Molecular Characterization of Auricularia Spp from South-Western Nigeria using Random Amplified Polymorphic DNA ( RAPD ) Markers

This study was conducted to investigate the genetic diversity in 48 samples of Auricularia species randomly collected from secondary forests in Osun (11), Oyo (10), Ondo (9), Ekiti (8), Ogun (8) and Lagos States (8) of Nigeria. Fourteen Random Amplified Polymorphic DNA (RAPD) markers were used for molecular characterization of Auricularia species. Phylogenetic relations were determined by cluster analysis and Polymorphic Information Content (PIC) and gene diversity determined using standard procedures. The Auricularia species were grouped into 6 distinct clusters based on morphological traits. The PIC value ranged from 0.5594 (OPH-15) to 0.7819 (OPB-12) and gene diversity from 0.5930 (OPH-15) to 0.7977 (OPB-12). Primer OPB-12 was the most informative for genetic diversity of Auricularia species. However, primer OPB-21 gave the highest number of alleles while OPB-12 showed the highest range of gene diversity and accounted for the diversity of the Auricularia species. The dendrogram and the principal coordinate analysis exhibited similar clustered patterns, revealing that all the tested strains could be divided into six distinct groups, each of which correlated with different geographical regions. Molecular characterisation is essential in genetic diversity studies and has proven useful in the classification of Auricularia spp.


Introduction
levels have been reported in mushrooms which Mushroom is a macro-fungus with a could be useful in the discovery of anti-cancer distinctive fruiting body, that can be easily seen genes and pathways (Bechtel et al., 2002; with the naked eye and large enough to be Borchers et al., 2004).Wood ear mushrooms of picked by hand, which can be either epigeous or the genus Auricularia have been domesticated hypogenous (Chang and Miles, 2004).
for cultivation in different parts of the world (Kirk Mushrooms can be identified by their distinctive et al., 2017).Auricularia is the fourth most umbrella-like fruiting bodies and are consumed important cultivated genus of mushrooms after as mushroom sauce, a special delicacy in Agaricus, Lentinula and Pleurotus (Yan et al., southern Nigeria. Mushrooms are rich in protein, 2004).Auricularia mushrooms have a distinct vitamins (B1, B2, C) and minerals in addition to taste and morphological features when other nutrients (Alofe et al., 1996; Ekpo and compared to other cultivated mushrooms due to Aluko, 2002;Daodu, 2003).Low cholesterol their jelly-like texture and horizontally septated numerous benefits The present study therefore basidium (Veeralakshmi et al., 2014).Auricularia investigated the genetic diversity of cultivated auricula (commonly called"jelly Ear) is mushroom Auricularia spp in six South Western basidiomycetes of the family Auriculaceae.
States of Nigeria using random amplified Auricularia polytricha is an edible polymorphic DNA (RAPD) markers.mushroom cultivated in tropical regions because its mycelium can grow at temperatures ranging Materials and Methods from 10 to 40ºC (Jonathan et al., 2009).It is the Forty-eight (48) mushroom samples of most appropriate species to cultivate in tropical Auricularia spp.were collected from, Oyo, Osun, regions where temperatures are high Ondo, Ogun, Ekiti and Lagos States in South (Veeralakshmi et al., 2014).Auricularia auricula-Western Nigeria (Table 1).Three species were Judae is used as food and folk medicine in Benin, identified (A.(Paterson et al., 1991) and (1.25 %), 2 % CTAB and 0.2 % âpharmacological programmes (Du et al., 2011).
supernatant was transferred to fresh sterile Nevertheless, most of the researches on Eppendorf tubes without disturbing the pellets.Auricuaria sp were mainly on cultivated strains 400ìl of ice-cold isopropanol was added to the with almost no study on the genetic variation supernatant and mixed by inverting the tubes 2among wild strains except the study of Du et al. 5 times to precipitate the DNA and subsequently (2011).Due to destruction of forest habitats, kept at -80°C for 1h.The DNA was pelleted down mushrooms are being threatened of depletion in by centrifugation at 12000 rpm for 10 min and the forests in South Western Nigeria (Gateri et the dried DNA pellets obtained, were re- al., 2004).It is important to cultivate wild suspended in 100 ìl of GIBCO water (Invitrogen, mushrooms for domestic and commercial values. Carlsbad, CA, USA) and 2 ìl of 10 mg/ml RNase There is paucity of information on the molecular (Qiagen Valencia, CA, USA) added to each of the characterization of Auricularia spp in the forests samples and kept at 4°C for 30 min to get rid of in Nigeria.Till date there is no record of RNA.The DNA quality of the extracted DNA samples was checked on 1.5% agarose gel.characterization of Auricularia spp in Nigerian Following the high level of concentration of the forests.Much emphasis has been on Pluerotus sp.
(OY3 and OY4|) as was also observed in the dendrogram.Principal coordinate analysis ( Group 1 consisted of 8 locations (OD1, future studies in relation to their nutritional and OD8, OY1, OG1, OG2, LA6, LA7, and LA8) which agronomic characteristics.This study has also must have been due to the traders moving the provided relevant information for cultivation mushroom from one place to the other.Group 2 purposes to mushroom farmers on the type and consisted of 6 locations (OD2, OD3, OD4, OD5, genetic variability of Auricularia mushroom OD6 and OD7).From this group, this particular source in South Western Nigeria and the genotype was localized in Ondo State.Group 3 usefulness of RAPD markers for DNA genotypes of mushroom were found in 22 fingerprinting of Auricularia spp which is relevant locations (OG3, OG4, OG5, OG6, OS5, OG7, EK8, to drug production due to its pharmacological E k 6 , O G 8 , benefits.The dendrogram and the principal EK4,EK3,OS1,EK1,OS8,OS7,OS6,EK7,OS4,OS3, coordinate analysis exhibited similar clustered OS2,EK2,EK5).This report shows that the patterns, revealing that all the tested strains genotype in Group 3 was prominent in three could be divided into six distinct groups, each of states and might be due to the similarity in the which correlated with different geographical soil and weather conditions.In Group 4, the regions.This is the first report on molecular genotype was found in only one location (LA5

