Acta Botanica Sinica
植 物 学 报
2004, 46 (1): 110－115
http://www.chineseplantscience.com
Heredity of Chloroplast and Nuclear Genomes of Asymmetric Somatic
Hybrid Lines Between Wheat and Couch Grass
CHEN Sui-Yun, LIU Shu-Wei, XU Chun-Hui , CHEN Yong-Zhe, XIA Guang-Min*
(College of Life Sciences, Shandong University, Jinan 250100, China)
Abstract: Chloroplast microsatellite primers of wheat (Triticum aestivum L.) and random primers were
employed to identify the chloroplast and nuclear genomes of three selfed linesⅡ-2, Ⅱ-Ⅰ-8 (F2-F6) and 8-1
(F3-F6) ( segregated from Ⅱ-Ⅰ-8 of F2), which were derived from the same asymmetric somatic hybrid clone
between Triticum aestivum L. cv. Jinan 177 and Agropyron elongatum (Host) Nevski. The results showed
that the chloroplast genomic components of the three lines were consistent and dominated by that of
wheat. Bands characteristic to both parents were only detected in the sequence of the intergenic region
between rpl14 and rpl16 of the chloroplast genome, suggesting the existence of chloroplast DNA of A.
elongatum in those hybrids. Furthermore, this exogenous integration of chloroplast DNA was passed to F6
stably. RAPD analysis showed that there were different DNA fragments of A. elongatum in different lines.
However, the nuclear genome basically remained stable during passage.
Key words:
somatic hybrid lines between Triticum aestivum and Agropyron elongatum ; nuclear genome;
chloroplast genome; chloroplast microsatellite; RAPD
Somatic hybridization is a new technique for plant breeding due to its possibility to combine sexually incompatible
species and both the nuclear and cytoplasmic genomes of
the fusion parents. There were some reports in breeding
studies through the use of plant somatic hybrids (Glimelius
et al., 1991), e.g. transfer of plant resistance against nematodes (Austin et al., 1988), bacteria (Hansen and Earle, 1995)
and fungi (Guo et al., 2000), practical applications of somatic hybrids between wild rice (Oryza officinalis) and cultivated rice (Oryza sativa), as well as cytoplasmic male
sterility (CMS) transfer of rice nuclear-cytoplasmic hybrids
(Bijoya et al., 1999). But there were quite few reports in
studies on the nuclear-cytoplasmic genome component of
somatic hybrid progenies, and no systematic researches
on hybrid heredities.
In most somatic hybrids reported, chloroplasts of both
parents were segregated randomly, and the chloroplast component tended to be uniparental (Derks et al., 1991;
Mohapatra et al.,1998; Guo and Deng, 2000; Liu and Deng,
2000), and there were only a few examples of chloroplast
recombination (Kisaka et al., 1997; Cardi et al., 1999; Zhou
et al., 2001). In these experiments, hybrids were analyzed
by Southern-based RFLP or direct cpDNA restriction
(August et al., 1992; Kisaka et al., 1997; Mohapatra et al.,
1998; Cardi et al., 1999; Zhou et al., 2001), which need lots
of DNA samples (Panaud et al., 1996). But hybrid samples
were usually rare, only a few loci in somatic hybrids had
been analyzed, thereby cpDNA patterns of most somatic
hybrids has not been actually defined. The PCR-based chloroplast microsatellite method, being less DNA-sample-consuming and more efficient, is able to detect more polymorphism and achieve stable and reliable results (Panaud et al.,
1996), which has been utilized in the analysis of somatic
hybrids between eggplant (Solanum melongena) and
Solanum aethiopicum (Collonnier et al., 2001), as well as in
Citrus somatic hybrids (Cheng et al., 2003).
Protoplasts derived from common wheat (Triticum
aestivum) (receptor) were fused with UV-treated protoplast
of couch grass (Agropyron elongatum) (donor) by polyethylene (PEG) method, and fertile asymmetric somatic hybrid
plants (similar to wheat in appearance) were obtained and
also have been raised to the F6. Analysis for chromosome
and isozyme of F0 and F1 has confirmed their hybrid properties (Xia et al., 1999). Some agronomic traits and protein
components of F2 lines were investigated (Xiang et al., 2001).
