Plant Cell Rep (2008) 27:261271

DOI 10.1007/s00299-007-0452-2

GENETIC TRANSFORMATION AND HYBRIDIZATION

Chromosome elimination and fragment introgression

and recombination producing intertribal partial hybrids
from Brassica napus Lesquerella fendleri crosses
Xue-Zhu Du Xian-Hong Ge Zhi-Gang Zhao
Zai-Yun Li

Received: 18 July 2007 / Revised: 3 September 2007 / Accepted: 4 September 2007 / Published online: 25 September 2007

Springer-Verlag 2007

Abstract The intertribal sexual hybrids between three

Brassica napus (2n = 38) cultivars and Lesquerella fendleri (2n = 12) with the latter as pollen parent were obtained
and characterized for their phenotypes and chromosomal
and genomic constitutions. F1 plants and their progenies
mainly resembled female B. napus parents, while certain
characters of L. fendleri were expressed in some plants,
such as longer flowering period, basal clustering stems and
particularly the glutinous layer on seed coats related to
drought tolerance. Twenty-seven F1 plants were cytologically classified into five types: type I (16 plants) had
2n = 38, type II (2) had 2n = 3842, type III (3) had
2n = 3138, type IV (5) had 2n = 2531, and type V (1)
had 2n = 1922. Some hybrids and their progenies were
mixoploids in nature with only 12 chromosomes or some
chromosomal fragments of L. fendleri included in their
cells. AFLP (Amplified fragments length polymorphism)
analysis revealed that bands absent in B. napus, novel for
two parents and specific for L. fendleri appeared in all F1
plants and their progenies. Some progenies had the modified fatty acid profiles with higher levels of linoleic,
linolenic, eicosanoic and erucic acids than those of B. napus parents. The occurrence of these partial hybrids with
phenotypes, genomic and fatty acid alterations resulted
possibly from the chromosome elimination and doubling
accompanied by the introgression of alien DNA segments
Communicated by K. Toriyama.
X.-Z. Du
X.-H. Ge
Z.-G. Zhao
Z.-Y. Li (&)
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,
Peoples Republic of China
e-mail: lizaiyun@mail.hzau.edu.cn; zaiyunli@yahoo.com.cn

and genomic reorganization. The progenies with some

useful traits from L. fendleri should be new and valuable
resource for rapeseed breeding.
Keywords Brassica napus
Lesquerella fendleri

Genomic in situ hybridization (GISH)

Amplified fragments length polymorphism (AFLP)

Fatty acids

Introduction
Wide hybridization, an efficient way to generate novel
genetic variability has been widely applied for the
improvement of Brassica crops (Voss et al. 2000; Warwick
et al. 2000) and breeders are increasingly interested in
introgressing genes conferring desirable traits from wild
species to cultivated crops through sexual hybridizations.
B. napus L. (2n = 38, AACC), which is grown worldwide
due to its superior seed yield and quality (Harlan and de
Wet 1971; Prakash et al. 1999), has obtained genetic
modification and genetic improvement through wide
hybridization, while Brassica species germplasm is still
very narrow because during these years emphasis was
focused mainly on the betterment of some limited traits
(Chrungu et al. 1999; Snowdon et al. 2000; Wang et al.
2003). There is thus an urgent need to broaden the oilseed
rape gene pool through introgression of desirable genes
from new and valuable sources.
Lesquerella fendleri Gray (Wats.) that belongs to the
tribe Drabae of the family Brassicaceae is an annual native
to the arid and semiarid regions of Southwestern America
and has been proposed as an important industrial seed oil
crop (Thompson and Dierig 1988). Its meal after oil
extraction is rich in protein and has an excellent

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distribution of amino acids being particularly high in lysine

(Carlson et al. 1990). L. fendleri is one new valuable
genetic resource for the rapeseed breeding of industrial
purpose, as it possesses favorable fatty acid compositions
containing high amounts of hydroxy fatty acids, about 60%
lesqueroloc acid (20:1D11, 14-OH) and 1.5% densipolic acid
(18:2D9, D15, 12-OH) (Barclay et al. 1962). Hydroxy fatty
acids are utilized for the production of biodegradable
lubricants, novel plastics, lithium greases, protective coatings and cosmetics (Roetheli et al. 1991). In addition, the
high tolerance to drought of L. fendleri is also useful for the
genetic improvement of rapeseed, especially the thick
glutinous polysaccharide layer on its seed coats. The
intertribal somatic hybrids between B. napus and L. fendleri have been obtained via protoplast fusions
(Skarzhinskaya et al. 1996). Due to unstable integration of
the donor genes in the rapeseed genomes or gene silencing
by methylation of the promoter regions, no hydroxy fatty
acids could be detected in any of the F6 plants (SchroderPontoppidan et al. 1999). In present study, the sexual
intertribal hybridizations between B. napus and L. fendleri
were successfully made for the first time, in order to
introduce into B. napus some useful traits of L. fendleri.
The hybrids and their progenies were characterized for
their chromosome complements using the methods of
genomic in situ hybridization (GISH) and amplified fragments length polymorphism (AFLP). Furthermore, the
progenies with glutinous layer on seed coats and the variations in fatty acid compositions were found.

