Karyotype analysis of Astragalus effusus Bunge (Fabaceae)

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1 CARYOLOGIA Vol. 63, no. 3: , 2010 Karyotype analysis of Astragalus effusus Bunge (Fabaceae) Yousefzadeh Kazem, Saadolah Houshmand* and Gelavij Zamani Dadane College of Agriculture, Shahrekord University, Shahrekord, Iran, p.o. box 115. Abstract In this study, the karyotype analysis of Astragalus effusus Bunge species was studied using Image Analysis System. The protocol assayed consisted of the use of α-bromonaphthalene for pretreatment, Lewitsky solution for fixation, 1N NaOH for hydrolysis and finally, aceto iron-hematoxylin for staining stage. The species chromosome number was 2n = 4x = 32, chromosome lengths range from 1.57 to 2.99 µm and the length of the whole genome was 75.8 µm; also, the chromosome length and arm lengths showed this species was most likely natural autotetraploid and observed one pericentric inversion in chromosomes of the VIII group. Regarding the karyotypic symmetry, this species belongs to the 2A karyotypic symmetry class with a 7m + 2m sat + 7sm karyotype formula. Asymmetrical parameters emphasize on rather symmetry karyotype in this species. In this paper, we assess the karyotype of this species for the first time. Key words: Astragalus effusus, autotetraploid, karyotype analysis. INTRODUCTION Astragalus L. (Gavan in Persian) is probably the largest genus of flowering plants, containing up to 3000 species (LOCK and SIMPSON 1991). This genus is a member of the legume family (Fabaceae) and is traditionally classified in the tribe Galegeae. The centre of development seems to be in the arid and semi-arid mountainous parts of the Northern Hemisphere (POLHILL 1981). It is most diverse in the Irano-Turkish region of south-western Asia, the Sino-Himalayan Plateau of south-central Asia, the Central Asian region, and the Great Basin and Colorado Plateau of western North America (POLHILL 1981). The species is characterised by perennial growth and the presence of bifurcate hairs (BUNGE 1868). Iran is one of the largest centers of diversity for this genus, with approximately 700 species and an endemism rate of 57% (GHAH- REMAN et al. 2002; PODLECH 2001). Astragalus effusus Bunge is a valuable perennial forage species of the Leguminosae family in the rangelands of Iran (ZAMANI 2008). It is known as a palatable plant, grazing tolerant, nutrient rich and prostrate species that have an undeniable role in soil conservation and animal feeding, hence it deserves serious consideration. An extensive number of cytological studies has been performed in Astragalus throughout the world, including Iran (LEDINGHAM 1960; LEDINGHAM and REVER 1963; MAASSOUMI 1987; BADR et al. 1996; MALALLAH et al. 2001; BADR and SHARAWY 2007; SHEIDAI et al. 2009), reporting the basic chromosome numbers of x = 7, 8, and diploid, tetraploid, hexaploid and octaploid levels for the genus. The present study reports somatic chromosome characteristics, including morphology and number of Astragalus effuses from Isfahan, Iran. MATERIALS AND METHODS *Corresponding author: phone and fax ; Houshmand@agr.sku.ac.ir or s_hoshmand@yahoo. com In order to study the karyotypic characteristics of Astragalus effusus Bunge species, seeds were collected from the Zayandeh Rud dam studies station, Isfahan province, Iran. The seeds were germinated at 20 C and roots with

