Partial Sequences Characteristics Analysis Of RRNA Genes From The Burrowing Nematode Radopholus Similis

The buowing nematode, Radopholus similis, was one of the most important quarantine pests in China. In order to identify the species, understanding the dynamic of phylogentic systems and the relationship between R. similis and related species, the partial rRNA gene sequences from nine populations of Radopholus similis detected from imported seedling or horticultural plants by the CIQ in China and four root lesion nematodes Pratylenhus were investigated. Results were as follows:D2 and D3 region of 28S rRNA gene from nine R. similis populations (RSHL,RSHN13,RSSZ,RSLZ,RSSH,RSHN12p,RSHN3,RSHN12r and RSHK) were amplified using universal primers D2A and D3B. After cloning and sequencing, nucleotide sequence length of each fragments were detected as 776bp,776bp,778bp,778bp,778bp,776bp,779bp,776bp,778bp respectively. The sequence of each population was submitted to GeneBank and the access numbers were EU555406,EU555411,EU555412,EU555409,EU555404,EU555407,EU555405,EU555408,EU555410 respectively. The sequence analysis and alignment analysis showed that the similarity between them was 99.13%. Phylogentic analysis of 9 populations with UPGMA program clustered 8 populations the same group and the other population RSHN3 in another group. It suggested that the host plant had great influence to the phylogentic development of R. similis.In order to further explore the sequence characteristics of 28S-rDNA-D2D3 district between populations of R. similis, we introduced two populations of Pratylenchus coffeae (PLD and PHN3) and two populations of Pratylenchus pseudocoffeae (PXM and PXM2). One fragment was generated by the amplification of 28S-rRNA-D2D3 district from the four populations using universal primers D2A and D3B. After cloning and sequencing, nucleotide sequence length of each fragments were detected as 781bp,778bp,785bp,786bp and these sequences were submitted to GeneBank.Seven restriction enzymes ( DdeI,Hpaâ…¡,Hinfâ… ,RsaI,Sau3AI,TaqI and MspI) were used for PCR-RFLP anlysis of 28S-rDNA-D2D3 district from nine populations of R. similis and four populations of root rot nematodes. The results showed that DdeI could identify RSHN3 population from other eight populations of R. similis, Hpaâ…¡, HinfI, Sau3AI, TaqI and MspI could identify three nematode species easily. RsaI could identify R. similis from root rot nematodes directly.The rDNA-ITS of three R. similis populations (RSHN3, RSHK and RSHZ) were amplified using universal primers FerF and FerR. After cloning and sequencing, the nucleotide sequence length of these populations were identified as 706bp. The sequence number submited to GeneBank were EU728660, EU728659, EU728661 respectively.Eight restriction enzymes were used for the PCR-RFLP analysis of rDNA-ITS from ten populations of R. similis. HinfI and RsaI could identify RSHN3 from other nine populations of R. similis. RSHN3 might either exist differences in genotypes with other populations or have mixed genotypes between these populations. Haeâ…¢, CfoI, Pvuâ…¡and TaqI digested the amplified fragments in the same way . However, BamHI and MspI could not digest the amplified fragments.The rDNA-IGS area of RSHN3 and RSHK populations were amplified using universal primers 5S and 18S. After cloning and sequencing, two nucleotide fragments were detected as 1123 bp, and 571bp in RSHN3, only one nucleotide fragment were detected as 1123bp in RSHK. These sequences were submitted to GeneBank.