Materials and methods

Plant materials
Three Brassica napus (2n = 38) cultivars Oro, Qingyou 14 and Zhongyou 821, were used as female parents
in crosses with Lesquerella fendleri Gray (Wats.) (2n = 12)
as pollen parent. Oro, the first cultivar in the world with
low content of erucic acid was introduced into China from
Canada in 1974. Qingyou 14, the spring B. napus cultivar
with double-low quality (low content of erucic acid in seed
oil and glucosinolates in seed meal) was developed in 1994
by Qinghai Academy of Agricultural and Forestry Sciences
(QAAFS), Xining, Qinghai Province, China. Qinghai
Province is the region of spring rapeseed in China. The
elite variety Zhongyou 821 with double-high quality, high
yield and good disease resistance was developed in 1986
by Institute of Oil Crops Research, Chinese Academy of
Agricultural Sciences, Wuhan, Hubei Province. L. fendleri
(PI293028, 596464) was supplied by Western Regional
Plant Introduction Station, Pullman, WA, USA. The

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Plant Cell Rep (2008) 27:261271

crosses were performed by hand emasculations and pollinations in the experiment field of QAAFS at Xining in July
2004 and were repeated at Wuhan in spring 2005 with the
immature embryo rescue. When sown in field at Xining in
May, L. fendleri grew well and flowered in July. But L.
fendleri should be planted in unheated greenhouse to
control the water in soil and to shelter it from the humid
weather in Wuhan to produce flowers (Fig. 1k). About 2
3 weeks after pollination, some immature embryos were
cultured on Murashige and Skoog (1962) (MS) agar medium and some mature seeds were harvested directly. F2 /
BC1 plants were obtained from F1 plants by selfing or
backcrossing to B. napus cultivars.

Cytological methods and genomic in situ hybridization

(GISH)
Cytological preparations were made from ovaries of young
flower buds and the root tips of young seedlings. The
determination of chromosome numbers and meiotic
observations were performed according to the method of Li
et al. (1995). The chromosome preparations for GISH were
made following the procedures of Zhong et al. (1996). The
anthers selected with PMCs at suitable stages were digested at 37C for approximately 60 min in an enzyme
mixture containing 0.6% cellulase Onozuka RS (Japan),
0.2% pectinase (MERCK, Germany) and 0.5% Snailase
(China). Total genomic DNA was extracted and purified
from young leaves according to the method by Dellaporta
et al. (1983). The DNA from L. fendleri was fluorescently
labeled with Bio-11-dUTP by the nick translation method
and used as the probe, and unlabeled genomic DNA from
B. napus was sheared by boiling for 15 min to produce
fragments about 200500 bp used for block. In situ
hybridization was carried out according to the protocols by
Leitch et al. (1994). Hybridization signals of the L. fendleri
probe were detected using Cy3-labelled streptavidin
(Sigma, St Louis, MO, USA).

Amplified fragments length polymorphism (AFLP)

analysis
AFLP analysis was performed on F1 plants/progenies and
parents according to the procedures of of Vos et al. (1995)
with some modifications. In brief, 50 ng of purified genomic DNA was digested to completion using the restriction
endonucleases EcoRI and MseI. Digested DNAs were then
ligated to EcoRI and MseI adapters, and the resulting
ligation products were amplified by PCR with primers
matching the adapters. After two steps of PCR

Plant Cell Rep (2008) 27:261271

263

Fig. 1 Phenotypes of F1, F2 plants from B. napus L. fendleri cross.

ac Flowering plants of B. napus cv. Oro (a), hybrid No.26 (b) and
No.27 (c). Bar 15 cm. d, e Leaves (d) (bar 5 cm) and flowers (e) (bar
1 cm ) of B. napus cv. Qingyou 14, hybrid plant No.27 and L.
fendleri (from left to right). f Flower morphology of B. napus cv.
Oro, hybrids Nos. 11, 8, 14, 1 and L. fendleri (from left to right).
Bar 1 cm. g The selfed seeds of B. napus cv. Qingyou 14, hybrid
plant No.22 and L. fendleri after absorbing enough water on wet filter

paper (from left to right). The color of seed coat of L. fendleri was
yellow and that of Qingyou 14 and plant No.22 was black. The seed
of L. fendleri was covered by a thick glutinous layer, the one of the
hybrid by a thin layer, but not the one of Qingyou 14. Bar 1 mm. hj
Young plants of B. napus cv. Qingyou 14 (h) and two F2 plants (i,
j) from the selfed seeds with the glutinous layer of F1 plant No.22.
Bar 5 cm. k Flowering plants of L. fendleri in a pot kept in green
house. Bar 15 cm

(pre-selective PCR and selective PCR in turn), the PCR

products were loaded on the gel and resolved. At last the
AFLP profile was obtained with silver staining (Bassam
et al. 1991) and bands with 801,000 bp were scored.

