Theor Appl Genet (1995) 91:346-352

9 Springer-Verlag 1995

V. S i m o n s e n 9 W. K. H e n e e n

Genetic variation within and among different cultivars and landraces of Brassica campestris L. and B. oleracea L. based on isozymes

Received: 22 April 1994 / Accepted: 27 January 1995

Abstract Genetic variation based on isozymes was studied in 43 landraces and cultivars of Brassica campestris from China, 4 cultivars of B. campestris from Sweden and 1 from India, and 5 cultivars of B. oleracea from Sweden and 1 from China (B. alboglabra). A total of 17 isozyme loci was studied, 10 of these were polymorphic in B. campestris and 6 were polymorphic in B. oIeracea. The level of heterozygosity seemed to be reduced in the Swedish cultivars compared to the Chinese landraces and cultivars of B. campestris. The level of heterozygosity in B. oleracea was even lower than that in the Swedish cultivars of B. campestris. A phylogeny of the cultivars and landraces ofB. campestris showed that the B. campestris var 'yellow sarson' cultivar, originating from India, deviated significantly from the other cultivars of B. campestris. A phylogeny of the cultivars of B. oleracea confirmed the expectations that the cultivar B. alboglabra was not closely related to the cultivated forms of B. oleracea.

Key words Brassica campestris 9Brassica oleracea 9 Isozymes - Genetic variation 9 Heterozygosity 9 Phylogeny

Communicated by G. Wenzel V. Simonsen (~)1 National Institute of Animal Science, Animal Physiology and Biochemistry, Foulum, RO. Box 39, DK-8830 Tjele, Denmark W. K. Heneen Department of Plant Breeding Research, The Swedish University of Agricultural Sciences, S-268 31 Sval6v, Sweden Present address: 1 National Environmental Research Institute, Department of Terrestrial Ecology, VejlsCvej 25, P.O. Box 314, DK-8600 Silkeborg, Denmark

Introduction Ever since U (1935) suggested the existence of a relationship among Brassica species B. campestris L. (genomes: AA), B. carinata A. Br. (BBCC), B. juncea (L.) Czern. (AABB), B. napus L. (AACC), B. nigra (L.) Koch (BB), and B. oleracea L. (CC), various analytical methods have been applied that have confirmed the validity of this theory. R6bbelen (1960) used chromosomal analyses to support the theory. Studies on isozymes and restriction fragment length polymorphisms (RFLP) have provided further evidence for the theory, as documented by Vaughan and Denford (1968), Song et al. (1990) and Warwick and Black (1991). A deviating result was obtained by Yadava et al. (1979), who suggested that B. nigra and B. campestris are the parental species of B. carinata, based on electrophoretic studies of proteins and enzymes. Within B. campestris, McGrath and Quiros (1991) observed reduced pollen and seed fertility when Indian B. campestris cultivars were used as parents in crosses with subspecies ofB. campestris from other areas. Song et al. (1990) have shown in a phylogenetic tree based on information from nuclear RFLP that cultivars from East Asia form one group, cultivars from Europe another group and finally that a cultivar from India is to some extent aberrant but still within the main cluster of B. campestris. In a recent work on B. campestris subspecies, McGrath and Quiros (1992) showed that the Indian variety 'yellow sarson' constitutes its own cluster and hence has its own specific origin. Within B. oleracea, Song et al. (1990) have found that B. alboglabra Bailey (a subordinate of B. oleracea) is more closely related to a wild accession of B. oleracea than to the cultivated B. oleracea accessions. The work of Warwick and Black (1991) supported the hypothesis that B. oleracea and B. oleracea ssp. alboglabra Bailey are rather closely related and belong to the same species. The aim of the present investigation was to use isozymes for studying the genetic variation within and among accessions of B. campestris and B. oleracea.

347 Table 1 List of the Chinese accessions of Brassica campestris analysed, their sample size, gene bank number and local name. Accessions marked with * are cultivars, all others are landraces

Table 2 List of the cultivars and breeding lines of Brassica campestris and B. oleracea used, their sample size, origin and further in-

Accession Sample no. size

Genebank no.

