Jan Cordes, Julie Stubbs, and Kimberly Reece

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 HealthVirginia Institute of Marine Science

College of William and Mary

Republic of China

India

Japan

• 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 cucullataS. commercialisLopha cristigalliCrassostrea virginica

C. gigasC. plicatula C. sikameaC. ariakensis

C. iredaleiC. gryphoidesC. madrasensisC. 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 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

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. belcheri

C. gigas

C. iredalei (China)

C. plicatula


C. virginica


Protocols

Sampling Locations

C. madrasensis

C. gryphoides

Lopha cristagalli

S. cucullata


Republic of China

India

Japan




C. belcheri

C. gigas

C. iredalei (China)

C. plicatula


C. virginica


Protocols

Sampling Locations

C. madrasensis

C. gryphoides

Lopha cristagalli

S. cucullata


Results

ITS-1

Parsimony Bootstrap

Analysis of ITS-1 Sequences


Cvir15 04Cvir23 04Cvir21 04Cvir22 05Cvir22 06ScucIndia82 05ScucIndia82 03ScucIndia52 01ScucIndia51 03ScucIndia51 01BL163 3Scom02 02Scom01 01BL166 2BL165 4LcrystIndia215 02LcrystIndia215 01LcrystIndia214 02LcrystIndia214 01LcrystIndia213 08CsikA25 04CsikA15 04CsikA10 01CsikA02 02CsikA02 01CsikA08 05CsikA08 04Csik02 04Csik02 03Csik01 04CplicChina03 10CplicChina01 05CariakWM4 3CariakWM4 1Cgig01 02Cgig01 01CplicChina01 03Cgig GENBANKCariakWM01 04CariakWM01 02Cgig04 03Cgig04 02CariakWM03 04CsikA10 05CsikA10 02CariakD19 04CariakD19 03CariakD19 02CariakD17 04CariakD17 03CariakD17 01CariakD10 02CariakD07 02CariakD07 01CariakC09 02CariakC09 01CariakC04 02CariakC04 01CariakC03 03CariakC03 02CariakC03 01CariakC02 02CariakC02 01CariakC01 05CariakB12 02CariakB12 01CariakB02 03CariakB11 04CariakB11 03CariakB04 02CariakB04 01CrivIndia212 02Criv GENBANKCariakI05 01CariakYR08 01CariakYR07 01CariakVi05 03CariakVi05 02CariakVi05 01CariakI07 01CariakI06 04CariakI06 03CariakI04 05CariakYR03 01CariakYR02 02CariakVi07 04CariakVi07 03CariakVi07 01CariakVi04 02CariakVi04 01CariakI04 04CariakVi02 02CariakVi02 01CariakVi02CariakI07 03CariakI07 02CariakI06 02RCariakI05 03CariakI05 02CariakI02 02CariakB03 03CariakB03 02CariakB03 01CariakB02 02Criv206 01Criv192 02CgryphIndia101 06CgryphIndia101 05CgryphIndia103 3tempCbelchSR03 05CbelchSR03 03CbelchSR03 02CbelchSr02 07CbelchRN01 05CariakMal02 02CariakMal01 02CariakMal01 01Be1 5Be2 6Be2 5Be1 4CmadIndia108 6CmadIndia01 02CmadIndia01 04CiradelliPN02 09CiradelliPN02 07CiradelliCb03 03CiradelliCb03 02CiradelliCb03 01CiradelliCb02 04CiradelliCb02 03CiradelliCb02 02CiradeliPN1 4CiradeliPN1 3CiradeliPN1 1CmadIndia02 02CmadIndia02 01CiradeliChina8 26CiradeliChina3 5CiradelChina3 2CiradelChina2 3CiradelChina2 2

