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Cytogenetic analysis of hybrids derived from wheat and Tritipyrum using conventional staining and genomic in situ hybridization G. MIRZAGHADERI1, G. KARIMZADEH1*, H.S. HASSANI2, M. JALALI-JAVARAN1 and A. BAGHIZADEH3 Plant Breeding and Biotechnology Department, Faculty of Agriculture, Tarbiat Modares University, P.O. Box 14115-336, Tehran, Iran1 Agronomy and Plant Breeding Department, Faculty of Agriculture, University of Shahid Bahonar, Kerman, Iran2 International Center of High Technology and Environmental Sciences, Mahan, Iran3 Abstract The new salt tolerant cereal, Tritipyrum (2n=6x=42, AABBEbEb) offers potential to introduce desirable characters for wheat improvements. This study was aimed to generate a segregating population from Iranian local wheat cultivars (2n=6x=42, AABBDD) and Tritipyrum crosses, study of the meiotic behaviour in F2 hybrids and identification of Eb chromosomes in F3 individuals. Results showed meiotic abnormalities in F2 plants and different pairing frequency in the meiosis among F2 plants. Genomic in situ hybridization revealed that total and Eb chromosome number of F3 seeds ranged from 39 to 45 and 0 to 10, respectively. A significant prevalence of hyper-aneuploidy was observed among F3 genotypes. C-banding patterns identified Eb chromosomes in Tritipyrum, indicating that it also can be useful to study wheat-Tritipyrum derivatives. Additional key words: alien chromosomes, meiotic abnormalities, Thinopyrum bessarabicum, Triticum aestivum. Introduction Wheat (Triticum spp.) is one of the most important cereals in the world. Salt tolerant species are available among wheat relatives. For example Thinopyrum bessarabicum (2n=2x=14, EbEb) is a perennial weed native to the Black Sea and Mediterranean region and can complete its life cycle in the presence of 350 mM NaCl (Gorham et al. 1986). Tritipyrum (2n=6x=42, AABBEbEb) is a new salt tolerant amphiploid, produced from a cross between T. durum (2n=4x=28, AABB) and Th. bessarabicum to introgress salt tolerance for wheat improvement (King et al. 1997a,b). Generation of a segregating population by crossing is important for further use in wheat improvement which is usually followed by the study of meiotic behaviour and chromosome composition of the progenies. Although generating a segregated population from wheat and

synthetic allohexaploids usually involves backcrossing of F1 plants to one of the parents, few attempts were reported for the production of segregating population without backcrossing (Hassani et al. 2003) because the F1 plants are highly sterile. Cytological markers such as C-banding, fluorescence in situ hybridization (FISH), genomic in situ hybridization (GISH) and molecular markers can be used in chromosome identification and screening addition, substitution and recombinant lines (Mullan et al. 2005, Silkova et al. 2006, Qi et al. 2007, Anugrahwati et al. 2008, Hřibová 2008, Landjeva et al. 2008). GISH is among the most powerful techniques to identify alien chromatin in the wheat background. In the case of wheat-Thinopyrum derivatives, GISH is some-what difficult due to the near relationship of the two genomes (Zhang et al. 2002).

⎯⎯⎯⎯ Received 24 July 2008, accepted 15 January 2009. Abbreviations: FISH - fluorescence in situ hybridization; GISH - genomic in situ hybridization. Acknowledgements: This work was supported by a grant of the Ministry of Science and Technology of Iran. John Innes Centre, UK is appreciated for providing of Tritipyrum materials. The first author acknowledges Dr. M.G. Francki, Department of Agriculture and Food Western Australia, South Perth, WA 6152; State Agricultural Biotechnology Centre, Murdoch University, Murdoch, WA 6150, Australia, for his technical training. We acknowledge Dr. J. Doležel, Institute of Experimental Botany, Olomouc, Czech Republic, for his review on the manuscript and helpful idea. * Corresponding author; e-mail: karim_gh@modares.ac.ir

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In the present study, the possibility of generating a segregating F2 and F3 population by crossing Iranian bread wheat (T. aestivum L., 2n=6x=42, AABBDD) cultivars with Tritipyrum was examined. Meiotic behavior of 42-chromosome F2 plants was evaluated at

meiosis and the chromosomal constitution of obtained F3 seeds was studied by genomic in situ hybridization at mitosis. The results are important for the further use in wheat breeding programs.

