Abstract: Heterodera avenae is distributed in 13 provinces of China and causes considerable yield losses of wheat. Inter-Simple Sequence Repeats (ISSR) fingerprint patterns and sequence analysis of the ITS region were used for the comparative study of genetic variation of H. avenae. DNAs of second-stage Juveniles from 16 populations of H. avenae and one population of Heterodera filipjevi in 4 provinces were analysed with 3 ISSR primers. Results of ISSR revealed a high genetic diversity (the percentage of polymorphic bands was 78.6%) within H. avenae on a large geographical scale. The analysis of molecular variance (AMOVA) indicated that about 74.8% of the variations resulted from genotypic variations within region and the remaining 25.2% were due to variances among regions. Base transition of ITS-rDNA sequence revealed low intraspecific variation in rDNA of H. avenae. The alignment of 16 sequences in H. avenae covered altogether 1045 nucleotide positions with 4 polymorphic bases. ISSR dataset grouped all populations into two clusters according to their geographical origin. These results suggested two different pathotypes or biological entities existing in the H. avenae species. Hybridization, multiple introductions, passive dispersal by anthropogenic activities and some natural means would probably be responsible for the genetic variation of H. avenae.
INTRODUCTION
The cereal cyst nematode, Heterodera avenae Wollenweber is distributed worldwide in most wheat growing areas, including Asia, Europe, America and Pacific Northwest states of the USA (Rivoal and Cook, 1993; Nicol et al., 2003). Heterodera avenae parasitized over 30 plant species and caused considerable yield losses of wheat (Chen et al., 1992; Rivoal and Cook, 1993; Smiley et al., 1994). The nematode was first detected in Hubei province, China, in 1989 and now it is widespread in Anhui, Beijing, Hebei, Henan, Jiangshu, Shandong, Shanxi, Sanxi, Qinghai, Ningxia and Inner Mongolia provinces (Chen et al., 1992; Zheng et al., 2000; Ou et al., 2008). Pathotypes of the nematode detected in China are different from those reported in Europe and Australia (Abdollahi, 2008; Peng and Cook, 1996; Zheng et al., 1997). The wide distribution and different pathotypes of H. avenae has required a series of management methods, such as rotational control, biological control and resistant cultivars (Zheng et al., 1997). Furthermore, it is difficult for most farmers to apply chemical pesticides because of the cost and environmental concern in China. In the search for ecological and economically suitable management strategies, the identification of genetic relationships at the species level and intra and interspecific variability is an essential step (Kaplan et al., 1999; Castagnone-Sereno, 2002).
Molecular information, mainly based on specific parts of the genome, was shown to provide significant insights into the degree of diversity existing among individuals and populations of plant-parasitic nematodes (Patra et al., 2011; Hlaoua et al., 2008; Powers et al., 1997; Peng et al., 2003; Madani et al., 2004). Several molecular techniques had been applied to study genetic diversity of cyst nematode populations (Zhang et al., 1998; Rivoal et al., 2003; Swofford, 2002; Madani et al., 2007). The Inter-simple Sequence Repeats (ISSR) PCR technique has been shown to have a wide application for the analysis of genetic variation at subspecies level, particularly in investigations of population structure and differentiation (Zietkiewicz et al., 1994). The ISSR technique was applied to study genetic diversity of populations of plant-parasitic nematodes of Bursaphelenchus xylophilus (Metge and Burgermeister, 2006), Nacobbus aberrans (Lax et al., 2007), Ditylenchus destructor (Huang et al., 2010) and to analyze the population structure of entomopathogenic nematodes and crop resistance of nematodes (Zietkiewicz et al., 1994; Berner and Schnetter, 2002; Metge and Burgermeister, 2006; Dayteg et al., 2008).
The Internal Transcribed Spacer region (ITS) of ribosomal DNA (rDNA), a well-conserved region controlled by concerted evolution (Baldwin and Mundo-Ocamp, 1991; Sridhar et al., 2010), has been demonstrated to be highly informative as a taxonomic marker at species level within nematodes (Powers et al., 1997; Subbotin et al., 2001). The phylogenetic relationships of cyst-forming nematodes were analyzed by ITS rDNA gene sequences (Subbotin et al., 2001; Peng et al., 2003; Maafi et al., 2003). The heterogeneity of the ITS-rDNA was revealed by RFLP analysis in many species of cyst forming nematodes (Szalanski et al., 1997; Zheng et al., 2000; Peng et al., 2003; Madani et al., 2004; Ou et al., 2008). It was revealed that intraspecific polymorphism of H. avenae populations in different countries can be distinguished by rDNA-RFLP studies of rDNA-ITS regions (Subbotin et al., 1999). Peng and Cook (1996) concluded that pathotypes of cereal cyst nematode in China may differ from those of other countries and regions but they did not allocate numbers for pathotypes using the scheme of, Andersen and Andersen (1982) because some host reactions still need to be validated. Zheng et al. (1997) showed the Taigu and Guzhen populations were distinctive new pathotypes. Several management methods may have specific genotype-level interactions with nematodes from different origins (Blok, 2005; Castagnone-Sereno, 2002). Therefore, evaluation of polymorphism among the populations of H. avenae by combining ISSR with ITS-rDNA gene analysis is needed to generate effective control strategies.
