* The research is supported by the natural Science Foundation of China

1. Gansu Academy of Agricultural Science

2. Huazhong Agricultural University

ABSTRACT

China is regarded as one of the possible origin centres of Brassica juncea and has abundant germplasm. The genetic diversity estimations of the germplasm is the base of exploiting and using them properly in rapessed genetic improvement. The paper reported on the genetic diversity of 68 Chinese landraces and 4 Canadian lines by RAPD markers. A total of 211 DNA bands were observed with 31 arbitrary 10-base primers and 180 of them were polymorphic. Average proportion of polymorphic loci was 85.31%. Abundant genetic diversity were found within landraces and lines. On the basis of the genetic distance, a phylogenetic tree was constructed by UPGMA. All of them were divided into 6 groups. It was shown that the geographic distribution and ecological environment was one of the important factors affecting genetic diversity. Genetic differences existed both within and between winter landraces and spring’s in B.juncea. Greater genetic diversity was found within winter B.juncea than within spring’s. High similarity was revealed among Hubei, Jiangsu and Guizhou landraces. Small genetic distances were observed between Henan and Shaanxi landraces. All the above areas have winter B.juncea. In some spring B.juncea places, for example, Shanxi and Gansu, their landraces had also close relationship.Another result was that there was very close relationship between spring landraces of Xinjiang Autonomous Region of China and the Canadian B.juncea breeding lines. These results provide valuable information for properly selecting parents of crosses and enhancing breeding efficiency.

KEYWORDS Genetic diversity Chinese landraces Brassica juncea RAPD UPGMA

INTRODUCTION

Brassica juncea is one of the three major oilseed Brassica species cultivated in China. It is mostly grown in northwest and southwest parts of China. China is regarded as one possible origin centres of Brassica juncea, and has very abundant germplasm. 1, 132 landraces were collected until 1995 (Qian, 1996). It is more than one fifth of the total Brassica resources. To identify and use them properly, the research had to be done about them by diverse methods. RAPD, one of the recently developed PCR-based multiple-locus marker technique, has been successfully carried out for estimating of genetic diversity in many crops. For example, Link et al (1995) revealed the genetic diversity of faba bean germplasm in European and Mediterranean by RAPD markers. Chan (1997) detected genetic diversity of wild species of Amaranthus. The object of present study here is to estimate the genetic diversity of Chinese B. juncea landraces and obtain reliable information for further study and serving rapeseed breeding.

MATERIALS AND METHODS

Plant materials

This study comprised 68 Chinese landraces and 4 Canadian lines. Chinese landraces were collected from northwest (Gansu (A), Shaanxi (F) and Xinjiang (J)); Southwest (Sichuan (C), Yunnan (D) and Guizhou (E)) and other regions (Shanxi (B), Hubei (I), Jiangsu (G) and Henan(H)). 6 plants per landrace were selected at random and approximately equal amount young leaves were taken from each plant, stored at -70for future use.

Arbitrary primers

Thirty one ten-base oligonucleotide primers (parts of OPI, OPU and OPX) were used.

RAPD methods

DNA isolation DNA was isolated according to the procedure described by Li et al (1994).

RAPD reaction system The total volume of DNA amplification reactions was 20μμl. It contained 10X Reaction Buffer, 1.25mM MgCl₂, 1U Taq polymerase, 100μμM each dNTP, 0.4μμM Primer, about 50ng template DNA. DNA amplification were performed in a Perkin Elmer Cetus DNA thermal cycler 480 using the following conditions:94for 2 min, 50for 1 min, then 72for 1.5min, followed by 38 cycles at 94for 1 min, 36for 1 min, 72for 2 min. Finally cycle was followed by an additional 10 min at 72. Amplification products were seperated by electrophoresis in 1.4% (W/V) agarose gels in 1TAE, then stained with 0.5μμg/ml ethidium bromide about 15-20 min, and were then photographed under UV.

Data analysis The presence or absence of each specific band was scored for each genotype. ‘1’ stands for presence and ‘0’ stands for absence. Similarity coefficients (F) and genetic distances (D) were calculated by the following formulas (Yu et al 1994):

F=2X₁₂/(X₁+X₂) D=-LnF

X₁ and X₂ means the total band number of No.1 and No.2 landraces or lines respectively. X₁₂ means common band number of both landraces, the dendrogram showing the relationships among the landraces was produced from the distances using the UPGMA.

RESULTS

Efficiency of amplification and rate of polymorphic loci

Thirty one primers were selected according to the following conditions: (1) discernible band patterns. (2) rich polymorphism. (3) stable repeatability. Those 31 primers were used in 72 landraces or lines for estimating the genetic diversity. Totally, there were 211 DNA bands in 72 landraces or lines using 31 primers and 180 of them were polymorphic. The average rate of polymorphism was 85.31% (Table 1). From table 1 it can be seen that different primer produced various number of bands. For example, OPU20 and OPX09 produced the least number bands, which was only 4, while OPI20 produced 12 bands. Table 1 also showed different rate of polymorphic loci number in 72 landraces of Brassica juncea with different primers. An example of this is that the rate of polymorphic loci of OPX07 was only 43%, while that of some other primers, e.g. OPI04 and OPI05 were 100%.

Table 1 Efficiencies of amplification and rates of polymorphic loci of 72

landraces or lines in Brassica juncea

Genetic diversity analysis

The dendrogram was obtained using the distance data and UPGMA method (Figure 1). From Figure 1, it can be concluded that all landraces and lines were obviously divided into 6 group (Table 2). On the basis of Figure 1 and Table 2, the following conclusions can be drawn: (1) There were very close relationship between genetic diversity and geographic distribution. Every group included almost all the landraces (lines) which came from similar ecological region. In such a case Groupcontained all the landraces of Xinjiang Autonomous Region. (2) Genetic differences existed both within and between winter landraces and springs in B. juncea. It also showed that there were greater genetic diversity within winter B. juncea than within spring B. juncea. (3) Landraces coming from lower region of Yangzi River had close relationship with the landraces of Guizhou. (4) Relationship of spring B. juncea of Gansu and Shanxi was very close, but spring B. juncea of Xinjiang was different form them. (5) Ruicheng landraces were different from others. They formed a special group. But others landraces of Shaanxi had similar genetic background to Henan landraces. (6) There was very close relationship between spring landraces of Xinjiang Autonomous Region of China and the Canadian B.juncea breeding lines.

Table 2 The 6 groups of the tested landraces and lines of B.juncea generated by UPGMA

DISCUSSION

The present study showed that geographic distribution and ecological environment were among the important factors affecting genetic diversity. This is in agreement with the study in B. campestris and B. juncea by Zhu et al (1998) and Wu et al (1997), respectively. The results showed that extensive genetic diversity existed among the 68 Chinese landraces and the 4 Canadian lines of B. juncea. The average rate of polymorphic loci was 85.3%. The various landraces were clustered together according to the different geoecotypes. The landraces of the lower regions of Yangzi River belonged to the same group as Guizhou’s. This may be explained by the fact that there are similar climates in the two regions. Similarly, the spring landraces of Gansu and Shanxi formed one group. This knowledge is useful for properly selecting parents of the crosses and thus enhancing the breeding efficiency.

ACKNOWLEDGEMENTS

The authors are grateful to Huazhong Agricultural University, Gansu and Xinjiang Academies of Agricultural Science for offering the seeds and to Dr.J.X. Tu and C.Z. Ma et al for helps.

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