Corresponding author: Wellington Ronildo Clarindo ( email@example.com )
Academic editor: Manoj Dhar
© 2017 Leonardo Luís Artico, Ana Cristina Mazzocato, Juliano Lino Ferreira, Carlos Roberto Carvalho, Wellington Ronildo 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: Artico LL, Mazzocato AC, Ferreira JL, Carvalho CR, Clarindo WR (2017) Karyotype characterization and comparison of three hexaploid species of Bromus Linnaeus, 1753 (Poaceae). Comparative Cytogenetics 11(2): 213-223. https://doi.org/10.3897/CompCytogen.v11i2.11572
Chromosome morphometry and nuclear DNA content are useful data for cytotaxonomy and to understand the evolutionary history of different taxa. For the genus Bromus Linnaeus, 1753, distinct ploidy levels have been reported, occurring from diploid to duodecaploid species. The geographic distribution of Bromus species has been correlated with chromosome number and ploidy level. In this study, the aims were to determine the nuclear genome size and characterize the karyotype of the South American Bromus species: Bromus auleticus Trinius ex Nees, 1829, Bromus brachyanthera Döll, 1878 and Bromus catharticus Vahl, 1791. The mean nuclear 2C value ranged from 2C = 12.64 pg for B. catharticus to 2C = 17.92 pg for B. auleticus, meaning a maximum variation of 2C = 5.28 pg, equivalent to 41.70%. Despite this significant difference in 2C value, the three species exhibit the same chromosome number, 2n = 6x = 42, which confirms their hexaploid origin. Corroborating the genome size, the chromosome morphometry (total, short- and long-arm length) and, consequently, the class differed among the karyotypes of the species. Based on the first karyograms for these Bromus species, some morphologically similar and several distinct chromosome pairs were found. Therefore, the karyotype characterization confirmed the hexaploid origin of the studied Bromus species, which differ in relation to the karyogram and the nuclear 2C value. Considering this, cytogenetics and flow cytometry can be used to discriminate Bromus species, contributing to taxonomy and systematic studies and providing information on the evolutionary history of this taxa.
Karyogram, nuclear genome size, polyploidy, forage grasses
The genus Bromus Linnaeus, 1753, family Poaceae comprises more than 160 species of annual and perennial grasses (
The basic chromosome number of the genus Bromus is x = 7, and its species possess karyotypes varying from 2n = 2x = 14 (diploid) to 2n = 12x = 84 (duodecaploid) (
Karyotype characterization showed that the chromosomes of the Bromus species are similar in relation to total length and class (
Differentiation of allohexaploid Bromus species in South America proceeded in the Pleistocene. Meanwhile, in North America, allopolyploidy events also occurred, leading to new ones with higher ploidy level (8x, 12x) (
According to current knowledge, the South American species (as B. auleticus, B. brachyanthera and B. catharticus) have chromosome number of 2n = 6x = 42 (
Numerical and structural chromosomal rearrangements have been reported to trigger changes in karyotype in various plant taxa. Due to these changes, the nuclear genome size varies between phylogenetically related species (
Hence, karyotype and nuclear 2C value are relevant data for the taxonomy and systematics of Bromus, as well as for understanding the evolutionary history of the genus and the relationships within the taxa. Thus, the aims of the present study were to measure the nuclear 2C value, determine the chromosome number and characterize the karyotype of the South American Bromus species B. auleticus, B. brachyanthera and B. catharticus.
Seeds of B. auleticus, B. brachyanthera and B. catharticus were provided by the South Forage Germplasm Bank (BAG) of Embrapa South Livestock, Brazil (BRA 00059183-4, 00080317-1 and 00059197-4, respectively). The seed samples were collected from several individuals of each species, occurring in the Brazilian Pampa biome, state of Rio Grande do Sul, Brazil. Copies of the species were deposited in the Herbarium CNPO Embrapa (voucher numbers CNPO 4408 for B. auleticus, CNPO 4412 for B. brachyanthera, and CNPO 4408 for B. catharticus).
Nuclear suspensions were prepared from leaf fragments (2 cm2) obtained from each specimen of B. auleticus, B. brachyanthera or B. catharticus (samples), together with the internal standard Pisum sativum L. (2C = 9.16 pg;
Wherein: 2CD: value of 2C DNA content (pg) of each Bromus species; C1: average G0/G1 peak channel of the Bromus species; C2: average G0/G1 peak channel of P. sativum; 2CS: value of 2C DNA content of P. sativum (2C = 9.16 pg).
