rDNA ITS Sequence Analysis and Molecular Phylogeny of Four Fungal Strains Assigned to the Family Rus

rDNA ITS Sequence Analysis and Molecular Phylogeny of Four Fungal Strains Assigned to the Family Rus1 Biotechnology Institute, Zhejiang Academy of Forestry Science, Hangzhou, Zhejiang?A310023，China；2Lishui Edible Fungi Research and Development Center，Zhejiang Essence Fungi DevelopmentCompany Ltd，Lishui，Zhejiang?A323000，ChinaInternal transcribed spacer (ITS) sequences of four edible and medicinal fungi belonging to the Russulaceae, collected from the Lishui mountainous area of Zhejiang Province, were cloned and sequenced. Comparisons with sequence data in GenBank were undertaken using BLAST, and the four ITS sequences, together with 15 reference sequences obtained from GenBank, were used to construct a phylogenetic tree. rDNA ITS sequences of the four strains ranged from 673 to 766 bp in length and between 47.4% and 49.48% in GC content. Phylogenetic data indicated strain R57 to beRussula cyanoxantha. Differences in the size and GC content of ITS sequences from strains R32 and R57 (R. cyanoxantha) and strain L37 (Lactarius sanguifluus) were significant, whereas thecorresponding differences between strains R32 and R57were relatively small. A close kinship, with only small detectable differences in their respective ITS sequences, existed betweenR. mustelina, R. virescensandR. parazurea. A similar situation applied to strains ofL. sanguifluusandL. salmonicolor.Russulaceae；?ArDNA；?AInternal Transcribed Spacer；?ASequence analysis；?APhylogeny；?AMacrofungiThe family Russulaceae is assigned to the Basidiomycotina, Hymenomycetes and Agaricales, and includes twogenera,RussulaandLactarius. According to WANG and SHI[1] and JIet al[2], 300 species ofRussulaand 400 species ofLactariushave been identified worldwide. Abundant macrofungal resources exist in the Lishui mountainous area located in southwest Zhejiang Province. According to YANGet al[3], 911 different kinds of wild fungi can be found in the Qingyuan County region of Lishui, 43 of which belong to the family Russulaceae. Most fungi assigned to the Russulaceae are edible and possess medicinal functions[4-8]. In order to produce fruiting bodies, it is essential for these fungi to form a symbiotic relationship with host plant roots and, since they have not yet been cultivated artificially, the supply of fruit bodies is limited.Although the Russulaceae has been widely studied with respect to available resources, environmental ecology, tissue isolation, nutritional and functional components, artificial cultivation and substrate fermentation[9-12], relatively little research has been undertaken on the molecular phylogeny and the extent of genetic polymorphism within the family. In this report, ITS sequences of four edible/medicinal fungi belonging to the Russulaceae and recurrently found in Lishui mountainous area, were cloned and sequenced. BLAST searches were conducted to identify homologous sequences in the GenBank database in order to provide a basis for future research on phylogenetic relationships and genetic polymorphism.1 Materials and Methods1.1Fungal strainsStrain no., and the site where fruit body samples were collected, are shown in Table 1 in the Chinese version. Strains R29, R32 and L37 have been identified to species level and designatedR. mustelinaFr.,R. cyano-xanthaSchaeff. : Fr. andL. sanguifluus(Paul.) Fr., respectively. Strain R57 has still to be identified. After collection, fruit bodies were cleaned of residualsediment, transported to the laboratory in liquid nitrogen, and thereafter preserved at-20 ℃.FU Lizhong,?ALI Haibo,?AWEI Hailong,?Aet al1.2Methods1.2.1Extraction of genomic DNAGenomic DNA was extracted from pilei (including gills) by the SDS-CTAB method[13] and purified using DNA purification kits (Hangzhou Bioer Technology Co. Ltd.). Purity was evaluated by 1.5% (w/v) agarose gel electrophoresis, and DNA concentrations were determined with a DNA/RNA UV spectrophotometer (GE Healthcare, Amer-sham, England).1.2.2rDNA ITS analysisITS-PCR amplification was performed in a programmable TC-XP thermocycler (Hangzhou Bioer Technology Co. Ltd.) in 25 μL reaction mixtures containing: 2.5 μL 10×PCR Buffer, 2 μL dNTPs (10 mmol/L), 2 μL MgCl??2 (25 mmol/L), 0.3 μLTaqDN A polyme-rase (5U/μL), 1 μL ITS5/ITS4primers[14] (10 μmol/L), 3 μL template DNA (17 ng/μL) and 14.2 μL ddH2O to volume. Amplification conditions were as follows: 1 cycle at 94 ℃for 1 min；30 cycles at 92 ℃for 15 s，61 ℃for 15 s and 72 ℃for 1 min；then a final extension at 72 ℃for 5 min.1.2.3Recovery, cloning and sequencing of ITS fragmentsITS fragments in 1.5% agarose gels were recovered using a DNA Gel Recovery Kit (Hangzhou Bioer Technology Co. Ltd), and nucleic acid concentrations were determined with aDNA/RNA ultraviolet spectro-photometer. The recovered fragments were ligated into pMD-18T Vector and transformed into competentE. coli(DH5α) cells. After blue-white spot screening and identification using PCR amplification, three random positive clones in each sample were sequenced (TaKaRa Biotechnology Co. Ltd., Dalian).1.3Comparison of ITS sequences and analysis of phylogenyAfter rem oval of the carrier sequences from the 5’and3’ends, ITS