59

100

10061

5762

100 78

64

1005278

96

60

50

97

10065

100

6194

6989

64 95

100

60

63

100

76

63

5666

76

10055 71

94

100

7766

100

72

93

66

60

100

100 100100

S. cucullata

S. commercialis

C. sikamea


C. gryphoides

L. crystigalli


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




59

100

10061

5762

100 78

64

1005278

96

60

50

97

10065

100

6194

6989

64 95

100

60

63

100

76

63

5666

76

10055 71

94

100

7766

100

72

93

66

60

100

100 100100

S. cucullata

S. commercialis

C. sikamea


C. gryphoides

L. crystigalli


C. belcheri


C. virginica

C. iredalei (China)


C. belcheri


Results

ITS-1

Parsimony Bootstrap




59

100

10061

5762

100 78

64

1005278

96

60

50

97

10065

100

6194

6989

64 95

100

60

63

100

76

63

5666

76

10055 71

94

100

7766

100

72

93

66

60

100

100 100100

S. cucullata

S. commercialis

C. sikamea


C. gryphoides

L. crystigalli


C. belcheri


C. virginica

C. iredalei (China)


C. belcheri


Results

COI

S. commercialis

C. sikamea

C. gigas

C. madrasensis


C. belcheri

C. ariakensis (southern China)

C. ariakensis (northern China)

C. gryphoides

S. cucullata

O. chilensis (outgroup)

C. plicatula

L. crystigalli

Cvirg46 05Cvirg46 04Cvirg05 02OchilensisCO1GENBANKOchilensis3CO1GENBANKOchilensis2CO1GENBANKOangasiCO1GENBANKScuc60 03Scuc60 02Scuc60 01OaupouriaCO1GENBANKScucu55 02Scom02 03Scom02 02Scom02 01Scom01 04Scom01 03Scom01 02Scom01 01CrivIndia195 06CplicBeihai02 13CplicBeihai03 13CiradeliChina05 11CiradeliChina05 09Lcryst214 04Lcryst214 03Lcryst17 01CsikA12 01CsikA10 4CsikA08 01RCsikA02 02Csik03 02Csik03 01Csik02 03Csik01 01Csik02 04Csik01 02Csik GENBANKCplicChina02 04CplicChina01 02CariakWM04 01Cang GENBANKCgigVi05 02CgigVi01 02CgigJg04 04CgigJg04 03Cgigas GENBANK 2Cgig GENBANKCrivIndia196 10CrivIndia196 07CrivIndia195 11CrivIndia195 03CrivIndia195 02CrivIndia194 02Cgryph103 02Cgryph101 03Cgryph01 02Cmad08 01Cmad07 01Cmad06 01Cmad01 03Cmad01 02CO1CiradeliCb03 01CiradeliCb01 03CiradeliCb01 02Ciradeli GENBANKCrivIndia195 01CbelchSR03 01CbelchSR02 03CbelchSR02 02CbelchRN02 02CbelchRN02 01CbelchRN01 01Cbelch GENBANKCariakMal05 02CariakMal04 03CariakMal04 02Cnippona GENBANKCariakYR06 03CariakYR06 02CariakYR06 01CariakI04 04CariakD08 04CariakD20 02CariakD20 01CariakD19 03CariakD07 04CariakC09 04CariakC09 01CariakC02 02rCariakC02 01CariakD18 02CariakD18 01CariakD17 02CariakC04 03CariakC04 02fCariakB12 01CariakB04 01CgigJg2 3CariakYR11 04CariakYr09 04CariakYr09 03CariakVi07 01CariakVi05 01CariakVi04 01CariakVi02 03CariakVi02 02CariakVi02 01CariakVi01 02CariakI06 03CariakI05 03CariakI05 01CariakI04 03fCariakI04 02fCariakI04 01CariakI02 04CariakI02 01Cariak GENBANK 2Cariak GENBANKCariakB03 02CariakB02 02rCariakB02 01Cariak GENBANK 5Cariak GENBANK 4Cariak GENBANK 3Cvirg05 01Cvirg GENBANK

51

100

92

8375

66 100 83

100

93

50100

100 99100

59

100 51

93

10099

68

10078

10088

70

100

100100

100

100

66

100

64

58

100

66

C. virginica

C. iredalei (China)



Parsimony Bootstrap

Analysis of COI Sequences

Results

COI

S. commercialis

C. sikamea

C. gigas

C. madrasensis


C. belcheri



C. gryphoides

S. cucullata


C. plicatula

L. crystigalli


51

100

92

8375

66 100 83

100

93

50100

100 99100

59

100 51

93

10099

68

10078

10088

70

100

100100

100

100

66

100

64

58

100

66

C. virginica

C. iredalei (China)



Parsimony Bootstrap


Results

COI

S. commercialis

C. sikamea

C. gigas

C. madrasensis


C. belcheri



C. gryphoides

S. cucullata


C. plicatula

L. crystigalli


51

100

92

8375

66 100 83

100

93

50100

100 99100

59

100 51

93

10099

68

10078

10088

70

100

100100

100

100

66

100

64

58

100

66

C. virginica

C. iredalei (China)



Parsimony Bootstrap


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

Comparison of Virtual Restriction Enzyme Digests on ITS Region of 6 Species of CrassostreaI. NEB 4, 5, & 6 Base Cutters (Cont'd)

