Even says this on CO website. They also appear with those GM messages which appear every 15 mins. Just FYI. Originally Posted by Tiny. OK to get exp pots you need to kill a boss ''Ganorderma'' ''Titan'' they all spawn at certain times. The time now is User Name: Password: Remember Me?
Register for your free account! Forgot your password? Recent Entries. Best Entries. The worldwide trade value of Ganoderma lucidum and its derived products is approximately 2. Due to the high demand of medical use and the potential profits, the identification and quality control of Ganoderma lucidum is a critical point for further industry development. A debate appears to persist in the delineation of Ganoderma species.
Karsten [ 19 ] from the UK first established the Ganoderma genus in After that, morphologically similar fungi were identified in Asia and Europe and given the same name of Ganoderma lucidum [ 20 ]. Due to geographical isolation, there are several morphological discrepancies between the Asian and European species, such as color, thickness and context in the fruit body [ 6 ]. Thus, Chinese scholars considered the Ganoderma lucidum species of Asia to differ from those of Europe.
The typical Ganoderma in China is proposed as Ganoderma lingzhi [ 6 , 21 ] by taxonomists, whereas the traditional Ganoderma species were named as Ganoderma lucidum Chi-Zhi in the Chinese Pharmacopoeia. However, the Ganoderma species delineation is still disputed. Traditional identification of Ganoderma lucidum is based on fruit body identification and culture description, which collectively are time-consuming.
In the attempt to overcome the difficulties of basidiocarp morphology in species identification, alternative methods have been investigated [ 22 ], including culture characteristics [ 20 , 23 , 24 ], isozyme profiles [ 25 ] and DNA-based techniques [ 26 — 28 ].
While antagonism is stably used in fungus breeding [ 29 ] and in fungal pathogen defense [ 30 ], which occurs when fungal hyphae with distinct genetic backgrounds come into contact with each other. DNA molecular technologies are widely used and considered as the most effective tools in the delineation of fungi, given their genetic stability and variance among species.
The ITS2 region was identified as the barcode for the delineation of Ganoderma lucidum by Liao et al. However, regarding ITS1, which is also considered as a barcode candidate for fungi [ 38 ]. Kinge et al. These studies indicated that ITS1 could be another barcoding marker candidate in fungi delineations. Here, we reported that the ITS1 region could differentiate Ganoderma lucidum into three geography-originated groups that are associated with secondary-structure variance.
And we also compared the difference between ITS1 and ITS2 regions with phylogenetic analysis and large-scale secondary structures prediction. In a specific analysis with Chinese spawn strains of Ganoderma lucidum , we found that strain compatibility is correlated with the single-nucleotide polymorphism SNP site in ITS1. These findings indicate that ITS1 could be a barcode marker candidate and thus support a novel method for Ganoderma lucidum delineation. These strains were used as spawns for commercial Ganoderma lucidum fruit bodies.
Mycelium agar discs 1 cm were obtained with a self-designed cutter and were used as inoculum in mL shake flasks that contained 50 mL of PDA liquid medium. The mycelia were harvested by vacuum filtration. Mycelium agar discs 1 cm were obtained from fresh PDA plates and transferred to accelerating medium plates 1 L, sucrose 35 g, peptone 5 g, yeast extract 2. Antagonistic streaks were photographed using a digital single lens reflex camera Canon, 70D.
All of the downloaded sequences were annotated following the above procedure. Other parameters followed the default settings. Trametes versicolor EF The Weblogo program [ 52 ] was used to generate sequence logos for assessing sequence conservation within ITS1 and ITS2 and the relative frequencies of the nucleotides at each position.
Outgroups were removed from the alignment prior to generating the sequence logos. Briefly, the latter method divides a sequence into several parts according to the presumed locations of the helices.
Each part was folded separately and was integrated afterward to build the full structure. Generally, the species of Ganoderma lucidum could be separated into three groups by phylogenetic analysis of ITS1 sequences Fig 1.
Seven Ganoderma lucidum strains were differentiated in the same group Group 3. B Three groups 1—3 are identified among Ganoderma lucidum sequences. Branch support is noted on branches only for the collapsed branches.
