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  • DNAbarcoding$for$iden0fica0on$of ...for$iden0fica0on$of$$ consumerrelevantfungisoldinNewYork! Authors:!Emily!Jensen2Vargas1, AhlenneAbreu 2! Mentor:!Chris
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DNA barcoding for iden0fica0on of consumerrelevant fungi sold in New York Authors: Emily JensenVargas 1 , Ahlenne Abreu 2 Mentor: Chris<ne Marizzi 3 Ph.D. 1 Francis Lewis High School, 2 Washington Heights Expedi<onary Learning School, 3 Cold Spring Harbor Laboratory Funded by: • White TJ, Bruns TD, Lee S, Taylor J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH. (eds). PCR Protocols: A Guide to Methods and Applications. Academic Press: London, pp. 315 – 322. • Altschul, Stephen; Gish, Warren; Miller, Webb; Myers, Eugene; Lipman, David (1990). "Basic local alignment search tool". Journal of Molecular Biology. 215 (3): 403–410 • Dentinger BTM, Suz LM. (2014) What’s for dinner? Undescribed species of porcini in a commercial packet. PeerJ 2:e570 Diaz, James H. "Mistaken Mushroom Poisonings." Official Journal of the Wilderness Medical Society”"Mushrooms." Official Website of the Department of Homeland Security. N.p., Mar. 2008. Web. 01 Feb. 2017. Moyer, Douglas C., Jonathan W. DeVries, and John Spink. "The Economics of a Food Fraud Incident – Case Studies and Examples including Melamine in Wheat Gluten." Science Direct. Division of Public Health, Michigan State University. • Now, Season Stepp More Content. "Season Stepp: Mushrooms Rising in Popularity." Wicked Local Plymouth. Wicked Local Plymouth, 11 Oct. 2016. Web. 17 Jan. 2017. <http://plymouth.wickedlocal.com/news/20161014/season-stepp-mushrooms-rising-in-popularity • Raja, Huzefa A. "DNA Barcoding for Identification of Consumer-relevant Mushrooms: A Partial Solution for Product Certification?" Science Direct. N.p., 9 July 2016. Web. 06 May 2017. <http://www.sciencedirect.com/science/article/pii/S0 Yahr R, Schoch CL, Dentinger BTM. 2016 Scaling up discovery of hidden diversity in fungi: impacts of barcoding approaches. Phil. Trans. R. Soc. B 371: 20150336. Acknowledgements Results References The DNA Barcoding Workflow Project Summary Aim: Inves&ga&ng dried fungi (mis)labeling in New York City. Intended Outcomes: Many foods in the US have been mislabeled in order to gain a greater profit for cheaper products replacing expensive ones (Moyer et al., 2017). We are interested to see if we can iden<fy mislabeled species of fungi sold in New York, including a commercial packet of dried fungi. Descrip3on: Reliably iden<fying wild collected foods can be a challenge, especially when they belong to poorly known groups like Fungi (UNITE). Although significant progress has been made in our understanding of fungi diversity, iden<fica<on based on phenotype can be difficult for even trained experts. Fungi typically have a cryp<c nature and can have a similar appearance to distantly related species (Yahr et al., 2016). What contributes to the mislabeling of fungi may be the phenotypical appearance of the dried fungi being unclear. Our goal was to collect and analyze sequences and data from dried and fresh fungi sold for commercial uses. We used DNA technology for species iden<fica<on of 10 samples that were collected from local supermarkets in two New York City boroughs. Although there were no cases of mislabeling, we found that our singlelocus ITS DNA barcoding approach allowed to iden<fy only 44% of all samples at the species level. This highlights the need for a curated, centralized ITS reference database that allows thirdparty annota<ons. Dried and fresh fungi samples were collected from local supermarkets in two New York City boroughs. All dried samples were obtained from a single bag of dried of edible “Mixed Mushrooms”. Samples were visually sorted by phenotype, documented and vouchered. While fresh samples were easy to iden<fy, many of the dried samples were broken down into smaller pieces and lacked key morphological characteris<cs that could be used for iden<fica<on. The DNA was extracted from approximately 0.1 g of specimen using the standard silica DNA extrac<on method (www. dnabarcoding101.org), with one minor modifica<on: for each sample, a small piece was removed and presoaked in dH 2 0 before being ground in lysis buffer with a sterile pestle. The extracted DNA was amplified using fungispecific ITS primers (ITS1F/ITS4 as described by White et al., 1990) and NEB Taq 2X master mix (New England Biolabs). Then, the amplified DNA was stained with SYBR green (Invitrogen) and confirmed using gel electrophoresis. Posi<ve amplicons were sent to GENEWIZ Inc. for sequencing in both direc<ons. Aier the DNA was sequenced, DNA Subway soiware (hjp://www.cyverse.org/dnasubway ) was used to analyze the DNA sequences. To verify species iden<fica<ons through DNA Subway sample consensus sequences were downloaded and submijed directly to Basic Local Alignment Search Tool (BLAST) in NCBIGenBank (Altschul et al., 1990) and UNITE Ver. 7.1, an openaccess, curated fungispecific database (hjps://unite.ut.ee ). Finally a phylogene<c tree was constructed based on DNA Subway’s MUSCLE and PHYLIP maximum