RDNA-ITS Sequence Characterization And Genetic Diversity Based On ISSR Of Heterodera Elachista From Hunan Province

Upland cyst nematode(Heterodera elachista) is one of cyst nematode pathogenic nematodes which has economic importance. It was first found in Tochigi, Japan. It was a major factor caused obstacles on mountain rice cropping, thus, upland cyst nematode is also called Japanese cyst nematode. In recent years, Upland cyst nematode has been found in hilly rice fields of Hunan, Guangxi provinces. Upland cyst nematode is a kind of migrating parasitic nematode on rice, it can cause 7-19% loss of rice yield. In order to clarify the distribution and genetic diversity of upland cyst nematode in Hunan province, there molecular methods, ITS,28S-D2D3 and ISSR, were used to analyze the genetic diversity of 12 upland rice cyst nematode populations in Hunan province. The main results were as follows:28S rDNA-D2D3, rDNA-ITS sequences of upland cyst nematode have been obtained by PCR, and genetic analysis of 28S rDNA-D2D3, rDNA-ITS sequences of 12 upland cyst nematode geographic groups in Hunan Province were studied in this paper. The results showed that 28S rDNA-D2D3, rDNA-ITS can distinguish H. elachista from other species, but 28S-D2D3 sequences were more conservative, 28S-D2D3 in different geographic groups of H. elachista only have Several different bases, similarity up to 99.7%. The mutation rate of rDNA-ITS was higher than 28S rDNA-D2D3, the Similarity of H. Elachista in different geographic groups was up to 98%, there were only Several different bases, but the similarity to cyst nematodes in different species was very low, less than 60%.polymorphic primers were seleted from 100 universal primers, four factors (including Taq DNA polymerase, template DNA, dNTPs, primers) of H. Elachista ISSR-PCR reaction system were optimized in 4 dosage levels by orthogonal design. The results showed that H. elachista optimal ISSR-PCR reaction system was as follows:TaqDNA polymerase (5 U/?L) 0.3 ?L, template DNA 1?L, dNTPs (2.5 mmol/L) 2?L, primer (10?mol/L) 1?L,10 × PCR Buffer (20 mmol/L Mg2+ plus) 2.5 ?L in 25?L reaction system. After annealing temperature test to high polymorphism primer, we found that the most suitable annealing temperature was 48 ?. The results proved that the new optimized ISSR-PCR reaction system and procedures of H. Elachista has good reproducibility and stability. After the genetic diversity analysis with appropriate primers of H. Elachista, we found the average percentage of polymorphic bands of H. Elachista was 96%. And the effective number of alleles (Ne) was 1.2132, expected heterozygosity (H) was 0.1521, Shannon's diversity index (I) was 0.2527. The overall genetic diversity Ht index of 12 different geographical populations was 0.1518, the genetic diversity Hs index of H. elachista within populations was 0.1008, the genetic differentiation coefficient Gst index was 0.3357, the gene flow (Nm) was 0.9895. Based on the above data, it showed that H. elachista had high genetic diversity, moreover, there was gene flow existed among populations and a certain degree of genetic differentiation.