Download Determination of n+1 Gamete Transmission Rate of Trisomics and

Journal of Integrative Plant Biology 2008
Determination of n+1 Gamete Transmission Rate of
Trisomics and Location of Gene Controlling 2n Gamete
Formation in Chinese Cabbage (Brassica rapa)
∗
Cheng-He Zhang , Xiao-Feng Li, Shu-Xing Shen, He Yuan and Shu-Xin Xuan
(College of Horticulture, Agricultural University of Hebei, Baoding 071001, China)
Abstract
A set of trisomics of Chinese cabbage was used for determining the n+1 gamete transmission rate and locating the gene
controlling 2n gamete formation on the corresponding chromosome. The results showed that the transmission rates of extra
chromosomes in different trisomics varied from 0% to 15.38% by male gametes and from 0% to 17.39% by female gametes.
Of the nine F 2 populations derived from the hybridizations between each trisomic and Bp058 (2n gamete material), only Tri4×Bp058 showed that the segregation ratio of plants without 2n gamete formation to plants with 2n gamete formation was
10.38:1, which fitted the expected segregation ratio of the trisomics (AAa) based on the 7.37% of n+1 gamete transmission
through female and 5.88% through male. In other populations the segregation ratios varied from 2.48:1 to 3.72:1, which
fitted the expected 3:1 segregation ratio of the bisomics (Aa). These results suggested that the gene controlling 2n gamete
formation in Chinese cabbage Bp058 was located on chromosome 4. Further trisomic analysis based on the chromosome
segregation and the incomplete stochastic chromatid segregation indicated that the gene locus was tightly linked to the
centromere.
Key words: Chinese cabbage; gene location; n+1 gamete transmission; primary trisomics; 2n gametes.
Zhang CH, Li XF, Shen SX, Yuan H, Xuan SX (2008). Determination of n+1 gamete transmission rate of trisomics and location of gene controlling
2n gamete formation in Chinese cabbage (Brassica rapa). J. Integr. Plant Biol. doi: 10.1111/j.1744-7909.2008.00765.x
Available online at www.jipb.net
The spontaneous formation of 2n gametes or unreduced
gametes is a comparatively prevalent phenomenon in higher
plants (Veilleux 1985), which plays an important role in plant
polyploidy and evolution (Bretagnolle and Thompson 1995).
Many studies have shown that it is advantageous for plant
polyploid breeding using natural 2n gametes (Ortiz et al. 1992;
Wang 1992; Levin 1993), and the cytological mechanism of 2n
gamete formation in plants has been studied clearly (Ramanna
1979; Guijun et al. 1997; Zhang et al. 1999; Shi et al. 2002).
So far the inheritance fashion of 2n gamete formation has
also been studied in some crops. Mok (1975) suggested that
recessive genes ps, pc1, pc2 and pc3 determined the 2n gamete
formation in potatoes. Qu et al. (1995) further proposed that
Received 29 Dec. 2007 Accepted 9 Jun. 2008
Supported by the Natural Science Foundation of Hebei Province
(C2006000450)
∗
Author for correspondence.
Tel: +86 312 752 8315;
Fax: +86 312 752 8309;
E-mail: <[email protected]>.
C 2008 Institute of Botany, the Chinese Academy of Sciences
doi: 10.1111/j.1744-7909.2008.00765.x
the number of genes for 2n gamete formation in potatoes was
two or four. Zhang et al. (2000) indicated that a single major
recessive gene determined the 2n gamete formation in Chinese
cabbage. However, the study of the chromosomal location of
gene controlling 2n gamete formation in higher plants has not
been conducted.
Chinese cabbage is one of the most important vegetable
crops in the world. Wang (1992) first discovered a Chinese
cabbage material, named Bp058, which can naturally produce
2n gametes with higher frequency. The cytological mechanism
and the inheritance pattern of the 2n gamete formation were
further studied by Zhang et al. (1999, 2000) who proved that the
triad formation due to spindle orientation change at metaphase II
was the main way of 2n gamete production, and the inheritance
fashion obeyed the segregation ratio (3:1) of Mendel.
