Taxonomy of Portuguese Tronchuda cabbage and Galega kale landraces using morphological characters,nuclear RFLPs,and isozyme analysis: A review

Summary Morphological characters, nuclear RFLPs, and isozyme analysis were used to study the similarity between 32 Portuguese Tronchuda cabbage and Galega kale landraces, and some cabbage cultivars traditionally grown in Portugal. Forty-six morphological characters observed in two consecutive years, RFLP data from 55 nuclear probes, detecting 291 polymorphic nuclear DNA restriction fragments, and allelic frequencies in 21 putative loci, generated by nine isozymes, were analyzed by the unweighted pair group method, using arithmetic averages (UPGMA), in order to present the results in the form of a phenogram. The three methods resulted in different clustering patterns of the 32 cole accessions. Morphological characters gave consistent clustering according to the traditional landrace definition and denomination, producing clear separation between Tronchuda cabbages and Galega kales. RFLPs were unable to separate Tronchuda cabbages from Galega kales and defined five landrace groups corresponding to their geographic origins rather than to their morphological similarities. Isozymes showed poor accession discrimination and an intermediate clustering pattern with some accessions being clustered according to their geographic origins and others according to their morphological similarities. Portuguese Tronchuda cabbages and Galega kales constitute a distinct and relatively homogenous group within Brassica oleracea, sharing the same genetic background. It is concluded that Portuguese coles have evolved independently from a common ancestor to the present cultivated forms. Portugal should be considered as an important region of domestication of specialized leafy coles.     

Numerical taxonomy of Portuguese Tronchuda cabbage and Galega kale landraces using morphological characters

Summary A morphological study was carried out to determine the relationships among Portuguese cole landraces using 58 accessions belonging to: (i) Portuguese Tronchuda cabbage (Brassica oleracea var. tronchuda Bailey syn. var. costata De Candolle, Couve Tronchuda); (ii) Portuguese Galega kale (B. oleracea var. acephala De Candolle, Couve Galega); and (iii) other economically less important coles such as Algarve cabbages (B. oleracea var. capitata L., Couve do Algarve or B. oleracea var. sabauda L., Couve Repolho-lombarda do Algarve). The cole accessions were collected from the growers, studied under field conditions during two consecutive years, and characterized using 46 morphological characters from seedling stage to ripe silique. Morphological data were analysed by numerical taxonomy techniques using UPGMA (Unweighted Pair Group Method Using Arithmetic Averages) and by principal component analysis (PCA). Phenograms based on correlation and distance coefficients showed the existence of 8 main groups (A-H). Five of these groups correspond to Tronchuda cabbage landraces: (A) Couves de Trás-os-Montes; (B) Couves do Minho; (C) Couves from Central Portugal; (D) Couve Portuguesa; (E) Couves from Southern inland Portugal. The three other groups correspond to: (F) cabbages; (G) miscellaneous coles; and (H) Galega kales. Landrace groups are primarily associated with morphological differences among accessions and secondly with accession geographical origin. The interannual character variation did not affect the clustering patterns of the accessions and therefore the stability of landrace classification. PCA was congruent with the landrace groups defined by the phenograms and gave supplementary information on the usefulness of the characters for the definition of the various groups.     

Screening of Portuguese cole landraces (Brassica oleracea L.) with Peronospora parasitica and Plasmodiophora brassicae