DpnII EaeI EagI EciI Fnu4HIC. virginica 7 38 46 554 500 500 \ 219 222 497 500C. belcheri 7 116 165 554 474 474 \ 90 182 201 497C. gigas 7 116 165 554 474 474 435 497C. sikamea 7 116 165 554 474 474 435 497C. ariakensis N. 7 116 554 474 474 \ 90 273 497C. ariakensis S. 7 116 165 554 474 474 \ 90 497

FokI HaeII HaeIII HhaI HindIIIC. virginica 289 206 122 212 282 502 150 205 494 496 \C. belcheri \ \ 159 344 476 205 427 454 494 496 \C. gigas \ 206 159 212 476 205 494 496 219C. sikamea \ 206 159 212 344 446 476 205 494 496 \C. ariakensis N. \ 206 159 212 275 344 476 205 494 496 \C. ariakensis S. \ 206 159 212 344 476 205 494 496 \

HinfI HinP1I HpaII HphI Hpy188IC. virginica \ 148 203 492 494 119 182 200 278 477 73 79 116 288C. belcheri \ 203 425 452 492 494 141 278 286 413 477 \ 116C. gigas 423 203 492 494 278 286 413 477 \ 116C. sikamea 423 203 492 494 278 286 413 477 \ 116 232C. ariakensis N. 423 203 492 494 114 278 286 413 477 \ \C. ariakensis S. 423 203 492 494 278 286 413 477 \ 116

Results

ITS-1

Species Size Cuts Positions Band Sizes

Crassostrea iredalei (China) A 476 0 -- 476

B 2 364, 368 4, 108, 364

Sacostrea commercialis A 525 1 452 73, 452

S. cucullata A 491 3 121, 134, 379 13, 112, 121, 245

B 4 53, 121, 134, 379 13, 53, 68, 112, 245

C 4 121, 134, 176, 379 13, 42, 112, 121, 203

D 5 53, 121, 134, 379, 466 25, 13, 53, 68, 87, 245

C. virgnica A 518 3 197, 259, 439 62, 79, 180, 196

B 4 112, 197, 259, 439 62, 79, 85, 112, 180

L. cristagalli A 572 1 474 98, 474

B 2 339, 474 98, 135, 339

C. madrasensis A 592 2 242, 493 99, 242, 251

B 4 177, 242, 374, 493 65, 99, 119, 132, 177

C. iredalei (Thailand) A 603 4 177, 241, 371, 502 64, 101, 130, 131, 177

B 5 177, 190, 241, 371, 502 13, 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

100 200 300 400 500 600

364 368

53 121 134 379

112 259 439197

339 474

242 374 493177

241 371 502177

514183 369

190

475

463171 333

202 437151

445165 336209 422

453160 332269211172

= 2nd morphotype = 3nd morphotype

466

= 4th morphotype

175

264

551

452476 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

459169 339218 270

* Includes flanking

primers in size estimates

202 437151

541 bp

= 1nd morphotype

Results

ITS-1

Results

ITS-1

Restriction Enzyme Digests of ITS-1 Region with Hae III

S. c

omm

erci

alis

S. c

ucu

llat

a

L. c

rist

agal

li

C. b

elch

eri

C. i

red

alei

CH

C. m

adra

sen

sis

C. g

ryp

hoi

des

C. g

igas

C. p

lica

tula

C. s

ikam

ea

C. a

riak

ensi

s N

.

C. i

red

alei

TH

C. a

riak

ensi

s S

.

Results

ITS-1


S. c

omm

erci

alis

S. c

ucu

llat

a

L. c

rist

agal

li

C. b

elch

eri

C. i

red

alei

CH

C. m

adra

sen

sis

C. g

ryp

hoi

des

C. g

igas

C. p

lica

tula

C. s

ikam

ea

C. a

riak

ensi

s N

.

C. i

red

alei

TH

C. a

riak

ensi

s S

.

S. c

omm

erci

alis

S. c

ucu

llat

a

L. c

rist

agal

li

C. b

elch

eri

C. i

red

alei

CH

C. m

adra

sen

sis

C. g

ryp

hoi

des

C. g

igas

C. p

lica

tula

C. s

ikam

ea

C. a

riak

ensi

s N

.

C. i

red

alei

TH

C. a

riak

ensi

s S

.

Results

ITS-1


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 cucullataS. commercialisLopha cristigalliCrassostrea virginicaC. iredalei CHC. iredalei THC. gryphoides

C. madrasensisC. belcheriC. gigasC. plicatula C. sikameaC. 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

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 cucullataS. commercialisLopha cristigalliC. virginicaC. iredalei CHC. iredalei THC. gryphoides

C. madrasensisC. belcheriC. gigasC. plicatulaC. sikameaC. 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

Documents

Jan Cordes, Julie Stubbs, and Kimberly Reece