Phenotype, cytology and GISH analyses of hybrids

and progenies

Fatty acids analysis

Seed oil was extracted and its composition was analyzed on
gas chromatography machine (HP 6890, Germany) (Chen
et al. 2007).

Results
Crossability and production of hybrids
Totally, more than 10,000 of flower buds on the inflorescences of the three B. napus cultivars were emasculated
and pollinated by the pollen grains of L. fendleri, and 27 F1
plants (Table 1) were produced by the seeds harvested
(Nos. 5, 6, 9, 17, 20, 21, 27) and the rescued immature
embryos (the remaining 20 plants). So the crossability
between these two species was quite low.

As L. fendleri probe was applied to the preparations of

B. napus cv. Qingyou 14 (the DNA of itself as block),
signals of large size and strong intensity were mainly
located at terminals of one bivalent and one chromosome in
each polar group of AI PMCs (Fig. 2I, J). The GISH signals on the L. fendleri chromosomes were distributed along
their entire lengths (Skarzhinskaya et al. 1998), which was
different from strong centromeric signals and very weak
hybridization on chromosome arms in Brassica. Additionally, the L. fendleri chromosomes were larger than
those in B. napus, averagely 1.5 times longer. These
cytological characteristics made it more reliable to detect
L. fendleri chromosomes/chromosomal segments in these
hybrids produced here.
According to the chromosome numbers recorded in
ovary cells and pollen mother cells (PMCs) (Table 2), 27
F1 plants were classified into five types: I (Nos. 116), II
(Nos. 17, 18), III (Nos. 1921), IV (Nos. 2226) and V
(No. 27). Plants of type I, II and III quite resembled their
female B. napus parents in morphology, including leaves,
stems, and branching habits, except for the smaller and

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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
26
27

145.2
133.3
128.4
136.1
109.0
122.4
159.5
132.6
148.2
149.8
133.4
149.1
141.2
154.3
123.0
152.0
136.2
157.9
116.8
109.5
114.8
127.0
140.2
116.2
120.4
147.3
114.3
165.7 7.8
148.9 5.6
154.6 3.5
27.1 1.9

heightb(cm)

parenta

Q
Q
Z
Z
Z
Z
O
O
O
O
O
O
O
O
O
O
Z
O
O
O
O
Q
Q
Q
Q
O
Q

Plant

Female

0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
6
9
5
10
5
5
0
0
0
12.3 3.4

basic brachesc

No. of

5.7 0.6
8.2 0.4
6.7 0.3
7.1 0.6
5.0 0.8
5.3 0.6
6.5 0.6
7.8 0.7
7.4 0.5
6.9 0.9
5.6 0.8
7.0 0.8
6.6 0.7
5.7 0.3
8.0 0.4
6.1 0.5
8.2 0.6
6.8 0.4
5.8 0.9
4.9 0.8
4.4 0.3
4.6 0.4
4.5 0.8
4.0 0.6
3.9 0.3
4.4 0.4
5.0 0.6
7.2 0.5
7.7 0.8
7.9 0.7
0.8 0.1

Podsd (cm)

Length of

S Specific for L. fendleri, N novel for two parents, D deleted in B. napus

Chro chromosome, F fragment, No labeled L. fendleri chromosomes

Seeds from 100 flowers pollinated with B.napus

N normal petals, S small petal, W wrinkle petal

Means and standard errors from twenty pods

Ten plants of each parental variety were observed

Q Qingyou 14, Z Zhongyou 821, O Oro

b, c

V
Q
Z
O
L. fendleri

IV

III

II

Plant No.

Types

W,S
W
N
N
N
N
N
W
N
W
Five petals
N
S
W,S
N
N
N
N
N
N
N
W
W
W
W
N
S

Flower

93.2
98. 7
96. 4
91. 6
0
0
96.9
95.9
94.3
97. 8
96. 4
92.5
99 .1
95.8
98.3
97. 6
89.5
94. 8
45.7
0
0
48.5
47.4
22.1
26.1
55. 4
12.1

stainability (%)

Pollen

83
95
112
54
2

224
362

315
59

Seedsf

Back-cross

38
38
38
38
38
38
38
38
38
38
38
38
38
38
38
38
3840
3842
3238
3238
3138
2531
2531
2531
2531
2531
1922

ovary cells

2n in

1Chro + F
F

12 Chro
12 Chro
12Chro + F
12 Chro
12 Chro
1 Chro

Chromosomeg

L. fendleri

Table 1 Phenotypes, cytology and polymorphic AFLP bands (number and percentage) of individual F1 plants from B. napus L. fendleri cross