likelihood (ML) methods. DNA Subway BLAST, GenBank UNITE Sample ID Loca0on Fungi sp. Per Label Common Name Fungi Barcode ID % Match Fungi Barcode ID % Match Fungi Barcode ID % Match S1 Supermarket, LIC, NY Cloudear (dried) Cloud Ear Auricularia heimuer 100 Auricularia heimuer 100 Auricularia heimuer 100 S2 Supermarket, LIC, NY Shiitake (dried) Shiitake Len&nula edodes 86 Len&nula edodes 99 Len&nula edodes 99.9 S3 Supermarket, LIC, NY Wood ear (dried) Wood ear N/A N/A N/A S4 Supermarket, LIC, NY Oyster (dried) Oyster Pleurotus geesterani 95 Pleurotus geesterani 94 Pleurotus sp. 94 S5 Supermarket, LIC, NY White Bujon (dried) White Bujon Agaricus bisporus 100 Agaricus bisporus 100 Agaricus bisporus 100 S6 Supermarket, LIC, NY Oyster (dried) Oyster Pleurotus sp. 90 Pleurotus ostreatus 91 Pleurotus ostreatus 91 S7 Supermarket, LIC, NY White Bujon (fresh) White Bujon Agaricus bisporus 100 Agaricus bisporus 100 Agaricus bisporus 100 S8 Supermarket, LIC, NY Korean Black (dried) Korean Black Auricularia heimuer 100 Auricularia heimuer 100 Auricularia auriculajudae 100 S9 Supermarket, NYC, NY Yellow foot (fresh) Yellow foot Craterellus tubaeformis 100 Craterellus tubaeformis 100 Craterellus sp. 100 S 10 Supermarket, NYC, NY Yellow foot (fresh) Yellow foot Craterellus tubaeformis 100 Craterellus tubaeformis 100 Craterellus sp. 100 There were no cases of mislabeling. For one sample (sample S3) we were not able to obtain a DNA sequence because of unsuccessful PCR amplifica<on in spite of several ajempts (Table 1). This may be due to poten<ally high degrada<on of the sample’s DNA due to heat treatment prior packaging sample S3 was very dry. Once DNA is broken into short fragments, genera<ng a ITS barcodes can be difficult, as primer binding sites may be degraded. Moreover, secondary metabolites can inhibit PCR by decreasing the Taqpolymerase’s ac<vity. Failure to generate a ITS barcode may also be because the primers used were not suited for amplifica<ons of all species. Sample S2 only yielded a single sequencing read (reverse direc<on). Samples S1, S2, S5, S6, and S7 were labeled correctly in both the species and genus level. Samples S4, S8, S9, and S10 were labeled at the correct genus level but we were unable to resolve them down to the species level as there were discrepancies between databases (see Figures 2 and 3). Species with highest % match/max score/bit score were considered the taxonomic iden<ty (Table 2). UNITE Ver. 7.1 serial BLAST search was u<lized in addi<on to GenBank because it has been found that 27% of fungal ITS sequences entered into GenBank were inadequately iden<fied and 20% were incorrectly labeled (Raja and Oberlies et al., 2016). Future studies would benefit highly from a curated, centralized ITS reference database that allows rapid thirdparty annota<ons. 90% 10% Sequencing Success Rates ITS sequence obtained (9/10) No ITS sequence obtained (1/10) 44% 56% Sample Iden0fica0on Rates Sample iden<fied at species level (4/9) Sample idenpied at genus level (5/9) Materials and Methods This project was supported by the Urban Barcode Research Program, funded by the Pinkerton Founda<on. We thank Melissa Lee, Alison Cucco and Jenna Dorey (DNA Learning Center, Cold Spring Harbor Laboratory) for their helpful assistance. Discussion Table 1 (above): List of fungi sampled in this study for ITS barcoding. Figures 2 and 3 (right): ITS sequencing and sample iden<fica<on success rates. Figure 1: How DNA barcoding works. Figure 4: Sample sequences (S1 S10) aligned with corresponding BLAST sequences hits using the MUSCLE mul<ple alignment soiware. The same data set is depicted as gene tree using PHYLIP ML to generate a phylogene<c tree using the maximum likelihood (ML) method. Sample S2 (Shiitake) was chosen as outgroup as only one sequencing read was available for species iden<fica<on. Table 2: Species iden3fica3on based on ITS DNA barcoding from all samples analyzed (n=10). Each consensus sequence of samples S1 – S10 was subjected to three search tools to verify iden<ty: 1) DNA Subway; 2) direct submission to BLAST search in NCBIGenBank; and 3) a serial BLAST search in the curated database termed UNITE. Sample Date Purchased Location of purchase Labeled on Package Picture Identified using UNITE serial BLAST search S1 February 2nd, 2017 Supermarket, LIC, NY Cloud<ear (dried) Auricularia heimuer [Cloud<ear] S2 February 2nd, 2017 Supermarket, LIC, NY Shiitake (dried) Lentinula edodes [Shiitake] S3 February 2nd, 2017 Supermarket, LIC, NY Wood ear (dried) No sequence S4 February 2nd, 2017 Supermarket, LIC, NY Oyster (dried) Pleurotus [Oyster] S5 February 2nd, 2017 Supermarket, LIC, NY White Button (dried) Agaricus bisporus [White button] S6 February 2nd, 2017 Supermarket, LIC, NY Unknown (dried) Pleurotus ostreatus [Oyster] S7 February 2nd, 2017 Supermarket, LIC, NY White Button (fresh) Agaricus bisporus [White button] S8 February 2nd, 2017 Supermarket, LIC, NY Korean Black (dried) Auricularia auricula6 judae [Wood<ear] S9 February 24, 2017 Supermarket, Manhattan, NY Yellow foot (fresh) Craterellus [Chanterelle] S 10 February 24, 2017 Supermarket, Manhattan, NY Yellow foot (fresh) Craterellus [Chanterelle]

