Molecular Characterization Of Auricularia Heimuer Strains And Development Of SSR Molecular ID System For Differentiating Germplasm

Auricularia heimuer is the fourth cultivated edible fungus in the world,which is next only to Agaricus bisporus,Lentinus edodes,Pleurotus ostreatus.It plays an important role in the edible fungus industry system.In this study,we used one hundred and twenty-seven cultivated and wild species come from different areas in China.SSR markers were used to establish molecular identity of Auricularia heimuer.Eleven SSR primer pairs,selected from a pool of one hundred and fourteen primer pairs,have been employed to differentiate a total of one hundred and twenty-seven Auricularia heimuer cultivars and wild-type strains whichbased on amplification patterns.About the analysis of the result,SSR molecular ID system to construct the molecular identities for the resource and new varieties of Auricularia heimuer.The experimental research mainly from the following parts:(1)We collected strains from different areas in China,separation and purification,we have one hundred and seven cultivated species,twenty wild species,The genomic DNA of the testing strains was extracted,DNA as the sample,we get the 740 bp was amplified by using the universal primer ITS1 / ITS4.The sequencing results of the PCR were compared with database in NCBI.aThe results of BLAST showed that the ITS1 / ITS4 could accurately identify the genus of Auricularia heimuer.and build the phylogenetic tree,which build the foundation for the establishment of the global DNA database of edible fungi.(2)Antagonism is a traditional method for the identification of genetic differences between strains.We can clearly observe,there is no obvious antagonism about Au1 to Au11,Au89 and Au127,Au112 and Au113,Au22 and Au82,Au43 and Au85,Au46 and Au88,according to the results we speculate that the strains relationship closely,other strains have obvious or more obvious antagonistic lines,According to the antagonism test,we can roughly classification of the one hundred and twenty-seven strains,it’s prove the conclusion of molecular biology.(3)Extraction of genome from the hypha,using Polymerase Chain Reaction(Polymerase Chain Reaction,PCR)for SSRprimers screening.From one hundred and fourteen pairs of primers,we get forty specific primers.PIC about 0.103-0.908.Eleven pairs of primers of best polymorphism and stability wered used as core primers for constructing the molecular identification caeds,PIC about 0.564-0.908.And then according to experimental results of SSR-PCR reaction system,the system was optimized,and get the optimum temperature of eleven pairs of primers.Data analysis was performed by NTSYS software.According to UPGMA cluster analysis,the genetic similarity coefficient is about 0.44-1.0.The results showed that theAuricularia heimuer derived from different regions were clustered together.which indicated that it was possible From the common primitive ancestors,or have a close relationship.The trend of the introduction of the Auricularia heimuer resulted in the clustering of species derived from northeast and Hubei.At the similarity coefficient of 0.55,some wild strains were separated from the cultivars,indicating that the genetic relationship between the strains was far.Brought more reference to breeding work in the future.(4)A molecular ID system based on SSR markers for identifying and differentiating.This experiment screening of eleven specific primers to establish molecular identity of Auricularia heimuer.We used the methods to constructing Auricularia heimuer germplasm resources molecular ID,and set up a more accurate database of strains information.With the development of information technology,qr code has been widely applied in the product label,we will trans the molecular ID of strains information into qr code.We can quickly identify the code,by mobile phone or other machineWe will combine the DNA bar code sequence,the corresponding species Latin name,the corresponding molecular identity card,the strain growth latitude and longitude,the growth environment into a qr code picture.It’s good for the identification and protection of germplasm resources,which provides a reference for the establishment of the identification system of edible fungus DNA bar code.It provides a more convenient way to identify the synonym and homonym.