Karyotype Studies on Pseudoroegneria gracillima and P. kosaninii (Poaceae: Triticeae)
In order to obtain more cytological data, the karyotypes of Pseudoroegneria gracillima and P. kosaninii were investigated. Root tips of P. gracillima and P. kosaninii were pretreated in an ice bath, fixed in a mixture of 95% ethanol: glacial acetic acid and treated in 1 M HCl at 60°C in a water bath. Somatic cells were stained in Schiff at room temperature and the meristematic portions of the root tips were squashed in 45% acetic acid. The results show that: (1) P. gracillima is diploid with two pairs of satellites and P. kosaninii is octoploid with three pairs of satellites. The karyotypes of diploid P. gracillima and octoploid P. kosaninii are first reported, (2) the karyotype formulas of P. gracillima and P. kosaninii are 2n = 2x = 14 = 12 m (2sat)+2sm (2sat) and 2n = 8x = 56 = 42 m (6sat)+12sm+2st, respectively and (3) the karyotype of P. gracillima is 1A type, while P. kosaninii is 2B type. This demonstrated that there are great variations between the karyotypes of P. gracillima and P. kosaninii.
Pseudoroegneria is a genus in Triticeae (Poaceae) with Pseudoroegneria
strigosa (M. Bieb.) a. Löve as the type species (Love,
1980). The genus contains a basic St genome, with diploid (2n = 2x = 14,
StSt) and auto- and allo-polyploid species. St genome is one of the most important
genomic components, present in more than half of the perennial Triticeae species
(Love, 1984; Dewey, 1984; Yen
and Yang, 1990; Yen et al., 2005a, b).
Morphologically, the species in this genus are caespitose, long-anthered and
cross-pollinating perennials. They are distributed in the Northern Hemisphere,
with its species occurring on open rocky hillsides from the Middle East and
Transcaucasia across Central Asia and Northern China to Western North America
(Love, 1984). Pseudoroegneria grasses have exceptionally
drought tolerant and excellent forage quality, which are precious germplasm
resources in crop forage breeding (Dewey, 1984).
Pseudoroegneria gracillima (Nevski) a. Löve and Pseudoroegneria
kosaninii (Nabelek) a. Löve are two species of Pseudoroegneria which
distributed in Russian Federation and Turkey, respectively. Love
(1984) treated them into Pseudoroegneria based on the morphological
study and he noted that the taxonomic status of the two species is temporary
with lacking of cytological data. Ding et al. (2004)
reported the karyotype of P. gracillima, whereas no cytological
data about P. kosaninii are reported. Pseudoroegneria kosaninii
is inferred to be tetraploid and the relationship of P. gracillima
and P. kosaninii is close in the RAPD and RAMP analysis (Ding
et al., 2005a, b). Based on genome specific
RAPD markers, P. gracillima and P. kosaninii contained
at least one St or slightly modified St genome (Ding et
al., 2005c). Yu et al. (2008) suggested
that P. gracillima and P. kosaninii contain one
St genome in the analysis of the ITS data of the species in Pseudoroegneria.
Therefore, the chromosome numbers and genomic constitutions of P. gracillima
and P. kosaninii are still obscure. To obtain more cytological
data, karyotypes of the two species were investigated. The aims of this study
are (1) to report the karyotypes of P. gracillima and P. kosaninii
and (2) to provide more cytological data for the appropriate taxonomic treatments
of the two species.
MATERIALS AND METHODS
The study was conducted in July 2007 at Dujiangyan City, Triticeae Research Institute of Sichuan Agricultural University. The materials used in this study are shown in Table 1. Seeds of P. gracillima and P. kosaninii were kindly provided by American National Plant Germplasm System (Pullman, Washington, USA). The two species are currently growing at Triticeae Research Institute, Sichuan Agricultural University, China (SAUTI) and the mature plants were carefully identified and determined by Chi Yen, Junliang Yang and Yonghong Zhou.
Seeds were scarified and germinated in Petri dishes at 22°C on filter paper.
Root tips were obtained from roots that were 1.0-1.5 cm in length and pretreated
in an ice bath for 24 h before fixation in a mixture of 95% ethanol: glacial
acetic acid (3:1, v/v) for 24 h. They were then treated for 8-10 min in 1 M
HCl at 60°C in a water bath. Somatic cells were stained in Schiff at room temperature
(20-25°C) for about 30 min. The meristematic portions of the root tips were
squashed in 45% acetic acid. Microphotographs were taken from metaphase cells
with a complete chromosome complement by the Olympus BX-51 camera system. Five
metaphase cells were analyzed for each species (Li and Chen,
1985). Idiograms were constructed based on the chromosome lengths and relative
arm ratios. Index of the karyotypic asymmetry and karyotype type analyses were
basically the same as described by Arano (1963) and
Stebbins (1971), respectively.
