Rapid Molecular Diagnosis Of Cyst Nematodes On Cereal Crops

Cyst nematodes on cereal crops is an important pest that caused economic losses of crops inChina and it is widely distributed in the world. In this study, we took work on the rapid moleculardiagnosis of the cyst nematodes on cereal crops, as the species of cyst nematodes on cereal crops aresimilar to the form, and the identification often encountered difficult problems.In this study, we detected and identified the samples from the occurrence regional of cyst nematodeson cereal crops and legume crops using universal primers TW81and AB28, and the amplificationresults of rDNA-ITS sequence were about1000bp. It could be determined that in our country26cystnematode samples were detected in Heterodera avenae,4samples were detected in H. filipjevi,14samples were detected in H. glycines,10samples were detected in H. goettingiana, and4samples weredetected in H. elachista. Multiple sequence alignment showed that H. avenae and H. filipjevi hadvariable sites at rDNA-ITS region. The phylogenetic analysis provided the relationship of the fivespecies of cyst nematode in China, and it provided the basis for the rapid molecular detection.The RFLP (Restriction Fragement Length polymorphism) results showed that the restrictionendonuclease TaqI could make different digested results of H. avenae, H. filipjevi and H. glycines,however, it can not be used to distinguish H. goettingiana and H. elachista.We screened out five specific RAPD (Random Amplified Polymorphism DNA) primers OPA06,OPD13, OPC06, OPK16and OPB15from the13Operon series of random primers containing10bases. Inthese five primers, OPA06and OPD13can specificly amplify H. avenae, as the same, OPA06was also thespecific RAPD primers to H. goettingiana. OPC06å'ŒOPK16was specific primers to H. filipjevi, andOPB15was specific primer to H. elachista. Through analysis of the RAPD sequence, we transformed theRAPD sequence into SCAR (Sequence Characterized Amplified Region) markers. We obtained twopairs of SCAR marker primers OPA06-Ha and OPD13-Ha, OPD13-Ha was applied for nationalpatent (application number：CN201210032018.0), and the amplification fragment was452bp and1010bp. Duplex PCR to H. avenae can be implemented by OPA06-Ha combined with universal primersTW81and AB28. We designed two pairs of SCAR marker primers OPC06-Hf, and OPK16-Hf to H.filipjevi, the amplification fragment was313bp and646bp. Duplex PCR to H. filipjevi can beimplemented by OPC06-Hf combined with universal primers TW81and AB28, and it was applied fornational patent (application number: CN201110338657.5). In addition, we designed a pair of primerOPB15-He to H. elachista and a pair of primer OPA06-Hgo to H. goettingiana, the amplificationfragment was434bp and544bp.The cDNA and gDNA sequences of β-actin from H. avenae was cloned by RACE (RapidAmplification of cDNA Ends) and RT-PCR methods, and it was named Ha-actin-1(GenBank AccessionNumber: JQ074059). The full cDNA length of Ha-actin-1was1308bp, and the ORF was1054bpencoding350amino acid, and the full gDNA length was2204bp. It can be concluded that the fullgenomic sequence of Ha-actin-1contained7introns and the splice-site was obeyed the GT/AG rule.Homology search showed that the amino acid sequence was highly homologous with actin protein of H. glycines,the similarity was100percent. Phylogenetic analysis of the actin protein from H. avenaeshowed that it had closer relationship with other cyst nematode. The homology analysisand bioinformatics analysis showed that the genomic sequence of actin gene was highly conserved andit can be a good housekeeping gene in the molecular detection.