IC4R012-GWAS-2016-26552884

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Project Title

  • A genome-wide association study of a global rice panel reveals resistance in Oryza sativa to root-knot nematodes


The Background of This Project

  • The root-knot nematode Meloidogyne graminicola is one of the most serious nematode pests worldwide and represents a major constraint on rice production. While variation in the susceptibility of Asian rice (Oryza sativa) exists,so far no strong and reliable resistance has been reported. Quantitative trait loci for partial resistance have been reported but no underlying genes have been tagged or cloned. Here, 332 accessions of the Rice Diversity Panel 1 were assessed for gall formation, revealing large variation across all subpopulations of rice and higher susceptibility in temperate japonica accessions.


Plant Culture & Treatment

  • The seeds of the rice accessions were obtained from the National Rice Research Centre, USA, and bulked in Aberdeen, UK. The nematode screening method was adopted from Shrestha et al., (2007). The accessions were assessed in batches of 40 using 10 temporally separated runs. Each run consisted of three plug trays (LBS, Colne, UK) of 84 wells each [36.5 × 36.5×50 mm (length × width × height)] with each tray containing two replicates of 40 accessions from the RDP1 plus Azucena (tropical japonica rice variety) and Bala (indica rice variety) as reference genotypes. All genotypes were replicated six times,making a total of 252 plants per screening run. Within the three plug trays, a randomized complete block design was used where a block was one half of a tray. The trays were filled with sand and sown directly with two seeds per plug, which were then thinned to one seed per plug after 1 week. Every 2 weeks, another batch of 40 RDP1 accessions plus Azucena and Bala reference plants were sown in a separate screening run. J2 were collected from the roots of 50 mature rice plants as described in Shrestha et al. (2007), except that where cut galls from these stock plant roots were incubated for 7 days at 30°C in Shrestha et al., (2007), we incubated them for only 3 days at 28°C.
  • The M. graminicola inoculum used was originally obtained from CABI (Egham, UK) and has been maintained on rice for 10 years in Aberdeen. The plants were inoculated with 200 J2 per plant 2 weeks after planting. Two weeks after inoculation, the roots were carefully removed and washed with tap water, and the nematode galls counted. An incubation period of 2 weeks was chosen to erminate the experiment near the end of one nematode life cycle to avoid secondary galling. The life cycle of M. graminicola is completed in 19 days at 22–29°C in well-drained soil (Pokharel et al., 2010; Kyndt et al., 2012; Ji et al., 2013). The temperature in the growth chamber ranged from 28°C (day) to 25°C (night), with 50–70% humidity, and light of 350 µmol m−2 s−1 photosynthetically active radiation at plant height for 12 h d−1. The plants were watered daily to field capacity and fertilized twice a week with Yoshida’s nutrient solution (Yoshida et al., 1976). One of the runs included 30 randomly selected plants of RDP1 assessed for a second time to confirm the repeatability of the method.


Research Findings

  • Figure 1 shows the frequency distribution of the relative gall numbers, while all values are given in Supplementary Table 1.


'Fig. 1. Frequency distribution of gall numbers in 332 RDP1 accessions relative to the check genotypes Azucena and Bala.'


'Supplemrnt Fig.1. qq plots for all data with navie and mixed modle results'


  • As an example, the results for some accessions of the RDP1 in one run that show low, medium, and high galls numbers are presented in Fig. 2. Within most runs there was highly significant variation due to genotype (significant in 9 out of 10 runs), indicating genetic variation for gall numbers is readily detectable. The result of the repeat run with the 30 randomly selected rice accessions revealed a strong correlation with the initial assessment (r = 0.730, P < 0.001) (data not shown),indicating the method is highly repeatable.


'Fig. 2. Plot of example accessions in one run, including reference cultivars Azucena and Bala and 12 RDP1 accessions grouped as four low, four medium, and four high. Bar is standard error.'


  • There was a significant difference (P < 0.001) in gall number between rice subpopulations that explained 21% of the variation in relative gall number (Fig. 3). The temperate japonica had the highest number of galls, followed by tropical japonica, aromatic, indica, and aus. A Tukey’s test indicated that the number of galls in the temperate japonicas was significantly higher than in the other subpopulations.


'Fig. 3. Box plot of relative gall numbers in the different subgroups of rice.Temperate japonicas were significantly higher.'


  • The number of galls observed in the 10 selected accessions of the RDP1 5 weeks after inoculation was highly significantly different between accessions (P < 0.001, R2 = 79%) and correlated very strongly (r = 0.970, P < 0.001) with the nematode gall numbers in the initial 2-week nematode screening of 332 diverse cultivars (Fig. 4).


'Fig. 4. Plot of mean and standard error of gall numbers in 10 RDP1 accessions inoculated for 5 weeks in large pots versus the relative gall numbers obtained after 2 weeks when RDP1 was screened. Note no galls were detected in Khao Pahk Maw and LD 24. Correlation r = 0.967.'


Labs working on this Project

  • Institute of Biological and Environmental Sciences, University of Aberdeen, Aberdeen, AB243UU, UK
  • Department of Molecular Biotechnology, Ghent University (UGent), Coupure Links 653, B-9000, Ghent, Belgium
  • Current address: Department of Crop and Soil Science, Rivers State University of Science and Technology, Nigeria


Corresponding Author

  • Adam H. Price:a.price@abdn.ac