Short Communication |
Corresponding author: Wellington Clarindo ( welbiologo@gmail.com ) Academic editor: Vladimir Lukhtanov
© 2016 Anelise Marques, Amélia Carlos Tuler, Carlos Roberto Carvalho, Tatiana Carrijo, Marcia Flores da Silva Ferreira, Wellington Clarindo.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Marques AM, Tuler AC, Carvalho CR, Carrijo TT, Ferreira MRS, Clarindo WR (2016) Refinement of the karyological aspects of Psidium guineense (Swartz, 1788): a comparison with Psidium guajava (Linnaeus, 1753). Comparative Cytogenetics 10(1): 117-128. https://doi.org/10.3897/CompCytogen.v10i1.6462
|
Euploidy plays an important role in the evolution and diversification of Psidium Linnaeus, 1753. However, few data about the nuclear DNA content, chromosome characterization (morphometry and class) and molecular markers have been reported for this genus. In this context, the present study aims to shed light on the genome of Psidium guineense Swartz, 1788, comparing it with Psidium guajava Linnaeus, 1753. Using flow cytometry, the nuclear 2C value of P. guineense was 2C = 1.85 picograms (pg), and the karyotype showed 2n = 4x = 44 chromosomes. Thus, P. guineense has four chromosome sets, in accordance with the basic chromosome number of Psidium (x = 11). In addition, karyomorphometric analysis revealed morphologically identical chromosome groups in the karyotype of P. guineense. The high transferability of microsatellites (98.6%) further corroborates with phylogenetic relationship between P. guajava and P. guineense. Based on the data regarding nuclear genome size, karyotype morphometry and molecular markers of P. guineense and P. guajava (2C = 0.95 pg, 2n = 2x = 22 chromosomes), P. guineense is a tetraploid species. These data reveal the role of euploidy in the diversification of the genus Psidium.
Psidium , polyploidy, karyotype evolution, cytogenetic, flow cytometry, SSR markers
Psidium Linnaeus, 1753 is a genus of Myrtaceae that comprises about 92 species (
Karyotypic characterization has been applied to better understand the changes that occur during genome evolution (
According to current knowledge, few Psidium species are diploid (2n = 22), such as Psidium chinense Loudon, 1830 (
Euploid species are key models for evolution because they provide evidence of the polyploidization event that promoted diversification and speciation. Considering that, this study aimed to refine the knowledge about karyological aspects of Psidium guineense. Besides, a comparison was performed with the diploid species (2x = 22) P. guajava, because this species is the only of the Psidium genus characterized from flow cytometry (FCM), cytogenetic (
Psidium guajava fruits were obtained from 50 plants growing in orchards located in different regions of the Brazil. Psidium guineense fruits were obtained from indigenous populations occurring in Atlantic Forest remnants located in the Municipalities of Alegre (four individuals), Itapemirim (three individuals), Santa Teresa (seven individuals), and Conceição da Barra (six individuals), all located in Espírito Santo state. The sampling was done between 2012 and 2014.
FCM and molecular analyses were conducted with the same 50 individuals of P. guajava and 20 of P. guineense. Due to FCM results, karyotype characterization was performed using seeds obtained from ten distinct plants of the two species. Solanum lycopersicum Linnaeus, 1753, ‘Stupické’ (reference standard for FCM, 2C = 2.00 picograms – pg;
Leaves were collected from S. lycopersicum (standard), P. guajava and P. guineense (samples). Nuclei suspensions were obtained from leaf fragments of the standard and of each sample, according to a previously described protocol (
Seeds of P. guineense and P. guajava were germinated in Petri dishes containing distilled water (dH2O) at 30 °C. The roots showing 1.0–2.0 cm in length were treated for a period of 4, 15 or 19 h with the microtubule-inhibiting agents amiprophos-methyl (APM, Nihon Bayer Agrochem K. K.®) or oryzalin (ORY, Sigma®) at a final concentration of 4 µM. Subsequently, the roots were washed with dH2O for 20 min, then fixed in fresh methanol:acetic acid (Merck®) solution (3:1). The fixative was changed three times, and the roots were stored at -20 °C for 24 h. The roots were washed and incubated for 2:00, 2:15 or 2:30 h at 34 °C in pectinase solution (Sigma®, E6287) at ratios of 1:8, 1:10, 1:12 or 1:15 (enzyme:water). Next, the roots were washed for 10 min in dH2O, fixed once more, and stored at -20 °C (
The genomic DNA was extracted from young leaves according to
The FCM protocol, using isolation buffer for 10 min and staining buffer for 30 min, provided peaks relative to G0/G1 nuclei with coefficient of variation (CV) lower than 3.46%, and thus high resolution. This result indicates that the suspensions contained sufficient number of intact, isolated and stoichiometrically stained nuclei.
Based upon the large number of plant samples of distinct genotypes evaluated in this study, the mean nuclear 2C value is 0.95 pg for all P. guajava plants (Fig.
Representative histograms obtained from FCM analysis of nuclear suspensions stained with propidium iodide. A G0/G1 nuclei peak of the sample P. guajava (2C = 0.95 pg), positioned in channel 95, and the standard S. lycopersicum (2C = 2.00 pg) in channel 200 B G0/G1 nuclei peak of the sample P. guineense (2C = 1.85 pg), positioned in channel 185, and the standard S. lycopersicum (2C = 2.00 pg) in channel 200.
