SCAR Marker And Molecular Detection Technique Of Bursaphelenchus Xylophilus

For the limitation of morphological identification of Bursaphelenchus xylophilus, four research works have been studied in this paper. The first work was the study on the detection technique of B. xylophilus and B. mucronatus by RAPD. The second thing was the SCAR molecuar maker on B. xylophilus and B. mucronatus .The third study was about rDNA restriction fragment length polymorphism of B. xylophilus and B. mucronatus. The last work was to conceive of the kit for quick detection of B. xylophilus . Thirty-five strains of nematodes were used for this study. Nineteen strains of B. xylophilus originated from America, Canada, Japan and China, and thirteen strains of B. mucronatus came from Japan, Korea, China and Taiwan province of China, and three interminatae species of nematodes were separated from the dead pines which came from main pine distribution in our country. The results were as follows: 1. We used worm lysis buffer for extracting genomic DNA of nematodes for a great deal of nematodes instead of grinding nematodes. The advantage of this method is decreasing the loss of gross DNA. The quality of DNA is very high and the ratio of absorbancy at 260nm and 280nm was between 1.7 to 1.9, and the content of DNA between 300~630μg/mL; Incision way has been used for extracting genomic DNA from a few even single nematode which were treated with liquid nitrogen for ten minutes, or -70℃for fifteen minutes or -20℃for forty minutes. 2. By random amplified polymorphic DNA technique, seven of the primers were screened from 140 arbitray oligonuleotide primers, namely OPC18, OPC19, OPC08, OPK09, OPM05, OPN09, and OPY01. Of total twelve specific fragments which were amplified using seven primers above, seven were the specific fragments of B.xylophilus, and five were that of B. mucronatus. (1) OPC08 and OPK09 were the specific primers of B. xylophilus. A approximate 760bp specific fragment was obtained with OPC08 and the length of two specific fragments obtained by OPK09 were about 1200bp, 2400bp. (2) OPN09, OPM05, OPY01 were the common specific primers of B. xylophilus and B. mucronatus. Two specific about 480bp and 880bp fragments of B. xylophilus and a 620bp specific fragment of B. mucronatus were obtained by OPN09. The specific fragment about 2100bp of B. xylophilus and one about 1300bp specific fragment of B. mucronatus were obtained with OPM05. The specific fragment about 730bp of B. xylophilus and one about 850bp specific fragment of B. mucronatus were obtained with OPY01. (3) OPC18, OPC19 were the specific primers of B. mucronatus, and one specific fragment about 970bp and the other specific fragment about 1350bp were obtained by OPC18 and OPC19 respectively. Experiment indicated that these random primers had high specificity and sensitivity on all strains of B. xylophilus and B. mucronatus. So B. xylophilus and B. mucronatus could be distinguished by these primers. 3. Candidate RAPD fragments of B. xylophilus and B. mucronatus were excised from agarose gels and purified which were ①OPC19-M1350, ②OPK09-X2400, ③OPC18-M970, ④OPM05-X2100,⑤OPN09-X480,⑥OPM05-M1300,⑦OPK09-X1200,⑧OPY01-M850,⑨OPY01-X730. Then,the gel-purified fragments were cloned into the pGEM?-T Vector, sequencing was done. Based on the sequences of RAPD markers, a number of sequences characterized amplified region (SCAR) primers were designed by the aid of the software Oligo5.0. It's successful to convert four of nine specific fragments to SCAR marker. (1) OPK09-X1200 could be converted to SCAR-K09-X837 by K09F1/R and SCAR-K09-X340 using K09F2/R. Both of them were the specific markers for B. xylophilus. (2) OPM05-X2100 could be converted to SCAR-M05-X1127 using primer M05F1/R1, and SCAR-M05-X600 using M05F2/R1, SCAR-M05-X1127 and SCAR-M05-X600 also were the specific markers of B. xylophilus. (3) OPY01-M850 could be converted to SCAR-Y01-M609 using Y01F/R, which was the specific markers of B. mucronatus (4) OPC18-M950 could be converted to SCAR-C18-M931 using Y01F/R, which also was the specific marker of B. mucronatus. Primers sets K09F1/R, K09F2/R, M05F2/R1 and Y01F/R had high specificity that could be used for the identification of B. xylophilus and B. mucronatus, but primer set M05F1/R1 wasn't fit for the identification of B. xylophilus and B. mucronatus, and B. xylophilus can't be separated from B. mucronatus using primer set C18F/R. 4. A ploymerse chain reaction-restriction fragment polymorphism (PCR-RFLP) analysis was used for discrimination of isolates of B. xylophilus and B. mucronatus. The amplications of isolates of B. xylophilus yielded one fragment of approximately 890bp. But that of B. mucronatus was about 930bp. Digestion of amplified products of each nematode isolate with five restriction endonucleases showed: (1) DraI digestion of its products of B. xylophilus populations yielded two fragments, 510 and 380bp. But DraI couldn't digest ITS products of B. mucronatus populations. (2) SalI can't digest the ITS products of all B. xylophilus populations. But it can digest that of B. mucronatus populations to two fragments, which are 720 and 220bp; (3) Digesting production of four B. xylophilus populations by MspI yielded two fragments, 530 and 360bp, except GZ02 whichcan't be digested. But B. mucronatus populations yielded three fragments, 340, 290and 180bp; (4) All populations of B. xylophilus and B. mucronatus can't be digested by ApaI; (5) Digestions of the products of B. xylophilus and B. mucronatus yielded two fragments respectively, 520 and 370bp, 530 and 400bp. The restriction endonucleases, DraI and SalI, could be used for identification of B. xylophilus and B. mucronatus. The results of digestion of B. xylophilus and B. mucronatus were such sharp difference that it was very easy to identify them and it was very convenient to be applied. MspI and XhoI were not fit for the identification of B. xylophilus and B. mucronatus; ApaI could not distinguish and identify B. xylophilus and B. mucronatus. 5. B. xylophilus, B. mucronatus and the other nematodes separated from dead pine were detected using primer set K09F2/R. The results indicated that the PCR product of all B. xylophilus isolates was a clear and bright fragment about 340bp. But B. mucronatus and other nematodes had not any fragment. So pairwise K09F2/R was used for constructing identification kit of B. xylophilus. It costs a short time to identify nematodes by adopting this kit. The whole process of detection need three and half hours, implementing the aim of quick detection. Besides, the sensitivity of this kit reached to 1/7 of a nematode, achieving the purpose of identify juvenile successfully. The result is easy to estimate, and the cost of detection is bargain, so it's suit for applying.