Materials and methods Plants: The allohexaploid Tritipyrum (AABBEbEb) lines: Azb, StbCrb and KabCrb (King et al. 1997b) were crossed reciprocally with Triticum aestivum spring cultivars Roushan, Falat and Navid. All the growth room grown F1 progenies of wheat as female were sterile but F1 plants derived from wheat (male) × Tritipyrum (female) were able to produce F2 seeds. Mitotic and meiotic chromosome preparations: F2 seeds from cv. Navid (male) × line Azb (female) crosses were germinated on moist filter paper in Petri dishes for 3 d at room temperature. The root tips (1.5 cm) were cut and pretreated in ice cold water for 24 h in order to arrest the cells in metaphase. Root tips were then fixed in ethanol:acetic acid (3:1, v/v) for 48 h at 4 °C. Chromo-some preparations were carried out using Feulgen staining method. For securing meiotic preparations, 42-chromosomes F2 seeds were planted in growth room to grow and flower. Immature spikes prior to emerging from flag leaf were harvested and fixed in ethanol:acetic acid for 24 h at 4 °C. Immature anthers were squashed in a drop of acetocarmine (1 %, m/v) on slide with gentle heating. Chiasmata per pollen mother cells (PMCs) in metaphase I of meiosis for each of the F2 plants was estimated by (2 × ring bivalents) + rod bivalents + (2 × trivalents) + (3 × quadrivalents) (Fedak and Han 2005). Chiasma frequencies in metaphase I of F2 plants were compared by one-way ANOVA using Minitab software (Ryan and Joiner 2001) after normality test and Bartlett's test of homogeneity. DNA isolation and labeling: Th. bessarabicum DNA was extracted from leaves using phenol-chloroform according to Pallotta et al. (2000) and labelled with biotin-16-dUTP (Roche Applied Science, Penzberg, Germany), using a nick translation kit according to the manufacturer's instructions. Probes were purified with ethanol precipitation. Efficiency of labelling was determined using a dot-blot detection kit (Fermentas, Vilnius, Lithuania). Total genomic DNA of cv. Navid was autoclaved for 5 min to give fragments of 100 - 200 base pairs and used at different concentrations as blocking DNA. Chromosome preparation for in situ hybridization: F3 seeds were placed on moist filter paper in Petri dishes at room temperature for 2 - 3 d in dark. Root tips were incubated in ice-cold water overnight and fixed in ethanol:acetic acid fixative for up to 3 d at 4 °C. They

were subsequently squashed in a drop of acetic acid (45 %, v/v) on microscope slide. The coverslips were removed after freezing in liquid nitrogen and the slides were dried by gentle heating. Slides were selected for well spread preparations by phase contrast microscopy. Selected slides were stored at room temperature for 1 - 2 d before in situ hybridization (Francki and Langridge 1994). In situ hybridization: GISH was adopted from standard protocols (Schwarzacher and Heslop-Harrison 2000). Slides were incubated in RNase A (10 µg cm-3 in 2× SSC) for 1 h at 37 °C and washed in 2× SSC for 5 min. Slides were hydrolyzed in 10 mM HCl for 5 min at room temperature and treated by pepsin (10 µg cm-3 in 10 mM HCl) for 10 min at 37 °C. After washing in 2× SSC then the slides were stabilized in 4 % (m/v) paraformaldehyde in 1× PBS for 10 min at room temperature, followed by washing 2 × 5 min and dehydrating in ethanol series (70, 90 and 100 %). Slides were then denatured in 50 % formamide in 2× SSC for 2.5 min at 70 °C and dehydrated in cold ethanol series. The hybridization solution, containing 50 % (v/v) formamide, 2× SSC, 10 % (m/v) dextran sulfate, 0.3 mg cm-3 of sheared salmon testes DNA, about 3 mg cm-3 of labelled probes and 75 mg cm-3 autoclaved genomic DNA of wheat was denatured in boiling water for 6 min. After chilling on ice, 0.03 cm3 of the hybridization mixture was applied to each slide and covered with a coverslip. Slides were placed for 10 min at 80 °C and left overnight at 37 °C for hybridization in a closed humidified container. After removing the coverslips in 2× SSC, post-hybridization washing was performed in 50 % (v/v) formamide in 2× SSC for 2 × 10 min at 42 °C followed by rinsing in 2× SSC for 2 × 10 min at room temperature. The biotin-labelled probe was detected with 5 µg cm-3 fluorescein avidin in blocking buffer (3 % BSA in washing buffer). After incubation at 37 °C for 30 min, the slides were washed in washing buffer (0.1 M sodium bicarbonate, 0.05 % igepal CA 630, pH 8) for 4 × 5 min and amplified using 5 µg cm-3 fluorescein anti-avidin, followed by a second round of washing. The slides were air-dried and mounted in 0.03 cm3 of mounting medium containing 1 µg cm-3 propidium iodide as counterstain. Slides were analyzed with an epifluorescence Olympus BX51 (Tokyo, Japan) microscope and images were captured, using a DP12 digital camera. C-banding: C-banding of Tritipyrum chromosomes followed the method described by Gill et al. (1991).

