PCR-DGGE Analysis: Unravelling Complex Mixtures of Badnavirus Sequences Present in Yam Germplasm
Abstract
:1. Introduction
2. Materials and Methods
2.1. Plant Material
2.2. Total Nucleic Acid Extraction from Yam Leaves and PCR Amplification of Badnavirus Sequences
2.3. Denaturing Gradient Gel Electrophoresis (DGGE)
2.4. Sequence Analysis and Phylogeny
3. Results
3.1. DGGE Resolves a Complex Mixture of Badnavirus Sequences Present in Dioscorea Species
3.2. Phylogenetic Diversity of Dioscorea Badnavirus Sequences
3.3. Monophyletic Group Assignment of Sequences Identified in This Study
3.4. Conservation of Amino Acid Motifs in Partial RT-RNaseH Badnavirus Sequences
4. Discussion
4.1. Potential of DGGE in Badnavirus Diversity Studies and Identification of Potential Integrated Sequences
4.2. DGGE-Captured Badnavirus Diversity
4.3. Endogenous Badnavirus Partial RT-RNaseH Sequences
5. Conclusions
Supplementary Materials
Acknowledgments
Author Contributions
Conflicts of Interest
Abbreviations
BF | Badna-forward primer |
BLAST | basic local alignment search tool |
BR | Badna-reverse primer |
BSOLV | Banana streak OL virus |
BSV | banana streak virus |
ComYMV | Commelina yellow mottle virus |
CSSV | Cacao swollen shoot virus |
CTAB | cetyltrimethylammonium bromide |
DBALV | Dioscorea bacilliform alata virus |
DBRTV | Dioscorea bacilliform rotundata (RT) virus |
DBV | Dioscorea bacilliform virus |
DGGE | denaturing gradient gel electrophoresis |
DOAJ | Directory of open access journals |
eDBV | endogenous Dioscorea bacilliform virus |
GC | GC clamp |
ICTV | International Committee on Taxonomy of Viruses |
IITA | International Institute of Tropical Agriculture |
ISEM | immunosorbent electron microscopy |
LD | linear dichroism |
MAFFT | Multiple Alignment using Fast Fourier Transform |
NCBI | National Centre for Biotechnology Information |
NG | Nigeria |
NGS | next-generation sequencing |
NRI | Natural Resources Institute |
o.p. | open pollinated |
ORF | open reading frame |
PCR | polymerase chain reaction |
RCA | rolling circle amplification |
RFLP | restriction fragment length polymorphism |
RNaseH | ribonuclease H |
RT | reverse transcriptase |
RTBV | Rice tungro bacilliform virus |
SCBMOV | Sugarcane bacilliform MO virus |
TaBV | Taro bacilliform virus |
TAE | Tris-acetate-EDTA |
TBE | Tris-Boric acid-EDTA |
TDa | Dioscorea alata accession |
TDb | Dioscorea bulbifera accession |
TDc | Dioscorea cayenensis accession |
TDe | Dioscorea esculenta accession |
TDr | Dioscorea rotundata accession |
Appendix A
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Plant Accession a | DGGE Sequence b | Accession | Primers c | Size (bp) | NCBI Nearest Match | Identity (%) | Species Group d |
---|---|---|---|---|---|---|---|
TDr 89/02475 | NGb1aDr | KY555456 | BF-GC + BR | 528 | eDBV9_S1h6Dr (KF829977) | 99 | K9 |
