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Annals of Botany 97: 993–998, 2006
doi:10.1093/aob/mcl073, available online at www.aob.oxfordjournals.org
Parental Genome Separation and Elimination of Cells and
Chromosomes Revealed by AFLP and GISH analyses in a
Brassica carinata · Orychophragmus violaceus Cross
Y U - W E I H U A , M I N L I U and Z A I - Y U N L I *
National Key Laboratory of Crop Genetic Improvement, National Center of Crop Molecular Breeding Technology,
National Center of Oil Crop Improvement (Wuhan), Huazhong Agricultural University, Wuhan 430070, China
Received: 5 January 2005 Returned for revision: 8 June 2005 Accepted: 10 February 2006 Published electronically: 19 April 2006
Background and Aims The phenomenon of parental genome separation during the mitotic divisions of hybrid
cells was proposed to occur under genetic control in intergeneric hybrids between cultivated Brassica species
and Orychophragmus violaceus (2n = 24). To elucidate further the cytological and molecular mechanisms behind
parental genome separation, Brassica carinata (2n = 34) · O. violaceus hybrids were resynthesized and their
chromosome/genomic complements analysed.
Methods F1 hybrids of the cross were obtained following embryo rescue, and were investigated for their
cytological behaviour and subjected to genomic in situ hybridization (GISH) and amplified fragment length
polymorphism (AFLP) to determine the contribution of parental genomes.
Key Results All the F1 plants with high fertility closely resembled B. carinata in morphological attributes. These
were mixoploids with 2n chromosome numbers ranging from 17 to 35; however, 34, the same number as in
B. carinata, was the most frequent number of chromosomes in ovary and pollen mother cells (PMCs). GISH
clearly identified 16 chromosomes of B. nigra in ovary cells and PMCs with 2n = 34 and 35. However, no
O. violaceus chromosome was detected, indicating the presence of the intact B. carinata genome and elimination
of the entire O. violaceus genome. However, some AFLP bands specific for O. violaceus and novel for the two
parents were detected in the leaves. Cells with fewer than 34 chromosomes had lost some B. oleracea chromosomes.
F2 plants were predominantly like B. carinata, but some contained O. violaceus characters.
Conclusions The cytological mechanism for the results involves complete and partial genome separation at mitosis
in embryos of F1 plants followed by chromosome doubling, elimination of cells with O. violaceus chromosomes and
some introgression of O. violaceus genetic information.
Key words: Brassica carinata, Orychophragmus violaceus, intergeneric hybrids, genomic in situ hybridization, amplified
fragments length polymorphism, genome separation, chromosome doubling, chromosome elimination, introgression.
INTROD UCTION
Hybridization between two different species with differentiated genomes is a major mechanism for the origin of plant
species leading to the formation of allopolyploids (Soltis
and Soltis, 1995; Otto and Whitton, 2000). Sometimes it
also leads to the formation of a new species with features
derived from both parents but without including the whole
genomes of both parents through the process of introgressive hybridization (Rieseberg et al., 2000; Guttman, 2001),
where one or more chromosomes or chromosomal segments
of one parent species are incorporated into the genome of
the other (Guttman, 2001). Wild and cultivated allopolyploids are well adapted; however, man-made ones are typically unstable and are rarely used as crops (Comai, 2000).
In artificial crosses, chromosome instability may result in
the production of partial hybrids with unexpected chromosome numbers (Riera-Lizarazu et al., 1996), mixoploids
consisting of cells with different chromosome numbers
(Schulz-Schaeffer, 1980) and haploids after elimination
of one of the parental genomes (Kasha and Kao, 1970).