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Karyogram and the idiogram of the C-banded chromosomes of Eb genome were generated using Adobe Photoshop software and arranged in order of decreasing length. As the homologous relationship of Eb genome with wheat chromosomes was unknown to us, homo-logous chromosomes were arranged and numbered in order of decreasing size regardless of their homologous relationship with A, B or D chromosomes of wheat. The nomenclature used for the description of Eb chromosome morphology is that proposed by Levan et al. (1964). The

chromosome parameters were: long arm (L), short arm (S), total chromosome length (TL = S + L), arm ratio (AR = L/S), r - value (S/L), relative length of chromosome [RL % = (TL/∑TL) × 100], centromeric index (CI = S/TL), form percentage of chromosome [F % = (S/∑TL) × 100], total chromatin length (X = 2 × ∑TL) and total form percentage of karyotype [TF % = (∑S/∑TL) × 100].

Results In the reciprocal crosses of wheat and Tritipyrum parents, when wheat cultivars were used as female parents, eighty five F1 seeds were obtained from 23 parents. Forty F1 progenies were grown in growth room. These F1 plants were completely sterile and no F2 seeds were produced. On the other hand, when wheat cultivars were used as male parents, obtained 150 F1 plants could produce some F2 seeds. Genomic in situ hybridization on a sample of F1 genotypes confirmed the presence of 7 Eb chromosomes (Fig. 2A). The 42-chromosome F2 seeds from Navid

(male) × Azb (female) crosses were selected by chromosome counting (Fig. 1A) and planted in growth room for meiotic studies. The morphology of F2 plants was similar to Tritipyrum parent: the mean height was equal and the spikes, awn length, mean number of spikelets in each spike (12 spikelets per spike) as well as leaf width were similar. Meanwhile, variation was observed among F2 plants especially in plant height and spike morphology (Fig. 1B).

Fig. 1. Mitotic and meiotic study of F2 progenies of wheat cv. Navid and Tritipyrum line Azb crosses. Feulgen metaphase chromosome staining in root tip cells of a F2 genotype with 42 chromosomes (A). Morphological variations were observed among F2individuals (B). Aceto-carmine staining showed anaphase bridge (C), lagging chromosomes (D), micronuclei or chromosome segments (E and F) in the microspores of F2 plants. Aceto-carmine staining of the chromosomes at meiosis metaphase I in the PMC of a F2 plant with 8 univalents, 5 rod bivalents and 11 ring bivalents (G). Bar = 20 µm.

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Table 1. Average metaphase I configuration per meiocyte in wheat (cv. Navid; male), Tritipyrum (line Azb; female) and F2 plants from and crosses between Navid and Azb. I - univalents, II - bivalents, III - trivalents, IV - quadrivalents. Means ± SE. Means with different letters showed significant differences (P < 0.05). MS between F2 plants = 14.26 (P < 0.01), MS among cells within F2 plant = 0.55. Numbers in parentheses show the range number of observed chromosome configuration.