In the present study, the ISSR technique and ITS-rDNA gene sequences were used to analyze the genetic variation of H. avenae populations in China at two different levels. Based on molecular phylogenetic relationships, the genetic structure of this nematode was analyzed at regional level.
MATERIALS AND METHODS
Nematode populations: The sample set used in the present study included H. avenae populations from 16 different locations of 4 provinces throughout wheat-producing regions in China (Table 1, Fig. 1).
| Table 1: | Location and genetic diversity of 16 populations of H. avenae and one population of H. filipjevi |
| Altitude, latitude and longitude data were recorded by a GPS locator, PPB: Percentage of polymorphic bands | |
| Fig. 1: | Geographic location of the 16 H. avenae populations and one H. filipjevi from 4 provinces in China |
One population of H. filipjevi from Henan province was also included as an outgroup. Ten soil cores (10 cm diameter and 25 cm deep) were collected from each location using a cylindrical sampling tube and mixed uniformly. Cysts were isolated from soil by the routine sieving-decanting method and identified by examining the vulval cone as described by Yan and Smiley (2010). Five cysts were selected randomly from each population and put under 4°C for six weeks then under 15°C for five weeks for the hatching of second-stage juveniles (J2s).
DNA isolation: For each population, genomic DNA was isolated from 50 J2s of the mixed cysts. DNAs of J2s from the same population were used to test the repeatability of the amplified products obtained. Each specimen was crushed with a glass pestle inside the tube after it was immersed in liquid nitrogen for 30 sec. Then 32 μL Worm Lysis Buffer (500 mM KCl, 10 mM Tris-HCl, 15 mM MgCl2, 1.0 mM DTT, 4.5% Tween20) and 8 μL Proteinase K (20 mg mL-1) were added to each tube. The tubes were incubated at 65°C for 2 h, followed by 10 min at 94°C in a Master cycler 5332 PCR thermal sequencer (Eppendorf Inc). DNA extracted from each population that produced reproducible bands selected for the next analysis and they were kept at -20°C until use.
ISSR-PCR amplification: Several DNA and primer concentrations used in the reaction mix, number of cycles, annealing temperature and evaluation of multiple 2 μL aliquots from DNA extraction were tested to optimize PCR reaction conditions and guarantee the repeatability of the amplified products obtained. A total number of 10 ISSR primers were tested (Zietkiewicz et al., 1994; Tikunov et al., 2003) those that produced clear, reproducible and polymorphic bands were selected for the analysis. Each ISSR reaction was carried out as the system of Lax et al. (2007) with the programme as follows. An initial denaturation at 94°C for 5 min, followed by 38 cycles of 30 sec at 94°C, 45 sec at 52°C, 1 min at 72°C and final extension of 10 min at 72°C. The PCR products were separated electrophoretically on 2% agrose gels in 0.5xTAE buffer. Gels were stained with ethidium bromide and photographed with Gel Doc XR image analysis system (Bio-Rad).
ISSR data analysis: Amplified bands were scored as 1/0 (presence/absence) of homologous bands for all samples. Only bands that were reproducible were included in the study. Similarity matrices were analyzed using the unweighted pair group arithmetic average (UPGMA) clustering method with the NTSYS program, version 2.1 and dendrograms were created (Rohlf, 2000). The robustness of the dendrogram was tested by bootstrapping with 1000 permutations. The resulting presence/absence data matrix of regional group of H. avenae was analyzed using POPGENE version 1.32 (Yeh et al., 1999) to estimate the level of genetic diversity by the Percentage of Polymorphic Bands (PPB), average Heterozygosity (H) and Shannon information index (I).
ITS-rDNA-PCR and sequencing: The protocol for ITS-rDNA-PCR was essentially performed as described by Madani et al. (2007) with some minor modifications. The PCR reaction consisted of 2 μL of DNA, 2.5 μL 10xreaction buffer (500 mM KCl, 100 mM tris-Cl, pH 9.0; 25 mM MgCl2), 2 μL of each dNTP (2.5 mM each), 2 μL of each primer (10 μM) and 0.5 μL of Taq DNA Polymerase (5 U μL-1) and distilled water to a total volume of 25 μL. PCR reactions were programmed for an initial denaturation at 94°C for 5 min, followed by 34 cycles of 30 sec at 94°C, 45 sec at 55°C, 1 min at 72°C and final extension of 10 min at 72°C.
