Linkage between sexual and asexual lineages: genome evolution in Bacillus stick insects

The sexually reproducing stick insects Bacillus rossius and B. grandii are sharply differentiated in terms of allozyme gene alleles; B. atticus is a polyclonal automictic parthenogen sister to B. grandii grandii. Although well differentiated for coding genes, these hybridize to produce diploid (B. whitei=rossius/grandii) or triploid (B. lynceorum=rossius/grandii/atticus) clonal forms which reproduce apomictically. Allozyme analyses of unisexual Bacillus clearly establish their relationships from bisexual ancestor species as does the existence in all of them of several clones (especially in B. atticus) whose egg maturation allows regular recombination to occur. Bacillus taxa share the Bag320 satellite DNA family within different reproductive frameworks, allowing satellite variant homogenization to be uncoupled from fixation. The nested analysis of monomers reveals different patterns of sequence diversity: sexual reproduction includes both homogenization and variant fixation, whereas the slowing of molecular turnover processes and the absence of syngamy in the parthenogens realizes a similar range of sequence diversity at the level of the individual and supra‐individual, but with no fixation. On the other hand, the actual values of sequence diversity appear mostly linked to species traits – range size, copy number of repeats, number of hybrid crosses – and possibly transposon activity, rather than to the reproductive mode. In addition, the mitochondrial genome reveals a comparable level of cox2 sequence variability in sexual and parthenogenetic taxa, thus adding to clonal variability. From Bacillus and other stick insect complexes, an overall picture of genomic diversification of parthenogens is therefore beginning to emerge. To define those animals that reproduce by non‐canonical sexual modes (i.e. parthenogenesis, hybridogenesis), but make use of egg and meiotic mechanisms, the term meta‐sexual is proposed. © 2003 The Linnean Society of London. Biological Journal of the Linnean Society, 2003, 79 , 137–150.     

Amplification of repetitive DNA from Nicotiana plumbaginifolia in asymmetric somatic hybrids between Nicotiana sylvestris and Nicotiana plumbaginifolia

Summary Asymmetric somatic hybrids were obtained between a chlorophyll-deficient mutant of Nicotiana sylvestris (V42) and a nitrate-reductase (NR)-deficient line of N. plumbaginifolia (cnx20 or Nia26), using each of the parents alternately as the irradiated donor. Irradiation doses applied ranged from 10 to 1,000 Gy of gamma-rays. Hybrid selection was based on complementation of NR deficiency with wild-type NR genes. To aid in the analysis of somatic hybrids, species-specific repetitive DNA sequences from N. plumbaginifolia (NPR9 and NPR18) were cloned. NPR18 is a dispersed repetitive sequence occupying about 0.4% of the N. plumbaginifolia genome. In turn, NPR9, which is part of a highly repetitive DNA sequence, occupies approximately 3% of the genome. The species-specific plant DNA repeats, together with cytological analysis data, were used to assess the relative amount of the N. plumbaginifolia genome in the somatic hybrids. In fusion experiments using irradiated N. plumbaginifolia, an increase in irradiation dose prior to fusion led to a decrease in N. plumbaginifolia nuclear DNA content per hybrid genome. For some hybrid lines, an increase in the quantity of repetitive sequences was detected. Thus, hybrid lines 1NV/21, 100NV/7, 100NV/ 9, and 100NV/10 (where N. plumbaginifolia was the irradiated donor) were characterized by amplification of NPR9. In the reverse combination (where N. sylvestris was the irradiated donor), an increase in the copy number of NPR18 was determined for hybrid clones 1VC/2, 1VC/3, 100VC/2 and oct100/7. Possible reasons for the amplification of the repeated sequences are discussed.     

Analysis of nuclear and organellar DNA of somatic hybrid calli and plants between Lycopersicon spp. and Nicotiana spp.

Protoplast fusion experiments between Lycopersicon esculentum or L. peruvianum and Nicotiana tabacum or N. plumbaginifolia were performed to investigate the possibility of producing symmetric and asymmetric somatic hybrids between these genera. These fusions, which involved 1.7 × 10⁸ protoplasts, yielded 35 viable hybrid calli. Plant regeneration was successful with two calli. One of these regenerants flowered, but developed no fruits. Analysis of the nuclear DNA by means of dot blot hybridization with species-specific repetitive DNA probes combined with flow cytometry, revealed that the nuclei of most hybrid calli contained the same absolute amount of Nicotiana DNA as the Nicotiana parent or (much) less, whereas the amount of Lycopersicon DNA per nucleus was 2–5 times that of the parental genotype. Eighteen of the 34 hybrids analyzed possessed Lycopersicon chloroplast DNA (cpDNA), whereas the other 16 had DNA from Nicotiana chloroplasts. The cpDNA type was correlated with the nuclear DNA composition; hybrids with more than 2C Nicotiana nuclear DNA possessed Nicotiana chloroplasts, whereas hybrids with 2C or less Nicotiana nuclear DNA contained Lycopersicon chloroplasts. Mitochondrial DNA (mtDNA) composition was correlated with both nuclear DNA constitution and chloroplast type. Hybrids possessed only or mainly species-specific mtDNA fragments from the parent predominating in the nucleus and often providing the chloroplasts. The data are discussed in relation to somatic incompatibility which could explain the low frequency at which hybrids between Lycopersicon and Nicotiana species are obtained and the limited morphogenetic potential of such hybrids.     

