**2. Methods**

#### **2.1. Cataloging techniques for Basidiomycete identification-**

Basically, scientists use three different markers for Basidiomycete identification including macroscopic, microscopic, and molecular analyses.

#### *2.1.1. Macroscopic features for Basidiomycete identification-*

To be arranged appropriately, valid recognizable proof is required. There are numerous conventional techniques for distinguishing proof of these fungi, yet not every one of them are solid and reliable [9, 10]. Prior, the gilled fungi were recognized and named based on certain macroscopic features, that is, longevity, texture, color of internal tissues, form, spore and basidia bearing surface, dimensions, host and nature of deterioration accompanying with a sporocarp on wood. Generally, Basidiomycetes (mostly mushrooms) are identified morphologically by their spore print color, ring and volva on stipe (presence/absence), substrate type, surface texture, and gill/hymenium attachment to the stipe. As we can observe that all these characters are variable to some extent with environmental conditions and cannot be used as prime features for identification purpose [11] (**Figure 1**).-

#### *2.1.2. Microscopic features for Basidiomycete identification-*

Traditionally, microscopic features are also used for the identification of these fungi [11]. Microscopic characters taken into consideration by many scientists include (a) hyphal composition of basidioma tissues which are of three types *viz.*, generative, skeletal, and binding hyphae. These hyphae form three different types of basidioma monomitic, dimitic, or trimitic; (b) nature of hymenium, basidia, cystidia, basidiospores, their shapes, dimensions, and color reaction in different reagents, and (c) clamp connections (presence/absence) [12] (**Figure 2**).-

#### *2.1.3. Misleading identification factors-*

The taxonomy of Basidiomycetes has been controversial because of the limited number of distinguish morphological characters, and there is uncertainty for sorting out of different sections- and species. Environmental factors and substrate have great influence on phenotypic variation may cause troublesome in morphological identification of edible mushroom. One of the- major issues for mushroom reproducers is the absence of an orderly consensus contrivance to segregate diverse species, which are occasionally morphologically indistinguishable [13].

Hence, they have to build up a proper strategy for distinguishing taxa [14]. The implements of molecular approaches are essential to confirm species delimitation. Traditional morphological strategies are less credible than cutting edge techniques that give more dependable approaches to distinguishing proof.

**Figure 1.** Some Basidiomycetes showingdifferent morphological characters. Thephotos in the figure are the original- collection by the authors of this chapter from Pakistan.

**Figure 2.** Different microscopic features of-Basidiomycetes.-(A) Basidiospores, (B)-Basidia, (C) Cystidia, and (D) Pileipellis.- These are the line drawings (anatomicalstructures) of *Agaricus* spp. prepared by the authors.

#### **3. Advanced molecular methods for Basidiomycete identification-**

#### **3.1. Molecular techniques**

The recent improvement in DNA technology has been regarded as a prerequisite procedure- provided a powerful addition to traditional taxonomic methods. Due to the limitations of conventional methods, molecular techniques are used to investigate the problems related to identification and classification of species. For fungal diagnosis, a high variety of molecular methods- are progressively becoming important tools in all aspects for identification. There are several- advanced level techniques that can be used for the identification of these fungi [15]. However,- the use of DNA marker is base for all methods which provide connection between unknown fungi and fully described, morphologically characterized herbarium specimen. Fungal identification is somewhat dependent upon reference species that have been identified by mycological- taxonomist for specific class of fungi that was taken into consideration with appropriate skills.- Additional sources of information can be obtained from public DNA sequence databases for tentative identifications but should not totally relied upon these database sequences, as authenticating the distinctiveness of source material is rarely possible. Important molecular techniques include Southern blotting, PCR restriction fragment length polymorphism (PCRRFLP), RAPD,- PCR, DNA sequencing, microarrays, etc. DNA extraction and purification is the first step for- any of these methods, for which many protocols and prepared kits are existing [16].

