Jan Cordes, Julie Stubbs, and Kimberly Reece Genetic Identification of Oyster Species Based on PCR-RFLP Analysis of ITS-1 and COI Gene Regions Department.

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Jan Cordes, Julie Stubbs, and Kimberly Reece Genetic Identification of Oyster Species Based on PCR-RFLP Analysis of ITS-1 and COI Gene Regions Department of Environmental and Aquatic Animal Health Virginia Institute of Marine Science College of William and Mary

Traditional species identification and taxonomy of Crassostrea and related oysters based on highly variable, environmentally influenced morphometric characters Existing molecular genetic keys usually include a relatively small number of species Unable to rely on correct identification of samples sent from the field, especially when receiving samples from widely separated locations This can greatly impact emerging efforts to introduce exotic oyster species into U.S. waters Background

Objectives Construct a reliable genetic key for 12 species of commercially important oysters Base key on at least one nuclear and one mitochondrial marker Base key on PCR/RFLP analysis to simplify use and reduce amount of tissue needed Optimize the RFLP analysis to minimize the number of restriction enzymes needed for complete differentiation of all 12 species Saccostrea cucullata S. commercialis Lopha cristigalli Crassostrea virginica C. gigas C. plicatula C. sikamea C. ariakensis C. iredalei C. gryphoides C. madrasensis C. belcheri

Since DNA from type specimens is not available, chose voucher samples from our archives based on phylogenetic analyses PCR amplified ITS-1(Hedgecock et al. 1999) and COI (Folmer et al. 1994) gene regions using published primers in individuals of each target species Sequenced and aligned PCR products for the 12 putative species of oysters (representative sequences from GenBank were included when possible) Identified the restriction enzyme(s) that distinguished among the largest number of species for each marker via virtual digestions using MacVector Tested the restriction enzymes identified as informative by traditional RFLP analysis using agarose gels Protocols

Republic of China India Japan C. ariakensis (northern) C. ariakensis (Malaysia) C. belcheri C. gigas C. iredalei (China) C. plicatula C. iredalei (Thailand) S. commercialis (Australia) Protocols Sampling Locations C. ariakensis (southern) C. rivularis (ariakensis) C. virginica C. madrasensis C. gryphoides Lopha cristagalli S. cucullata C. sikamea

Republic of China India Japan C. ariakensis (northern) C. ariakensis (southern) C. ariakensis (Malaysia) C. belcheri C. gigas C. iredalei (China) C. plicatula C. iredalei (Thailand) C. virginica S. commercialis (Australia) Protocols Sampling Locations C. madrasensis C. gryphoides Lopha cristagalli S. cucullata C. rivularis (ariakensis)

Republic of China India Japan C. ariakensis (northern) C. ariakensis (southern) C. ariakensis (Malaysia) C. belcheri C. gigas C. iredalei (China) C. plicatula C. iredalei (Thailand) C. virginica S. commercialis (Australia) Protocols Sampling Locations C. madrasensis C. gryphoides Lopha cristagalli S. cucullata C. rivularis (ariakensis)

