Intergeneric hybrids between Enarthrocarpus lyratus,a wild species,and crop brassicas

Summary Attempts were made to produce intergeneric hybrids between Enarthrocarpus lyratus, a wild species, and several species of crop brassicas: B. campestris, B. nigra, B. oleracea, B. juncea, B. napus and B. Carinata. Hybrids using E. lyratus as female parent were realized by means of embryo rescue in four combinations — E. lyratus x B. campestris, E. lyratus x B. oleracea, E. lyratus x B. napus and E. lyratus x B. carinata. Reciprocal crosses showed strong pre-fertilization barriers and yielded no hybrids except in one combination — B. Juncea x E. Lyratus — in which a single hybrid could be realized. All of the hybrids were multiplied in vitro through the multiplication of axillary shoots. Morphological and cytological studies confirmed hybridity. All hybrids were completely pollen sterile except for E. lyratus x B. carinata, which showed 2% pollen fertility. Attempts to double the chromosome number through the in vitro application of colchicine to axillary meristems of F₁ hybrids were successful in only one hybrid, E. lyratus x B. oleracea. Cytological studies of the hybrids indicated the presence of a partial homology between the genomes of E. lyratus and crop brassicas. Backcross progenies were raised from all of the five F₁ hybrids to develop malesterile alloplasmic lines.     

Identification of the different Brassica nigra chromosomes from both sets of B. oleracea-B. nigra and B. napus-B. nigra addition lines with a special emphasis on chromosome transmission and self-incompatibility

 The F₁ hybrids produced after crosses between B. gra and B. oleracea were backcrossed two or three times to B. oleracea. Among the 14 plants analysed, five were monosomic addition lines (2n=19), six were double monosomic addition lines (2n=20) and three had three or four additional chromosomes. From these lines, 14 isozyme and 80 RAPD loci were localized on the eight chromosomes of B. nigra. The comparison between B. napus-B. nigra, from which five B. nigra chromosomes were already described, and the new set of B. oleracea-B. nigra addition lines was performed using five isozyme and 22 common RAPD loci. The homology of the common RAPD loci was confirmed by hybridization of the two sets of addition lines as well as the presence of duplicated loci on different chromosomes. For the five added chromosomes available on the two genetic backgrounds, i.e. B. napus and B. oleracea, using isozyme markers, the chromosome transmission rate was studied from backcross progeny using the recurrent parent either as male or as female and from the selfing of monosomic addition lines. For each chromosome, no difference was detected between male and female transmission except for chromosome 3. This latter presented a percentage of female transmission of around 20%, close to the ones observed for the other chromosomes, but a very low male transmission (1.3%). The analysis from restriction enzyme digests of PCR products, obtained from primers selected in highly conserved regions of self-incompatible genes, suggested that the chromosome 3 probably carried the SLG-B. nigra locus. Received: 25 September 1996 / Accepted: 18 October 1996     

Somatic hybrids with substitution type genomic configuration TCBB for the transfer of nuclear and organelle genes from Brassica tournefortii TT to allotetraploid oilseed crop B. carinata BBCC

Oilseed crop Brassica carinata BBCC is a natural allotetraploid of diploid species B. nigra BB and B. oleracea CC. To transfer the nuclear and organelle genes in a concerted manner from an alien species, B. tournefortii TT, to B. carinata, we produced somatic hybrids with genomic configuration TCBB using B. nigra and B. oleracea stocks that carried selectable marker genes. B. tournefortii TT was sexually crossed with hygromycin-resistant B. oleracea CC. Protoplasts isolated from shoot cultures of hygromycin-resistant F₁ hybrids of B. tournefortiixB. oleracea TC were fused with protoplasts of kanamycin-resistant B. nigra BB. In two different fusion experiments 80 colonies were obtained through selection on media containing both hygromycin and kanamycin. Of these, 39 colonies regenerated into plants. Analysis of 15 regenerants by random amplified polymorphic DNA (RAPD) markers showed the presence of all three genomes, thereby confirming these to be true hybrids. Restriction fragment length polymorphism (RFLP) analysis of organelle genomes with heterologous chloroplast (cp)and mitochondrial (mt) DNA probes showed that the chloroplast genome was inherited from either of the two parents while mitochondrial genomes predominantly showed novel configurations due to either rearrangements or intergenomic recombinations. We anticipate that the TCBB genomic configuration will provide a more conducive situation for recombination between the T and C genomes during meiosis than the TTCCBB or TCCBB type configurations that are usually produced for alien gene transfer. The agronomic aim of producing TCBB hybrids is to transfer mitochondrial genes conferring cytoplasmic male sterility and nuclear genes for fertility restoration from B. tournefortii to B. carinata.     

