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Jan Cordes, Julie Stubbs, and Kimberly Reece

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

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Jan Cordes, Julie Stubbs, and Kimberly Reece

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

  2. Background • 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

  3. Saccostrea cucullata S. commercialis Lopha cristigalli Crassostrea virginica C. iredalei C. gryphoides C. madrasensis C. belcheri C. gigas C. plicatula C. sikamea C. ariakensis 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

  4. Protocols • 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 20-60 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

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

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

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

  8. Results 59 C. virginica Cvir15 04 Cvir23 04 Cvir21 04 Cvir22 05 Cvir22 06 57 S. cucullata ScucIndia82 05 61 ITS-1 ScucIndia82 03 ScucIndia52 01 100 62 ScucIndia51 03 ScucIndia51 01 S. commercialis BL163 3 100 Scom02 02 Scom01 01 78 BL166 2 52 BL165 4 L. crystigalli LcrystIndia215 02 100 LcrystIndia215 01 78 LcrystIndia214 02 LcrystIndia214 01 LcrystIndia213 08 CsikA25 04 CsikA15 04 C. sikamea 65 CsikA10 01 CsikA02 02 100 CsikA02 01 CsikA08 05 CsikA08 04 Csik02 04 Csik02 03 Csik01 04 97 CplicChina03 10 69 CplicChina01 05 94 CariakWM4 3 C. gigas/ C. plicatula 61 89 CariakWM4 1 Cgig01 02 Cgig01 01 CplicChina01 03 100 Cgig GENBANK CariakWM01 04 95 CariakWM01 02 64 Cgig04 03 Cgig04 02 CariakWM03 04 CsikA10 05 CsikA10 02 CariakD19 04 CariakD19 03 CariakD19 02 CariakD17 04 CariakD17 03 50 CariakD17 01 CariakD10 02 C. ariakensis (southern) CariakD07 02 CariakD07 01 CariakC09 02 CariakC09 01 60 CariakC04 02 CariakC04 01 CariakC03 03 CariakC03 02 64 CariakC03 01 CariakC02 02 CariakC02 01 CariakC01 05 63 CariakB12 02 CariakB12 01 CariakB02 03 CariakB11 04 CariakB11 03 CariakB04 02 CariakB04 01 100 CrivIndia212 02 76 Criv GENBANK 100 CariakI05 01 CariakYR08 01 CariakYR07 01 CariakVi05 03 CariakVi05 02 63 CariakVi05 01 CariakI07 01 CariakI06 04 60 CariakI06 03 C. ariakensis (northern) CariakI04 05 CariakYR03 01 CariakYR02 02 CariakVi07 04 100 CariakVi07 03 CariakVi07 01 56 CariakVi04 02 CariakVi04 01 Parsimony Bootstrap Analysis of ITS-1 Sequences CariakI04 04 66 CariakVi02 02 CariakVi02 01 CariakVi02 CariakI07 03 CariakI07 02 CariakI06 02R CariakI05 03 CariakI05 02 CariakI02 02 CariakB03 03 CariakB03 02 C. rivularis (India) CariakB03 01 CariakB02 02 71 Criv206 01 55 96 C. gryphoides Criv192 02 94 100 CgryphIndia101 06 CgryphIndia101 05 CgryphIndia1033temp C. belcheri CbelchSR03 05 CbelchSR03 03 76 CbelchSR03 02 77 CbelchSr02 07 C. ariakensis (Malaysia) 66 CbelchRN01 05 CariakMal02 02 CariakMal01 02 100 CariakMal01 01 C. belcheri Be1 5 Be2 6 Be2 5 Be1 4 93 CmadIndia108 6 CmadIndia01 02 CmadIndia01 04 60 C. madrasensis/ C. iredalei (Thailand) 72 CiradelliPN02 09 CiradelliPN02 07 CiradelliCb03 03 CiradelliCb03 02 66 CiradelliCb03 01 100 CiradelliCb02 04 CiradelliCb02 03 CiradelliCb02 02 CiradeliPN1 4 CiradeliPN1 3 CiradeliPN1 1 100 CmadIndia02 02 CmadIndia02 01 100 C. iredalei (China) CiradeliChina8 26 100 CiradeliChina3 5 CiradelChina3 2 100 CiradelChina2 3 CiradelChina2 2