25,
35,
28,
27,
25,
25,
19,
18,
10,
21,
15,
23,
12,
22,
26,
18,
33,
28,
24,
26,
20,
34,
38,
41,
35,
34,
33,

S
4.3
3.9
5.0
4.6
4.3
4.2
3.3
3.1
1.7
3.6
2.5
4.1
2.0
3.9
4.5
3.3
5.6
4.7
4.0
4.4
3.5
5.8
6.2
6.9
6.0
5.7
5.5

34,
45,
38,
35,
36,
37,
27,
29,
28,
32,
28,
30,
22,
29,
34,
24,
46,
37,
52,
39,
33,
57,
46,
49,
56,
36,
54,

AFLP bandsh

5.9
7.4
6.7
6.0
6.3
6.3
4.6
5.0
4.7
5.4
4.7
5.4
3.8
5.1
5.9
4.3
7.8
6.3
8.6
6.6
5.8
9.7
7.5
8.2
9.6
6.3
9.0

33,
24,
46,
43,
34,
13,
15,
18,
14,
13,
19,
33,
19,
31,
27,
40,
36,
25,
32,
38,
35,
28,
29,
44,
45,
38,
35,

D
5.7
5.7
8.1
6.0
5.9
2.6
2.6
3.1
2.4
2.2
3.2
5.9
3.2
5.4
4.7
7.2
6.1
4.2
5.3
6.5
6.1
4.8
4.7
7.4
7.7
6.6
5.8

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Plant Cell Rep (2008) 27:261271

265

Fig. 2 Cytogenetic analysis of hybrids from B. napus L. fendleri

cross. AJ DAPI and merged (signals from the L. fendleri are
indicated by arrows) images of each PMC were shown. A, B Two
diakinesis PMCs with 17II+4 I (arrow) +1F (fragment) (A) and
18II+2 I (arrow) +1F (B) from plant No.5, having one chromosome of
one bivalent and one chromosomal fragment (arrow) were fully
labeled, respectively. The univalents are indicated by arrowheads.
C One PMC at diakinesis with 19 II + 1F from plant No. 6, and the
fragment (arrow) was fully labeled. D One diakinesis PMC with 13
II + 5 I (arrow) + +1F one from plant No. 22, and the fragment
(arrow) was fully labled. E One diakinesis PMC with12 II+7 I

(arrow) from plant No. 24, and one chromosome (arrow) of one
bivalent was fully labeled. F One PMC with14 II from plant No. 25,
one chromosome (arrow) of one bivalent was fully labeled. G One AI
PMC with 14:15 segregation from plant No. 26, and one chromosome
(arrow) was fully labeled. H One PMC with 19 I from plant No. 27,
and one chromosome (arrow) was fully labeled and the other signals
covered centromeric or terminal parts of some chromosomes. I, J The
distribution of GISH signals of L. fendleri probe on chromosomes of
one diakinesis (I) and AI (J) PMC of B. napus cv. Qingyou14.
K One deeply stained chromosome (arrowed) in one AI PMC with
19:19 segregation from one BC1 plant of plant No. 25. Bar 5 lm

wrinkled petals and the lower pollen fertility of some plants

(Table 1; Fig. 1f). Plants of type I grew vigorously and
showed nearly normal seed-set by selfing, but two plants
Nos. 5, 6 produced no pollen grains but had good seed-set
after being pollinated by female B. napus. They had
2n = 38 (the same number as B. napus) in all ovary cells
observed. The majority of their pollen mother cells (PMCs)
had 19II at diakinesis and 19:19 segregations at anaphaseI
(AI), while a few had 17II+4I, 17II+4I+1F (fragment),
18II+2I+1F, and 20:18, 21:17 segregations. No lagging
chromosomes were observed. GISH observations on some
diakinesis PMCs from plants Nos. 56 showed that only
one chromosome of one bivalent and the fragments in
PMCs with various pairing configurations were wholly
labeled by the L. fendleri probe (Fig. 2A, B, C).

