MOLECULAR BASED IDENTIFICATION OF BOLETUS EDULAIS FOUND IN JHARKHAND

1. Research Scholar, PG Department of Botany, Ranchi University, Ranchi, Jharkhand. 2. University Professor,PG Department of Botany, Ranchi, Jharkhand. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History Received: 05 June 2021 Final Accepted: 09 July 2021 Published: August 2021

Food industry is dynamic and in search of innovation and nutrition content. Nutrition and food supplements demand can be fulfilled by mushroom.Agaricus(Button mushroom), Pleurotus (Oyster mushroom)and Volvariella(Paddy straw mushroom)are only popularly species cultivated and commercialized. There are many other wild mushrooms which can be commercialized with ethnomedicinal and rich nutrition content. In Jharkhand many wild mushroom species are found but they identified with many synonyms. (Srivastava and Soreng 2014) Identification is done on the basis of morphological structures and microscopic study of spores. (Pegler 1981 This study is to identify the wild mushroom locally called as -Jamun khukhari‖ through molecular characterization and report molecular data to NCBI GeneBank.
Boletus edulais is known asJamun khukhari by local and tribal people in Jharkhand. This name was given because it is belief that it is ectomycorrhizal associated with roots of Syzygiumcumini(black plum) Jamun in Hindi.Thisspecies of mushroom is entitledas king mushroom. This was placed in family Boletaceae. This is easily noticed and distinguished by the absence of gills, colourful caps, pores hard thick stipe. (Verma and Pandro, 2018) Wounded parts of some species of Boletaceae turns blue due oxidation of pulvinic acid derivatives (Nelson, 2010). Few species of this groups are found to be poisonous but many are safe for consumption. (Singer 1986) Wild mushroom can be commercialised after proper identification and phytochemical analysis. Morphological features are misleading the identification of closely related mushrooms reported from different region of India. In present era molecular characterization for fungal identification is made easy and accurate with ITS (Internal transcribed spacer) regions by finding similarities in Genetic database BLAST (Basic Local for Alignment Search Tool). (Rajaratnam, et. al., 2012)The present study describes the DNA sequencing for attempting to identify the wild 181 edible mushrooms collected from Jharkhand, using genomic DNA from fruiting bodies and also looking for a match of sequences for molecular based Boletus edulais on NCBI database.

Materials And Methods:-Collection and survey
Jamun khukhari was collected form weekly village market of Khunti district, Jharkhand. The Sample was photographed and survey was conducted with the tribal people belonging to around villages during collection.

DNA isolation and Purification
Sample were brought to lab cleansed with tap water distilled for removing muddy content in outer surface. Then it was cleansed with 70 % of alcohol. Sample were treated with Liquid nitrogen and crushed in motor pestle making fine powder. Powdered sample were mixed with CTAB and Centrifuged as per Doyle and Doyle protocol. (Doyle and Doyle 1987) The genomic DNA was checked by agarose gel electrophoresis in 1.5 % agarose gel. The obtained DNA content was purified by RNase treatment at 37℃ for 60 minutes followed by Phenol-Chloroformisomylalcohol (25:24:1 ratio). Centrifugation at 12,000 rpm for 10 min of purified supernatant with equal volume of 70% ethanol. The resultant DNA pellet were air dried and suspended to ice box after dissolving in 1X TE buffer. (Aamir, et. al., 2015) Amplification PCR reactions was performed for amplification with Forward Primer 5'-ACT GAA CCT TAT CAT TTA GAG-3' and Reverse Primer 5'-AAG TCC ACT GAA CCT TAT CAT-3' in a 50 µl reaction volume with 30 ng template DNA and ran on programme with initial denaturation at 94℃ for 5 minutes, denaturation at 94℃ for 30 seconds, annealing at 60℃ for 40 seconds, extension at 72℃ for 1 minute and final extension at 72℃ for 2 minutes. The amplicons were eluted and Agarose gel run with Ethidium Bromide stain for 90 minutes 50V and photographed.

Results:-
The present study survey reviled different report from earlier research documentation. Jamun khukhari found in Jharkhand are having dark black colour cap with large hard stipe in scales. The size of cap is found to in maximum size than reported in this study. The local villagers denied the fact about the association with the roots of Syzygiumcumini they notified the location of this species near the edges ofSal (Shorearobusta) forest.Local name Jamun Khukhari is given due to its black colour of pileus. It is been reported and bitter tasting mushroom often cooked with tamarind to enhance the bitter taste. It having high ethnomedicinal importance as remedy for stomach disorders.The photograph of market-place of collection and morphological structure were recorded (Fig 1).
The sample collected was processed for DNA isolated and ITS was amplified by PCR for sequencing (Fig 2). The resultant ITS sequence was matched with BLAST Database and found to be 99.71% similarities and 0 E value (Fig  3). (Table 1) and identified as Phlebopusportentosus. ITS sequence of length 905 bp was deposited in NCBI GENBANK and obtained accession number MT611108.

Conculsion:-
This study went through morphological features to previous research document of boletus edulis, different reports were found. The accurate identitywas obtained by ITS for conserved region through sequencing. The mushroom species known to be boletus edulis was concluded as Phlebopusportentosus.Phlebopusearlier was placed under the family Boletinellaceae (suborderSclerodermatineae of the Boletales) (Binder and Hibbett2006) as a Boletus by Heim (1936), and gave the status of genus by Singer (1936)This mushroom has recorded associated with Salforest of Central India (Verma and Pandro 2018). In southern China, it is commonly known as -Black Boletus‖. It is found under shade of Ficus Sp. and Eriobotrya japonicapeople of China has successful in cultivating and established profitable market. (Zhang et al., 2017)Now this mushroom can be further processed for culture and commercialization as identification by molecular characterization has concluded this mushroom species as Phlebopusportentosus.