Experimental evidence on the affinities ofPapaver somniferum (Papaveraceae)

Results obtained from crossing experiments betweenP. somniferum subsp.somniferum (2n = 22) and subsp.setigerum (2n = 44),P. glaucum (2n = 14) andP. gracile (2n = 14) and from the observation of meiotic chromosome pairing in the various hybrids obtained do not provide straightforward evidence for the hypothesis thatP. somniferum originated as a triploid hybrid between taxa similar toP. glaucum andP. gracile (Kadereit 1986a, b).—On the one hand, the pattern of crossability found reflects the closer similarity of subsp.somniferum toP. glaucum and of subsp.setigerum toP. gracile, which was interpreted as segregation of parental characters, and the high frequency of 2n = 28 chromosomes among F₂-progeny from the hybrid subsp.somniferum × subsp.setigerum (2n = 33) might reveal n = 7 as the base number also ofP. somniferum. On the other hand, however, the general difficulty of obtaining hybrids, and the low incidence of bivalent formation in their meiosis, probably indicating a lack of chromosome homology between the different species, do not fit the above hypothesis.—These results are in marked contrast to the morphological similarity between the three species involved.     

Fertile somatic hybrids between Petunia hybrida and a wild species,Petunia variabilis

Somatic hybrid plants were regenerated following electrofusion between leaf mesophyll protoplasts of P. hybrida (2n = 14) and a wild sexually incompatible species, P. variabilis (2n = 18). The selection of hybrids was based on the hybrid vigour, expressed both in the growth of the callus and at the shoot formation stage, resulting from the combination of parental genomes. Calli exhibiting vigorous growth were selected, and upon transfer to regeneration medium gave rise to shoots. Four regenerated plants from three calli had morphological characteristics intermediate between those of the parents. The hybrid nature of these plants was confirmed by chromosome counts as well as isozyme and DNA analyses. They had amphidiploid chromosome numbers (2n = 32) and were fertile. Following self-pollination and backcrossing with P. variabilis, large numbers of F₂ and BC₁ seedlings were obtained.     

Somatic hybridization between Brassica juncea and Moricandia arvensis by protoplast fusion

Intergeneric somatic hybrids have been produced between Brassica juncea (2n=36, AABB) cv. RLM-198 and Moricandia arvensis (2n=28, MM) by protoplast fusion. Hypocotyl protoplasts of B. juncea were fused with mesophyll protoplasts of M. arvensis using polyethylene glycol. Fusion frequency, estimated on the basis of differential morphological characterstics of parental protoplasts was about 5%. Of the 156 calli obtained, four calli produced shoots intermediate in morphology between the parents. Hybrid nature of the plants was confirmed using wheat nuclear rDNA probe. Hybridization of total DNA with a mitochondrial DNA probe carrying 5s–18s rRNA genes of maize showed that the mitochondria of the somatic hybrids were derived from the wild species M. arvensis. Meiosis in the only hybrid that produced normal flowers revealed the occurrence of 64 chromosomes, the sum of chromosomes of parental species. Inspite of complete pollen sterility, siliquas were produced in this hybrid by back-crossing with B. juncea. These siliquas on in vitro culture produced 12 seeds.     

Hybridization and evolution inCardamine (Brassicaceae) at Urnerboden, Central Switzerland: Biosystematic and molecular evidence

Hybridization between two diploid (2n = 2x = 16) species ofBrassicaceae, Cardamine rivularis andC. amara, at Urnerboden, Central Switzerland, resulted in the rather unusual triploid hybridC. insueta (2n = 3x = 24), and later on in the amphiploidC. schulzii (2n = 6x = 48). The hybrid and the neopolyploid species colonized successfully some man-made biotopes. Plants ofC. insueta are mostly functional females with non-dehiscent anthers, but true hermaphrodite individuals with partly sterile pollen grains also occur within the population. Analyses of cpDNA and nuclear DNA permitted to establish the parentage of the hybrid: the maternal parent which contributed unreduced egg cells proved to beC. rivularis whereas the normally reduced pollen originated fromC. amara. The pronounced genetic variability inC. insueta revealed by isozyme and RAPD analyses, at variance with the polarized segregation, heterogamy and strong vegetative reproduction of the hybrid, is possibly influenced by recurrent formation ofC. insueta which party results from backcrosses betweenC. insueta andC. rivularis but may also proceed by other pathways. The amphiploidCardamine schulzii has normally developed anthers but its pollen is sometimes highly sterile. The surprisingly uniform genetic make-up of the new amphiploid species might be related to its possible monotopic origin and/or young phylogenetic age but should be further assessed. Site management seems to be very important to a further development of hybridogenous populations and their parent species. In conclusion, the evolution at Urnerboden is discussed in the context of the traditional concept of multiple plant origins.     