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Page 1: DNAbarcoding$for$iden0fica0on$of ...for$iden0fica0on$of$$ consumerrelevantfungisoldinNewYork! Authors:!Emily!Jensen2Vargas1, AhlenneAbreu 2! Mentor:!Chris

DNA  barcoding  for  iden0fica0on  of    consumer-­‐relevant  fungi  sold  in  New  York  

Authors:  Emily  Jensen-­‐Vargas1,  Ahlenne  Abreu2  

Mentor:  Chris<ne  Marizzi3  Ph.D.  1Francis  Lewis  High  School,  2  Washington  Heights  Expedi<onary  Learning  School,  3Cold  Spring  Harbor  Laboratory  

Funded  by:  

 •  White TJ, Bruns TD, Lee S, Taylor J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH. (eds).

PCR Protocols: A Guide to Methods and Applications. Academic Press: London, pp. 315 – 322. •  Altschul, Stephen; Gish, Warren; Miller, Webb; Myers, Eugene; Lipman, David (1990). "Basic local alignment search tool". Journal of Molecular Biology. 215 (3):

403–410 •  Dentinger BTM, Suz LM. (2014) What’s for dinner? Undescribed species of porcini in a commercial packet. PeerJ 2:e570

Diaz, James H. "Mistaken Mushroom Poisonings." Official Journal of the Wilderness Medical Society”"Mushrooms." Official Website of the Department of Homeland Security. N.p., Mar. 2008. Web. 01 Feb. 2017. Moyer, Douglas C., Jonathan W. DeVries, and John Spink. "The Economics of a Food Fraud Incident – Case Studies and Examples including Melamine in Wheat Gluten." Science Direct. Division of Public Health, Michigan State University.