Primary trisomics can be used to locate a gene on a particular
chromosome, to verify the independence of linkage groups,
and to associate linkage groups with individual chromosomes.
This method has been used to determine gene-chromosome
relationships in several plant species, such as rice (Dong et al.
2001), soybean (Gardner et al. 2001; Zhou et al. 2003), foxtail
millet (Wang et al. 2002; Gao et al. 2003; Wang et al. 2007)
and so on. The chromosomal location of a specific locus can be
2 Journal of Integrative Plant Biology
2008
determined by the altered segregation ratios in the F 2 offspring
of trisomics. In these progenies, the segregation ratios will be
modified from the expected 3:1 F 2 ratio for a dominant gene.
The new ratio depends on the genotype of the F 1 primary
trisomic plants, the type of chromosome segregation, and the
transmission rate of the n+1 gametes. However, it was not until
2006 that primary trisomic series were established in Chinese
cabbage, yet n+1 gamete transmission rates of this set of
primary trisomics have not been exploited. The objective of
our study is to determine the extra chromosome transmission
rates of the trisomics in order to use them for gene orientation
correctly, and to locate the gene controlling 2n gamete formation
on a corresponding chromosome so as to establish a basis for
its clone and application.
Results
n+1 gamete transmission rate of the trisomics
When using primary trisomic analysis to locate genes on corresponding chromosomes, it is necessary to determine the
n+1 gamete transmission rate, because it directly affects the
segregation ratio of the F 2 or testcross progenies. The examined
results of n+1 gamete transmission rate are shown in Table 1.
Of 10 trisomics, the extra chromosome transmission rate of
trisomic-10 (Tri-10) was 0% due to it being seedless, and others
varied from 1.61% to 15.38% through male gametes and from
5.43% to 17.39% through female gametes. The Tri-7 had the
highest transmission rate of n+1 male gametes, and Tri-1 had
the highest transmission rate of n+1 female gametes. The
average transmission rates of the nine trisomics (Tri-1–Tri-9)
were 8.02% through male gametes and 10.35% through female
gametes.
Controlling 2n gamete formation is a recessive trait
Pollen examination of F 1 plants showed differences in pollen
amount per anther and that pollen viability existed among
different trisomic combinations, but there was no F 1 offspring
with 2n male gamete formation (Table 2). This indicated that the
2n gamete formation was a recessive trait in Chinese cabbage
Bp058.
Segregation of 2n gamete formation in F2 and testcross
populations
The segregation ratios of the plants without 2n gamete formation
to the plants with 2n gamete formation in the F 2 populations derived from Tri-1×Bp058, Tri-2×Bp058, Tri-3×Bp058,
Tri-5×Bp058, Tri-6×Bp058, Tri-7×Bp058, Tri-8×Bp058, Tri9×Bp058 and bisomic (CK)×Bp058 varied from 2.48:1 to
3.72:1. These ratios were consistent with the expected
Mendelian segregation of the 3:1 ratio (Table 3). However, in
the F 2 population derived from Tri-4×Bp058, the segregation
ratio was revealed as 10.83:1, being very different from the
Mendelian ratio of 3:1. The above results were also verified
by the testcrosses (Table 4). Therefore, the gene controlling 2n
gamete formation in Chinese cabbage Bp058 should be located
on chromosome 4 (Tri-4, Figure 1A).
To further clarify the location of the gene controlling 2n gamete
formation, the trisomic genetic analysis was carried out with
Tri-4. For convenience, “a” stands for the gene controlling 2n
gamete formation and “A” for its dominant allele. Thereafter, the
cross between Tri-4 and Bp058 can be shown as AAA×aa, and
the genotype of trisomic hybrid in F 1 is AAa. The gamete types
and ratios produced from AAa are 2A: 1a: 1AA: 2Aa based
on the chromosomal segregation, and are 6A: 3a: 5AA: 6Aa:
1aa based on the incomplete stochastic chromatid segregation.