Two plants of type II had predominantly 2n = 38 in

ovary cells and occasionally 2n = 39, 40, 42. The majority
of PMCs had 19II and 19II+ 1I and showed segregations
19:19, 19:20 and 21:19.
The hybrid plants of group III had 2n = 3138 but
mainly 2n = 38 and produced PMCs with 19II and 19:19
segregation in predominance and with 18II, 18II+2I and
17II+2I and segregations of 19:17, 16:17 and 15:16 at very
low frequency. Signals of different sizes and intensities
from the L. fendleri probe were located mainly at terminal
or small centromeric parts of some bivalents or chromosomes and no whole chromosomes/bivalents were labeled
in these two groups.
The plants of types IV and V expressed some obvious
characters from the male parent L. fendleri, such as a

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Table 2 Chromosome numbers recorded in ovary cells and PMCs of F1 plants from B. napus L. fendleri cross
Types

Plant numbers

Number of cells with chromosome numbersa

42

16

40

39

Ovary

III

Ovary
PMCs

36

33

32

31

24

29

28

25

148
140

32
10

45
14

22

21

20

346
4

11

18

43

36

119

Ovary

33

19

PMCs

67

32

16

IV

Ovary
PMCs

Ovary

14

PMCs

26

22

19

171

PMCs
II

38

31
72

86
154

The data for PMCs from the sum of pairings and segregations

longer flowering period, elongated leaves, basic clustering

stems, and petal shape (Fig. 1be, k). They had very low
pollen fertility and poor seed-set after selfing or even
backcrossing to B. napus. Particularly, some selfed seeds of
F1 plant No. 22 had the glutinous layer on seed coats after
absorbing water, as the seeds of L. fendleri did, but the
layer was thinner (Fig. 1g).
The five plants of type IV mainly possessed 2n = 25, 28,
29 and 31 with 2n = 29 being much more frequent in ovary
cells (Table 2). In their PMCs, several pairing configurations at diakinesis and AI segregations appeared and the
frequency of the 14:15 pattern was much higher. One to
three lagging chromosomes were observed at A/T (telophase) I frequently. One chromosome of one bivalent and
chromosomal fragments in a few PMCs at diakinesis and
12 chromosomes in AI polar groups were wholly labeled
by L. fendleri probe (Fig. 2DG).
The plant (No. 27) in type V had 2n = 1922 in ovary
cells with a predominance of 2n = 19 (76.8%). In its
PMCs, the 10:9 segregation was frequently observed,
together with others (7:12, 6:13, 8:11, 10:11 and 11:11).
The laggards and chromosomal bridges at AI/AII were
often encountered. One chromosome in some PMCs with
2n = 19 was wholly labeled (Fig. 2H), while no fully
labeled chromosomes were found in PMCs with 2n [ 19.
The chromosome numbers in ovary cells of F2 and BC1
progenies were determined. All the F2/BC1 plants from F1
plants in types IIII showed that good seed-set had 2n = 38
predominantly in ovary cells. Fourteen F2 plants and 90
BC1 plants obtained from F1 hybrids of group IV showed
variable fertility (42.1098.21%) and some of them
expressed the male parent traits as their F1 plants, such as
longer flowering period, basal clustering of stems and
shorter plants. The F2 plants produced by the seeds with the
glutinous layer exhibited hairy and elongated leaves and

123

grew very weakly (Fig. 1i, j), and some even died as young
plants. Their ovary cells had 2n = 29, 31 with the first one
being much more frequent, and their PMCs mainly showed
the AI segregations 15:14 and 15:16. Most of 90 BC1
plants from type IV were mixoploids, consisting of cells
with different chromosome numbers (2n = 2954), mostly
2n = 3135. Eight plants had 2n = 38 in all ovary cells and
showed mainly normal pairings (19II) and segregations
(19:19) in PMCs, while the pairing 18II+2I and segregations (20:18, 21:17, in 36.9% AI PMCs) were observed.
They showed poor seed-set after selfing, though pollen
fertility was high (85%), one possible reason was that the
self-incompatible trait of L. fendleri (Mitchell 1997) was
introgressed into these plants. Two BC1 plants produced by
plant No. 27 in typeV had 2n = 3857 but mainly 2n = 38,
suggesting the formation of unreduced gametes (n = 19).
They showed the higher number of first branches on main
stems and lower branching positions than in the female
Qingyou 14.
In PMCs of three F2 plants from F1 plant No. 22 (other
than those from the seeds with the glutinous layer) and
eight BC1 plants from F1 plant Nos. 24, 25 of group IV, no
whole chromosomes or large chromosomal fragments from
L. fendleri were detected, while darker staining and more
condensed chromosomes were encountered in PMCs at AI
(Fig. 2K).

AFLP analyses of hybrids and progenies

From AFLP analysis with 26 pairs of primers randomly
selected, a total of 1,271 bands from 80 to1,000 bp were
scored in F1 plants (Nos. 127) and their parents (Fig. 3).
Three kinds of bands (absent in B. napus, novel for two
parents and specific for L. fendleri) were detected in all F1

Plant Cell Rep (2008) 27:261271

Fig. 3 Representative AFLP profiles of hybrids (top) between B.