•  Now, Season Stepp More Content. "Season Stepp: Mushrooms Rising in Popularity." Wicked Local Plymouth. Wicked Local Plymouth, 11 Oct. 2016. Web. 17 Jan. 2017. <http://plymouth.wickedlocal.com/news/20161014/season-stepp-mushrooms-rising-in-popularity

•  Raja, Huzefa A. "DNA Barcoding for Identification of Consumer-relevant Mushrooms: A Partial Solution for Product Certification?" Science Direct. N.p., 9 July 2016. Web. 06 May 2017. <http://www.sciencedirect.com/science/article/pii/S0 Yahr R, Schoch CL, Dentinger BTM. 2016 Scaling up discovery of hidden diversity in fungi: impacts of barcoding approaches. Phil. Trans. R. Soc. B 371: 20150336.  

Acknowledgements  

Results                                            

                                               

References  

The  DNA  Barcoding  Workflow                                          

                                                   

Project  Summary  Aim:  Inves&ga&ng  dried  fungi  (mis)labeling  in  New  York  City.      Intended  Outcomes:  Many  foods  in  the  US  have  been  mislabeled  in  order  to  gain  a  greater  profit  for  cheaper  products  replacing  expensive  ones  (Moyer    et  al.,  2017).  We  are  interested  to  see  if  we  can  iden<fy  mislabeled  species  of  fungi  sold  in  New  York,  including  a  commercial  packet  of  dried  fungi.      Descrip3on:    Reliably  iden<fying  wild  collected  foods  can  be  a  challenge,  especially  when  they  belong  to  poorly  known  groups  like  Fungi  (UNITE).  Although  significant  progress   has   been   made   in   our   understanding   of   fungi   diversity,  iden<fica<on  based  on  phenotype  can  be  difficult  for  even  trained  experts.  Fungi   typically  have  a  cryp<c  nature  and  can  have  a  similar  appearance  to  distantly   related   species   (Yahr   et   al.,   2016).   What   contributes   to   the  mislabeling  of  fungi  may  be  the  phenotypical  appearance  of  the  dried  fungi  being  unclear.  Our  goal  was  to  collect  and  analyze  sequences  and  data  from  dried  and  fresh  fungi  sold  for  commercial  uses.  We  used  DNA  technology  for  species   iden<fica<on   of   10   samples   that   were   collected   from   local  supermarkets  in  two  New  York  City  boroughs.  Although  there  were  no  cases  of  mislabeling,  we  found  that  our  single-­‐locus  ITS  DNA  barcoding  approach  allowed   to   iden<fy   only   44%   of   all   samples   at   the   species   level.   This  highlights   the   need   for   a   curated,   centralized   ITS   reference   database   that  allows  third-­‐party  annota<ons.            Dried   and   fresh   fungi   samples   were   collected   from   local   supermarkets   in  two  New  York  City  boroughs.  All  dried  samples  were  obtained  from  a  single  bag  of  dried  of  edible  “Mixed  Mushrooms”.  Samples  were  visually  sorted  by  phenotype,  documented  and  vouchered.  While  fresh  samples  were  easy  to  iden<fy,  many  of  the  dried  samples  were  broken  down  into  smaller  pieces  and   lacked   key   morphological   characteris<cs   that   could   be   used   for  iden<fica<on.  The  DNA  was  extracted  from  approximately  0.1  g  of  specimen  us ing   the   s tandard   s i l i ca   DNA   ext rac<on   method   (www.  dnabarcoding101.org),   with   one   minor   modifica<on:   for   each   sample,   a  small   piece  was   removed   and   pre-­‐soaked   in   dH20   before   being   ground   in  lysis   buffer   with   a   sterile   pestle.   The   extracted   DNA   was   amplified   using  fungi-­‐specific  ITS  primers  (ITS1F/ITS4  as  described  by  White  et  al.,  1990)  and  NEB  Taq  2X  master  mix  (New  England  Biolabs).  Then,  the  amplified  DNA  was  stained   with   SYBR   green   (Invitrogen)   and   confirmed   using   gel  electrophoresis.   Posi<ve   amplicons   were   sent   to   GENEWIZ   Inc.   for  sequencing  in  both  direc<ons.  Aier  the  DNA  was  sequenced,  DNA  Subway  soiware     (hjp://www.cyverse.org/dna-­‐subway)   was   used   to   analyze   the  DNA   sequences.   To   verify   species   iden<fica<ons   through   DNA   Subway  sample   consensus   sequences  were   downloaded   and   submijed   directly   to  Basic  Local  Alignment  Search  Tool  (BLAST)  in  NCBI-­‐GenBank  (Altschul  et  al.,  1990)  and  UNITE  Ver.  7.1,  an  open-­‐access,  curated  fungi-­‐specific  database    (hjps://unite.ut.ee).   Finally   a  phylogene<c   tree  was   constructed  based  on    DNA  Subway’s  MUSCLE  and  PHYLIP  maximum  likelihood  (ML)  methods.      