Chromosomal characteristics and chromosomal parameters are shown in Table
2 and 3, respectively. The morphology of somatic chromosomes
and karyotypes are shown in Fig. 1A-D. The idiograms are shown
in Fig. 2A and B. Results of karyotype
analysis of two species studied are presented below.
The karyotype formula is 2n = 2x = 14 = 12 m (2sat)+2sm (2sat), which
belongs to 1A type. The average arm ratio is 1.40, with a longest chromosome/shortest
chromosome ratio of 1.62. Percentage of chromosomes with arm ratio >2 is
0 and index of the karyotypic asymmetry is 58.20. The relative length of chromosomes
ranges in size from 11.07 to 17.94. All the chromosomes are metacentric with
the exception of chromosome 3, which is submetacentric. One pair of minute satellites
and one pair of large satellites are located on the short arms of chromosome
3 and 7, respectively (Fig. 1A, 2A).
||Materials used in the karyotype analysis
|| Chromosomal parameters of two species in the karyotype analysis
|*Satellite chromosome, with the satellite length included
in the chromosome length
||The morphology of somatic chromosomes and karyotypes of two species in
Pseudoroegneria (A, B) P. gracillima and (C, D) P.
kosaninii. Bar = 10 μm
|| Chromosomal characteristics of two species in the karyotype
|AAR: Average arm ratio; Lc: Longest chromosome; Sc: Shortest
chromosome; PCA: Percentage of chromosomes with arm ratio >2; As.k (%):
Index of the karyotypic asymmetry
| Idiograms of two species in Pseudoroegneria (A) P.
gracillima and (B) P. kosaninii
The karyotype formula is 2n = 8x = 56 = 42 m (6sat)+12sm+2st, which
belongs to 2B type. The average arm ratio is 1.45, with a longest chromosome/shortest
chromosome ratio of 2.35. Percentage of chromosomes with arm ratio >2 is
0.07 and index of the karyotypic asymmetry is 57.98. The relative length of
chromosomes ranges in size from 2.24 to 5.27. All the chromosomes are metacentric
or submetacentric, with the exception of chromosome 28, which is subtelocentric.
Three pairs of large satellites are located on the short arms of chromosomes
2, 3 and 12, respectively (Fig. 1B, 2B).
Based on the cytological data, Dewey (1984) pointed out
that some Pseudoroegneria species may have diploidy and tetraploidy in
different populations, such as the diploid (2n = 2x = 14, StSt) P. strigosa
and tetraploid (2n = 2x = 28, StStStSt) P. strigosa. The previous analysis
indicated that P. gracillima is tetraploid with one pair of satellites
(Ding et al., 2004). Pseudoroegneria gracillima
and P. kosaninii is closely related and P. kosaninii is inferred
to be tetraploid based on molecular data (Ding et al.,
2005a, b). However, the ploidy of P. kosaninii
is lack of cytological evidence. In the present study, the karyotypes of
diploid P. gracillima and octoploid P. kosaninii were first reported.
Combined with the earlier study, P. gracillima have diploidy and tetraploidy
in different populations. The results added new cytological data of Pseudoroegneria
Stebbins and Pun (1953) indicated that different versions
of the St genome exist in diploid Pseudoroegneria species, which suggested
the differentiations of the St genome. Hsiao et al.,
(1986) reported that five diploid Pseudoroegneria species have two
pairs of satellites. One pair of large satellites are on the short arms of chromosomes
5, the other pair of small satellites are on the short arms of chromosomes 1,
2, or 3. In this study, diploid P. gracillima have one pair of small
satellites on the short arms of chromosomes 3 and one pair of large satellites
on the short arms of chromosomes 7, which is different to the reported five
Pseudoroegneria species. Satellites on different chromosomes displayed
the variations in karyotype formulas among diploid Pseudoroegneria species.
It is valid that different versions of the St genome have different karyotype
formulas. The karyotype of diploid P. gracillima is 1A type and most
of the chromosomes are metacentric or submetacentric and the similar results
were obtained in diploid Pseudoroegneria species and tetraploid
P. gracillima (Hsiao et al., 1986; Ding
et al., 2004).
The karyotype analyses indicated that P. kosaninii is octoploid
species, which is a higher ploidy than that of other Pseudoroegneria
species (Dewey, 1984; Hsiao et
al., 1986). Also, there are great variations between the karyotypes
of P. kosaninii and other Pseudoroegneria species, especially
in sizes and positions of the satellites (Hsiao et al.,
1986; Liu and Wang, 1993; Ding
et al., 2004). Therefore, more evidence needs to be obtained
to make clear the taxonomic status and phylogenetic relationships of P. kosaninii
The authors are thankful to the National Natural Science Foundation of China (No. 30670150, 30470135). We particularly thank American National Plant Germplasm System for providing seeds.
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