Individuals 2n Number Meiotic configuration Chiasmata of PMCs I Rod II Ring II III IV

Navid 42 29 0 0.17±0.38 (0- 1) 20.83±0.38 (20-21) 0 0 41.83±0.30 Azb 42 31 0.68±1.50 (0-6) 0.97±0.91 (0- 3) 19.60±1.40 (16-21) 0.03 (0-1) 0.064 (0-1) 40.42±2.02 Azb × Navid. F2 (1) 42 24 2.66±1.73 (0-6) 6.91±2.92 (3-14) 12.79±2.45 ( 7-16) 0 0 32.50±2.22b Azb × Navid. F2 (2) 42 16 3.37±1.40 (2-6) 4.62±1.63 (2- 8) 14.69±1.49 (12-17) 0 0 34.00±1.67a Azb × Navid. F2 (3) 42 25 4.80±1.85 (2-8) 4.92±1.60 (0- 7) 13.48±2.04 ( 9-18) 0.08 (0-1) 0 32.04±2.63b Azb × Navid. F2 (4) 42 43 6.49±1.42 (2-8) 5.21±1.86 (1- 9) 12.41±1.87 ( 9-16) 0.07 (0-1) 0 30.17±2.01c Azb × Navid. F2 (5) 42 31 7.09±1.23 (3-9) 6.35±1.24 (4- 8) 10.71±1.70 ( 8-14) 0.26 (0-3) 0 28.29±1.83d

Table 2. The Eb and A,B and D chromosome number of individual F3 seeds from Navid (male) × Azb (female) crosses revealed by genomic in situ hybridization in mitotic metaphase preparations.

Number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Mean

Eb 4 5 4 6 5 8 6 9 0 6 6 10 6 7 8 4 3 5 5 9 6 7 6 8 9 6.08 A, B and D 39 36 38 36 38 37 38 31 42 33 37 34 36 35 35 38 41 39 38 34 36 35 35 34 34 36.36 Total 43 41 42 42 43 45 44 40 42 39 43 44 42 42 43 42 44 44 43 43 42 42 41 42 43 42.44

Fig. 2. Genomic in situ hybridization on mitotic metaphase preparations of a F1 root tip from Navid × Azb using Th. bessarabicum probe indicating 7 chromosomes of Eb genome (A). Genomic in situ hybridization of mitotic metaphase chromosomes of a F3 plant root tip using Th. bessarabicum probe revealed four Eb and 40 wheat chromosomes (B). Bar = 20 µm. The number of univalents, bivalents, trivalents and quadrivalents at metaphase I in PMCs of plants were counted for parents and five F2 individuals (Fig. 1G; Table 1). The number of univalents ranged from 0 to 9 in metaphase I of each PMC of F2 plants (Table 1). The study of chromosome pairing in the meiosis of F2 individuals showed that they are unstable having a mean number of univalents between 2.37 and 7.09 per cell. The mean comparisons revealed significant differences in the rate of chiasmata among F2 plants (Table 1). Anaphase I showed the majority of chromosomes migrating to the poles while some anaphase bridges were observed (Fig. 1C) and some chromosomes were lagging in the metaphase plate of the cells (Fig. 1D). A special type of micronuclei was observed inside some microspores of F2 plants which resembled chromosomes or chromosome segments, ranging from 0 to 3 in each microspore (Fig. 1E,F).

The chromosome constitution of F3 seeds was characterized by GISH in root tip cells of twenty five F3 Table 3. Number and percentage of F3 seeds with the same chromosome number.

