Analysis of the meiotic recombination frequency in transgenic tomato hybrids expressing <Emphasis Type="Italic">recA</Emphasis> and <Emphasis Type="Italic">NLS-recA-licBM3</Emphasis> genes

To study and induce meiotic recombination in plants, we generated and analyzed transgenic tomato hybrids F₁-RecA and F₁-NLS-recA-LicBM3 expressing, respectively, the recA gene of Escherichia coli and the NLS-recA-licBM3 gene. It was found that the recA and NLS-recA-licBM3 genes are inherited through the maternal and paternal lineages, they have no selective influence on the pollen and are contained in tomato F₁-RecA and F₁-NLS-RecA-LicBM3 hybrids outside the second chromosome in the hemizygous state. The comparative analysis of the meiotic recombination frequency (rf) in the progenies of the transgenic and nontransgenic hybrids showed that only the expression of the recA gene of E. coli in cells of the F1-RecA plants produced a 1.2–1.5-fold increase in the frequency of recombination between some linked marker genes of the second chromosome of tomato.     

Polyploidization increases meiotic recombination frequency in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Background  

Polyploidization is the multiplication of the whole chromosome complement and has occurred frequently in vascular plants. Maintenance of stable polyploid state over generations requires special mechanisms to control pairing and distribution of more than two homologous chromosomes during meiosis. Since a minimal number of crossover events is essential for correct chromosome segregation, we investigated whether polyploidy has an influence on the frequency of meiotic recombination.     

Cytogenetic analysis of hybrids derived from wheat and <Emphasis Type="Italic">Tritipyrum</Emphasis> using conventional staining and genomic <Emphasis Type="Italic">in situ</Emphasis> hybridization

The new salt tolerant cereal, Tritipyrum (2n=6x=42, AABBE^bE^b) offers potential to introduce desirable characters for wheat improvements. This study was aimed to generate a segregating population from Iranian local wheat cultivars (2n=6x=42, AABBDD) and Tritipyrum crosses, study of the meiotic behaviour in F₂ hybrids and identification of E^b chromosomes in F₃ individuals. Results showed meiotic abnormalities in F₂ plants and different pairing frequency in the meiosis among F₂ plants. Genomic in situ hybridization revealed that total and E^b chromosome number of F₃ seeds ranged from 39 to 45 and 0 to 10, respectively. A significant prevalence of hyper-aneuploidy was observed among F₃ genotypes. C-banding patterns identified E^b chromosomes in Tritipyrum, indicating that it also can be useful to study wheat-Tritipyrum derivatives.     

Taxonomic genetics of <Emphasis Type="Italic">Zygowilliopsis</Emphasis> yeasts

Genetic hybridization analysis was conducted with 16 natural Zygowilliopsis strains isolated in different geographical regions and maintained in collections under species names Z. californica, Hansenula dimennae, and Pichia populi. Genetic relatedness was determined on the basis of mating, viability of hybrid progeny, and meiotic recombination of markers. Four new biological species are recognized in the former monotypic genus Zygowilliopsis. Species Z. californica and Zygowiliopsis sp. 3 probably include divergent geographical populations. It is necessary to reconsider the species composition of the genus Zygowiliopsis and generic assignment of P. populi yeasts. Genetic and molecular identifications of the Zygowiliopsis species are in perfect agreement.     

Two <Emphasis Type="Italic">CLE</Emphasis> genes are induced by phosphate in roots of <Emphasis Type="Italic">Lotus japonicus</Emphasis>

Genes of CLE (CLAVATA3/ESR-related) family encode peptide ligands that regulate plant development in response to external stimuli such as rhizobial infection and the nitrate application as well as various internal stimuli. To investigate whether LjCLE gene(s) may involve in plant response to inorganic phosphate (Pi), we analyzed Pi responses of 39 LjCLE genes in hydroponically grown Lotus japonicus plants (ecotype Miyakojima ‘MG-20’). Two LjCLE genes, LjCLE19 and 20, were up-regulated specifically and greatly in roots of L. japonicus by Pi addition to the hydroponic solution. When the external Pi level increased, expressions of LjCLE19 and 20 increased before the increase in the Pi content in plants. On the other hand, when the external Pi level decreased, the Pi content in plants decreased first, then expression levels of LjCLE19 and 20 decreased. Based on our results, we discuss the relationship between LjCLE19 and 20 and the tissue Pi levels in plants. This is the first report showing induction of specific CLE genes by phosphate.     