Gel purified ITS-rDNA-PCR products were ligated into a pMD-18T vector and transferred to Escherichia coli DH5a according to the manufacturer’s instructions (Invitrogen, America). Both DNA-strands were sequenced from all samples by an ABI 377 sequencer in Beijing Genomic Institute.
Phylogenetic data analysis: A total 16 newly obtained ITS-rDNA sequences of H. avenae and one sequence of H. filipjevi were used for the final analysis. Four ITS Sequences (AY148360, AY148362, AY148367, EF153843) of H. avenae from other countries were downloaded from GenBank for the phylogenetic analysis. Sequences were aligned with Clustal (W) and Minimum-Evolution (ME) analyses were performed with Mega4 software by Tamura et al. (2007). The robustness of the dendrogram was tested by bootstrapping with 1000 permutations.
RESULTS
The cuticle of mature cysts was dark brown to black for H. avenae in color and golden to light brown for H. filipjevi. A distinct underbridge with bifurcated arms was present in vulval cones of the cysts of H. filipjevi whereas no underbridge was found in H. avenae.
ISSR analysis: Sequences of the three anchored primers selected to amplify ISSR in all populations were shown in Table 2. High polymorphism and genetic variability were observed using selected primers (Table 1, Fig. 2). A total number of 42 unambiguous fragments were scored, among which 33 were polymorphic (78.6% polymorphism). For the three selected primers (Table 2), the number of scored bands were 15, 14, 13 and the polymorphism was 80.0, 78.6, 76.9%, respectively. No population-specific band of H. avenae was detected. Similarity matrix generated by Jacard’s coefficient showed that the average genetic similarity of H. avenae was 0.69 and the genetic similarity of H. filipjevi was 0.65. The highest genetic similarity coefficient was observed between HNPY and HNXC with the value of 0.93. The lowest genetic similarity coefficient was observed between HNAY and HBXT with the value of 0.48.
Within the four studied geographical regions, the PPB of H. avenae was varied from 33.3 and 54.8% in Hebei and Shandong province, respectively (Table 1). Average heterozygosity (H) value and I index showed similar trends, which ranged from 0.14 to 0.23 and 0.21 to 0.33 in Hebei and Shandong province, respectively. At the species level of H. avenae, the average PPB was 78.6%, heterozygosity (H) was 0.30 and Shannon information index (I) was 0.44. At the region level of H. avenae, the hierarchical AMOVA revealed small genetic divergence among regions from different locations.
| Table 2: | Sequence of ISSR primers used for the analysis of H. avenae populations and polymorphism of primers |
| Table 3: | Hierarchical analysis of molecular variance (AMOVA) of the 16 H. avenae populations |
| Fig. 2(a-c): | ISSR bands of 16 different populations of H. avenae and one population of H. filipjevi to indicate the selected bands (arrows) for the polymorphism analysis, (a) Primer ISSR2, (b) Primer ISSR10 and (c) Primer ISSR16 |
| Fig. 3: | Phylogenetic relationships among H. avenae populations based on their Jacard’s genetic similarity values using ISSR markers. UPGMA clustering was constructed using the NTSYS program, version 2.1. Confidence levels over 60% from bootstrap analysis (1000 permutations) are indicated at the nodes. A: Clade A, B: Clade B, Grouped on the basis of Jacard’s diversity level of 0.75 |
The results also showed that the majority of genetic variation (74.8%) occurred within regions, whereas small genetic variation (25.2%) was due to regional differences (Table 3). Values for two hierarchical levels were significantly different.
Sixteen populations of H. avenae were grouped into two main clades at the Jacard’s diversity level of 0.75 (Fig. 3). Clade A comprises all the populations of H. avenae in Anhui and Henan province and three populations in Shandong province. The SDLW, SDLY population and all populations of Hebei province of H. avenae were grouped in clade B. Most H. avenae populations were grouped in the same clade which was corresponded to their geographic distributions. However, five populations in Shandong province were clearly separated to two different clades.
ITS analysis: The ITS sequences of H. avenae and H. filipjevi reported here have been deposited in GenBank: HM370406-HM370411, HM370414-HM370420, HM370426-HM370429. The ITS-rDNA sequence length of H. avenae was 1045 bp and the ITS-rDNA sequence length of H. filipjevi was 1054 bp. Except for ten extra bases existing in H. filipjevi, thirty parsimony-informative sites occurred between sequences of H. avenae and H. filipjevi. The alignment of 16 sequences in H. avenae covered 1045 nucleotide positions with 4 polymorphic sites.