Regeneration of plants from protoplasts of Capsella bursa-pastoris and somatic hybridization with rapid cycling Brassica oleracea

Fertile rooted plantlets were recovered from leaf mesophyll protoplasts of Capsella bursa-pastoris. Protoplasts cultured over a feeder layer of Brassica napus cells produced 221 colonies, 7 of which regenerated multiple plantlets. The nuclear DNA content of most regenerates varied from 0.89 to 1.0 pg/nucleus, close to the value for seed-grown C. bursa-pastoris (0.94±0.03 pg/nucleus). Two regenerants had a tetraploid DNA content (1.8– 2.0 pg). Plants with a DNA content close to Capsella produced seeds, both in vitro and in soil. Intertribal somatic hybrids were obtained by polyethylene glycol-mediated fusion of untreated C. bursa-pastoris protoplasts with iodoacetate-treated protoplasts of rapid-cycling B. oleracea. Plants were confirmed as somatic hybrids by isozyme and RAPD analysis. The nuclear DNA content of the hybrids ranged from 3.2 to 6.4 pg, higher than the sum of the parental genomes. One of two hybrids tested was resistant to Alternaria brassicicola, like the Capsella fusion partner. Hybrids rooted easily and produced sterile flowers when transplanted to soil. Received: 13 April 1998 / Revision received: 25 August 1998 / Accepted: 31 August 1998     

Production and characterization of interspecific somatic hybrids between Brassica oleracea var. botrytis and B. nigra and their progenies for the selection of advanced pre-breeding materials

Somatic hybridization is a potential method for gene transfer from wild relatives to cultivated crops that can overcome sexual incompatibilities of two distantly related species. In this study, interspecific asymmetric somatic hybrids of Brassica oleracea var. botrytis (cauliflower) and Brassica nigra (black mustard) were obtained by protoplast fusion and their backcrossed (BC₃) and selfed (S₃) offspring were analyzed. Cytological analysis showed that the B. nigra chromosomes were successively eliminated in the backcrosses with cauliflower. The fertility of the hybrid progenies was quite different due to the asynchronous and abnormal chromosome behavior of pollen mother cells (PMC) during meiosis. Analysis of sequence-related amplified polymorphism (SRAP) showed that all of these hybrids mainly had the DNA banding pattern from the two parents with some alterations. Genetically, the selfed generations were closer to B. nigra, while the backcrossed generations were closer to the cauliflower parent. Analysis of cleaved amplified polymorphic sequences (CAPS) and restriction fragment length polymorphisms (RFLP) showed that all somatic hybrids in this study contained chloroplast (cp) DNA of the donor parent black mustard, while mitochondrial (mt) DNA showed evidence of recombination and variations in the regions analyzed. Furthermore, three BC₃ plants (originated from somatic hybrids 3, 4, 10) with 2–8 B. nigra-derived chromosomes shown by genomic in situ hybridization (GISH) displayed a more cauliflower-like morphology and high resistance to black-rot. These plants were obtained as bridge materials for further analysis and breeding.     

