Great Britain: Devon: Seaton, Beer. 10. GB235 ... Great Britain: Cornwall: West Looe. 450 ..... mining genetic similarity among Brassica oleracecl L. geno- types.
Genetic Resources and Crop Evolution 43: 13-23, 1996. @ 1996 KluwerAcadernicPublishers. Printedin the Netherlands.
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Diversity in natural populations of wild Brassica oleracea as estimated by isozyme and RAPD analysis C. L a n n 6 r - H e r r e r a t , M. G u s t a f s s o n , A.-S. F~ilt & T. B r y n g e l s s o n Department of Plant Breeding Research, The Swedish University of Agricultural Sciences, S-268 31 SvalOv, Sweden Ipresent address: Department of Agronomy and Horticulture, New Mexico State University, Las Cruces, New Mexico, 88003-0003, USA Received 9 June 1994;accepted9 November1994
Key words." wild cole, population survey, intrapopulational variation, interpopulational variation, genetic distance
Abstract Naturally occurring populations of wild Brassica oleracea were collected in Spain, France, and Great Britain. Allele frequencies at four isozyme loci were determined for 18 populations, while five populations were screened using five random primers to generate RAPDs. Levels of homozygosity and gene diversity, H, were computed for each population using isozyme data and RAPD data when applicable. Homozygosity levels tended to be higher in smaller populations, which could also be observed as increased numbers of homozygous loci in smaller populations. Gene diversity values based on isozymes indicated considerable within population variation regardless of population size. The RAPID based gene diversities were significantly higher and the two exceptional populations displayed diversity levels more in keeping with the rest. The coefficient of gene differentiation, GsT, for populations in each region showed that the Spanish populations were more homogeneous than the French or British. When the GsT" for all populations was calculated using isozymes vs. RAPD data, the RAPD data gave a significantly lower value, a plausible result of the higher within population variation detected using RAPDs. Genetic distances between populations from different regions were also calculated from both data sets and used to produce phenograms. Clustering according to geographic region was not evident using either isozyme or RAPD data.
Introduction Brassica oleracea L. comprises both crop plants, cultivated throughout the world, and wild populations, which are distributed along the Atlantic coasts of northern Spain, France and Great Britain. B. oleracea belongs to the 2n = 18 cytodeme (Harberd, 1972) and is most related to the Mediterranean species B. montana Pourret and B. bourgeaui (Webb) O. Kuntze endemic to the Canary islands (Snogerup et al., 1990). The wild populations orB. oleracea grow in steep parts of maritime cliffs consisting of limestone or chalk, but in situations protected from grazing, plants can be found below the cliffs in scree and among shrubs. Usually, a single population is restricted in size and distribution, mostly due to limited areas of suitable cliffs. Adjacent populations are isolated from each oth-
er by unsuitable habitats like sandy shores, woodland and human settlements. Consequently, the number of plants comprising a population may vary from one location to another. Hence populations of B. oleraeea are characterized by large variation in size and fluctuations in reproductive capacity from year to year_ The species B. oleracea presents an excellent opportunity to study the degree of intra- and interpopulational varlation in populations of various sizes. It is possible to investigate whether small populations of B. oleracea are capable of maintaining genetic variation. Small populations are more or less continuously subject to genetic drift, which may lead to loss or fixation of alleles in a population. In such cases inbreeding depression may occur, although this can be counteracted by factors promoting equilibrium with permanent heterozygosity (Darlington, 1958; Carson, 1967).
