Eur J Plant Pathol (2012)133:899-910 903 Table 1 Primers used for RACE of cndoglucanase Rs it-/6 Primer Sequence Source hain rea Actin- S-GAAAGAGGGCCGGAAGAG-3 Jacob et al.(2007 Actin-R 5-AGATCGTCCGCGACATAAAG-3 Jacob et al.(2007) on in Rs lation NEST S 5-ACGAGACCTACAATGAGC-3 NEST S2 5-AGCCTGCCCGTGTTCGTGAC-3 NEST Al S-GGCAGGTACTCGGTCACG-3 (ds)RNA of Rs-eng-Ib NEST A2 5-CATTGTAGGTCTCGTACC-3 OPCR-E 5-AATCTCTTACGTGAACTGGGC-3 OPCR-R SGGTCGCTCCAGATTTAGTCG-3 cds-E 5.TCCGCTTTCACCGCTTTCA-3 cds-R SCAGACATTCAGCATCCA3 T7S S-TAATACGACTCACTATAGGGGCTGTI A TCATTGTAG-3 NGGTGGGCTCATTGTAG-3 G-T7S G-A 5-CGATGCGGTTCACCAGGGTGTCG-3 G-T7A Gs 5CACAAGTTCAGCGTGTCCGGCG-3 standard curves.Melt curves were done routinely.and nd G)(Table)o gp)was generated with desig necific nrimer this allowed the possibility of hoth contamination and primer dimers to be discounted.Actin was used as a cloning vector PYL 322-d1-GFPn supplied by QL positive control in all experiments.All experiments Zhu,College of Life-Science,South China Agricultural were performed in triplicate. University. Synthesis of Rs-eng-Ib dsRNA Rs-eng-/b's RNAi treatment and silence detection A fragment of about 450 bp from the ORF of Rs-eng Five hundred nematodes from the Rs-C population Ib was cloned into the vector PMD18-T (TAKARA) cultivated on carrot callus were collected and trans The constructed vector was further confirmed by se ferred in an Eppendorf tube,treated with DEPC quencing.Based on the fragment,specific primers water and soaked in 50 ul rs-eng-lb dsRNa solution (T7S.A T7A.and S)(Table 1)with a T7 promoter (2 ug/ul)at room temperature for 12 h.24 h.36 h were designed to amplify the sense and anti-sense and 48 h,respectively.Non-endogenous gfp dsRNA product.Sense and antisense rNA were transcribed solution (2 ug/ul)was used as a control.The using T7 transcription kit(TOYOBO)according to the treatment times used for the control were same as manufacturer's instructions.Sense and antisense Rs-eng-/b dsRNAs.Meanwhile,untreated nematodes transcripts were annealed for 30 min at 37 C and were used as a blank control.Treated nematodes were analyzed by agarose gel electrophoresis.The dsRNA cleaned with DEPC water three times and the total RNA was purified by equal amount of LiCl(3 mol/l) was then extracted.aPCR was used to analyze transcript ovemight and washed by 70%ethylalcohol three times suppression of Rs-eng-/b in the nematodes after the finally stored at-80C for later use.Non-endogenous RNAi treatments.All experiments were performed control dsRNA (125 bp)(green fouorescent protein three times. Springe standard curves. Melt curves were done routinely, and this allowed the possibility of both contamination and primer dimers to be discounted. Actin was used as a positive control in all experiments. All experiments were performed in triplicate. Synthesis of Rs-eng-1b dsRNA A fragment of about 450 bp from the ORF of Rs-eng- 1b was cloned into the vector PMD18-T (TAKARA). The constructed vector was further confirmed by se￾quencing. Based on the fragment, specific primers (T7S, A, T7A, and S) (Table 1) with a T7 promoter were designed to amplify the sense and anti-sense product. Sense and antisense RNA were transcribed using T7 transcription kit (TOYOBO) according to the manufacturer’s instructions. Sense and antisense transcripts were annealed for 30 min at 37 °C and analyzed by agarose gel electrophoresis. The dsRNA was purified by equal amount of LiCl (3 mol/l) overnight and washed by 70 % ethylalcohol three times, finally stored at −80 °C for later use. Non-endogenous control dsRNA (125 bp) (green fouorescent protein gene, gfp) was generated with designed specific primers (G-T7S, G-A, G-T7A, and G-S) (Table 1) from the cloning vector PYL 322-d1-GFPn supplied by QL Zhu, College of Life-Science, South China Agricultural University. Rs-eng-1b’s RNAi treatment and silence detection Five hundred nematodes from the Rs-C population cultivated on carrot callus were collected and trans￾ferred in an Eppendorf tube, treated with DEPC water, and soaked in 50 μl Rs-eng-1b dsRNA solution (2 μg/μl) at room temperature for 12 h, 24 h, 36 h and 48 h, respectively. Non-endogenous gfp dsRNA solution (2 μg/μl) was used as a control. The treatment times used for the control were same as Rs-eng-1b dsRNAs. Meanwhile, untreated nematodes were used as a blank control. Treated nematodes were cleaned with DEPC water three times and the total RNA was then extracted. qPCR was used to analyze transcript suppression of Rs-eng-1b in the nematodes after the RNAi treatments. All experiments were performed three times. Table 1 Primers used for RACE of endoglucanase Rs-eng-1b gene sequence, quantitative polymerase chain reaction (qPCR) for Rs-eng-1b expression in Rs-C population and Rs-P population and different development stage of Radopholus similis, and to generate double-stranded (ds) RNA of Rs-eng-1b and non-endogenous gfp dsRNA control Primer Sequence Source Actin-F 5′-GAAAGAGGGCCGGAAGAG-3′ Jacob et al. (2007) Actin-R 5′-AGATCGTCCGCGACATAAAG-3′ Jacob et al. (2007) NEST S1 5′-ACGAGACCTACAATGAGC-3′ NEST S2 5′-AGCCTGCCCGTGTTCGTGAC-3′ NEST A1 5′-GGCAGGTACTCGGTCACG-3′ NEST A2 5′-CATTGTAGGTCTCGTACC-3′ qPCR-F 5′-AATCTCTTACGTGAACTGGGC-3′ qPCR-R 5′-GGTCGCTCCAGATTTAGTCG-3′ cds-F 5′-TCCGCTTTCACCGCTTTCA-3′ cds-R 5′-CAGACATTCAGCATCCA-3′ T7S 5′-TAATACGACTCACTATAGGGGCTGTT CTGGTCGCAATG-3′ A 5′-CAGAGGTGGGCTCATTGTAG-3′ T7A 5′-TAATACGACTCACTATAGGGCAG AGGTGGGCTCATTGTAG-3′ S 5′-GCTGTTCTGGTCGCAATG-3′ G-T7S 5′-GGATCCTAATACGACTCACTATAGGG CACAAGTTCAGCGTGTCCGGCG-3′ G-A 5′-CGATGCGGTTCACCAGGGTGTCG-3′ G-T7A 5′-GGATCCTAATACGACTCACTATAGGG CGATGCGGTTCACCAGGGTGTCG-3′ G-S 5′-CACAAGTTCAGCGTGTCCGGCG-3′ Eur J Plant Pathol (2012) 133:899–910 903