  9. Results 59 C. virginica Cvir15 04 Cvir23 04 Cvir21 04 Cvir22 05 Cvir22 06 57 S. cucullata ScucIndia82 05 61 ITS-1 ScucIndia82 03 ScucIndia52 01 100 62 ScucIndia51 03 ScucIndia51 01 S. commercialis BL163 3 100 Scom02 02 Scom01 01 78 BL166 2 52 BL165 4 L. crystigalli LcrystIndia215 02 100 LcrystIndia215 01 78 LcrystIndia214 02 LcrystIndia214 01 LcrystIndia213 08 CsikA25 04 CsikA15 04 C. sikamea 65 CsikA10 01 CsikA02 02 100 CsikA02 01 CsikA08 05 CsikA08 04 Csik02 04 Csik02 03 Csik01 04 97 CplicChina03 10 69 CplicChina01 05 94 CariakWM4 3 C. gigas/ C. plicatula 61 89 CariakWM4 1 Cgig01 02 Cgig01 01 CplicChina01 03 100 Cgig GENBANK CariakWM01 04 95 CariakWM01 02 64 Cgig04 03 Cgig04 02 CariakWM03 04 CsikA10 05 CsikA10 02 CariakD19 04 CariakD19 03 CariakD19 02 CariakD17 04 CariakD17 03 50 CariakD17 01 CariakD10 02 C. ariakensis (southern) CariakD07 02 CariakD07 01 CariakC09 02 CariakC09 01 60 CariakC04 02 CariakC04 01 CariakC03 03 CariakC03 02 64 CariakC03 01 CariakC02 02 CariakC02 01 CariakC01 05 63 CariakB12 02 CariakB12 01 CariakB02 03 CariakB11 04 CariakB11 03 CariakB04 02 CariakB04 01 100 CrivIndia212 02 76 Criv GENBANK 100 CariakI05 01 CariakYR08 01 CariakYR07 01 CariakVi05 03 CariakVi05 02 63 CariakVi05 01 CariakI07 01 CariakI06 04 60 CariakI06 03 C. ariakensis (northern) CariakI04 05 CariakYR03 01 CariakYR02 02 CariakVi07 04 100 CariakVi07 03 CariakVi07 01 56 CariakVi04 02 CariakVi04 01 Parsimony Bootstrap Analysis of ITS-1 Sequences CariakI04 04 66 CariakVi02 02 CariakVi02 01 CariakVi02 CariakI07 03 CariakI07 02 CariakI06 02R CariakI05 03 CariakI05 02 CariakI02 02 CariakB03 03 CariakB03 02 C. rivularis (India) CariakB03 01 CariakB02 02 71 Criv206 01 55 96 C. gryphoides Criv192 02 94 100 CgryphIndia101 06 CgryphIndia101 05 CgryphIndia1033temp C. belcheri CbelchSR03 05 CbelchSR03 03 76 CbelchSR03 02 77 CbelchSr02 07 C. ariakensis (Malaysia) 66 CbelchRN01 05 CariakMal02 02 CariakMal01 02 100 CariakMal01 01 C. belcheri Be1 5 Be2 6 Be2 5 Be1 4 93 CmadIndia108 6 CmadIndia01 02 CmadIndia01 04 60 C. madrasensis/ C. iredalei (Thailand) 72 CiradelliPN02 09 CiradelliPN02 07 CiradelliCb03 03 CiradelliCb03 02 66 CiradelliCb03 01 100 CiradelliCb02 04 CiradelliCb02 03 CiradelliCb02 02 CiradeliPN1 4 CiradeliPN1 3 CiradeliPN1 1 100 CmadIndia02 02 CmadIndia02 01 100 C. iredalei (China) CiradeliChina8 26 100 CiradeliChina3 5 CiradelChina3 2 100 CiradelChina2 3 CiradelChina2 2

