Collect SFE specimens

Extract DNA

PCR amplify ~5 barcode

regionsDNA

sequencingAssemble database

Building a Comprehensive and AccessibleDNA Barcode Database for Fish and Invertebrates

in the San Francisco Estuary

Ravi Nagarajan1, Ann Holmes1, Danielle Myers1, Julia Mouat2, Alisha Goodbla1, Mallory Bedwell3, Melinda Baerwald3, Brian Schreier3, Andrew Rypel4, Larry Brown5, & Andrea Schreier1

Author Affiliations

1. Genomic Variation Laboratory, UC Davis2. Integrative Genetics and Genomics Graduate Group, UC Davis3. CA Dept of Water Resources4. Department of Wildlife, Fish, and Conservation Biology, UC Davis5. US Geological Survey

Acknowledgements Funding: CDFW/CA Prop 1 Cory Saltzman (CDFW) Ted Grosholz (UC Davis)Sakura Evans (CDFW) Betsy Wells (DWR)Martha Volkoff (CDFW) Jesse Adams (DWR)Heather Perry (CDFW) Suisun Marsh Fish Survey Ian Ralston (CDFW) Teejay O’Rear (UC Davis)Chelsea Callahan (CDFW) Dylan Stompe (UC Davis)Jessica Weidenfeld (UC Davis) John Durand (UC Davis)Shayan Kaveh (UC Davis) Lisa Thompson (Sacramento Area SewerKhyana Yearwood (UC Davis) District & Sacramento Regional CountySerra Perry (UC Davis) Sanitation District)Victor Martinez (UC Davis) Cara Wademan (UC Davis)Samantha Barnum (UC Davis) Grace Auringer (UC Davis)

Background• Environmental DNA (eDNA) metabarcoding methods rely on DNA

barcode reference sequences for each species.

• Some SFE species already have DNA barcode reference sequencesavailable in public databases, but some do not have have barcode data and many have only partial data.

• We are building a custom DNA barcode reference sequence database for SFEfish and invertebrates.

• This database is the first phase of a multiyear Prop 1 funded project to developeDNA metabarcoding methods to complement existing SFE monitoring.

• The custom database will be made public as a resource to all scientists and will enable accurate taxonomic assignments from eDNA metabarcoding sequencing data. The data will also be useful for other genetic applications.

Approach

• Database expected completion by December 2020.• The database will improve the accuracy and utility of eDNA

metabarcoding. • Will enable biodiversity monitoring using eDNA metabarcoding,

complementing and augmenting existing IEP monitoring.

Summary

Gene Type Primer names (Forward/Reverse) ReferenceCytochrome C Oxidase 1 (COI) mitochondrial jgLCO1490/jgHCO2198 Geller et al 2013

12S rRNA mitochondrial tRNA-Phe/teleo_R Doble et al 2019

12S rRNA mitochondrial MiFish-U-F/MiFish-U-R Miya et al. 2015

16s rRNA mitochondrial Ac16s-F/Ac16s-R Evans et al. 2015

16s rRNA mitochondrial 16s_Metazoa_fw/16s_Metazoa_rev Shelton et al 2016

16s rRNA mitochondrial 16S-V5-F/16S-V5-F Riaz et al. 2011

Cytochrome B (CytB) mitochondrial L14912/H15149c Burgener and Hubner 1998

18S rRNA nuclear SSU-F1/SSU-R568 Tanabe et al 2015

18S rRNA nuclear SSU-F1289/SSU-R1772 Tanabe et al 2015

Target Species

Collection of invasive zebra mussels (Dreissena polymorpha)from San Justo Reservoir, CA. Photos credit:Heather Perry, CDFW

• Specimen collection and DNA extractions ongoing

• PCR primer testing complete (see below)

• Goal for database is to sequence ~5 PCR products for each specimen• Some barcoding genes contain multiple barcodes

• eDNA metabarcoding accuracy is improved by sequencing >1 barcode for each sample

• For reference database sequencing, we are prioritizing PCR primers that will give us sequence information for multiple barcodes from one PCR/sequencing reaction

• DNA sequencing started July 2020 (delayed due to COVID-19 lab shutdown)• Some extracted DNAs will also be used to create known DNA mixtures to test

eDNA metabarcoding protocol prior to using with eDNA samples

PCR primers tested for Sanger DNA sequencing:

Fish

Invertebrates

Publicly available reference sequence(not necessarily from SFE)

