Telomeric heterochromatin, seeds and meiotic characteristics in two tricepiro lines/Heterocromatina telomerica, semillas y caracteristicas meioticas en dos lineas de tricepiro.
Tricepiro is a synthetic forage crop obtained in Argentina in 1972 by crossing a hexaploid triticale (AABBRR 2n=6x=42) and an octoploid trigopiro (AABBDDJJ, 2n=8x=56) (Covas, 1976, 1989, 1995). Different lines, all with 2n=42 chromosomes, were obtained after several years of breeding (Tosso et al., 2000). Two of these lines, named Don Rene INTA and FA-L2, were studied in the present work. The genomic composition of tricepiro in different lines is quite similar to triticales, but tricepiros have Thinopyrum introgression (Ferrari et al., 2005; Fradkin et al., 2009a). Both lines of tricepiro analyzed here differ in their kernel characteristics: Don Rene has shriveled kernels while FA-L2 has smooth ones.
Kernel shriveling and meiotic instability have been considered important problems of synthetic amphiploid triticales. These characteristics have been frequently related to the presence of large terminal heterochromatin blocks on the rye chromosomes (Thomas & Kaltsikes, 1974; Merker, 1976; Bennett, 1977; Roupakias & Kaltsikes, 1977; Gustafson & Bennet, 1982; Naranjo & Lacadena, 1982; Soler et al., 1990; Jouve & Soler, 1996) . However, in some cases, the size of the terminal heterochromatin blocks seems not to be associated with kernel shriveling and meiotic instability (Varghese & Lelley, 1983; Papa et al., 1990).
Triticale meiotic instability can be originated by causes that are not related to the large rye terminal heterochromatin bands, such as mutations, that could disturb the correct rye meiotic mechanisms (Jenkins et al., 2005, 2008; Sosnikhina et al., 2005, 2007).
The aim of the present work is to analyze the relationship between the percentage of the rye terminal heterochromatin bands and the presence and number of univalents in Metaphase I, the Meiotic Index, and the kernel characteristics (shriveled or smooth kernels and weight) in the tricepiro lines Don Rene and FA-L2.
MATERIALS AND METHODS
Seeds of the tricepiro Don Rene INTA, tricepiro FA-L2 and Secale cereale L. were provided by Ing. G. Covas. Plant samples from which seeds were obtained were deposited at Facultad de Agronomia de la Universidad Nacional de La Pampa, Argentina. Plants were grown in irrigated beds in the greenhouse of the Instituto Fitotecnico de Santa Catalina, Llavallol, Buenos Aires Province, Argentina.
Young spikes were fixed in absolute alcohol: acetic acid (3:1). The anthers from immature flowers were squashed in 2% acetic hematoxylin as stain and 1% ferric citrate as mordant. Slides were made permanent by freezing with CO2, removing the cover slip, dehydrating in absolute alcohol and mounting in Euparal.
The number of tetrads with and without micronuclei (normal) of each line was determined from the observation of at least 100 tetrads. The Meiotic Index (MI) was obtained according to the following formula:
MI = (number of normal tetrads / number of total tetrads analyzed) x 100 (Love, 1949).
C-banding was performed according to Tito et al. (1991) with minor modifications. Roots (1 cm long) were pre-treated in ice-cold water for 36 hours and fixed in absolute alcohol: acetic acid (3:1) for 24 hours at room temperature and stored at -20[degrees]C. Fixed roots were washed in 0.01 M citric acid-sodium citrate buffer (pH 4.6) to remove fixative, and transferred to an enzyme solution containing 2% cellulase and 20% liquid pectinase. The softened material was washed again in buffer solution. Finally, slides were prepared using the squash technique in a drop of 45% acetic acid and chromosomes were stained with 4'-6-Diamidino-2phenylindole (DAPI).
The lengths of rye chromosomes and their terminal heterochromatin bands were measured with the computer application MicroMeasure version 3.01, available on the Internet at http://www.cohstate.edu/Depts/Biology/MicroMeasure.
The lengths of the rye terminal heterochromatin bands were normalized dividing the length of each band by the total length of the corresponding chromosome.
The percentage of normalized heterochromatin present in the rye chromosomes of each line was calculated using the following formula: (X NLH / n) x 100 (NLH=normalized length of heterochromatin terminal bands, n=total number of rye chromosomes in each line). Twenty cells in each line were analysed.
One hundred seeds of each line were individually weighed in a digital scale and the seed average weight of each line was obtained.
Differences in the seed weight and size of telomeric heterochromatin between the two lines were tested through the t-Test. The number of cells with and without univalents at Metaphase I and the number of tetrads with and without micronuclei were tested using the Chi-square test. All the statistical analyses mentioned above were carried out with the program INFOSTAT (Di Rienzo et al., 2008).
Different superscript letters in each column denote significant differences (p<0.05)
We confirmed that individuals belonging to the two tricepiro lines studied had the same chromosome number (2n=42) (Fig. 1A, B) and 14 rye chromosomes with conspicuous DAPI (+) terminal heterochromatic bands (Fig. 1A-C). No differences were found in the heterochromatin percentage of the two lines (p<0.05) (Table 1).
A complete pairing of chromosomes, forming 21 bivalents, up to late diakinesis was observed in both lines (Fig. 2A, B). However, differences in the number of univalents outside the equatorial plate in Metaphase I were found between Don Rene and FA-L2 (Fig. 2C, D). FA-L2 had a lower percentage of cells with univalents outside the equatorial plate in Metaphase I and a narrower univalent range (Table 1).
