Vol. XXX Issue 2 / ARTICLE 5 - Journal of Basic & Applied Genetics

Vol. XXX Issue 2
Article 5

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd"><html xmlns="http://www.w3.org/1999/xhtml"><head><meta http-equiv="Content-Type" content="text/html; charset=iso-8859-1" /><title>Documento sin título</title></head><body>ARTÍCULOS ORIGINALESGenome size in three species of Glandularia and their hybridsTamaño del genoma en tres especies de Glandularia y sus híbridos Ferrari M.R.1, Greizerstein E.J.2,3, Poggio L.4*1 Facultad de Ciencias Veterinarias,INITRA, UBA, CABA, Argentina. 2 Catedra de Mejoramiento Genetico, Facultad de Ciencias Agrarias, UNLZ, Buenos Aires, Argentina.  3 Instituto de Investigaciones en Produccion Agropecuaria, Ambiente y Salud (IIPAAS-FCA-CIC), Argentina.  4 IEGEBA (UBA-CONICET) Dpto. de Ecologia, Genetica y Evolucion, FCEN, CABA, Argentina.  Corresponding author:Lidia Poggio <a href="mailto:lidialidgia@yahoo.com.ar">lidialidgia@yahoo.com.ar</a>DOI: 10.35407/bag.2019.xxx.02.05Received: 07/19/2019  Revised version received: 11/23/2019  Accepted: 12/10/2019<hr />ABSTRACTIn this work the relationship between genome size of Glandularia species and the meiotic configurations found in their hybrids are discussed. Glandularia incisa (Hook.) Tronc., growing in two localities of Corrientes and Córdoba provinces, Argentina, with different ecological conditions, showed inter-population variability of the 2C-value. The DNA content found in the Corrientes locality (2.41 pg) was higher than that obtained in the Córdoba locality (2.09 pg) which has more stressful environmental conditions than the former. These values are statistically different from those that were found in Glandularia pulchella (Sweet) Tronc. from Corrientes (1.43 pg) and in Glandularia perakii Cov. et Schn from Córdoba (1.47 pg). The DNA content of the diploid F1 hybrids, G. pulchella × G. incisa and G. perakii × G. incisa, differed statistically from the DNA content of the parental species, being intermediate between them. Differences in the frequency of pairing of homoeologous chromosomes were observed in the hybrids; these differences cannot be explained by differences in genome size since hybrids with similar DNA content differ significantly in their meiotic behavior. On the other hand, the differences in the DNA content between the parental species justify the presence of a high frequency of heteromorphic open and closed bivalents and univalents with different size in the hybrids.Key words: Intra-specific DNA content variability; Homoeologous pairing; Heteromorphic  bivalents.RESUMENEn el presente trabajo se discute la relación entre el tamaño del genoma en especies de Glandularia y las configuraciones meióticas encontradas en sus híbridos. El valor 2C mostró variabilidad interpoblacional en muestras de Glandularia incisa (Hook.) Tronc. coleccionadas en dos localidades con diferentes condiciones ecológicas (provincias de Corrientes y Córdoba, Argentina). El contenido de ADN encontrado en Corrientes (2,41 pg) fue mayor que el obtenido en Córdoba (2,09 pg) donde se registran condiciones ambientales más estresantes. Estos valores son estadísticamente diferentes de los determinados en Glandularia pulchella (Sweet) Tronc. de Corrientes (1.43 pg) y en Glandularia perakii Cov. et Schn de Córdoba (1.47 pg). El contenido de ADN de los híbridos diploides F1, G. pulchella × G. incisa y G. perakii × G. incisa, difirió estadísticamente del contenido de ADN registrado en las especies parentales siendo intermedio entre ellas. Las diferencias observadas en la frecuencia de apareamiento de cromosomas homeólogos no pueden explicarse por diferencias en el tamaño del genoma, ya que híbridos con un contenido de ADN similar difieren significativamente en su comportamiento meiótico. Sin embargo, la diferencia en el contenido de ADN entre las especies parentales explica la presencia de una alta frecuencia de bivalentes heteromórficos tanto abiertos como cerrados y univalentes con diferentes tamaños.Palabras clave: Variabilidad intra-específica del contenido de ADN; apareamiento homoeólogo; Bivalentes heteromórficos.<hr /> INTRODUCTION Genome size varies among species and its diversification accompanies the evolution of many groups of plants (Bennett and Leitch, 2005; Leitch et al., 2005; Gregory et al., 2007; Leitch and Leitch, 2013; Poggio et al., 2014). Significant variation in DNA content was found among species of the same genera, among populations of one species or even among individuals belonging to the same population or cultivar (Cavallini and Natalli, 1991; Grehilhuber and Leitch, 2013; Realini et al., 2016). Variation in genome size arises by increase and/ or decrease of DNA content. The increase arises predominantly through polyploidy and amplification of non-coding repetitive DNA heterochromatin and retrotransposons. Moreover, recombination-based processes are mechanisms involved in decrease in genome size or genome downsizing (Soltis et al., 2003; Bennetzen et al., 2005; Grover and Wendel, 2010; Hidalgo et al., 2017). Besides, numerical polymorphism of B-chromosomes can modify the size of the genome (Kalendar et al., 2000; Gregory, 2004; Bennet and Leitch, 2005; Grehilhuber and Leitch, 2013; Fourastié et al., 2018).  