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DAPI heterochromatin in cultivated and wild Amaranthus species
Scientia Horticulturae 164 (2013) 249–255
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Localization of ribosomal DNA and CMA3/DAPI heterochromatin in cultivated and wild Amaranthus species Bozena Kolano ∗ , Katarzyna Saracka, Anna Broda-Cnota, Jolanta Maluszynska Department of Plant Anatomy and Cytology, University of Silesia, Jagiellonska 28, 40-032 Katowice, Poland
a r t i c l e
i n f o
Article history: Received 26 March 2013 Received in revised form 13 August 2013 Accepted 12 September 2013 Keywords: rDNA Amaranthus Chromosomes FISH CMA/DAPI banding
a b s t r a c t The distributional pattern of GC-rich regions and the physical mapping of ribosomal DNA (location of 35S and 5S rDNA) in the chromosomes of fourteen Amaranthus species were established using fluorochrome banding and fluorescence in situ hybridization (FISH). The karyotypes are composed of 2n = 32 or 2n = 34 chromosomes. Some species were also shown to exhibit infraspecific polymorphism in the chromosome number and consisted of accessions with both of these chromosome numbers. Two families of ribosomal genes, 35S and 5S rDNA, were separated onto different pairs in chromosome complements of most of the species examined. Both 35S and 5S rDNA sites were always located in the terminal part of the chromosomes and usually 35S rDNA sites were present in a lower number than the 5S rDNA sites in most of the species that were analyzed. Polymorphism of the rDNA site number was observed in three species. Fluorochrome banding revealed that CMA+ /DAPI− bands were associated with the 35S rDNA sites in all of the species that were analyzed. In some amaranth accessions, 5S rDNA regions were also GC-rich. The possible mechanisms of the evolution of rDNA loci are discussed. © 2013 Elsevier B.V. All rights reserved.
1. Introduction The genus Amaranthus (family Amaranthaceae) consists of about 60–70 herbaceous species grouped into three subgenera (Acnida, Amaranthus, Albersia) (Mosyakin and Robertson, 1996, 2003). Most of these are annual weeds and only a limited number are of cultivated types (Trucco and Tranel, 2011); however, Amaranths are valued as a source of pseudocereals, vegetables and ornamentals (Sauer, 1967). The current interest in amaranths is based on the fact that they have a high nutritional value due to a higher amount of protein with a balanced essential amino acid content, high photosynthetic efficiency, low input requirements, high yield potential for grain, vegetable and fodder production and a relatively high tolerance to drought, diseases and pests (Trucco and Tranel, 2011). The three most economically important species, A. caudatus, A. cruentes and A. hypochondriacus, belong to the subgenus Amaranthus and have been domesticated for grain production (Mosyakin and Robertson, 2003). The three grain amaranths are classified along with their putative progenitor species (A. hybridus L., A. quitensis H.B.K. and A. powellii S. Wats.) in what is termed the A. hybridus complex (Greizerstein and Poggio, 1994, 1995; Pal et al., 1982). The evolutionary origin of the grain amaranths is still under debate, although two hypotheses were proposed by Sauer (1967). The first
∗ Corresponding author. Tel.: +48 322009468; fax: +48 322562434. E-mail address: [email protected] (B. Kolano). 0304-4238/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.scienta.2013.09.016
hypothesis is based on geography and suggests that all three grain amaranths evolved independently, while the second hypothesis is based on morphological features and proposes that all three grains are descended mainly from A. hybridus. Molecular studies support Sauer’s second hypothesis of a monophyletic evolution of each of the three grain amaranth species from A. hybridus but also suggest an alternative hypothesis to explain their origins – multiple, independent domestication events from geographically diverse populations of A. hybridus (Chan and Sun, 1997; Mallory et al., 2008; Transue et al., 1994; Xu and Sun, 2001) To date cytological investigations of Amaranthus species have been somewhat random and have mainly been restricted to chromosome number reports. Two gametic numbers have been reported in this genus (n = 16, n = 17) and in some cases both numbers occur in the same species (Greizerstein and Poggio, 1994; http://www.tropicos.org/Project/IPCN). Amaranths themselves appear to be paleopolyploid and their genomes behave like diploids during meiosis with the exception of Amaranthus dubius, an allotetraploid with n = 32 (Pal et al., 1982). The results of the few karyotype structure and evolution studies that have been published showed that Amaranthus chromosomes were very small (ca. 1 m); however, Greizerstein and Poggio (1994) were able to show that amaranth karyotypes consisted of metacentric, submetacentric and telocentric chromosomes, possessed one pair of NOR chromosomes and demonstrated a wide variety of C banding patterns. Small B chromosomes were reported in some individuals of A. caudatus (Greizerstein and Poggio, 1994).
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A serious weakness in the analysis of the Amaranthus karyotype is the paucity of chromosome markers, which has limited the identification of the chromosome pairs and makes a comparative analysis very difficult. To date, fluorescent in situ hybridization analysis has only been done for A. caudatus, where two or four sites of 35S rDNA were observed depending on the accession (Kolano et al., 2001). Techniques such as chromosome banding or fluorescent in situ hybridization (FISH) have proven to be useful tools for additional chromosome differentiation, often allowing for conclusions about chromosomal rearrangements (Morales et al., 2012; Sheng and Wang, 2010; Weiss-Schneeweiss and Schneeweiss, 2003; Weiss-Schneeweiss et al., 2008). In an evolutionary context, the markers most often used, especially in non-model organisms, are the 5S and 35S ribosomal genes due to their abundance as ‘house-keeping genes’ and their relatively conserved nature (Maluszynska et al., 1998). They have been used for the construction of physical maps of chromosomes and for phylogenetic studies in many plant species (Hasterok et al., 2006; Morales et al., 2012; Weiss-Schneeweiss et al., 2008). Thus, there is a clear need to find out more about the karyotype structure in the Amaranthus genus. The present study used fluorochrome banding to characterize AT- and GC-rich chromosome regions and FISH to obtain the patterns of rRNA gene loci distribution in this genus as well as differentiation among species. In order to contribute to the understanding of the evolution of the rDNA repetitive fraction in Amaranthus species, the number and localization of 5S and 35S rDNA sites in 20 accessions of 14 species was determined. Additionally, double staining with CMA and DAPI was used to identify the spatial relationships between rDNA sites and CMA+ bands. The results are discussed in relation to the chromosomal evolution of the genus. 2. Materials and methods 2.1. Plant material All of the Amaranthus accessions analyzed in this study are listed in Table 1. Plants were cultivated in pots in the greenhouse of the Silesian University. Shoot meristems and young leaves were pretreated with 2 mM 8-hydroxyquinoline for 4 h at 20◦ C, fixed in ethanol/acetic acid (3:1) for at least 12 h and stored in fixative at −20◦ C until used. 2.2. Chromosome preparation Metaphase spreads were prepared as described earlier (Kolano et al., 2011) with minor modifications. The fixed materials were rinsed thoroughly with a citric buffer and macerated in an enzyme mixture containing 4% cellulase “Onozuka R-10” (Serva, Heidelberg, Germany) and 20% pectinase (Sigma, St. Louis, USA) for 4 h at 37 ◦ C. After pelleting, the protoplasts were washed three times with 45% acetic acid. The pellet was resuspended in 20–30 l of 45% acetic acid and incubated 1 min at 65 ◦ C then 5 l of the suspension was applied to a slide and covered with a coverslip. After freezing, the coverslips were removed and the preparations were immediately post-fixed in cold ethanol–glacial acetic (3:1), dehydrated in absolute ethanol and air-dried. 2.3. Fluorochrome banding Double fluorescent staining with chromomycin A3 (CMA) and 4 ,6-diamidino-2-phenylindole (DAPI) was used, following the techniques of Schweizer (1976) with minor modifications. Slides were stained with 0.5 mg/ml of CMA (Serva, Heidelberg, Germany) in a McIlvaine buffer, pH 7.0 containing 2.5 mM MgCl2 for 1.5 h, briefly rinsed in distilled water, air dried and mounted