According to the extra chromosome transmission rates of 5.36%
through male gametes and 9.37% through female gametes in
Tri-4 (Table 1), the expected phenotypic segregation ratio of
the plants without 2n gamete formation to the plants with 2n
gamete formation should be 9.34:1 in F 2 and 2.24:1 in testcross
based on the chromosomal segregation; and 8.93:1 in the F 2
and 2.51:1 in the testcross based on the incomplete stochastic
chromatid segregation. The χ2 test indicated that the expected
segregation ratios fit the observed segregation ratios in both
cases (Table 3, F 2 population; Table 4, testcross population).
Table 1. Transmission rates of n+1 gamete in Chinese cabbage trisomics
Combinations
Total plants
identified
2n+1
plants
Transmission rates of
n+1 male gametes
Combinations
Total plants
identified
2n+1
plants
Transmission rates of
n+1 female gametes
17.39
Bp058×Tri-1
121
3
2.48
Tri-1×Bp058
92
16
Bp058×Tri-2
124
2
1.61
Tri-2×Bp058
104
8
7.69
Bp058×Tri-3
114
6
5.26
Tri-3×Bp058
101
10
9.91
Bp058×Tri-4
102
6
5.36
Tri-4×Bp058
95
7
9.37
Bp058×Tri-5
120
10
5.88
Tri-5×Bp058
92
5
5.43
Bp058×Tri-6
104
16
8.33
Tri-6×Bp058
102
16
15.69
Bp058×Tri-7
102
15
15.38
Tri-7×Bp058
92
14
15.22
Bp058×Tri-8
106
14
14.71
Tri-8×Bp058
98
6
6.12
Bp058×Tri-9
116
4
13.21
Tri-9×Bp058
95
6
6.31
Bp058×Tri-10
0
0
0
Tri-10×Bp058
0
0
0
Location Gene Controlling 2n Gamete Formation in Brassica
3
Table 2. Pollen characteristics of F 1 hybrids
Number of pollen
Pollen
Number of
Number of pollen
Pollen
Number of
Combinations
grains per anther
viability %
2n pollen
Combinations
grains per anther
viability %
2n pollen
Bp058×Tri-1
8 561
78.63
0
Tri-1×Bp058
8 236
79.47
0
Bp058×Tri-2
10 333
83.91
0
Tri-2×Bp058
9 859
88.53
0
Bp058×Tri-3
9 063
88.67
0
Tri-3×Bp058
8 847
85.97
0
Bp058×Tri-4
9 442
90.33
0
Tri-4×Bp058
9 033
89.69
0
Bp058×Tri-5
9 861
87.56
0
Tri-5×Bp058
8 337
84.21
0
Bp058×Tri-6
8 374
86.67
0
Tri-6×Bp058
8 017
81.13
0
Bp058×Tri-7
8 167
70.03
0
Tri-7×Bp058
7 973
75.67
0
Bp058×Tri-8
9 023
90.49
0
Tri-8×Bp058
9 123
90.17
0
9 833
88.42
0
Tri-9×Bp058
9 446
86.59
0
10 028
94.39
0
2n×Bp058(CK) 2n(CK)
10 673
93.83
0
Bp058×Tri-9
Bp058×2n(CK)
Table 3. Segregation of 2n gamete formation in F 2 populationa
Combinations
Total plants
Plants of non-2n
gamete formation
Plants of 2n
gamete formation
Ratios
χ2 values (3:1)
χ2 values (9.34:1)
χ2 values (8.93:1)
Tri-1×Bp058
86
62
24
2.58:1
0.21
32.56
30.04
Tri-2×Bp058
90
69
21
3.29:1
0.13
19.24
16.74
Tri-3×Bp058
101
79
22
3.59:1
0.86
16.82
14.90
Tri-4×Bp058
91
83
8
10.38:1
12.75
0.08
0.17
Tri-5×Bp058
92
71
21
3.38:1
0.23
12.73
15.85
Tri-6×Bp058
120
86
34
2.53:1
0.71
47.84
44.04
Tri-7×Bp058
118
86
32
2.69:1
0.28
41.12
37.81
Tri-8×Bp058
87
62
25
2.48:1
0.65
36.19
33.46
Tri-9×Bp058
85
67
18
3.72:1
0.72
12.88
11.56
124
91
33
2.75:1
0.17
–
–
2n×Bp058(CK)
a
Degrees of freedom (d.f.) = 1, χ 0.05 = 3.84; d.f. = 1, χ 0.01 = 6.64.
