ISSN 00954527, Cytology and Genetics, 2015, Vol. 49, No. 5, pp. 322–327. © Allerton Press, Inc., 2015. Original Ukrainian Text © M.O. Twardovska, I.O. Andreev, V.A. Kunakh, 2015, published in Tsitologiya i Genetika, 2015, Vol. 49, No. 5, pp. 55–61.
Intraspecific Chromosomal Polymorphism of Iris pumila L. from the Territory of Ukraine M. O. Twardovska, I. O. Andreev, and V. A. Kunakh Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine, Kyiv, 03680 Ukraine email:
[email protected] Received July 17, 2014
Abstract—Cytogenetic analysis of Iris pumila L. plants from different habitats in Ukraine revealed the intraspecific chromosomal polymorphism. The chromosome number 2n = 32 was established for plants of this species. Root apical meristem of I. pumila shoots of all populations showed a high level of mixoploidy. The differences in the percentage of aneuploid cells and anaphase aberration were found between the sampled populations. The anaphase analysis revealed the level of chromosomal rearrangements that reached 9.2% in some populations. Keywords: Iris pumila L., chromosome number, mixoploidy, aneuploidy DOI: 10.3103/S0095452715050096
INTRODUCTION Iris pumila L. is a typical steppe xerophyte of Ukrainian flora. It is spread in the most of country’s territory, except of the Carpathians, Polissia and the south of Steppe [1]. In eight regions, it is under regional protection and considered to be a candidate to enter into the Red Book of Ukraine [2]. The eco nomic activity in steppe creates a threat of shortening the number of I. pumila, causes areal fragmentation, loss of genetic resources of this species, and also decreases a number of its local populations. Such areas are much isolated from each other and they are affected by anthropogenic load of different force. It has a bad influence on specific gene pool and requires estimation of genetic resources in order to forecast the state of these populations in future. The cytogenetic analysis is an important compo nent of study of genetic variety of species. The analysis of karyotype polymorphism in general, as well as poly morphism of structure and morphology of individual chromosomes, allow one to establish the presence of chromosome races, and to determine the structure of species and particular populations and the variety level. The cytogenetic findings extend the existing understandings about population dynamics, natural selection, and adaptation of species to different spe cific conditions of environment and effect of stressful factors. Also, they help to realize the mechanisms of appearance and accumulation of genetic changes in populations. Chromosome variations can hinder sex ual propagation of plants and usually cause sterility of mature individuals. That is why species that are capa ble of clonal propagation, including I. pumila, are
considered to be the most prospective for searching and studying possible mechanisms of chromosome variability. The cytological studies of I. pumila was started in 1900 [3], but the chromosome number for species was established only in 1932. It was 2n = 32 but also other values of diploid set are found: 2n = 30 and 36, which has been described in detail earlier [4]. We found only one statement about study on kary otype I. pumila from the territory of Ukraine and it concerns Crimean populations, which are character ized by diploid set 2n = 32 [5]. Some karyotypes of these plants contained a heteromophous pair of chro mosomes that differed in position of centromere, prob ably, due to exchange of segments. This may suggest that different geographical races of the same species can carry the same chromosome number [5]. The sim ilar changes of chromosome morphology in different geographical races were found and they were typical for I. pseudopumila Tineo, I. mellita Janka, I. pallida Lam. and I. variegata L. [6, 7]. Considering the necessity of estimation of gene pool of natural populations of I. pumila and limitation and ambiguity of data concerning karyological studies of this species, we conducted the cytogenetic analysis of I. pumila plants from different habitats in Ukraine. In particular, the aims of our work were to establish the chromosome number of I. pumila and to study intraspecific chromosomal polymorphism of these plants from different habitats in Ukraine.
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Cherkasy
1 day. The material for anaphase analysis (root tips) were fixed without preliminary processing. The fixed roots were kept at –20°C. The samples were stained with 1% solution of acetoorceine and then squashed preparations were made. Each preparation was used to analyze those metaphase plates where chromosomes can be counted. We studied at least 20 shoots from each pop ulation. The number of metaphases of different shoots which are available for analysis was different and it varied within 2–63 metaphases. In our work, we used an NU2E Carl Zeiss micro scope. Photomicrography was carried out with the use of a Canon 1000D digital camera. The obtained data were processed statistically [9].
Poltava
1
Kirovohrad Pervomaisk 2
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Zaporizhzhia
Mykolaiv 3 Kherson
RESULTS AND DISCUSSION
Kerch Simferopol 0
50
100 150 km
4
Fig. 1. Habitats of I. pumila populations: (1) village of Prydniprovske (Chornobaivsk rayon, Cherkasy province), (2) village of Muhiia (Pervomaisk rayon, Mykolaiv province), (3) village of Dmytrivka (Ochakiv rayon, Mykolaiv province), (4) town of Balaklava (Autonomous Republic of Crimea).