Table 1 Origins, chromosome numbers, numbers of 5S and 35S rDNA sites and thee number CMA3 + bands of the 20 accessions of the Amaranthus species studied. Taxon
Cultivar/Accession number
Number of rDNA site CMA+
2n
35S
Kiwicha 3 Kiwicha Molinera ornamental form 12
34 32 32 32
2 4 2 2
6 8 8 12
2 4 2 2
Ames 5324 95
32 32
6 2
10 10
6 2
b
Ames 15197 120 Pygmy Torch PI 604570 92 PI 615696
34 32 32 34 32 32
2 2 2 2 2 2
8 8 6 8 8 8
2 4 4 2 4 2
b
Ames 15306
34
2
8
2
b
Section: Dubia Amaranthus dubius
PI 642737
34
2
6
2
b
Section Centrusa Amaranthus spinosus
PI 500293
34
4
2*
4
b
PI 606281
34
2
4
6
b
Ames 5150
34
2
2
2
b
87 PI 608661
32 34
4 2
2 2*
6 2
Ames 5160
34
2
6
2
Subgenus: Amaranthus Section: Amaranthus Subsection Amaranthus Amaranthus caudatus
Amarantus caudatus var. purpurosum Amaranthus quitensis Amarantus retroflexus Subsection: Hybrida Amaranthus cruentus
Amaranthus hybridus Amaranthus hypochondriacus Amaranthus powellii subsp. powellii
Subgenus Albersia Section Blitopsis Amaranthus blitum subsp. oleraceus Amaranthus viridis Section Pyxidium Amaranthus albus Amaranthus graecizans subsp. silvestris Amarantus tricolor
5S
Origin
c c d e
a
f g b a b
a b
b
*
Colocalization of 35S and 5S rDNA loci. Botanical Garden Berlin, Dahlem, Germany. b USDA North Central Regional Plant Introduction Station of the US National Plant Germplasm System. c Seeds kindly provided by Dr. Luz Gomez Pando from National Agrarian University – La Molina, Lima, Peru. d Torseed – Seed Company, Torun, Poland. e BG & Museum Natural History Museum of Denmark, Copenhagen, Denmark. f The Botanical Garden of the University of Coimbra, Portugal. g PNOS – Seed Company Ozarów Mazowiecki, Poland. a
in Vectashield (Vector Laboratories, Peterborough, UK) containing 2 g/ml DAPI (Serva, Heidelberg, Germany). Then the slides were incubated at 37 ◦ C for 4–5 days prior to examination. After taking the images, the slides were washed in distilled water, de-stained in ethanol–glacial acetic (3:1), dehydrated in absolute ethanol and used for FISH.
2.4. DNA probe labeling and fluorescent in situ hybridization The probe used for the detection of 35S rRNA gene sites was a 2.3 kb fragment of the 25S rDNA coding region from Arabidopsis thaliana (Unfried and Gruendler, 1990), labeled with dioxygenin-11-dUTP using nick translation. For detection of 5S rDNA sites, a 410 bp clone isolated from Triticum aestivum (Gerlach and Dyer, 1980) was amplified and labeled with rhodamine-4-dUTP using PCR as described (Kolano et al., 2008). Fluorescence in situ hybridization was performed according to the
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protocols described by Schwarzacher and Heslop-Harrison (2000) with minor modifications. Briefly, the hybridization mixture – consisting of 100 ng of labeled DNA probe, 50% formamide, 10% dextran sulfate, 0.1% SDS and 10 g of sheared salmon sperm DNA – was denatured for 10 min at 85 ◦ C, then chilled on ice and applied to the chromosome preparation. The slides and hybridization mixture were denatured together at 72 ◦ C for 5 min in an in situ Thermal Cycler (Thermo Hybaid, Franklin, USA) and then allowed to hybridize for 48–72 h in a humid chamber at 37 ◦ C. Stringent washes (10% formamide in 0.1× SSC at 37 ◦ C) were followed by the immunodetection of digoxigenin-labeled DNA probe using fluorescein-conjugated primary anti-digoxigenin antibodies and signal amplification with FITC-conjugated anti-sheep secondary antibodies (Jackson ImmunoResearch, Suffolk, UK). The preparations were mounted in Vectashield (Vector Laboratories, Peterborough, UK) containing 2 g/ml DAPI.
3. Results The Amaranthus species that were analyzed revealed the chromosome number 2n = 32 or 2n = 34. Intraspecific variations of the chromosome number were observed for three species: A. caudatus, A. cruentes and A. hybridus. Depending on the accessions, 2n = 32 or 2n = 34 chromosomes were observed. The chromosome number of the analyzed amaranths are summarized in Table 1. The distribution of rRNA gene sites was analyzed using FISH with 5S and 25S rDNA as probes. The results of the double-target FISH to the mitotic metaphase of the Amaranthus species are presented in Figs. 1 and 2 and the total numbers of 5S rDNA and 35S rDNA sites in 20 accessions of the 14 species studied are summarized in Table 1. In most of the Amaranthus accessions that were analyzed, hybridization signals of 35S rDNA and 5S rDNA were observed in separate chromosomes; however, two species, A. spinosus and A. greacizans subsp. silvestris, showed a co-localization between 35S rDNA and 5S rDNA sites (Figs. 1S and 2F). In all chromosomes carrying 35S rDNA loci or/and 5S rDNA loci these were observed in terminal position (Figs. 1 and 2). Most of the species from the subgenus Amaranthus sec. Amaranthus that were analyzed exhibited two sites of 35S rDNA. The only exceptions were A. quitensis, which had six sites, and one accession of A. caudatus, which had four sites of 35S rDNA (Table 1), both from the subsection Amaranthus. The number of 5S rDNA sites was relatively most abundant among amaranths from the sect. Amaranthus. In this section between 6 and 12 sites of 5S rDNA were observed depending on the species; however, those with eight sites were present more often (Fig. 1, Table 1). In the subsection Hybrida nearly all accessions showed eight sites of 5S rDNA although species from the subsection Amaranthus exhibited a much more variable pattern of 5S rDNA sites. In accessions with more than two sites of 35S rDNA, one pair of sites was major with much more intense hybridization signals and the rest were minor. Also, in the case of 5S rDNA, major and minor sites could be distinguished in every accession of amaranths from the sect. Amaranthus (Fig. 1). Two species from the section Amaranthus showed intraspecific polymorphism in loci number. The A. caudatus accessions that were analyzed showed polymorphism in a number of 35S rDNA sites and 5S rDNA. Each analyzed accession revealed a different pattern of rDNA sites (Table 1, Fig. 1A–G). A. cruentus showed polymorphism in a number of 5S rDNA loci and depending on the accession six or eight loci were observed (Table 1, Fig. 1H–K). The next two sections, Dubia and Centrusa (subgenus Amaranthus), were represented by one species each. A. dubius exhibited two sites of 35S rDNA and six sites of 5S rDNA (Fig. 1R). A. spinosus (sec. Centrusa) revealed the lowest number of 5S rDNA sites among the analyzed species from the subgenus Amaranthus. This species revealed only two sites of 5S rDNA and four sites of 35S rDNA. Interestingly, two