The seeds were aseptically scarified, disinfested, inoculated into medium composed of half-strength MS salts, 10 ml l-1 MS vitamins (
FCM nuclear suspensions resulted in G0/G1 fluorescence peaks with a coefficient of variation of less than 5% for Bromus species and P. sativum. Thereby, FCM procedures provided suspensions with adequate amount of isolated, intact and stoichiometrically stained nuclei. The 2C nuclear DNA content was measured for the Bromus species through analysis of the histograms. 2C value of B. catharticus was 2C = 12.64 ± 0.00 pg, B. brachyanthera was 2C = 16.73 ± 0.16 pg, and B. auleticus was 17.92 ± 0.44 pg. Mean value for B. auleticus was 41.70% higher than for B. catharticus, and 7.10% greater than for B. brachyanthera. In turn, B. brachyanthera presented 2C value 32.36% higher than that of B. catharticus. These values reflect interspecific variation among the nuclear genome sizes of the analyzed species.
Root meristems treated with amiprophos-methyl and macerated in enzyme pool solution resulted in adequate metaphase chromosomes. Metaphases were chosen based on the following criteria: well-spread chromosomes with well-defined constriction, no chromatin deformations and no cytoplasmic background noise. These features allowed accurate chromosome counting, karyotype measurements, chromosome class determination and karyogram assembly. All three Bromus species showed a conserved number of 2n = 42 chromosomes (Figure
Based on the morphometric data, the chromosome class was determined and the differences between the karyotypes of the three species were verified. B. auleticus presented eleven metacentric (1, 3, 4, 5, 6, 8, 9, 11, 15, 18 and 19) and ten submetacentric chromosomes (2, 7, 10, 12, 13, 14, 16, 17, 20 and 21). B. brachyanthera exhibited 13 metacentric (1, 2, 4, 5, 6, 9, 10, 13, 15, 16, 17, 19 and 21) and eight submetacentric chromosomes (3, 7, 8, 11, 12, 14, 18 and 20). Finally, B. catharticus displayed eleven metacentric (1, 4, 5, 7, 8, 9, 10, 11, 14, 17 and 20) and ten submetacentric chromosomes (2, 3, 6, 12, 13, 15, 16, 18, 19 and 21) (Table
The three Bromus species presented only metacentric and submetacentric chromosomes. Despite belonging to the same class, the chromosomes differed intra- and interspecifically based on their morphology, which was characterized by occurrence of well-defined telomere and centromere portions and relatively low chromatin compaction level (Figure
First karyograms of Bromus species, displaying 2n = 6x = 42 chromosomes: a B. catharticus b B. brachyanthera and c B. auleticus. a B. catharticus displayed eleven metacentric (1, 4, 5, 7, 8, 9, 10, 11, 14, 17 and 20) and ten submetacentric chromosomes (2, 3, 6, 12, 13, 15, 16, 18, 19 and 21) b B. brachyanthera exhibited 13 metacentric (1, 2, 4, 5, 6, 9, 10, 13, 15, 16, 17, 19 and 21) and eight submetacentric chromosomes (3, 7, 8, 11, 12, 14, 18 and 20) c B. auleticus presented eleven metacentric (1, 3, 4, 5, 6, 8, 9, 11, 15, 18 and 19) and ten submetacentric chromosomes (2, 7, 10, 12, 13, 14, 16, 17, 20 and 21). Note the morphologically similar chromosomes: a 12–13 in B. catharticus b 11–12 and 15–16 in B. brachyanthera, and c 3–4 in B. auleticus. Bar = 5 µm.
Morphometry of the metaphasic chromosomes of B. auleticus, B. brachyanthera and B. catharticus.
|Chrom.||B. auleticus||B. brachyanthera||B. catharticus|
According to the chromosome number found here and the complement set x = 7 (
The mean nuclear 2C value divergences were corroborated by chromosome morphometry. B. catharticus clearly differed from the other species, being that its relatively small genome size correlated with the sum of the total chromosome length. Differently, for B. auleticus and B. brachyanthera this relation was not observed, as a result of the low compaction level of the chromatin in the latter species (Figure
Despite the similar morphology of some chromosome pairs (12–13 in B. catharticus, 11–12 and 15–16 in B. brachyanthera, and 3–4 in B. auleticus; Figure
The nuclear 2C value and karyotype characterization allowed differentiating the three Bromus species, thus contributing to the cytotaxonomy and evolutional understanding in this taxon. As also demonstrated by other authors, these data provide insights about the evolutionary process and diversification of the polyploid Bromus species.
We thank the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brasília – DF, Brazil), the Fundação de Amparo à Pesquisa do Espírito Santo (FAPES, Vitória – ES, Brazil), and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brasília – DF, Brazil) for financial support. Scholarship holder associated to the project MP1 – 01.15.02.002.05.08 of the Rede Nacional de Recursos Genéticos, Vertente Vegetal, Projeto Componente “Bancos ativos de Germoplasma de Forrageiras”.