Overexpression of <Emphasis Type="Italic">Petunia SOC1</Emphasis>-like Gene <Emphasis Type="Italic">FBP21</Emphasis> in Tobacco Promotes Flowering Without Decreasing Flower or Fruit Quantity

FBP21 is one of the SOC1-like genes isolated from Petunia hybrida. Based on sequence analysis, FPB21 is suggested to have a role in promoting flowering. In this study, FBP21 was expressed in a tobacco host plant under the control of the CaMV 35S promoter. Our results showed that the transgene accelerated flowering, i.e. the transgenic plants flowered just 3 months after germination, in comparison to the wild-type tobacco which flowered after 5 months. Plant morphology was also affected, with the transgenic tobacco plants developing at least five robust lateral branches, while the control plants generally had just three. Total leaf area was significantly reduced in the transgenic tobacco compared to wild-type tobacco. By contrast, there was no significant difference between transgenic and control plants for the total number of flowers or fruits. Thus, the flower or fruit yield expressed per unit leaf area was higher in transgenic tobacco than in wild-type plants. Semi-quantitative RT-PCR analysis indicated that overexpression of FBP21 in tobacco resulted in the up-regulation of some flowering-related genes. The results of this study in tobacco indicate that the Petunia FBP21 gene may permit the engineering of early-flowering and short-growth habits without compromising flower or fruit yields.     

Isolation of <Emphasis Type="Italic">madA</Emphasis> homologs in <Emphasis Type="Italic">Pilobolus crystallinus</Emphasis>

Pilobolus crystallinus shows unique photoresponses at various growing stages. cDNAs for putative photoreceptors were cloned from this fungus. Three genes named pcmada1, pcmada2, and pcmada3 were identified from the PCR fragments, and amplified with degenerated primers for the LOV domain, which is conserved in many blue-light receptors. Deduced amino acid sequences for PCMADA1, PCMADA2, and PCMADA3 had one light-oxygen-voltage (LOV)-sensing and two PER-ARNT-SIM (PAS) domains. A zinc finger DNA-binding motif was conserved in the C-terminals of PCMADA1 and PCMADA3. However, PCMADA2 lacked the zinc finger motif. Expression of pcmada1 was suppressed by blue light whereas that of pcmada3 was promoted by blue-light irradiation.     

Influence of co-evolution with a parasite, <Emphasis Type="Italic">Nosema whitei,</Emphasis> and population size on recombination rates and fitness in the red flour beetle, <Emphasis Type="Italic">Tribolium castaneum</Emphasis>

The high prevalence of meiotic recombination—an important element of sexual reproduction—represents one of the greatest puzzles in biology. The influence of either selection by a co-evolving parasite alone or in combination with genetic drift on recombination rates was tested in the host-parasite system Tribolium castaneum and Nosema whitei. After eight generations, populations with smaller genetic drift had a lower recombination rate than those with high drift whereas parasites had no effect. Interestingly, changes in recombination rate at one site of the chromosome negatively correlated with changes at the adjacent site on the same chromosome indicating an occurrence of crossover interference. The occurrence of spontaneous or plastic changes in recombination rates could be excluded with a separate experiment.     

Molecular genetic mapping of the <Emphasis Type="Italic">sy1</Emphasis> and <Emphasis Type="Italic">sy9</Emphasis> asynaptic genes in rye (<Emphasis Type="Italic">Secale cereale</Emphasis> L.) using microsatellite and isozyme markers

Studies of phenotypical expression of synaptic mutations in combination with the localization of corresponding genes on a genetic map permit individual stages of the meiotic process to be differentiated. Two rye asynaptic genes, sy1 and sy9, were mapped with the use of microsatellite markers (SSR) in the pericentromeric regions of the long chromosome arms 7R and 2R, respectively. The sy9 gene cosegregated with two SSR markers Xscm43 and Xgwm132. The asynaptic gene sy1 was mapped within the interval between the isozyme locus Aat2 and two cosegregating loci Xrems1188 and Xrems1135 that are located at a distance of 0.4 cM proximally and 0.1 cM distally with respect to the gene lous. Possible evolutionary relationships of the mapped genes with homeological loci of the Triticeae species and more distant cereal species, such as maize and rice, are discussed.     