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sites of 35S rDNA were colocalised with 5S rDNA sites in A. spinosus (Fig. 1S). The second studied subgenus Albersia was represented by five species belonging to two sections: Blitopsis and Pyxidium (Table 1). These species showed a relatively lower abundance of 5S rDNA loci when compared with the species from the subgenus Amaranthus (Fig. 2). 5S rDNA sequences were most often localized in two sites, except for A. blitum, which had four sites and A. tricolor which had six sites of 5S rDNA (Fig. 2, Table 1). In subgenus Albersia in one karyotype the size of the hybridization signals for different 5S rDNA sites were similar, in contrast to the subgenus Amaranthus (Figs. 1 and 2). The hybridization signals of 35S rDNA were only observed on one pair of chromosomes of most of the species from the subgenus Albersia that were analyzed except for A. albus, which had two pairs of sites that showed an equal intensity of hybridization signals. The sites of rDNA sequences were located on separate chromosomes except for A. graecizans subsp. silvestris. In this genome, the loci of 35S and 5S rDNA were located in two sites in the same chromosome and the hybridization signals of the two rRNA genes were located adjacent to each other at the most terminal position of 5S rDNA sites (Fig. 2F). Double fluorescent staining with DAPI/CMA is used to localize the chromosome regions that are rich in AT and GC base pairs, respectively (Schweizer, 1976). In all of the species examined, no DAPI+ bands were found, although DAPI− bands that corresponded to CMA+ regions were observed. After CMA/DAPI staining, all of the species revealed CMA+ /DAPI− bands (i.e. brighter with CMA and duller with DAPI) in the terminal part of chromosomes. The number of CMA+ /DAPI− bands per karyotype differed among analyzed accessions and two, four or six bands were observed depending on the accession (Figs. 1B, D, G and I, 2B and 3). When more than one pair of CMA+ /DAPI− bands were observed, one pair of chromosomes carried brighter bands than the other two (Figs. 1D and I and 3B, G, L and O) except for A. albus, which had six CMA+ bands of approximately equal brightness. The maximum number, six CMA+ /DAPI− bands, was observed in three accessions (A. quitensis A. blitum subsp. oleraceus and A. albus); however, two CMA+/DAPI− bands were present most often (Table 1). In many of the analyzed accessions, CMA+ /DAPI− bands colocolised with a secondary construction. (e.g. Fig. 3D and H). Sequential CMA/DAPI banding and FISH with rDNA sequences were performed for some of the species and these indicated that the CMA+ /DAPI− bands colocalised with 35S rDNA sites (Figs. 1A–I and 2A and B). Some species, however, showed more CMA+ /DAPI− bands than the number of 35S rDNA sites (two accessions of A. cruentes, A. spinosus, A. blitum and A. albus) (Table 1). Sequential CMA/DAPI banding and FISH with rDNA sequences were performed for selected species and these indicated that the CMA+ /DAPI− bands colocalised not only with 35S rDNA sites but also could colocalise with 5S rDNA sites (Fig. 1H and I and 2A and B). 4. Discussion Somatic chromosome numbers of 32 or 34 were observed for the analyzed amaranth accessions and they are mainly in accordance with those available at the Index to Plant Chromosome Number (www.tropicos.org/Project/IPCN) or those that were reported earlier by Grant (1959). The exception was A. dubious, which according to Grant (1959) was a polyploid species with 64 chromosomes, however the accession analyzed in this study has 34 chromosomes. The basic chromosome numbers in the genus are n = 16 and n = 17 and both numbers are found in the subgenus Amaranthus as well as in the subgenus Albersia. Pal et al. (1982), based on a cytogenetic analysis of interspecific hybrid, suggested that the gametic number n = 17 originated from the n = 16 through primary trisomy. Cytogenetic research has demonstrated that a variation between n = 16
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Fig. 1. Distribution of 5S (red fluorescence; the smallest 5S rDNA sites were marked by red arrows) and 35S (green fluorescence) ribosomal DNA sites and CMA+ bands (yellow fluorescence) in Amaranthus species from subgenus Amaranthus: A. caudatus Kiwicha 3 (A and B); A. caudatus Kiwicha Molinera (C and D); A. caudatus var. purpurosum (E); A. caudatus ornamental form (F and G); A. cruentus Pygmy Torch (H and I); A. cruentus Ames 15,197 (J); A. cruentus 120 (K); A. hypochondriacus (L); A. powellii subsp. powellii (M); A. quitensis (N); A. hybridus PI 604570 (O); A. hybridus 92 (P); A. retroflexus (Q); A. dubius (R); A. spinosus (S). White arrows: CMA+ bands associated with 35S rDNA sites; white arrowheads: CMA+ bands associated with 5S rDNA sites. Scale bar = 5 m.
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Fig. 2. Distribution of 5S (red fluorescence; the smallest 5S rDNA sites were marked by red arrows) and 35S (green fluorescence) ribosomal DNA sites and CMA+ bands (yellow fluorescence) in Amaranthus species from subgenus Albersia: A. albus (A and B); A. blitum subsp. oleraceus (C); A. viridis (D); A. tricolor (E); A. graecizans subsp. silvestris (F). White arrows: CMA+ bands associated with 35S rDNA sites; white arrowheads: CMA+ bands associated with 5S rDNA sites. Scale bar = 5 m.
and n = 17 can occur even within one species (e.g. accessions of A. caudatus, A. cruentus and A. hybridus). Earlier, intraspecific polymorphism in chromosome numbers was also reported for Amaranths by Grant (1959) and Popenoe et al. (1989). Amaranthus species have rather small chromosomes, which in many cases are similar in shape and size, and are therefore difficult to distinguish. For this reason, additional landmarks are required in order to enable chromosome identification. rDNA sequences can be localized by FISH and can provide markers that are useful for the identification of individual chromosomes. A comparison of the chromosome number and the distribution of rDNA sites can provide valuable information about the phylogenetic relationship between related taxa, for example, in various Brassicaceae (Hasterok et al., 2006), Hypocheris (Weiss-Schneeweiss and Schneeweiss, 2003) and Paphiopedilum species (Lan and Albert, 2011). The present report gives the first description of rDNA localization for 11 species and confirms the mapping of 35S rDNA sites in A. caudatus, A. cruentus and A. hypochondriacus (Kolano et al., 2001; Bonasora et al., 2013). The chromosomal positions of 35S and 5S rDNA sites are relatively conservative among species analyzed. Most of them showed 35S rDNA and 5S rDNA signals in the terminal position of chromosome arms and most often 35S rDNA sites and 5S rDNA sites were located on different chromosomes. There were only two exceptions, A. spinosus and A. greacizans subsp. silvestris, where the one pair of 35S rDNA sites colocalised with 5S rDNA sites in one chromosome pair. These more unique patterns of rDNA distribution might be a trace of chromosomal rearmaments or could be connected with translocation events during the evolution of the species (Raskina et al., 2008; Thomas et al., 2001). The analyzed Amaranthus species showed a high variability in the number of rDNA sites. Simultaneous FISH with 5S and 35S rDNA probes identified between one and eight chromosome pairs carrying rDNA sequences depending on the accessions. This phenomenon is commonly observed in many different plant genera including Brassica Paphiopedilum, Aristolochia (Berjano et al., 2009; Hasterok et al., 2006; Lan and Albert, 2011). However, the strong conservation of rDNA site number has been described in many plant species or even in an entire genera, e.g. Glycine and Daucus (Iovene et al., 2008; Singh et al.,