Allotetraploid origin ofAllium altyncolicum (Alliaceae, Allium sect.Schoenoprasum) as investigated by karyological and molecular markers

The tetraploidAllium altyncolicum (2n = 4x = 32) is considered to be of hybrid origin, because most of its morphological characters are intermediate between those of its putative parents,A. schoenoprasum andA. ledebourianum. In the present work an attempt has been made to ascertain its parentage by several methods: Giemsa C-banding, genomic in situ hybridization (GISH), PCR-RFLP of cpDNA, restriction enzyme mapping of the rDNA, and RAPDs. C-banding and GISH indicates clearly thatA. altyncolicum is a segmental allopolyploid.Allium schoenoprasum andA. ledebourianum are the most likely the parental species and the larger part of the genome ofA. altyncolicum (26 chromosomes) is derived fromA. schoenoprasum. The low genetic divergence between these three species was confirmed by the lack of sequence variation in the ITS sequences of nuclear rRNA genes and of the plastid rbcL-atpB intergenic spacer. Both parental species andA. altyncolicum could be distinguished by RFLP of the rDNA repeats. The geographic origin of the putative parental species was investigated using RAPDs.Dedicated to emer. Univ.-Prof. DrFriedrich Ehrendorfer on the occasion of his 70th birthday     

Hybridization,transgressive segregation and evolution of new genetic systems in<Emphasis Type="Italic">Drosophila</Emphasis>

Introgressive hybridization facilitates incorporation of genes from one species into the gene pool of another. Studies on long-term effects of introgressive hybridization in animal systems are sparse.Drosophila nasuta (2n = 8) andD. albomicans (2n = 6)—a pair of allopatric, morphologically almost identical, cross-fertile members of thenasuta subgroup of theimmigrans species group-constitute an excellent system to analyse the impact of hybridization followed by transgressive segregation of parental characters in the hybrid progeny. Hybrid populations ofD. nasuta andD. albomicans maintained for over 500 generations in the laboratory constitute new recombinant hybrid genomes, here termed cytoraces. The impact of hybridization, followed by introgression and transgressive segregation, on chromosomal constitution and karyotypes, some fitness parameters, isozymes, components of mating behaviour and mating preference reveals a complex pattern of interracial divergence among parental species and cytoraces. This assemblage of characters in different combinations in a laboratory hybrid zone allows us to study the emergence of new genetic systems. Here, we summarize results from our ongoing studies comparing these hybrid cytoraces with the parental species, and discuss the implications of these findings for our understanding of the evolution of new genetic systems. This paper is dedicated to the memory of our teacher, Prof. N. B. Krishnamurthy.     

Somatic hybrid plants between the forage legumes Medicago sativa L. and Medicago arborea L.

Interspecific somatic hybrid plants were obtained by symmetrical electrofusion of mesophyll protoplasts of Medicago sativa with callus protoplasts of Medicago arborea. Somatic hybrid calli were picked manually from semi-solid culture medium after they were identified by their dual color in fluorescent light. Twelve putative hybrid calli were selected and one of them regenerated plants. The morphogenesis of the somatic hybrid calli was induced by the synthetic growth regulator 1,2 benzisoxazole-3-acetic acid. Somatic hybrid plants showed intensive genome rearrangements, as evidenced by isozyme and RFLP analysis. The morphology of somatic hybrid plants was in general intermediate between the parents. The production of hybrids by protoplast fusion between sexually incompatible Medicago species is related to the in vitro respon siveness of the parental protoplasts. The possibility of using somatic hybrid plants in alfalfa breeding is discussed.     