Table 4. Segregation of 2n gamete formation in testcross populationsa
Combinations
Total plants
Plants of non-2n
gamete formation
Plants of 2n
gamete formation
Observed ratios
χ2 values (1:1)
χ2 values (2.24:1)
χ2 values (2.51:1)
Tri-1×Bp058
78
35
43
1:1.23
0.82
11.01
27.24
Tri-2×Bp058
86
46
40
1.15:1
0.42
11.4
13.72
Tri-3×Bp058
80
37
43
1:1.16
0.45
19.62
25.02
Tri-4×Bp058
77
52
25
2.08:1
9.47
0.09
0.61
Tri-5×Bp058
76
36
40
1:1.11
0.21
11.90
21.72
Tri-6×Bp058
81
43
38
1.13:1
0.31
9.76
13.44
Tri-7×Bp058
76
41
35
1.17:1
0.47
8.21
11.51
Tri-8×Bp058
77
37
40
1:1.08
0.12
16.03
20.91
Tri-9×Bp058
69
30
39
1:1.30
1.17
21.31
26.58
a
Degrees of freedom (d.f.) = 1, χ 0.05 = 3.84; d.f. = 1, χ 0.01 = 6.64.
Hence, it further verified that the gene controlling 2n gamete
formation was located on chromosome 4. As the χ2 values (0.08
in F 2 ; 0.09 in testcross) based on chromosomal segregation
were much less than those (0.17 in F 2 ; 0.61 in testcross) based
on incomplete stochastic chromatid segregation, this indicated
that the gene locus was tightly linked to the centromere.
Discussion
The spontaneous occurrence of 2n gametes is a common
phenomenon in higher plants. 2n gametes include 2n female
gametes (2n eggs) and 2n male gametes. Because of its
difficulty to identify the 2n gamete formation by egg examination,
4 Journal of Integrative Plant Biology
2008
Figure 1. Process of 2n gamete formation and karyotype of trisomic-4 in Chinese cabbage.
(A) Karyotype of Tri-4.
(B) Triangle orientation spindle at MetaphaseII.
(C) Tripolar body.
(D) A triad, including one 2n microspore and two n microspores.
(E) Pollen grains. The arrow indicates a 2n pollen grain.
(F) A 2n pollen grain with 20 chromosomes.
(G) An n pollen grain with 10 chromosomes.
it is usually identified by pollen examination under a microscope.
Morphologically, the 2n pollen grains are much bigger than
n pollen grains. According to Ma et al. (1999), the mean
size of the fresh pollen grains in Chinese cabbage is about
38.75 μm × 24.25 μm for 2n, and 30.23 μm × 15.67 μm for n.
Thus, it is quite easy to distinguish the 2n pollen grains from
n pollen grains. Our previous research indicated the formation
of 2n male gamete in Chinese cabbage Bp058 was mainly due
to the spindle abnormal orientation at metaphase II, such as
forming triangle spindles (Figure 1B) that led to form tripolar
body (Figure 1C) and triad (Figure 1D). Each triad produced
one 2n pollen grain and two n pollen grains, and the 2n pollen
grain was much bigger than the n pollen grain (Figure 1E,
pollen grains became round after staining). The 2n pollen grain
contained 20 chromosomes (Figure 1F) and the n pollen grain
with 10 chromosomes (Figure 1G). Although a difference in
pollen amount and viability existed among the different trisomic
combinations, there was no plant with 2n pollen formation in all
F 1 hybrids derived from Tri-1×Bp058 to Tri-9×Bp058 or from
Bp058×Tri-1 to Bp058×Tri-9. This demonstrated that the gene
controlling 2n gamete formation was a recessive trait.