TDr 89/02475 | NGb1bDr | KY555457 | BF-GC + BR | 528 | 432B39Ds (AM503361) | 99 | K9 |
TDr 89/02475 | NGb2aDr | KY555458 | BF-GC + BR | 528 | 432B39Ds (AM503361) | 100 | K9 |
TDr 89/02475 | NGb2bDr | KY555459 | BF-GC + BR | 528 | 432B39Ds (AM503361) | 99 | K9 |
TDr 89/02475 | NGb3aDr | KY555460 | BF-GC + BR | 527 | 432B39Ds (AM503361) | 99 | K9 |
TDr 89/02475 | NGb3bDr | KY555461 | BF-GC + BR | 528 | 432B39Ds (AM503361) | 99 | K9 |
TDr 89/02475 | NGb4Dr | KY555462 | BF-GC + BR | 528 | GyJT2Dt (AM503389) | 93 | K8 |
TDr 89/02475 | NGb5Dr | KY555463 | BF-GC + BR | 528 | BfA103Dc (AM503393) | 99 | K8 |
TDr 89/02475 | NGb6aDr | KY555464 | BF-GC + BR | 528 | GyJT2Dt (AM503389) | 92 | K8 |
TDr 89/02475 | NGb6bDr | KY555465 | BF-GC + BR | 528 | GyJT2Dt (AM503389) | 93 | K8 |
TDr 89/02475 | NGb7aDr | KY555466 | BF-GC + BR | 528 | GyJT2Dt (AM503389) | 92 | K8 |
TDr 89/02475 | NGb7bDr | KY555467 | BF-GC + BR | 528 | GyJT2Dt (AM503389) | 92 | K8 |
TDr 89/02475 | NGb8aDr | KY555468 | BF-GC + BR | 528 | DBRTV1-[3RT] (KX008598) | 99 | T13 |
TDr 89/02475 | NGb8bDr | KY555469 | BF-GC + BR | 528 | GyJT2Dt (AM503389) | 92 | K8 |
Pona | NGl9aDr | KY555470 | BF-GC + BR | 528 | eDBV12_S1a4Dr (KF829956) | 99 | U12 |
Pona | NGl9bDr | KY555471 | BF-GC + BR | 528 | eDBV12_S1a4Dr (KF829956) | 99 | U12 |
Pona | NGl10aDr | KY555472 | BF + BR-GC | 528 | BfA103Dc (AM503393) | 99 | K8 |
Pona | NGl10bDr | KY555473 | BF + BR-GC | 528 | BfA103Dc (AM503393) | 99 | K8 |
Pona | NGl11aDr | KY555474 | BF + BR-GC | 528 | BfA103Dc (AM503393) | 99 | K8 |
Pona | NGl11bDr | KY555475 | BF + BR-GC | 528 | BfA103Dc (AM503393) | 99 | K8 |
Pona | NGl12aDr | KY555476 | BF + BR-GC | 528 | eDBV12_S1a4Dr (KF829956) | 99 | U12 |
Pona | NGl12bDr | KY555477 | BF + BR-GC | 528 | eDBV12_S1a4Dr (KF829956) | 100 | U12 |
Pona | NGl13aDr | KY555478 | BF + BR-GC | 528 | eDBV12_S1a4Dr (KF829956) | 100 | U12 |
Pona | NGl13bDr | KY555479 | BF + BR-GC | 528 | eDBV12_S1a4Dr (KF829956) | 99 | U12 |
Pona | NGl14aDr | KY555480 | BF + BR-GC | 528 | BfA103Dc (AM503393) | 99 | K8 |
Pona | NGl14bDr | KY555481 | BF + BR-GC | 528 | BfA103Dc (AM503393) | 100 | K8 |
Pona | NGl15aDr | KY555482 | BF + BR-GC | 528 | BfA103Dc (AM503393) | 99 | K8 |
Pona | NGl15bDr | KY555483 | BF + BR-GC | 528 | BfA103Dc (AM503393) | 99 | K8 |
TDr 89/02475 | NGb16aDr | KY555484 | BF + BR-GC | 528 | GyJT2Dt (AM503389) | 92 | K8 |
TDr 89/02475 | NGb16bDr | KY555485 | BF + BR-GC | 527 | 432B39Ds (AM503361) | 99 | K9 |
TDr 89/02475 | NGb17aDr | KY555486 | BF + BR-GC | 528 | GyJT2Dt (AM503389) | 92 | K8 |
TDr 89/02475 | NGb17bDr | KY555487 | BF + BR-GC | 528 | GyJT2Dt (AM503389) | 92 | K8 |
TDr 89/02475 | NGb18aDr | KY555488 | BF + BR-GC | 528 | BfA103Dc (AM503393) | 99 | K8 |
TDr 89/02475 | NGb18bDr | KY555489 | BF + BR-GC | 528 | GyJT2Dt (AM503389) | 93 | K8 |
TDr 89/02475 | NGb19aDr | KY555490 | BF + BR-GC | 528 | BfA103Dc (AM503393) | 99 | K8 |