Intergeneric hybridization between Diplotaxis siettiana and crop brassicas for the production of alloplasmic lines

Summary Intergeneric hybrids were produced between Diplotaxis siettiana and Brassica campestris through embryo rescue. The hybrids were completely pollen sterile and backcrosses with pollen of B. campestris did not yield any seeds. Induction of colchiploidy restored pollen fertility and backcross pollinations yielded viable seeds. Cytological details of the hybrid, amphidiploid and backcross progenies were studied. Both pollen-sterile and pollen-fertile plants have been obtained in backcross 2 progeny. This hybrid (D. siettiana x B. campestris) was used as a bridge cross to transfer the cytoplasm of D. Siettiana to two other incompatible cultivars of Brassica — B. juncea and B. napus. Pollinations of the amphidiploid (D. siettiana x B. campestris, 2n = 36) with pollen of B. juncea/B. napus readily produced seeds without embryo rescue. These hybrids were grown to flowering and their cytological details were studied. Seeds have been produced from backcross pollinations of both these hybrids with the pollen of the respective cultivars. The results clearly show the feasibility of producing alloplasmic lines in all the three oilseed brassicas.     

Identification and genetic diversity of Fusarium sacchari associated with pokkah boeng disease of sugarcane in Northeast Brazil

Isolates from the Fusarium fujikuroi species complex, mainly F. sacchari, have been reported to be the causal agents of pokkah boeng in sugarcane in Brazil. However, inadequate information was available on the occurrence and genetic diversity of F. sacchari in Northeast Brazil, which is a limiting factor on management. Thus, isolates of F. subglutinans sensu lato from sugarcane plants with symptoms of pokkah boeng were evaluated using the sexual cross-fertility to determine species. All the isolates produced black perithecia when they were crossed with the test isolates of F. sacchari. Three weeks after the crossing, the formation of fertile ascospores cirri was observed. Thirty-four isolates were self-sterile hermaphrodites, while 21 were fertile only as males. Five isolates were homothallic. The effective size [N_e(f)] of the population as a function of the frequency of hermaphrodites and female sterile strains was 95.5%. The F. sacchari isolates were separated into four genetic groups independent of geographic location. The mean of polymorphism among all populations was 79%, and the average unbiased genetic diversity (uh) was considered moderate (0.31). This study in addition to confirming that F. sacchari as the main species associated with pokkah boeng in sugarcane in Northeast Brazil, reveals the relationship of mating type and genetic diversity of F. sacchari. The unrestricted gene flow between regions is probably the best explanation for the low geographic correlation. This knowledge will help in the adoption of management measures with fungicides or resistant cultivars.     

Vigorous growth of fusion products allows highly efficient selection of interspecific potato somatic hybrids: molecular proofs

Summary An early identification of fusion products was based on the presumed vigorous growth of hybrid calluses after fusion between Solanum brevidens and S. tuberosum leaf protoplasts. The S. brevidens protoplasts were unable to form multicellular colonies under the applied culture conditions. Three size groups of calluses were separated and analyzed at two different early phases of culture period. Squash blot hybridization with a S. brevidens specific repetitive DNA probe showed that the group of the largest calluses consisted of putative somatic hybrids with a frequency of 80–100% in three independent experiments. Furthermore, approximately 80–95% of the middle sized calluses and 33–90% of the smallest ones were shown to be hybrid. The unexpectedly high percentage of fusion products, even in the case of the smallest calluses, may result from the suppression of the development of parental potato colonies in cultures with mixed cell population. Till this time 120 independent colonies selected as putative hybrids have been regenerated into plants. All of them exhibited hybrid phenotype, and their hybrid origin was proved by cytological and restriction fragment length polymorphism analyses.Abbreviations BA N⁶-benzyladenine - NAA -naphthaleneacetic acid - RFLP restriction fragment length polymorphism - UV ultraviolet     

Response of wheatgrasses and wheat × wheatgrass hybrids to barley yellow dwarf virus

