Molecular Diversity And Genetic Divergence Of Heterodera Glycines Detected By Inter-simple Sequence Repeats (ISSRs)

Heterodera glycines(Soybean cyst nematode,SCN)is one of the most serious diseases in soybean production area in China,which affects the yield and quality of soybean seriously.It has been a bottleneck that restricting the development of soybean industry in China.In the present study,the ISSR technique was used to characterize genetic variability and partition of H.glycines populations from 17 geographic patterns in China.Furthermore,the results may be helpful to interpret the invasive mechanism of H.glycines,and thus can be a guideline in sustainable controlling its further spread and expansion.The main results were shown as follows.(1)A total of 336 samples were collected from 116 counties of seventeen provinces including Heilongjiang,Jilin,Liaoning,Inner Mongolia,Hebei,Shandong,Shanxi,Henan,Jiangsu,Anhui,Hubei,Zhejiang,Fujian,Jiangxi,Guangdong,Guangxi and Guizhou in 2009-2017.Cyst nematodes had been found in 256 samples from 105 counties of sixteen provinces,and the total detection rate was 76.20%.The highest density was 366.8 cysts per 200 g soil averagely from the Qiqihaer soybean producing area in the northeast China,and followed by 283.0 cysts per 200 g soil from Songyuan.The average number of 28.3 cysts per 200 g of soil was from Shanxi Province,and was the highest among the total Huanghuai region.The cysts had been detected sporadically and occasionally,and 0.8-3.5 cysts per 200 g soil in several provinces of the southern China.(2)The nineteen geographical populations of cyst nematodes were isolated and purified.Seventeen populations of soybean cyst nematode and two populations of rice cyst nematode were identified by morphology.The rDNA-ITS fragments of 146 cysts were amplified and sequenced and then uploaded and registered in NCBI.The phylogenetic tree by Neighbor-Joining was built,and seventeen populations were 98% similar to soybean cyst nematode,and the two populations were the rice cyst nematode,and the molecular results accords with the morphological identifications.The differences of the ITS fragments were less than 2% in seventeen soybean cyst nematode populations and 1% of those in two rice cyst nematode populations,indicating that ITS molecular characteristics were suitable for the identification and potential DNA barcodes for cyst nematode,but that did not reflected in the genetic diversity and genetic differentiation among populations.(3)The molecular diversity and genetic divergence of H.glycines were detected by three inter-simple sequence repeats(ISSRs)primers PTY26,PTY188 and PTY888,that amplified polymorphically and clearly and selected from twelve ISSRs primers by references.The ISSRs amplification program and system of soybean cyst nematode were explored and optimized and established eventually.The results showed that the amplification of ISSR-PCR system had been the best stability and repeatability that included 1.5?L dNTPs,0.8?L primers,0.34?L Taq polymerase,2.5?L 10×PCR Buffer and 1.2?L DNA temple when the annealing temperature of the program held 55 ?.The clear and polymorphic ISSRs bands of soybean cyst nematode could be obtained for further analysis.(4)Total different lengths of fifty-eight bands(18,19 and 21 bands respectively)were amplified by three primers PTY26,PTY188 and PTY888 for fourteen geographical populations.Fifty(fifteen,sixteen,and nineteen,respectively)of which were polymorphic bands were amplified by three primers,and the percentage of polymorphic loci was 86.21%(83.33%,84.21% and 90.48%,respectively).The results showed that the rich ISSRs polymorphisms had been in tested nematode populations.(5)The genetic distance was calculated from the genetic distances of the tested SCN populations,and the genetic diversity was analyzed.The results showed that the genetic distances of SCN in 14 geographical populations ranged from 0.0984 to 0.4098.The genetic similarity between Qiqihar and Daqing was the lowest,and the genetic distance was the largest;Songyuan and Zhalantun in Inner Mongolia had the highest genetic similarity and the smallest genetic distance.Genetic related data were calculated by software.The effective number of alleles(Ne)was 1.3377,the Nei's gene diversity index(He)was 0.2087,Shannon's diversity index(I)was 0.3287,and the total genetic diversity of 14 populations,the index of genetic diversity of genetic diversity between the population(Ht)was 0.2087,the index of genetic diversity in the population(Hs)was 0.0648,the gene differentiation coefficient index(Gst)was 0.6893,and the gene flow(Nm)was 0.2255.The results of polymorphism analysis showed that the geographical distance of SCN populations had irrelevance to genetic distance of those populations,and there was high genetic differentiation and genetic diversity among the 14 populations.The result provide proof to reveal the evolution mechanism of H.glycines,thus may be as groundwork in sustainable study of its changes of physiological races and virulence.