Results ITS-1 Parsimony Bootstrap Analysis of ITS-1 Sequences C. ariakensis (Malaysia) Cvir15 04 Cvir23 04 Cvir21 04 Cvir22 05 Cvir22 06 ScucIndia82 05 ScucIndia82 03 ScucIndia52 01 ScucIndia51 03 ScucIndia51 01 BL163 3 Scom02 02 Scom01 01 BL166 2 BL165 4 LcrystIndia LcrystIndia LcrystIndia LcrystIndia LcrystIndia CsikA25 04 CsikA15 04 CsikA10 01 CsikA02 02 CsikA02 01 CsikA08 05 CsikA08 04 Csik02 04 Csik02 03 Csik01 04 CplicChina03 10 CplicChina01 05 CariakWM4 3 CariakWM4 1 Cgig01 02 Cgig01 01 CplicChina01 03 Cgig GENBANK CariakWM01 04 CariakWM01 02 Cgig04 03 Cgig04 02 CariakWM03 04 CsikA10 05 CsikA10 02 CariakD19 04 CariakD19 03 CariakD19 02 CariakD17 04 CariakD17 03 CariakD17 01 CariakD10 02 CariakD07 02 CariakD07 01 CariakC09 02 CariakC09 01 CariakC04 02 CariakC04 01 CariakC03 03 CariakC03 02 CariakC03 01 CariakC02 02 CariakC02 01 CariakC01 05 CariakB12 02 CariakB12 01 CariakB02 03 CariakB11 04 CariakB11 03 CariakB04 02 CariakB04 01 CrivIndia Criv GENBANK CariakI05 01 CariakYR08 01 CariakYR07 01 CariakVi05 03 CariakVi05 02 CariakVi05 01 CariakI07 01 CariakI06 04 CariakI06 03 CariakI04 05 CariakYR03 01 CariakYR02 02 CariakVi07 04 CariakVi07 03 CariakVi07 01 CariakVi04 02 CariakVi04 01 CariakI04 04 CariakVi02 02 CariakVi02 01 CariakVi02 CariakI07 03 CariakI07 02 CariakI06 02R CariakI05 03 CariakI05 02 CariakI02 02 CariakB03 03 CariakB03 02 CariakB03 01 CariakB02 02 Criv Criv CgryphIndia CgryphIndia CgryphIndia103 3temp CbelchSR03 05 CbelchSR03 03 CbelchSR03 02 CbelchSr02 07 CbelchRN01 05 CariakMal02 02 CariakMal01 02 CariakMal01 01 Be1 5 Be2 6 Be2 5 Be1 4 CmadIndia108 6 CmadIndia01 02 CmadIndia01 04 CiradelliPN02 09 CiradelliPN02 07 CiradelliCb03 03 CiradelliCb03 02 CiradelliCb03 01 CiradelliCb02 04 CiradelliCb02 03 CiradelliCb02 02 CiradeliPN1 4 CiradeliPN1 3 CiradeliPN1 1 CmadIndia02 02 CmadIndia02 01 CiradeliChina8 26 CiradeliChina3 5 CiradelChina3 2 CiradelChina2 3 CiradelChina S. cucullata S. commercialis C. sikamea C. ariakensis (northern) C. gryphoides L. crystigalli C. ariakensis (southern) C. madrasensis/ C. iredalei (Thailand) C. belcheri C. gigas/ C. plicatula C. virginica C. iredalei (China) C. rivularis (India) C. belcheri