napus cv. Qingyou 14 (P2) and L. fendleri (P1), their progenies
(bottom). Bands novel for two parents, absent in female parent and L.
fendleri-specific are indicated by arrows, arrowheads and circles,
respectively. Primer combinations: 50 -GACTGCGTACCAATTC
AGC-30 and 50 -GATGAGTCCTGAGTA ACCG-30 (top), 50 -GACTG
CGTACCAATTCACA-30 and 50 -GATGAGTCCTGAGTAACTT-30
(bottom)

plants at the frequencies of 2.27.7, 3.89.7, and 1.76.9%

(Table 1). The average frequency of L. fendleri -specific
bands in plants of types IV and V was higher than that in
other plants, indicating that there was a positive correlation
between the morphological traits of L. fendleri expressed
and its chromosomes detected by GISH/the AFLP bands.
The maximal number and frequency of novel bands (9.7%)
appeared in plant No. 22 and those of absent bands in B.
napus (8.1%) were observed in plant No. 3 with 2n = 38.
Twenty F2 plants from F1 plants No. 18 (six plants) in
type II, and No. 22 (14 plants) in type IV and 25 BC1 plants
from F1 plants in type IV were subjected to AFLP analysis
with ten pairs of randomly selected primers (Fig. 3). Of the
total 472 AFLP bands in these progenies, the average frequency of L. fendleri-specific bands, absent bands in B.
napus, and novel bands for two parents were 2.8, 6.6, and
5.7%, respectively. Unexpectedly, the F2 plants from the
selfed seeds with the glutinous layer of F1 plant No. 22 had
the highest number of L. fendleri-specific bands (6.4%),
even higher than that in F1 plant No. 22 (Table 1), this
maybe due to unequal segregation and combination of
genetic elements from L. fendleri during meiosis of
hybrids. But whether intact L. fendleri chromosomes were
included in these plants needed confirmation, for we failed
to obtain suitable tissues for GISH investigations.

Fatty acid composition analysis of F1 plants and

progenies
The fatty acid compositions in seeds of F1 plants and
progenies were analyzed (Table 3). The majority of progenies by selfing or by backcrossing to B. napus parents
showed the fatty acid profiles biased towards their female

267

parents (data not shown), while the decrease in oleic acid

content and the increase in the contents of linoleic, linolenic and eicosanoic acids were observed in the partial
progenies of types I, IV and V. The highest levels of linolenic and eicosanoic acids reached 13.17 and 13.66%,
respectively, in progenies of types I and IV, higher than
those of their female parents. Moreover, 4.2615.66% erucic acid was detected in the progenies of types I, IV and
V, whereas the content of erucic acid was nearly zero in
their parents B. napus cvs. Qingyou 14, Oro and L.
fendleri.

Discussion
The sexual intertribal crosses between Brassica crops and
the species of the other tribes were rarely reported for their
very distant phylogenetical relationships (Luo et al. 2003;
Chen et al. 2007). In the crosses between B. napus cv.
Oro and Mathiola incana (L.) R. Br. (2n = 14) of the
tribe Matthioleae, only two mature embryos were obtained
from 750 pollinated ovaries cultured in vitro and the hybrid
plants were mixoploids in nature, consisting of cells with
19, 26 (the expected number) and 38 chromosomes (Luo
et al. 2003). In the crosses between two Brassica species
(B. rapa, B. napus) as female and Capsella bursa-pastoris
of the tribe Lepidieae as male parent, majority of F1 plants
resembled female parents in morphology and chromosome
numbers, and only a few expressed some characters of
male parent and contained one to two chromosomes of C.
bursa-pastoris, while AFLP analysis indicated the introgressions at various levels from C. bursa-pastoris and
genomic alterations following hybridization (Chen et al.
2007). Intertribal somatic hybrids between the cultivated
Brassica species in the tribe Brassiceae and the species in
other four tribes Arabideae, Drabeae, Lepideae and Sisymbrieae have been produced by symmetric and asymmetric
protoplast fusions (Glimelius 1999). A higher degree of
asymmetric hybrids were obtained among the intertribal
hybrids than among the intrageneric and intergeneric
hybrids. Moreover, the intertribal somatic hybrids were, in
general, more difficult to root and culture to mature plants
outside in vitro conditions and foreign chromosomes are
easier to be eliminated in the following generations.
Although B. napus and L. fendleri have been considered
to be sexually incompatible (Skarzhinskaya et al. 1998), in
the present study a number of sexual hybrid plants were
obtained after B. napus were pollinated by L. fendleri and
the hybridity was revealed at the morphological, cytological and molecular levels (Figs. 1, 2, 3; Tables 1, 2, 3). All
F1 plants in five groups were non-Mendelian partial hybrids
with variable chromosome numbers, individual chromosome or chromosomal fragments from L. fendleri at very

123

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Plant Cell Rep (2008) 27:261271

Table 3 Fatty acid compositions (%) of seeds from some F1 plants and progenies
Plant
types