Summary Forty-four landraces of Portuguese coles (Brassica oleracea L.), representing different morphotypes, were screened at the cotyledon stage for downy mildew resistance, caused by Peronospora parasitica. Two isolates of the pathogen, PHW 630 and PHW 828, were used in the experiment. The mean disease interaction phenotype varied among the landraces. Overall, the Portuguese coles have shown resistance to both isolates, particularly to isolate PHW 828. Some landraces were identified with differential reaction to the two P. parasitica isolates. Some of the Portuguese coles are, therefore, good sources of resistance genes to P. parasitica, and could be used in breeding programs or in the development of a downy mildew host differential set. Among the morphotypes tested, Couve Algarvia (ISA 207), Couve Galega (ISA 243 and 187) and Couve Glória de Portugal (ISA 84) have presented the highest resistance interaction. For the development of host differential sets, the landraces ISA 2 (Penca de Mirandela), ISA 35 (Couve de Valhascos) and ISA 600 (Penca Espanhola) could be further selected for the differentiation of P. parasitica isolates. They are resistant to PHW 828 and susceptible to PHW 643.The same morphotypes and landraces were also screened at the seedling stage for resistance to clubroot, caused by Plasmodiophora brassicae Woron.. Although some variation for resistance could be noticed in some the landraces tested, mainly Galega kale types, none of them presented a sufficient level of resistance as to be directly be useful in breeding for resistance to race 6 of P. brassicae.     

The exploration of genetic resources of Portuguese cabbage and kale for resistance to several Brassica diseases

Summary Twenty three accessions of nine Portuguese cabbage and kale land races from different geographic origins were tested at the seedling stage for resistance to several important brassica diseases. Resistance to downy mildew (Peronospora parasitica), expressed as necrosis of the cotyledon mesophyll, was found in all the accessions. Type A resistance to cabbage yellows (Fusarium oxysporum f. sp. conglutinans race 1) was present in most of the landraces. Resistance to clubroot (Plasmodiophora brassicae race 6) was found in one accession of the Portuguese tree kale. High resistance to blackleg (Leptosphaeria maculans) and white rust (Albuco candida) was not detected, although several accessions showed 20 to 30% of plants with intermediate expression of resistance. All Portuguese cole accessions were susceptible to blackrot (Xanthomonas campestris pv. campestris).     

Half-sib family selection for yield of digestible organic matter in kale (Brassica oleraceal L.)

Summary Four generations of half-sib family selection for yield of digestible organic matter have been completed, from an initial population which included marrow-stem kales, thousand-head kales, curled kales, Brussels sprouts and cabbages. Relative to the mean yields of two control cultivars the population means were: 106% (gen₀), 122% (gen₁), 128% (gen₂), 111% (gen₃) and 103% (gen₄). These initially encouraging and then disappointing results are discussed along with suggestions for improvements in the population improvement scheme, particularly with respect to the assessment of genotype-environment interactions.     

Evaluation of French Brassica oleracea landraces for resistance to Plasmodiophora brassicae

A total of 240 kale, 38 cabbage and 126 winter cauliflower French landraces from the B. oleracea genepool of INRA were assessed for resistance to clubroot caused by Plasmodiophora brassicaeWoron. Two French isolates of the pathogen (K and SJ) were used in the experiments under controlled conditions. The reaction of the 126 cauliflower accessions to naturally occurring clubroot was also evaluated in field trials. Kales exhibited considerable variation for expression of disease resistance and high levels of resistance were found in several accessions. In this group, single resistant plants were observed in most of the morphological types and from quite different geographical origins. Cabbage accessions were moderately to highly susceptible to both isolates. All cauliflower populations proved to be highly susceptible to K isolate and moderately susceptible to SJ isolate. In field trials, cauliflowers were also severely infected. Two lines selected from a resistant kale population were highly resistant against a large range of pathotypes of the pathogen. These lines presented a sufficient level of resistance to be directly useful in the breeding program in order to develop cauliflower and broccoli hybrids resistant to clubroot. This revised version was published online in July 2006 with corrections to the Cover Date.     