Localname

Species

l 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 28 29 30 31 32 33 34* 35* 36 37 38 39 40 41 42 43

0003 0052 0060 0070 0053 0082 0109 0133 0134 0091 0171 0173 0175 0179 0068 0181 0199 0195 0206 0208 0222 0233 0294 0358 0362 0372 0382 0391 0197 0368 0352 0386 0401 0418 0422 0423 0414 0430 0461 0476 0532 0535 0536

Deng Long Zhong Huang Yan Ling Bo Zhong Zhe You Yi Hao Wu Xian Cang Cai Zi Xiao Jiao Bei You Cai Li Yang Tu Zhong Lu An Da Wu Zi Feng Yang You Cai Shang Dang You Cai Tai Xian You Cai Xi Shui You Cai Bei Bei Guo Tian You Cai Ma Cheng Gui Shan You Cai Shang Ba He You Bei Cai Bei You Yi Hao Hong An Nong Chang You Cai Tian Men Da Ye You Cai Tian Men You Cai Tian Men Hong Gan You Cai Tian Men Wu Ye Bei Wu Chang Bei You Cai Tong Shan You Cai Dong Kou Tian You Cai Fen Yi You Cai Gao An Tian You Cai Bo Yang Tian You Cai Guang Feng Tu You Cai Jin Xian You Cai Tian Men You Cai Bei Ping Xiang Ben Di You Cai Nan Chang Tian You Cai Yi Huang Tian You Cai Ji An Ben Di You Cai Chuan You Ba Hao Xie Zuo Er Hao Xin Dou Ti Zi Shuan Guang Chang Hei Cai Zi Cheng Du Ai You Cai Wen Jiang Zhu Sha Hong Yi Bing Hei You Cai De Jiang Bei You Cai Si Nan Bei You Cai Shi Qian Bei You Cai

25 24 24 25 23 24 24 24 24 25 25 25 24 25 25 25 25 25 25 24 25 25 23 23 25 25 25 25 25 24 25 24 25 24 25 25 25 25 25 24 25 25 25

formation on the accessions Accession no.

Sample Origin Size

Information

B. campe- 44 stris

32

Sweden

45 46 47 48

24 32 25 22

Sweden India Sweden Sweden

Breeding line Sv 88-39339 Cultivar Kova Yellow sarson K-151 Breeding line WW 1722 Cultivar Emma

B. olera- 49 cea 50

32 23 19 25 23 24

China Sweden Sweden Sweden Sweden Sweden

51 52 53 54

B. alboglabra, no. 4003 White cabbage Brussels sprouts Savoy cabbage Cauliflower Broccoli

aminopeptidase (LAR E.C. 3.4.1 t. 1), malate dehydrogenase (MDH, E.C. 1.1.1.37), 6-phosphogluconate dehydrogenase (PGD, E.C. 1.1.1.44), phosphoglucomutase (PGM, E.C. 5.4.2.2) and shikimate dehydrogenase (SDH, E.C. 1.1.1.25).

Isozyme and allozyme nomenclature The nomenclature follows the outlines given by Chen et al. (1989, 1990) and Simonsen and Heneen (1995).

Statistical analyses Fit to Hardy-Weinberg proportions was tested with the testator F,~N as described by Brown (1970) using the programme G-FSTAT from the G-STAT package, version 3.0 (Siegismund 1992). The program G-FSTAT was used for estimating Wright's statistics F~T,Fis and FST as well as the genetic distance according to Nei (1972). Similarity in genetic and geographical distances was tested with the test developed by Mantel (1967). A programme for this test was available in the G-STAT package. The phylogenic tree was estimated by applying the programme CONTML, and the depiction of the phylogeny by the programme DRAWGRAM from the PHYLIP package (Felsenstein 1989).