14 In the present paper, results concerning isozyme polymorphism and degree of homozygosity in relation to population size and location are presented. The advent of DNA based molecular methodologies in the measurement of genetic variation has highlighted differences in the amount and type of variation assayed by alternate methods. Often, more variation is disclosed by marker systems like RFLPs (Restriction Fragment Length Polymorphisms) and RAPDs (Random Amplified Polymorphic DNAs) (Chahners et al,, 1992; McGrath & Quiros, 1992; Gonzales & Ferrer, 1993: Liu & Furnier, 1993). The RFLP markers are determined by detecting shared DNA fragments of up to several kilobasepairs in size while RAPDs consist of smaller DNA fragment patterns generated from shared primer hybridization sites in the genome. The agreement between genotypic variation patterns and deduced genetic relationships, determined from isozyme, RFLP and RAPD assays, is usually good (Chase et al., 1991 ; Thormann & Osborn, 1992; dos Santos et al., 1994), although minor discrepancies may arise at the species level and higher. Accordingly, a comparison of population attributes determined by isozyme analysis with those determined by RAPD analysis among wild B. oleracea was also conducted. Materials and methods
Germplasm of 44 wild populations ofB. oleracea were collected in Spain, France, and Great Britain in 1988. Field observations were recorded at the collection site, among others, population habitat, companion species and the actual population size. Seeds were collected from as many plants as possible and throughout the entire location, thus representing a good population sample. Sixty seeds from these original samples were taken at random, were sown in pots containing a mixture of clay and peat. The seedlings were transplanted to individual pots and placed in a heated greenhouse, with a temperature of about 20°C and 16 h of light. The isozyme and RAPD analysis were based on 38 to 60 plants of 18 populations. The origins of the 18 investigated populations are listed in Table 1. Collection locations of the investigated populations are presented on a map in Fig. 1. lsoz, v n w a t m h ' s i s
The isozyme systems assayed were glucose phosphate isomerase (GPI, EC 5.3.1.9), phosphoglucomutase (PGM, EC 2.7 5.1), and diaphorase (DIA, EC
f
l-ig, 1. Map of distribution of 18 wild B. oleracea populations along Spanish, French and British coasts. Arrows indicate populations also investigated using RAPDs.
1.6.4.3). Cotyledons or leaf samples were ground in 5(1 l;1 Tris-HCl pH 7.2, 0.05% fr-mercaptoethanol (Soltis et al., 1983) using a glass rod mounted in a rotary-homogenizer. Samples were taken up on paper wicks and run on 12% starch gels. Electrophoresis was carried out in a modified buffer system 1 of Soltis et al. (1983) containing 0.02 M histidine-HC1 pH 8.0 in the gel buffer and 0.4 M citric acid pH 7.0 as the electrode buffer. Electrophoresis was performed for about 4 h at 125 V. Gels were sliced horizontally into three layers and stained according to Soltis et aL (1983) for GPI and Vallejos (1983) for DIA. The PGM stain was based on the protocol of Wendel & Weeden (1989) with modified amounts of a-D-glucose-l-phosphate (33 rag/50 ml), glucose-6-phosphate dehydrogenase (16 U/50 ml), MgC12 (208 rag/50 ml) and NADP (2.5 rag/50 ml).
15 Table 1.
Description of the wild Brassica oleracera populations
investigated Pop. number
Collectionsite
Pop. size
ES200 ES209 ES210 FR211 FR212 FR218 FR224 GB225 GB226 GB227 GB228 GB233 GB235 GB236 GB237 GB241
Spain: Asturias: Cabo Penas Spain: Guipozcoa: Guetana Spain: Guipozcoa: San Sebastian France: Charante maritime: Mortange France: Manche: Granville France: Seine Maritime: Fecamp France: Seine Maritime: Mers-les-Baines Great Britain: Kent: Dover Great Britain: Kent: Folkstone Great Britain: Kent: Folkstone Great Britain: Kent: St. Margaret's Bay Great Britain: Devon: Seaton, Beer Great Britain: Devon: Dartmouth Great Britain: Cornwall: West Looe Great Britain: Cornwall: Polruan Great Britain: Wales: Llandudno Great Ormes Head Great Britain: Wales: Llandudno Little Ormes Head Great Britain: Wales: Llandulas
5000 80 75 550 4000 3000 8000 11 000 400 900 9000 10 2000 450 10 000 12 000
GB242 GB243
R A P D analysis
D N A was isolated from leaf tissue according to Edwards et al_ (1991). Primers were purchased from Operon Technologies (Alameda, Ca., USA), RAPD patterns were established for each using 5 primers: A06 ( G G T C C C T G A C ) , A- 18 ( A G G T G A C C G T ) , B-08 ( G T C C A C A C G G ) , B-16 ('FI'TGCCCGGA) and F-06 ( G G G A A T I ' C G G ) . Taq polymerase along with reaction buffer (10 m M Tris pH 9.0, 50 m M KCI, 1.5 mM MgCI2, 0.1% Triton X- 100, 1 mg/ml gelatin) was purchased from H T Biotechnologies (Cambridge, England). PCR reaction mixtures contained 2.5/zl template (approx. 40 ng DNA), 15 ng primer, 100 # M each dATP, dCTP, dGTP, and dTTP, 0.5 U Taq polymerase, 1X supplied buffer and sterile water in a total volume of 20 #1. The samples were prepared in microtiter plates in a sterile bench and each sample was overlaid with 60 #1 mineral oil. Reactions were run in a Hybaid Omnigene thermal cycler. The PCR cycle included a 93 °C denaturation for 3 min, followed by 45 cycles of 93 °C for 40 sec, 36 °C for 1 min and 72 °C for 2 min, finished with a 10 min fill-in polymerization at 72 °C, after which samples were kept on a 25 °C