2001). In general, the FISH patterns of 35S rDNA loci are reported to be more polymorphic than those of the 5S rDNA (Adams et al., 2000; Datson and Murray, 2006; Kwon and Kim, 2009). In contrast, Amaranthus species showed a higher variability in the number of 5S rDNA; however, differences in the number of 35S rDNA sites were also observed. Similarly, higher diversification of 5S rDNA distribution patterns and lower degree of numeric variation in 35S rDNA sites were also observed for Paphilopedilum and Byblis liniflora complex (Fukushima et al., 2011; Lan and Albert, 2011). Generally, the number of 5S rDNA loci was higher in Amaranths from the subsection Amaranthus and Hybrida (subgenus Amaranthus, section Amaranthus) than in species from other taxa that were analyzed. The accessions from the Amaranthus subsection also showed a very high numeric variability in the number of 5S rDNA sites, whereas it could be shown that for the four species of the subsection Hybrida it was relatively stable in relation to the number of rDNA sites. In addition, intraspecific polymorphism in the number of both 5S and 35S rDNA sites was shown in a few species: A. caudatus, A. cruentus. The phenomenon of numerical variation in rDNA sites has been observed in several plant species (Dydak et al., 2009; Kolano et al., 2012; Ksiazczyk et al., 2010;), with a fourfold variation in the number of 35S rRNA loci being reported within the diploid Phaseolus vulgaris as an extreme example (Pedrosa-Harand et al., 2006). Variation in the number and location of rDNA sites could indicate their mobility in the recent evolutionary history of the species. Different mechanisms have been postulated to account for this phenomena and polymorphism in the number, size and position of rDNA sites, such as a transposon-mediated transpositional event or chromosome rearrangements caused by a homologue or a non-homologous unequal crossing-over and gene conversion (Altinkut et al., 2006; Datson and Murray, 2006; Raskina et al., 2008; Thomas et al., 2001). Thus, the high polymorphism of the chromosomal organization of the rDNA clusters that was observed among the Amaranthus accession could also indirectly suggest a relatively high level of microevolutionary genomic changes during speciation within this genus. It is obvious that speciation-related chromosomal repatterning might further increase/decrease the number of rDNA sites or their
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Fig. 3. CMA/DAPI fluorescent stained chromosome complements of Amaranthus species: A. caudatus var. purpurosum (A); A. quitensis (B); A. retroflexus (C); A. cruentus Ames 15197 (D); A. cruentus 120 (E); A. hypochondriacus (F); A. hybridus 92 (G); A. hybridus PI 604570 (H); A. powellii subsp. powellii (I); A. dubius (J); A. viridis (K); A. blitum subsp. oleraceus (L); A. greacizans subsp. silvestris (M); A. tricolor (N); A. spinosus (O). DAPI – blue fluorescence, CMA3 – yellow fluorescence. The arrows point out CMA+ bands. Scale bar = 5 m. (For interpretation of the references to color in the artwork, the reader is referred to the web version of the article.)
repositioning. The dynamics of rDNA clusters may also be regarded as a strong indicator for significant intra-genomic processes (Jiang and Gill, 1994; Raskina et al., 2004). Double fluorescent staining with DAPI/CMA was used to localize chromosome regions rich in AT and GC base pairs, respectively (Schweizer, 1976). The organization of GC rich DNA is similar in all of the amaranths that were analyzed. CMA+ /DAPI− bands were located in the terminal position in the chromosomes of all of the analyzed accessions and could be associated with rDNA sites, both 35S and 5S. Most of the analyzed Amaranthus accessions showed the same number of 35S rDNA sites as the number CMA+/ DAPI− bands, which were usually observed in a secondary construction. As was indicated for A. caudatus, the polymorphism of the number of CMA+ /DAPI− bands corresponds to the polymorphism in the 35S rDNA sites. So for most analyzed accessions the regions occupied by genes for 35S rRNA were the only large GC rich blocks
of chromatin as was shown earlier for many plants (Moraes and Guerra, 2010). Other amaranth accessions showed a higher number of CMA+ /DAPI− bands than the number of 35S rDNA loci. As was shown for A. albus and A. cruentus, the CMA+ bands could correspond not only to 35S rDNA sites but also to 5S rDNA sites. However, not all 5S rDNA sites in A. cruentus Pygmy Torch correspond to CMA+ bands, possibly because the sites were too small to be detected with CMA. The association of GC-rich regions with 35S rDNA sites is commonly found in plants (Dydak et al., 2009; Guerra, 2000; Penas et al., 2009) although there is little data on the occurrence of such heterochromatin with 5S rRNA genes being available for plants since few studies have simultaneously combined banding techniques and 5S rDNA in situ hybridization (Cabral et al., 2006; Hajdera et al., 2003; Hamon et al., 2009; Sijak-Yakovev et al., 2003). This could reflect both the composition of the 5S rDNA sequences and the nature of the adjacent heterochromatin.
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The amount and localization of rDNA sequences and CMA+ /DAPI− bands seem to be useful cytological markers and allowed us to identify between one and eight chromosome pairs in Amaranthus. All analyzed accessions showed similar number of chromosomes (2n = 32 or 2n = 34) however their numbers of rDNA sites and CMA+ /DAPI− bands were very different. The interspecific as well as intraspecific karyotypic differences in the number of rDNA sites and CMA+ /DAPI− bands suggest, that divergence and speciation in this genus was accompanied by genome restructuring, and that reduction and possible gains in rDNA sites provided one of the mechanisms. References Adams, S.P., Leitch, I.J., Bennet, M.D., Chase, M.W., Leitch, A.R., 2000. Ribosomal DNA evolution and phylogeny in Aloe (Asphodelaceae). Am. J. Bot. 87, 1578–1583. Altinkut, A., Raskina, O., Nevo, E., Belyayev, A., 2006. En/Spm-like transposons in Poaceae species: transposase sequence variability and chromosomal distribution. Cell Mol. Biol. Lett. 11, 214–229. Berjano, R., Roa, F., Talavera, S., Guerra, M., 2009. Cytotaxonomy of diploid and polyploid Aristolochia (Aristolochiaceae) species based on the distribution of CMA/DAPI bands and 5S and 45S rDNA sites. Plant Syst. Evol. 280, 219–227. Bonasora, M.G., Poggio, L., Greizerstein, E.J., 2013. Cytogenetic studies in four cultivated Amaranthus (Amaranthaceae) species. Comp. Cytogenet. 7, 53–61. Cabral, J.S., Felix, L.P., Guerra, M., 2006. Heterochromatin diversity and its colocalization with 5S and 45S rDNA sites in chromosomes of four Maxillaria species (Orchidaceae). Genet. Mol. Biol. 29, 659–664. Chan, K.F., Sun, M., 1997. Genetic diversity and relationships detected by isozyme and RAPD analysis of crop and wild species Amaranthus. Theor. Appl. Genet. 