Results Materials and methods Genetic variation within the accessions Plant material The Chinese cultivars and landraces of B. campestris are listed in Table 1, and the accessions of B. campestris from Sweden and India, as well as the accessions ofB. oleracea, are listed in Table 2. Sample preparation and electrophoresis The method for sample preparation was described by Chen et al. (1989). The electrophoretic method used was horizontal starch gel electrophoresis (Chen et al. 1989; Simonsen and Heneen 1995). The enzymes studied were esterase (EST, E.C. 3.1.1 .-), fructose-l,6-biphosphatase (FB R E.C. 3.1.3.11), glutamate oxaloacetate trans-aminase (GOT, E.C. 2.6.1.1), glucose phosphate isomerase (GPI, E.C. 5.3.1.9), iso-citric acid dehydrogenase (ICD, E.C. 1.1.1.42), leucine

The following loci, all of which were described by Chen et al. (1989, 1990) and S i m o n s e n and H e n e e n (1995), were scored in all the accessions: Est-1, Fbp-l, Fbp-3, Got-l,

Got-2, Gpi-2, Icd-l, Icd-2, Lap-l, Mdh-1, Mdh-2, Pgd-1, Pgd-2, Pgm-1, Pgm-2, Pgm-3 and Sdh-1. Est-2 and Sdh-2 were not used for further analyses as silent alleles were found in both loci. Lap-2 and Sdh-3 were often too faint to score, especially in the Chinese accessions. The variation in Fbp-2 and Gpi-1 caused smeary z y m o g r a m s , which did not allow further interpretation. Out of the 17 loci scored, 10 in the B. campestris accessions and 6 in the B. oleracea material studied were found to be polymorphic.

348 Table 3 Frequencies of the overall most frequent allele for the polymorphic loci studied in the accessions of Brassica cam-

pestris

Number

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 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

Locus

Est-1

Fbp-3

Got-1

Gpi-2 Icd-1

L a p - 1 Mdh-2

Pgm-1

Pgm-2

Sdh-1

1.00 1.00 1.00 0.88 1.00 0.85 0.85 0.81 0.79 0.86 0.68 0.90 0.85 1.00 0.88 0.26 0.32 0.16 0.16 0.40 0.06 0.04 0.20 0.50 0.12 0.92 0.52 0.36 0.28 0.29 0.76 0.02 0.38 0.61 0.88 0.86 0.02 0.32 0.02 0.44 0.98 0.50 0.24 0.81 0.85 0.68 0.86 0.77

0.92 0.79 0.59 0.32 0.71 0.79 0.56 0.36 0.52 0.34 0.52 0.50 0.38 0.56 0.44 0.50 0.42 0.38 0.78 0.50 0.65 0.77 0.41 0.12 0.86 0.62 0.65 0.42 0.50 0.61 0.52 0.56 0.54 0.63 0.46 0.84 0.79 0.56 0.48 0.76 0.86 0.66 0.76 0.70 0.42 1.00 0.42 0.17

0.82 0.60 0.48 0.88 0.67 0.83 0.67 0.83 0.92 0.84 0.84 0.88 0.81 0.82 0.90 0.90 0.76 0.80 0.72 0.71 0.78 0.84 0.74 0.87 0.74 0.62 0.86 0.80 0.85 0.96 0.96 0.85 0.88 0.90 0.86 0.74 0.64 0.74 0.94 0.85 0.88 0.82 0.84 0.92 1.00 0.95 1.00 1.00

0.94 0.88 0.71 0.70 0.54 0.96 0.65 0.79 0.85 0.82 0.90 0.68 0.69 0.94 0.80 0.86 0.72 0.64 0.84 0.71 0.82 0.80 0.70 0.74 0.68 0.76 0.82 0.84 0.88 0.67 0.68 0.81 0.76 0.90 0.74 0.82 0.72 0.82 0.76 0.81 0.66 0.90 0.52 1.00 1.00 0.31 0.98 1.00

0.54 0.48 0.56 0.40 0.63 0.60 0.54 0.71 0.79 0.40 0.54 0.78 0.63 0.60 0.58 0.70 0.66 0.70 0.52 0.46 0.58 0.62 0.63 0.74 0.60 0.30 0.58 0.70 0.66 0.58 0.54 0.63 0.60 0.46 0.52 0.68 0.70 0.60 0.40 0.63 0.66 0.66 0.56 0.08 0.83 0.11 0.40 0.00