Summary Resistance to barley yellow dwarf virus (BYDV), manifested by low enzyme-linked immunosorbent assay (ELISA) values in plants exposed to viruliferous aphids, was identified in several wheatgrasses (Agropyron spp.). ELISA results were similar for root and leaf extracts of infested plants. No difference in reaction to BYDV was found between plants grown in the field and those in the growth chamber. Interspecific hybrids were generated using pollen from single resistant plants of Agropyron spp. to pollinate soft red winter wheat spikes. Resistance in hybrids appeared to be at the level of virus replication rather than at the level of vector inoculation. The hybrids varied in their reaction to BYDV. Expression of BYDV resistance in hybrids was influenced not only by wheat genotype and Agropyron species but, in some cases, reaction varied even among hybrids between the same wheat genotype and Agropyron plant. Implications of these results are discussed.Contribution from the Purdue Univ. Agric. Exp. Stn., West Lafayette, IN 47907, and USDA-ARS. The research was supported in part by Public Varieties of Indiana. Purdue Univ. Agric. Exp. Stn. Journal No. 11656     

Embryo-callus-regenerated hybrids and their colchicine-induced amphiploids between Elymus canadensis and Secale cereale

Summary Intergeneric hybrids recovered through plant regeneration from embryo callus culture and their colchicine-induced amphiploids were obtained from a cross of Elymus canadensis with Secale cereale (cv Gazeller). The embryo-callus-regenerated F₁ plants grew vigorously to maturity and regrew well after clipping, while the embryo-rescued F₁ died of hybrid necrosis before maturity. Most of the morphological characters of the sterile F₁ hybrids were intermediate between the parents, but tiller number and dry matter yield were higher than the parents. Amphiploids from these F₁ plants had improved fertility but were less vigorous than the F₁ plants. The predominance of univalents in the F₁ and bivalents in the amphiploids indicated that the genomes S, H, and R were distinct. However, the occasional occurrence of multivalents reflected a random, intergenomic or intragenomic pairing. The mean chromosome associations of the F₁ (2n=21, SHR), the C₀ amphiploids (2n=42, SSHHRR), and the C₁ amphiploid (2n=40) at metaphase I (MI) were 16.51_I+2.05_II+0.06_III+0.02_IV+0.02_V, 2.20_I+19.87_II +0.02_IV, and 7.10_I+16.37_II+0.04_III+0.02_IV, respectively. These amphiploids could be exploited as new germplasm for forage crop improvement by controlled introgression and backcrosses to the parents.     

Transmission of organelles in triploid hybrids produced by gametosomatic fusions of two Nicotiana species

Summary Gametosomatic hybrids produced by the fusion of microspore protoplasts of Nicotiana tabacum Km⁺Sr⁺ with somatic cell protoplasts of N. rustica were analysed for their organelle composition. For the analysis of mitochondrial (mt)DNA, species-specific patterns were generated by Southern hybridization of restriction endonuclease digests of total DNA and mtDNA with four DNA probes of mitochondrial origin: cytochrome oxidase subunit I, cytochrome oxidase subunit II, 26s rDNA and 5s-18s rDNA. Of the 22 hybrids analyzed, some had parental-type pattern for some probes and novel-type for the others, indicating interaction between mtDNA of the two parent species. For chloroplast (cp)DNA analysis, species-specific patterns were generated by Southern hybridization of restriction endonuclease digests of total DNA with large subunits of ribulose bisphosphate carboxylase and cpDNA as probes. All the hybrids had N. rustica-specific patterns. Hybrids were not resistant to streptomycin, a trait encoded by the chloroplast genome of N. tabacum. In gametosomatic fusions of the two Nicotiana species, mitochondria but not the chloroplasts are transmitted from the parent contributing microspore protoplasts.     

Somatic hybrid plants produced by electrofusion between Solanum melongena L. and Solanum torvum Sw

Summary Somatic hybrid plants between eggplant (Solanum melongena) and Solanum torvum have been produced by the electrofusion of mesophyll protoplasts in a movable multi-electrode fusion chamber. Using hair structure as a selection criteria, we identified a total of 19 somatic hybrids, which represented an overall average of 15.3% of the 124 regenerated plants obtained in the two fusion experiments. Several morphological traits were intermediate to those of the parents, including trichome density and structure, height, leaf form and inflorescence. Cytological analyses revealed that the chromosome numbers of the somatic hybrids approximated the expected tetraploid level (2n=4x=48). Fifteen hybrid plants were homogeneous and had relatively stable chromosome numbers (46–48), while four other hybrids had variable chromosome numbers (35–48) and exhibited greater morphological variation. The hybridity of these 19 somatic hybrid plants was confirmed by analyses of phosphoglucomutase (Pgm) and esterase zymograms.