 .    

     

 

DNA  Subway   BLAST,  GenBank   UNITE  

Sample  ID   Loca0on   Fungi  sp.  Per  Label    

Common  Name   Fungi  Barcode  ID   %  Match   Fungi  Barcode  ID   %  Match   Fungi  Barcode  ID   %  Match  

S  1   Supermarket,  LIC,  NY  

Cloud-­‐ear  (dried)   Cloud  Ear   Auricularia  heimuer   100   Auricularia  heimuer   100   Auricularia  heimuer   100  

S  2   Supermarket,  LIC,  NY  

Shiitake  (dried)   Shiitake     Len&nula  edodes   86   Len&nula  edodes   99   Len&nula  edodes   99.9  

S  3   Supermarket,  LIC,  NY  

Wood  ear  (dried)   Wood  ear     N/A       N/A       N/A      

S  4   Supermarket,  LIC,  NY  

Oyster  (dried)   Oyster   Pleurotus  geesterani   95   Pleurotus  geesterani   94   Pleurotus  sp.   94  

S  5   Supermarket,  LIC,  NY  

White  Bujon  (dried)  

White  Bujon     Agaricus  bisporus   100   Agaricus  bisporus   100   Agaricus  bisporus   100  

S  6   Supermarket,  LIC,  NY  

Oyster  (dried)   Oyster   Pleurotus  sp.   90   Pleurotus  ostreatus   91   Pleurotus  ostreatus   91  

S  7   Supermarket,  LIC,  NY  

White  Bujon  (fresh)  

White  Bujon     Agaricus  bisporus   100   Agaricus  bisporus   100   Agaricus  bisporus   100  

S  8   Supermarket,  LIC,  NY  

Korean  Black  (dried)  

Korean  Black     Auricularia  heimuer   100   Auricularia  heimuer   100   Auricularia    auricula-­‐judae   100  

S  9   Supermarket,  NYC,  NY  

Yellow  foot  (fresh)   Yellow  foot   Craterellus  

tubaeformis   100   Craterellus  tubaeformis   100   Craterellus  sp.   100  

S  10   Supermarket,  NYC,  NY  

Yellow  foot  (fresh)   Yellow  foot     Craterellus  

tubaeformis   100   Craterellus  tubaeformis   100   Craterellus  sp.   100  

There  were  no  cases  of  mislabeling.  For  one  sample   (sample  S3)  we  were  not  able   to  obtain   a   DNA   sequence   because   of   unsuccessful   PCR   amplifica<on   in   spite   of   several  ajempts  (Table  1).  This  may  be  due  to  poten<ally  high  degrada<on  of  the  sample’s  DNA  due   to   heat   treatment   prior   packaging   -­‐   sample   S3   was   very   dry.   Once   DNA   is  broken   into   short   fragments,   genera<ng   a   ITS   barcodes   can   be   difficult,    as  primer  binding  sites  may  be  degraded.  Moreover,  secondary  metabolites  can  inhibit  PCR  by  decreasing  the  Taq-­‐polymerase’s  ac<vity.  Failure  to  generate  a  ITS  barcode  may  also  be  because  the  primers  used  were  not  suited  for  amplifica<ons  of  all  species.  Sample  S2  only  yielded  a  single  sequencing  read  (reverse  direc<on).  Samples  S1,  S2,  S5,  S6,  and  S7  were   labeled  correctly   in  both  the  species  and  genus   level.  Samples  S4,  S8,  S9,  and  S10  were  labeled  at  the  correct  genus  level  but  we  were  unable  to  resolve  them  down  to  the  species  level  as  there  were  discrepancies  between  databases  (see  Figures  2  and   3).   Species   with   highest   %   match/max   score/bit   score   were   considered   the  taxonomic  iden<ty  (Table  2).  UNITE  Ver.  7.1  serial  BLAST  search  was  u<lized  in  addi<on  to  GenBank  because   it  has  been   found  that  27%  of   fungal   ITS  sequences  entered   into  GenBank   were   inadequately   iden<fied   and   20%   were   incorrectly   labeled   (Raja   and  Oberlies  et  al.,   2016).   Future   studies  would  benefit  highly   from  a   curated,   centralized  ITS  reference  database  that  allows  rapid  third-­‐party  annota<ons.    