Several studies reported some relationships between the DNA content and phenotypic characteristics such as cell size, duration of the cell cycle, growth rate, leaf expansion, flowering time, weediness, invasiveness, seed weight, and minimum generation time (Grime and Mowforth, 1982; Bennett, 1987; Ohri and Pistrick, 2001; Beaulieu et al., 2007; Greihuber and Leitch, 2013; Leitch and Leitch, 2013; Fourastie et al., 2018). Genome size was also associated with ecological parameters (temperature, precipitation and length of the growing season) and geographical parameters (altitude and latitude) (Greihuber and Leitch, 2013; Fourastie et al., 2018). These correlations suggest a biological role for genome size or “nucleotype”, term coined to describe the condition of the nucleus that affects the phenotype independently of the informational content of the DNA (Bennett, 1971; 1972). Glandularia J.F. Gmel is a genus of the Verbenaceae family composed of ca. 100 species with a North-South American disjoint distribution (O´Leary and Peralta, 2007; Peralta and Múlgura, 2011). Many of these species, their hybrids and polyploids have great ornamental potential due to their colourful flowers, long flowering period and low water requirements (Imhof et al., 2013; González Roca et al., 2015). The chromosome numbers of numerous species have been studied, and it was found that the South American species are mostly diploid (2n=2x=10) whereas the North American species are hexaploid (2n=6x=30) or tetraploid (2n=4x=20) (Schnack and Covas, 1945; Solbrig et al., 1968; Umber, 1979; Poggio et al., 1993; 2016; Turner and Powell, 2005).  Schnack and Solbrig (1953) and Solbrig et al. (1968) carried out an extensive hybridization program between South American species of the genus Glandularia and recently, many artificial hybrids were obtained with ornamental purposes (Imhof, 2014). Poggio et al. (2016) analysed G. pulchella × G. incisa F1 natural hybrids observing variability in the frequency of bivalents and univalents.  They also reported the presence of heteromorphic bivalents and differences in the size of univalents. In the present work, DNA content and its variations are reported for the first time in G. pulchella, G. incisa, G. perakii and their F1 hybrids, G. pulchella × G. incisa and G. perakii × G. incisa, species and their hybrids have the same chromosome number (2n=10) (Poggio et al. 2011; Poggio et al., 2016). Moreover, the effect of DNA content of the parental species on meiotic pairing of homoeologous chromosome in the natural hybrids is discussed. These studies could shed light on the biological importance of variation in genome size and processes of hybrid speciation.MATERIALS AND METHODSGlandularia incisa, G. pulchella and their natural hybrids (HA1, HA2, HA3, and HA4): Argentina, province of Corrientes, Dpto. Capital, Aeropuerto Cambá Punta. G. incisa, G. perakii and their natural hybrids (HB1): Argentina, province of Córdoba, Embalse Río Tercero. Taxonomic identification of the species and natural hybrids was made according to morphological criteria described by Poggio et al. (1993).  Herbarium materials were deposited in Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Autónoma de Buenos Aires, Argentina. The distance between Corrientes Dpto. Capital and Embalse Río Tercero is 726 Km. Geografical and ecological differences between the two localities are presented in <a href="#tab1">Table 1</a> (Cabrera, 1976).<a name="tab1" id="tab1"></a>Table 1. Ecological conditions in Corrientes Dpto. Capital, Province of Corrientes, Argentina and in Embalse Rio Tercero Province of Cordoba,Argentina. <img src="https://sag.org.ar/jbag/wp-content/uploads/2020/02/a05tab1.jpg" width="278" height="333" />Meiotic studies  These studies were done in inmature flowers fixed in 3:1 (ethanol: acetic acid). The anthers were squashed in 2% acetic haematoxylin as stain and 1% ferric citrate as mordant (Nuñez, 1968). In the present paper at least 50 Metaphase I were studied in the parental species and in G. perakii × G. incisa natural hybrid (HB1). The meiotic determinations made in G. pulchella × G. incisa were taken from Poggio et al. (2016). Hybrids HA1, HA2, HA3 and HA4 in the present paper correspond, respectively to the hybrids named H5, H8, H9, H4 by Poggio et al. (2016).Feulgen staining and cytophotometry  DNA content was measured in meiotic cells stainedwith the Feulgen Reaction. Inmature flowers were fixedin 3:1 (ethanol: acetic acid). The staining method wasperformed as described in Naranjo et al. (1998) and the measurements of DNA