Results ITS-1 Parsimony Bootstrap Analysis of ITS-1 Sequences C. ariakensis (Malaysia) Cvir15 04 Cvir23 04 Cvir21 04 Cvir22 05 Cvir22 06 ScucIndia82 05 ScucIndia82 03 ScucIndia52 01 ScucIndia51 03 ScucIndia51 01 BL163 3 Scom02 02 Scom01 01 BL166 2 BL165 4 LcrystIndia LcrystIndia LcrystIndia LcrystIndia LcrystIndia CsikA25 04 CsikA15 04 CsikA10 01 CsikA02 02 CsikA02 01 CsikA08 05 CsikA08 04 Csik02 04 Csik02 03 Csik01 04 CplicChina03 10 CplicChina01 05 CariakWM4 3 CariakWM4 1 Cgig01 02 Cgig01 01 CplicChina01 03 Cgig GENBANK CariakWM01 04 CariakWM01 02 Cgig04 03 Cgig04 02 CariakWM03 04 CsikA10 05 CsikA10 02 CariakD19 04 CariakD19 03 CariakD19 02 CariakD17 04 CariakD17 03 CariakD17 01 CariakD10 02 CariakD07 02 CariakD07 01 CariakC09 02 CariakC09 01 CariakC04 02 CariakC04 01 CariakC03 03 CariakC03 02 CariakC03 01 CariakC02 02 CariakC02 01 CariakC01 05 CariakB12 02 CariakB12 01 CariakB02 03 CariakB11 04 CariakB11 03 CariakB04 02 CariakB04 01 CrivIndia Criv GENBANK CariakI05 01 CariakYR08 01 CariakYR07 01 CariakVi05 03 CariakVi05 02 CariakVi05 01 CariakI07 01 CariakI06 04 CariakI06 03 CariakI04 05 CariakYR03 01 CariakYR02 02 CariakVi07 04 CariakVi07 03 CariakVi07 01 CariakVi04 02 CariakVi04 01 CariakI04 04 CariakVi02 02 CariakVi02 01 CariakVi02 CariakI07 03 CariakI07 02 CariakI06 02R CariakI05 03 CariakI05 02 CariakI02 02 CariakB03 03 CariakB03 02 CariakB03 01 CariakB02 02 Criv Criv CgryphIndia CgryphIndia CgryphIndia103 3temp CbelchSR03 05 CbelchSR03 03 CbelchSR03 02 CbelchSr02 07 CbelchRN01 05 CariakMal02 02 CariakMal01 02 CariakMal01 01 Be1 5 Be2 6 Be2 5 Be1 4 CmadIndia108 6 CmadIndia01 02 CmadIndia01 04 CiradelliPN02 09 CiradelliPN02 07 CiradelliCb03 03 CiradelliCb03 02 CiradelliCb03 01 CiradelliCb02 04 CiradelliCb02 03 CiradelliCb02 02 CiradeliPN1 4 CiradeliPN1 3 CiradeliPN1 1 CmadIndia02 02 CmadIndia02 01 CiradeliChina8 26 CiradeliChina3 5 CiradelChina3 2 CiradelChina2 3 CiradelChina S. cucullata S. commercialis C. sikamea C. ariakensis (northern) C. gryphoides L. crystigalli C. ariakensis (southern) C. belcheri C. gigas/ C. plicatula C. virginica C. iredalei (China) C. rivularis (India) C. belcheri C. madrasensis/ C. iredalei (Thailand)

Results ITS-1 Parsimony Bootstrap Analysis of ITS-1 Sequences C. ariakensis (Malaysia) Cvir15 04 Cvir23 04 Cvir21 04 Cvir22 05 Cvir22 06 ScucIndia82 05 ScucIndia82 03 ScucIndia52 01 ScucIndia51 03 ScucIndia51 01 BL163 3 Scom02 02 Scom01 01 BL166 2 BL165 4 LcrystIndia LcrystIndia LcrystIndia LcrystIndia LcrystIndia CsikA25 04 CsikA15 04 CsikA10 01 CsikA02 02 CsikA02 01 CsikA08 05 CsikA08 04 Csik02 04 Csik02 03 Csik01 04 CplicChina03 10 CplicChina01 05 CariakWM4 3 CariakWM4 1 Cgig01 02 Cgig01 01 CplicChina01 03 Cgig GENBANK CariakWM01 04 CariakWM01 02 Cgig04 03 Cgig04 02 CariakWM03 04 CsikA10 05 CsikA10 02 CariakD19 04 CariakD19 03 CariakD19 02 CariakD17 04 CariakD17 03 CariakD17 01 CariakD10 02 CariakD07 02 CariakD07 01 CariakC09 02 CariakC09 01 CariakC04 02 CariakC04 01 CariakC03 03 CariakC03 02 CariakC03 01 CariakC02 02 CariakC02 01 CariakC01 05 CariakB12 02 CariakB12 01 CariakB02 03 CariakB11 04 CariakB11 03 CariakB04 02 CariakB04 01 CrivIndia Criv GENBANK CariakI05 01 CariakYR08 01 CariakYR07 01 CariakVi05 03 CariakVi05 02 CariakVi05 01 CariakI07 01 CariakI06 04 CariakI06 03 CariakI04 05 CariakYR03 01 CariakYR02 02 CariakVi07 04 CariakVi07 03 CariakVi07 01 CariakVi04 02 CariakVi04 01 CariakI04 04 CariakVi02 02 CariakVi02 01 CariakVi02 CariakI07 03 CariakI07 02 CariakI06 02R CariakI05 03 CariakI05 02 CariakI02 02 CariakB03 03 CariakB03 02 CariakB03 01 CariakB02 02 Criv Criv CgryphIndia CgryphIndia CgryphIndia103 3temp CbelchSR03 05 CbelchSR03 03 CbelchSR03 02 CbelchSr02 07 CbelchRN01 05 CariakMal02 02 CariakMal01 02 CariakMal01 01 Be1 5 Be2 6 Be2 5 Be1 4 CmadIndia108 6 CmadIndia01 02 CmadIndia01 04 CiradelliPN02 09 CiradelliPN02 07 CiradelliCb03 03 CiradelliCb03 02 CiradelliCb03 01 CiradelliCb02 04 CiradelliCb02 03 CiradelliCb02 02 CiradeliPN1 4 CiradeliPN1 3 CiradeliPN1 1 CmadIndia02 02 CmadIndia02 01 CiradeliChina8 26 CiradeliChina3 5 CiradelChina3 2 CiradelChina2 3 CiradelChina S. cucullata S. commercialis C. sikamea C. ariakensis (northern) C. gryphoides L. crystigalli C. ariakensis (southern) C. belcheri C. gigas/ C. plicatula C. virginica C. iredalei (China) C. rivularis (India) C. belcheri C. madrasensis/ C. iredalei (Thailand)