The basic principle of using primary trisomics to locate a gene
on a particular chromosome is the altered segregation ratios in
the F 2 or testcross populations and the new ratios are mainly
based on the n+1 gamete transmission rate of the trisomics.
Therefore, it is necessary to determine the n+1 gamete transmission rate before carrying out the gene location by primary
trisomic analysis. The extra chromosome of the trisomics is
generally transmitted through female gametes, and the transmission rate is always less than 50%. For example, the average
female transmission rate of the 10 primary maize trisomics was
38% (Li and Song 1999), that of the 12 primary rice trisomics was
31.3% (Khush et al. 1984), and that of the nine primary cabbage
trisomics was 17.58% (Zhang et al. 2007). In this study, of the
10 primary Chinese cabbage trisomics, except the trisomic-10
(SAT-chromosome; the SAT-chromosome was arranged last in
the karyotype of trisomics, Figure 1A) with 0% transmission rate
because of the abnormal anther and ovary, the remaining nine
trisomics could transmit their extra chromosomes through both
the female gametes and male gametes. Although the average
transmission rate by male gametes was lower than the gametes,
it was available to conduct gene location when these trisomics
were used as male parents.
2n gamete formation is an important trait in plants. In this
study, the chromosomal location of the gene controlling 2n
gamete formation was conducted. Using the full set of Chinese
cabbage trisomics, the gene controlling 2n gamete formation
was suggested to be located on chromosome 4, which was
Location Gene Controlling 2n Gamete Formation in Brassica
tightly linked to the centromere. Although the present research
is a preliminary work, it would be helpful to further screen the
molecule markers tightly linked to the gene and to clone this
gene.
Materials and Methods
Chinese cabbage Bp058 was offered by Wang (1992), and
can naturally produce 2n male gametes with a percentage
of about 16.14%. A set of primary trisomics (from Tri-1 to
Tri-10) of Chinese cabbage was established through isolated
microspore culture of tetraploid Chinese cabbage and chromosome identification (Shen et al. 2006). The trisomic lines were
propagated and kept by subculture in Murashige-Skoog medium
(MS medium) with 6-benzyl aminopurine (BA) 1.0 mg/L and
indoleacetic acid (IAA) 0.1 mg/L.
5
Location of the 2n gamete formation gene
The trisomic plants identified from each F 1 hybrid of
Tri-1×Bp058, Tri-2×Bp058, Tri-3×Bp058, Tri-4×Bp058, Tri5×Bp058, Tri-6×Bp058, Tri-7×Bp058, Tri-8×Bp058 and Tri9×Bp058 were selfed and back-crossed to Bp058, respectively. At florescence, the pollen grains were examined from
the plants one by one for each F 2 population or testcross
population, and the segregation ratios of plants without 2n male
gamete formation to plants with 2n male gamete formation
were calculated. Furthermore, based on the bisomic and and
trisomic segregation fashion the tests of goodness of fit between
observed and excepted segregation ratios were conducted, respectively. The trisomic analysis was also carried out according
to the chromosomal segregation (supposing the gene is tightly
linked to the centromere) or the incomplete stochastic chromatid
segregation (supposing the distance between the gene and
centromere is >50 cM).
Determination of n+1 female gamete transmission rate of
Chinese cabbage trisomics
The complete set of Chinese cabbage trisomic lines (Tri-1–Tri10) as female parents was crossed with Bp058, respectively.
Then the chromosome number of each F 1 offspring was examined by the pollen mother cell squash method. The n+1
female gamete transmission rate was estimated by the 2n+1
plants/total plant ratio for each F 1 offspring.
Acknowledgements
We thank Dr Jianguang Zhang, Dr Jianjun Zhao and Yingxiang
Zhang for improving this manuscript.
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