Camalexin induction in intertribal somatic hybrids between Camelina sativa and rapid-cycling Brassica oleracea

  Camelina sativa, a wild relative of Brassica crops, is virtually immune to blackspot disease caused by Alternaria brassicicola. Intertribal somatic hybrids were produced between C. sativa and rapid-cycling Brassica oleracea as a step toward the transfer of resistance to this disease into Brassica vegetable crops. The plants recovered were confirmed as somatic hybrids by flow cytometry and RAPD analysis. All hybrids showed a morphology intermediate between the two parents. Rooted plants grew in soil up to 4–5 weeks, and some produced sterile flowers. Two of three hybrids tested showed a high level of resistance to  A. brassicicola. Resistance was correlated with the induction of high levels of the phytoalexin camalexin 48 h after inoculation, as in the resistant Camelina fusion partner. In contrast, susceptible somatic hybrids produced much lower levels of camalexin. Received: 2 April 1998 / Accepted: 14 July 1998     

Production and cytogenetics of intergeneric hybrids between the three cultivated Brassica diploids and Orychophragmusviolaceus

It has been proposed that both complete and partial separation of the parental genomes during mitosis and meiosis occurs in the intergeneric hybrids between Orychophragmus violaceus (2n=24) and the three cultivated Brassica tetraploids (B. napus, B. carinata and B. juncea). The hypothesis has been that this and the variations in chromosome numbers of these hybrids and their progenies result from the different roles of the A, B and C genomes originating from Brassica. To test this hypothesis, we produced hybrids between O. violaceus and the cultivated Brassica diploids. The hybrids with B. oleracea (2n=18, CC) had an intermediate morphology, but their petals were purple like those of O. violaceus. They were sterile and had the expected chromosome number (2n=21) in their mitotic and meiotic cells. The hybrid with B. campestris (2n=20, AA) was morphologically intermediate, except for its partial fertility and its yellow petals, which were similar to those of B. campestris. It was mixoploid (2n=23–42), and cells with 2n=34 were most frequent. Partial separation of parental genomes during mitosis, leading to the addition of O. violaceus chromosomes to the B. campestris complement, was proposed to explain the findings in the mitotic and meiotic cells of the hybrid and its progeny. In crosses with B. nigra (2n=16, BB), the majority of the F₁ plants were of the maternal type (2n=16), a small fraction had B. nigra morphology but were mixoploids (2n=16–18), predominantly with 2n=16 cells and three plants, each with a specific morphology, were mixoploids consisting of cells with varying ranges of chromosome numbers (2n=17–26, 11–17 and 14–17). The origin of these different types of plants was inferred to be a result of the complete and partial separation of parental genomes and the loss of O. violaceus chromosomes. Our findings in the three crosses suggest that the A genome was more influential than the C genome with respect to complete genome separation during mitosis and meiosis of the hybrids with B. napus. Possible complete and partial genome separation during mitotic divisions of the hybrids with B. carinata was mainly attributed to the role of the B genome. The combined roles of the A and B genomes would thus contribute to the most variable chromosome numbers of mitotic and meiotic cells in the hybrids with B. juncea and their progenies. The possible cytological mechanisms pertaining to these hybrids and the potential of genome separation in the production of Brassica aneuploids and homozygous plants are discussed. Received: 8 February 1998 / Accepted: 12 March 1999     

Incorporation of hygromycin resistance in Brassica nigra and its transfer to B. napus through asymmetric protoplast fusion

Summary With the idea to develop a selection system for asymmetric somatic hybrids between oilseed rape (Brassica napus) and black mustard (B. nigra), the marker gene hygromycin resistance was introduced in this last species by protoplast transformation with the disarmed Agrobacterium tumefaciens strain C58 pGV 3850 HPT. The B. nigra lines used for transformation had been previously selected for resistance to two important rape pathogens (Phoma lingam, Plasmodiophora brassicae). Asymmetric somatic hybrids were obtained through fusion of X-ray irradiated (mitotically inactivated) B. nigra protoplasts from transformed lines as donor with intact protoplasts of B. napus, using the hygromycin resistance as selection marker for fusion products. The somatic hybrids hitherto obtained expressed both hygromycin phosphotransferase and nopaline synthase genes. Previous experience with other plant species had demonstrated that besides the T-DNA, other genes of the donor genome can be co-transferred. In this way, the produced hybrids constitute a valuable material for studying the possibility to transfer agronomically relevant characters — in our case, diseases resistances — through asymmetric protoplast fusion.     