  10. Results 59 C. virginica Cvir15 04 Cvir23 04 Cvir21 04 Cvir22 05 Cvir22 06 57 S. cucullata ScucIndia82 05 61 ITS-1 ScucIndia82 03 ScucIndia52 01 100 62 ScucIndia51 03 ScucIndia51 01 S. commercialis BL163 3 100 Scom02 02 Scom01 01 78 BL166 2 52 BL165 4 L. crystigalli LcrystIndia215 02 100 LcrystIndia215 01 78 LcrystIndia214 02 LcrystIndia214 01 LcrystIndia213 08 CsikA25 04 CsikA15 04 C. sikamea 65 CsikA10 01 CsikA02 02 100 CsikA02 01 CsikA08 05 CsikA08 04 Csik02 04 Csik02 03 Csik01 04 97 CplicChina03 10 69 CplicChina01 05 94 CariakWM4 3 C. gigas/ C. plicatula 61 89 CariakWM4 1 Cgig01 02 Cgig01 01 CplicChina01 03 100 Cgig GENBANK CariakWM01 04 95 CariakWM01 02 64 Cgig04 03 Cgig04 02 CariakWM03 04 CsikA10 05 CsikA10 02 CariakD19 04 CariakD19 03 CariakD19 02 CariakD17 04 CariakD17 03 50 CariakD17 01 CariakD10 02 C. ariakensis (southern) CariakD07 02 CariakD07 01 CariakC09 02 CariakC09 01 60 CariakC04 02 CariakC04 01 CariakC03 03 CariakC03 02 64 CariakC03 01 CariakC02 02 CariakC02 01 CariakC01 05 63 CariakB12 02 CariakB12 01 CariakB02 03 CariakB11 04 CariakB11 03 CariakB04 02 CariakB04 01 100 CrivIndia212 02 76 Criv GENBANK 100 CariakI05 01 CariakYR08 01 CariakYR07 01 CariakVi05 03 CariakVi05 02 63 CariakVi05 01 CariakI07 01 CariakI06 04 60 CariakI06 03 C. ariakensis (northern) CariakI04 05 CariakYR03 01 CariakYR02 02 CariakVi07 04 100 CariakVi07 03 CariakVi07 01 56 CariakVi04 02 CariakVi04 01 Parsimony Bootstrap Analysis of ITS-1 Sequences CariakI04 04 66 CariakVi02 02 CariakVi02 01 CariakVi02 CariakI07 03 CariakI07 02 CariakI06 02R CariakI05 03 CariakI05 02 CariakI02 02 CariakB03 03 CariakB03 02 C. rivularis (India) CariakB03 01 CariakB02 02 71 Criv206 01 55 96 C. gryphoides Criv192 02 94 100 CgryphIndia101 06 CgryphIndia101 05 CgryphIndia1033temp C. belcheri CbelchSR03 05 CbelchSR03 03 76 CbelchSR03 02 77 CbelchSr02 07 C. ariakensis (Malaysia) 66 CbelchRN01 05 CariakMal02 02 CariakMal01 02 100 CariakMal01 01 C. belcheri Be1 5 Be2 6 Be2 5 Be1 4 93 CmadIndia108 6 CmadIndia01 02 CmadIndia01 04 60 C. madrasensis/ C. iredalei (Thailand) 72 CiradelliPN02 09 CiradelliPN02 07 CiradelliCb03 03 CiradelliCb03 02 66 CiradelliCb03 01 100 CiradelliCb02 04 CiradelliCb02 03 CiradelliCb02 02 CiradeliPN1 4 CiradeliPN1 3 CiradeliPN1 1 100 CmadIndia02 02 CmadIndia02 01 100 C. iredalei (China) CiradeliChina8 26 100 CiradeliChina3 5 CiradelChina3 2 100 CiradelChina2 3 CiradelChina2 2