0.71 0.77 1.00 0.77 0.67 0.75 0.31 0.30 0.08 0.38 0.44 0.42 0.81 0.78 0.88 0.33 0.62 0.70 0.76 0.63 0.66 0.64 0.46 0.74 0.62 0.55 0.64 0.70 0.58 0.46 0.86 0.46 0.63 0.75 0.76 0.71 0.56 0.58 0.82 0.40 0.98 0.68 0.52 0.00 0.04 0.00 0.32 0.32

0.74 0.81 0.79 0.68 0.80 0.52 0.71 0.67 0.55 0.62 0.42 0.58 0.85 0.68 0.64 0.64 0.54 0.56 0.30 0.40 0.70 0.64 0.63 0.35 0.52 0.66 0.54 0.72 0.50 0.57 0.42 0.39 0.40 0.71 0.40 0.80 0.40 0.58 0.80 0.50 0.58 0.64 0.56 0.30 0.57 1.00 0.90 0.82

0.22 0.88 0.94 0.90 0.76 1.00 0.86 0.85 0.69 0.88 0.88 0.72 0.85 0.62 0.73 0.82 0.54 0.75 0.76 0.88 0.90 0.78 0.91 0.78 0.62 0.80 0.86 0.78 0.68 0.73 0.80 0.77 0.68 0.77 0.80 0.90 0.74 0.68 0.56 0.65 0.72 0.86 0.80 0.66 0.80 0.02 0.88 0.80

The f r e q u e n c i e s of the o v e r a l l m o s t c o m m o n allele for each o f the p o l y m o r p h i c loci in the two species are listed in Tables 3 and 4. Tests for fit to H a r d y - W e i n b e r g proportions were done w h e n possible. F o u r hundred and sixtyt w o test values were obtained for the B. carnpestris accessions, and 28 o f these d e v i a t e d significantly, w h i c h was m o r e than the e x p e c t e d 5% o f the total n u m b e r o f values. W h e n the n u m b e r o f samples with an excess o f h e t e r o z y gores was c o m p a r e d to the samples with a d e f i c i e n c y of h e t e r o z y g o t e s , the o b s e r v e d distribution 208:254 did not fit with the e x p e c t e d ratio 1:1 (Z2=4.58 with 1 df, P=0.03). W h e n the B. campestris accessions w e r e split into C h i n e s e landraces, C h i n e s e cultivars and S w e d i s h cultivars, an

0.26 0.75 0.69 0.64 0.46 0.85 0.63 0.10 0.90 0.40 0.56 0.56 0.65 0.50 0.70 0.58 0.46 0.46 0.34 0.67 0.66 0.76 0.52 0.50 0.36 0.70 0.38 0.50 0.48 0.54 0.58 0.67 0.40 0.56 0.36 0.46 0.40 0.46 0.46 0.31 0.46 0.30 0.40 0.14 0.44 0.00 0.70 0.59

0.66 0.85 0.88 0.60 0.67 0.63 0.92 0.79 0.56 0.74 0.54 0.52 0.65 0.74 0.74 0.58 0.58 0.64 0.70 0.65 0.72 0.56 0.74 0.63 0.60 0.56 0.48 0.56 0.74 0.54 0.72 0.69 0.46 0.71 0.62 0.68 0.50 0.82 0.62 0.78 0.78 0.74 0.74 0.86 0.56 0.75 0.72 0.52

Table 4 Frequencies of the overall most common allele for the polymorphic loci studied in the accessions of Brassica oleracea

Accession no. Locus

49 50 51 52 53 54

Est-1

Fbp-3

Gpi-2 Lap-1 Pgm-2 Pgm-3

0.23 0.46 0.56 0.82 1.00 0.90

0.94 0.81 1.00 1.00 1.00 0.48

0.63 0.00 0.05 1.00 0.98 0.17

1.00 0.87 0.77 1.00 0.00 0.31

0.92 0.00 0.58 0.00 1.00 0.63

1.00 0.41 1.00 1.00 0.00 0.00

349 Table 5 Heterozygosity (Hobs) in the Brassica campestris accessions studied

Table 7 Values of Wright's F-statistics for Brassica campestris and B. oIeracea

Accession no.