Analysis of chloroplast and mitochondrial segregation in three different combinations of somatic hybrids produced within Brassicaceae

Summary Mitochondrial and chloroplast DNA were characterized in three different combinations of somatic hybrids produced between different species within Brassicaceae. The fusions were made between B. campestris and B. oleracea, B. napus and B. nigra and between B. napus and Eruca sativa. The combinations represent interspecific hybridizations, but the phylogenetic distance between the species used in each instance is different. Whereas the B. campestris (+) B. oleracea and the B. napus (+)B. nigra hybrids are both examples of intrageneric hybrids, B. campestris is more closely related to B. oleracea than B. napus is to B. nigra. The fusion of B. napus and E. sativa represents an intergeneric hybridization. Since hybrids were produced with reproducible and uniform fusion and culture methods, a comparison of chloroplast and mitochondrial segregation and mitochondrial DNA (mt-DNA) rearrangements could be made between the combinations. The segregation of both chloroplasts and mitochondria was biased in the B. napus (+)B. nigra and the B. napus (+)E. sativa combination. The nonrandom segregation of chloroplasts and mitochondria could be due to the different ploidy levels of the fusion partners and/or reflect differences in organelle replication rate. Furthermore, segregation of mitochondria was correlated to the differences in phylogenetic distance between the species used in the fusions. However, mitochondrial segregation, in contrast to chloroplast segregation, could in all combinations also have been affected by the cell type used as protoplast source in the fusions. All different chloroplast types could be established within each combination. Hybrids containing chloroplast from one parent together with mitochondria from the other parent were found in two of the combinations, although the majority of the hybrids had mt-DNA that was altered compared to the parental species. The rearranged mt-DNA found in most hybrids was an effect of the heteroplasmic state following protoplast fusion rather than of the tissue culture methods, since no mt-DNA rearrangements were found in B. napus plants regenerated from protoplast culture. The mtDNA restriction patterns of the hybrids with rearranged mt-DNA indicated that specific regions of the mt-DNA were involved in the rearrangements following protoplast fusion.     

Synthesis of Brassica carinata from Brassica nigra x Brassica oleracea hybrids obtained by ovary culture

Summary Brassica carinata was synthesized by hybridization between its diploid progenitor species B. nigra and B. oleracea followed by chromosome doubling. Up to 40 times more hybrids were obtained, using ovary culture than with conventional hand pollination. Two hybrids from the cross B. nigra x B. oleracea var alboglabra were raised to maturity. High chromosome pairing was observed in both the hybrids and the amphidiploid. A range of variability for desirable characters is reported in the synthesized amphidiploids.     

Accessing and exploiting genes of breeding value of distant relatives of crop Brassicas

The Brassicas are an important group of crops in India yielding edible oils and many vegetables. For improving cultivated Brassicas, the wild relatives are of considerable value. The Brassica group of seed oil and vegetables comprises six cultivated species, out of which three are diploids and three are digenomic tetraploids. Brassica juncea is the major seed oil crop in India which can be improved for several traits by incorporating genes from its distant relatives. The early work in India relating to genome manipulation consisted of synthesis of B. juncea by crossing B. campestris with B. nigra, experimental resynthesis of Brassica species and non-homologous pairing and genetic exchange at the interspecific level. The alloploid species B. napus and B. carinata have not been successful in India due to agrometereological limitations. However, synthetic forms of B. napus have been produced which have a desirable maturity period with good yield potential. Also, through non-homologous pairing, pod shatter resistant B. napus has been obtained, B. napus ordinarily suffers from pod shattering. Similarly, synthetic forms of B. carinata have been derived from reciprocal crosses between morphotypes of B. oleracea and B. nigra and also through protoplast fusion of B. nigra with B. oleracea. Molecular analysis has revealed that one of the somatic hybrids had a novel cytoplasmic combination which carried B. nigra mitochondrial and B. oleracea chloroplast genomes. A range of wild and weedy species related to crop Brassicas possess extensive genetic variability. Work for utilizing this variability included hybridization between wild and crop species, analysis of chromosome pairing and induction of alloploidy. Among Brassicas of interest to India, protoplast culture and regeneration has been successful in the case of B. oleracea, B. juncea, B. nigra and B. carinata (cultivated species) and Eruca sativa and Diplotaxis muralis (related wild species). Polyethylene glycol mediated protoplast fusion has been the most commonly used method in India for producing somatic hybrids involving Brassicas. The eight somatic hybrids produced and studied showed that in the majority of cases the fusions led to symmetric hybrids combining the complete genomes of the donor species. For developing suitable male sterile lines, B. juncea, B. campestris and B. napus nuclei have been combined with the cytoplasm of six wild species and stable male steriles have been developed. Protoplast fusion methodology has been used extensively for improving these CMS by manipulating cytoplasmic organelles, including production of new combinations of cp and mt.     