[FIGURE 1 OMITTED]
Although both lines had tetrads with micronuclei (Fig 2E, F), the number of tetrads with micronuclei was higher in Don Rene than in FA-L2. Besides, Don Rene had the highest number of micronuclei per tetrad (Table 1).
The meiotic index value was different in the two lines, being lower in Don Rene than in FA-L2. The seed weight also differed significantly between both lines, being lower in Don Rene than in FAL2 (Table 1).
DISCUSSION AND CONCLUSIONS
The same chromosome number and similar rye chromosomes in both lines are a consequence of their common origin (Ferrari, 2004; Ferreira et al., 2007). The rye chromosomes were recognized by their typical terminal C-bands, in accordance with other authors (Papa et al., 1990; Cuadrado & Jouve, 1994; Fradkin et al., 2009b). The common origin of the two lines can explain the similarity in heterochromatin percentage presented in the rye chromosomes. However, some of their morphological, agronomical and biochemical characteristics are different (Bertoni et al., 1995 a, b; Gros et al., 1995; Ferrari et al., 2001).
In the present work, these lines showed differences in the meiotic behavior: tricepiro Don Rene had a greater number of univalents in Metaphase I and micronuclei in the tetrads than FA-L2. Besides, Don Rene and FA-L2 differed in their kernel characteristics: the former had shriveled seeds whereas the latter had smooth ones with greater average weight.
The rye heterochromatic telomeric bands have been associated with abnormalities in the meiotic behavior, the shriveling and the weight of the seeds (Bennet, 1977; Soler et al., 1990). However, in the present study, the two tricepiro lines differed in their meiotic behavior and the weight and texture of their kernel, but had similar heterochromatin content of the terminal areas of the rye chromosomes. This suggests that other mechanisms may be involved in the origin of these differences. We thus hypothesize that one of the possible reasons is the presence of genes that modify the meiotic stability of the rye chromosomes. This idea has been supported by different authors who suggested that genes that induce asynapsis or desynapsis of homologous chromosomes could account for the origin of univalents in Metaphase I and of micronuclei in tetrads (Jenkins et al., 2005; Sosnikhina et al., 2005; Mikhailova et al., 2006).
[FIGURE 2 OMITTED]
The lower frequency of meiotic abnormalities in FA-L2 would enable this line to produce more balanced gametes than Don Rene. Therefore, the well-developed endosperm of FA-L2 would produce seeds with a smoother texture and a greater weight than those of Don Rene. We cannot rule out that physiological factors are involved in the shriveling of the seed, as postulated by several authors (Klassen et al., 1971; Branlard et al., 1985; Heneen & Brismar, 1987; Bernardo et al., 1990).
As a consequence of the trigeneric origin of tricepiro (wheat, rye and wheatgrass), the genetic complexity could justify the variability found in the lines. Recent studies have shown that the allopolyploid formation in triticale and wheat is accompanied by diverse genetic and epigenetic events (Ma et al., 2004; Feldman & Levy 2005; Ma & Gustafson, 2006, 2008).
We postulate that the origin of the abnormalities in the meiotic behaviour and the shriveling and weight of the kernels observed in the tricepiro lines studied are not related to the heterochromatin content of the rye chromosomes. The analysis carried out in the present work illustrates the importance of cytogenetic evaluation and multidisciplinary studies when analyzing different parameters in breeding programs.
This research was supported by grants from Consejo Nacional de Investigaciones Cientificas y Tecnicas (CONICET-PIP 112-200801-00342) and Universidad de Buenos Aires (X178 and X446). In memory of Dr. Naranjo.
Original recibido el 9 de septiembre, aceptado el 11 de mayo de 2011.
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Maia Fradkin (1), Eduardo Greizerstein (1,4), Lidia Poggio (1), Hector Paccapelo (2) & Maria Rosa Ferrari (3)
(1) Departamento de Ecologia, Genetica y Evolucion, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Intendente Guiraldes 2160, Ciudad Universitaria, C428EHA Ciudad Autonoma de Buenos Aires, Argentina; email@example.com (author for correspondence)
(2) Facultad de Agronomia, Universidad Nacional de La Pampa, Cnel. Gil N[degrees]353, L6300DUF Santa Rosa, La Pampa, Argentina.
(3) Facultad de Ciencias Veterinarias, Universidad de Buenos Aires, Chorroarin 280, C'427CWO Ciudad Autonoma de Buenos Aires, Argentina.
(4) Facultad de Ciencias Agrarias, Universidad Nacional de Lomas de Zamora, Ruta 4, km 2, '846 Lavallol, Prov. de Buenos Aires, Argentina.
Table 1. Seed and meiotic characteristics of Don Rene INTA and FA-L2 tricepiro lines.Abbreviations: n1, number of cells; n2, number of tetrads; n3, number of seeds; SD, standard deviation. Don Rene INTA FA-L2 Kernel Shriveled Smooth Rye chromosome heterochromatin mean 12.55[+ or -] 12.79[+ or -] (%)[+ or -]SD 0.[17.sup.a] 0.[23.sup.b] [n.sub.1] 20 20 Cells with I (%) in MI 42. [85.sup.a] 14.[00.sup.b] Range of I/cell 1-11 1-4 [n.sub.1] 77 40 Meiotic index 66.47 87.90 Range of micronuclei/tetrad 1-9 1-6 [n.sub.2] 173 211 Seed weight average 0.029[+ or -] 0.038[+ or -] (g)[+ or -]SD 0.[000.sup.a] 0.[001.sup.b] [n.sub.3] 100 100