Results COI S. commercialis C. sikamea C. gigas C. madrasensis C. iredalei (Thailand) C. belcheri C. ariakensis (southern China) C. ariakensis (northern China) C. gryphoides S. cucullata O. chilensis (outgroup) C. plicatula L. crystigalli C. virginica C. iredalei (China) C. ariakensis (Malaysia) C. rivularis (India) Parsimony Bootstrap Analysis of COI Sequences

Results COI S. commercialis C. sikamea C. gigas C. madrasensis C. iredalei (Thailand) C. belcheri C. ariakensis (southern China) C. ariakensis (northern China) C. gryphoides S. cucullata O. chilensis (outgroup) C. plicatula L. crystigalli C. virginica C. iredalei (China) C. ariakensis (Malaysia) C. rivularis (India) Parsimony Bootstrap Analysis of COI Sequences

Results COI S. commercialis C. sikamea C. gigas C. madrasensis C. iredalei (Thailand) C. belcheri C. ariakensis (southern China) C. ariakensis (northern China) C. gryphoides S. cucullata O. chilensis (outgroup) C. plicatula L. crystigalli C. virginica C. iredalei (China) C. ariakensis (Malaysia) C. rivularis (India) Parsimony Bootstrap Analysis of COI Sequences

Comparison of Virtual Restriction Enzyme Digests on ITS Region of 6 Species of Crassostrea Results ITS-1

SpeciesSizeCutsPositionsBand Sizes Crassostrea iredalei (China) A B2364, 3684, 108, 364 Sacostrea commercialis A , 452 S. cucullata A , 134, 37913, 112, 121, 245 B453, 121, 134, 37913, 53, 68, 112, 245 C4121, 134, 176, 37913, 42, 112, 121, 203 D553, 121, 134, 379, 46625, 13, 53, 68, 87, 245 C. virgnica A , 259, 43962, 79, 180, 196 B4112, 197, 259, 43962, 79, 85, 112, 180 L. cristagalli A , 474 B2339, 47498, 135, 339 C. madrasensis A , 49399, 242, 251 B4177, 242, 374, 49365, 99, 119, 132, 177 C. iredalei (Thailand) A , 241, 371, 50264, 101, 130, 131, 177 B5177, 190, 241, 371, 50213, 51, 101, 130, 131, 177 Virtual Digests of ITS-1 Intraspecific Aligned Sequences – Hae III Restriction Sites and Fragment Sizes- Including Flanking Primer Sites Results ITS-1

