| Species in Thelephoralesare a group of macrofungi in forest ecosystems.They can form ectomycorrhizas with host plants and play an important role in improving the stability of forest ecosystems and promoting forest vegetation restoration.The species diversity of this group is very rich.The study of taxonomy and phylogeny mainly depends on morphology classification and molecular systematics.The genes used in molecular phylogenetic analysis mainly include ITS(nuclear ribosomal DNA internal transcribed spacer),n LSU(The 28 S nuclear ribosomal large subunit r RNA gene),n SSU(The 18 S nuclear ribosomal small subunit r RNA gene),RPB1(The largest subunit of RNA polymerase II),RPB2(The second largest subunit of RNA polymerase II)and mitochondrial mt SSU(Mitochondrial ribosomal small subunit).The gene fragment amplification mainly depends on fungal universal primers.However,some of the current fungal universal primers are not suitable for studying phylogeny of these fungi.Therefore,it is of great significance to design corresponding primers for Thelephorales to understand the species diversity in this order.In this study,metagenomic sequencing technology was used to sequence the mixed genomic DNA from 48 specimens which belong to 41 species in 9 genera of Thelephorales.The short sequences of the gene fragments of the primers to be designed were extracted.After comparison and assembly,the conserved regions for primer design were determined.A total of 11 pairs of primers for 5 gene fragments of Thelephorales were designed using Primer Premier 5.The annealing temperature of each pair of primers was further determined by landing polymerase chain reaction.The designed primers and their annealing temperature(AT)as follow: n LSU primer LSU116F/(LSU1218A and LSU1218A1,AT = 58.3°C);primer NS217F/NS763R(AT=57.1°C)for n SSU;the primers of mt SSU were MS3F/MS4R(AT=58.0°C)and MSph F/MSph R(AT=57.6°C).RPB1 primers RPB1-Tf/RPB1-Tr and RPB1-f/RPB1-r(AT=57.6°C and 57.0°C,respectively);RPB2 primers RPB2-Tf/RPB2-Tr(AT=58.0°C),RPB2-BPf/RPB2-BPr(AT=58.2°C),RPB2-Pf/RPB2-Pr(AT=58.0°C)and RPB2-SHr/RPB2-SHf(AT=58.0°C).These primers match all the potential SNPs contained in the corresponding gene fragments and are highly conserved among the major genera of the Thelephorales.Using the above primers and reaction conditions,the sequences of some corresponding gene fragments were successfully amplified.The amplification success rates of n SSU,RPB2,and RPB1 were 83 %,73 %,and30 %,respectively,which were better than those of the existing universal primers.It provides a new choice for amplifying the corresponding gene fragments of the fungi in Thelephorales.Using the primers designed in this study,the classification and systematics of some specimens of Thelephorales were studied which are preserved in the Northeast Biological Herbaria(IFP)of Shenyang Institute of Applied Ecology,Chinese Academy of Sciences.Based on the single gene and combined data of multiple genes,the phylogenetic trees were constructed using Maximum Likelihood method(ML),the phylogenetic status of these species were determined,and the phylogenetic relationships among the potential new species and other species were analyzed.The illustrated descriptions were provided in detail by macromorphological and micromorphological methods. |