Seeds
origin

Oleic acid
C18:1

Linoleic acid
C18:2

2a

Linolenic acid
C18:3

Eicosenoic acid
C20:1

Erucic acid
C22:1

54.71

17.32

8.39

9.50

6.11

13

49.61

18.94

8.20

10.41

7.06

16a

49.36

18.05

6.38

13.66

6.30

22a

38.23

21.56

7.31

10.95

15.66

22b

43.97

17.00

8.55

12.46

12.23

23a

42.26

21.06

9.27

8.53

12.18

23
23c

48.19
53.34

15.10
17.92

8.39
13.17

13.19
4.83

9.78
4.26

23c

46.42

17.76

9.40

10.32

10.88

23d

48.56

16.78

11.75

6.70

8.21

24a

49.32

17.62

7.99

11.20

7.95

24

56.37

18.30

7.92

5.88

4.43

26b

34.37

17.42

8.73

7.74

12.52

26d

52.26

19.65

10.73

5.01

5.33

27e

47.32

19.99

9.51

8.33

8.98

P1

69.06

15.55

7.55

0.42

0.67

P2

67.92

15.47

7.76

1.78

0.81

15.2

7.6

0.0

0.0

IV

P3

13.1

P1 B. napus cv. Qingyou 14, P2 B. napus cv. Oro, P3 L. fendlei

Seeds from F1 plants by selfing

Seeds from F2 plants by selfing

Seeds from BC1 plants by selfing

Seeds from BC1 plants by backcrossing to their original female parents

Selfed seeds from F1 plants with chromosome doubled

the data from Kleiman (1990)

low frequency and some polymorphic AFLP bands (L.

fendleri-specific, novel for two parents and absent in B.
napus). It was necessary to distinguish most of B. napustype F1 plants in type I from false hybrids or unisexual
reproduction having 2n = 38 by the combination of
chemical, cytological and genomic analyses (Tables 1, 2,
3). The cytological events leading to those plants in IIV
types probably involved the complete or partial elimination
of chromosomes and fragments from L. fendleri during the
mitotic divisions of embryos and plants, and chromosome
doubling accompanied by genome rearrangements consecutive to genomic shock (McClintock 1984; Faure et al.
2002; Madlung and Comai 2004; Liu and Li 2007; Chen
et al. 2007). For the formation of 16 plants of type I with
2n = 38 in all cells, complete elimination should occur in
majority; and for the production of two plants in type II
with 2n = 3842, one or two L. fendleri chromosomes were
retained and then eliminated in most cells after doubling. In
three plants of type III, some cells had partial B. napus
complements and lost several chromosomes. The five
mixoploids of type IV had all cells with partial B. napus

123

complements, which might result from the elimination of

almost all chromosomes from L. fendleri and some ones
from B. napus. The only plant in type V was obviously the
outcome of the successive elimination of male chromosomes without doubling. The elimination of L. fendleri
chromosomes and some of B. napus occurred most likely
prior to the doubling, for B. napus mixoploids at haploid
and diploid levels were produced. The production of BC1
plants from F1 plants in groups IV and V with 2n 38
probably were attributable to the formation of unreduced
gametes during the meiotic divisions of these hybrids.
Whether the darkly stained chromosomes observed in AI
PMCs of some progenies (Fig. 2K) were from L. fendleri
needs further study, as the situation observed in crosses
between Brassica species and Orychophragmus (Li et al.
1998).
The rate and mechanism of alien chromosome elimination in plant wide crosses are not clearly known now. The
recent results from wheat and pearl millet crosses show that
uniparental chromosome elimination at mitosis and interphase involves micronucleus formation, progressive

Plant Cell Rep (2008) 27:261271

heterochromatinization, and DNA fragmentation (Gernand

et al. 2006). The success of hybridization and gene transfer
depends on the level of genetic and structural relatedness
between the genomes of crops and wild plants (Leflon et al.
2006). Riera-Lizarazu et al. (1996) proposed that the maize
chromosome retention in oat haploids was probably related
to the similarity between maize and oat centromere structure, further maybe related to the expression of factors
necessary for the relevant function of the alien chromosomes in a foreign genetic background. In the derived oatmaize addition line with one maize chromosome in which
two CenH3 genes were present, one from oat and one from
maize, the oat CENH3 (centromeric histone H3, replacing
the regular histone H3 in centromeric chromatin) was
consistently incorporated by the maize centromeres (Jin
et al. 2004). The presence of oat CENH3 on the additional
maize chromosomes may provide some molecular basis for
their retention in oat background. While chromosome
elimination prevailed in present hybrids and only individual
chromosomes or chromosomal segments of L. fendleri
origin were retained, a closer homoeologous relationship
should exist between one chromosome in B. napus and L.
fendleri, which paired and formed one bivalent (Fig. 2E, F).
The production of partial hybrids with the same chromosome number as one parent but with morphological
variations has been reported in several wide crosses covering rice (Liu et al. 1999), sunflower (Faure et al. 2002)
and rapeseed (Hua et al. 2006; Liu and Li 2007; Chen et al.
2007, present study). The genomic compositions of such
hybrids were deviated from the parents by having
polymorphic DNA segments at certain frequencies, introgressed from donor parent, novel for two parents and
absent in the recipient parent (Table 1; Fig. 3). Extensive
and genomic wide de novo variations occurred in up to
30% of the loci in rice recombinant inbred lines with
\0.1% alien introgressed DNA, and the loss of parental
bands was more frequent than the gain of novel bands
(Wang et al. 2005). Inducd by alien DNA integration,
significant changes such as alteration in DNA methylation
patterns and mobilization of transposable elements were
also demonstrated in the rice recombinant inbred lines (Liu
and Wendel 2003; Wang et al. 2004; Shan et al. 2005).
This suggested that extensive genomic and epigenomic
variations facilitating genome evolution and speciation
might also be generated in the progenies of our wide
hybridizations (Fig. 3). The male sterility of two F1 plants
Nos. 5 and 6 with 2n = 38 probably resulted from the
disturbance of normal function of genes for pollen development by the insertion of alien DNA fragments.
Modification of the phenotype and reduced fertility of
the hybrids were correlated with increased dosages of the
L. fendleri genome (Skarzhinskaya et al. 1998). At least
one genome complement of L. fendleri was required to