Hob s

Accession no.

Hobs

Species

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

0.17 0.19 0.16 0.21 0.20 0.17 0.23 0.21 0.21 0.20 0.25 0.24 0.21 0.19 0.22 0.24 0.25 0.24 0.20 0.25 0.20 0.20 0.25 0.24

25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

0.20 0.24 0.25 0.24 0.23 0.24 0.23 0.21 0.23 0.21 0.22 0.19 0.21 0.22 0.2t 0.24 0.16 0.22 0.25 0.13 0.16 0.11 0.17 0.15

A v e r a g e Hob s

0.21

Table 6 Heterozygosity (Hobs) in the Brassica oleracea accessions studied Accession no.

Hobs

49 50 51 52 53 54

0.07 0.12 0.09 0.02 0.00 0.12

Average

0.07

overall deficiency of heterozygotes was seen. The test values for the B. oleracea accessions were in all 20 of which only 1 deviated significantly from Hardy-Weinberg proportions: 14 test values indicated an excess of heterozygotes and 6 denoted a deficiency ofheterozygotes. The figures for B. oleracea fit the 1:1 expectation (%2=3.20 with 1 df, P=0.07). The averages of the observed heterozygosity (Hobs) for the accessions of the two species are listed in Tables 5 and 6. The 17 loci used for these estimations comprised both monomorphic and polymorphic loci. The average of the heterozygosity for all the B. campestris accessions was 0.21, and the individual values varied from 0.11 to 0.25. The average of heterozygosity was 0.22 for the Chinese landraces, 0.19 for the Chinese cultivars, 0.16 for the Swedish cultivars and 0.11 for the Indian variety. The lower values obtained for the cultivars indicated that they had less genetic variation than the landraces. The B. oleracea

Groupof accessions Number F~T of accessions

B. campe-Chineselandraces stris Chinesecultivars Chinese accessions Swedish cultivarsa All accessions B. olera- Swedishcultivars cea All accessions

40 3 43 3 48 5 6

0.174 0.262 0.178 0.300 0.218

Fm

FST

0.068 0.114 0.071 0.144 0.072

0.114 0.167 0.115 0.182 0.157

0.627 0.016 0.621 0.611 0.005 0.610

a Only accessions nos. 45, 47 and 48 were pure Swedish accessions

accessions had indeed a lower level of heterozygosity than B. campestris. The overall value for B. oleracea was 0.07, which was the same as that for the Swedish accessions and for B. alboglabra. The values for B. oleracea as such varied from 0.00 to 0.12.

Genetic variation among the accessions For 8 of the Chinese B. campestris accessions, the place of origin was known. Accessions nos. 3, 6 and 7 originated from Zhejiang province, nos. 18, 19 and 20 from Hubei province and nos. 34 and 40 from Sichuan province. When homogeneity among accessions within an area was tested, no homogeneity was found. With the objective of investigating a possible correlation between the genetic and the geographical distances, we performed the test described by Mantel (1967). The genetic distances were estimated according to Nei (1972), and the approximate geographic distances in kilometers between the sites were determined and used for the test. The result showed that there was no correlation between the genetic and the geographic distances (r=-0.24). Table 7 shows the F-statistics for the two species and their subgroups based on geographical area and classification as landrace or cultivar. Ten polymorphic loci were used for the estimation of the statistics for B. campestris and 6 for B. oleracea. The differentiation in B. campestris was due to variation both within and among the accessions. The FsT value for the Swedish cultivars was remarkably higher than the values for the Chinese material, but the trends in the values were similar: FST was always higher than Fm. Phylogenetic relationships among the studied accessions of the two Brassica species are depicted in Figs. la, b and 2. The phylogenetic trees were based on the same polymorphic loci as those mentioned above. The trees were estimated using the restricted maximum likelihood method (Felsenstein 1989). Similar trees were obtained using genetic distances and the Fitch-Margolias method in the same programme package. Figure la shows that B. campestris accession no. 46, var. 'yellow sarson', originating from India, is quite different from all the other accessions. The breeding line no. 44, originating from crosses between

