STUDIES ON THE RECESSIVE
GENIC MALE STERILITY AND ITS GENETIC MARKERS IN RAPESEED(Brassica napus
L.)
Tu Jinxing1; Fu
Tingdong1; Zhang yi2 and Tian Shaohua3
1:Dept. Of Agronomy,Huazhong Agri.Univ. Wuhan,Hubei ,China,430070
2: National Agrotech.Extension and Service Center, Ministry of Agri.
Beijin,China,100026
3:Hubei Province Planning Committee,Wuhan, Hubei ,China,430000
ABSTRACT
A more efficient and
economical method is necessary for removing 50% male fertile plants from the
female lines in the utilization of heterosis by a genic male sterile(GMS) line.
It is a useful method to employ the linkage relationship between a
morphological character and GMS. Eight characters were tested, but only the
purple stem was linked to the male fertility. In this paper, the BC1F1
population from S45A x Zi‑1968 was investigated by bulked segregant analysis(BSA),
and it was found that two RAPD ( random amplified polymorphic DNA)
fragments, UBC158.580 and UBC187.880, are linked to the purple
stem gene and the male fertile gene(Ms1). The results show that these four
genes are located in the same linkage group, and the order is:
Pur‑‑‑Ms1‑‑‑‑‑‑‑‑‑UBC158.580‑‑‑‑‑‑‑‑‑UBC187.880
3.75cM 8.67cM
8.78cM
KEYWORD:Brassica napus, GMS,
RAPD markers
Employing plant male sterility
is one of the most economical methods for utilizing heterosis. Plant male
sterility include cytoplasmic male sterility(CMS)and genic male sterility(GMS).
The advantage of using CMS system is that it can generate a whole male sterile
population economically. On the other hand, this system needs three lines, it
takes many years to breed them, and it is complicated to produce and spread
their hybrid seeds,In addition, most CMS systems have the limitation in their
utilization because they have stringent restoring-maintaining relationships and
their male sterile cytoplasms may have negative effects on yields, and some CMS
may not give stable male sterility under all environments. In contrast,
utilizing recessive GMS has many advantage. Firstly, it needs only two lines
and therefore shorten the breeding cycles. Secondly, for recessive GMS, any
good lines can be used as resterors, so it is easy to get the combinations with
strong heterosis. Thirdly, GMS doesn’t have the negative cytoplasmic effect on
yield as CMS may do. But GMS system has its fatal shortcoming of being
difficult to drive a whole male sterile population. About 50% male fertile
plants need to be remove from the female lines. In order to develop the
advantages of GMS, an efficient and economical method is necessary for removing
these plants from the lines. There is a useful method to employ the linkage
relationship between morphological character and GMS. Eight characters were
tested (data not shown), but only the purple stem was linked to the male fertility(Tu,et
al.1999)..
The PCR technique developed by Williams et.al.[1990]using
arbitrary primers for random amplication of polymorphic DNA sequences (RAPD)
was found many applications on genetic research. Combined with the ‘Bulked segregant
analysis’(BSA) method developed by Michelmore et al[1991], RAPD markers appear
to be suitable for mapping of genes. Hu et al [1995] and Tanhuanpaa et al.[1995]
found some RAPD markers with linolenic acid and palmitic acid concentration in
the seed oil of rapeseed with BSA, respecitively. Foisset et al.[1996] found
the RAPD markers with drwarf BREIZH(Bzh) gene in a double haploid population. Diederichsen
et al.[1995] used a backcross population to identify one RAPD marker with the clubroot
resistant genes in B. napus. Delourme et al.[1994] identified four RAPD
markers linked to a fertility restorer gene for the Ogura radish cms of
rapeseed(B.napus). Jean et al.[1997] mapped the nuclear fertility
restorer genes for the ‘Polima’CMS in canola(B.napus) and got only one
RAPD marker(among the 11 markers directly linked to the Rfp1 locus. In this
paper, RAPD markers were employed to identify the linkage relationship between
the purple stem and male fertility.