Chromosome number of F3 seeds 45 44 43 42 41 40 39

Number of F3 seeds 1 4 7 9 2 1 1 [%] 4 16 28 36 8 4 4

individuals in which the Eb chromosomes were clearly highlighted (Fig. 2B). Eb chromosome number ranged from 0 to 9 among F3 genotypes (Table 2). F3 seeds with 42 mitotic chromosome number appeared at highest frequency (36 %; Table 3). A significant frequency of

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hyper-aneuploidy in comparison with hypo-aneuploidy was observed among F3 progenies: 48 and 16 % of which had more and less than 42 chromosomes, respectively (χ2 = 4, P < 0.05). In order to further characterization of Tritipyrum, chromosomes of Th. bessarabicum (Eb genome) in Tritipyrum generally were identified by their distinctive C-banded pattern in comparison with standard C-banding pattern of wheat chromosomes (Gill et al. 1991). Homologous chromosomes were identified according to their banding pattern and arm ratio. For well spread chromosomes and high quality karyotypes, the karyogram and the idiogram of the chromosomes of Eb genome were generated (Fig. 3). In chromosomal

parameters, the mean total length of chromosome (TL) in Eb genome ranged from 10.86 to 14.48 µm (Table 4), showing relative lengths of 12.2 to 16.3 %, respectively. Total chromatin length (X) was 176.44 µm, the centromeric index (CI) of the chromosomes in the complement varied between 0.33 to 0.48 and the form percentage of chromosomes (F %) ranged from 4.3 to 7.3 (Table 4). In karyotypic parameters, Eb genome karyotype had a predominance of 'm' chromosomes and showed a karyotype formula of 12m + 2sm based on the nomenclature proposed by Levan et al. (1964). Total form percentage of the karyotype (TF %) was 42 %.

Fig. 3. C-banding pattern of the Tritipyrum (line Azb) chromosomes (A and B). Bar = 20 µm. Karyogram and idiogram of Eb

chromosomes which distinguished by comparison of wheat and Azb chromosomes pattern (C). Values are in µm. Table 4. Chromosome characteristics [μm] of Eb genome (Th. bessarabicum; 2n=2x=14, EbEb). L - length of long arm, S - length of short arm, TL - total chromosome length, AR - arm ratio, r-value - S/L, RL % - relative length of chromosome, CI - centromeric index, F % - form percentage of chromosome, m - medium region, sm - submedium region, X (total chromatin length) = 176.44, TF (total form percentage) = 42 %, Stebbins category of asymmetry = 2A. Means ± SE.

No. L S TL AR r-value RL % CI F % Type

1 6.43 ± 0.27 8.00 ± 0.30 14.48 ± 0.562 1.24 ± 0.02 0.80 ± 0.02 16.33 ± 0.010 0.44 ± 0.005 7.27 ± 0.316 m 2 6.44 ± 0.18 7.77 ± 0.19 14.22 ± 0.362 1.21 ± 0.01 0.82 ± 0.01 16.09 ± 0.004 0.45 ± 0.003 7.29 ± 0.204 m 3 5.06 ± 0.18 8.05 ± 0.15 13.09 ± 0.302 1.59 ± 0.04 0.63 ± 0.01 14.83 ± 0.003 0.38 ± 0.007 5.72 ± 0.209 m 4 6.10 ± 0.20 6.53 ± 0.29 12.60 ± 0.448 1.06 ± 0.04 0.94 ± 0.03 14.27 ± 0.005 0.48 ± 0.009 6.90 ± 0.229 m 5 4.64 ± 0.15 7.02 ± 0.30 11.65 ± 0.461 1.51 ± 0.02 0.66 ± 0.01 13.20 ± 0.005 0.40 ± 0.003 5.25 ± 0.176 m 6 3.80 ± 0.10 7.73 ± 0.33 11.53 ± 0.303 2.03 ± 0.14 0.49 ± 0.03 13.05 ± 0.003 0.33 ± 0.130 4.30 ± 0.122 sm 7 4.56 ± 0.13 6.23 ± 0.26 10.86 ± 0.385 1.36 ± 0.04 0.73 ± 0.02 12.21 ± 0.004 0.42 ± 0.007 5.16 ± 0.154 m

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Discussion It has been argued that the transfer of a whole genome may not be desirable, since all chromosomes in a genome may not carry useful traits and some of them may actually carry harmful traits. Therefore, it would be better to transfer some alien chromosomes or chromosome segments to wheat through hybridization. In the present study, it has been achieved by crossing bread wheat (AABBDD) and salt tolerant allohexaploid Tritipyrum (AABBEbEb) which was crossed to produce AABBDEb