Analysis had been done on the salt-tolerance of Ⅱ-Ⅰ-8
and Ⅱ-2 of F3 to F4 (Chen et al., 2000); Chromosomal behavior of pollen mother cell (PMC) (Zhao et al., 2002) and
chromosomal translocation of A. elongatum (by Genome in
situ Hybridization (GISH) analysis) (Xia et al., 2003) of F5
lines were observed, and the glutenin subunit components
were assayed (Zhao et al., 2003). As for cytoplasmic genome,
including chloroplast genome, the component and hereditary stability have not been investigated yet. In order to
Received 30 Jun. 2003 Accepted 23 Aug. 2003
Supported by the Hi-Tech Research and Development (863) Program of China (001AA241032) and the National Natural Science Foundation
of China (30070397).
* Author for correspondence. E-mail:<[email protected]>.
CHEN Sui-Yun et al.: Heredity of Asymmetric Somatic Hybrid Lines Between Wheat and Couch Grass
understand the nuclear-chloroplast genome components
and hereditary stability in asymmetric somatic hybrid progenies between wheat and couch grass, chloroplast
microsatellite primers and random primers were employed
to identify the chloroplast and nuclear genome of three
selfed linesⅡ-2,Ⅱ-Ⅰ-8 (F2-F6) and 8-1 (F3-F6) ( segregated
from Ⅱ-Ⅰ-8 of F2)，which were derived from one asymmetric somatic hybrid clone. The selected materials possess the following various features:Ⅱ-2 has taller stems
(80 cm) with large ears and grains, and exhibits potential for
enhanced yield and salt tolerance; Ⅱ-Ⅰ-8 has short stems
(55 cm on average) with a strong tillering ability, smaller
ears and grains as well as elevated seed protein content
(about 17%－22%) compared with that of the parent wheat
(about 14%) (Zhao et al., 2003); 8-1 has taller stems (about
75 cm) with the ear size between the formers.
1 Materials and Methods
1.1 Plant materials
Plants used in this study are as follows: common wheat
( Triticum aestivum L. cv. Jinan 177) ; couch grass
(Agropyron elongatum (Host) Nevski). Three somatic hybrid selfed linesⅡ-2,Ⅱ-Ⅰ-8 ( F2 , F4, F6 ), 8-1 (F3, F4, F6).
1.2 Microsatellite assay of chloroplast genome
1.2.1 Wheat chloroplast primers Seven wheat chloroplast microsatellite primers WCt6, WCt7, WCt9, WCt11,
WCt13, WCt20 and WCt23 (Ishii et al., 2001) were used in
this experiment (Table 1). Primers were synthesized by
SIGMA company (USA).
1.2.2 DNA extraction and chloroplast microsatellite assay DNA extraction was performed following Xia et al.
(2001). PCR reaction was performed in a volume of 20 µL in
a thermocycler (MJ Research, PTC-100, USA,). The reaction mixture contained 2 µL 10×buffer, 1.5 µmol/L MgCl2,
100 µmol/L of each primer, 1U r-Taq (TaKaRa, China), with
50－100 ng sample DNA. After 5 min at 95 ℃, 35 cycles were
performed with 1 min at 95 ℃, 1 min at 55 ℃, and 2 min at 72
℃, followed by a final extension step of 10 min at 72 ℃.
The amplification products were resolved on 6%
polyacrymide denatured gel stabilized at 80 W, and the
banding patterns were visualized using silver staining as
described by Panaud et al. (1996). The gel was photographed after being dried at room temperature.
1.3 RAPD analysis of nuclear genome DNA
Sixty random primers (OPERON, USA) were used in these
tests. The PCR reaction system was mentioned above. After 5 min at 95 ℃，
45 cycles were performed with 1 min at
95 ℃, 50 s at 40 ℃，
and 50 s at 72 ℃, followed by a final
extension step of 10 min at 72℃.
The amplification products were electrophoresed in
1.5% agarose gel. Gel was stained with 0.5% ethidium bromide and analyzed with the syngene gel imaging system
(Syngene, USA)
2
Results
2.1 Chloroplast microsatellite marker assay
As shown in Fig.1, there were consistent amplified bands
in the three hybrid lines of F6 on the seven sites selected,
of which both parents and the three hybrid lines all exhibited consistency on the sites of WCt6 (trnC-rpoB), WCt7
(ropC2), WCt11 (atpF) and WCt20 (infA). The band patterns from the three hybrid lines were consistent with parental wheat on sites WCt9 (atpI-atpH) and WCt13 (trnFndhJ). Profiles of the three hybrid lines were from both
parents on the site of WCt23 (rpl14-rpl16). Results of F2,
F4 and F6 were consistent with the WCt23 (rpl14-rpl16)
primer screened (Fig.2).