Orychophragmus violaceus (2n = 24, genomes OO),
a member of Cruciferae, is cultivated as an ornamental
plant in China. This species has been reported to be valuable
* For correspondence. E-mail [email protected]
for the genetic improvement of Brassica crops, because
of its high seed yield potential and good quality seed oil
(Luo et al., 1994). Intergeneric hybrids between the six
cultivated Brassica species in the U-triangle (U, 1935)
and O. violaceus were obtained previously and the cytogenetics were documented (Li et al., 1995, 1998; Li and
Heneen, 1999; Li and Liu, 2001). As a continuation of these
studies, B. carinata · O. violaceus hybrids were resynthesized. F1 plants and their progeny were investigated cytologically and were subjected to genomic in situ hybridization
(GISH) and amplified fragment length polymorphism
(AFLP) analyses, and the results are reported here.
MATERIALS AND METHODS
Plant material and crosses
The cross Brassica carinata (accession no. GO-7) ·
Orychophragmus violaceus (L.) O. E. Schulz [syn.
Moricandia sonchifolia (Bunge) Hook Fil.] was made by
hand emasculation and pollination as reported earlier
(Li et al., 1998); the reciprocal cross was not attempted
as previous experiments showed it to be unsuccessful.
About 2–3 weeks after pollination, the immature embryos
were cultured on MS agar medium (Murashige and Skoog,
1962). The leaves of young plants were collected for AFLP
The Author 2006. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved.
For Permissions, please email: [email protected]
Hua et al. — Parental Genome Seperation in a B. carinata · O. violaceus Cross
994
T A B L E 1. Phenotypes, cell components and AFLP patterns of F1 plants from a B. carinata · O. violaceus cross
Type
I
II
F1 plant
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
III
B. carinata
O. violaceus
a
Stem
coloura
Seed
coat
coloura
Percentage
of ovary cells
(2n = 34)
Types
of ovary
cells
Percentage
of PMCs
(2n = 34)
Types
of
PMCs
g
p
p
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g/p
y
y
b
y
y
y
y
y
y
y
y
y
y
y
y
y
y
y
y
y
y
y
y
y
y
y
b
34.8
44.0
88.9
37.5
56.0
50.0
53.3
45.5
51.5
57.1
30.0
48.4
51.2
50.0
46.9
31.9
45.2
38.7
30.0
23.3
40.5
37.5
43.4
39.4
27.3
8
10
2
12
9
11
11
8
7
6
8
7
9
7
12
16
8
13
11
12
10
9
12
9
9
62.4
89.3
100
80.9
93.8
85.4
86.5
92.0
91.9
85.5
66.7
96
91.4
100
100
72.7
100
90.2
83.3
91.7
72.2
94.4
88.2
75.7
86.4
4
2
1
3
2
3
3
3
3
3
3
2
2
1
1
3
1
4
2
3
3
2
3
3
2
Pollen
stainabiltity
No. of
O. violaceus
bands
No. of
deleted
bands in
B. carinata
No. of
novel
bands
89.9
90.1
87.9
94.2
82.4
92.9
98.4
95.6
91.5
89.3
95.9
75
96.3
95.2
71.9
93.1
90.1
89.1
83.5
85.4
91
83.5
91.5
79.7
94.6
1
16
16
1
0
2
3
4
2
9
3
1
5
1
4
14
7
1
5
3
0
3
2
4
3
0
4
3
0
0
0
0
8
0
7
1
0
0
0
1
13
0
0
0
0
2
2
0
2
0
0
4
4
0
0
0
1
2
0
1
0
1
0
0
4
7
1
0
0
0
0
0
0
1
0
g = green; p = purple; y = yellow, b = brown.
analyses. A population of ten F2 plants was raised from
each F1 plant.
DNA extraction and probe labelling
DNA was extracted and purified from young leaves
according to the method of Dellaporta et al. (1983). The
DNA from B. oleracea var. alboglabra and B. carinata
(GO-7) was sheared by boiling for 15 min and used as a
block. The DNA from O. violaveus was labelled with
digoxigenin-11-dUTP (Roche) or biotin-11-dUTP (Sabc
in China) by the nick translation method, and the DNA
from B. nigra ‘Giebra’ was labelled with biotin-11-dUTP
using the same method, after purification, and dissolved in
TE (10 M Tris.HCl, 0.5 M EDTA) at a concentration of
100 ng mL–1. The length of probe DNA fragments averaged
approx. 500 bp.