With the outgroup of H. filipjevi, seventeen equally most parsimonious trees was generated by ME analysis of ITS-rDNA gene alignment. Sixteen H. avenae populations were grouped into two main clades, which were distinctively divergent from the H. filipjevi population (Fig. 4). The clade I contained all the populations of China and the clade II contained populations from India, Gemany, Morocco and USA.
| Fig. 4: | Phylogenetic relationships with the ITS-rDNA sequences of H. avenae. Posterior probabilities greater than 60% are given for appropriate clades. I: Clade I containing all the population of China, II: Clade II containing population of India, Morroco, Germany and USA |
Within clades I, close relationship was observed according to their geographical origin in most subclades. However, SDLW and SDSG were somewhat divergent from other H. avenae populations.
DISCUSSION
ISSR results: To our knowledge, this is the first study for the genetic structure of H. avenae populations revealed by ISSR markers at a macro geographical level. It was proved that ISSR markers were efficient for detecting genetic variation among the different geographic populations of H. avenae in China. At the species level, genetic diversity within population of H. avenae was higher than those of H. glycines, H. schachtii and Bursaphelenchus xylophilus, regardless of molecular markers (Zhang et al., 1998; Plantard and Porte, 2004; Madani et al., 2007; Vieira et al., 2007). Genetic similarity index is reliable for the evaluation of genetic variation level among different populations. In the present study, the genetic similarity index ranged from 0.41 to 0.86, which indicated H. avenae had high genetic diversity. One possible source of the high genetic polymorphisms found in H. avenae populations might be related to the reproduction mode. Because of their small body size, active dispersal is probably limited to a few centimeters or decimeters. Individual male could mate with different females and occasionally several males would attempt to mate with the same female at the same time (Anderson and Darling, 1964). The multiple matings would favor offspring with higher genetic diversity of H. avenae. Based on the investigation of Zheng et al. (1997) and Peng et al. (2003) on the race of H. avenae, we speculate there are at least two different pathotypes within H. avenae in China. The outcrossing of different pathotypes at the same field will favor the increase of the genetic variation among populations. In addition, the genetic variation may largely result from multiple introductions of H. avenae from different geographical origins (Da Conceicao et al., 2003; Plantard et al., 2008). In addition, the nematodes can move only short distances in the soil and have no natural means of long-range movement, gene flow among populations could be generated by passive transport of nematodes across fields by human activities (e.g. transport of soil by farm machinery, combine harvester and cultivation) or by water (flood, irrigation or drainage) and wind (Meagher, 1977; Plantard and Porte, 2004). These dispersal mechanisms would be favored for the maintenance of highly effective population sizes which contribute to the increase of the genetic diversity of natural populations within this species (Lax et al., 2007).
ITS results: Although, ITS repeat units are generally considered to be rapidly homogenized by concerted evolution, the occurrence of intra-specific and intra-individual, ITS polymorphism were reported for Meloidogyne, Globodera and Heterodera spp. (Szalanski et al., 1997; Subbotin et al., 2000). Our sequence analysis of ITS-rDNA clone variants revealed low intraspecific variation in rDNA of H. avenae. Base transition and transvrersion of rDNA may result from different individuals of the same population or from different operons of the same nematode. A more possible explanation for the observed ITS variation might be related to the presence of several major rDNA gene loci in the genome of H. avenae (Madani et al., 2007). Since it was demonstrated that intrachromosomal homogenization was faster than interchromosomal recombination (Parkin and Butlin, 2004), the processes of concerted evolution might not have enough time to homogenize rDNA sequences across different loci. In addition, the outcrossing nature of H. avenae would also lead to hybridizations between nematodes and subsequently resulted in homogenization of the variants by concerted evolution.
Comparison between ISSR and ITS data: From the results of this study, it was clear that the ISSR molecular marker, which covers multiple loci across the genome, can provide a higher resolution of clustering between the analyzed populations of H. avenae at large scale. Based on the ISSR and ITS data we conclude that H. avenae showed some degree of differentiation at large geographical scale. High level of variability and gene flow within and between populations of different regions in China was also observed. ISSR and ITS-rDNA dataset grouped most populations into two clusters separately. These results would be confirmed by two different pathotypes or biological entities existing within the species. Given the clear genetic differentiations between these two groups considered, it would be interesting to conduct intensively investigation to make a genetic comparison between these two biological entities.
ACKNOWLEDGMENTS
This study was funded by the Special Project for Public Agriculture Research and Development of Ministry of Agriculture (Project No. 200903040) and the Key International Science and Technology Cooperation Project (2009DFB30230).