2.2 RAPD assay
Visible amplified bands were obtained in 45 of the 60
random primers selected, but among the hybrids, stable
Table 1 Wheat chloroplast microsatellite primers used in this experiment
Locus
WCt6
WCt7
WCt9
WCt11
WCt13
WCt20
WCt23
Lo c a t i o n （g e n e ）
Intergenic region
(trnC-rpoB)
Coding region
(ropC2)
Intergenic region
(atpI-atpH)
Intron
(atpF)
Intergenic region
(trnF-ndhJ)
Coding region
(infA)
Intergenic region
(rpl14-rpl16)
Repeat
(C)10
(A)12
(T)12
(A)14
(A)15
(T)10
(T)10
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Primer sequence
5 '- TCACAGGCTGCAAAATTCAG-3 '
5 '-GGATAATAATGCTGTCGGACC-3 '
5'-ATCGTTCCCCACAAGACAAG-3 '
5 '-AGGGTTAAATGTTAAATGGGGG-3 '
5 '-CGCAGCCTATATAGGTGAATCC-3 '
5 ' -TTGCAACCAAGCAGATTATCC-3 '
5 ' -TTTTATCTAGGCGGAAGAGTCC-3 '
5 ' -TCATTTGGCTCTCACGCTC-3 '
5 ' -TGAAAATCTCGTGTCACCA-3 '
5 ' -TGTATCACAATCCATCTCGAGG-3 '
5 ' -TTCCATTGGGTAGGGCTTC-3 '
5'-GTAATCGCCCCCGCCTATAGT-3 '
5'-TCCAGAAAGAAAAACCGGG-3'
5 '-TAGCTGCCAGTAAAAATGCC-3 '
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Fig.1. Amplification profiles using seven wheat chloroplast microsatellite primer pairs WCt 6, WCt7, WCt9, WCt11, WCt13, WCt23.
Lane A, Agropyron elongatum; lane T, Triticum aestivum; lane H1,Ⅱ-Ⅰ-8; lane H2,Ⅱ-2; lane H3, 8-1; lane M, size marker; arrow, A.
elongatum-specific band (about 120 bp).
Fig.2. Amplification profiles in the progenies and their parents
of different somatic hybrids using wheat chloroplast microsatellite
primer WCt23. Lane T, Triticum aestivum; lane A, Agropyron
elongatum; lane H1,Ⅱ-Ⅰ-8; lane H2,Ⅱ-2; lane H3, 8-1; arrow, A.
elongatum-specific band (about 120 bp).
polymorphism was only detected in seven primers, (OPA2
(5'- TGC CGAGCTG-3'), OPA10 (5'-GTGATCGCAG-3'),
OPA17 (5'-GACCGCTTG T-3'), OPG18 (5'-GGCTCATGTG3'),OPH4 (5'-GGAAGTCGCC-3'),OPH9(5'-TGTAGCTGGG3'), and OPH19 (5'-CTGACCAGCC-3'), three of which amplified different specific bands of A. elongatum in different
somatic hybrid lines (Figs.3－5). The amplified bands of other
primers were most like parent wheat except for a few different bands among the three hybrid lines. Three specific
bands of A. elongatum were amplified in 8-1 with OPA17
primer, one of which existed in Ⅱ-Ⅰ-8, another in Ⅱ-2
(Fig.3); and one specific band of A. elongatum was amplified inⅡ-2 and 8-1 with OPH19 primer, the other existed in
Ⅱ-Ⅰ-8 and 8-1 (Fig.4). One specific band of A. elongatum
was amplified in all lines and another was amplified inⅡⅠ-8 with OPH9 (Fig.5). The results suggested that nuclear
genome exhibited some segregation in the three hybrid lines
and different lines maintained different nuclear genetic
materials of A. elongatum. In addition, using OPH19 as
primer, F6 had one band less than F2 inⅡ-2 (Fig.4), indicating that a few changes occurred from F2 to F6 generation.
Fig.3. RAPD result using primer OPA17. Lane M, size marker
(λDNA/EcoRⅠ + Hind Ⅲ); lane A, Agropyron elongatum; lane
T, Triticum aestivum; lane H1,Ⅱ-Ⅰ-8; lane H2,Ⅱ-2; lane H3, 81; arrows, specific bands of A. elongatum.
Fig.4. RAPD result using primer OPH19. Lane M, size marker
(λDNA/EcoRⅠ + Hind Ⅲ); lane A, Agropyron elongatum; lane
T, Triticum aestivum; lane H1,Ⅱ-Ⅰ-8; lane H2,Ⅱ-2; lane H3, 81; thin arrows, specific bands of A. elongatum; fat arrows, different bands between F2 and F6 in Ⅱ-2 line.