Table 1 :
Areas of collection of Auricularia samples in South Western Nigeria sample was run twice for PCR amplification and Random Amplified Polymorphic DNA (RAPD) PCR only reproducible, relatively intense bands were amplification scored.Fourteen arbitrary RAPD decamer primers obtained from Operon Technology

Table 2 :
RAPD markers used for the amplification of DNA samples from Auricularia

Table 4 :
The major allele frequency, number of alleles, genetic diversity and polymorphic information content (PIC) obtained from 48 accessions of Auricularia collected from South West Nigeria analysis of 48 Auricularia spp from South Auricularia spp from 6 States in South Western West of Nigeria Nigeria (Fig.2).The 48 Auricularia spp were The dendrogram constructed using jaccard's grouped into six distinct clusters with a similarity similarity coefficient summarized the coefficient of 0.646.interrelationships observed among the 48

Ekun et al./ Nig. J. Biotech. Vol. 35 (2018): 34-43
Groups 4 and 6 were brown in colour An update.Exp.Biol.Medici.229(5):393 -406.with flattened shape and rubbery in texture.https://doi.org/10.1177/153537020422900507Both the dendrogram and the principal Chandra, S., Ghosh, K. and Acharya, K. (2010).coordinate analysis grouped the accessions into Comparative studies on the Indian cultivated 6 distinct groups based on states and Pleurotus species by RAPD fingerprinting.Nat.morphological characters.The RAPD analysis in Sci.8(7):90 -94.this study has proven to be useful in d i s c r i m i n a t i o n , c h a r a c t e r i z a t i o n a n d Chang, S. T. and Miles, P. G. (2004).Mushrooms: differentiation of Auricularia genotypes and Cultivation, Nutritional Value, Medicinal Effect grouped them according to similarity.and Environmental Impact, CRC Press, Inc.In conclusion, the study revealed the Florida, p. 451.extent of genetic diversity among the genotypes of Auricularia spp evaluated.Genotypes EK2 and Daodu, O.O. (2003).Effect of different lime EK5 from Ekiti State were the most closely concentrations on the cultivation of an edible related specie with 100% similarity and fungus (Pleurotus sajor caju; Oyster mushroom).genotype LA5 the most distinct from the others in M.Sc.Thesis, University of Ibadan, Nigeria.all evaluations.This variability makes possible https://doi.org/10.1007/s00216-011-5171-4