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, 12 (2), e0170799
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Genome Size, Cytogenetic Data and Transferability of EST-SSRs Markers in Wild and Cultivated Species of the Genus Theobroma L. (Byttnerioideae, Malvaceae)

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Genome Size, Cytogenetic Data and Transferability of EST-SSRs Markers in Wild and Cultivated Species of the Genus Theobroma L. (Byttnerioideae, Malvaceae)

Rangeline Azevedo da Silva et al. PLoS One.

Abstract

The genus Theobroma comprises several trees species native to the Amazon. Theobroma cacao L. plays a key economic role mainly in the chocolate industry. Both cultivated and wild forms are described within the genus. Variations in genome size and chromosome number have been used for prediction purposes including the frequency of interspecific hybridization or inference about evolutionary relationships. In this study, the nuclear DNA content, karyotype and genetic diversity using functional microsatellites (EST-SSR) of seven Theobroma species were characterized. The nuclear content of DNA for all analyzed Theobroma species was 1C = ~ 0.46 pg. These species presented 2n = 20 with small chromosomes and only one pair of terminal heterochromatic bands positively stained (CMA+/DAPI- bands). The small size of Theobroma ssp. genomes was equivalent to other Byttnerioideae species, suggesting that the basal lineage of Malvaceae have smaller genomes and that there was an expansion of 2C values in the more specialized family clades. A set of 20 EST-SSR primers were characterized for related species of Theobroma, in which 12 loci were polymorphic. The polymorphism information content (PIC) ranged from 0.23 to 0.65, indicating a high level of information per locus. Combined results of flow cytometry, cytogenetic data and EST-SSRs markers will contribute to better describe the species and infer about the evolutionary relationships among Theobroma species. In addition, the importance of a core collection for conservation purposes is highlighted.

Conflict of interest statement

The organization EMBRAPA did not play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Thus, this does not alter our adherence to PLOS ONE policies on sharing data and materials.

Figures

Fig 1
Fig 1. Cytogenetic analysis of the genus Theobroma.
(a-a’) metaphase and caryogram of the T. cacao ssp leicocarpum stained with CMA (yellow) and DAPI (blue). (b-b’) Metaphase and caryogram of T. cacao cv. Scavina 6. (c) metaphase of T. bicolor stained with CMA+/DAPI. (d) diakinesis of T. bicolor showing the 10 pair sofbivalent. (e) prometaphase of T. grandiflorum, bars = 5 μm. Arrows in a, b, c points to the CMA+ bands.
Fig 2
Fig 2. Histogram of relative nuclear DNA content (genome size).
(A) Nuclear DNA content in T. cacao cv. ICS100. (B) S. lycopersicum cells, included as an internal standard. Nuclei isolated from cocoa leaves were stained with propidium iodide and analyzed by flow cytometry.

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Grant support

This work was supported by Cocoa Reserarch Center (CEPEC/CEPLAC); Brazilian Agricultural Research Corporation (EMBRAPA); Santa Cruz State University, (UESC), Coordination for the Improvement of Higher Education Personnel (CAPES); FINEP/CNPq. The funder provided support in the form of either research material/ or salaries for authors KPG, UVL, RMA, and LHM, and CAPES provided scholarship to RAS, but they did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.
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