content were done in telophase I nuclei (2C). The amount of Feulgen staining per nucleus, expressed in arbitrary units, was measured at a wavelength of 570 nm using the scanning method in a Zeiss Universal Micro spectrophotometer (UMSP 30). The DNA content expressed in picograms was calculated using Allium cepa Ailsa Craig as a standard (2C-DNA=33.55 pg; Bennett and Smith, 1976).Statistical analysis  Differences in DNA content were tested through analysis of variance (ANOVA) and multiple contrasts were performed with the LSD Fisher method (Fisher, 1932). These statistical analyses were considered significant if P values were <0.05 and were performed using the Infostat program, FCA, National University of Córdoba (Di Rienzo et al., 2015).RESULTSThe 2C-DNA content of G. pulchella and G. incisa and their hybrids collected in the Province of Corrientes showed significant differences (F6.76=20.83; P<0.0001).  Contrasts performed with the LSD Fisher method are presented in <a href="#tab2">Table 2</a>. The 2C-DNA content of G. perakii, and G. incisa, and their hybrids collected in the Province of Córdoba showed significant differences (F9.127=44.85; P<0.0001). Contrasts performed with the LSD Fisher method are presented in <a href="#tab3">Table 3</a>. Numerical polymorphism for B chromosomes was detected in G. incisa, G. perakii and hybrids collected in the Córdoba locality. DNA content was determined only in plants without B chromosomes for a better comparison with plants from other localities. Intra-populational significant differences (P<0.001) were detected in the three species and in their hybrids studied in the present work. Inter-populational significant differences (P<0.001) were detected between G. incisa collected in the provinces of Corrientes and Córdoba. In <a href="#fig1">Figure 1</a>, a graphic representation is presented of 2C-DNA content of the parental species and their hybrids in collections made in the provinces of Corrientes (A) and Córdoba (B). The DNA content of the hybrids, G. pulchella × G. incisa and G. perakii × G. incisa was intermediate between the parental species (<a href="#tab1">Table 1</a>, <a href="#tab2">Table 2</a> and <a href="#fig1">Figure 1</a>). DNA content of HA1, HA2, and HA3, did not show significant differences among them, but they differed from that of HA4. <a name="tab2" id="tab2"></a>Table 2. Nuclear DNA content in G. pulchella, G. incisa, and G. pulchella x G. incisa, collected in Province of Corrientes, Argentina. Means with the same letters are not significantly different (P≤0.05). Pu= G. pulchella,In= G. incisa and HA1 - HA4= G. pulchella x G. incisa. <img src="https://sag.org.ar/jbag/wp-content/uploads/2020/02/a05tab2.jpg" width="279" height="423" /><a name="tab3" id="tab3"></a>Table 3. Nuclear DNA content in G. perakii, G. incisa and G. perakii x G. incisa collected in Province of Cordoba, Argentina. Means with the sameletters are not significantly different (P≤0.05). Pe = G. perakii, In = G. incisa and HB1 = G. perakii x G. incisa. <img src="https://sag.org.ar/jbag/wp-content/uploads/2020/02/a05tab3.jpg" width="275" height="444" /><a name="fig1" id="fig1"></a><img src="https://sag.org.ar/jbag/wp-content/uploads/2020/02/a05fig1.jpg" width="509" height="287" />  Figure 1. Graphic comparison of 2C-DNA content in G. pulchella, G. perakii, G. incisa and the hybrids G. pulchella x G. incisa and G. perakii x G. incisa. A) Materials collected in the Province of Corrientes: G. pulchella (Pu, grey); G. Incise (In, black), hybrids (HA, white). B) Materials collected in the Province of Cordoba. G. perakii (Pe, grey); G. incisa (In, black); hybrid (HB, white). Different numbers indicatedifferent individuals in each taxa. The three parental species presented five homomorphic bivalents in all Metaphase I (more than n=50 cells were studied in each species) (<a href="#fig2">Figure 2 a and b</a>). The hybrids had univalents of different size and up to five heteromorphic bivalents (<a href="#fig2">Figure 2 c-f</a>). <a name="fig2" id="fig2"></a><img src="https://sag.org.ar/jbag/wp-content/uploads/2020/02/a05fig2.jpg" width="402" height="643" />  Figure 2. Metaphase I in Glandularia species and F1 hybrids. a) G. incisa (collected in Cordoba) with five homomorphic bivalents and six B chromosomes. b) G. pulchella with five homomorphic bivalents. c-f) hybrids with univalents and heteromorphic bivalents. c) G. perakii x G. incisa with two B chromosomes and five heteromorphic bivalents. d) G. perakii x G. incisa with five heteromorphic bivalents. e) G. pulchella x G. incisa with five heteromorphic bivalents. f) G. pulchella x G. incisa with one heteromorphic bivalent and eight univalents. Arrows show B chromosomes and arrow heads indicatesome notorious heteromorphic bivalents. Bars 10 μm.The mean frequency and standard deviation of bivalents in the natural hybrids G. pulchella × G. incisa was HA1: 4.98±0.16, HA2: 5.00±0.00, HA3: 