Production of hybrids, amphiploids and backcross progenies between a cold-tolerant wild species, Erucastrum abyssinicum and crop brassicas

Three intergeneric hybrids were produced between a cold-tolerant wild species, Erucastrum abyssinicum and three cultivated species of Brassica, B. juncea, B. carinata and B. oleracea, through ovary culture. The hybrids were characterized by morphology, cytology and DNA analysis. Amphiploidy was induced in all the F₁ hybrids through colchicine treatment. Stable amphiploids and backcross progenies were obtained from two of the crosses, E. abyssinicum x B. juncea and E. abyssinicum x B. carinata. The amphiploid, E. abyssinicum x B. juncea was successfully used as a bridge species to produce hybrids with B. napus, B. campestris and B. nigra. These hybrids and backcross progenies provide useful genetic variability for the improvement of crop brassicas.     

An alginate-layer technique for culture of Brassica oleracea L. protoplasts

Ten accessions belonging to the Brassica oleracea subspecies alba and rubra, and to B. oleracea var. sabauda were used in this study. Protoplasts were isolated from leaves and hypocotyls of in vitro grown plants. The influence of selected factors on the yield, viability, and mitotic activity of protoplasts immobilized in calcium alginate layers was investigated. The efficiency of protoplast isolation from hypocotyls was lower (0.7 ± 0.1 × 10⁶ ml⁻¹) than for protoplasts isolated from leaf mesophyll tissue (2 ± 0.1 × 10⁶ ml⁻¹). High (70–90%) viabilities of immobilized protoplasts were recorded, independent of the explant sources. The highest proportion of protoplasts undergoing divisions was noted for cv. Reball F1, both from mesophyll (29.8 ± 2.2%) and hypocotyl (17.5 ± 0.3%) tissues. Developed colonies of callus tissue were subjected to regeneration and as a result plants from six accessions were obtained.     

An Agrobacterium tumefaciens transformation protocol effective on a variety of cottonwood hybrids (genus Populus)

 We describe a protocol for Agrobacterium tumefaciens-mediated transformation of hybrid cottonwoods (Populus sections Tacamahaca Spach. and Aigeiros Duby). The protocol has allowed routine transformation of several economically important cottonwood hybrids (Populus trichocarpa Torr. & Gray×P. deltoides Bartr. ex. Marsh. and P. deltoides×P. nigra L.) that were previously difficult to transform. The procedure was applied to 11 different hybrid cottonwood genotypes and one P. deltoides genotype using kanamycin as the selection agent. Additional experiments showed a very strong interaction between auxin preculture and the effectiveness of various cytokinins for induction of shoot organogenesis. The data also demonstrated the superiority of Agrobacterium strain EHA105 over C58 and LBA4404 for T-DNA transfer based on transient assays with a reporter gene. Received: 16 June 1998 / Revision received: 5 February 1999 / Accepted: 14 April 1999     

Genomic regions involved in productivity of two interspecific poplar families in Europe. 1. Stem height, circumference and volume

