Abstract
The aim of this study was to develop simple sequence repeat (SSR) markers for genetic studies on G. chacoensis, as well as to evaluate their transferability to other bamboo species. Genomic DNA was isolated from G. chacoensis and its partial sequencing was used to find SSR loci. The obtained sequencing data were de novo assembled using the software CLC Genomics Workbench® 8.0v. The SSR loci primers were identified and designed with the software SSRLocator. The selected markers were validated using 56 plants sampled in seven populations from southern Brazil. The markers with potential polymorphism were selected and fluorescently labeled for characterization by capillary electrophoresis. In total, 92 SSR loci were found in G. chacoensis contigs. Suitable primers were designed for 70 SSR loci, and the remaining 22 SSR loci did not have sequences for primer development. Out of 35 selected SSR markers, after PCR optimization, 10 with high polymorphism potential were characterized. These loci can be used in genetic analyses of G. chacoensis and all of them were successfully transferred to other bamboo species. Non-polymorphic loci require further tests with additional plants, from different populations, to identify possibilities of their use.
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Acknowledgements
The authors thank Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) (Grant no. Finance Code 001) for the scholarship awarded to MDR and LNV, and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for the financial support (Proc. 457726/2013-0) and scholarships awarded to MPG, RON, TCT, GHFK, and RP. The authors would also like to thank to Núcleo de Fixação Biológica de Nitrogênio/UFPR for sequencing. We are also grateful to the Parque Nacional do Iguaçu (ICMBio/Brazil), and to the farms Sarakura and Vagalume, for supporting and allowing the collection of plant material.
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MDR, MPG, RP and RON conceived the research. MDR and RON designed the experiments. MDR, TCT, GHFK and RFS conducted the lab and statistical analyses. MDR and RON wrote the manuscript. RP, RFS, GHFK, TCT, and LNV revised the draft of the manuscript. MPG coordinated and supported the research and revised the manuscript. All authors read and approved the final manuscript version.
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Figure S1.
PCR products amplified on different annealing temperatures (52-62°C) from simple sequences repeat markers (Gcha01–Gcha 5) of Guadua chacoensis. Agarose gels were stained with GelRed. (PDF 144 kb)
Figure S2.
Genotyping image of multiplex “A” amplicons in ABI 3500 XL platform. a) Amplicons of loci Gcha04 (black) and Gcha18 (red) of Guadua chacoensis, compared with GeneScan 600 LIZ® dye size standard (orange); b) Locus Gcha04 in multiplex amplification reaction; c) Locus Gcha18 in the multiplex “A” amplification reaction. (PDF 253 kb)
Figure S3.
Genotyping image of multiplex “B” amplicons in ABI 3500 XL platform. a) Amplicons of loci Gcha08 (blue), Gcha10 (black), Gcha21(green) and Gcha33 (red) of Guadua chacoensis, compared with GeneScan 600 LIZ® dye size standard (orange); b) Locus Gcha08 in multiplex “B” amplification reaction; c) Locus Gcha10 in multiplex “B” amplification reaction; d) Locus Gcha21 in multiplex “B” amplification reaction; e) Locus Gcha33 in multiplex “B” amplification reaction. (PDF 368 kb)
Figure S4.
Genotyping image of multiplex “C” amplicons in ABI 3500 XL platform. a) Amplicons of the loci Gcha02 (blue), Gcha05 (black) and Gcha06 (green) loci of Guadua chacoensis, compared with GeneScan 600 LIZ® dye size standard (orange); b) Locus Gcha02 in multiplex “C” amplification reaction; c) Locus Gcha05 in multiplex “C” amplification reaction; ) Locus Gcha06 in multiplex amplification reaction. (PDF 349 kb)
Figure S5.
Genotyping image of multiplex “D” amplicons in ABI 3500xL platform. a) Amplicons of loci Gcha01 (blue) and Gcha07 (black) of Guadua chacoensis, compared with GeneScan 600 LIZ® dye size standard (orange); b) Locus Gcha01 in multiplex “D” amplification reaction; c) Locus Gcha07 in multiplex “D” amplification reaction. (PDF 308 kb)
Figure S6.
Multiplex “C” transferability test from Guadua chacoensis to G. paniculata amplicons in ABI 3500 XL platform. a) Amplicons of loci Gcha02 (blue), Gcha05 (black) and Gcha6 (green), compared with GeneScan 600 LIZ® dye size standard (orange); b) Locus Gcha02; c) Locus Gcha05; d) Locus Gcha06. (PDF 298 kb)
Figure S7.
Multiplex “C” transferability test from Guadua chacoensis to a) Bambusa oldhamii, b) Chusquea tenella, c) Dendrocalamus latifolius, d) Fargesia yunnanensis, e) G. angustifolia, and f) G. paraguayana. in ABI 3500 XL platform. Amplicons of the loci Gcha02 (blue), Gcha05 (black) and Gcha06 (green) loci, compared with GeneScan 600 LIZ® dye size standard (orange) (PDF 273 kb)
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Rossarolla, M.D., Tomazetti, T.C., Vieira, L.N. et al. Identification and characterization of SSR markers of Guadua chacoensis (Rojas) Londoño & P.M. Peterson and transferability to other bamboo species. 3 Biotech 10, 273 (2020). https://doi.org/10.1007/s13205-020-02268-4
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DOI: https://doi.org/10.1007/s13205-020-02268-4