  11. Cvirg46 05 Cvirg46 04 Cvirg05 02 83 OchilensisCO1GENBANK 100 66 Ochilensis3CO1GENBANK Ochilensis2CO1GENBANK 75 OangasiCO1GENBANK 100 83 Scuc60 03 Scuc60 02 Scuc60 01 OaupouriaCO1GENBANK Scucu55 02 50 Scom02 03 Scom02 02 100 Scom02 01 92 Scom01 04 93 Scom01 03 Scom01 02 Scom01 01 99 100 CrivIndia195 06 CplicBeihai02 13 CplicBeihai03 13 51 100 CiradeliChina05 11 CiradeliChina05 09 51 100 Lcryst214 04 Lcryst214 03 Lcryst17 01 CsikA12 01 CsikA10 4 CsikA08 01R CsikA02 02 99 Csik03 02 68 Csik03 01 100 Csik02 03 Csik01 01 Csik02 04 Csik01 02 93 Csik GENBANK CplicChina02 04 78 CplicChina01 02 CariakWM04 01 100 Cang GENBANK 88 100 CgigVi05 02 CgigVi01 02 100 CgigJg04 04 CgigJg04 03 Cgigas GENBANK 2 Cgig GENBANK CrivIndia196 10 CrivIndia196 07 CrivIndia195 11 100 CrivIndia195 03 CrivIndia195 02 CrivIndia194 02 Cgryph103 02 Cgryph101 03 70 Cgryph01 02 Cmad08 01 100 Cmad07 01 Cmad06 01 100 Cmad01 03 Cmad01 02CO1 CiradeliCb03 01 100 CiradeliCb01 03 CiradeliCb01 02 Ciradeli GENBANK CrivIndia195 01 CbelchSR03 01 CbelchSR02 03 CbelchSR02 02 59 100 CbelchRN02 02 CbelchRN02 01 CbelchRN01 01 Cbelch GENBANK CariakMal05 02 CariakMal04 03 CariakMal04 02 Cnippona GENBANK CariakYR06 03 CariakYR06 02 CariakYR06 01 64 CariakI04 04 66 CariakD08 04 CariakD20 02 CariakD20 01 CariakD19 03 CariakD07 04 100 58 CariakC09 04 CariakC09 01 CariakC02 02r CariakC02 01 CariakD18 02 CariakD18 01 CariakD17 02 CariakC04 03 CariakC04 02f CariakB12 01 CariakB04 01 CgigJg2 3 CariakYR11 04 CariakYr09 04 CariakYr09 03 CariakVi07 01 CariakVi05 01 CariakVi04 01 CariakVi02 03 CariakVi02 02 66 CariakVi02 01 CariakVi01 02 CariakI06 03 CariakI05 03 CariakI05 01 CariakI04 03f CariakI04 02f CariakI04 01 100 CariakI02 04 CariakI02 01 Cariak GENBANK 2 Cariak GENBANK CariakB03 02 CariakB02 02r CariakB02 01 Cariak GENBANK 5 Cariak GENBANK 4 Cariak GENBANK 3 Cvirg05 01 Cvirg GENBANK C. virginica Results O. chilensis (outgroup) COI S. cucullata S. commercialis C. iredalei (China) L. crystigalli C. sikamea C. plicatula C. gigas C. rivularis (India) C. gryphoides C. madrasensis C. iredalei (Thailand) C. belcheri C. ariakensis (Malaysia) Parsimony Bootstrap Analysis of COI Sequences C. ariakensis (southern China) C. ariakensis (northern China)

