNAs I -VI) differ in size. These scmtDNA contained mitochondrial gene coding sequences and about 2kb non-coding region common to all small circular mitochondrial DNAs (Armstrong et al., 2000). The authors suggested that scmtDNAs are present in all G. pallida but their frequencies vary between different populations (Armstrong et al., 2007). For example, while scmtDNAs I and III were not detected in several UK populations, scmtDNA IV was present in all population analyzed. Last study shows that scmtDNA IV seems to be the most evolutionary stable than other subgenomic circles and may be used for studying genetic interactions between populations. Moreover, only scmtDNA IV contains rRNA genes which are necessary for the translation of mitochondrial protein. The scmtDNA IV region can decise populations as monophyletic what indicates this DNA region may be a useful marker for G. pallida. Analysis of scmtDNA sequences variation can be a powerful tool to research relationship between European G. pallida populations and their South American ancestors (Armstrong et al., 2007).
On base of maternal mode of mtDNA inheritance and theory that recombinant products are indistinguishable from their progenitor molecules, Hoolahan in 2012 investigated past and contemporary recombination of British population of G. pallida. Past recombination was detected and confirmed between a South American and several UK population of white potato cyst nematode. In their study, progeny from experimental crosses of tested population of G. pallida had no evidence of contemporary recombination between the mtDNA of the maternal and paternal populations (Hoolahan et al., 2012) what support current arguments that animal mtDNA recombination events are rare (Kivisild et al., 2000;Innan and Nordberg, 2002).

RFLP
Molecular analysis based on restriction digest of total DNA (Restriction Fragment Length Polymorphisms -RFLP) are being used to observe genetic variation both between and within species. Investigations of variability within population are based on a sequence information from different populations and on using various clones. Inter-and intraspecific polymorphism between reference populations of G. rostochiensis and G. pallida has been observed for the first time by Burrows and Boffey in 1986. This method was used several times to examine genetic variation between different population of G. pallida but the results did not correlate with a type of pathotype (De Jong et al., 1989;Schnick et al., 1990;Phillips et al., 1992).
The studies of rDNA using PCR-RFLP method are attractive in case of small organisms like nematodes. In 1998 Blok reported variation in the multiple copies of rDNA of eighteen populations of G. pallida from Europe and South America. Authors conducting RFLP analysis of PCR products of ribosomal cistron out of six digestion enzymes found one that discriminated among most of the tested populations. The results support already existing studies that the majority of European population of G. pallida derived from one source with few exceptions (Blok et al., 1998).
RFPLs analysis of ITS-PCR products were carry out by Subbotin in 2000 on group of Russian populations of Globodera rostochiensis and the other Globodera species (Subbotin et al., 2000). Researchers used RFLP catalogue and sequence information from different populations or species and set of digestion enzymes to compare PCR products of nematode populations. Results of work revealed that rDNA in the genomes of G. rostochiensis populations is present as a mixture of haplotypes with different sequences and RFLP profiles and sequences of ITS region can be used rather for inter-than for intra-specific identification of Globodera species.

RAPD
Random Amplified Polymorphic DNA (RAPD) is one of the high sensitive PCR-based technique which involves the use of single 8-12 nucleotide length primers to amplification of many discrete and random DNA segments in the target genome (Welsh and McClelland, 1990). Genetic relationships between Dutch populations of G. rostochiensis and G. pallida analyzed by Folkertsma et al. (1994) with the use of RAPDs has shown pathotypes designation within the first species but not with the second. They found that isolates of G. rostochiensis classified as one pathotype were distinguishable by a number of unique RAPD fragments. Intra-species differences in European and South American populations of G. pallida and two populations of G. rostochiensis was examined by Blok et al. (1997). In their study both populations of golden PCN, identificated as Ro1, were similar but they reported higher dissimilarity of white PCN isolates then in a Dutch surveys. Research carried out by Bendezu et al. in 1998 on nine populations of G. rostochiensis from UK, Bolivia, the Netherlands and Germany with the use of four primers shown genomic similarity among European populations of yellow potato cyst nematode in 82% and among UK populations in 89%. Intra-species differences in virulence were investigated by Pastrik et al. (1995) on German G. pallida populations and the partially resistant potato cultivar Darwina. RAPD patterns with the use of 40 primers showed generally high similarity of selected and unselected for virulence PCN populations except of two non-homologous DNA fragments present in selected ones. Pastrik interpreted that one of this DNA fragment indicates a correlation with the particular type of virulence in individual population (Pastrik et al., 1995). Populations were analyzed and compared using random amplified polymorphic DNA and sixteen primers. Results of study showed that two populations of G. rostochiensis appeared to be distinct from the main group of this species as well as one population of G. pallida. Distinct clusters were observed within both species but the clusters could not be related to the geographic proximity of the populations.