Screening of Portuguese cole landraces (Brassica oleracea L.) with Leptosphaeria maculans and Xanthomonas campestris pv. campestris

Summary Fifty-six landraces of Portuguese coles (Brassica oleracea l.), representing fourteen morphotypes, were screened for resistance to Leptosphaeria maculans at the cotyledon stage. Three isolates of the pathogen belonging to three pathogenicity groups were used in the screening. The mean disease interaction phenotype of the B. oleracea landraces with the L. maculans isolates confirmed their differences in pathogenicity. When compared to pathogenicity grouping using a B. napus differential set, a different classification was observed on B. oleracea for the three L. maculans isolates, suggesting possible differences in the genetic interaction between the two plant species and the pathogen. Most of the Portuguese cole landraces were found to be susceptible to all three isolates of L. maculans, although a few have shown intermediate interaction phenotype. Some accessions, on the other hand, elicit a differential reaction with the three isolates tested, and can be further selected to be used in host differential sets or in breeding for resistance to black leg.Fifty-four landraces of Portuguese coles, representing eleven morphotypes, were also screened in the field for resistance to Xanthomonas campestris pv. campestris. A good level of resistance to the bacterium was found in several of the morphotypes.Accessions of the Penca type, particularly, were identified as highly resistant. Landraces belonging to the same Portuguese cole group presented a similar range of mean interaction phenotype with the bacterium. Some Portuguese landraces of B. oleracea are potential sources for use in breeding programs for black rot resistance.     

Association of RFLP markers with trait loci affecting clubroot resistance and morphological characters in Brassica oleracea L.

Summary Resistance to Plasmodiophora brassicae Wor. race 7, the causal agent of the disease clubroot, was examined in an F₂ population of a cross between a clubroot resistant broccoli (Brassica oleracea var. italica) and a susceptible cauliflower (B. oleracea var. botrytis). A genetic linkage map was constructed in the same population based on the segregation of 58 dispersed restriction fragment length polymorphism (RFLP) markers. Associations between the inheritance of RFLP marker genotypes and segregation for disease resistance, morphological and maturity characteristics were examined. For each triat examined, several chromosomal regions marked by RFLP probes appeared to contain trait loci, suggesting that each trait was under polygenic control. RFLP marker linkage to a major factor imparting dominance for clubroot resistance from the broccoli parent was observed in this population. Additionally, RFLP marker linkage to an independently segregating factor contributing clubroot resistance from the cauliflower parent was observed, indicating that it should be possible to use RFLP markers to facilitate selection of transgressive segregants having the combined resistance from both parental sources. In some instances, RFLP markers from the same or closely linked chromosomal regions were associated with both clubroot resistance and morphological traits. Analysis of RFLP marker genotypes at linked loci should facilitate the selection of desired disease resistant morphotypes.     

Genetic analysis of resistance to clubroot (Plasmodiophora brassicae Woron) in two Brassica oleracea groups (ssp. acephala and ssp. botrytis) through diallel analysis

Clubroot, caused by Plasmodiophora brassicae Woron, is a major disease of cruciferous crops and few sources of resistance have been detected and genetically studied in the Brassica oleracea species. In this study, two diallel mating systems using resistant kale lines and susceptible cauliflower lines were performed. Resistance was assessed from a disease index evaluated on young plants artificially inoculated with local isolates of the parasite. Both general and specific combining ability effects (GCA and SCA) and partly reciprocal effects were demonstrated. Resistance inheritance was, however, characterized by a predominance of additive genetic effects (GCA). Three groups of parent lines could be separated; kale lines with very high resistance, kale lines with intermediate resistance and susceptible cauliflower lines. In the two kale groups, two genetically different resistance types were suggested and various recurrent selection procedures are proposed following the diallel results.     

Productivity of F hybrids of botanical varieties ofBrassica oleracea L.

Summary F₁ hybrids were obtained by making pair-crosses between nine botanical varieties ofBrassica oleracea. Although non-inbred parents were used, their yield was surpassed by all F₁ hybrids. The highest yields were shown by the crosses marrow-stem kale × sprouting broccoli and marrow-stem kale × cauliflower while the highest consumable yield (assessed by feeding plants to sheep) was that of F₁ hybrids marrow-stem kale × cauliflower and wild cabbage × cauliflower. A scheme for the production of F₁ hybrid seed is suggested, based upon an adaptation ofThompson's (1964) Triple-cross technique.     

