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.    


3:Hubei Province Planning Committee,Wuhan, Hubei ,China,430000   



 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:


 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[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.



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].



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      





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.



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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