AFLP
Amplified Fragment Lenght Polymorphism (AFLP) is a fingerprinting technique used to detection of polymorphism in different genomic regions of DNA. This method, highly sensitive and reproducible, is based on the PCR amplification of restriction fragments from a total digest of genomic DNA (Zabeau and Vos, 1993). It based on the ligation of adapters to ends of restriction fragments and a selective PCR-based amplification with adapter-specific primers. The standard procedures of AFLP described by Vos et al. (1995) become widely used for the identification of genetic variation in closely related species and populations. Like RFLP and RAPD analysis, AFLPs give an information about mutation that are dispersed over the genome. The comparative study of Globodera species and populations using AFLP revealed greater inter-and intra-specific variability than obtained by RAPD (Subbotin and Moens, 2006). AFLP technique were used to amplify genomic DNAs extracted from cysts of 16 Swedish and 20 other European populations of PCN, both G. rostochiensis and G. pallida from UK, Germany, Norway and the Netherlands (Manduric and Andersson, 2003). The results of analysis revealed that Swedish Ro1 populations were very similar to corresponding populations from other parts of Europe. The rest of G. rostochiensis populations appeared as a genetically heterogeneous group with two Swedish populations being most dissimilar. Characteristic of 9 G. rostochiensis populations carried out by Folkertsma et al. (1996) by AFLP analysis clustering them into three groups based on variants expressed by the presence or absence of polymorphic DNA fragments. These groups were distinguished by 3,7 and 12 unique DNA fragments, respectively.

Microsatellites
Microsatellites such as Simple Sequence Repeats (SSR) or Short Tandem Repeats (STR) or Simple Sequence Length Polymorphisms (SSLP) are repetitive DNA in which short DNA motifs are repeated many times in tandem (TRs). They are unstable and can mutate at rates between 10 3 and 10 6 per cell (Gemayel et al., 2012). TRs are present in coding and non-coding regions of nematode genome (Castagnone-Sereno et al., 2010;Pérez-Jiménez et al., 2013;Phumichai et al., 2015). Particular alleles differ in number of tandem repeats and give an information about differences in nematode populations (Jarne and Lagoda, 1996). In 2013 Boucher et al. with the used of three sets of microsatellite markers was looking for genetic diversity and the origin of introduction of 15 populations of G. rostochiensis distributed worldwide. Obtained results confirm influence of genetic drift on losing of genetic diversity of tested nematode populations and indicate populations from South America less diverse than European ones.
Microsatellites markers were also used to explain of allelic richness of G. pallida populations introduced from South America to Europe, Africa, North America and Asia. Plantard et al. (2008) carried out comparative analysis of the allelic richness at seven microsatellite loci observed in the Western European populations. They found only one-third of them observed in this part of southern Peru comparable to the allelic richness observed in the northern region of Peru. The authors give an explanation that genetic variability can be a result of a single introduction of infected plant and can influence on control of quarantine nematodes on the field.
Many molecular techniques based on DNA/RNA analysis is used to searching for variations among nematode populations but for practical use there is a need to distinguish species and pathotypes. Shields et al. (1996) used the polymerase chain reaction to amplify a region between the 5S rRNA and spliced leader RNA genes in G. rostochiensis and G. pallida. Isolates of G. rostochiensis amplified a single 914 bp product and were distinguishable from G. pallida isolates which amplified 914 and 853 bp products or a single 853 bp product. Concordant identifications of G. pallida and G. rostochiensis isolates were obtained by using 5S-SL PCR for specific DNA probes and differential host plant tests. Later the multiplex PCR analysis with primers GroR-GroF and PaR-PaF has been developed to distinguish the species Globodera rostochiensis and G. pallida by Fullaondo et al. (1999). Using a approach based on melting peak analysis of PCR products Bates et al. (2002) developed a semi-quantitative assay to measure the relative proportions of Globodera pallida and G. rostochiensis in a sample. The method depends on a multiplex PCR where the products of each species can be separated by their individual melting temperatures (Tm). 2% of G. pallida cysts in a mixture could be detected. Nakhla et al. (2010) developed the multiplex real-time PCR assays for the identification of the potato and tobacco cyst nematodes. They used a set of primers (PITSpf and PITS4) and a TaqMan probe (GFAMp) for the specific detection of G. pallida, and another set of primers (PGrtf and Prostor) for the detection of G. rostochiensis and the specific for G. tabacum primer set (PGrtf and PITSt3mr) with a TaqMan probe (GTETp).
However DNA/RNA markers could not be used to distinguish the PCN pathotypes. Hinch et al. (1998) developed a technique of high performance capillary electrophoresis (CE) that allows to obtain polypeptide profiles of each of the pathotypes of G. rostochiensis Rol-5, and G. pallida Pal, and mixture of Pa2 and Pa3. CONCLUSION Understanding of genetic diversity between populations and way of its dispersal in-and outside of origin geographic region is essential to prevent further spreading and create new methods of protection strategies. Both host range and genetic variation are important to have access to methods that enable inter-and intra-species identification of nematode populations. Classical methods of identification of PCN population based on morphological characterization and biotests identification are time-consuming and are more often replaced by molecular methods which are independent of environmental influence and stage of nematode. DNA-based diagnostic techniques are relatively quicker and the results seems to be more reliable in assessments of similarity of populations. For the practical purposes it is possible to distinguish the species of the evaluated nematodes on the basis of the DNA/RNA markers but up to now it is not possible to distinguish the pathotypes in a such way.