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

Vol. XXX Issue 2
Article 2

<!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 ORIGINALESKaryological study in the chilean rhatany Krameria  cistoidea Hook. & Arn. (Krameriaceae)Estudio cariológico en el pacul chileno Krameria cistoidea Hook. & Arn. (Krameriaceae) Palma Rojas C.1*, Jara Seguel P.2, García M.1, von Brand E.3, Araya Jaime C.1,41 Departamento de Biologia,  Facultad de Ciencias, Universidad  de La Serena, Casilla 599, La  Serena, Chile.  2 Departamento de Ciencias Biologicas y Quimicas, Nucleo de  Estudios Ambientales, Facultad de  Recursos Naturales, Universidad  Catolica de Temuco, Casilla 15-D,  Temuco, Chile.  3 Departamento de Biologia  Marina, Facultad de Ciencias del  Mar, Universidad Catolica del Norte,  Casilla 117, Coquimbo, Chile.  4 Instituto de Investigacion  Multidisciplinar en Ciencia y  Tecnologia, Universidad de La  Serena, La Serena, Chile.  Corresponding author:  Claudio Palma-Rojas  <a href="mailto:cpalma@userena.cl">cpalma@userena.cl</a>DOI: 10.35407/bag.2019.xxx.02.02Received: 06/19/2019  Revised version received: 09/27/2019  Accepted: 11/05/2019<hr />ABSTRACTThe karyotype of the plant species Krameria cistoidea Hook. & Arn. was studied by assessing chromosome characters such as morphology, size, and C-banding pattern. The karyotype of K. cistoidea was composed only by metacentric chromosomes in the two populations studied. The haploid set length was 51.9±2.3 μm and the mean chromosome size was 8.68±0.78 μm. Some similarities in chromosome morphology and size can be observed among K. cistoidea and K. triandra, in addition to the chromosome number 2n=12 which is conserved within the genus. K. cistoidea exhibited a symmetric banding pattern with large C-bands in the telomeres of the short and long arms of all chromosomes, except the short arm of pair 1. The relative length of the C-bands was 23.5% of the total haploid set length. These cytological results on K. cistoidea are the first data on quantitative karyotype morphology and C-banding patterns in the genus Krameria.Key words: Krameria; Karyotype; C-banding.RESUMENEl cariotipo de la especie vegatal Krameria cistoidea Hook. & Arn., 2n=12, se estudió en individuos de dos poblaciones considerando las variables de tamaño, morfología y patrón de bandas C. La longitud del set haploide fue de 51,9±2,3 μm con un tamaño cromosómico promedio de 8,68±0,78 μm. Se encontraron algunas similitudes de morfología y tamaños cromosómicos entre el cariotipo de K. cistoidea y el descrito para K. triandra, ambas con 2n=12 guarismo conservado dentro del género. Los cromosomas de K. cistoidea muestran un patrón simétrico de grandes bandas C en los telómeros de todos ellos, excepto en el brazo corto del par 1 y con una longitud relativa de los segmentos con bandas C de un 23,5% del set haploide. Estos resultados son los primeros datos cuantitativos relativos al cariotipo y patrón de bandas C en el género Krameria.Palabras clave: Krameria; Cariotipo; Bandas-C.<hr /> INTRODUCTIONKrameria cistoidea (Krameriaceae) Hook. & Arn., is a plant species endemic to Chile with a center of distribution located between Huasco (28º S) and Limari rivers basins (30º S) in the coastal and pre-Andean slopes of a semiarid zone (Squeo et al., 2001). Along its geographical range K. cistoidea shares the habitat with K. lappacea. At present, almost 16 Krameria species constitute the monogeneric family Krameriaceae which is distributed across the Americas, but only two species are present in Chile. The taxonomic classification of Krameria has been principally based upon morphology, anatomy, pollen ultra-structure, wood anatomy and DNA sequences (Heusser, 1971; Robertson, 1973; Simpson and Skvarla, 1981; Soltis et al., 2000; Simpson et al., 2004; Carlquist, 2005). Nevertheless, since its description by Loefling in 1758, the genus Krameria has presented a problem to taxonomists as to its placement within the dicotyledons (Robertson, 1973; Simpson and Skvarla, 1981). Currently, Krameria is considered within the Zygophyllales order together with other genera belonging to the Zygophyllaceae family (Soltis et al., 2000; Simpson et al., 2004). Historical reports have described that the roots of K. cistoidea have had a variety of uses such as medicinal herb, for liqueur production, and as an important source of dye (Muñoz, 1985). However, despite of its extraction and habitat degradation by anthropic and natural effects, information on its conservation is scarce, but this species does not meet the criteria to be considered vulnerable (Benoit, 1989; Squeo et al., 2001). Krameria species show a haploid chromosome number n=6 (Turner, 1958; Lewis et al., 1962), which later was corroborated with the count of the diploid number 2n=12 described in K. triandra (Teppner, 1984). Recently, the chromosome