(7"A+7"B+7'D+7'Eb). The sterility of the F1 hybrids from wheat (as female parent) and Tritipyrum offers the need for their backcrossing onto the wheat parent. The morphology of F2 plants from Tritipyrum (as female parent) was mainly Tritipyrum-like. In other research work, similar morphology between Tritipyrum and F2 individuals was also identified among F1 progenies, synthesized by crossing the male Thinopyrum ponticum (2n=10x=70) parent and a hexaploid bread wheat (Jauhar 1995). Meiosis in the progenies generated from crosses between wheat and wild relatives usually shows abnormalities such as anaphase bridges, lagging chromosomes, chromosome segments and micronuclei (Brasileiro-Vidal et al. 2005). In agreement to the latter report, similar results were detected in the present study (Fig. 1). The micronuclei which were similar to chromosome segments (Fig. 1) probably originated from acentric chromosome fragments which were the result of unbalanced translocations in meiosis. Because of the delayed migration to the poles, these acentric fragments might locate far from the other chromosomes during the next phases of meiosis and can be seen around the nucleus inside the microspore. In agreement to this, a similar result was found among F1 hybrids of wheat-Thinopyrum partial amphiploids (Chen et al. 1992, Fedak and Han 2005). Significant difference in chiasmata among F2 individuals (Table 1) confirmed the possibility of production of plants with different chromosome composition such as either addition or substitution lines in the next generations. In the present study, the variation of chromosome number among individual F3 progenies may be partially due to the variable chromosome number of D and Eb genomes caused by meiotic instability of F1 and F2 hybrids. The significant prevalence of hyper-aneuploidy in comparison with hypo-aneuploidy among F3 genotypes in the present study was similar to the tendency which

was found among the BC1F3 progeny of crosses derived from T. aestivum and different Agropyron species (Chen et al. 1992, Jauhar 1995). The observation of aneuploid cells of F3 plants can be due to non-disjunction of bivalents, inclusion of univalents in the same gamete or loss of lagging chromosomes during meiosis which is in agreement with the Evans (1964) and Sharma and Gill (1983). Chromosome alteration in interspecific hybrids may also be brought about by modifications in gene expression due to parental genome interactions and/or different ploidy levels (Comai 2000). On the other hand, no translocation was detected in the counted F3 plants in the present study while some was reported in BC1F4-BC1F6 progenies of wheat and wheatgrasses in other different studies (Ellneskog-Staam and Merker 2002, Fedak and Han 2005). Eb genome of Th. bessarabicum and different E genomes of Th. ponticum and Th. intermedium has been identified by GISH previously (Wang and Zhang 1996, Zhang et al. 1996, Zhang et al. 2002), using blocking-probe DNA ratio of 80-150:1. These authors used genomic DNA from Pseudoroegneria stipifolia (St genome) as a probe for discrimination between E-genome and ABD-genome chromosomes. In our study, the ability to distinguish Th. bessarabicum chromosomes was reproducible, using Th. bessarabicum DNA as a probe and with ratio of 25:1. The ratio used here was close to the 20:1 blocking-probe ratio which was applied by Jauhar and Peterson (2006), using Th. bessarabicum DNA as a probe. C-banding procedure (Fig. 3) showed that Eb chromosomes are characterized by symmetrical karyotype, with a predominance of 'm' chromosomes. When C-banding pattern and karyotypic data are considered together, all Eb chromosomes can be differentiated, indicating that it also can be used to study wheat-Tritipyrum derivatives. In conclusion, F1 to F3 seeds were obtained from crosses between Iranian bread wheat and Tritipyrum. Chromosome pairing was studied in F2 plants. Both GISH (on F1 and F3 seeds) and C-banding (on Tritipyrum) techniques were made an accurate identification of Eb chromosomes which can be applied in the study of wheat-Tritipyrum derivatives. A significant prevalence of hyper-aneuploidy in comparison with hypo-aneuploidy was observed among F3 progenies. The production of such wheat lines (containing Eb chromatin) is aimed to assess their potential tolerance in local dry and salty areas.

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