Random amplified polymorphic DNA (RAPD) variation in Fagopyrum tataricum Gaertn. accessions from China and the Himalayan region

The diversity among 52 landraces and cultivars of tartary buckwheat (Fagopyrum tataricum Gaertn.) and one accession of its wild ancestor, F. tataricum ssp. potanini Batalin, from diverse geographic origins was examined using random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) markers. Eighteen primers produced a total of 240 fragments, of which 153 (63.75%) were monomorphic and 87 (36.25%) polymorphic bands. UPGMA-based pairwise Jaccard’s coefficient of similarity was used to deduce the relationships among 53 genetically diverse accessions. The similarity between cultivated tartary buckwheat accessions ranged from 0.61 to 1.00. Four distinct clusters were formed which corresponded well with the geographic distribution of the tartary buckwheat. Nepalese accessions showed maximum diversity followed by Chinese accessions. Tartary buckwheat accessions from the Himalayan region of northwestern India revealed a narrow gene pool. The wild buckwheat accession did not group with any of the three cultivated tartary buckwheat groups, and formed its own single-entry group. Genetic similarity (0.59) of Chinese buckwheat accessions with the wild ancestor reaffirmed that cultivated tartary buckwheat originated in the Yunnan province of northwestern China. Consistent with some earlier reports, our study demonstrated the usefulness of the RAPD technique for the characterization of plant genetic resources and assessment of diversity between species. This revised version was published online in August 2006 with corrections to the Cover Date.     

Evaluation of a core collection of Brassica oleracea accessions for resistance to Brevicoryne brassicae, the cabbage aphid

A rapid, simple and reliable technique was developed for evaluating Brassica accessions in the glasshouse for resistance to Brevicoryne brassicae, the cabbage aphid. Brassica accessions were grouped into crop types with similar habit and vigour for evaluation. 401 Brassica oleracea accessions, representing the genetic and geographic diversity of the species, were evaluated for resistance to a population of B. brassicae collected originally from HRI, Wellesbourne. Ninety eight accessions were identified as being moderately resistant (grade 3) or partially resistant (grade 4). Re-testing of accessions in the glasshouse identified 12 moderately resistant and 43 partially resistant accessions. The most promising gene pool was kale, where a higher than expected number of resistant accessions were found. Three of the kales were consistently rated as moderately resistant in all tests. In contrast the broccoli gene pool was a poor source of resistance with less (one out of 46) resistant accessions than expected. Eleven of the kales graded 3 were tested in the field in 1995 and resistance to B. brassicae was confirmed. Ten accessions were also tested for resistance to two additional populations of B. brassicae, one from Greece and the other from Portugal. The results provided no evidence of aphid biotype/host specific differences in the resistance of these B. oleracea accessions. This revised version was published online in August 2006 with corrections to the Cover Date.     

The exploitation of genetic resources of Brassica oleracea in breeding for resistance to clubroot (Plasmodiophora brassicae)

Summary About 1000 Brassica oleracea accessions were evaluated in glasshouse tests for response to Plasmodiophora brassicae (clubroot). Resistance was confirmed in some north and west European kales and cabbage. A new source of resistance in cabbage, from Eire, is reported. Most other accessions were highly susceptible but lower levels of susceptibility were observed in open pollinated Brussels sprouts and forms of south European cabbage, cauliflower and broccoli. Modern breeding (as in the production of hybrid cultivars) appears to have resulted in increased susceptibility in several crop types. The implications of these results for the exploitation of germplasm are discussed.     

RFLP mapping of an aphid resistance gene in cowpea (Vigna unguiculata L. Walp)

Summary Restriction fragment length polymorphism (RFLP) analysis has several advantages over traditional methods of genetic linkage mapping, one of these being the starting point for map-based cloning. The recent development of an RFLP map of cowpea (Vigna unguiculata L. Walp) has allowed the investigation of associations between genes of interest and RFLP markers. A cross between an aphid (Aphis craccivora Koch) resistant cultivated cowpea, TT84S-2246-4, and an aphid susceptible wild cowpea, NI 963, was screened for both aphid phenotype and RFLP marker segregation. One RFLP marker, bg4D9b, was found to be tightly linked to the aphid resistance gene (Rac ₁) and several flanking markers in the same linkage group (linkage group 1) were also identified. The close association of Rac ₁ and RFLP bg4D9b presents a real potential for cloning this insect resistance gene.     