95, 865–873. Datson, P.M., Murray, B.G., 2006. Ribosomal DNA locus evolution in Nemesia: transposition rather than structural rearrangement as the key mechanism? Chromosome Res. 14, 845–857. Dydak, M., Kolano, B., Nowak, T., Siwinska, D., Maluszynska, J., 2009. Cytogenetic studies of three European species of Centaurea L. (Asteraceae). Hereditas 146, 152–161. Fukushima, K., Imamura, K., Nagano, K., Hoshi, Y., 2011. Contrasting patterns of the 5S and 45S rDNA evolutions in the Byblis liniflora complex (Byblidaceae). J. Plant Res. 124, 231–244. Gerlach, W.L., Dyer, T.A., 1980. Sequence organization of the repeating units in the nucleus of wheat which contain 5S ribosomal-RNA genes. Nucleic Acids Res. 8, 4851–4865. Grant, W., 1959. Cytogenetic studies in Amaranthus III. Chromosome numbers and phylogenetic aspects. Can. J. Bot. 1, 313–328. Greizerstein, E., Poggio, L., 1994. Karyological Studies in grain Amaranthus. Cytologia 59, 25–30. Greizerstein, E., Poggio, L., 1995. Meiotic studies of spontaneous hybrids of Amaranthus: genome analysis. Plant Breed. 114, 448–450. Guerra, M., 2000. Patterns of heterochromatin distribution in plant chromosomes. Genet. Mol. Biol. 23, 1029–1041. Hajdera, I., Siwinska, D., Hasterok, R., Maluszynska, J., 2003. Molecular cytogenetic analysis of genome structure in Lupinus angustifolius and Lupinus cosentinii. Theor. Appl. Genet. 107, 988–996. Hamon, P., Siljak-Yakovlev, S., Srisuwan, S., Robin, O., Poncet, V., Hamon, S., de Kochko, A., 2009. Physical mapping of rDNA and heterochromatin in chromosomes of 16 Coffea species: a revised view of species differentiation. Chromosome Res. 17, 291–304. Hasterok, R., Wolny, E., Hosiawa, M., Kowalczyk, M., Kulak-Ksiazczyk, S., Ksiazczyk, T., Heneen, W.K., Maluszynska, J., 2006. Comparative analysis of rDNA distribution in chromosomes of various species of Brassicaceae. Ann. Bot. 97, 205–216. Iovene, M., Grzebelus, E., Carputo, D., Jiang, J.M., Simon, P.W., 2008. Major cytogenetic landmarks and karyotype analysis in Daucus carota and other Apiaceae. Am. J. Bot. 95, 793–804. Jiang, J.M., Gill, B.S., 1994. New 18S-26S ribosomal-RNA gene loci – chromosomal landmarks for the evolution of polyploid wheats. Chromosoma 103, 179–185. Kolano, B., Gardunia, B.W., Michalska, M., Bonifacio, A., Fairbanks, D., Maughan, P.J., Coleman, C.E., Stevens, M.R., Jellen, E.N., Maluszynska, J., 2011. Chromosomal localization of two novel repetitive sequences isolated from the Chenopodium quinoa Willd. genome. Genome 54, 710–717. Kolano, B., Pando, L.G., Maluszynska, J., 2001. Molecular cytogenetic studies in Chenopodium quinoa and Amaranthus caudatus. Acta Soc. Bot. Pol. 70, 85–90. Kolano, B., Plucienniczak, A., Kwasniewski, M., Maluszynska, J., 2008. Chromosomal localization of a novel repetitive sequence in the Chenopodium quinoa genome. J. Appl. Genet. 49, 313–320. Kolano, B., Tomczak, H., Molewska, R., Jellen, E.N., Maluszynska, J., 2012. Distribution of 5S and 35S rRNA gene sites in 34 Chenopodium species (Amaranthaceae). Bot. J. Linn. Soc. 170, 220–231.
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Localization of ribosomal DNA and CMA3/DAPI heterochromatin in cultivated and wild Amaranthus species Bozena Kolano ∗ , Katarzyna Saracka, Anna Broda-Cnota, Jolanta Maluszynska Department of Plant Anatomy and Cytology, University of Silesia, Jagiellonska 28, 40-032 Katowice, Poland
a r t i c l e
i n f o
Article history: Received 26 March 2013 Received in revised form 13 August 2013 Accepted 12 September 2013 Keywords: rDNA Amaranthus Chromosomes FISH CMA/DAPI banding
a b s t r a c t The distributional pattern of GC-rich regions and the physical mapping of ribosomal DNA (location of 35S and 5S rDNA) in the chromosomes of fourteen Amaranthus species were established using fluorochrome banding and fluorescence in situ hybridization (FISH). The karyotypes are composed of 2n = 32 or 2n = 34 chromosomes. Some species were also shown to exhibit infraspecific polymorphism in the chromosome number and consisted of accessions with both of these chromosome numbers. Two families of ribosomal genes, 35S and 5S rDNA, were separated onto different pairs in chromosome complements of most of the species examined. Both 35S and 5S rDNA sites were always located in the terminal part of the chromosomes and usually 35S rDNA sites were present in a lower number than the 5S rDNA sites in most of the species that were analyzed. Polymorphism of the rDNA site number was observed in three species. Fluorochrome banding revealed that CMA+ /DAPI− bands were associated with the 35S rDNA sites in all of the species that were analyzed. In some amaranth accessions, 5S rDNA regions were also GC-rich. The possible mechanisms of the evolution of rDNA loci are discussed. © 2013 Elsevier B.V. All rights reserved.
1. Introduction The genus Amaranthus (family Amaranthaceae) consists of about 60–70 herbaceous species grouped into three subgenera (Acnida, Amaranthus, Albersia) (Mosyakin and Robertson, 1996, 2003). Most of these are annual weeds and only a limited number are of cultivated types (Trucco and Tranel, 2011); however, Amaranths are valued as a source of pseudocereals, vegetables and ornamentals (Sauer, 1967). The current interest in amaranths is based on the fact that they have a high nutritional value due to a higher amount of protein with a balanced essential amino acid content, high photosynthetic efficiency, low input requirements, high yield potential for grain, vegetable and fodder production and a relatively high tolerance to drought, diseases and pests (Trucco and Tranel, 2011). The three most economically important species, A. caudatus, A. cruentes and A. hypochondriacus, belong to the subgenus Amaranthus and have been domesticated for grain production (Mosyakin and Robertson, 2003). The three grain amaranths are classified along with their putative progenitor species (A. hybridus L., A. quitensis H.B.K. and A. powellii S. Wats.) in what is termed the A. hybridus complex (Greizerstein and Poggio, 1994, 1995; Pal et al., 1982). The evolutionary origin of the grain amaranths is still under debate, although two hypotheses were proposed by Sauer (1967). The first
∗ Corresponding author. Tel.: +48 322009468; fax: +48 322562434. E-mail address: [email protected] (B. Kolano). 0304-4238/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.scienta.2013.09.016
hypothesis is based on geography and suggests that all three grain amaranths evolved independently, while the second hypothesis is based on morphological features and proposes that all three grains are descended mainly from A. hybridus. Molecular studies support Sauer’s second hypothesis of a monophyletic evolution of each of the three grain amaranth species from A. hybridus but also suggest an alternative hypothesis to explain their origins – multiple, independent domestication events from geographically diverse populations of A. hybridus (Chan and Sun, 1997; Mallory et al., 2008; Transue et al., 1994; Xu and Sun, 2001) To date cytological investigations of Amaranthus species have been somewhat random and have mainly been restricted to chromosome number reports. Two gametic numbers have been reported in this genus (n = 16, n = 17) and in some cases both numbers occur in the same species (Greizerstein and Poggio, 1994; http://www.tropicos.org/Project/IPCN). Amaranths themselves appear to be paleopolyploid and their genomes behave like diploids during meiosis with the exception of Amaranthus dubius, an allotetraploid with n = 32 (Pal et al., 1982). The results of the few karyotype structure and evolution studies that have been published showed that Amaranthus chromosomes were very small (ca. 1 m); however, Greizerstein and Poggio (1994) were able to show that amaranth karyotypes consisted of metacentric, submetacentric and telocentric chromosomes, possessed one pair of NOR chromosomes and demonstrated a wide variety of C banding patterns. Small B chromosomes were reported in some individuals of A. caudatus (Greizerstein and Poggio, 1994).