Karyological and chemotaxonomical studies of the hybridPelargonium incarnatum ×P. patulum (Geraniaceae) and its parents

Pelargonium incarnatum is a tetraploid species (2n = 40) andP. patulum is diploid (2n = 22). A natural hybrid, collected at the same locality as the parent species, has 2n = 42 chromosomes. The meiosis of the hybrid is practically normal and its pollen fertility corresponds to that ofP. incarnatum. The flavonoid patterns of the parent species and the hybrid are very similar. The origin and phylogenetic implications of this intersectional hybrid are discussed.     

Resynthesis of Brassica carinata by protoplast fusion and recovery of a novel cytoplasmic hybrid

Brassica carinata (2n=34, BBCC), was synthesized by fusing dark grown etiolated hypocotyl protoplasts of B. nigra (2n=16, BB) with green mesophyll protoplasts of B. oleracea (2n=18,CC) using polyethylene glycol. Heterokaryons could be microscopically distinguished from the parental types by their dark green chloroplasts in the colourless hypocotyl protoplast background. The mean heterokaryotic fusion frequency estimated on the basis of this morphological distinction was about 16%. A total of 626 calli were obtained, of which 92 regenerated shoots after transfer to zeatin (2 mg/l) supplemented MS medium. Of these, 81 calli differentiated into plants morphologically similar to naturally occurring B. carinata and 11 calli yielded plants resembling parental types. Meiosis in seven hybrid plants showed the chromosome number to be 2n=34 the sum of B. nigra and B. oleracea chromosomes. Molecular confirmation of the amphidiploid nature of hybrids was obtained by probing with a B. juncea derived genomic clone. The use of chloroplast and mitochondrial specific gene probes, revealed that one plant was a cytoplasmic hybrid having cp DNA sequences of both B. oleracea and B. nigra and mt DNA sequences of B. nigra.Abbreviations PEG Polyethylene glycol - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA Naphthaleneacetic acid - IBA Indole-3-butyric acid - BAP 6-Benzylaminopurine - MS Murashige and Skoog (1962)     

Spontaneous hybridization between a male-sterile oilseed rape and two weeds

Spontaneous interspecific hybrids were produced under natural conditions (pollination by wind and bees) between a male-sterile cybrid Brassica napus (AACC, 2n = 38) and two weeds Brassica adpressa (AdAd, 2n = 14) and Raphanus raphanistrum (RrRr, 2n = 18). After characterization by chromosome counts and isozyme analyses, we observed 512 and 3 734 inter-specific seeds per m² for the B. napus-B. adpressa and B. napus-R. raphanistrum trials respectively. Most of the hybrids studied had the expected triploid structure (ACX). In order to quantify the frequency of allosyndesis between the genomes involved in the hybrids, their meiotic behavior was compared to a haploid of B. napus (AC). For the B. napus-B. adpressa hybrids, we concluded that probably no allosyndesis occurred between the two parental genomes, and that genetic factors regulating homoeologous chromosome pairing were carried by the B. adpressa genome. For the B. napus-R. raphanistrum hybrids, high chromosome pairing and the presence of multivalents (in 9.16% of the pollen mother cells) indicate that recombination is possible between chromosomes of different genomes. Pollen fertility of the hybrids ranged from 0 to 30%. Blackleg inoculation tests were performed on the three parental species and on the interspecific hybrids. BC₁ production with the weeds and with rapeseed was attempted. Results are discussed in regard to the risk assessment of transgenic rapeseed cultivation, F₁ hybrid rapeseed variety production, and rapeseed improvement.     