350 '-

39 41

~ 3 7 7

7

11

~

........... ,38

47

48

...... 40

~

-

11 d;';3 Bd

1831 ,,4~ 3~ 22 ~7 19

3 ~ 2 0 9 1 ~ 23 23

ii ~" _[

42

46

24

9

33 6

,33 58 '6

---•13

4

'14

14

"-•

32

I

..........

12~

11

12

3 10 8

I12

b Fig. 1 a Phylogeny ofBrassica campestrisaccessions (see Tables 1 and 2) based on 10 polymorphic isozyme loci, estimated with the CONTML programme from Felsenstein (1989) and depicted using the DRAWGRAM programme for phenograms, b Phylogeny of the Chinese Brassica campestris accessions (see Table 1) based on 10 polymorphic isozyme toci, estimated with the CONTML programme from Felsenstein (1989) and depicted using the DRAWGRAM programme for phenograms

Fig. 2 Phylogeny of Brassica oleracea accessions (see Table 2) based on 6 polymorphic isozyme loci, estimated with the CONTML programme from Felsenstein (1989), and depicted using the DRAWGRAM programme for phenograms

5t

dendrogram for the Chinese material is depicted in Fig. lb. The overall relationship among the Chinese accessions was alike in the two dendrogrmns, and reflected the limited differentiation among the lines. Figure 2 shows the dendrogram for the B. oIeracea accessions. No. 49, B. albogtabra, seems to be separated from the other accessions, which might be expected due to its geographical origin.

Discussion

52

Genetic variation within accessions

54 9 53

-

49

Swedish cultivars and no. 46 has yellow seeds as has no. 46. The similarity between these two accessions was confirmed by the dendrogram. Accessions nos. 45, 47 and 48, representing pure European material, ought to cluster together, but only nos. 47 and 48 did this. However, the length of the branches indicated that the 3 Swedish accessions are not closely related to the Chinese accessions. A

The number of samples, irrespective of species, which deviated from Hardy-Weinberg proportions was about 6%, which was close to the 5% expected. This result might be due to the sample size, to the propagation regimes of the different accessions (Lande and Barrowclough 1987) or to self-fertilization. Becker et al. (1992) found around 30% self-fertilization in B. napus, and it might be expected that a similar self-fertifization rate is likely for B. campestris. In a review on self-incompatibility in Brassica Nasrallah and Nasrallah (1989) mentioned that although self-incompatibility exists in B. eampestris, self-compatible strains may be generated from self-incompatible ones, so that selfing may play a role in B. campestris. However, the over-

351 all deviation was not due to an excess or a deficiency of heterozygotes as the ratio between samples with an excess and samples with a deficiency (222:260) was close to the expected 1:1 ratio (Z2=3.00 with 1 df, P= 0.08). When the accessions were separated into species, B. campestris had more samples with a deficiency of heterozygotes than expected, which might be due to a certain amount of self-fertilization. For B. oleracea, more samples with an excess of heterozygotes were seen, which might be due to the propagating regime or selection. However, the sample size was small and hence this might be the most likely explanation for observing more significant test values than expected. From the level of heterozygosity (Tables 5 and 6), it was clear that the Swedish accessions ofB. campestris had lost genetic variation compared to the Chinese accessions. A comparison of the Chinese cultivars with the landraces indicated that the landraces in general had a higher level of heterozygosity. Chinese landraces have not been subjected to as intensive breeding selection as the cultivars, and this was reflected in the heterozygosity values. An intensive propagation regime based on a small sample size might cause less heterozygosity in the accession due to random genetic drift, and this might be the explanation for the lower level of heterozygosity in the cultivars of both species. The situation in highly specialized cultivars of B. oleracea clearly supports the above statement by the fact that the level of heterozygosity was low.