GISH and AFLP analyses
The determination of chromosome numbers and
meiotic observations were carried out according to Li
et al. (1995). The slide preparation of chromosomes for
GISH mainly followed the procedures of Zhong et al.
(1996). In situ hybridization was carried out according
to the protocols of Leitch et al. (1994). AFLP fingerprints
were generated based on the protocol of Vos et al. (1995),
and DNA bands were visualized by silver staining (Bassam
et al., 1991).
RESULTS
Morphology of F1 plants and progeny
At least three F1 plants from each of the 25 surviving
embryos from the B. carinata · O. violaceus cross were
established in the field. The plants derived from the same
embryo were morphologically indistinguishable. Based on
the morphology and chromosome number in ovary cells,
F1 plants were classified into three types: type I (2n =
24–35), type II (2n = 20–34) and type III (2n = 17–34)
(Table 1). All F1 plants and their progeny had good
seed-set on selfing or open pollination, although certain
differences in pollen stainability were observed (Table 1).
Plants of type I (no. 1) and III (nos 4–25) had matroclinous
morphology. F2 plants were morphologically similar to the
respective F1 plants. However, two plants of type II (nos 2
and 3) had morphological traits of B. carinata in addition to
some characters from O. violaceus, such as the light purple
stems in both plants and brown seed coat in plant no. 3.
These characters were also observed in F2 plants, as among
ten F2 plants in type II, half still maintained the light purple
stems and two from plant no. 3 produced brown seeds.
Cytogenetics and GISH analyses of F1 plants
and their progeny
All the F1 plants were mixoploids (2n=17–34), with
2n = 34 being the most frequent chromosome number as
Hua et al. — Parental Genome Seperation in a B. carinata · O. violaceus Cross
A1
A2
B1
995
B2
C1
C2
C3
D1
D2
D3
E1
E2
E3
F1
F2
F3
F I G . 1. Genomic in situ hybridization analyses on the ovary cells and PMCs in the hybrids between B. carinata and O. violaceus. Blue signals were due to
40 ,6-diamidino-2-phenylindole (DAPI) staining, red signals were due to Cy3 and green signals were due to fluorescein isothiocyanate (FITC). (A1and A2) The
metaphase chromosomes in one root tip cell (2n = 24) of O. violaceus (A1) were fully labelled by its own genomic DNA probe (A2). (B1 and B2) In one
AI PMC of plant no. 1 with 17 : 17 segregation and two lagging fragments (B1), the terminal parts of two chromosomes in each polar group were labelled by the
O. violaceus probe (B2). In the four cells below, the B. nigra (red) and O. violaceus probes (green) were used and the green signals overlapped with the red
signals. (C1–C3) In one root tip cell (2n = 35) from selfed seeds of plant no. 1 (C1), 16 chromosomes were labelled red (C2), but the minor chromosome
(C1, red arrow) was unlabelled by both probes (C2 and C3). Large signals of both types appeared on satellites (C1, white arrows). (D1–D3 and E1–E3) One
metaphase (2n = 34/30) and one interphase nucleus (D1 and E1) in one ovary of plant nos 2 and 7, respectively, with 16 and 15 chromosomes, respectively,
labelled red (D2 and E2), and four and three chromosomes, respectively, labelled green at their terminal parts (D3 and E3) and both signals in the nucleus
(D2, D3, E2 and E3). (F1–F3) One AI PMC of plant no. 9 with 17 : 17 segregation (F1) had eight large red signals (F2) and two small ones at terminals in each
polar group (F3). Scale bar = 5 mm.