Ectopic expression of two MADS box genes from orchid (<Emphasis Type="Italic">Oncidium</Emphasis> Gower Ramsey) and lily (<Emphasis Type="Italic">Lilium longiflorum)</Emphasis> alters flower transition and formation in <Emphasis Type="Italic">Eustoma grandiflorum</Emphasis>

Lisianthus [Eustoma grandiflorum (Raf.) Shinn] is a popular cut flower crop throughout the world, and the demand for this plant for cut flowers and potted plants has been increasing worldwide. Recent advances in genetic engineering have enabled the transformation and regeneration of plants to become a powerful tool for improvement of lisianthus. We have established a highly efficient plant regeneration system and Agrobacterium-mediated genetic transformation of E. grandiflorum. The greatest shoot regeneration frequency and number of shoot buds per explant are observed on media supplemented with 6-Benzylaminopurine (BAP) and α-Naphthalene acetic acid (NAA). We report an efficient plant regeneration system using leaf explants via organogenesis with high efficiency of transgenic plants (15%) in culture of 11 weeks’ duration. Further ectopic expression of two MADS box genes, LMADS1-M from lily (Lilium longiflorum) and OMADS1 from orchid (Oncidium Gower Ramsey), was performed in E. grandiflorum. Conversion of second whorl petals into sepal-like structures and alteration of third whorl stamen formation were observed in the transgenic E. grandiflorum plants ectopically expressing 35S::LMADS1-M. 35S::OMADS1 transgenic E. grandiflorum plants flowered significantly earlier than non-transgenic plants. This is the first report on the ectopic expression of two MADS box genes in E. grandiflorum using a simple and highly efficient gene transfer protocol. Our results reveal the potential for floral modification in E. grandiflorum through genetic transformation.     

Isolation of a <Emphasis Type="Italic">Ve</Emphasis> homolog, <Emphasis Type="Italic">mVe1</Emphasis>, and its relationship to verticillium wilt resistance in <Emphasis Type="Italic">Mentha longifolia</Emphasis> (L.) Huds

As a step toward greater understanding of the genetics of verticillium wilt resistance in plants, we report the sequencing of a candidate wilt resistance gene, mVe1, from the mint diploid model species, Mentha longifolia (Lamiaceae). mVe1 is a putative homolog of tomato (Solanum lycopersicum L.) verticillium wilt (Ve) resistance genes. The mVe1 gene has a coding region of 3,051 bp. The predicted mVe1 protein contains a leucine-rich repeat domain, a common feature of plant disease resistance proteins. We compared 13 mVe1 alleles from three mint species. These alleles shared 96.2–99.6% nucleotide identity. We analyzed four M. longifolia populations segregating with respect to mVe1 alleles and wilt resistance versus susceptibility and found one association between mVe1 genotype and wilt phenotype. We conclude that mVe1 may play a role in mint verticillium wilt resistance, but variation for resistance in our segregating progenies is likely polygenic. Therefore, further investigations of mVe1 and identification of additional candidate genes are both warranted.     

Analysis of a <Emphasis Type="Italic">c</Emphasis><Subscript>0</Subscript><Emphasis Type="Italic">t-1</Emphasis> library enables the targeted identification of minisatellite and satellite families in <Emphasis Type="Italic">Beta vulgaris</Emphasis>

Background  

Repetitive DNA is a major fraction of eukaryotic genomes and occurs particularly often in plants. Currently, the sequencing of the sugar beet (Beta vulgaris) genome is under way and knowledge of repetitive DNA sequences is critical for the genome annotation. We generated a c ₀ t-1 library, representing highly to moderately repetitive sequences, for the characterization of the major B. vulgaris repeat families. While highly abundant satellites are well-described, minisatellites are only poorly investigated in plants. Therefore, we focused on the identification and characterization of these tandemly repeated sequences.     

