2022 Volume 87 Issue 2 Pages 119-122
Two accessions were recognized in the species of Cousinia lactifelora Rech. f. Accession A had n=12 and accession B showed the presence of 0 to 10 B-chromosomes at various stages of meiosis in addition to n=12 chromosomes. These B-chromosomes were smaller than the A-chromosomes and there existed as univalent and bivalents. Association of B-chromosomes with A-chromosomes occurred in less than 0.5% at metaphase I. This phenomenon is reported here for the first time. Analysis of chromosome behavior at meiosis showed that the presence of B-chromosomes decreases chiasma frequency in A-chromosomes. The effects of B-chromosomes in odd and even numbers are different. Comparison between plants with B-chromosomes and plants without B-chromosomes suggested that the effect of B-chromosomes was on pollen stainability. It seems that the presence of B-chromosomes may be responsible for the reduction of pollen stainability in C. lactiflora.
B-chromosomes also named accessory or supernumerary chromosomes represented one of many cases of numeric variation described in more than 1,500 species of plants, 500 species of animals, and some fungi (Jones and Rees 1982, Jones 1995, Puertas 2002, Jones and Houben 2003, Camacho 2004, Burt and Trivers 2006). B-chromosomes are either euchromatic or partially or entirely heterochromatic (Jones 1995). The question of the usefulness of supernumerary B-chromosomes has long fascinated cytogeneticists.
The genus Cousinia of the tribe Cardueae is in its current circumscription one of the largest genera in the Asteraceae, with 600–700 species (Mehregan and Kadereit 2008). This genus is one of the most diverse genera in the Irano-Turanian region and central Asia (Rechinger 1972, 1979, 1986, Knapp 1987). About 250 species of the genus are distributed throughout Iran, 165 of which are endemic (Ghahreman and Attar 1999). According to the current understanding, Cousinia sect. Cynaroideae Bunge is the largest section of the genus with 110 species, is characterized by a basic chromosome number of x=12 (Ghaffari et al. 2006, Mehregan and Kadereit 2008). In the genus Cousinia, B-chromosomes are reported for five species without any effects presentation viz: C. kotschyi (2n=24+0-2B), C. lucida (2n=26+0-2B), C. komarowii (n=13+2B), C. bienerti (n=13+1B), C. tabriziana (2n=26+1B) (Afzal-Rafii 1980, Djavadi 2007, Sheidai et al. 2012). Cousinia lactiflora is an endemic species to C and CW parts of Iran and distributed on stony slopes and in an open area. During the present study, B-chromosomes were found for the first time in some accessions of C. lactiflora.
Cousinia lactiflora Rech. f. is an endemic species to Iran and found in a limited area of Lorestan and Markazi provinces. Two chromosomal accessions were recognized in this species that was collected from the same and different area in Iran. Accession A showed n=12 chromosomes and accession B showed the presence of 0 to 10 B-chromosomes in addition to n=12. To ensure the validity of our results concerning the chromosome number and meiotic analysis, we used a collection of 2–5 plants within each accession (Table 1). For meiotic studies, floral buds of appropriate size were fixed in a mixture (ethanol : chloroform : propionic acid=6 : 3 : 2) for at least 48 h at 4°C, then were stored in 70% ethanol at 4°C until used. Staining was carried out with 2% acetocarmine. The staining technique with cotton blue was used to estimate pollen viability (Dafni and Firmage 2000). Voucher specimens are preserved in the Central Herbarium of Tehran University (TUH).
| Accession | No. of plant | n | Location | Coordinate | Altitude (m) |
|---|---|---|---|---|---|
| A | 2 | 12 | Luristan: Aligudarz, 6 km towards Khomain | 33°27′48.34″N, 49° 42′14.09″E | 2,100 |
| A | 4 | 12 | Markazi: Khomain, 10 km towards Arak | 33°43′20.07″N, 50°0′11″E | 1,900 |
| B | 9 | 12+0-10B | Luristan: Aligudarz, 6 km towards Khomain | 33°27′48.34″N, 49°42′14.09″E | 2,100 |
Meiosis in accession A was regular with 12 bivalents at metaphase I and 12-12 segregation at anaphase I (Fig. 1A, B). Metaphase II indicated 12 dyads and anaphase II showed 12 monads in each pole (Fig. 1C, D). Analysis of 112 cells at metaphase I, indicated the mean number of chiasmata was 1.70 for each bivalent. Occasionally, in some cells, overlapping of bivalents was observed (Fig. 1E). Three bivalents of chromosomes were associated with nucleolus at diakinesis (Fig. 1F). These chromosomes are known as nucleolus organizer chromosomes. This phenomenon is also reported for nine other species of the genus Cousinia previously by Ghaffari and Djavadi (1998).