CHEN Sui-Yun et al.: Heredity of Asymmetric Somatic Hybrid Lines Between Wheat and Couch Grass
Fig.5. RAPD result using primer OPH9. Lane M, size marker
(λDNA/EcoRⅠ + Hind Ⅲ); lane A, Agropyron elongatum; lane
T, Triticum aestivum; lane H1,Ⅱ-Ⅰ-8; lane H2,Ⅱ-2; lane H3, 81; arrows, specific bands of A. elongatum.
3 Discussion
According to current chloroplast physical maps and the
known localized genes, e.g. that of rDNA, tRNA, some proteins and chloroplast genes are very conservative in different species. All nucleotide sequences analyzed on Nicotiana tabacum and Marchantia polymorpha, have revealed
that they contain almost identical chloroplast genes although the two species belong to Angiospermae and
Bryophyta respectively (Wang and Dai, 1999). In the seven
sites detected in this experiment, both parents had the same
bands inside the genes, including two sites of the gene
coding regions (WCt7, WCt20) and one site of the intron
region (WCt11). However, wheat and couch grass have
different bands on three sites (WCt9, WCt13, WCt23) of
the four sites (WCt6, WCt9, WCt13, WCt23) located in the
intergenic regions. The results showed that chloroplast
genes of both wheat and A. elongatum were also
conservative, perhaps the differences between them were
mostly in the intergenic regions.
The three somatic hybrid lines here studied were derived from the same somatic hybrid clone. They all have
different and obvious properties in morphology, cytology,
subunit component of glutenin and gliadin (Xia et al., 1999;
Xiang et al., 2001; Chen et al., 2000; Zhao et al., 2002; Xia
et al., 2003; Zhao et al., 2003), which was the same as that
of the RAPD results. However, there were consistently amplified bands in F6 of the three hybrid lines on the seven
chloroplast sites analyzed. Theoretically, exogenous integration of nuclear-cytoplasmic DNA in the period of cell
１１３
fusion should have occurred before division of somatic
hybrid cell or during the events of early hybrid cell division.
Because of the technique limitation in our laboratory before,
the cytoplasmic component of somatic hybrid clones and
F0 -F1 progenies had not been detected. However, as shown
in Figs.1 and 2, components of chloroplast DNA from different lines were consistent, indicating that the integration
of chloroplast DNA occurred before the segregation of the
afore-mentioned lines, and moreover, no segregation of
chloroplast genome was found in generation propagation.
As an extra nuclear inheritance system, chloroplast genome controls some inheritance traits, for example, resistance to herbicides and antibiotics (Guo and Deng, 2000).
Cytoplasmic sterility is also controlled both by mitochondrion and chloroplast genes (Wang and Dai, 1999). It is
known that almost all cytoplasmic genes are maternally inheritable in angiosperm, and hybrids derived from distant
sexual cross possess only maternal cytoplasmic genome.
Furthermore transfer of cytoplasmic genes of interspecies
also needs backcrossing continually even in classical breeding (Glimelius et al., 1991). So, chloroplast gene transfer is
of importance with somatic fusion technique for plant
breeding. However, there are a few reports about existence
and inheritary stability of donor chloroplast gene in somatic hybrid progeny. Our results showed that donor chloroplast DNA could be transferred directly and passed stability (Fig.2) using this technique, which provides a new
potential method for introducing chloroplast genes into
wheat.
RAPD analysis also demonstrated that segregation of
nuclear genome occurred in early generation and different
lines retained different nuclear genetic materials of A.
elongatum. In addition, RAPD assay in different generations showed that somatic hybrid lines were relatively stable
on most loci in different generations. Although not so many
primers have been used, in combination with other analytical results from studying these hybrid lines (showing their
phenotypes and hereditary stability), it was proposed that
changes might happen in only certain loci during passage.
Furthermore, it is also suggested that the lines, which were
derived either from the same somatic clone with different
phenotypes (Ⅱ-2 and Ⅱ-Ⅰ-8) or from re-segregation of
early generation (8-1, segregated from F2 ), could become
stabilized quickly in later passage, and could be used in
breeding. Such exogenous nuclear-cytoplasmic genetic
material had been passed to F6, which may offer evidence
for the use of the above-mentioned hybrid lines in breeding practice.
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(Managing editor: ZHAO Li-Hui)