Complex regulation and multiple developmental functions of <Emphasis Type="Italic">misfire</Emphasis>, the <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>ferlin gene

Background  

Ferlins are membrane proteins with multiple C2 domains and proposed functions in Ca²⁺ mediated membrane-membrane interactions in animals. Caenorhabditis elegans has two ferlin genes, one of which is required for sperm function. Mammals have several ferlin genes and mutations in the human dysferlin (DYSF) and otoferlin (OTOF) genes result in muscular dystrophy and hearing loss, respectively. Drosophila melanogaster has a single ferlin gene called misfire (mfr). A previous study showed that a mfr mutation caused male sterility because of defects in fertilization. Here we analyze the expression and structure of the mfr gene and the consequences of multiple mutations to better understand the developmental function of ferlins.     

<Emphasis Type="Italic">IXR1</Emphasis> and <Emphasis Type="Italic">HMO1</Emphasis> genes jointly control the level of spontaneous mutagenesis in yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The yeast genes IXR1 and HMO1 encode proteins belonging to the family of chromatin nonhistone proteins, which are able to recognize and bind to irregular DNA structures. The full deletion of gene IXR1 leads to an increase in cell resistance to the lethal action of UV light, γ-rays, and MMS, increases spontaneous mutagenesis and significantlly decreases the level of UV-induced mutations. It was earlier demonstrated in our works that the hmo1 mutation renders cells sensitive to the lethal action of cisplatin and virtually does not affect the sensitivity to UV light. Characteristically, the rates of spontaneous and UV-induced mutagenesis in the mutant are increased. Epistatic analysis of the double mutation hmo1 ixr1 demonstrated that the interaction of these genes in relation to the lethal effect of cisplatin and UV light, as well as UV-induced mutagenesis, is additive. This suggests that the products of genes HMO1 and IXR1 participate in different repair pathways. The ixr1 mutation significantly increases the rate of spontaneous mutagenesis mediated by replication errors, whereas mutation hmo1 increases the rate of repair mutagenesis. In wild-type cells, the level of spontaneous mutagenesis was nearly one order of magnitude lower than that obtained in cells of the double mutant. Consequently, the combined activity of the Hmo1 and the Ixr1 proteins provides efficient correction of both repair and replication errors.     

Rice Gene <Emphasis Type="Italic">OsDSR-1</Emphasis> Promotes Lateral Root Development in <Emphasis Type="Italic">Arabidopsis</Emphasis> Under High-Potassium Conditions

Rice gene Oryza sativa Drought Stress Response-1 (OsDSR-1) was one of the genes identified to be responsive to drought stress in the panicle of rice at booting and heading stages by both microarray and quantitative real-time PCR analyses. OsDSR-1 encodes a putative calcium-binding protein, and its overexpression in Arabidopsis rendered transgenic plants to produce much shorter lateral roots (LRs) than wild-type (WT) plants in the medium supplemented with abscisic acid (ABA), suggesting that OsDSR-1 may act as a positive regulator during the process of ABA inhibition of LR development. No significant difference was observed in the total LR length between WT and transgenic plants in the media with the increase of only osmotic stress caused by NaCl, LiCl, and mannitol, while transgenic Arabidopsis seedlings appeared to produce larger root systems with longer total LR lengths under high-potassium conditions than WT seedlings. Further analysis showed that external Ca²⁺ was required for the production of larger root systems, indicating that the promotion by OsDSR-1 of the LR development of transgenic Arabidopsis seemed to occur in a Ca²⁺-dependent manner under high-potassium conditions. We propose that OsDSR-1 may function as a calcium sensor of the signal transduction pathway controlling the LR development under high-potassium conditions.     

The order of the <Emphasis Type="Italic">bs,Skdh</Emphasis>, and <Emphasis Type="Italic">Aadh1</Emphasis> genes in chromosome 5R of rye <Emphasis Type="Italic">Secale cereale</Emphasis> L.