Accession B showed the presence of zero to ten B-chromosomes in addition to n=12 chromosomes. These B-chromosomes were smaller than the A-chromosomes and were in the form of univalents and bivalents. When more than one B-chromosome (2-10) was present, some of them paired with one another, as bivalent. In PMCs with four B-chromosomes, they appeared as two bivalents (Fig. 2A) or two univalent and one bivalent (Fig. 2B). When ten B-chromosomes were present, they appeared as three bivalents and four univalents (Fig. 2C) or four bivalents and two univalents (Fig. 2D). Analysis of 390 cells at metaphase I indicated that the percentage of univalent B and bivalents B was 62.10 and 37.9 respectively. The A-chromosomes in each case showed normal bivalent formation in the vast majority of cells. The bivalents B generally undergo non-disjunction at first anaphase. Non-disjunction Bs at anaphase I were found in 56.53% of the cells. The non-disjunction bivalents Bs were seen most clearly at metaphase II (Fig. 2E). In this study, an association of B-chromosomes with A-chromosomes occurred less than 0.5% at metaphase I (Fig. 2F). This rare observation needs to be repeated in high magnification. Previous reports indicated that in plants with more than two Bs multivalents can occur (Jones 1995), but in C. lactiflora with 10 Bs, we did not observe any Bs multivalent. The mean chiasmata frequency of A-chromosomes in pollen mother cells were 1.35, 1.27, 1.41, 1.34, 1.45, 1.31, 1.44, 1.33, 1.46, 1.40, and 1.56 when B-chromosomes number were 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 respectively (Table 2). These results indicated that the variation in B-chromosome has led to variation in chiasma frequency. Analysis of variance indicated that the difference in chiasma frequency was statistically significant (Table 3). Three types of effects on B-chromosome carriers have been reported such as decrease in chiasmata frequency (Simchen et al. 1971, Bakshi et al. 1987), increase in chiasmata frequency (Ghaffari and Bidmeshkipoor 2002, Kumar and Singh 2005) and no effect has been observed by Carter and Smith-White (1972). Comparison between mean numbers of chiasmata in the plants without Bs (1.70) with plants with Bs (1.34–1.57) indicated that the B-chromosomes decrease in chiasmata frequency in A-chromosomes.
| No. of cellsanalyzed | No. of analyzed B-chromosomes | Chiasma frequencyper bivalent |
|---|---|---|
| 28 | 0 | 1.35±0.01 |
| 20 | 1 | 1.27±0.03 |
| 22 | 2 | 1.41±0.01 |
| 34 | 3 | 1.34±0.02 |
| 24 | 4 | 1.45±0.04 |
| 12 | 5 | 1.31±0.01 |
| 20 | 6 | 1.44±0.02 |
| 8 | 7 | 1.33±0.01 |
| 6 | 8 | 1.46±0.03 |
| 2 | 9 | 1.40±0.02 |
| 8 | 10 | 1.56±0.05 |
| Source of variation | Degree of freedom (df) | Sum of squares (SS) | Mean of squares (MS) | E (MS) F value |
|---|---|---|---|---|
| Between groups | 10 | 0.513 | 0.0513 | 2.07* |
| Within groups | 81 | 2.004 | 0.0247 | |
| Total | 91 | 2.517 |
*Significant at 5% level of probability
Previous reports indicated that the presence of B- chromosomes did not affect the qualitative characters in the phenotype, the same result was also found in our study which no morphology changes were observed between accessions A and B. Phenotypic effects are usually unfavorable, especially with high numbers (Jones 1995). In this study, plants with B-chromosomes have phyllaries with ca. three pairs of spines at the margin but plants without B-chromosomes showed phyllaries with indistinct spines at the margin. Comparison between plants with B-chromosomes and without B suggested that the effect of B-chromosomes was on pollen stainability. The pollen stainability obtained from 2,370 grains for plants with B, and 2,572 grains for the plant without B was 61.18 and 94.88 respectively (Table 4). Thus, it seems that the presence of B-chromosomes may be responsible for the reduction of pollen stainability in C. lactifelora. These same effects of B-chromosomes have been reported in the rye (Muntzing 1963, Kishikowa 1965, Bakshi et al. 1987), in which the presence of B-chromosomes has been associated with a reduction in the pollen fertility and seed set. The inverse effect of B-chromosomes has been reported in Acanthophyllum laxiusculum by Ghaffari and Bidmeshkipoor (2002).
| No. of Pollen | No. of grains stained | No. of grains unstained | Pollen stainability (%) |
|---|---|---|---|
| 2370 (with B) | 1,450 | 920 | 61.18 |
| 2572 (without B) | 2,440 | 102 | 94.88 |
This work was supported by the Research Council, the University of Tehran.