90%  

10%  

Sequencing  Success  Rates  ITS  sequence  obtained  (9/10)  

No  ITS  sequence  obtained  (1/10)  

44%  

56%  

Sample  Iden0fica0on  Rates  

Sample  iden<fied  at  species  level  (4/9)  

Sample  idenpied  at  genus  level  (5/9)  

Materials  and  Methods                                            

                                         

       

This  project  was  supported  by  the  Urban  Barcode  Research  Program,   funded   by   the   Pinkerton   Founda<on.  We   thank  Melissa  Lee,  Alison  Cucco  and  Jenna  Dorey  (DNA  Learning  Center,   Cold   Spring   Harbor   Laboratory)   for   their   helpful  assistance.    

 Discussion                                              

                                               

       

Table  1  (above):  List  of  fungi  sampled  in  this  study  for  ITS  barcoding.    Figures  2  and  3  (right):    ITS  sequencing  and  sample  iden<fica<on  success  rates.    

Figure  1:  How  DNA  barcoding  works.    

Figure   4:  Sample   sequences   (S1   -­‐   S10)   aligned  with   corresponding   BLAST   sequences   hits   using   the  MUSCLE  mul<ple  alignment  soiware.    The  same  data  set  is  depicted  as  gene  tree  using  PHYLIP  ML  to  generate  a  phylogene<c  tree  using  the  maximum  likelihood  (ML)  method.  Sample  S2  (Shiitake)  was  chosen  as  out-­‐group  as  only  one  sequencing  read  was  available  for  species  iden<fica<on.    

     

Table   2:   Species   iden3fica3on   based   on   ITS   DNA   barcoding   from   all   samples   analyzed   (n=10).   Each   consensus  sequence   of   samples   S1   –   S10   was   subjected   to   three   search   tools   to   verify   iden<ty:   1)   DNA   Subway;   2)   direct  submission  to  BLAST  search  in  NCBI-­‐GenBank;    and  3)  a  serial  BLAST  search  in  the  curated  database  termed  UNITE.    

Sample' Date'Purchased'

Location'of'purchase'

Labeled'on'Package'

Picture' Identified'using'UNITE'serial'BLAST'

search'

S'1' February(2nd,(2017(

Supermarket,(LIC,(NY(

Cloud<ear((dried)(

(

Auricularia(heimuer([Cloud<ear](

S'2' February(2nd,(2017(

Supermarket,(LIC,(NY(

Shiitake((dried)(

(

Lentinula(edodes([Shiitake](

(

S'3' February(2nd,(2017(

Supermarket,(LIC,(NY(

Wood(ear((dried)(

'

No(sequence(

S'4' February(2nd,(2017(

Supermarket,(LIC,(NY(

Oyster((dried)(

(

Pleurotus([Oyster](

(

S'5' February(2nd,(2017(

Supermarket,(LIC,(NY(

White(Button((dried)(

(

Agaricus(bisporus([White(button](

S'6' February(2nd,(2017(

Supermarket,(LIC,(NY(

Unknown((dried)(

(

Pleurotus(ostreatus([Oyster](

S'7' February(2nd,(2017(

Supermarket,(LIC,(NY(

White(Button((fresh)(

(

Agaricus(bisporus([White(button](

S'8' February(2nd,(2017(

Supermarket,(LIC,(NY(

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