4500 2500
hold step until the machine could be turned off, Sample electrophoresis was carried out on 1.4% TAE agarose gels at 80 V for approximately 5 h in TAE buffer. Statistical treatment
Individual genotypes were analyzed from the isozyme gels and allele frequencies for each population sample were calculated. Bands in RAPD profiles were treated as independent loci. Gene diversity, H, as described by Nei (1973), was calculated for all populations. The proportion of the total genetic diversity due to variation among populations was computed as G S T , the coeffictent of gene differentiation, also according to Nei (1973). Genetic distance between populations was estimated using Nei's distance, D, (Nei, 1972)_ Genetic distances were displayed as phenograms using the U P G M A (unweighted pair group method, arithmetic average) clustering method of NTSYS-pc (Rohlf, 1993). The analysis of variance of the morphological data was performed using the procedure ANOVA from the statistical package SAS (1990).
16 Table 2_ Size of 44 populationsof wild Brassica oleracea, all estimated in August 1988. N indicates the number of populations from each region
Origin
Population size (number of individuals) l 50 100 500
Spain France Britain Total % Studied pop. Category
4 0 1 5 11 1
4 0 3 7 16 2
1 1 2 4 9 2
small
Results
-
1000
-
0 3 2 5 11 2
5000
-
1 2 6 9 20 5
1000
N
0 4 3 7 16 2
11 14 19 44 99 18
1 4 2 7 16 4
medium
E E
>101300
large
13
Size of petal
Population size 12
H[] D H
Q.
The sizes of the collected populations were estimated as to the total number of individuals and the area occupied. The distribution of populations of various sizes is presented in Table 2. The Spanish populations tend to be small in number, mostly due to small maritime cliff sites. The situation for the French populations is quite the opposite, many of the populations are large and distributed over fairly large distances. In Great Britain, the population sizes range from very small (10 individuals in population GB233) to quite large (up to 12 000 plants in GB241).
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9 []
8 20
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Significance iength P< 0,01 width P< 0,01
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i
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22
24
2'o
2'8
3'o
Length of petal (mm) Morphological variation
Cultivation experiments were conducted with 44 populations of wild B. oleracea. Each population was represented by 60 plants, split into three replicates in a heated greenhouse with 16 h of light. Not all plants sprouted, which means that the generative traits are based on in average 45 plants, but never less than 25. In total, eleven characters were analysed. One of these, the variation (population means) in tepal size, is shown in Fig. 2. Statistical analysis of three other characters are shown in Table 3. The variation can be summarized as follows: there is a considerable variation in all the characters studied; the largest part of the variation exists among populations; there is no correlation between morphological difference and the geographical distance separating the populations - even adjacent populations may differ significantly in many charac-
Fig. 2. Mean values for size of petals (length and width) of 44 wild populations ofB. oleracea. The measurementshave been carried out on plants cultivted in a heated greenhouse with 16 h of light.
ters; small populations (less than 100 plants) may also exhibit a large variation; principal component analysis does not indicate clustering, which means that no regional differentiation is obvious. In other words, a British population is, from a morphological point of view, not more closer related to the other British populations than to the Spanish or French ones. Isozyme variation
The three isozyme systems assayed encoded 4 and a total of 13 alleles. In this material GPI dimer at one locus with 5 alleles. In the material homozygotes were represented (AA, BB, CC, DD,
loci is a five and
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Table 3. Statistical analysis of three generative characters based on comparative cultivation experiments of 44 populations of wild B. oleracea Anova tables Pedicel length Between populations Within populations Sepal length Between populations Within populations Length of filament Between populations Within populations
df
MS
F-value P