Both trees are rooted to Trametes versicolor. Group 1 is composed of 56 strains originally determined as Ganoderma lucidum , including 12 strains from Italy, 8 strains from India, 6 strains from China, 5 strains from Russia, 4 strains from Armenia, 3 strains from France, 2 strains from the UK, and 1 strain from Canada, Poland, Sweden, Slovenia, Czech Republic, the USA, Norway and Finland, respectively. Group 2 is composed of 73 strains, with 54 strains originating from India, 11 strains originating from Taiwan, 3 strains originating from mainland China and 1 strain originating from the Philippines.
Seven spawn Ganoderma lucidum strains were in the same group. Regarding ITS2 sequences, the species of Ganoderma lucidum could only be separated into two groups by phylogenetic analysis Fig 2. Seven Ganoderma lucidum spawn strains were differentiated in the same group Group 3. B Two groups 1,3 and 2 are identified among Ganoderma lucidum sequences. Group 1 blue part and Group 3 as the subgroup formed the same group. Group 1 and Group 3 together formed one group Fig 2A and 2B.
Regarding Group 1, it is composed of 30 Ganoderma lucidum strains, including 7 strains from Italy, 8 strains from India, 2 strains from Armenia, 4 strains from France, and 1 strain from China, UK, Canada, Poland, and Sweden, respectively.
Group 2 is composed of 38 strains, with 33 strains originated from India and 3 strains originated from Taiwan. There were two GC-rich regions within helix 1 positions 3—62 and helix 2 positions 70— These regions were potential hotspots for compensatory changes, including nucleotide substitutions and indels. Notably, the indels were responsible for the expansion or contraction of the helix structure Fig 4. There were two main variable regions, both located within helix 2, including nucleotide positions 97 to and to The nucleotide substitutions mainly consisted of the 13th C to T and the 9th A to G changes.
The C to T substitutions were scattered within the ITS1 region, whereas A to G substitutions mainly occurred in the variable region of helix 2. There are helical-loop regions near the ITS1 starts and termini in which the nucleotides are highly conserved. Regions represent potential nucleotide variance.
Major variance regions are enclosed in boxes. Three helices commonly found in the 2D structure of Groups 2 and 3 are numbered from Helix 1 to Helix 3. Four helices found in the 2D structure of Group 1 are numbered from Helix 1 to Helix 4. All substitutions recorded among three groups of Ganoderma lucidum are mapped on the 2D models.
There were three GC-rich regions within helix 1 positions 26—43 , helix 2 positions 48—75 , and helix 3 positions 94— However, these regions were less potential hotspots for compensatory changes compared to corresponding ITS1 segments indicating in Fig 5. Notably, the major potential hotspots for compensatory changes were mainly located in helix 4, which formed the main variable region from positions — The nucleotides are highly conserved in helical-loop regions near the ITS2 starts and termini.
Four helices commonly found in the 2D structure of Groups 1, 2 and 3 are numbered from Helix 1 to Helix 4. ITS1 sequences of three groups of Ganoderma lucidum were folded in the RNAfold program following the energy minimization method. Three representative structures are presented in Fig 4. All these sequences in each group shared the same secondary structures. Therefore, a sequence from these sharing parts of each group was chosen as the representative to compare secondary structures and the nucleotide variances of three groups were analyzed based on multi-alignments.
The sequences used as representative secondary structure analysis for Group 1, Group 2, and Group3 were EU For ITS1 secondary structures, all three structures consisted of a central core formed by three Groups 2 and 3 or four Group 1 helices.
Specifically, helix 1 of three Ganoderma lucidum groups is approximately 60 nt long and consists of a long hairpin structure with four Groups 1 and 2 or five Group 3 bulges and a small loop.
The primary sequence of helix 1 was conserved in length. Furthermore, the 2D structure was also conserved, with only one or two variable nucleotides appearing in bulges indicated in Fig 4 among the three groups of Ganoderma lucidum.
Notably, one hemi compensatory base change hemi-CBC site appeared in helix 1 of Group 3. Helix 2 was the longest among all of the helices.