Genetic relationships of Brassica vegetables determined using database derived simple sequence repeats

Sequence databases were screened to identify simple sequence repeats (SSRs) in Brassica oleracea sequences. A total of 512 B. oleracea DNA sequences were screened and 43 potential SSRs were identified. Thirty-six primer pairs were designed to amplify target sequences. Of the 36 primer pairs, six failed to amplify fragments of expected sizes, and 17 primer pairs failed to generate polymorphisms. Thirteen SSRs were used to assess genetic similarity between 54 B. oleracea cultivars, belonging to 3 variteal groups (cabbage, cauliflower, and broccoli). Pairwise genetic similarities were calculated for cultivars, and a dendrogram of relationships was produced. All cabbage cultivars were distinguished from each other and clustered in two separate groups. Five cauliflower cultivars could not be distinguished with SSR markers used in the study. Three broccoli cultivars clustered with cauliflower cultivars, and two cauliflower cultivars grouped with broccoli cultivars. The varietal group with the narrowest genetic variation in the study was cauliflower (B. oleracea var. botrytis) followed by broccoli (B. oleracea var. italica) and cabbage (B. oleracea var. capitata) groups. Polymorphism information content (PIC) values and number of alleles produced per marker ranged between 0.25 to 0.86 and 1 to 8, respectively, for database derived SSR markers.     

Cluster analysis of RFLP data from related maize inbred lines of the BSSS and LSC heterotic groups and comparison with pedigree data

Summary In this study, 31 elite inbred lines of maize (Zea mays L.) were analyzed with 149 clone-enzyme combinations for their respective RFLP profiles. Objectives were (1) to determine the utility of RFLPs for estimation of genetic similarties among 16 inbred lines from the Iowa Stiff Stalk Synthetic (BSSS) and among 15 inbred lines from the Lancaster Sure Crop (LSC) heterotic groups and (2) to compare genetic similarities based on molecular markers with those based on pedigree information. Coefficients of genetic similarity (GS) and coancestry (f) between pairs of lines from the same heterotic group were calculated from RFLP and pedigree data, respectively. For lines from the BSSS heterotic group, cluster analyses based on RFLP and pedigree data revealed similar associations. GS and f values were closely correlated (r=0.70) for related BSSS lines. For lines from the LSC heterotic group, considerable discrepancies existed between the GS and f values, especially for those pairs involving inbreds Va22 and Lo924. Effect of selection and/or erroneous pedigree records are discussed as possible explanations for the low correlation of GS and f values (r=0.07) for related LSC lines. RFLPs seem useful for investigation of relationships among maize inbreds, verification of pedigree records, and quantification of the degree of relatedness.     

Organic varieties for cauliflowers and cabbages in Brittany: from genetic resources to participatory plant breeding

Plant breeding for organic agriculture (OA) was stimulated when it came under the European Organic Agriculture Regulation (2092/91) in 2004. In Brittany, the need for specific varieties for organic farming arose early for the Brassica species because of the unsuitability of most of the modern varieties to the principles of OA. Moreover, the private sector of plant breeding finds it economically difficult to satisfy the demands of OA. The aim of the present study is to provide varieties and seed for organic farmers for two vegetable Brassica crops, and to show how genetic resources can contribute to this purpose in the framework of a Participatory Plant Breeding (PPB) programme. The emergence of PPB in Brittany is the result of several concomitant and favourable circumstances: the will of the professionals (represented by IBB, Inter Bio Bretagne), their organization (an experimental station, the PAIS, Plateforme Agrobiologique d’IBB à Suscinio), the research initiative in INRA and the availability of genetic resources. From genetic resource observations, our experience showed several breeding situations: reviving a traditional activity (Roscoff cauliflower and local cabbages), extending tradition (autumn cauliflower), diversifying production by new introductions (coloured cauliflowers), and creating new forms of population varieties (broccoli and coloured cauliflowers). Farmers have taken charge of population breeding by mass selection and the PAIS, with INRA scientific support, has taken up innovative selection and the improvement of varieties completing the farmers’ initiatives. The PAIS remained the central point for information and for providing the seed for trials. Seed production will be managed in a collective way. Until now, the exchange of seed remained an experimental dimension of PPB. French seed legislation represents a limitation on the development of seed exchange by PPB.     