2.80±1.21 and HA4: 3.50±0.72. HA1 and HA2 did not present significant differences between them (Poggio et al., 1993).DISCUSSIONSignificant differences in the DNA content of G. incisa with G. pulchella and G. perakii were found. The mean DNA 2C-values and the variation ranges were determined in G. incisa 2.25 pg (2.00 pg - 2.71 pg), G. pulchella 1.43 pg (1.31 pg - 1.52 pg) and G. perakii 1.47 pg (1.36 pg - 1.57 pg). According to the Kew DNA C-values, these are the first reports for genus Glandularia. These values are between the minimum value (Tectona grandis 0.96 pg) and the maximum value (Lantana camara 5.50 pg) determined in five species belonging to different genera of the Verbenaceae family (RBG Kew DNA C-values, 2017). Inter-population variability of the 2C-value was observed in G. incisa growing in two localities with different geographical and ecological conditions (Corrientes and Córdoba Provinces). The values of DNA content obtained in both localities differed significantly and the 2C values determined in the Corrientes population were 15% greater than that reported in the Córdoba population. The population of G. incisa with the smaller genome was found in drier and more stressful conditions. These results are in agreement with the reported in different studies, suggesting that variation in DNA amount has adaptive significance related to environmental, climatic and phenological parameters such as temperature, precipitation, length of growing season and type of soil (Bennett, 1987, reviewed in Grehilhuber and Leitch, 2013). Another important difference between the two populations is that G. incisa growing in Córdoba presented polymorphism for B chromosomes (0-6 B´s) whereas B chromosomes were not detected in plants growing in Corrientes. However, this fact does not affect the comparisons performed because measurements in the Córdoba population were carried out in cells without B chromosomes.  In addition to the interpopulation variation reported in this paper, the three species showed intrapopulation variability. The origin of this variation in the nuclear DNA content would be the result of a fraction potentially unstable such as transposable elements subject to environmental and/or genetical events that induce deletion and amplification of sequences (Grover and Wendel, 2010). It is usually expected that the DNA content of interspecific hybrids were in an intermediate range between the respective parental species, although different authors have found, in some cases, that hybrids have more or less DNA content than their parents. This would be indicating the occurrence of genetic and epigenetic changes that were reported in newly-formed hybrids in several groups of plants (Rayburn et al., 1993; Grattapaglia and Bradshaw, 1994; Ma and Gustafson, 2005). The natural F1 hybrids G. pulchella × G. incisa and G. perakii × G. incisa, analyzed in the populations of Corrientes and Córdoba, have intermediate 2C-DNAvalues between the parental values and differedstatistically from them.Differences in DNA content are usually correlatedwith karyotype parameters and can affect the entirechromosome complement or they may be restrictedto a subset of chromosomes. So far, the G. incisa, G.pulchella and G. perakii karyotypes have been analysed(data not published) and all of them show metacentricchromosomes, being larger the chromosomes of G. incisa.This leads us to suggest that changes in DNA contentoccurred in the whole-chromosome complement addingor losing equal DNA amounts to both arms or in thepericentromeric region, maintaining their metacentricmorphology (in preparation). Solbrig et al. (1968) made artificial crosses betweenspecies of Glandularia and observed homoeologouspairing in F1 hybrids and suppression of homoeologouspairing in the allotetraploids. Poggio et al. (2016)reported that the F1 hybrids G. pulchella × G. incisa hadvariability in homoeologous pairing forming from one tofive heteromorphyc bivalents and univalents of differentsize. These authors explained the homoeologous pairingin the F1 hybrids by suggesting the presence of a pairingregulator gene/s that precluded homoeologous pairingwhen homologous genomes are in two doses in thepolyploids, and display incomplete penetrance whenhomologous genomes are in one dose in the diploids. The differences in DNA content found in the parentalgenomes could explain the presence of heteromorphicbivalents in the F1 hybrids. However, the genomesize cannot explain the differences observed inhomoeologous pairing since hybrids with similar DNAcontent differed significantly in their meiotic behavior.The differences in the frequency of pairing ofhomoeologous chromosomes that were observed in thehybrids cannot be explained by the genome, size of theparental species, since hybrids with similar DNA contentdiffered significantly in their meiotic behavior. 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