MATERIALS AND METHODS To conduct the cytological analysis, we used I. pumila seeds from four remote from each other pop ulations in Ukraine. Figure 1 shows their location. The roots of shoots 0.8–1 cm long obtained from I. pumila seeds in aseptic conditions were used as the research material. The conditions of sterilization and sprouting of seeds were described in details in the work [8]. Shooting roots, which were used for accumulation and synchronization of mitoses, were kept in water at 4°C for 24 hours or in 0.2% solution of colchicine at 37°C for 2 hours. The samples were fixed in the mix ture of ethanol : glacial acetic acid in ratio of 3 : 1 for
Using the karyological analysis of root apical mer istem cells of I. pumila shoots from different ecotopes in Ukraine, we established the chromosome number 2n = 32. Figure 2 shows metaphase plates. Many sam pled shoots were mixoploid—along with diploid cells in such shoots, cells with other chromosome numbers were found. The variability according to the chromo some number from different shoots ranged (from 16 to 50 chromosomes). The chromosome numbers varied both between individual plants and in populations of meristem cells of individual plants. The modal chromosome number in cells of root tips of shoots was 2n = 32 in the analyzed samples, but we found the high level of mixoploidy for studied I. pumila plants. Table 2 shows that the preportion of mixoploid plants was big in all studied habitats and was 60–80%. For I. pumila (village of Prydniprovske), 12 from 20 shoots were mixoploid, which was approximately 60% (Table 1). Only diploid cells made up modal class in five mixoploids, the rest had most of aneuploid cells with 20, 24, 26 and 28 chromosomes, and also the cells with 16 chromosomes (Table 2). For I. pumila shoots (village of Dmytrivka), 16 of 20 studied contained cells with a chromosome number that was different from the diploid one. One mixoploid shoots was characterized by modal class with haploid,
Table 1. Number of diploid and mixoploid shoots of I. pumila from different habitats Number of shoots Habitat
Studied shoots, ea
Village of Prydniprovske Village of Dmytrivka Village of Myhiia Town of Balaklava CYTOLOGY AND GENETICS
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diploid
mixoploid
ea
%
ea
%
8 4 7 7
40.0 ± 11 20.0 ± 8.9 30.4 ± 9.6 31.8 ± 9.9
12 16 16 15
60.0 ± 11 80.0 ± 8.9 69.6 ± 9.6 68.2 ± 9.9
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(a)
(b)
(d)
(e)
(c)
Fig. 2. Metaphase plates with different chromosome numbers in root apical meristem of I. pumila shoots cells: (a–c) 32 chromo somes, (d) 18 chromosomes, and (e) 28 chromosomes. Scale is 10 µm.
six mixoploids with diploid, and the rest of shoots with hypodyploid cells (Table 2). For I. pumila (village of Myhiia), 16 of 23 sampled shoots were mixoploid. Diploid cells prevailed in ten mixoploids, and the remaining cells contained from 24 to 50 chromosomes. Three shoots had modal class that was represented by hypodyploid cells with 28 chromosomes (Table 2). For I. pumila shoots (town of Balaklava), 15 of 22 studied were mixoploid. Only four of them had pre vailing number of diploid cells, while the remaining four shoots contained aneuploid cells along with dip loid metaphases (Table 2). Hypodyploid cells with 28 chromosomes made up modal class in four I. pumila shoots (town of Balaklava). Other scientists discovered mixoploidy while con ducting the cytogenetic analysis of cells of root apical meristem of I. pumila and also other iris species: I. set osa, I. ensata, I. oxypetata, I. pseudacorus, I. laevigata [10]. The chromosome number of these species ranged from 6 to 60 and cells with diploid chromosomes pre vailed (35–60%). The preportion of cells with chro mosome number, distinctive from diploid set, and their level of ploidy differed. Thus, the percentage of diploid cells in cell populations of root meristem of I. pumila was 43%, while aneuploid ones were 51%. The studied cell populations of mentioned species of Iris L. genus were characterized by high preportion of aneuploid cells (28–54%) [10]. Mixoploidy was described for other species of plants—Plukenetia volubilis L. [11], Santalum album L. [12], many species of Brassicaceae, particularly Bras
sica nigra, B. rapa, B. cretica, B. napus, Raphanus sati vus var. niger [13], B. crops [14], B. oleracea var. botrytis [15], Panax ginseng C. A. Mey [10, 16], and for species of genera Morus L. [17] and Bromus L. [18]. According to literature data, the preportion of cells with the num ber of chromosomes different from diploid set can reach 77% in meristems of different plant species. It is assumed that mixoploidy increases adaptive potential of plants [19]. Probably, this is a reflection of increased adaptive organism ability regarding survival of individ uals, including many cells with changed number of chromosomes. During the anaphase analysis root apical meristem of I. pumila shoots cells, we found that plants are char acterized by a comparatively high level of aberrations that reached 9.2% in some sampled populations. Fig ure 3 shows that chromosomal rearrangements were fragments and bridges among which single (chroma tid) bridges prevailed. The presence of one or some acentric fragments in anaphase is an indication of “fresh” chromosomal break [19]. Only 6% of frag ments among all aberrations were found in samples of I. pumila from the village of Dmytrivka. The bridges without fragments are the results of some mechanisms: (1) preservation of dicentric chromosomes in cell gen erations in consequence of bridge cycle of break fusionbridge, (2) involvement of acentric chromo somal fragment and its movement to one of poles with further loss, (3) movement of both dicentric cen tromeres to one pole and its detection as a bridge in the next cycle. But the first type of mechanisms of appear ance of bridges without fragments is considered to be the main one [19, 20]. The appearance of single CYTOLOGY AND GENETICS
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Table 2. Number of chromosomes in root tips of mixoploid shoots of I. pumila Shoot no.