number 2n=12 was also found in K. cistoidea, which was complemented with data on DNA C-value (1C=9.3 pg) (Palma Rojas et al., 2017), thus supplying new cytological data for the genus. However, despite these advances, the karyotype morphology has not been described for species of the genus, and there are no reports on specific chromosome markers, for example, C-banding patterns, which show the location of constitutive heterochromatin. It is remarkable that the Chilean taxa of Krameria form part of the most southern species along the geographical range of the genus in America (Simpson et al., 2004). In this sense karyotype studies including heterochromatin location may be fundamental to understand patterns on genetic variation, genome evolution and speciation in these plants (Stebbins, 1971; Guerra, 2000; Levin, 2002; Jara Seguel et al., 2010; Jara Arancio et al., 2012; Jara Seguel and Urrutia, 2012), and may contribute significantly to establish the cytological relationships among North American and South American Krameria species, supplying also additional evidence for its taxonomic status within the Zygophyllales. For this reason, we describe the karyotype morphology and the distribution of constitutive heterochromatin in K. cistoidea, the most representative species of the genus present in the Chilean flora.MATERIALS AND METHODSPlants of Krameria cistoidea Hook. & Arn. were collected from two naturally growing populations, in Punta Colorada (28º 30´ S; 70º 48´ W, altitude 585 m above sea level), and Cuesta Buenos Aires (30º 2´ S; 70º 49´ W, altitude 380 m above sea level), both spaced at a distance of approximately 170 km in Central Chile. Voucher specimens for both populations were deposited at the Herbarium of the Universidad de La Serena, La Serena, Chile (Herbario ULS). Roots of germinated seeds were pre-treated with 8-Hydroxiquinoline 2 mM at 7º C for 3 h, fixed in ethanol-glacial acetic acid (3:1 v/v) at 4º C for 24 h, and stored in ethanol 70% (v/v) at 4º C until use. To determine chromosome morphology, the roots were stained with Feulgen reaction and chromosome preparations were made by squashing the root tips. For Giemsa C-banding, the fixed roots were washed in distilled water and treated with a solution of pectinasecellulase (Fluka; 2:1 w/w) at 7.5% (w/v) in 0.2 M citrate buffer pH 4.2 at 37º C for 30 minutes. The procedure used to obtain C-bands was based on the technique described by Summer (1972). In photomicrographs of ten metaphase plates (Feulgen preparations) obtained from ten plants, the short and long arms were measured and the total relative length of each chromosome pair (expressed as percentage of the total haploid set length) was calculated. Additionally, total haploid set length (THL in μm), and mean chromosome size (in μm) were estimated. The karyotype was constructed according to decreasing chromosome length and chromosome morphology, using the nomenclature by Levan (Levan et al.,1964; Spotorno, 1985). The C-bands were classified according to their chromosome location as centromeric, pericentromeric, interstitial or telomeric. Relative length values of C-bands (RLC) were calculated by using the follow equation described by Linde-Larsen et al. (1980): RLC= (C-band length of the haploid set / Total haploid set length) x 100.RESULTSThe Feulgen stained karyotype of K. cistoidea is shown in <a href="#fig1"> Figure 1a</a>, and chromosome measurements are presented in <a href="#tab1">Table 1</a>. Both populations of K. cistoidea studied here showed a diploid chromosome number of 2n=12, with a karyotype composed only by metacentric chromosomes. Satellites and secondary constrictions were not observed in these chromosomes. The chromosomes of K. cistoidea are large, with a mean chromosome size of 8.68±0.78μm and a total haploid set length of 51.9±2.3 μm. <a name="fig1" id="fig1"></a><img src="https://sag.org.ar/jbag/wp-content/uploads/2020/02/a02fig1.jpg" width="294" height="105" /> Figure 1a. Karyotype of Krameria cistoidea, 2n=12. Feulgen stain. Bar=10 μm.<img src="https://sag.org.ar/jbag/wp-content/uploads/2020/02/a02fig1b.jpg" width="304" height="118" /> Figure 1b. Karyotype of Krameria cistoidea with Giemsa C-banding. Bar=10 μm. <a name="tab1" id="tab1"></a>Table 1. Karyotype characters of Krameria cistoidea. SA, short arm (%); LA, long arm (%); TL, total chromosome length (%); CS, absolutechromosome size (μm), CI, centromeric index; SD, standard deviation; m, metacentric. <img src="https://sag.org.ar/jbag/wp-content/uploads/2020/02/a02tab1.jpg" width="553" height="224" /> The C-banded karyotype of K. cistoidea is shown in <a href="#fig1">Figure 1b</a>. K. cistoidea exhibited C-bands located in the telomeres of the short and long arms of all chromosomes, except the short arm of pair 1. These large regions of constitutive heterochromatin in the karyotype were concurrent with the presence of many conspicuous and large chromocenters in the interphase nuclei (<a href="#fig2">Figure 2</a>). Homologous chromosomes exhibited similar C-banding patterns and pairing was possible. The centromeres were also evident in all chromosomes. The relative length of the C-bands was equivalent to 23.5% of the total haploid set length. Polymorphism in C-banding pattern was not observed in both studied populations.