Somatic hybridization between birdsfoot trefoil (Lotus corniculatus L.) and L. conimbricensis Willd

Summary Somatic hybrid plants were produced by fusion of birdsfoot trefoil (Lotus corniculatus) cv Leo and L. conimbricensis Willd. protoplasts. Birdsfoot trefoil etiolated hypocotyl protoplasts were inactivated with iodoacetate to inhibit cell division prior to fusion with L. conimbricensis suspension culture protoplasts. L. conimbricensis protoplasts divided to form callus which did not regenerate plants. Thus, plant regeneration from protoplast-derived callus was used to tentatively identify somatic hybrid cell lines. Plants regenerated from three cell lines exhibited additive combinations of parental isozymes of phosphoglucomutase, and L. conimbricensis-specific esterases indicating that they were somatic hybrids. The somatic chromosome number of one somatic hybrid was 36. The other somatic hybrid exhibited variable chromosome numbers ranging from 33 to 40. These observations approximate the expected combination of the birdsfoot trefoil (2n=4x=24) and L. conimbricensis (2n=2x=12) genomes. Somatic hybrid flowers were less yellow than birdsfoot trefoil flowers and had purple keel tips, a trait inherited from the white flowered L. conimbricensis. Somatic hybrids also had inflorescence structure that was intermediate to the parents. Fifteen somatic hybrid plants regenerated from the three callus lines were male sterile. Successul fertilization in backcrosses with birdsfoot trefoil pollen has not yet been obtained suggesting that the hybrids are also female sterile. This is the first example of somatic hybridization between these two sexually incompatible Lotus species.Formerly USDA-ARS, St. Paul, Minn, USA     

Production,morphology, and cytogenetics of Triticum aestivum (L.) Thell × Elymus scabrus (R. Br.) Love intergeneric hybrids obtained by in ovulo embryo culture

Summary Intergeneric hybrids were produced between common wheat, Triticum aestivum (2n=6x=42, AABBDD), and an apomictic Triticeae species, Elymus scabrus (syn. Agropyron scabrum) (2n=6x=42, HHSSSS), the first successful report of this cross. Nine tiny, underdeveloped, and structureless embryos were obtained in vitro only by in ovulo embryo culture at 4 days after pollination, which gave rise to five mature hybrid plants. All the hybrid plants were vigorous and possessed a phenotype intermediate to the two parents. There were 2n=6x=42 (ABDHSS) somatic chromosomes in the hybrids. There was little or no homology between the parental genomes, as shown by an overall meiotic chromosome association of 32.83 I + 4.08 rod II + 0.21 ring II + 0.18 III + 0.02 IV. The hybrids were completely sterile and so far backcrosses to wheat parent have not been successful. Alternate approaches to induce gene transfer(s) from E. scabrus to wheat are being attempted.Agriculture Canada Contribution No. 398.     

Haploidy from Hordeum interspecific crosses

Summary Interspecific crosses of Hordeum brachyantherum (2n = 28) and H. depressum (2n = 28) with H. bulbosum (2n = 14 or 28) and H. vulgare (2n = 14 or 28) were made. Crosses between brachyantherum and diploid bulbosum resulted in dihaploids (2n = 14) of brachyantherum and hybrids (2n = 21), whilst the crosses of brachyantherum by tetraploid bulbosum or vulgare gave hybrid progeny. Similarly, crosses between H. depressum and diploid bulbosum resulted in dihaploids (2n = 14) of depressum and hybrids (2n = 21), whereas depressum by tetraploid bulbosum or vulgare invariably produced hybrids.Cytological observations on 12 day old embryos obtained from these crosses revealed chromosome variability down to 14 in crosses with diploid bulbosum indicating thereby that chromosome elimination leads to haploid formation. Embryonic cells from the brachyantherum by diploid vulgare cross also exhibited a certain degree of chromosomal instability as micronuclei.The results indicate that the ratio of parental genomes in the zygote determines whether haploids or hybrids will be produced in crosses of brachyantherum or depressum with bulbosum. Furthermore, brachyantherum appears to be more efficient in eliminating bulbosum chromosomes in comparison with depressum.     