MATERIALS AND METHODS
Plant material:one fertile parent,
Zi-1968, has a purple stem and two male fertile genes;another parent,S45A,has
two recessive sterile genes and the green stem. the backcross population
derived from the combination S45A´Zi-1968
had a ratio of 3 male fertile and 1 male sterile plants
( 348:130, Χ2=1.135)
DNA extraction:Total DNA was extracted
according to Horn and Rafalski[1992]. Three~four gram freeze-dried leaves were
ground to powder in a mortar with liquid nitrogen. The powder was pured into
the tube containing 12 ml of extraction buffer(100mM Tris-HCl, pH8.0, 500mM NaCl,
10mM β-ME, 50mM EDTA pH8.0) and 1.5ml of SDS(20%). The mixture was
incubated at 65°C for 25~30min. After adding
3ml of 5M KAc liquid, the tube was transferred into ice-bathing for 30min with
intermittent shaking gently, and then centrifuged for 8min at 5,000rpm. The
supernatant was transferred into a new tube and precipitate with a two-third
volume of isopropanol at -20°C. After 20min of
centrifugation at 3,000rpm, the supernatant was discarded, and the DNA resuspended
in 5ml of 70% ethanol. Thirty minutes later, the tube was centrifuged for 15min
and the pellets obtained were placed in sterile tube containing 1ml of TE
buffer, 10μl Rnase A(10mg/ml) was added and the suspension was incubated
at 37°C. About one hour later, the
suspension was treated with one volume phenol/chloroform and repeated twice
with one volume of chloroform/isoamyl alcohol. The DNA was reprecipitated in
one-tenth volume of 5M ammonium acetate and 2 volume of ethanol, centrifuged for
30min at 5,000rpm, and washed three times with 70% ethanol. The DNA was redissolved
and stored in TE buffer(1.5ml) at 4°C until
use.
RAPD analysis:The protocol using RAPD
primer to detect polymorphism was first applied to the DNA from the backcross
population of S45A´(S45A´Zi-1968)
as follows: equal amounts of DNA from the 10 male fertile individual were
pooled to create one bulk and equal amounts of DNA from the 10 male sterile
individual were pooled to form the second bulk. RAPD primers were tested in a
random manner on each of these two bulks. The primers that gave the expected
amplification bands were selected and tested on the individual sampling from
the population randomly.
The primers were obtained from Operon
Company and the University of British Columbia.Concentrations of primer, template and Taq polymerase(the
National Key Lab of Crop Genetic Improvement) were optimized to give maximum
band intensity. The final reaction mixture included 1´reaction
buffer, 1.5mM MgCl2, 0.08mM dNTP, 0.45μM primer,1.2unit of Taq
polymerase, 70ng of genomic DNA made of a volume of 20μl with sterilized
double-distilled water. Enzymatic amplifications, on a Perkin Elmer 480 machine,were
subjected to: 3min at 95°C, 1min at 50°C, 1.5min at 72°C,
followed by 38 cycles of 1min at 94°C, 1min
at 40°C and 1.5min at 72°C, and a final stage of 10min at 72°C.
The amplification products plus blue mix(3μl,
stop buffer/1.5% bromophenol blue/ glycerol:200/400/200) were separated by
electrophoresis using 1.5% agarose in 1´TAE
buffer. A1-kb ladder was included as a size marker, and the bands detected with
ethidium bromide staining [2.5μl(10mg/ml)100ml].
RESULTS
Selection of primers:Of the 980 primers
used, about 30 percent primers were polymorphic between the two parents. But
only two primers, UBC158 and UBC187, generated the expected polymorphism
between the two bulks. According to Fig.1, Zi-1968, male fertile bulk, and the
male fertile individual having purple stem(line15~22) have the bands of
UBC158.580(Fig1a) and UBC187.880(Fig1b). On the contrary, S45A, male sterile
bulk and most male sterile plants have not these fragments. Among of the male
sterile progeny, only one plant had these fragments.This result indicated that
these fragments might be linked to the male fertile gene.
