Analysis of Genome Size of Sixteen Coffea arabica Cultivars Using Flow Cytometry

in HortScience

Coffee is an important crop worldwide, grown on about 10 million hectares in tropical regions including Latin America, Africa, and Asia. The genus Coffea includes more than 100 species; most are diploid, except for C. arabica, which is allotetraploid and autogamous. The genetic diversity of commercial coffee is low, likely due to it being self-pollinating, in addition, the widespread propagation of few selected cultivars, such as Caturra, Bourbon, and Typica. One approach is the analysis of genome size in these cultivars as a proxy to study its genetic variability. In the present work, genome size of 16 cultivars was assessed through high-resolution flow cytometry (FCM). Nuclear DNA was analyzed using a modified procedure that uses propidium iodide (PI) and 4′,6′-diamino-2-phenylindole dihydrochloride hydrate (DAPI) staining. The C. arabica cultivars investigated possessed a nuclear DNA content ranging from 2.56 ± 0.016 pg for Typica, to 3.16 ± 0.033 pg for ICATU, which had the largest genome size. All cultivars measured using both fluorochromes had greater estimates with DAPI than PI. The proportion of the genome composed of guanosine and cytosine (GC%) among the cultivars evaluated in this study ranged from 37.03% to 39.22%. There are few studies of genome size by FCM of distinct important C. arabica cultivars, e.g., hybrids and artificial crosses. Thus, this work could be valuable for coffee breeding programs. The data presented here are intended to expand the genomic understanding of C. arabica and could link nuclear DNA content with evolutionary relationships such as diversification, hybridization and polyploidy.

Contributor Notes

The authors thank Victor Rosales for his technical assistance and members of the laboratory for helpful discussions. This work was supported by a PhD fellowship provided by CONACyT to JO-O and CONACyT grants (105985, 158801, and FC-2015-2, 01234 to B.X.C. and 156162 to R.R.M.).

Corresponding author. E-mail: bxoconos@cinvestav.mx.

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Article Figures

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    Representative cytograms of fluorescence intensity by flow cytometry of G0/G1 nuclei from some C. arabica cultivars and standards. Representative cytograms showing G0/G1 peaks with cvs ranging between 3.76% and 4.58%, obtained from propidium iodide–stained nuclear suspensions prepared from leaves of comparison standards; 1) soybean (channel 163.6) (2C = 2.30 pg) and 2) C. canephora (channel 92.85) (2C = 1.29 pg). Four representative C. arabica cultivars are shown. (A) Catuai 3) channel fluorescence 204.83 (2C value = 2.88 pg); (B) ICATU 4) channel fluorescence 224.35 (2C value = 3.16 pg) (C) Oro Azteca 5) channel fluorescence 224.04 (2C value = 3.15 pg); and (D) Typica 6) channel fluorescence 181.7 (2C value = 2.56 pg).

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    Cytograms of relative DNA content of nuclei isolated from 16 C. arabica cultivars associated to propidium iodide fluorescence. (A) ‘Typica’-related family. As controls, soybean and Coffea canephora var. Robusta also are shown. (B) Introgressed (artificial crosses and hybrids) cultivars. Controls similar to (A) were employed.

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    DNA 2C-values experimentally measured of tested cultivars with two dyes. Genome size of all C. arabica cultivars arranged by ascending order when nuclei were stained with PI and when nuclei were stained with DAPI. PI = propidium iodide; DAPI = 4′,6′-diamino-2-phenylindole dihydrochloride hydrate.

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    Genealogy of the coffee cultivars analyzed in this study. Different lineages are distinguished in commercial cultivars giving rise to the extant cultivars: Pluma Hidalgo and Typica, Typica and Bourbon, Bourbon and Caturra, and Typica and Robusta. The cultivars indicated with a dashed arrow were obtained as natural mutations (‘Pluma Hidalgo’, ‘Pacamara’, ‘Maragogype’, ‘Bourbon’, ‘Villa Sarchi’, and ‘Caturra’). The Bourbon and Typica cultivars generated ‘Mundo Novo’, which with ‘Caturra’ generated ‘Catuai’, ‘Garnica’, and ‘Garena’, whereas ‘Villa Sarchi’ and ‘Timor’ hybrid 832/2 generated ‘IAPAR 59’. Another identified group resulted from the ‘Typica’ and ‘Robusta’ genetic cross, thus generating the ‘Timor’ hybrid 832/1, which with ‘Caturra’ produced ‘Oro Azteca’ and ‘Costa Rica’ cultivars. The ‘Timor’ hybrids 832/1 and 1343 generated the commercial cultivar Colombia. Cultivars showing partial resistance to coffee rust are highlighted in shaded boxes. International code for Timor cultivars accessions are indicated in parenthesis.

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    Lineal comparison of DNA nuclear content among C. arabica cultivars using two different standards. Similar slope (R2 = 0.98) in all evaluated cultivars was found when nuclei was stained with propidium iodide.

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