TDr 89/02475 | NGb19bDr | KY555491 | BF + BR-GC | 528 | BfA103Dc (AM503393) | 99 | K8 |
TDr 89/02475 | NGb20aDr | KY555492 | BF + BR-GC | 528 | GyJT2Dt (AM503389) | 92 | K8 |
TDr 89/02475 | NGb20bDr | KY555493 | BF + BR-GC | 528 | GyJT2Dt (AM503389) | 93 | K8 |
TDr 95/18544 | NGb21aDr | KY555494 | BF + BR-GC | 528 | BfA103Dc (AM503393) | 99 | K8 |
TDr 95/18544 | NGb21bDr | KY555495 | BF + BR-GC | 528 | BfA103Dc (AM503393) | 99 | K8 |
TDr 95/18544 (o.p.) | NGb22aDr | KY555496 | BF-GC + BR | 528 | eDBV5_S1g6Dr (KF829974) | 99 | K5 |
TDr 95/18544 (o.p.) | NGb22bDr | KY555497 | BF-GC + BR | 528 | eDBV5_S1g6Dr (KF829974) | 99 | K5 |
TDr 95/18544 (o.p.) | NGb23aDr | KY555498 | BF-GC + BR | 528 | eDBV5_S1g6Dr (KF829974) | 99 | K5 |
TDr 95/18544 (o.p.) | NGb23bDr | KY555499 | BF-GC + BR | 528 | BfA103Dc (AM503393) | 97 | K8 |
TDr 95/18544 (o.p.) | NGb24aDr | KY555500 | BF-GC + BR | 528 | eDBV5_S1g6Dr (KF829974) | 99 | K5 |
TDr 95/18544 (o.p.) | NGb24bDr | KY555501 | BF-GC + BR | 528 | BfA103Dc (AM503393) | 99 | K8 |
TDr 97/00917 × TDr 99/02607 | NGb25aDr | KY555502 | BF-GC + BR | 528 | eDBV9_S1h2Dr (KF829975) | 99 | K9 |
TDr 97/00917 × TDr 99/02607 | NGb25bDr | KY555503 | BF-GC + BR | 528 | BN4Dr (AM944586) | 99 | K9 |
TDr 97/00917 × TDr 99/02607 | NGb26aDr | KY555504 | BF-GC + BR | 528 | BN4Dr (AM944586) | 99 | K9 |
TDr 97/00917 × TDr 99/02607 | NGb26bDr | KY555505 | BF-GC + BR | 528 | eDBV9_G1Dr (KF830002) | 99 | K9 |
TDr 97/00917 × TDr 99/02607 | NGb27aDr | KY555506 | BF-GC + BR | 528 | eDBV12_S2a7Dr (KF829978) | 99 | U12 |
TDr 97/00917 × TDr 99/02607 | NGb27bDr | KY555507 | BF-GC + BR | 528 | BN4Dr (AM944586) | 99 | K9 |
TDr 97/00917 × TDr 99/02607 | NGb28aDr | KY555508 | BF-GC + BR | 528 | BN4Dr (AM944586) | 99 | K9 |
TDr 97/00917 × TDr 99/02607 | NGb28bDr | KY555509 | BF-GC + BR | 528 | BN4Dr (AM944586) | 99 | K9 |
TDr 97/00917 × TDr 99/02607 | NGb29aDr | KY555510 | BF-GC + BR | 528 | BN4Dr (AM944586) | 99 | K9 |
TDr 97/00917 × TDr 99/02607 | NGb29bDr | KY555511 | BF-GC + BR | 528 | eDBV9_S2f8Dr (KF829993) | 99 | K9 |
TDr 97/00917 × TDr 99/02607 | NGb30aDr | KY555512 | BF-GC + BR | 527 | BN4Dr (AM944586) | 99 | K9 |
TDr 97/00917 × TDr 99/02607 | NGb30bDr | KY555513 | BF-GC + BR | 528 | BfA103Dc (AM503393) | 100 | K8 |
TDr 99/02793 × TDr 1892 | NGb31Dr | KY555514 | BF-GC + BR | 528 | GyJT2Dt (AM503389) | 93 | K8 |
TDr 99/02793 × TDr 1892 | NGb32Dr | KY555515 | BF-GC + BR | 528 | eDBV9_S1e3Dr (KF829969) | 100 | K9 |
TDr 04/219 × TDr 98/02677 | NGb33aDr | KY555516 | BF-GC + BR | 527 | GyJT2Dt (AM503389) | 92 | K8 |
TDr 04/219 × TDr 98/02677 | NGb33bDr | KY555517 | BF-GC + BR | 528 | GyJT2Dt (AM503389) | 93 | K8 |
TDr 97/00917 (o.p.) | NGb34aDr | KY555518 | BF-GC + BR | 528 | GyJT2Dt (AM503389) | 92 | K8 |
TDr 97/00917 (o.p.) | NGb34bDr | KY555519 | BF-GC + BR | 528 | GyJT2Dt (AM503389) | 93 | K8 |