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A serious weakness in the analysis of the Amaranthus karyotype is the paucity of chromosome markers, which has limited the identification of the chromosome pairs and makes a comparative analysis very difficult. To date, fluorescent in situ hybridization analysis has only been done for A. caudatus, where two or four sites of 35S rDNA were observed depending on the accession (Kolano et al., 2001). Techniques such as chromosome banding or fluorescent in situ hybridization (FISH) have proven to be useful tools for additional chromosome differentiation, often allowing for conclusions about chromosomal rearrangements (Morales et al., 2012; Sheng and Wang, 2010; Weiss-Schneeweiss and Schneeweiss, 2003; Weiss-Schneeweiss et al., 2008). In an evolutionary context, the markers most often used, especially in non-model organisms, are the 5S and 35S ribosomal genes due to their abundance as ‘house-keeping genes’ and their relatively conserved nature (Maluszynska et al., 1998). They have been used for the construction of physical maps of chromosomes and for phylogenetic studies in many plant species (Hasterok et al., 2006; Morales et al., 2012; Weiss-Schneeweiss et al., 2008). Thus, there is a clear need to find out more about the karyotype structure in the Amaranthus genus. The present study used fluorochrome banding to characterize AT- and GC-rich chromosome regions and FISH to obtain the patterns of rRNA gene loci distribution in this genus as well as differentiation among species. In order to contribute to the understanding of the evolution of the rDNA repetitive fraction in Amaranthus species, the number and localization of 5S and 35S rDNA sites in 20 accessions of 14 species was determined. Additionally, double staining with CMA and DAPI was used to identify the spatial relationships between rDNA sites and CMA+ bands. The results are discussed in relation to the chromosomal evolution of the genus. 2. Materials and methods 2.1. Plant material All of the Amaranthus accessions analyzed in this study are listed in Table 1. Plants were cultivated in pots in the greenhouse of the Silesian University. Shoot meristems and young leaves were pretreated with 2 mM 8-hydroxyquinoline for 4 h at 20◦ C, fixed in ethanol/acetic acid (3:1) for at least 12 h and stored in fixative at −20◦ C until used. 2.2. Chromosome preparation Metaphase spreads were prepared as described earlier (Kolano et al., 2011) with minor modifications. The fixed materials were rinsed thoroughly with a citric buffer and macerated in an enzyme mixture containing 4% cellulase “Onozuka R-10” (Serva, Heidelberg, Germany) and 20% pectinase (Sigma, St. Louis, USA) for 4 h at 37 ◦ C. After pelleting, the protoplasts were washed three times with 45% acetic acid. The pellet was resuspended in 20–30 l of 45% acetic acid and incubated 1 min at 65 ◦ C then 5 l of the suspension was applied to a slide and covered with a coverslip. After freezing, the coverslips were removed and the preparations were immediately post-fixed in cold ethanol–glacial acetic (3:1), dehydrated in absolute ethanol and air-dried. 2.3. Fluorochrome banding Double fluorescent staining with chromomycin A3 (CMA) and 4 ,6-diamidino-2-phenylindole (DAPI) was used, following the techniques of Schweizer (1976) with minor modifications. Slides were stained with 0.5 mg/ml of CMA (Serva, Heidelberg, Germany) in a McIlvaine buffer, pH 7.0 containing 2.5 mM MgCl2 for 1.5 h, briefly rinsed in distilled water, air dried and mounted
Table 1 Origins, chromosome numbers, numbers of 5S and 35S rDNA sites and thee number CMA3 + bands of the 20 accessions of the Amaranthus species studied. Taxon
Cultivar/Accession number
Number of rDNA site CMA+
2n
35S
Kiwicha 3 Kiwicha Molinera ornamental form 12
34 32 32 32
2 4 2 2
6 8 8 12
2 4 2 2
Ames 5324 95
32 32
6 2
10 10
6 2
b
Ames 15197 120 Pygmy Torch PI 604570 92 PI 615696
34 32 32 34 32 32
2 2 2 2 2 2
8 8 6 8 8 8
2 4 4 2 4 2
b
Ames 15306
34
2
8
2
b
Section: Dubia Amaranthus dubius
PI 642737
34
2
6
2
b
Section Centrusa Amaranthus spinosus
PI 500293
34
4
2*
4
b
PI 606281
34
2
4
6
b
Ames 5150
34
2
2
2
b
87 PI 608661
32 34
4 2
2 2*
6 2
Ames 5160
34
2
6
2
Subgenus: Amaranthus Section: Amaranthus Subsection Amaranthus Amaranthus caudatus
Amarantus caudatus var. purpurosum Amaranthus quitensis Amarantus retroflexus Subsection: Hybrida Amaranthus cruentus
Amaranthus hybridus Amaranthus hypochondriacus Amaranthus powellii subsp. powellii
Subgenus Albersia Section Blitopsis Amaranthus blitum subsp. oleraceus Amaranthus viridis Section Pyxidium Amaranthus albus Amaranthus graecizans subsp. silvestris Amarantus tricolor
5S
Origin
c c d e
a
f g b a b
a b
b
*
Colocalization of 35S and 5S rDNA loci. Botanical Garden Berlin, Dahlem, Germany. b USDA North Central Regional Plant Introduction Station of the US National Plant Germplasm System. c Seeds kindly provided by Dr. Luz Gomez Pando from National Agrarian University – La Molina, Lima, Peru. d Torseed – Seed Company, Torun, Poland. e BG & Museum Natural History Museum of Denmark, Copenhagen, Denmark. f The Botanical Garden of the University of Coimbra, Portugal. g PNOS – Seed Company Ozarów Mazowiecki, Poland. a
in Vectashield (Vector Laboratories, Peterborough, UK) containing 2 g/ml DAPI (Serva, Heidelberg, Germany). Then the slides were incubated at 37 ◦ C for 4–5 days prior to examination. After taking the images, the slides were washed in distilled water, de-stained in ethanol–glacial acetic (3:1), dehydrated in absolute ethanol and used for FISH.
2.4. DNA probe labeling and fluorescent in situ hybridization The probe used for the detection of 35S rRNA gene sites was a 2.3 kb fragment of the 25S rDNA coding region from Arabidopsis thaliana (Unfried and Gruendler, 1990), labeled with dioxygenin-11-dUTP using nick translation. For detection of 5S rDNA sites, a 410 bp clone isolated from Triticum aestivum (Gerlach and Dyer, 1980) was amplified and labeled with rhodamine-4-dUTP using PCR as described (Kolano et al., 2008). Fluorescence in situ hybridization was performed according to the
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protocols described by Schwarzacher and Heslop-Harrison (2000) with minor modifications. Briefly, the hybridization mixture – consisting of 100 ng of labeled DNA probe, 50% formamide, 10% dextran sulfate, 0.1% SDS and 10 g of sheared salmon sperm DNA – was denatured for 10 min at 85 ◦ C, then chilled on ice and applied to the chromosome preparation. The slides and hybridization mixture were denatured together at 72 ◦ C for 5 min in an in situ Thermal Cycler (Thermo Hybaid, Franklin, USA) and then allowed to hybridize for 48–72 h in a humid chamber at 37 ◦ C. Stringent washes (10% formamide in 0.1× SSC at 37 ◦ C) were followed by the immunodetection of digoxigenin-labeled DNA probe using fluorescein-conjugated primary anti-digoxigenin antibodies and signal amplification with FITC-conjugated anti-sheep secondary antibodies (Jackson ImmunoResearch, Suffolk, UK). The preparations were mounted in Vectashield (Vector Laboratories, Peterborough, UK) containing 2 g/ml DAPI.