In fungal categorization, DNA strategies are fast and authentic to build up the individualities of wild collections. After the approach of cycle sequencing technique [16], direct sequencing of PCR products turned into a normal issue at least in organelle DNA loci or repetitive nuclear DNA such as ribosomal DNAs [17]. This innovation is thought to be a standout among the most great techniques for phylogenetic investigations [18, 19]. Internal transcribed spacer (ITS) region of rDNA is usually utilized region for molecular recognizable proof of Basidiomycetes growing in differing natural surroundings. The corelationship among phenotypes and genotypes has been archived as phylogeny [20].

#### **3.2. Fungal barcoding**

A barcode is a categorization of a definite country of the genome which encompasses approximately genetic discrepancy among species, so countenancing one species to be renowned from an additional. The foremost DNA section which encounters this criterion for fungi is the- "nuclear ribosomal internal transcribed spacer" or (ITS) expanse. Fungal DNA covers manifold- copies of the ITS region which safeguards a virtuous resource of appropriate substantial for- abstraction and examination. The barcode regions jumblesale for fungal taxonomy characteristically ranges from 400 to 1000 base pairs in distance. Comprehensive studies which engender- phylogenetic trees customarily expenditure arrangement evidence from supplementary than one barcode region. A barcode for an unidentified/unfamiliar species can be paralleled with- barcodes apprehended in intercontinental records including GenBank and UNITE.-Conversely,- inaccuracies such as imprecisions in credentials of the original material or certification errors at- a later date cast doubt on the validity of some records. A study by [21] nominated that more than 27% of all fungal ITS sequences were insufficiently identified in the International Nucleotide- Sequence Database and in many cases had "compromised taxonomic annotations" [22].

#### *3.2.1. Choice of primer*

Choice of primer is a very crucial step. Nevertheless, one should start amplifying the ITS region of Basidiomycetes because of two reasons: first of all, universal primer for fungi (ITS1F) can work on it favorably, and secondly, this region has occupied maximum data of all type of fungi, incomparable to other barcoding regions which are now being the interest of scientist (Mycologist). Especially in the case of nom. prov. (seems new) species where data based on one genetic region seems insufficient and unreliable. Moreover, the most suitable primer will be chosen according to the category of a Basidiomycete to which it belongs to. Mostly, universal primer for fungi, that is, ITS1F is used as a forward primer that reads from 5′ to 3′ direction of one template strand, while ITS4 is being used as reverse primer that reads the second template DNA strand from 3′ to 5′ direction. There are many other fungal specified primers that have been used for different groups of fungi [9] (**Figures 3** and **4**).-

#### *3.2.2. Fungal barcoding primers*

Following are some important primers that are under the use for molecular and phylogenetic study of Basidiomycetes.-


#### *3.2.3. Phylogenetics*

Phylogenetics is the learning of evolutionary associations among biological bodies often genes, individual or species and assists to classify the organism, finding pathogenies, forensic sciences or in bioinformatics. Sometimes, it provides base line to investigate the fundamental relationships among different taxa belonging to whether same or different class, while most of the time, it also helps in approaching application of a particular morphon [23].

**Figure 3.** Internal specified region of apart of genome. © Mishra RK, Verma DK,-Pandey BK, Pathak N and Zeeshan M- (2014) Direct Colony NestedPCR for the-Detection of Fusarium oxysporum f. sp. Psidii Causing Wilt Disease in Psidium- guajava L. J Horticulture 1:105.doi:10.4172/23760354.1000105.

**Figure 4.** Three regions and theirdirections to amplify. © Toju H,-Tanabe AS, Yamamoto S, Sato H (2012)-HighCoverage ITS Primers for the DNA-Based-Identification of Ascomycetes and-Basidiomycetes in Environmental Samples.-PLoS ONE 7(7): e40863.https://doi.org/10.1371/journal.pone.0040863 credited.