996
Hua et al. — Parental Genome Seperation in a B. carinata · O. violaceus Cross
in B. carinata. The majority of their pollen mother cells
(PMCs) exhibited regular formation of 17 bivalents and
17 : 17 segregation as in B. carinata. This factor contributed
to the high fertility of these F1 plants and production
of B. carinata-type progeny. However, plant no. 1 (2n =
24–35) was an exception where some cells had 2n = 35,
i.e. the full complement of B. carinata and an additional
minor chromosome. This minor chromosome remained as
univalent and was lagged or divided at anaphase I (AI)
(Fig. 1B1 and B2). The root tip cells (Fig. 1C1–C3)
and ovary cells of F2 plants also retained this minor
chromosome.
When the O. violaceus genomic probe was applied to
O. violaceus preparations, signals distributed along the
whole length of chromosomes (Fig. 1A1 and A2). In contrast, GISH in Brassica was normally characterized by
strong signals at centromeric heterochromatin and only
very weak hybridization on chromosome arms (Snowdon
et al., 1997). This made it easier to distinguish the parental
chromosomes in their hybrids.
In all, 219 ovary cells with different chromosome numbers were observed. Signals from the O. violaceus probe
showed the variation in size, number and intensity, but the
majority of signals were located at the terminal parts or on
one arm of the chromosome. This indicated that no intact
O. violaceus chromosomes were included in ovary cells.
When B. nigra and O. violaceus probes were used simultaneously, the signals from the B. nigra probe were at
centromeric parts and were generally larger and stronger
than those from the O. violaceus probe, which showed the
existence of chromosomes of B. nigra origin. In all cells
with 2n=34 and 35, 16 B. nigra chromosomes were easily
identified (Fig. 1D1–D3). In some cells (2n < 34), the 16
chromosomes of B. nigra origin were clearly identified,
suggesting the loss of some B. oleracea chromosomes,
while in other cells (2n < 34) more chromosomes of B.
nigra origin than B. oleracea origin were preferentially
maintained (Fig. 1E1–E3), showing that most of the chromosomes lost from the complements of B. carinata were
from the C genome of B. oleracea. In PMCs at diakinesis,
metaphase I (MI) and AI, signals from the O. violaceus
probe were again located at terminal or small and centrimeric parts of bivalents or chromosomes, and exhibited
much larger variation in size, number and intensity than
those of the B. nigra probe. The signals of the O. violaceus
probe again confirmed no inclusion of the bivalents or chromosomes from O. violaceus in all PMCs with various pairings and segregations. In PMCs with 17 bivalents and 17
bivalents plus one univalent, eight bivalents from B. nigra
were easily detected from the large and strong centromeric
signals, and the signals from the O. violaceus probe were
located at the end of the bivalents and overlapped with those
of the B. nigra probe. Some signals of the O. vioalceus
probe covered half of the bivalent. Eight bivalents of B.
nigra origin were maintained in some PMCs with fewer
than 17 bivalents. In PMCs at AI with 17 chromosomes
in each polar group and occasionally one or two extra laggards, eight chromosomes from B. nigra were identified and
two signals of different sizes from the O. violaceus probe
appeared, the stronger one overlapping one from the
M P1 P2
F1 Plants
M
F1 Plants
M
500bp
400bp
300bp
200bp
*
A
M P1 P2
300bp
200bp
B
F I G . 2. AFLP profiles generated from the primer combinations
50 -GACTGCGTACCAATTCATT-30 and 50 -GATGAGTCCTGAGTAACCC-30 (A) and 50 -GACTGCGTACCAATTCATG-30 and 50 -GATGAGTCCTGAGTAACAC-30 (B) in F1 plants nos 1–25. M = 100 bp DNA ladder,
P1 = B. carinata, P2 = O. violaceus. O. violaceus-specific and deleted for
B. carinata bands are indicated by black and white arrowheads, respectively
(A), and the novel bands for two parents by black arrowheads (B). The
asterisk marks the faint B. carinata band (A).