TDr 96/00629 × TDr 99/02607 | NGb35aDr | KY555520 | BF-GC + BR | 528 | 432B39Ds (AM503361) | 99 | K9 |
TDr 96/00629 × TDr 99/02607 | NGb35bDr | KY555521 | BF-GC + BR | 528 | 432B39Ds (AM503361) | 100 | K9 |
TDr 97/00917 × TDr 99/02607 | NGb36aDr | KY555522 | BF-GC + BR | 528 | BN4Dr (AM944586) | 99 | K9 |
TDr 97/00917 × TDr 99/02607 | NGb36bDr | KY555523 | BF-GC + BR | 527 | BN4Dr (AM944586) | 99 | K9 |
TDr 97/00917 × TDr 99/02607 | NGb37aDr | KY555524 | BF-GC + BR | 528 | eDBV9_S2f8Dr (KF829993) | 99 | K9 |
TDr 97/00917 × TDr 99/02607 | NGb37bDr | KY555525 | BF-GC + BR | 528 | eDBV9_S1h2Dr (KF829975) | 99 | K9 |
TDr 04/219 × TDr 04/219 | NGb38aDr | KY555526 | BF-GC + BR | 528 | GyJT2Dt (AM503389) | 93 | K8 |
TDr 04/219 × TDr 04/219 | NGb38bDr | KY555527 | BF-GC + BR | 528 | GyJT2Dt (AM503389) | 92 | K8 |
TDr 04/219 × TDr 04/219 | NGb39aDr | KY555528 | BF-GC + BR | 528 | GyJT2Dt (AM503389) | 92 | K8 |
TDr 04/219 × TDr 04/219 | NGb39bDr | KY555529 | BF-GC + BR | 528 | GyJT2Dt (AM503389) | 93 | K8 |
TDr 04/219 × TDr 04/219 | NGb40Dr | KY555530 | BF-GC + BR | 528 | GyJT2Dt (AM503389) | 93 | K8 |
TDr 97/00205 ×TDr 1892 | NGb41aDr | KY555531 | BF-GC + BR | 528 | eDBV12_S1a4Dr (KF829956) | 99 | U12 |
TDr 97/00205 ×TDr 1892 | NGb41bDr | KY555532 | BF-GC + BR | 528 | eDBV9_S1h2Dr (KF829975) | 99 | K9 |
TDr 96/00629 × TDr 99/02607 | NGb42aDr | KY555533 | BF-GC + BR | 528 | eDBV12_S1a4Dr (KF829956) | 99 | U12 |
TDr 96/00629 × TDr 99/02607 | NGb42bDr | KY555534 | BF-GC + BR | 528 | 432B39Ds (AM503361) | 99 | K9 |
TDr 95/18544 | NGb43Dr | KY555535 | BF-GC + BR | 528 | BfA103Dc (AM503393) | 99 | K8 |
TDr 95/18544 | NGb44Dr | KY555536 | BF-GC + BR | 528 | BfA103Dc (AM503393) | 99 | K8 |
TDr 95/18544 | NGb45Dr | KY555537 | BF-GC + BR | 528 | BfA103Dc (AM503393) | 99 | K8 |
TDr 96/00629 | NGb46aDr | KY555538 | BF-GC + BR | 528 | eDBV12_S2a7Dr (KF829978) | 99 | U12 |
TDr 96/00629 | NGb46bDr | KY555539 | BF-GC + BR | 528 | BfA103Dc (AM503393) | 99 | K8 |
TDr 89/02677 | NGb47aDr | KY555540 | BF-GC + BR | 528 | GyJT2Dt (AM503389) | 92 | K8 |
TDr 89/02677 | NGb47bDr | KY555541 | BF-GC + BR | 528 | GyJT2Dt (AM503389) | 93 | K8 |
TDr 89/02677 | NGb48aDr | KY555542 | BF-GC + BR | 528 | GyJT2Dt (AM503389) | 92 | K8 |
TDr 89/02677 | NGb48bDr | KY555543 | BF-GC + BR | 528 | GyJT2Dt (AM503389) | 92 | K8 |
TDr 89/02677 | NGb49Dr | KY555544 | BF-GC + BR | 528 | GyJT2Dt (AM503389) | 92 | K8 |
TDr 96/00629 | NGb50Dr | KY555545 | BF-GC + BR | 528 | eDBV12_S2a7Dr (KF829978) | 99 | U12 |
TDr 89/02677 | NGb51Dr | KY555546 | BF-GC + BR | 528 | GyJT2Dt (AM503389) | 91 | K8 |
TDr 97/00917 (o.p.) | NGb52Dr | KY555547 | BF-GC + BR | 528 | eDBV5_S1g6Dr (KF829974) | 98 | K5 |
TDr 97/00917 (o.p.) | NGb53Dr | KY555548 | BF-GC + BR | 528 | NC1 (KJ854414) | 79 | K13 |
TDr 97/00917 × TDr 99/02607 | NGb54Dr | KY555549 | BF-GC + BR | 528 | eDBV9_S1h2Dr (KF829975) | 99 | K9 |