Segregation analysis was performed in the progenies obtained in analyzing crosses of hybrids of spring and winter accessions of rye Secale cereale L. and wild S. montanum subsp. anatolicum (Grossh.) Tzvel. (syn. S. strictum (J. Presl) J. Presl). The test genes controlled the brittle stem (bs), the allelic variants of aromatic alcohol dehydrogenase (Aadh1) and shikimate dehydrogenase (Skdh), and the growth habit (Vrn1). A linkage was observed in the inheritance of the brittle stem and the aromatic alcohol dehydrogenase and shikimate dehydrogenase alloenzymes. The order of genes was established to be bs-Skdh-Aadh1, and the genetic distances were estimated to be bs-(9.0%)-Skdh, bs-(10.8%)-Aadh1, and Skdh-(5.3%)-Aadh1. The recombination coefficient between the Skdh and Aadh1 genes varied from 2.2 to 18.2%, averaging 5.3%. The growth habit was inherited independently of the bs-Skdh-Aadh1 linkage group.     

Photosynthetic and transpiration responses of <Emphasis Type="Italic">in vitro</Emphasis>-regenerated <Emphasis Type="Italic">Solanum nigrum</Emphasis> L. plants to <Emphasis Type="Italic">ex vitro</Emphasis> adaptation

In vitro regeneration of black nightshade (Solanum nigrum L.) plants was achieved through callus-mediated shoot organogenesis followed by 30 d indoor ex vitro adaptation to nutritional stress under environmental ambience and thereafter 6-d outdoor acclimatization in pots prior to field establishment. Relevant physiological parameters including pigment content, chlorophyll a fluorescence, net photosynthetic rate (P _N), transpiration rate (E), and stomatal conductance (g _s) of in vitro-regenerated plants were investigated during the course of ex vitro adaptation. During the first 4 d of indoor transplantation to potting substrate, there was a marginal reduction in the leaf chlorophyll and carotenoid contents but P _N and E were strongly reduced. The stomatal conductance and E/P _N ratio were significantly higher in plants up to 20 d of indoor adaptation than those of comparable age grown naturally from seeds. The shape of the OJIP fluorescence transient varied significantly with acclimatization, and the maximum change was observed at 2.0 ms. The 2.0 ms variable fluorescence (V _j), 30 ms relative fluorescence (M ₀), photon trapping probability (TR₀/Abs), and photosystem II (PSII) trapping rate (TR₀/RC) showed initial disturbance and subsequent stabilization during 30 d of indoor acclimatization. Energy dissipation (DI₀/RC) and electron transport probability (ET₀/TR₀) showed an initial phase of increase during the 4 d after plants were transplanted outdoors. During the 6-d outdoor acclimatization after transfer of plants to soil, no significant change in total chlorophylls and carotenoids, E, and g _s were observed, but P _N improved after reduction on the first d. The OJIP-derived parameters experienced change on the first d but were stabilized quickly thereafter. There was no significant difference between outdoor acclimatized plants and those of the seed-grown plants of comparable age with respect to photosynthetic and fluorescence parameters. Direct transfer of plants without indoor acclimatization, however, showed a completely different trend with respect to P _N, E, and OJIP fluorescence transients. The bearing of this study on optimizing micropropagation is discussed.     

Introduction of <Emphasis Type="Italic">OsglyII</Emphasis> gene into <Emphasis Type="Italic">Oryza sativa</Emphasis> for increasing salinity tolerance

Mature seed-derived embryogenic calli of indica rice (Oryza sativa L. cv. PAU201) were induced on semisolid Murashige and Skoog medium supplemented with 2.5 mg dm⁻³ 2,4-dichlorophenoxyacetic acid + 0.5 mg dm⁻³ kinetin + 560 mg dm⁻³ proline + 30 g dm⁻³ sucrose + 8 g dm⁻³ agar. Using OsglyII gene, out of 3180 calli bombarded, 32 plants were regenerated on medium containing hygromycin (30 mg dm⁻³). Histochemical GUS assay of the hygromycin selected calli revealed GUS expression in 50 % calli. Among the regenerants, 46.87 % were GUS positive. PCR analysis confirmed the presence of the transgene of 1 kb in 60 % of independent plants. Further, these plants have been grown to maturity in glasshouse. In vitro screening for salt tolerance showed increase in fresh mass of OsglyII putative transgenic calli (185.4 mg) as compared to control calli (84.2 mg) on 90 mM NaCl after 15 d. When exposed to 150 mM NaCl, OsglyII putative transgenic plantlets showed normal growth while the non-transgenic control plantlets turned yellow and finally did not survive.