2 258 YOUSEFZADEH, HOUSHMAND and DADANE a cm length were used to obtain mitotic metaphases and determine karyotype characteristics such as chromosome numbers, chromosome length and total chromosomes length. The root tips for pretreatment were placed into α-bromonaphthalene and kept for 6 h at 4 C. For the fixation stage, Lewitsky fixative (chromic acid 1%+formaldehyde 10%; 1:1) was used for 24 h and 1N NaOH for 14 min at 60 C was used for hydrolysis stage. Finally, aceto iron-hematoxylin was used for the staining stage for 75 min at 50 C. The squashed preparations of root tips were made with 45% acetic acid. Then, to study the karyotype, the 10 best metaphases were photographed by an Olympus BX51 microscope using a DP12 digital camera, and measuring the total and arms length was done by micromeasure software (REEVES 2001). The karyogram was constructed by arranging the chromosomes in homologous pairs by order of their length, r-value and centromer index as measured and calculated from micromeasure software. Chromosomes were identified according to Levan method (LEVAN et al. 1964). To estimate the karyotype asymmetry, three numerical parameters were used, following ROMERO ZARCO (1986): A 1 [intrachromosomal asymmetry index =1 [Σ(short arm/long arm)/n)], A 2 [interchromosomal asymmetry index=standard deviation(s)/mean length (X)] and PASZKO (2006): Asymmetry index (AI)=(CV cl *CV ci /100), that CV cl : chromosome length coefficient variability, and CV ci : centromer index coefficient variability. The length of chromosomes in A. effusus varied from 1.57 to 2.99 µm and the total haploid length of this species was µm (Table 1). With regard to karyotype asymmetry, the A. effusus karyotype is classified as category 2A in symmetry classes of STEBBINS (1971). The analysis of the karyotype asymmetry indexes showed a value of 0.34, 0.19 and 2.8 for A 1, A 2 and AI, respectively and the karyotype was rather symmetrical. In addition to absolute chromosome size, pair chromosomes with the same size in the group VIII differed in the position of the Fig. 1 Mitotic metaphase chromosomes of A. effusus Bunge 2n = 4x = 32. RESULTS AND DISCUSSION Karyotypic characters, mitotic metaphase chromosomes, monoploid ideogram and karyogram of Astragalus effusus Bunge are shown in Table 1 and Figs Analysis of somatic metaphases showed that the chromosome numbers of the species are 2n = 4x = 32. In haploid set, 7 groups of four similar chromosomes were observed in size and centromer position and two pairs in the VIII group with similar in size and different in centromer position, so this species is most likely to be a natural autotetraploid species with a basic chromosome number of X = 8 (Table 1 and Figs. 2 and 3). The karyotype consisted of nine metacentric and seven submetacentric chromosomes in haploid set with a 7m+2m sat +7sm karyotype formula, and observed satellite on the short arm of group I chromosomes. Fig. 2 monoploid ideogram of A. effusus Bunge (2n = 4x = 32) and arrow indicate pericentric inversion group.

3 KARYOTYPE ANALYSIS OF ASTRAGALUS EFFUSUS BUNGE (FABACEAE) 259 centromer (Table 1 and Fig. 2 and 3). This difference is usually brought about by unequal translocation, pericentric inversion, and centric fusion and fission (STEBBINS 1971). However, in this karyotype the pericentric inversion probably was the reason for the chromosomal rearrangement and arms difference, because this phenomenon has characters of inversion. But more study in meiosis and mitosis stages of this species is required to understand the subject and determine the actual ratio of gametes and progenies. The analysis of the mitotic stages indicated some irregularities such as delayed chromosomes in anaphase or telophase (Fig. 4), leading to cells with a variable chromosome number (2n = 34, 30, 28 and 22), in low frequency. Therewith several Fig. 3 Karyogram of A. effusus; 2n = 4x = 32. vertical and slant arrows indicate pericentric inversion and homologue group include satellite, respectively. Fig. 4 Delayed chromosomes in Mitotic anaphase chromosomes of A. effusus Bunge. the seeds were visibly non-viable, which may be due to reduced gamete fertility of autotetraploid plants, because autotetraploids are partially pollen and ovule abortive (SINGH 2002). The basic chromosome number of A. effusus was X=8. LEDINGHAM (1960) found that the Astragalus species from the old world have a basic chromosome number of X = 8, while those from the new world have X = 11, 12 and 13. This report was substantiated by LEDINGHAM and REVER (1963). Chromosome counts, based on X = 8 have been reported in the vast majority of old world in Astragalus genus while counts based on base numbers X = 7 or X = 6 have been encountered in few species (MAASSOUMI 1987; BADR et al. 1996; MALALLAH et al. 2001; BADR and SHARAWY 2007). The preponderance of Astragalus species with a basic number of X = 8 led BADR et al. (1996) to conclude that it is the primary basic number in Astragalus species. They remarked that the X = 7 and X = 6 numbers have been derived from X = 8 by aneuploid loss of chromosomes. Polyploidy in this species have been reported by BADR and SHARAWY (2007). They determined the chromosome number in a karyological study on 24 species of Egyptian Astragalus and found different ploidy levels such as diploid (2n = 16, 14 and 12), triploid (2n = 24), tetraploid (2n = 32, 30 and 28), pentaploid (2n = 30), hexaploid (2n = 48) and octaploid (2n = 64) and SHEIDAI et al. (2009) have reported 2n = 16, 32 & 48, in some species of Iranian Astragalus, indicating the role of polyploidy in the evolution of this genus. We found that the A. effusus is an autotetraploid species. In nature, most if not all polyploids have arisen by sexual polyploidization through unreduced (2n) gametes (DE WET 1982; RAMSEY & SCHEMSKE 1998), in which production is genetically controlled (BRETAGNOLLE and THOMPSON 1995). This has been reported in cultivated plants such as alfalfa (BARCACCIA et al. 2003), white clover (HUSSAIN and WILLIAMS 1997), and red clover (PARROTT and SMITH 1984, 1986; PARROTT et al. 1985), among others. On the other hand, these species had forage uses and therefore, polyploidy could exist in nature with adaptive advantages of lushness and largeness, and high biomass yield (BINGHAM et al. 1994). For this reason, somatic polyploidization (TAY- LOR et al. 1976) or sexual polyploids (RAMANNA and JACOBSEN 2003) are important tools in forage plant breeding. The induced polyploidy have been used in some forage species such as Lolium perenne, with the purpose of obtaining improved autotetraploid cultivars or generating