TDr 99/02793 (o.p.) | NGb55aDr | KY555550 | BF-GC + BR | 528 | BfA103Dc (AM503393) | 100 | K8 |
TDr 99/02793 (o.p.) | NGb55bDr | KY555551 | BF-GC + BR | 528 | BfA103Dc (AM503393) | 99 | K8 |
TDr 96/00621 (o.p.) | NGb56Dr | KY555552 | BF-GC + BR | 528 | eDBV12_S1a4Dr (KF829956) | 100 | U12 |
TDr 97/00917 × TDr 97/00777 | NGb57Dr | KY555553 | BF-GC + BR | 528 | GyJT2Dt (AM503389) | 93 | K8 |
TDr 96/00629 × TDr 99/02607 | NGb58Dr | KY555554 | BF-GC + BR | 528 | GyJT2Dt (AM503389) | 92 | K8 |
TDr 97/00917 × TDr 99/02607 | NGb59Dr | KY555555 | BF-GC + BR | 528 | eDBV12_S2h10Dr (KF829998) | 99 | U12 |
TDa 99/00240 × TDa 95/00310 | NGb60Da | KY555556 | BF-GC + BR | 528 | GyJT2Dt (AM503389) | 92 | K8 |
TDa 99/00240 × TDa 01/00012 | NGb61Da | KY555557 | BF-GC + BR | 528 | NG1Da (AM944571) | 93 | K8 |
TDa 01/00081 × TDa 02/00012 | NGb62Da | KY555558 | BF-GC + BR | 528 | GyJT2Dt (AM503389) | 93 | K8 |
TDa 1/00081×TDa 98/00150 | NGb63Da | KY555559 | BF-GC + BR | 528 | SB42Da (AM072696) | 99 | K1 |
TDa 00/00194 × TDa 98/00150 | NGb64Da | KY555560 | BF-GC + BR | 528 | eDBV9_S1b6Dr (KF829960) | 89 | K9 |
TDe 3049A | NGl65De | KY555561 | BF-GC + BR | 528 | WS31aDn (AM421696) | 73 | K12 |
TDd 4118B | NGl66Dd | KY555562 | BF-GC + BR | 528 | eDBV5_S1un5Dr (KF830000) | 93 | K5 |
TDd 4118B | NGl67Dd | KY555563 | BF-GC + BR | 528 | BN2Da (AM944584) | 98 | K8 |
TDr 1950B | NGl68Dr | KY555564 | BF-GC + BR | 528 | GN2Dr (AM944575) | 96 | K8 |
TDc 3808C | NGl69Dc | KY555565 | BF-GC + BR | 528 | eDBV9_S1b2Dr (KF829958) | 100 | K9 |
TDb 3045B | NGl70Db | KY555566 | BF-GC + BR | 528 | SB42Da (AM072696) | 99 | K1 |
TDd 3778B | NGl71Dd | KY555567 | BF-GC + BR | 528 | FJ65bDe (AM072660) | 99 | K1 |
TDa 1013C | NGl72Da | KY555568 | BF-GC + BR | 528 | SB42Da (AM072696) | 99 | K1 |
TDe 3028A | NGl73De | KY555569 | BF-GC + BR | 528 | FJ65bDe (AM072660) | 99 | K1 |
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Turaki, A.A.; Bömer, M.; Silva, G.; Kumar, P.L.; Seal, S.E. PCR-DGGE Analysis: Unravelling Complex Mixtures of Badnavirus Sequences Present in Yam Germplasm. Viruses 2017, 9, 181. https://doi.org/10.3390/v9070181
Turaki AA, Bömer M, Silva G, Kumar PL, Seal SE. PCR-DGGE Analysis: Unravelling Complex Mixtures of Badnavirus Sequences Present in Yam Germplasm. Viruses. 2017; 9(7):181. https://doi.org/10.3390/v9070181
Chicago/Turabian StyleTuraki, Aliyu A., Moritz Bömer, Gonçalo Silva, P. Lava Kumar, and Susan E. Seal. 2017. "PCR-DGGE Analysis: Unravelling Complex Mixtures of Badnavirus Sequences Present in Yam Germplasm" Viruses 9, no. 7: 181. https://doi.org/10.3390/v9070181
APA StyleTuraki, A. A., Bömer, M., Silva, G., Kumar, P. L., & Seal, S. E. (2017). PCR-DGGE Analysis: Unravelling Complex Mixtures of Badnavirus Sequences Present in Yam Germplasm. Viruses, 9(7), 181. https://doi.org/10.3390/v9070181