970 AJCS 6(6):970-979 (2012) ISSN:1835-2707 Morphological and molecular characterization of cereal cyst nematode (Heterodera avenae) populations from arid environments Ahmed A.M. Dawabah* 1,4 , Ahmad S. Al-Hazmi 1,4 , Soloiman M. Al-Rehiayani 3 , Ahmed L. Abdel- Mawgood 2,4 , Mohamed I. Motawei 3 , Soleman Al-Otayk 3 , Monther T. Sadder 4 , Abdallah M. Elgorban 4 , Hussein M. Migdadi 2 , Khaled A. Moustafa 2,4 and Abdullah A. Al-Doss 2,4 1 Plant Protection Department, College of Food and Agriculture Sciences, King Saud University, Saudi Arabia 2 Plant Production Department, College of Food and Agriculture Sciences, King Saud University, Saudi Arabia 3 Plant Production and Protection Department, College of Agriculture and Veterinary Medicine, Qassim University, Saudi Arabia 4 Center of Excellence in Biotechnology Research, King Saud University, Saudi Arabia *Corresponding author: adawabah@ksu.edu.sa Abstract The morphological and molecular characteristics of four cereal cyst nematode (Heterodera avenae) populations collected from the Qassim, Tabouk, Riyadh, and Hail regions, Saudi Arabia were comparatively investigated. A large number of soil samples were collected from a representative field (72 ha) in each region. The morphological and morphometric characteristics of the populations were determined. Morphometric data were subjected to multivariate canonical discriminant analysis to analyze the relationship between the studied populations and to identify the variables that show the highest multiple correlations with these populations. For molecular characterization, DNA was extracted and purified from five random white females from each population. The internal transcribed spacer (ITS1) regions were subjected to direct sequencing to study the diversity of these populations. Discriminant analysis of the morphometric traits indicated that the studied populations belong to one species (H. avenae). The ITS1 sequence alignments showed similarity between individuals, ranging from 87 to 99%. Based on the sequencing data, consensus parsimonious and maximum likelihood trees showed an overlap between the individuals of the four populations, suggesting that all four populations represented one species. However, based on the morphological and molecular analysis, the Hail population was somewhat different from the other three populations. Minor genetic and phenotypic differences between the four populations could indicate that these populations are heterogenic, probably mixed populations. This study also revealed the value of some J 2 morphometric traits such as J 2 midbody width, J 2 body width at the anus, J 2 head height and the J 2 ratios a, b, c and cʹ in determining intraspecific variation between H. avenae populations. Keywords: Discriminant analysis, Heterodera avenae, intraspecific variation, ITS, heterogenic, polymorphism, phylogenetic. Abbreviations: CCN – cereal cyst nematode, CDA - canonical discriminant analysis, DNA – deoxyribonucleic acid, dNTP – deoxyribonucleotide triphosphate, Ha – hectare, ITS – internal transcribed spacer, J 2 – second-stage juveniles, PCR – polymerase chain reaction, rDNA – ribosomal DNA, RFLP – restriction fragment length polymorphism. Introduction The cereal cyst nematode (CCN), Heterodera avenae Woll. (1924) has been reported on wheat and other cereal crops in many countries with different climatic types throughout the world (Handoo, 2002). In Saudi Arabia, the nematode was reported in an irrigated wheat (Triticum aestivum L.) field in the Qassim region in 1987 (Youssif, 1987). The nematode has since spread quickly to become a major limiting factor to wheat production in Saudi Arabia (Al-Hazmi and Dawabah, 2009, Ibrahim et al., 1999). Saudi populations of cereal cyst nematodes were identified as H. avenae, based on morphological and morphometric features (Al-Hazmi et al., 1994), and DNA markers (Al-Rehiayani, 2007). Isoelectric focusing of protein pattern analysis has shown that Saudi populations are typically H. avenae (Sturhan and Rumpenhorst, 1996; Subbotin et al., 1996; 1999; 2002). The pathotype of the nematode was characterized as being very close to the European pathotype Ha21 (Al-Hazmi et al., 2001). Heterodera avenae is one of several species comprising the H. avenae group, which contains 11-12 valid species plus other species (Handoo, 2002; Subbotin et al., 2003). These species can be differentiated from each other by some morphological and morphometric features (Abidou et al., 2005; Handoo, 2002; Subbotin et al., 1999). However, with the increasing number of species in this group, reliable identification based on morphology is becoming more difficult (Rivoal et al., 2003; Subbotin et al., 2003). DNA sequence variation in the internal transcribed spacer (ITS) regions of ribosomal DNA can be used to identify many nematode taxa and phylogenetic relations (Subbotin et al., 2001). The ribosomal genes flanking the ITS region are highly conserved, allowing for the construction of PCR primers. However, the ITS region is less conserved than the flanking regions, which allows for the detection of polymorphisms (Kumar et al., 2009; Madani et al., 2004).