269

express its morphological features. Only one plant with the

expected 2n = 50 from symmetric fusion had L. fendlerilike elongated leaves. However, this trait and the basal
clustering stems (Fig. 1c, d, k) were expressed mainly by
the haploid plant with 2n = 19 and only one L. fendleri
chromosome in some cells (Fig. 2G), indicating that the
alien genes were easier to express in the haploid background of the recipient parent. Moreover, the character of
the glutinous layer on seed coats of L. fendleri (Fig. 1g) for
drought tolerance was expressed by some selfed seeds from
plant No. 22 with partial B. napus complements, which
contrasted with the result that the transferred genes usually
failed to express, for they were affected by many factors in
some wide hybridizations, especially the hexaploid nature
of B. napus genomes buffered the expression of foreign
genes (Skarzhinskaya et al. 1998). Therefore, the expression of the glutinous layer hinted that few genes are
responsible for it.
In the offspring of asymmetric somatic hybridizations
between B. napus and L. fendleri, elongase and hydroxylase genes can be transferred to rapeseed and expressed,
which result in the production of very long chain and
hydroxylated fatty acids (Schroder-Pontoppidan et al.
1999). In the first generation with zero-erucic acid rapeseed
as recipient, one plant was found to produce a seed containing up to16.5% erucic acid and 15% eicosaenoic acid,
as well as a seed having 4.3% hydroxylated ricinoleic acid
(18:1D9, 12-OH) but no seeds with lesquerolic acid, the one
major hydroxy fatty acid in L. fendleri. Similarly, some
progenies of the sexual partial hybrids had much increased
contents of fatty acids with longer chains (eicosanoic and
erucic acids), especially some plants had 2n = 38 with
normal pairing and segregation. One reason leading to the
change of fatty acid compositions might be certain physiological irregularities due to genomic imbalances (Wang
et al. 2003) and chromosomes elimination. Another may be
correlated with the alien introgressed genetic elements
from L. fendleri. The production of erucic acid in some
progenies from the zero-erucic acid B. napus cultivar and
L. fendleri, as reported by Schroder-Pontoppidan et al.
(1999), suggested a contribution of fae1 gene copies from
L. fendleri. Moreover, the increase of linoleic and linolenic
acids in the offsprings might be attributed to LFAH12
introgression, an oleate 12-hydroxylase gene from L.
fendleri which has both hydroxylase and desaturase activities (Broun et al. 1998). Whether hydroxylase gene was
introgressed and hydroxy fatty acids from L. fendleri were
produced in progenies, especially B. napus-like ones needs
further analyses, for the standard samples of hydroxy fatty
acids in L. fendleri are still not available for us.
In conclusion, the success of the intertribal sexual
hybridizations between B. napus and L. fendleri was
achieved and the partial hybrids with variable phenotypes,

123

270

chromosome complements and genomic compositions

were produced. The possible mechanisms for these hybrids
involved the chromosome elimination and doubling
accompanied by the introgression of alien DNA segments
and genomic reorganization. The genomic and fatty acid
alterations of B. napus-like hybrids further showed that the
wide hybridization was an efficient way to intrigue genetic
modifications. The progenies with some useful traits from
L. fendleri should provide the new and valuable resource
for the genetic improvement of rapeseed, especially the
glutinous layer on seed coats after absorbing water was
obviously beneficial for the germination of seeds under the
drought stress, which is usually encountered in the sowing
seasons for winter and spring rapeseed in China.
Acknowledgments The study was supported by 948 Project of
Agricultural Ministry of China and by PCSIRT (IRT0442).

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