1 2 3 4 5
6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
A --580b
--880b
B
Fig.1 RAPD analysis with primers of UBC158
and UBC187 .
Line 1:fertile parent
Zi-1968, line2:fertile plants bulk, line3:sterile plants bulk,
line4:sterile parent
S45a, line5:1kb ladder, line6~14:green stem and male sterile plants,
line15~22:purple stem
and male fertile plants
RAPD analysis of the male fertile plants
from the population:The result on analyzing with the two primers to test the
male fertile plants from the population randomly was listed in Table 1. Of the
33 purple stem and fertile plants,about 87.87%(29 plants) have the amplified
fragments with the two primers. Whereas among the 14 green stem and male
fertile plants, about 85% and 78.5% do not have the specifc fragments with
UBC158 and UBC187, respectively. The data showed that the two RAPD loci, the
purple stem gene and one(MS1) of the two male fertile genes were likely to be
located in the same linkage group.
Table1:Result of RAPD analysis to the male
fertile plants
|
RAPD
Purple st fertile Green st fertile
fragments + -
+ -
|
|
UBC187.880
29 4 3 11
UBC158.580 29
4 2 12
|
+/-: presence/absence of
fragements
RAPD analysis of the male sterile plants
from the population:Because the male sterility was controlled by two pairs of
recessive duplicated genes[Tu et al. 1999], any plant only having one male
fertile gene must produce a normal anther.So it was difficult to discriminate
whether a plant was derived from the recombinant or not in the male fertile
groups by RAPD markers. But it was easy to do so in the male sterile groups. If
some male sterile plants happened to cross over between ms1 and RAPD loci, they
would have the amplified fragments. Therefore, a recombination frequency should
be calculated with the male sterile group.
Table2:Result of RAPD analysis to the
male sterile plants
|
RAPD Purple st
sterile Green st sterile
fragments
+ - + -
|
|
UBC187.880
0 3 16 76
UBC158.580 0
3 8 83
|
+/-: presence/absence of
fragemens
Of
the 95 male sterile plants from the same population randomly, most of them
didn’t have the amplified fragments, but 16 and 8 plants had the UBC187.880 and
UBC158.580, respectively (Table 2). The most likely map positions were shown in
Figure 2. Both RAPD loci were located at one side of MS1 locus, and Pur locus
was at another side.All of them covered about 20cM in length.
LOD 2.014 16.677 16.677
Pur MS1 UBC158.580
UBC187.880
Dist 3.252 8.671
8.778
Fig 2: linkage group of MS1 gene
It obtained from the
population of S45A´(S45A
´ Zi-1968).Map
distances in
cM were indicated under
the linkage group and LOD values were over the
linkage group.These
loci were located through the Joinmap analysis.
DISCUSSION
The
linkage relationship between the male fertile gene(MS1)and the purple stem gene
was identified by RAPD markers in the male fertile group from the population
with the BSA method. The distances among the RAPD loci and MS1 locus were
calculated exactly by analyzing the male sterile group.
In
this study, nearly 1000 primers were used to screen the two bulk, but only two
primers can generate the polymorphism. Why was such a low frequency observed?
The unexpected result is maybe due to three reasons: Firstly, the difference
between the two parents was very small, only 30% primers could generate the
polymorphic fragments between them.The previous results[Song and Osborn,1992]
indicated that this difference was low for identification some of genes.
Secondly, the backcross population for mapping gene was not better than the self
population, because its message was only half of that of the
latter[Allard,1956]. But the backcross population in this study had the
advantage of producing many male sterile plants. Thirdly, it is related to the Brassica
genome. There were three genomes:A,B and C. A lot of researches had shown a
high degree of homology among them. the high homology might lead to more
difficult mapping in B. napus than the diploid species
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