Molecular Characterisation Based on ITS and Genetic Diversity of the Heterodera Avenae Group on the Huang-huai Floodplain of China
|School||Henan Agricultural University|
|Keywords||Cereal cyst nematode Genetic diversity H. avenae H. filipjevi ITS-RFLP ISSR|
Cereal cyst nematode (CCN) has been a widespread disease in Huanghuai floodplain of China and caused serious losses to wheat production. Currently, there are 12 species. It has been confirmed that there are two species (H. avenae and H. filipjevi) distributed in China, since the disease was found in Hubei province in 1989. In order to define the regional distribution and genetic diversity of Heterodera avenae group in Huanghuai floodplain, this paper was to determine better molecular method for CCN through comparing and discussing common-used molecular methods such as ISSR and ITS. At the same time, 21 populations of Huanghuai floodplain was analyzed in species and genetic diversity with the method of ITS and ISSR. The results are as follows:Comparing and analyzing three sequences of rDNA(ITS、28S D2/D3、IGS) , we found that both of ITS and D2/D3 can distinguish H. avenae and H. filipjevi obviously. While the sequence of D2D3 in 28S is the most identity,there are only few bases different between H. avenae and H. filipjevi. The similarity of IGS is minimum and the similarity between H. avenae and H. filipjevi is just 60%. However, the similarity between different populations is 80% to 95%. Through comprehensive analysis, the sequence of ITS and 28S D2/D3 can be used for identification and diversity of species. Now, the datebase of ITS sequence of CCN is abundant, so experimental results obtained easily compared to database information.Cereal cyst nematode collected in Huanghuai floodplain of China were studied using restriction fragment length polymorphism and sequences of internal transcribed spacer (ITS) of rDNA. The result showed that AYAY, PYQF, BDNS, FYYS, HD, HZHL, KFQX, XFNS, LFXF, LYYI, XAYL, XCYZ and XXYJ populations were belong to H. avenae type“C”,ZZXY and ZZXS populations were the same as H. avenae“australis”, JZBA, XCXC, ZKSS, ZKHY population was identified as H. filipjevi, but JZBA population is a new ITS type of H. filipjevi. The result also showed that LYLU and SQSY populations were mixed with H. avenen and H. filipjevi.Analyzing restriction length of restriction fragments of ITS sequences using Vector NTI, the H. avenae and H. filipjevi ITS sequences include 10 types profiles. The sequences of H. avenae“australis”, ZZXY, ZZXS and JZBA populations has two types profiles. Neither two types are same as the gal profiles, but mixture of two types can appeared the same result because of the heterogeneity.Phylogenetic analyses of close related species and tested populations based on rDNA-ITS sequences using maximum parsimony (MP) method. The result showed that H. filipjevi in China was less diverse than H. avenae, with greatest similarity to specimens from Italy and the USA, which is consistent with a more recent introduction. Whereas, H. avenae in China was clearly distinct from H. avenae found elsewhere, except for the discovery of the Australian type. Although the Australian types clustered together, this fell within the variation found for the remainder of the specimens from China, which may represent a single species.Based on random marker of ISSR and RAPD, we found that the diversity of ISSR is much more abundant and steady than RAPD. So, this experiment used ISSR marker methods to analyze the genetic diversity of CCN of Huanghuai floodplain. The mantel test showed that there was significant positive correlation between the result of ISSR and RAPD.We selected 8 ISSR primers from 50 ISSR primers using for genetic diversity to analyze populations of Huanghuai floodplain. The result of genetic diversity analyzing showed that the percentage of polymorphic band (PPB) of H. avenae was 92.77% on average and the PPB of H. filipjevi was only 61%. It showed that the diversity of H. avenae is much more abundant than H. filipjevi. Phylogenic analysis of the ISSR showed XCYZ and XAYL population is mixed with H. avenae and H. filipjevi. The ZZXY population of H. avenae isminimum similarity to other populations of H. avenae, and the result is the same as the ITS Phylogenic analysis,but genetic distance is not related with geographic distance.Genetic diversity is not related with different hosts in the same field and the resistance of the hosts.