javanica appear to vary in pathogenicity to passion fruit. incognita and the ITS was also 99.7% identical (2 out of 630 bp difference) to OP745246 of M. Based on the BLAST sequence similarity search, the D2-D3 was found 99.7% identical (2 out of 660 bp difference) to MW551794 of M. Two representative sequences, one each for D2-D3 and ITS were submitted to GenBank and received the accession numbers OQ331163 and OQ331162, respectively. 1992) respectively and following the thermal profiles from Singh et al. The D2-D3 and the ITS of rDNA were amplified from the gDNA using the primer pairs D2A: 5´-ACA AGT ACC GTG AGG GAA AGT TG-3´/D3B: 5´-TCC TCG GAA GGA ACC AGC TAC TA-3´ (Nunn 1992), and Vrain2F: 5´-CTT TGT ACA CAC CGC CCG TCG CT-3´/Vrain2R: 5´-TTT CACT CGC CGT TAC TAA GGG AAT C-3´ (Vrain et al. For gDNA extraction from a J2, its cuticle was first punctured using a flamed metallic pin used as a ‘nematode picking tool’ and transferred to 10 μm of 0.05 M NaOH in a PCR tube followed by adding 1 μm of Tween 20 and subjecting the tube at 95 ☌ for 15 min in a thermocycler. The identification was also checked by amplifying the D2-D3 of 28 S and ITS of ribosomal DNA (rDNA) from individual (n = 5) of J2s. A fragment of approximately 290 bp DNA size was amplified only in Mi2F4/Mi1R1 primer, which confirmed the M. DNA samples were evaluated with a duplex PCR technique using two sets of species-specific primers including SEC-1 F/SEC-1R for M. Genomic DNA (gDNA) was isolated by smashing individual females (n = 5) in tubes containing PCR-grade water, followed by freezing at -20 ☌ overnight and thawing at 95 ☌ for 1 min.
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