Studied metaphases, ea
1 2 3 4 5 6 7 8 9 10 11 12
7 11 10 2 2 3 3 2 5 9 6 3
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
36 63 9 7 18 6 5 59 4 4 5 4 4 26 7 7
1 2 3 4 5 6 7 8 9 10 11 12
3 6 7 5 3 7 3 4 4 10 12 6
Found chromosome numbers* Village of Prydniprovske 30(1), 32(5), 34(1) 28(2), 32(4), 42(3), 48(2) 26(4), 32(5), 42(1) 24(1), 32(1) 16(1), 20(1) 28(1), 32(2) 32(2), 50(1) 28(1), 32(1) 24(3), 26(1), 32(1) 24(1), 26(1), 28(6), 32(1) 28(3), 32(3) 26(1), 28(1), 32(1) Village of Dmytrivka 8(2), 14(1), 16(8), 20(5), 22(9), 26(4), 28(3), 32(3), 46(1) 20(1), 24(17), 26(1), 28(20), 30(8), 32(16) 20(1), 22(1), 24(1), 26(3), 28(3) 22(1), 24(1), 28(1), 32(3), 38(1) 20(2), 24(4), 28(4), 32(7), 42(1) 22(1), 24(2), 32(2), 36(1) 16(1), 24(1), 32(2), 48(1) 22(1), 24(3), 26(6), 28(18), 30(6), 32(22), 36(2), 44(1) 24(1), 28(2), 30(1) 32(2), 34(1), 40(1) 16(1), 24(1), 28(1), 30(2) 16(2), 22(1), 24(1) 22(3), 32(1) 16(1), 22(1), 24(8), 26(4), 28(12) 16(1), 20(1), 24(3), 26(1), 28(1) 24(1), 28(2), 32(4) Village of Myhiia 32(2), 44(1) 28(2), 32(4) 26(1), 28(1), 30(1), 32(2), 42(1), 50(1) 28(1), 32(4) 28(2), 32(1) 32(4), 42(1), 44(1), 38(1) 38(2), 42(1) 28(2), 30(1), 32(1) 32(3), 38(1) 28(2), 32(8) 28(1), 32(4), 36(1), 40(4), 42(1), 48(1) 28(2), 32(4)
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Modal number
Number of metaphases with modal number, %
32 32 32 24, 32 16, 20 32 32 28, 32 24 28 28, 32 26, 28, 32
71.4 36.7 50 50; 50 50; 50 66.7 66.7 50; 50 60 66.7 50; 50 33.3; 33.3; 33.3
22 28 26, 28 32 32 24, 32 32 32 28 32 30 16 22 28 24 32
25 31.7 33.3; 33.3 42.9 38.9 33.3; 33.3 40 37.3 50 50 40 50 75 46.2 42.9 57.1
32 32 32 32 28 32 38 28 32 32 32, 40 32
66.7 66.7 28.6 80 66.7 57.1 66.7 50 75 80 33.3; 33.3 66.7
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Table 2. (Contd.) Shoot no.
