902 Eur J Plant Pathol (2012)133:899-910 nematodes were ground in liquid nitrogen.Total RNA non-redundant protein database(nr)and a non- was extracted from the nematodes using TRIZOL redundant nucleotide database (nt)in NCBL following manufacturer instructions (Invitrogen)and further treated with DNase I(Promega)for 15 min at Obtaining complete sequence of Rs-eng-/b 37 C.The RNA was verified by 1.0 agarose gel electrophoresis and was stored-80C for later use. The fragment candidate of Rs-eng-Ib was screened Only 500 mixed stages nematodes separated from out of the library with alignment analysis.To obtain carrot disks were treated by RNAi.Therefore,a the complete sequence of Rs-eng-/b,3RACE primers MicroElute total RNA kit (OMEGA)was used to (NEST-S1 and NEST-S2)(Table 1)and 5'RACE extract the total RNA according to the kit operation primers (NEST-Aland NEST-A2)(Table 1)were protocol. designed to amplify the 3'end and 5'end of Rs-eng /b using a SMART RACE cDNA amplification kit SSH library (Clontech),respectively.Finally,we spliced three frag ments of Rs-eng-Ib(5'end,middle fragment,and 3 A SMARTer PCR DNA Synthesis Kit (Clontech) end)into the complete sequence of Rs-eng-/6.Two tran tota RNA ron A-C an specific primers (cds-F and cds-R)(Table 1)from to cDN ording t complet sequences of Rs-eng-Ib were anu cture designed to form the complete sequence of Rs-eng-/b went using a PCR-S on ki Expression analysis of Rs-eng-Ib and qPCR secondary I amplifie produ nt pGEM-T easy ega and qPCR was used to asse the var on in the e expres tran plet Escherich a coli JM1 eng-Ib be etween Rs-Ca -P ne cells.Final single were ran omly selecte ato ent for later PCR detecting and sequencing. eggs carrot callus Reverse Northern blotting juvenile Total RNAs from Rs-Cand Rs-P nematode population ively wer R h synthesize P fro PCR de populati prime DNA la samples as three biol dete on th cDNA ed CR-R by ano-Dr DNA abe pro kit and P om SSH d Rs-p nd c Sequencing and alignment analysis d in trinlic CFX-96 (Bio-Rad)qPCR machir SYBR GO After hybridization the results of the hybridized sis lus kit (TOYOBO) the nale analysed by UV transilluminator (Alpha and 60C for 30eA0 verse orthern blotting signals wer elected for cing.After CEX-96 ted ct valu and adapter ea uences, alig extrap olated relative levels of PCR products from Springernematodes were ground in liquid nitrogen. Total RNA was extracted from the nematodes using TRIZOL following manufacturer instructions (Invitrogen) and further treated with DNase I (Promega) for 15 min at 37 °C. The RNA was verified by 1.0 % agarose gel electrophoresis and was stored −80 °C for later use. Only 500 mixed stages nematodes separated from carrot disks were treated by RNAi. Therefore, a MicroElute total RNA kit (OMEGA) was used to extract the total RNA according to the kit operation protocol. SSH library A SMARTer PCR cDNA Synthesis Kit (Clontech) was used to transcribe total RNAs from Rs-C and Rs-P nematode populations into cDNA according to the manufacturer’s protocol. Resulting cDNAs under￾went Suppression Subtractive Hybridization (SSH) using a PCR-Select cDNA subtraction kit (Clontech). The secondary PCR amplified product was cloned into the vector pGEM-T easy (Promega) and was then transformed into complete Escherichia coli JM109 cells. Finally, single clones were randomly selected for later PCR detecting and sequencing. Reverse Northern blotting Total RNAs from Rs-C and Rs-P nematode populations were transcribed into cDNA according to operation in￾struction of a ReverTra Ace qPCR RT kit (TOYOBO). To synthesize Probe-C and Probe-P from Rs-C and Rs-P nematode populations, a DIG high prime DNA labelling and detection starter kit I (Roche) was used to label the cDNA templates. The concentration of probes was quantified by a Nano-Drop spectrophotometer. According to operation protocol of DIG high prime DNA labelling and detection starter kit I (Roche), the PCR products from SSH were hybridized with Probe-C and Probe-P. Sequencing and alignment analysis After hybridization, the results of the hybridized sig￾nals were analysed by UV transilluminator (Alpha Innotech). Blots that had two-fold differences in re￾verse northern blotting signals were selected for se￾quencing. After removing vector sequences and adapter sequences, sequences were aligned against a non-redundant protein database (nr) and a non￾redundant nucleotide database (nt) in NCBI. Obtaining complete sequence of Rs-eng-1b The fragment candidate of Rs-eng-1b was screened out of the library with alignment analysis. To obtain the complete sequence of Rs-eng-1b, 3′RACE primers (NEST-S1 and NEST-S2) (Table 1) and 5′ RACE primers (NEST-A1and NEST-A2) (Table 1) were designed to amplify the 3′ end and 5′ end of Rs-eng- 1b using a SMART RACE cDNA amplification kit (Clontech), respectively. Finally, we spliced three frag￾ments of Rs-eng-1b (5′ end, middle fragment, and 3′ end) into the complete sequence of Rs-eng-1b. Two specific primers (cds-F and cds-R) (Table 1) from spliced complete sequences of Rs-eng-1b were designed to form the complete sequence of Rs-eng-1b. Expression analysis of Rs-eng-1b and qPCR qPCR was used to assess the variation in the expres￾sion levels of Rs-eng-1b between Rs-C and Rs-P nem￾atode populations, and among different development stages of Rs-C: females, males, juveniles and eggs. Rs￾C and Rs-P were isolated from infected carrot callus for total RNA extraction. One hundred females, males, juveniles and eggs respectively were used for RNA extraction. The RNA was then quantified and qualified using a Nano-drop spectrophotometer, and then stored at −80 °C for further analysis. All the RNA used for qPCR was prepared from three different samples as three biological replicates. Based on the complete sequence of Rs-eng-1b, spe￾cific primers qPCR-F and qPCR-R (Table 1) were designed to represent Rs-eng-1b expression. According to the method described by Jacob et al. (2007), the primers Actin-F and Actin-R were synthesized (Table 1) to amplify the reference gene, β-actin. qPCRs were performed on mixed life stages of Rs-C and Rs-P and females, males, juveniles, and eggs of Rs￾C, and reactions were performed in triplicate on CFX-96 (Bio-Rad) qPCR machine, using SYBR Green qPCR Master Mix-plus kit (TOYOBO) according to the man￾ufacture’s protocol with the following reaction condi￾tions: 95 °C for 15 s and 60 °C for 30 s (40 cycles). Initial data analysis was carried out using the Bio-Rad CFX-96 manager software, which created Ct values and extrapolated relative levels of PCR products from 902 Eur J Plant Pathol (2012) 133:899–910