Somatic hybrids between Solanum etuberosum and diploid,tuber bearing Solanum clones

Electrofusion was used to obtain somatic hybrids between Solanum etuberosum (2n=2x=24) and two diploid potato lines. These hybridizations were conducted to determine if haploidxwild species hybrids are better fusion partners than conventional S. tuberosumGp. Tuberosum haploids. Restriction fragment length polymerase (RFLP) analyses of the putative somatic hybrids confirmed that each parental genome was present. The somatic hybrids between S. etuberosum and a haploid S. tuberosum clone, US-W730, were stunted and had curled, purple leaves. In contrast, somatic hybrids between S. etuberosum and a haploidxwild species hybrid (US-W 730 haploidx S. berthaultii), were vigorous and generally tuberized under field conditions. These hybrids were designated as E+BT somatic hybrids. Analyses of 23 E+BT somatic hybrids revealed a statistically significant bias towards the retention of S. etuberosum chloroplasts. Stylar incompatibilities were observed when the E+BT somatic hybrids were used as pollen donors in crosses with S. tuberosum cultivars. Reciprocal crosses did not show this incompatibility. The progeny were vigorous and had improved tuber traits when compared to the maternal E+BT parent. RFLP analyses of three sexual progeny lines confirmed the presence of all 12 S. etuberosum chromosomes. In two of these lines, RFLPs that marked each of the 24 chromosome arms of S. etuberosum were present. However, RFLP markers specific for regions on chromosomes 2, 7, and 11 were missing from the third clone. Because other markers for these chromosomes were present in the progeny line, these results indicated the likelihood of pairing and recombination between S. etuberosum and S. tuberosum chromosomes.     

Origin and differentiation of Aegilops triuncialis L. as determined by esterase isozyme analysis

Summary Banding patterns of esterase isozymes in Aegilops triuncialis (2n = 28, genome formula C^uC^uCC) and its putative parental species, Ae. umbellulata (2n = 14, C^uC^u) and Ae. caudata (2n = 14, CC), were studied by the gel isoelectric focusing method using pH 6–8 carrier ampholite. Zymogram phenotypes of both parents were quite uniform. Seven zymogram phenotypes (designated as phenotypes 1 to 7) were found among the 260 strains of Ae. triuncialis examined. Of these phenotypes, phenotype 1 was identical to the zymogram phenotype produced by the ancestral species, Ae. umbellulata, and bands considered to have been derived from Ae. caudata were absent in this phenotype. Phenotype 3 had all bands of both parents. The other phenotypes differed greatly from phenotype 3. Therefore, phenotype 3 was considered to be most primitive of the 7 types, and the Ae. triuncialis strains which showed phenotype 3 to be the most primitive of the strains examined. If Ae. triuncialis originated as a hybrid between Ae. umbellulata and Ae. caudata, the zymogram phenotype must have been phenotype 3, in which the isozymes of both parental species are present. Whether the phenotypes other than type 3 were due to introgressive hybridization could not be verified, but they were considered in this article to be a consequence of a rearrangement of chromosomes.Contribution from the Laboratory of Genetics, Faculty of Agriculture, Kyoto University No. 432     

Characterisation of the double genome structure of modern sugarcane cultivars (Saccharum spp.) by molecular cytogenetics

Cultivated sugarcane clones (Saccharum spp., 2n=100 to 130) are derived from complex interspecific hybridizations between the speciesS. officinarum andS. spontaneum. Using comparative genomic DNA in situ hybridization, we demonstrated that it is possible to distinguish the chromosomes contributed by these two species in an interspecific F1 hybrid and a cultivated clone, R570. In the interspecific F1 studied, we observed n+n transmission of the parental chromosomes instead of the peculiar 2n+n transmission usually described in such crosses. Among the chromosomes of cultivar R570 (2n=107–115) about 10% were identified as originating fromS. spontaneum and about 10% were identified as recombinant chromosomes between the two speciesS. officinarum andS. spontaneum. This demonstrated for the first time the occurrence of recombination between the chromosomes of these two species. The rDNA sites were located by in situ hybridization in these two species and the cultivar R570. This supported different basic chromosome numbers and chromosome structural differences between the two species and provided a first bridge between physical and genetical mapping in sugarcane.     