C. gigas C. belcheri C. ariakensis S. C. ariakensis N. C. sikamea C. virginica Virtual Digests of ITS-1 Intraspecific Aligned Sequences* – Hae III 0 C. iredalei CH C. plicatula C. gryphoides S. cucullata S. commercialis C. madrasensis C. iredalei TH L. cristagalli = 2 nd morphotype= 3 nd morphotype 466 = 4th morphotype bp 525 bp 491 bp 518 bp 572 bp 592 bp 603 bp 611 bp 575 bp 541 bp 548 bp 556 bp 565 bp * Includes flanking primers in size estimates bp = 1 nd morphotype Results ITS-1

Results ITS-1 Restriction Enzyme Digests of ITS-1 Region with Hae III S. commercialisS. cucullata L. cristagalli C. belcheri C. iredalei CH C. madrasensis C. gryphoides C. gigas C. plicatulaC. sikamea C. ariakensis N. C. iredalei TH C. ariakensis S.

Results ITS-1 Restriction Enzyme Digests of ITS-1 Region with Hae III S. commercialisS. cucullata L. cristagalli C. belcheri C. iredalei CH C. madrasensis C. gryphoides C. gigas C. plicatulaC. sikamea C. ariakensis N. C. iredalei TH C. ariakensis S.

S. commercialis S. cucullata L. cristagalli C. belcheri C. iredalei CH C. madrasensis C. gryphoides C. gigas C. plicatulaC. sikamea C. ariakensis N. C. iredalei TH C. ariakensis S. Results ITS-1 Restriction Enzyme Digests of ITS-1 Region with Hae III

Flow Chart for Oyster Species Identification using RFLP Analysis of PCR-Amplified ITS-1 Nuclear DNA Fragments C. sikamea C. ariakensis S. Hae III Dde I C. sikamea / C. ariakensis S. Saccostrea cucullata S. commercialis Lopha cristigalli Crassostrea virginica C. iredalei CH C. iredalei TH C. gryphoides C. madrasensis C. belcheri C. gigas C. plicatula C. sikamea C. ariakensis S. C. ariakensis N. C. gigas / C. plicatula S. cucullata S. commercialis L. cristagalli C. virginica C. iredalei CH C. iredalei TH / C. madrasensis C. gryphoides C. belcheri C. ariakensis N. C. madrasensis C. iredalei TH C. plicatula C. gigas None Found Results ITS-1

Flow Chart for Oyster Species Identification using RFLP Analysis of PCR-Amplified COI MtDNA Fragments C. sikamea C. ariakensis S. Dde I Saccostrea cucullata S. commercialis Lopha cristigalli C. virginica C. iredalei CH C. iredalei TH C. gryphoides C. madrasensis C. belcheri C. gigas C. plicatula C. sikamea C. ariakensis S. C. ariakensis N. C. gigas /C. ariakensis N. S. cucullata S. commercialis L. cristagalli C. virginica C. iredalei CH C. iredalei TH / C. madrasensis/ C. plicatula C. gryphoides C. belcheri C. iredalei TH/ C. madrasensis C. plicatula Bgl II* C. gigas C. ariakensis N. C. iredalei TH C. madrasensis TspR I* * Still need to be verified on agarose gels Results COI Bgl II*

Verify utility of Bgl II and TspR I restriction enzymes and complete COI species key Refine species phylogenies by obtaining additional samples for questionable (C. iredalei from China, C. ariakensis from Malaysia, and C. rivularis from India) and missing (i.e. C. rhizophorae) species Compile tables with species-specific band patterns for each marker along with gel images and create an easy-to-follow genetic key that can be made available to others (perhaps via a website) Next Steps

Test accuracies of species identifications in archived samples currently housed in our laboratory Verify species identities of samples sent to us from other researchers Verify species identity of samples collected in the field for new project using microsatellite markers to investigate genetic variation in native and hatchery populations of C. ariakensis Applications