3. Results The Amaranthus species that were analyzed revealed the chromosome number 2n = 32 or 2n = 34. Intraspecific variations of the chromosome number were observed for three species: A. caudatus, A. cruentes and A. hybridus. Depending on the accessions, 2n = 32 or 2n = 34 chromosomes were observed. The chromosome number of the analyzed amaranths are summarized in Table 1. The distribution of rRNA gene sites was analyzed using FISH with 5S and 25S rDNA as probes. The results of the double-target FISH to the mitotic metaphase of the Amaranthus species are presented in Figs. 1 and 2 and the total numbers of 5S rDNA and 35S rDNA sites in 20 accessions of the 14 species studied are summarized in Table 1. In most of the Amaranthus accessions that were analyzed, hybridization signals of 35S rDNA and 5S rDNA were observed in separate chromosomes; however, two species, A. spinosus and A. greacizans subsp. silvestris, showed a co-localization between 35S rDNA and 5S rDNA sites (Figs. 1S and 2F). In all chromosomes carrying 35S rDNA loci or/and 5S rDNA loci these were observed in terminal position (Figs. 1 and 2). Most of the species from the subgenus Amaranthus sec. Amaranthus that were analyzed exhibited two sites of 35S rDNA. The only exceptions were A. quitensis, which had six sites, and one accession of A. caudatus, which had four sites of 35S rDNA (Table 1), both from the subsection Amaranthus. The number of 5S rDNA sites was relatively most abundant among amaranths from the sect. Amaranthus. In this section between 6 and 12 sites of 5S rDNA were observed depending on the species; however, those with eight sites were present more often (Fig. 1, Table 1). In the subsection Hybrida nearly all accessions showed eight sites of 5S rDNA although species from the subsection Amaranthus exhibited a much more variable pattern of 5S rDNA sites. In accessions with more than two sites of 35S rDNA, one pair of sites was major with much more intense hybridization signals and the rest were minor. Also, in the case of 5S rDNA, major and minor sites could be distinguished in every accession of amaranths from the sect. Amaranthus (Fig. 1). Two species from the section Amaranthus showed intraspecific polymorphism in loci number. The A. caudatus accessions that were analyzed showed polymorphism in a number of 35S rDNA sites and 5S rDNA. Each analyzed accession revealed a different pattern of rDNA sites (Table 1, Fig. 1A–G). A. cruentus showed polymorphism in a number of 5S rDNA loci and depending on the accession six or eight loci were observed (Table 1, Fig. 1H–K). The next two sections, Dubia and Centrusa (subgenus Amaranthus), were represented by one species each. A. dubius exhibited two sites of 35S rDNA and six sites of 5S rDNA (Fig. 1R). A. spinosus (sec. Centrusa) revealed the lowest number of 5S rDNA sites among the analyzed species from the subgenus Amaranthus. This species revealed only two sites of 5S rDNA and four sites of 35S rDNA. Interestingly, two
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sites of 35S rDNA were colocalised with 5S rDNA sites in A. spinosus (Fig. 1S). The second studied subgenus Albersia was represented by five species belonging to two sections: Blitopsis and Pyxidium (Table 1). These species showed a relatively lower abundance of 5S rDNA loci when compared with the species from the subgenus Amaranthus (Fig. 2). 5S rDNA sequences were most often localized in two sites, except for A. blitum, which had four sites and A. tricolor which had six sites of 5S rDNA (Fig. 2, Table 1). In subgenus Albersia in one karyotype the size of the hybridization signals for different 5S rDNA sites were similar, in contrast to the subgenus Amaranthus (Figs. 1 and 2). The hybridization signals of 35S rDNA were only observed on one pair of chromosomes of most of the species from the subgenus Albersia that were analyzed except for A. albus, which had two pairs of sites that showed an equal intensity of hybridization signals. The sites of rDNA sequences were located on separate chromosomes except for A. graecizans subsp. silvestris. In this genome, the loci of 35S and 5S rDNA were located in two sites in the same chromosome and the hybridization signals of the two rRNA genes were located adjacent to each other at the most terminal position of 5S rDNA sites (Fig. 2F). Double fluorescent staining with DAPI/CMA is used to localize the chromosome regions that are rich in AT and GC base pairs, respectively (Schweizer, 1976). In all of the species examined, no DAPI+ bands were found, although DAPI− bands that corresponded to CMA+ regions were observed. After CMA/DAPI staining, all of the species revealed CMA+ /DAPI− bands (i.e. brighter with CMA and duller with DAPI) in the terminal part of chromosomes. The number of CMA+ /DAPI− bands per karyotype differed among analyzed accessions and two, four or six bands were observed depending on the accession (Figs. 1B, D, G and I, 2B and 3). When more than one pair of CMA+ /DAPI− bands were observed, one pair of chromosomes carried brighter bands than the other two (Figs. 1D and I and 3B, G, L and O) except for A. albus, which had six CMA+ bands of approximately equal brightness. The maximum number, six CMA+ /DAPI− bands, was observed in three accessions (A. quitensis A. blitum subsp. oleraceus and A. albus); however, two CMA+/DAPI− bands were present most often (Table 1). In many of the analyzed accessions, CMA+ /DAPI− bands colocolised with a secondary construction. (e.g. Fig. 3D and H). Sequential CMA/DAPI banding and FISH with rDNA sequences were performed for some of the species and these indicated that the CMA+ /DAPI− bands colocalised with 35S rDNA sites (Figs. 1A–I and 2A and B). Some species, however, showed more CMA+ /DAPI− bands than the number of 35S rDNA sites (two accessions of A. cruentes, A. spinosus, A. blitum and A. albus) (Table 1). Sequential CMA/DAPI banding and FISH with rDNA sequences were performed for selected species and these indicated that the CMA+ /DAPI− bands colocalised not only with 35S rDNA sites but also could colocalise with 5S rDNA sites (Fig. 1H and I and 2A and B). 4. Discussion Somatic chromosome numbers of 32 or 34 were observed for the analyzed amaranth accessions and they are mainly in accordance with those available at the Index to Plant Chromosome Number (www.tropicos.org/Project/IPCN) or those that were reported earlier by Grant (1959). The exception was A. dubious, which according to Grant (1959) was a polyploid species with 64 chromosomes, however the accession analyzed in this study has 34 chromosomes. The basic chromosome numbers in the genus are n = 16 and n = 17 and both numbers are found in the subgenus Amaranthus as well as in the subgenus Albersia. Pal et al. (1982), based on a cytogenetic analysis of interspecific hybrid, suggested that the gametic number n = 17 originated from the n = 16 through primary trisomy. Cytogenetic research has demonstrated that a variation between n = 16
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Fig. 1. Distribution of 5S (red fluorescence; the smallest 5S rDNA sites were marked by red arrows) and 35S (green fluorescence) ribosomal DNA sites and CMA+ bands (yellow fluorescence) in Amaranthus species from subgenus Amaranthus: A. caudatus Kiwicha 3 (A and B); A. caudatus Kiwicha Molinera (C and D); A. caudatus var. purpurosum (E); A. caudatus ornamental form (F and G); A. cruentus Pygmy Torch (H and I); A. cruentus Ames 15,197 (J); A. cruentus 120 (K); A. hypochondriacus (L); A. powellii subsp. powellii (M); A. quitensis (N); A. hybridus PI 604570 (O); A. hybridus 92 (P); A. retroflexus (Q); A. dubius (R); A. spinosus (S). White arrows: CMA+ bands associated with 35S rDNA sites; white arrowheads: CMA+ bands associated with 5S rDNA sites. Scale bar = 5 m.