  12. Cvirg46 05 Cvirg46 04 Cvirg05 02 83 OchilensisCO1GENBANK 100 66 Ochilensis3CO1GENBANK Ochilensis2CO1GENBANK 75 OangasiCO1GENBANK 100 83 Scuc60 03 Scuc60 02 Scuc60 01 OaupouriaCO1GENBANK Scucu55 02 50 Scom02 03 Scom02 02 100 Scom02 01 92 Scom01 04 93 Scom01 03 Scom01 02 Scom01 01 99 100 CrivIndia195 06 CplicBeihai02 13 CplicBeihai03 13 51 100 CiradeliChina05 11 CiradeliChina05 09 51 100 Lcryst214 04 Lcryst214 03 Lcryst17 01 CsikA12 01 CsikA10 4 CsikA08 01R CsikA02 02 99 Csik03 02 68 Csik03 01 100 Csik02 03 Csik01 01 Csik02 04 Csik01 02 93 Csik GENBANK CplicChina02 04 78 CplicChina01 02 CariakWM04 01 100 Cang GENBANK 88 100 CgigVi05 02 CgigVi01 02 100 CgigJg04 04 CgigJg04 03 Cgigas GENBANK 2 Cgig GENBANK CrivIndia196 10 CrivIndia196 07 CrivIndia195 11 100 CrivIndia195 03 CrivIndia195 02 CrivIndia194 02 Cgryph103 02 Cgryph101 03 70 Cgryph01 02 Cmad08 01 100 Cmad07 01 Cmad06 01 100 Cmad01 03 Cmad01 02CO1 CiradeliCb03 01 100 CiradeliCb01 03 CiradeliCb01 02 Ciradeli GENBANK CrivIndia195 01 CbelchSR03 01 CbelchSR02 03 CbelchSR02 02 59 100 CbelchRN02 02 CbelchRN02 01 CbelchRN01 01 Cbelch GENBANK CariakMal05 02 CariakMal04 03 CariakMal04 02 Cnippona GENBANK CariakYR06 03 CariakYR06 02 CariakYR06 01 64 CariakI04 04 66 CariakD08 04 CariakD20 02 CariakD20 01 CariakD19 03 CariakD07 04 100 58 CariakC09 04 CariakC09 01 CariakC02 02r CariakC02 01 CariakD18 02 CariakD18 01 CariakD17 02 CariakC04 03 CariakC04 02f CariakB12 01 CariakB04 01 CgigJg2 3 CariakYR11 04 CariakYr09 04 CariakYr09 03 CariakVi07 01 CariakVi05 01 CariakVi04 01 CariakVi02 03 CariakVi02 02 66 CariakVi02 01 CariakVi01 02 CariakI06 03 CariakI05 03 CariakI05 01 CariakI04 03f CariakI04 02f CariakI04 01 100 CariakI02 04 CariakI02 01 Cariak GENBANK 2 Cariak GENBANK CariakB03 02 CariakB02 02r CariakB02 01 Cariak GENBANK 5 Cariak GENBANK 4 Cariak GENBANK 3 Cvirg05 01 Cvirg GENBANK C. virginica Results O. chilensis (outgroup) COI S. cucullata S. commercialis C. iredalei (China) L. crystigalli C. sikamea C. plicatula C. gigas C. rivularis (India) C. gryphoides C. madrasensis C. iredalei (Thailand) C. belcheri C. ariakensis (Malaysia) Parsimony Bootstrap Analysis of COI Sequences C. ariakensis (southern China) C. ariakensis (northern China)