Interspecific hybrids of Populus species are known for their superior growth. In this study, we examined the effect of the genetic background and contrasting environmental conditions on growth and searched for quantitative trait loci (QTL) affecting growth traits. To this end, two hybrid poplar families resulting from controlled crosses, Populus deltoides ‘S9-2’ × P. nigra ‘Ghoy’ (D × N, 180 F₁) and P. deltoides ‘S9-2’ × P. trichocarpa ‘V24’ (D × T, 182 F₁), were grown at two contrasting sites, Northern Italy and Central France. At the end of the second growing season, tree dimensions (stem height, circumference, and volume) were assessed. The performances of both families significantly differed within and between sites. Tree volume was significantly larger at the Italian site as compared to the French site. Genotype by environment interactions were significant but low for both families and for all growth traits. Tight correlations among the individual growth traits indicated that there may be a common genetic mechanism with pleiotropic effects on these growth traits. In line with previous studies, linkage groups I, VII, IX, X, XVI, XVII, and XIX appeared to have genomic regions with the largest effects on growth traits. This study revealed that (1) both families have high potential for selection of superior poplar hybrids due to the pronounced heterosis (hybrid vigor) and the large genetic variability in terms of growth and (2) the choice of site is crucial for poplar cultivation. Dillen and Storme contributed equally to the work. An erratum to this article can be found at     

Genetic distance as a predictor of heterosis and hybrid performance within and between heterotic groups in sunflower

Heterosis is significant for seed yield and is one of the driving forces behind the hybrid seed industry in cultivated sunflower (Helianthus annuus L). Heterotic groups in sunflower, if any other than the female and male inbred-line groups exist, have not been well studied or described. The primary aims of this study were to assess the utility and validity of a series of proposed heterotic groups and estimate correlations between genetic distance, heterosis, and hybrid performance for seed yield in sunflower. Fortytwo female by male heterotic group (A × R) and 81 female by female heterotic group (A × B) single-cross hybrids were grown in Corvallis, Ore., and Casselton, N.D., in 1996 and 1997. Heterosis was significant for seed yield and plant height but not for seed oil concentration and days to flowering. Genetic distances were significantly correlated with hybrid seed yield when estimated from AFLP fingerprints (G _D) (r = 0.63 for A × R and 0.79 for A × B hybrids), but not from coancestries (G _C) (r = -0.02 for A × R and 0.54 for A × B hybrids). G _D (R ² = 0.4) was a poor predictor of hybrid seed yield. The proposed heterotic groups in sunflower seem to have utility, but do not seem to be as strongly differentiated as those in corn (Zea mays L.). The highest-yielding hybrids were from the B_C× R_B heterotic pattern; however, several B_C× B_C hybrids (within-group hybrids) were among the top-yielding hybrids. The outstanding performance of certain B_C× B_C hybrids casts some doubt on the validity of the B_C group. Substantial genetic diversity seems to be present within and between heterotic groups in sunflower. Received: 1 September 1998 / Accepted: 14 September 1999     

Quantitative karyotyping of three diploid Brassica species by imaging methods and localization of 45s rDNA loci on the identified chromosomes

 Chromosomes of the three diploid Brassica species, B. rapa (AA), B. nigra (BB) and B. oleracea (CC), were identified based on their morphological characteristics, especially on the condensation pattern appearing at the somatic pro-metaphase stage. The morphological features of the pro-metaphase chromosomes of the three Brassica spp. were quantified by imaging methods using chromosome image analyzing system II (CHIAS 2). As a result, quantitative chromosome maps or idiograms of the three diploid Brassica spp. were developed. The fluorescence in situ hybridization (FISH) method revealed the location of 45s rDNA (the 26s-5.8s-18s ribosomal RNA gene cluster) on the chromosomes involved. The number of 45s rDNA loci in the B. rapa, B. nigra and B. oleracea are five, three and two, respectively. The loci detected were systematically mapped on the idiograms of the three Brassica spp. Received: 5 September 1997 / Accepted: 6 October 1997     

Ultrastructural and molecular study of plastid inheritance in Abies alba and some Abies hybrids