<a name="fig2" id="fig2"></a><img src="https://sag.org.ar/jbag/wp-content/uploads/2020/02/a02fig2.jpg" width="297" height="229" />  Figure 2. Meristematic interphase nuclei of Krameria cistoidea, with manychromocenters. Bar=10 μm.DISCUSSION The results of this study corroborate the chromosome number of 2n=12 described previously for K. cistoidea (Palma Rojas et al., 2017), which is also similar to other six species of the genus described some decades ago (Turner, 1958; Lewis et al., 1962; Teppner, 1984) (<a href="#tab2">Table 2</a>). In this work, quantitative karyotype morphology of one Chilean species of the genus Krameria, K. cistoidea (<a href="#fig1">Figure 1a</a>, <a href="#tab1">Table 1</a>) is reported, which is additional to the data on chromosome number and 2C-value previously reported for one population of the same species (Cuesta El Churque population, Chile) (Palma Rojas et al., 2017). The karyotype of K. cistoidea was uniform among both populations studied. However, at the interspecific level differences in chromosome morphology and size were observed between the karyotype of K. cistoidea and K. triandra from Perú. K. cistoidea, with a metacentric and unimodal karyotype, had all chromosomes with a centromeric index CI between 0.45 and 0.48 (mean CI=0.46±0.012), and a chromosome size that varied between 7.63 and 10.0 μm with an average size of 8.68 μm. In the case of K. triandra the centromeres are located in median or sub-median region as determined by mean qualitative analysis, and the range of chromosome size varied between 10 μm and 14 μm (Teppner, 1984). Such interspecific differences in chromosome morphology and size among these Krameria species may be preliminary evidence on the occurrence of mechanism of chromosome rearrangements (e.g., inversions, duplication, deletions) during the evolution of the genus as it has been described in various other Angiosperm groups (Stebbins, 1971; Levin, 2002). Future comparative karyotype studies in Krameria may give more evidence to corroborate this hypothesis. <a name="tab2" id="tab2"></a>Table 2. Chromosome number for Krameria species. n, gameticchromosome number; 2n, somatic chromosome number. <img src="https://sag.org.ar/jbag/wp-content/uploads/2020/02/a02tab2.jpg" width="296" height="256" /> The chromosome location of constitutive heterochromatin is another additional genome character for the first time studied here for one Krameria species. The banding pattern of both populations of K. cistoidea exhibited large blocks of constitutive heterochromatin, located only in telomeric regions in the short and long arms of the metacentric chromosomes with a symmetrical banding (Greilhuber, 1984). However, due to the large chromosome size (higher to 5.0 μm according to Guerra, 2000), it is possible that the entire constitutive heterochromatin content of the species has not been revealed through this method, as it has also been described in other plant groups (Schweizer and Loidl, 1987; Buitendijk and Ramanna, 1996; Guerra, 2000). In this way, the information on C-bands in Krameria can be a fundamental knowledge for the application of other modern molecular techniques (FISH, GISH, CMA3, and/ or DAPI) focused on describing genome organization, as it has been done in other flowering plants in which complex C-banding patterns have been performed (Joachimiak et al., 1997; Guerra, 2000; Zhou et al., 2003; She et al., 2007; Hamon et al., 2009).  Within the Zygophyllales, comparative karyological studies have been made within the genus Bulnesia belonging to Zygophyllaceae. Bulnesia species with highest (B. retama 2C=4.5 pg, and B. chilensis 2C=2.9 pg) and lowest 2C-values (B. sarmientoi 2C=0.7 pg) possess the most asymmetric karyotype (with metacentric, submetacentric, subtelocentric and telocentric chromosomes), whereas species with intermediate 2C-values (B. foliosa and B. schickendantzii, both with approximately 2C-Values of 1.1 pg) possess most symmetric karyotypes (with metacentric and submetacentric chromosomes) (Poggio et al., 1986). In addition, the species with the highest 2C-values (B. retama and B. chilensis) have the highest constitutive heterochromatin content as reveled by C-banding patterns (Poggio and Hunzkiker, 1986). All these karyotype studies in Bulnesia have been useful to elucidate interesting evolutionary trends within the genus. 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