Psidium guajava was one of the first Myrtaceae species for which the nuclear genome size was measured using Feulgen microdensitometry. With this method, mean values of 2C = 0.66 pg (
The distinct 2C values observed for P. guineense and P. guajava may be related to the different techniques, plant standards, nuclear isolation and staining procedures used. More inconsistent values of DNA content were found by
Based on DNA content, the occurrence of karyotype modifications that increased the genome size may have played a role in the origin of P. guineense. To confirm this hypothesis, karyotypic characterization was accomplished for P. guineense and P. guajava. The root tips that were treated with 4 µM APM for 15 h and macerated in 1:10 pectinase solution for 2 h provided the most adequate metaphases for morphometric analysis. Metaphases were chosen based on relevant characteristics: well-spread chromosomes with well-defined constriction, without chromatin deformations and cytoplasmic background noise. These features allowed accurate chromosome counting, morphometric characterization and assembly of the karyograms (Fig.
A P. guajava karyogram showing 2n = 2x = 22 chromosomes, being five metacentric (3, 4, 8, 9, 10) and six submetacentric pairs (1, 2, 5, 6, 7, 11) B P. guineense karyogram showing 2n = 4x = 44 chromosomes, with two metacentric (11, 12) and twenty submetacentric pairs (1–10, 13–22). Note groups of morphologically identical chromosomes, such as 1 and 2, 3 and 4, 21 and 22. Bar = 5 μm.
Morphometric data and classification of the chromosomes of P. guajava and P. guineense. The mean values of total length, short and long arms were measured using at least 10 metaphases of each species.
P. guajava | P. guineense | ||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|
Chroma | Totalb | Short armb | Long armb | rc | Classd | Chroma | Totalb | Short armb | Long armb | rc | Classd |
1 | 2.03 | 0.73 | 1.30 | 1.78 | SM | 1–2 | 1.82 | 0.63 | 1.20 | 1.91 | SM |
2 | 1.60 | 0.52 | 1.08 | 2.08 | SM | 3–4 | 1.60 | 0.60 | 1.00 | 1.67 | SM |
3 | 1.57 | 0.64 | 0.93 | 1.45 | M | 5–6 | 1.53 | 0.57 | 0.96 | 1.70 | SM |
4 | 1.52 | 0.73 | 0.79 | 1.08 | M | 7–8 | 1.44 | 0.55 | 0.89 | 1.62 | SM |
5 | 1.47 | 0.58 | 0.89 | 1.53 | SM | 9–10 | 1.30 | 0.50 | 0.80 | 1.60 | SM |
6 | 1.42 | 0.53 | 0.89 | 1.68 | SM | 11–12 | 1.25 | 0.58 | 0.68 | 1.17 | M |
7 | 1.37 | 0.53 | 0.84 | 1.58 | SM | 13–14 | 1.20 | 0.48 | 0.73 | 1.53 | SM |
8 | 1.15 | 0.56 | 0.59 | 1.05 | M | 15–16 | 1.17 | 0.43 | 0.74 | 1.70 | SM |
9 | 1.12 | 0.45 | 0.67 | 1.49 | M | 17–18 | 1.06 | 0.30 | 0.76 | 2.53 | SM |
10 | 1.05 | 0.50 | 0.55 | 1.10 | M | 19–20 | 0.93 | 0.25 | 0.68 | 2.73 | SM |
11 | 0.85 | 0.27 | 0.58 | 2.15 | SM | 21–22 | 0.66 | 0.20 | 0.46 | 2.29 | SM |
Totale | 15.15 | 13.96 |
The chromosome number of P. guajava and of P. guineense were accurately determined here as 2n = 2x = 22 and 2n = 4x = 44, respectively (Fig.
Psidium guajava exhibited metacentric (pairs 3, 4, 8, 9, 10) and submetacentric chromosomes (pairs 1, 2, 5, 6, 7, 11). This species had relatively small and morphologically similar chromosomes, two of which (1 and 11) were distinguished by their very distinct total length.
As observed for P. guajava, the karyotype of P. guineense also showed only metacentric (11, 12) and submetacentric (1–10, 13–22) chromosomes (Table
The karyomorphometric analysis also revealed groups of morphologically identical chromosomes in P. guineense: 1–2, 3–4, 5–6, 7–8, 9–10, 11–12, 13–14, 15–16, 17–18, 19–20 and 21–22 (Table
Considering the basic chromosome number of Psidium (x = 11) (
From meiotic analysis in P. guineense,
The variation in chromosome number seen in the genus Psidium can promote genetic isolation and possibly create barriers to gene flow (
Among the 142 SSR markers, 140 were amplified in P. guineense, representing 98.6% of transferability. The high amplification rate (98.6%) found for the P. guajavaSSR primers in P. guineense showed that the annealing regions are conserved in both species, revealing the high similarity between them. This result also evidenced that these DNA sequences of P. guineense are very similar in relation to P. guajava, since values of cross-amplification of approximately 73% have been reported for species of the same genus (
Of the 140 primers, 117 were chosen to determine the total number of alleles, which varied from 170 for P. guineense to 148 for P. guajava (Suppl. material
This study points to the tetraploidy origin of P. guineense. These results reveal the importance of combining cytogenetic and molecular markers for a better understanding of how euploid events have influenced the speciation process in angiosperms.
The authors are grateful to Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brasília – DF, Brazil), Fundação de Amparo à Pesquisa do Espírito Santo (FAPES, Vitória – ES, Brazil), Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG, Belo Horizonte – MG, Brazil), and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brasília – DF, Brazil) for financial support.
SSR locus, annealing temperature (TA), motif, and allele number amplified in the genome of P. guineense and P. guajava.
Data type: Word document.
Explanation note: The Supplementary material 1 summarizes the SSR locus, annealing temperature (TA), motif, and allele number amplified in the genome of P. guineense and P. guajava.