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Fig. 2. Distribution of 5S (red fluorescence; the smallest 5S rDNA sites were marked by red arrows) and 35S (green fluorescence) ribosomal DNA sites and CMA+ bands (yellow fluorescence) in Amaranthus species from subgenus Albersia: A. albus (A and B); A. blitum subsp. oleraceus (C); A. viridis (D); A. tricolor (E); A. graecizans subsp. silvestris (F). White arrows: CMA+ bands associated with 35S rDNA sites; white arrowheads: CMA+ bands associated with 5S rDNA sites. Scale bar = 5 m.
and n = 17 can occur even within one species (e.g. accessions of A. caudatus, A. cruentus and A. hybridus). Earlier, intraspecific polymorphism in chromosome numbers was also reported for Amaranths by Grant (1959) and Popenoe et al. (1989). Amaranthus species have rather small chromosomes, which in many cases are similar in shape and size, and are therefore difficult to distinguish. For this reason, additional landmarks are required in order to enable chromosome identification. rDNA sequences can be localized by FISH and can provide markers that are useful for the identification of individual chromosomes. A comparison of the chromosome number and the distribution of rDNA sites can provide valuable information about the phylogenetic relationship between related taxa, for example, in various Brassicaceae (Hasterok et al., 2006), Hypocheris (Weiss-Schneeweiss and Schneeweiss, 2003) and Paphiopedilum species (Lan and Albert, 2011). The present report gives the first description of rDNA localization for 11 species and confirms the mapping of 35S rDNA sites in A. caudatus, A. cruentus and A. hypochondriacus (Kolano et al., 2001; Bonasora et al., 2013). The chromosomal positions of 35S and 5S rDNA sites are relatively conservative among species analyzed. Most of them showed 35S rDNA and 5S rDNA signals in the terminal position of chromosome arms and most often 35S rDNA sites and 5S rDNA sites were located on different chromosomes. There were only two exceptions, A. spinosus and A. greacizans subsp. silvestris, where the one pair of 35S rDNA sites colocalised with 5S rDNA sites in one chromosome pair. These more unique patterns of rDNA distribution might be a trace of chromosomal rearmaments or could be connected with translocation events during the evolution of the species (Raskina et al., 2008; Thomas et al., 2001). The analyzed Amaranthus species showed a high variability in the number of rDNA sites. Simultaneous FISH with 5S and 35S rDNA probes identified between one and eight chromosome pairs carrying rDNA sequences depending on the accessions. This phenomenon is commonly observed in many different plant genera including Brassica Paphiopedilum, Aristolochia (Berjano et al., 2009; Hasterok et al., 2006; Lan and Albert, 2011). However, the strong conservation of rDNA site number has been described in many plant species or even in an entire genera, e.g. Glycine and Daucus (Iovene et al., 2008; Singh et al.,
2001). In general, the FISH patterns of 35S rDNA loci are reported to be more polymorphic than those of the 5S rDNA (Adams et al., 2000; Datson and Murray, 2006; Kwon and Kim, 2009). In contrast, Amaranthus species showed a higher variability in the number of 5S rDNA; however, differences in the number of 35S rDNA sites were also observed. Similarly, higher diversification of 5S rDNA distribution patterns and lower degree of numeric variation in 35S rDNA sites were also observed for Paphilopedilum and Byblis liniflora complex (Fukushima et al., 2011; Lan and Albert, 2011). Generally, the number of 5S rDNA loci was higher in Amaranths from the subsection Amaranthus and Hybrida (subgenus Amaranthus, section Amaranthus) than in species from other taxa that were analyzed. The accessions from the Amaranthus subsection also showed a very high numeric variability in the number of 5S rDNA sites, whereas it could be shown that for the four species of the subsection Hybrida it was relatively stable in relation to the number of rDNA sites. In addition, intraspecific polymorphism in the number of both 5S and 35S rDNA sites was shown in a few species: A. caudatus, A. cruentus. The phenomenon of numerical variation in rDNA sites has been observed in several plant species (Dydak et al., 2009; Kolano et al., 2012; Ksiazczyk et al., 2010;), with a fourfold variation in the number of 35S rRNA loci being reported within the diploid Phaseolus vulgaris as an extreme example (Pedrosa-Harand et al., 2006). Variation in the number and location of rDNA sites could indicate their mobility in the recent evolutionary history of the species. Different mechanisms have been postulated to account for this phenomena and polymorphism in the number, size and position of rDNA sites, such as a transposon-mediated transpositional event or chromosome rearrangements caused by a homologue or a non-homologous unequal crossing-over and gene conversion (Altinkut et al., 2006; Datson and Murray, 2006; Raskina et al., 2008; Thomas et al., 2001). Thus, the high polymorphism of the chromosomal organization of the rDNA clusters that was observed among the Amaranthus accession could also indirectly suggest a relatively high level of microevolutionary genomic changes during speciation within this genus. It is obvious that speciation-related chromosomal repatterning might further increase/decrease the number of rDNA sites or their
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Fig. 3. CMA/DAPI fluorescent stained chromosome complements of Amaranthus species: A. caudatus var. purpurosum (A); A. quitensis (B); A. retroflexus (C); A. cruentus Ames 15197 (D); A. cruentus 120 (E); A. hypochondriacus (F); A. hybridus 92 (G); A. hybridus PI 604570 (H); A. powellii subsp. powellii (I); A. dubius (J); A. viridis (K); A. blitum subsp. oleraceus (L); A. greacizans subsp. silvestris (M); A. tricolor (N); A. spinosus (O). DAPI – blue fluorescence, CMA3 – yellow fluorescence. The arrows point out CMA+ bands. Scale bar = 5 m. (For interpretation of the references to color in the artwork, the reader is referred to the web version of the article.)
repositioning. The dynamics of rDNA clusters may also be regarded as a strong indicator for significant intra-genomic processes (Jiang and Gill, 1994; Raskina et al., 2004). Double fluorescent staining with DAPI/CMA was used to localize chromosome regions rich in AT and GC base pairs, respectively (Schweizer, 1976). The organization of GC rich DNA is similar in all of the amaranths that were analyzed. CMA+ /DAPI− bands were located in the terminal position in the chromosomes of all of the analyzed accessions and could be associated with rDNA sites, both 35S and 5S. Most of the analyzed Amaranthus accessions showed the same number of 35S rDNA sites as the number CMA+/ DAPI− bands, which were usually observed in a secondary construction. As was indicated for A. caudatus, the polymorphism of the number of CMA+ /DAPI− bands corresponds to the polymorphism in the 35S rDNA sites. So for most analyzed accessions the regions occupied by genes for 35S rRNA were the only large GC rich blocks
of chromatin as was shown earlier for many plants (Moraes and Guerra, 2010). Other amaranth accessions showed a higher number of CMA+ /DAPI− bands than the number of 35S rDNA loci. As was shown for A. albus and A. cruentus, the CMA+ bands could correspond not only to 35S rDNA sites but also to 5S rDNA sites. However, not all 5S rDNA sites in A. cruentus Pygmy Torch correspond to CMA+ bands, possibly because the sites were too small to be detected with CMA. The association of GC-rich regions with 35S rDNA sites is commonly found in plants (Dydak et al., 2009; Guerra, 2000; Penas et al., 2009) although there is little data on the occurrence of such heterochromatin with 5S rRNA genes being available for plants since few studies have simultaneously combined banding techniques and 5S rDNA in situ hybridization (Cabral et al., 2006; Hajdera et al., 2003; Hamon et al., 2009; Sijak-Yakovev et al., 2003). This could reflect both the composition of the 5S rDNA sequences and the nature of the adjacent heterochromatin.
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