 The ultrastructure of egg cells in Abies alba was examined to elucidate the lack of maternal inheritance of plastids. Before fertilization, maternal plastids are absent in the perinuclar zone containing mainly mitochondria and smooth endoplasmic reticulum. During egg cell development the maternal plastids are transformed into large inclusions which are situated mostly towards the periphery of the egg cell, and finally disintegrate. As a consequence, they do not participate in zygote formation. RFLP analysis of cpDNA of parental trees and their F1 interspecific hybrids (A. alba×A. numidica, A. alba×A. nordmanniana, A. nordmanniana×A. Alba) using HindIII and BamHI showed a paternal mode of cpDNA inheritance. Paternal inheritance has also been found with PCR/RFLP analysis of cpDNA from parental trees and their hybrids (A. alba×A. pinsapo, A. pinsapo×A. alba, A. pinsapo×A. numidica) using ApaI and HaeIII digests, as well as in the crosses of A. cephalonica×A. nordmanniana, A. nordmanniana×A. cephalonica, A. cephalonica×A. numidica using TagI digests. Received: 1 June 1998 / Accepted: 14 August 1998     

Simple sequence repeats reveal uneven distribution of genetic diversity in chloroplast genomes of <Emphasis Type="Italic">Brassica oleracea</Emphasis> L. and (<Emphasis Type="Italic">n </Emphasis>= 9) wild relatives

Diversity in the chloroplast genome of 171 accessions representing the Brassica ‘C’ (n = 9) genome, including domesticated and wild B. oleracea and nine inter-fertile related wild species, was investigated using six chloroplast SSR (microsatellite) markers. The lack of diversity detected among 105 cultivated and wild accessions of B. oleracea contrasted starkly with that found within its wild relatives. The vast majority of B. oleracea accessions shared a single haplotype, whereas as many as six haplotypes were detected in two wild species, B. villosa Biv. and B. cretica Lam.. The SSRs proved to be highly polymorphic across haplotypes, with calculated genetic diversity values (H) of 0.23–0.87. In total, 23 different haplotypes were detected in C genome species, with an additional five haplotypes detected in B. rapa L. (A genome n = 10) and another in B. nigra L. (B genome, n = 8). The low chloroplast diversity of B. oleracea is not suggestive of multiple domestication events. The predominant B. oleracea haplotype was also common in B. incana Ten. and present in low frequencies in B. villosa, B. macrocarpa Guss, B. rupestris Raf. and B. cretica. The chloroplast SSRs reveal a wealth of diversity within wild Brassica species that will facilitate further evolutionary and phylogeographic studies of this important crop genus. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Differentiation of F1 hybrids P. nigra J. F. Arnold × P. sylvestris L., P. nigra J. F. Arnold × P. densiflora Siebold et Zucc., P. nigra J. F. Arnold × P. thunbergiana Franco and their parental species by needle volatile composition

The volatile composition of needles from three F₁ hard pine hybrids produced by the controlled hybridization and their parental species were researched with gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS) in order to explore the utility of terpenes in hybrid identification (their differentiation from the parental species) as well as confirmation of hybridity. The analysed hybrids were: 1. Pinus nigra J. F. Arnold × Pinus sylvestris L. (= nisy), 2. P. nigra × Pinus densiflora Siebold et Zucc. (= nide) and 3. P. nigra × Pinus thunbergiana Franco (= nith). A total of 55 compounds were identified. All identified compounds were terpenes, except trans-2-hexenal.Three analysed F₁ hybrids showed the same qualitative pattern of the needle volatile composition as their parental species. However, there were quantitative differences in several major terpenes. The volatile composition of the needles from the hybrids nisy were equally similar to both parents, the hybrids nide were more similar to the female parent (P. nigra), whereas the hybrids nith were more similar to the male parent (P. thunbergiana). According to the content of germacrene D, as the specific component of P. nigra (female parent of the three analysed F₁ hybrids), all hybrids were intermediary in relation to the parental species. The content of Δ-3-carene (the specific component of P. sylvestris) in the hybrids nisy was also intermediary. The hybrids nide had a higher content of thunbergol (specific component of P. densiflora) than the other analysed hybrids. In view of the content of β-pinene, the specific component of P. thunbergiana, the hybrids nith were intermediary to the parental species and that content was considerably higher than in the other analysed hybrids. The intermediary quality of F₁ hybrids for these specific components in relation to the parental species confirms their hybrid character.The needle volatile composition analysis as well as the previous morphometric analysis confirm the hybrid character of three F₁ hybrids, whose female parent is P. nigra, and male parents are P. sylvestris, P. densiflora, i.e. P. thunbergiana.