B. nigra probe and the weaker one located on the nonB. nigra chromosome (Fig. 1F1–F3). The minor chromosome in 2n=35 of ovary cells and PMCs was not labelled by
any probe. Among F2 plants, no chromosomes were wholly
labelled by the O. violaceus probe, and the minor chromosome also remained unlabelled with any probe. Judging
from the morphology of the fragment and its transmission
between generations, it might be that this minor chromosome belongs to the C genome of B. oleracea.
Moreover, in ovary cells and PMCs at diakinesis and AI,
the larger signals from the O. violaceus probe overlapped
with those from the B. nigra probe in most cases, and the
weak signals were located on the remaining chromosomes
of B. oleracea origin, which might result from the blocking
of DNA from B. oleracea var. alboglabra. More signals
from the O. violaceus probe overlapped with the chromosomes of B. nigra origin than those of B. oleracea origin,
suggesting the higher homology between the dispersed
repeats of B. nigra and O. violaceus. The strong signals
or unique signals came from the satellites and nucleolar
organizer regions (NORs) in all cells observed using
the O. violaceus probe, if the satellites appeared. In cells
(2n < 34) with recognizable satellites, the two satellite
chromosomes were always included.
AFLP analyses
Using 35 pairs of primers, bands between 80 and 1000 bp
were scored in the two parents and F1 plants. The numbers
of bands amplified in B. carinata and O. violaceus were
1780 and 1858, respectively; of these, 338 bands (approx.
20 %) were common for the two parents. The 1780 bands
Hua et al. — Parental Genome Seperation in a B. carinata · O. violaceus Cross
I
II
III
BB × OO
BB × OO
BB × OO
BO
BO
BO
B
O
B+1O
O–1
BB
OO
BB+2O
OO–2
B
O
BB
B O
BO
OO
B+1O
BB+2O
B–1B +1O
Hybrid cells
O–1O+1B
GS
BB–2B+2O OO –2O+2B
B–1B +1O
BB–2B+2O
997
CD
Meiosis
Selfing
F I G . 3. Possible situations of genome separation (GS) followed by chromosome duplication (CD) in the hybrids between Brassica (BB) species and
O. violaceus (OO). When the parental genomes were completely separated during the mitotic and meiotic divisions of the hybrid cells (I), theoretically three
types of plants, i.e. Brassica parents, hybrids and those with the O. violaceus genome and the Brassica cytoplasm, should be produced by the hybrids; however,
the third type was not found, probably due to the low frequency and viability of the cells and gametes with the O. violaceus genome. The addition (II) and
exchange (III) of some chromosomes between parental genomes during partial genome separation resulted in the production of Brassica species–O. violaceus
additions and substitutions (exemplified by one chromosome).
were averagely amplified in the leaves of each hybrid plant,
and three types of special bands appeared in the F1 plants:
O. violaceus-specific bands; deleted bands in B. carinata;
and novel bands for two parents (Table 1; Fig. 2). Plant no. 1
had one O. violaceus-specific band but no other bands.
Plants nos 2 and 3 with some O. violaceus characters
exhibited 16 O. violaceus-specific bands out of 1797 and
1796 bands, respectively, the highest number among all
F1 plants. In type III, plants nos 8, 10, 15, 16 and 24 had
three types of bands, others had one or two typess of bands
and plant no. 5 exhibited the same bands as B. carinata.
In particular, plant no. 16 with the maximal number (16)
of types of ovary cell showed 14 O. violaceus-specific,
13 deleted and seven novel bands, the latter two numbers
being the highest among all F1 plants and much higher than
those of others in this group.
DISCUSSION
The hybrids derived from the same cross of B. carinata
(GO-7) · O. violaceus in the present and previous studies
(Li et al., 1998) were cytologically similar and were
mixoploids, consisting of cells with various chromosome
numbers. The number of chromosomes with the highest
frequency in somatic cells of ovaries and PMCs was
2n=34, the same number as in the female parent B. carinata.