A comparative analysis of the genetic relationships between rye cultivars using RFLP and RAPD markers

Summary The genetic similarities of eight closely related rye cultivars were estimated using two molecular marking techniques: restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD). Cultivars were evaluated for variation by 11 random cDNA and genomic clones used in combination with four restriction enzymes and 40 decamer primers. A total of 53 polymorphic RFLP fragments and 94 polymorphic RAPD fragments were observed. Based on the presence/absence of fragments, two genetic similarity matrices were calculated which were then used in cluster analysis. Differences between pair of cultivars were observed in RFLP and RAPD dendrograms. RFLP analysis produced estimates of genetic relationships more in accordance with the partially known pedigree of the cultivars than did RAPD analysis. The use of bulk samples of DNA in these analyses affected the sensitivity of RAPD assays more strongly. Dendrograms which took into account all fragments produced, either by RFLP or RAPD, reflected better the relationships between cultivars than did dendrograms based on only one type of marker. This reflects the importance of the number of markers used in determining the genetic relationships between genotypes.     

A strict diploid-like pairing regime is associated with tetrasomic segregation in induced autotetraploids of kale

Fertility and reproductive stability in polyploids depend on the precise control of chromosome pairing at meiosis. In this study, meiotic pairing configurations are related to isozyme segregation ratios in F₁ hybrids between colchicine‐induced autotetraploids of kale. An immediate diploid‐like pairing regime is associated with tetrasomic segregation. Although the mechanisms driving such a behaviour have yet to be identified, pairing preferences are not responsible for the cytological diploidization of autotetraploid kales.     

RFLP variation and genetic relationships in cultivated cucumber

Summary Two sets of cucumber (Cucumis sativus L.) germplasm were used to determine the potential use of restriction fragment length polymorphisms (RFLPs) for estimating genetic relationships. Sixteen accessions [15 domesticated variety sativus and one feral variety hardwickii (PI 183967)] of diverse origin were used to assess RFLP variation in cucumber, and to determine if genetic relationships based on RFLPs were similar to those obtained by isozyme analysis. Additionally, 35 commercial lines or cultivars were surveyed to determine genetic relationships among and within common cucumber types (narrow genetic base). The 16 accessions were surveyed with 440 low copy clones from two libraries (Pst I partial genomic and cDNA) using two restriction enzymes. Data from a subset of 104 random (mapped and unmapped) and a set of 30 mapped RFLPs were used to estimate genetic relationships among the 16 cultigens. Variability was low among RFLPs (33% of all probes) and putative alleles ( 2.2 polymorphic fragments/probe). RFLP variation between sativus lines and hardwickii (21±4%) was greater than among sativus lines (12±2%). RFLPs among the 16 accessions revealed genetic relationships which agree with those obtained using isozymes. Genetic relationships estimated using mapped and unmapped RFLPs were similar. The 35 elite lines were surveyed using a set of 40 RFLPs from 3 libraries (Pst I and EcoR I partial genomic and cDNA) to evaluate the discriminatory value of RFLPs among and between commercial cucumber types. The RFLP-derived genetic relationships among this germplasm were in agreement with predictions based on fruit type and pedigree information. Thus, RFLPs are a useful addition to the morphological characters and isozyme loci currently used for taxonomic classification and plant variety protection of cucumber.