This helix consisted of a long hairpin structure with five Groups 1 and 3 or six Group 2 bulges and a small loop. The primary sequences of helix 2 in the three groups were 84, 83 and 82 nt in length, respectively. Variations of the 2D structure of helix 2 were observed, with 9- to nt polymorphisms among the three groups. Specific 2-, 2- and 1-nt indels could be observed in the alignment of ITS1 sequences of Groups 1, 2 and 3, respectively.
The major patterns of nucleotide variances were C to U and G to A and vice versa. Helix 3 of Group 1 was a short helix approximately 16 nt in length, including a short base-pair region and an 8-nt loop. A non-CBC appeared in the joint of base-pair and loop region. Helix 3 of Groups 2 and 3 and helix 4 of Group 1 varied in sequence length from 28 Group 3 to 29 nt Groups 1 and 2.
Bulge variances were also discovered 1 in Groups 1 and 2 and 2 in Group 3. Large-scale evaluation of the ITS1 rRNA secondary structure models exhibited that the majority of each subgroup within three groups shared the similar secondary structures Fig 6.
In specific, for Group 1 two subgroups Fig 6A , A1 JX, and A2 FJ exhibited the similar secondary structures with the representative model, while these two subgroups were all originated from India. And the other two subgroups Fig 6A , A3 FJ, and A4 JQ exhibited with the similar secondary structure with Group 2 model, while these two subgroups were all originated from China. Regarding Group 2, six subgroups shared the similar secondary structures with the slightly differences in bulges in helix 1 compared to model structure and they all originated from India Fig 6B.
In addition, for Group 3, all the subgroups shared the similar structure with model structures Fig 6C except one subgroup exhibited a slightly different structure Fig 6 , C1 HQ Three groups of ITS1 sequences exhibited obvious differences in helices of their secondary structure and the large-scale evaluations exhibited that for the majority of each subgroup, the ITS1 secondary structure could reflect originations of three groups of Ganoderma lucidum.
A Ganoderma lucidum Group 1 with four representative secondary structures of each subgroup. B Ganoderma lucidum Group 2 with eight representative secondary structures of each subgroup. C Ganoderma lucidum Group 3 with five representative secondary structures of each subgroup. Each of the subgroups of three groups A1-A4, B1-B8, C1-C5 chooses a sequence as the representative to predict the secondary structure. Differences mainly in bulges are indicated with arrows. ITS2 sequences of three groups of Ganoderma lucidum were folded in the ITS2 database following the energy minimization method.
Three representative structures are presented in Fig 5. Therefore, a sequence from these sharing part of each group was selected as the representative to compare secondary structures and analyzed the nucleotide variance based on multi-alignments. A Ganoderma lucidum Group 1 with one representative secondary structures of each subgroup.
B Ganoderma lucidum Group 2 with eleven representative secondary structure of each subgroup. C Ganoderma lucidum Group 3 with seven representative secondary structures of each subgroup. Each of the subgroups of three groups A1, B1-B11, C1-C7 chooses a sequence as the representative to predict the secondary structure. For ITS2 secondary structures, all three structures consisted of a central core formed by four helices. Specifically, helix 1 of three Ganoderma lucidum groups was approximately 36 nt long and consisted of a long hairpin structure with one bulge and a small loop.
Furthermore, the 2D structure was also conserved, with only one or two variable nucleotides appearing in bulges indicated in Fig 5 among the three groups of Ganoderma lucidum. Helix 2 of three Ganoderma lucidum groups was approximately 35 nt long Group 1 with only one nucleotide variance in length Group 2 and Group 3 and consisted of a long hairpin structure with one bulge and a small loop. Furthermore, the 2D structure was conserved with only one or two variable nucleotides appearing in bulges indicated in Fig 5 among the three groups of Ganoderma lucidum.
Helix 3 was the longest among all of the helices. This helix of three groups consisted of a long hairpin structure with six bulges and a small loop. The primary sequences of helix 2 in the three groups were conserved in length with 72nt.
Variations of the 2D structure of helix 2 were observed with only one or two nucleotide polymorphisms among the three groups. The primary sequences of helix 4 in the three groups were 16, 26 and 12 nt in length, respectively, including a short base-pair region, a bulge Group 2 , and a 4-nt loop. A non-CBC appeared in the base-pair region of Group 1. More for us. The only thing Membership is missing is YOU.
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