4 260 YOUSEFZADEH, HOUSHMAND and DADANE TABLE 1 The detailed features of monoploid somatic metaphase chromosomes of Astragalus effusus Bunge (2n = 4X = 32). CG CL (µm) L (µm) S (µm) r-value (L/S) CT I m II sm III m IV m V sm VI m VII sm VIII * m sm Trait TCL A 1 A 2 AI TF% Lc/Sc CP: Chromosome group, CL: Chromosome length, L: Long arm length, S: Short arm length, L/S: r-value (Long arm/short arm), CT: Chromosome type (STEBBINS 1971), TCL: Total haploid chromosome length, *: chromosome group with pericentric inversion. interspecific hybrids. In general, the induced autotetraploid show better establishment, higher in vitro digestibility and forage production, and better performance in response to such adverse factors as disease, frost and drought, than the corresponding diploids (SIMONSEN 1976), also in potato (CARPUTO and BARONE 2005) and alfalfa (SLEDGE et al. 2002) they are used to transfer desirable characteristics from the wild into the cultivated species. Therefore, polyploidy is recognized as a major mechanism in plant evolution and adaptation (RAMSEY and SCHEMSKE 1998; BENNETT 2004). Previous studies (AHMADI et. al. 2006; ZAMANI 2008) illustrated A. effusus as a valuable source of quantity and quality provender in the rangelands. Hence, the probability of using it in intera or inter species crossing for developing auto or allo-ploid perennial forage plants, originating new genetic combinations, more variability and improving of feed production capacity. Finally, the results of the present work, together with further research on the polyploidy and dysploidy processes, will improve the understanding of the organization, structure, and evolution of the genome of Astragalus. On the other hand, different types of polyploidy levels, especially autotetraploidy, in this genus probably indicates the existence of gene(s) which control pairing similar chromosomes through cell division, gametes production such as pairing the homologous gene in wheat (SLEPER and POEHLMAN 2006), so more study is needed to identify its characteristics and the roles of this gene(s). Acknowledgments We thank Dr. Pedro Martínez Gómez for the helpful discussions and review of the manuscript. REFERENCES AHMADI A., SHAH MORADI A.A., QAEMI T., HESARI B., MASOUMI A.A. and RASHIDI D., 2006 Autecological studies of Astragalus effusus in west Azerbaijan province. FAO report, R. N.: IR BADR A., HAMOUD M. and EL-RABEY H., 1996 Chromosomal studies in the Egyptian flora V. chromosomal relationships in the genus Astragalus L. (Fabaceae) and their taxonomic inferences. Cytologia, 61: BADR A. and SHARAWY S. M., 2007 Karyotype analysis and systematic relationships in the Egyptian Astragalus L. (Fabaceae). International J. Botany, 3(2): BARCACCIA G., TAVOLETTI S., MARIANI A. and VERO- NESI F., 2003 Occurrence, inheritance and use of reproductive mutants in alfalfa improvement. Euphytica, 133: BENNETT M.D., 2004 Perspectives on polyploidy in plants - ancient and neo. Biological Journal of the Linnean Society, 82: BINGHAM E.T., GROOSE R.W., WOODFIELD D.R. and KIDWELL K.K., 1994 Complementary gene interactions in alfalfa is greater in autotetraploids than in diploids. Crop Science, 34: BRETAGNOLLE F. and THOMPSON J.D., 1995 Gametes with the somatic chromosome number: mechanisms of their formation and role in the evolution of autopolyploid plants. New Phytologist, 129: BUNGE A. VON., 1868 Generis Astragali species gerontogeae. Par Prior Claves Diagnosticae. Mem

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