Cytogenetic results showed that the chromosome behaviour
in these hybrids was repeatable and not an occasional event.
The remarkable cytological characteristic of these
F1 mixoploids was that all ovary cells (2n = 17–34) and
PMCs contained B. carinata chromosomes without intact
O. violaceus chromosomes (Fig. 1B–F). The majority of
PMCs showed the same chromosome pairing and segregation as in normal B. carinata plants even though limited
O. violaceus DNA was detected by AFLP (Table 1; Fig. 2).
It is proposed that complete and partial separation of the
parental genomes during mitosis reported earlier for
the cross (Li et al., 1998) could well explain the present
results, as illustrated in Fig. 3. The GISH identification of
cells (2n = 34) with the whole B. carinata complement
substantiated the proposal that cells with the O. violaceus
complement or its chromosomes are eliminated during
the process of embryo development and plant growth.
The identification of cells with a complete and partial complement of B. carinata in one plant indicated the multiple
origins of cells from different events. In future, efforts
should be directed to determine the genomic complements
of hybrids at early stages using GISH and molecular
markers.
Although no O. violaceus chromosomes were detected
in ovary cells and PMCs, some O. violaceus-specific bands,
deleted bands in B. carinata and new AFLP bands in the
leaves of F1 plants indicated the existence of partial
O. violaceus DNA, loss of some B. carinata genetic material
and occasional recombination. Moreover, some hybrids
and progeny (2n=34) showed some characters specific for
O. violaceus, such as the brown seed coat and the purple
colour on the stems and leaves, suggesting the introgression
of O. violaceus genetic material or genes but beyond GISH
resolution.
The GISH results showed that more chromosomes from
B. oleracea than from B. nigra (B. carinata evolving from
the hybridization between B. oleracea and B. nigra) were
lost in cells with partial B. carinata complements (2n < 34),
which was the opposite to what was expected from the
cytology in the hybrids of O. violaceus with B. oleracea
and B. nigra (Li and Heneen, 1999). It is also interesting to
note that the intact B. nigra genome was maintained in
hybrids (B. carinata ·B. rapa, AABBCC) · O. violaceus
998
Hua et al. — Parental Genome Seperation in a B. carinata · O. violaceus Cross
(2n = 40–44) (X.-H. Ge and Z.-Y.Li, unpubl. res.). One
possible reason for this could be attributed to the dominance
of rRNA genes from the two ancestors of B. carinata
because the hierarchy of rRNA gene transcriptional dominance was B. nigra > B. rapa > B. oleracea, and B. nigra
rRNA transcripts were readily detected in both natural and
synthetic lines of B. carinata, but B. oleracea transcripts
were not detectable (Chen and Pikaard, 1997). The two
satellite chromosomes which were always observed in
the cells (2n < 34) with partial complements of B. carinata
were of B genome origin and their retention helped to stabilize other chromosomes of this genome.
The complete or partial elimination of the chromosomes from one parent during the mitotic divisions of
the zygotes in hybrids from widely separated species was
reported to result in haploid embryos and plants of one
parent (Kasha and Kao, 1970) or partial hybrids with a
haploid complement from one parent and some chromosomes of the other parent (Riera-Lizarazu et al., 1996).
The cytological mechanisms in the present investigation
are different from pseudogamy (Jörgensen, 1928) and
semigamy (Coe, 1953; Turcotte and Feaster, 1967), or
simply chromosome elimination during embryo development, and therefore seem to be intermediate between
the classical hybrids of allopolyploids and chromosome
elimination.
ACKNOWLEDGEMENTS
The study was supported by the grants from Hubei Province
(2002AC015) and National Natural Science Foundation
(30070413) and Education Ministry of the P.R. China
and by PCSIRT (IRT0442). The constructive suggestions
of two anonymous reviewers to revise the manuscript and
the critical reading and modifying by Professor Shyam
Prakash from the Indian Agricultural Research Institute,
New Delhi were greatly appreciated.
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