Genetic variation among the accessions The variation among accessions, at least for 8 of the Chinese B. campestris accessions, was not correlated with geographic distribution, as shown by the Mantel test. This lack of correlation might be caused by the fact that nos. 3 and 34 are cultivars while the other 6 accessions are landraces and hence less exposed to selection. However, the level of heterozygosity for these 8 accessions (Table 5), does not seem to support this theory. The estimation of allelic frequencies was presumably highly influenced by the small sample size (on average 25 individuals, see Tables 1 and 2). The values for Wright's F-statistics given in Table 7 clearly show that the main variation found in the two species was due to differentiation among the accessions. This result was most likely due to the propagation of genotypes selected for their valuable agronomic traits. It seems that there is a greater selection potential in B. campestris than in B. oleracea as there exists a greater within-population variation in the first-mentioned species. The cultivars of B. campestris, both the Chinese and the Swedish, had a higher FST value than the landraces. This observation indicates that differentiation among the cultivars was greater than among the landraces, which might be expected as a result of propagation and selection. The results obtained by McGrath and Quiros (1992) for genetic diversity within B. campestris showed an opposite trend, with a greater within- than between-population diversity. Their results were based on three isozymes and four RFLP markers.

The notably different morphology of the B. oleracea accessions fit nicely to the high value of FST. Without doubt, the cultivars ofB. oIeracea have been strongly selected for specific morphological traits, hence the genetic variation seen in isozymes. The low level of heterozygosity in the species supports this statement. The phylogenetic tree obtained for the B. campestris accessions (Fig. la) was similar to the one obtained by Song et al. (1990). In their study as in the present one, the accessions originating in India deviated clearly from the other Asian accessions. This observation is supported by the study of McGrath and Quiros (199 i). They found that the Indian accessions remarkably diverge from the other B. campestris subspecies with respect to pollen and seed fertility when crossed to other subspecies. This fact was not pronounced in the work by McGrath and Quiros (1992) on B. campestris subspecies, although var. 'yellow sarson' still formed its own cluster. A possible explanation for this phenomenon might be that the Indian 'yellow sarson' (no. 46) is an original Indian species that has been separated from other B. campestris subspecies in Eurasia by plate tectonics (Valentine and Moores 1974), and hence it has its own identity in evolution. The phylogenetic tree of B. oleracea (Fig. 2) was similar to the trees obtained by RFLP (Song et al. 1990; Warwick and Black 1991) with regard to B. alboglabra not being closely related to the cultivated forms of B. oleracea. A close relationship between accessions no. 53 (cauliflower) and no. 54 (broccoli) was expected due to the similarity of their morphology, whereas the similarity in morphology between Savoy cabbage and white cabbage was not reflected in the isozymes. However, any variation in morphology was not seen in the isozymes or viCe-versa (Simonsen 1976). In conclusion, despite the smal ! sample size, the present isozyme studies supported the hypotheses, based on DNA methodology, regarding the evolution of Brassica campestris and B. oleracea (Song et al. 1990; Warwick and Black 1991). Acknowledgements We wish to thank Drs. B. Y. Chen and B. F. Cheng for supplying the accessions from China and for comments on the manuscript. Drs. C. Persson and S. Lejon for supplying the remaining accessions, Mr. S. Svendsen for competent technical assistance, and the National Institute of Animal Science for providing laboratory facilities. Dr. H. Siegismund is thanked for statistical support and introduction to the programme packages G-STAT and PHYLIP. The study was financially supported by the Nordic Joint Committee for Agricultural Research (NKJ project No. 74).

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Genetic variation within and among different cultivars and landraces of Brassica campestris L. and B. oleracea L. based on isozymes.

Genetic variation based on isozymes was studied in 43 landraces and cultivars of Brassica campestris from China, 4 cultivars of B. campestris from Swe...
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