Inspection And Quarantine Technology To Prevent Loss Of Wild Soybean Germplasm Resources

According to <AQSIQ about enhancing the biological species resources of entry and exit inspection of quarantine work guidance>(CIQ [2013] no.1),AQSIQ will make the entry and exit species resources as the key work.Wild soybean as a rare and precious germplasm resources in China,a large number of wild soybean outflow with the exported soy products.In the inspection and quarantine work,due to the close relatives between the soybean and wild soybean,identifing wild soybean with the traditional morphological methods is difficult.The project gets the research materials from LNCIQ export goods,using the common PCR,real-time fluorescent quantitative PCR,DNA barcoding technology and loop-mediated isothermal amplification(LAMP)to rapid identified the wild soybean germplasm.(1)General PCR method: Based on the cDNA sequence of UNK2 gene,common PCR primers were designed to amplify the sample genome DNA and build the PCR reaction system.This method can distinguish soybean germplasm from non-soybean germplasm,but whether it can distinguish soybean germplasm from non-soybean germplasm remaining to be further studied.(2)Real-time fluorescence quantitative PCR method: With UNK2 gene as the target gene,design the specific primers,and construct a real-time fluorescence quantitative PCR reaction system.This method can specifically distinguish between soybean germplasm and other legumes,but whether it can distinguish soybean germplasm and non-soy germplasm remaining to be further studied.(3)DNA barcoding technology: With ITS2,trnh-psba,matK and rbcL as barcode genes,PCR amplification sequencing and sequence comparison analysis showed that matK had the largest mutation rate,but its PCR amplification efficiency and sequencing success rate were low.ITS2 and trnh-psba polymorphisms are not good.The results of genetic distance analysis showed that the matK gene sequence was significantly different between soybean species,and the genetic distance was much higher than the average of the other three genes,and ITS2,psbA,and rbcL decreased successively.Phylogenetic analysis showed that the four genes could not be effectively differentiated among soybean species,but rbcL was a potential candidate fragment in terms of the difficulty and size of amplification and could be used in combination with other fragments.(4)Loop-mediated isothermal amplification(LAMP): MatK and ITS2 genes were used as the target genes,and design three groups of primers that specific to wild soybean 453.The results of LAMP test showed that the LAMP reaction of the primers group was the earliest,and could be used as the optimal LAMP primer for wild soybean 453,and then make the optimal reaction system.