No reference sequence

Partial reference sequence

Common NameMitogenome Accession COI 12S 16S CytB 18S Scientific Name

Bay barnacle Amphibalanus improvisusAmphibalanus subalbidus Amphibalanus subalbidusYukon floater Anodonta beringianaCalifornia floater mussel Anodonta californiensisWoebegone floater Anodonta dejectaWestern floater Anodonta kennerlyiOregon floater Anodonta oregonensisWillamette floater Anodonta wahlamatensisChinese mystery snail NC_035734.1 Cipangopaludina chinensisJapanese mystery snail LC514194.1* Cipangopaludina japonicaAsian clam NC_046410.1 Corbicula flumineaCorophium Corophium spp.California bay shrimp Crangon franciscorumQuagga mussel Dreissena bugensisZebra mussel KY091877.1* Dreissena polymorphaEogammarus confervicolus Eogammarus confervicolusChinese mitten crab NC_006992.1 Eriocheir sinensisSiberian prawn MF687349.1 Exopalaemon modestusGammarus daiberi Gammarus daiberiWestern ridged mussel Gonidea angulataHyalella azteca NC_039403.1 Hyalella aztecaHyperacanthomysis shrimp Hyperacanthomysis longirostrisAtlantic Macoma Macoma petalumWestern pearlshell NC_015476.1 Margaritifera falcataFalse dark mussel Mytilopsis leucophaeataNeomysis shrimp Neomysis mercedisOriental shrimp Palaemon macrodactylusPea cockle or pea clam Pisidium casertanumRidged-beak peaclam Pisidium compressumGolden apple (mystery) snail NC_037691.1 Pomacea bridgesiiChanneled apple snail NC_024586.1 Pomacea canaliculataOverbite clam Potamocorbula amurensisNew Zealand mud snail NC_020790.1 Potamopyrgus antipodarumSynidotea laticauda Synidotea laticaudaBanded mystery snail Viviparus georgianus

TOTALS: 12 34 12 12 12 15

Barcode Genes

Common NameMitogenome Accession COI 12S 16S CytB 18S Scientific Name

Yellowfin Goby Acanthogobius flavimanusGreen Sturgeon NC_028405 Acipenser medirostrisWhite Sturgeon NC_004743 Acipenser transmontanusAmerican Shad NC_014690 Alosa sapidissimaWhite Catfish NC_037012 Ameiurus catusSacramento Perch Archoplites interruptusSacramento Sucker Catostomus occidentalisPrickly Sculpin NC_036145 Cottus asperRiffle Sculpin Cottus gulosusCommon (European) Carp NC_001606 Cyprinus carpioThreadfin shad NC_009580 Dorosoma petenenseWestern Mosquitofish NC_004388 Gambusia affinisThree-spined Stickleback NC_041244 Gasterosteus aculeatusCalifornia roach Hesperoleucus symmetricusWakasagi Smelt NC_015242 Hypomesus nipponensisDelta Smelt Hypomesus transpacificusTule Perch Hysterocarpus traskiiRiver Lamprey Lampetra ayresiPacific Lamprey Lampetra tridentata/Entosphenus tridentatusHitch NC_033930 Lavinia exilicaudaBluegill NC_015984 Lepomis macrochirusStaghorn Sculpin Leptocottus armatusBluefin Killifish Lucania goodeiInland (Mississippi) Silverside Menidia beryllinaSmallmouth Bass NC_011361 Micropterus dolomieuSpotted Bass Micropterus punctulatusLargemouth Bass NC_008106 Micropterus salmoidesStriped Bass NC_014353 Morone saxatilisHardhead Mylopharodon conocephalusSteelhead/Rainbow Trout NC_001717 Oncorhynchus mykissKokanee Salmon NC_008615 Oncorhynchus nerkaChinook Salmon NC_002980 Oncorhynchus tshawytschaSacramento Blackfish Orthodon microlepidotusLargescale Loach NC_023803 Paramisgurnus dabryanusBigscale Logperch NC_008111 Percina macrolepidaStarry Flounder NC_010966 Platichthys stellatusSacramento Splittail NC_033942 Pogonichthys macrolepidotusSacramento Pikeminnow Ptychocheilus grandisLongfin Smelt Spirinchus thaleichthysShimofuri Goby NC_015992 Tridentiger bifasciatus

TOTALS: 23 39 31 27 37 8

• Most SFE fish and invertebrate species have existing COI reference sequences

• References for other commonly used barcode genes (12S, 16S, Cytb, and 18S) are still needed for many SFE species

• Our objective is to fill in as many of these gaps as possible

References• Burgener and Hubner (1998) Mitochondrial DNA enrichment for species identification and evolutionary analysis. European Food Research and Technology 207(4) 261-3.• Doble et al (2019) Testing the performance of environmental DNA metabarcoding for surveying highly diverse tropical fish communities: A case study from Lake Tanganyika.

Environmental DNA 2(1) 24-41 DOI: 10.1002/edn3.43.• Evans et al (2015) Quantification of mesocosm fish and amphibian species diversity via environmental DNA metabarcoding. Mol Ecol Res 16, 29-41.• Geller et al (2013) Redesign of PCR primers for mitochondrial cytochrome c oxidase subunit I for marine invertebrates and application in all-taxa biotic surveys. Mol Ecol Res 13, 851–861• Miya et al (2015) MiFish, a set of universal PCR primers for metabarcoding environmental DNA from fishes: detection of more than 230 subtropical marine species. R. Soc. open sci. 2: 150088.• Riaz et al (2011) ecoPrimers: inference of new DNA barcode markers from whole genome sequence analysis. Nuc Acids Res 39(21). doi:10.1093/nar/gkr732.• Shelton et al (2016) A framework for inferring biological communities from environmental DNA. Ecological Applications, 26(6), 1645–1659.• Tanabe et al (2015) Comparative study of the validity of three regions of the 18S-rRNA gene for massively parallel sequencing-based monitoring of the planktonic eukaryote community.

Mol Ecol Res 16(2) 402-414 doi: 10.1111/1755-0998.12459.