Cytogenetical changes of offsprings from the induced tetraploid hybrid betweenRanunculus silerifolius (2n = 16) andR. chinensis (2n = 16) (Ranunculaceae)

Cytogenetical studies were carried out on the successive generations of offsprings from the induced tetraploid hybrid (2n = 32) betweenRanunculus silerifolius (2n = 16) andR. chinensis (2n = 16). Aneuploids, 2n = 30 to 35, frequently occurred. In latter subsequent generations the deviation of aneuploids increased, but the proportion of euploids decreased, accompanied by the reduction of fertility of pollen grains and seed sets. F₂ and F₄ PMCs constantly exhibited meiotic abnormality, i.e. formation of quadrivalents and univalents. The speciation process ofR. cantoniensis (2n = 32), which was presumed to arise from tetraploid hybrids between the above two species, is discussed on the basis of the above evidences.Former contributions of this series areOkada & Tamura (1977) andOkada (1984).     

Somatic hybrid plants obtained by protoplast fusion between Citrus sinensis and Poncirus trifoliata

Summary Somatic hybrid plants of Rutaceae were obtained by protoplast fusion between Citrus sinensis Osb. (Trovita orange) and Poncirus trifoliata. Protoplasts isolated from embryogenic cells of C. sinensis and from leaves of P. trifoliata, and the culture of fusion products in the presence of high concentrations of sucrose were essential requirements for the selection of hybrids. Green globular embryoids derived from protoplasts resulted in the regeneration of trifoliate plants. Other morphological characters of these plants were intermediate between both parents. The chromosome number in one of the hybrid plants was 36, which was the sum of C. sinensis (2n=18) and P. trifoliata (2n=18). EcoRI restriction analysis of rDNA confirmed the presence of parental nuclear DNAs in the hybrid.     

Effect of hybridization of the Quercus crassifolia×Quercus crassipes complex on the community structure of endophagous insects

In a previous study, we showed that the geographic proximity of hybrid plants to the allopatric areas of parental species increases their morphological and genetic similarity with them. In the present work, we explored whether the endophagous fauna of hybrid plants show the same pattern. We studied the canopy species richness, diversity and composition of leaf-mining moths (Lepidoptera: Tischeridae, Citheraniidae) and gall-forming wasps (Hymenoptera: Cynipidae) associated with two species of red oaks (Quercus crassifolia and Quercus crassipes) and their interspecific hybrid (Quercus×dysophylla Benth pro sp.) in seven hybrid zones in central Mexico, during four seasons in 2 years. The study was conducted on 194 oak trees with known genetic status [identified by leaf morphology and molecular markers (random amplified polymorphic DNAs)], and the results indicate a bidirectional pattern of gene flow. Hybrid plants supported intermediate levels of infestation of gall-forming and leaf-mining insects compared to their putative parental species. The infestation level of leaf-mining insects varied significantly following the pattern: Q. crassifolia>hybrids>Q. crassipes, whereas the gall-forming insects showed an inverse pattern. A negative and significant relationship was found between these two types of insect guilds in each host taxa, when the infestation percentage was evaluated. It was found that 31.5% (n=11) of the endophagous insects were specific to Q. crassipes, 22.9% (n=8) to Q. crassifolia, and 8.6% (n=3) to hybrid individuals. The hybrid bridge hypothesis was supported in the case of 25.7% (n=9) of insects, which suggests that the presence of a hybrid intermediary plant may favor a host herbivore shift from one plant species to another. Greater genetic diversity in a hybrid zone is associated with greater diversity in the endophagous community. The geographic proximity of hybrid plants to the allopatric site of a parental species increases their similarity in terms of endophagous insects and the Eje Neovolcánico acts as a corridor favoring this pattern. Electronic Supplementary Material Supplementary material is available for this article at     

Competition in natural populations of Daphnia

I investigated the competitive relationships between two species of Daphnia, D. galeata and D. cucullata, and their interspecific hybrid. The term hemispecific competition was introduced to describe competition between parental species and hybrids. In eutrophic Tjeukemeer both parental species were found to compete with the hybrid, whereas competition between D. galeata and D. cucullata seemed limited. Although the effect of competition on life history traits of daphnids may be profound, the influence of the competitors on the seasonal dynamics of the Daphnia species seems limited.