  13. Cvirg46 05 Cvirg46 04 Cvirg05 02 83 OchilensisCO1GENBANK 100 66 Ochilensis3CO1GENBANK Ochilensis2CO1GENBANK 75 OangasiCO1GENBANK 100 83 Scuc60 03 Scuc60 02 Scuc60 01 OaupouriaCO1GENBANK Scucu55 02 50 Scom02 03 Scom02 02 100 Scom02 01 92 Scom01 04 93 Scom01 03 Scom01 02 Scom01 01 99 100 CrivIndia195 06 CplicBeihai02 13 CplicBeihai03 13 51 100 CiradeliChina05 11 CiradeliChina05 09 51 100 Lcryst214 04 Lcryst214 03 Lcryst17 01 CsikA12 01 CsikA10 4 CsikA08 01R CsikA02 02 99 Csik03 02 68 Csik03 01 100 Csik02 03 Csik01 01 Csik02 04 Csik01 02 93 Csik GENBANK CplicChina02 04 78 CplicChina01 02 CariakWM04 01 100 Cang GENBANK 88 100 CgigVi05 02 CgigVi01 02 100 CgigJg04 04 CgigJg04 03 Cgigas GENBANK 2 Cgig GENBANK CrivIndia196 10 CrivIndia196 07 CrivIndia195 11 100 CrivIndia195 03 CrivIndia195 02 CrivIndia194 02 Cgryph103 02 Cgryph101 03 70 Cgryph01 02 Cmad08 01 100 Cmad07 01 Cmad06 01 100 Cmad01 03 Cmad01 02CO1 CiradeliCb03 01 100 CiradeliCb01 03 CiradeliCb01 02 Ciradeli GENBANK CrivIndia195 01 CbelchSR03 01 CbelchSR02 03 CbelchSR02 02 59 100 CbelchRN02 02 CbelchRN02 01 CbelchRN01 01 Cbelch GENBANK CariakMal05 02 CariakMal04 03 CariakMal04 02 Cnippona GENBANK CariakYR06 03 CariakYR06 02 CariakYR06 01 64 CariakI04 04 66 CariakD08 04 CariakD20 02 CariakD20 01 CariakD19 03 CariakD07 04 100 58 CariakC09 04 CariakC09 01 CariakC02 02r CariakC02 01 CariakD18 02 CariakD18 01 CariakD17 02 CariakC04 03 CariakC04 02f CariakB12 01 CariakB04 01 CgigJg2 3 CariakYR11 04 CariakYr09 04 CariakYr09 03 CariakVi07 01 CariakVi05 01 CariakVi04 01 CariakVi02 03 CariakVi02 02 66 CariakVi02 01 CariakVi01 02 CariakI06 03 CariakI05 03 CariakI05 01 CariakI04 03f CariakI04 02f CariakI04 01 100 CariakI02 04 CariakI02 01 Cariak GENBANK 2 Cariak GENBANK CariakB03 02 CariakB02 02r CariakB02 01 Cariak GENBANK 5 Cariak GENBANK 4 Cariak GENBANK 3 Cvirg05 01 Cvirg GENBANK C. virginica Results O. chilensis (outgroup) COI S. cucullata S. commercialis C. iredalei (China) L. crystigalli C. sikamea C. plicatula C. gigas C. rivularis (India) C. gryphoides C. madrasensis C. iredalei (Thailand) C. belcheri C. ariakensis (Malaysia) Parsimony Bootstrap Analysis of COI Sequences C. ariakensis (southern China) C. ariakensis (northern China)

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

  15. Results Virtual Digests of ITS-1 Intraspecific Aligned Sequences – Hae III Restriction Sites and Fragment Sizes- Including Flanking Primer Sites ITS-1

  16. = 2nd morphotype = 3nd morphotype = 4th morphotype Virtual Digests of ITS-1 Intraspecific Aligned Sequences* – Hae III Results ITS-1 * Includes flanking primers in size estimates = 1nd morphotype 364 368 C. iredalei CH 476 bp 452 S. commercialis 525 bp 53 121 134 175 379 466 S. cucullata 491 bp 112 197 259 439 C. virginica 518 bp 339 474 L. cristagalli 572 bp 177 242 374 493 C. madrasensis 592 bp 177 190 241 371 502 C. iredalei TH 603 bp 183 264 369 475 514 C. gryphoides 611 bp 171 333 463 551 C. belcheri 575 bp 151 202 437 C. gigas 541 bp 151 202 437 C. plicatula 541 bp 165 209 336 422 445 C. sikamea 548 bp 160 172 211 269 332 453 C. ariakensis N. 556 bp 169 218 270 339 459 C. ariakensis S. 565 bp 0 100 200 300 400 500 600

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

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

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

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

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

  22. Next Steps • 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)

  23. Applications • 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

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