Studied metaphases, ea
13 14 15 16
3 8 4 3
Modal number
Found chromosome numbers* 32(2), 36(1) 24(1), 28(4), 32(3) 28(2), 32(2) 24(1), 32(2)
Number of metaphases with modal number, %
32 28 28, 32 32
66.7 50 50; 50 66.7
28 32 20, 32 28 26, 28 32 30, 32 26, 32 24 26 28 28 32 28, 32 32
75 50 50; 50 45.5 40; 40 62.5 50; 50 50; 50 66.7 66.7 66.7 66.7 80 50; 50 71.4
Town of Balaklava 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
4 4 2 11 5 8 2 2 3 6 6 3 5 2 7
28(3), 32(1) 22(1), 26(1), 32(2) 20(1), 32(1) 26(2), 28(5), 30(1), 32(3) 26(2), 28(2), 32(1) 26(2), 32(5), 36(1) 30(1), 32(1) 26(1), 32(1) 24(2), 32(1) 24(1), 26(4), 32(1) 26(1), 28(4), 32(1) 28(2), 32(1) 22(1), 32(4) 28(1), 32(1) 24(1), 30(1), 32(5)
Number of metaphases with the given chromosome number is shown in the brackets.
bridges among anaphase aberrations shows that chro mosome affection occurs in the cells of root apical meristem of shoots cells mostly as a result of simple breaks at the stage of two effective threads. But rarely is aberration possible at the stage of G1 and also the appearance of complicated rearrangements in case of dual and plural breaks. The found types of aberrations prove that their totality is a summary result of “fresh” breaks and bridge cycles or aberrations that “go” through mitosis to mitosis.
(a)
(b)
The literature shows that the level of spontaneous anaphase aberrations of chromosomes in intact plants rarely exceeds 1% [19]. The reasons of high level of mixoploidy and per centage of structural rearrangements in I. pumila are not simple. We can assume that these phenomena deal with hybrid origin of species and peculiarities of its biology and propagation system [21]. It is considered that tertraploid I. pumila (2n = 32) is a natural hybrid of two Mediterranean diploid species: I. pseudopumila (2n = 16) and I. attica (2n = 16) [7, 22]. In particular,
(c)
Fig. 3. Typical structures of rearrangements of chromosomes which occur in root apical meristem of I. pumila shoots cells at the stage of anaphase of mitosis: (a) single (chromatid) bridge, (b) paired (chromosome) bridge, and (c) fragment. Scale is 10 µm. CYTOLOGY AND GENETICS
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the studies showed that I. pumila is amphidiploid (allotetraploid), whose karyotype can be formed resulting in combination of chromosome sets of these species. Thus, as a result of conducted cytogenetic analysis of I. pumila plants from the territory of Ukraine, we found the presence of intraspecific chromosomal polymorphism. The sampled populations differed in the preportion of aneuploid cells and percentage of anaphase aberrations. Thus, we found the highest level of mixoploidy and the lowest percentage of anaphase aber rations in the samples from the village of Dmytrivka. I. pumila shoots from the villages of Muhiia and Prydni provske were characterized by a high level of mixoploidy and structural rearrangements of chromosomes as well. The samples from the town of Balaklava showed a high level of mixoploidy and the twice lower level of anaphase aberrations in comparison with other ecotopes. We can assume that the causes of found dif ferences between populations can be as different pres sure of anthropogenic load as different climatic condi tions of growth. CONCLUSIONS We established the chromosome number 2n = 32 for I. pumila plants from different habitats in Ukraine. Mixoploidy in root apical meristem of I. pumila shoots was also found and its level was different for different populations. The differences between the studied pop ulations regarding the preportion of aneuploid cells and percentage of anaphase aberrations were discov ered. Their causes can be the following: different pres sure of anthropogenic load and different climatic con ditions of growth. The high level of anaphase aberra tions of chromosomes for intact plants (9.2%) was found. We can assume that the high level of mixop loidy and percentage of chromosomal aberrations detected at the stage of anaphase of mitosis is con nected with hybrid origin of sampled species. ACKNOWLEDGEMENTS We thank the director of Mykolaiv Region Ecolog icalNaturalistic Center of School Youth T.B. Troitska for the materials given. REFERENCES 1. Bairak, O.M., Shevel’, I.M., Gritsai, I.A., et al., Botan ichnii zakaznik “Drabinivka” (Botanical Reserve “Dra binivka”), Poltava: Verstka, 2006. 2. Parnikoza, I.Yu., Trots’ka, T.B., Trots’kii, M.O., and Kunakh, V.A., The state of Iris pumila L. populations from different regions of Mikolaiv region, in Materiali drugikh naukovikh chitan' pam’yati Sergiya Tarash chuka (Proc. Scientific Readings in Memory of Sergei Tarashchuk), Mikolaiv, 2011, pp. 112–115. 3. Strasburger, E., Uber Reduktionstheilung, Spindelbil dung, Centrosome und Cilienbildner in Pflanzenreich, Hist. Beitr., 1900, vol. 6, pp. 1–284. CYTOLOGY AND GENETICS
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Translated by N. Berestetska