sequences were transformed into FASTA format and multiple alignments between four ITS sequences and reference sequences obtained from a BLAST search of the GenBank database were confirmed using Clustal X（Version 1.81）[15]software.A phylogenetic tree was constructed with MEGA（Version2.1）software[16]. Genetic distances were calculated using the Kimura 2-parameter model. Evolutionary distances were determined using the neighbor-joining method, and thestatistical significance of each branch of the phylogenetic tree was confirmed using bootstrap values from 1000 replicates.2 Results2.1Analysis of rDNA ITS sequencesThe amplified rDNA ITS regions of strains R29, R32, L37, and R57 consisted of 701 bp, 673 bp, 766 bp and 673 bp respectively, and the corresponding GC contents were 49.22%, 47.85%, 49.48% and 47.4% (see Fig.1 in the Chinese version). The largest difference in sequence length (93 bp) was between strain L37 and strains R32 and R57, while the largest difference in GC content (2.08%) was between strains L37 and R57. Our data indicated that significant differences existed among different genera included in the family Russulaceae. It is noteworthy that the size of the ITS sequence in strains R32 and R57 was identical (673 bp), and that the GC content differed by only 0.45%.2.2Homologous BLAST retrieval of ITS sequencesFifteen reference strains with ITS sequences similar to those of the test strains were identified by a BLAST search of the GenBank database (see Table 2 in the Chinese version). Fourreference strains, two identified asR. virescensand theother two asR. parazureaandR. mustelina, exhibited relatively high similarity with strain R29. Six reference strains, four identified asR. cyanoxantha, one as aRussulasp. and the sixth an uncultured fungus, showed relatively high similarity to strains R32 and R57. Five reference strains, four identified asL. salmonicolorand one asL. sanguifluus, exhibited relatively high similarity to strain L37.2.3Phylogenetic analysisThe four ITS sequences, together with 15 reference sequences obtained from the GenBank database were used to construct a phylogenetic tree (see Fig.2 in the Chinese version). Thirteen strains ofRussulaand six strains ofLactariusrespectively were clustered in the two genera. Strain L37 was located in the same branch as AF249289 (L. sanguifluus), and these two strains were then clustered in a group withL. salmonicolorstrains AF140258, AF140259, AF140265 and AF249287 at the bootstrap value of 100%. This suggests a relatively small sequence difference and a closer kinship betweenL. sanguifluusandL. salmonicolor.Strains R32 and R57 were located in the same branch at a bootstrap value of 100%, and these two strains formed anothercluster withR. cyanoxanthastrains AF418608, AM087258,AY606960 and AY061669, and an uncultured fungus DQ054553, at a bootstrap value of 82%. Therefore, based on these data and those shown in Fig.1, R57 and R32 should be regarded as geographically different strains ofR. cyanoxantha. Strain R29 was located in the same branch as strain AY061693 (R. mustelina) at a bootstrap value of 97%, and these two strains were clustered in a group together withR. virescensstrainsAM087264 and AY061727, and strain AY061704 (R. parazurea) at a bootstrap value of 93%. This indicated small sequence differences and close kinship betweenR. mustelina,R. virescensandR. parazurea.3 AnalysisMost fungi belonging toRussulaandLactariusare ectomycorrhizae, and have not yet been grown in artificial culture. However, pure mycelial cultures of these macrofungi can be identified using ITS sequence analysis. ITS sequence analysis has been used to identify mycelia fromR. vinos[17],L. deliciosus[18],Amanita exitialis[19] andSuillus bovinus[20].Our data indicate that ITS sequences can be used not only for themolecular identification ofRussulaandLactarius, fungi that are not easy to accurately identify to species level using morphological characteristics, but also for preliminary estimates of phylogeny and kinship between different species of Russulaceae.Strain R57 was identified in this study asR. cyanoxanthabased on ITS sequence similarities and the close clustering of their respective ITS sequences in the phylogenetic tree. There are no standard definitions of species based on ITS sequences[21,22], although ITS sequences are good molecular indicators for comparative interspecies and intraspecies research. We consider ITS sequence analysis combined with fruit body morphological characterization to be more scientifically credible for identifying and classifying macrofungi, especially when those fungi cannot be easily and accurately identified based on fruit body morphology alone.WANG QY, SHI MB. Present and future ofRussulain China ［J］. Edible fungus of China, 2004，23（4）：10-12. (in Chinese with English abstract)［2］JI HF, YANG Q, SONG RQ. Progress in the application ofRussula［J］. Forestry Sciences & Technology,2006, 31（3）: 28-30. (in Chinese)［3］YAO PS, YAO QS, YAO CR. A survey of wild fungi resources in Zhejiang Province Qingyuan County［J］. Edible Fungi of China，2002，21(4)：27-29. (in Chinese)［4］LI HZ, HUANG DX, XU XP. Advances in the biochemical characteristics ofRussula［J］. 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