The rpl23 gene and pseudogene are hotspots of illegitimate recombination in barley chloroplast mutator seedlings.

Previously, through a TILLING (Targeting Induced Local Lesions in Genomes) approach applied on barley chloroplast mutator (cpm) seedlings a high frequency of polymorphisms in the rpl23 gene was detected. All the polymorphisms corresponded to five differences already known to exist in nature between the rpl23 gene located in the inverted repeats (IRs) and the rpl23 pseudogene located in the large single copy region (LSC). In this investigation, polymorphisms in the rpl23 gene were verified and besides, a similar situation was found for the pseudogene in cpm seedlings. On the other hand, no polymorphisms were found in any of those loci in 40 wild type barley seedlings. Those facts and the independent occurrence of polymorphisms in the gene and pseudogene in individual seedlings suggest that the detected polymorphisms initially arose from gene conversion between gene and pseudogene. Moreover, an additional recombination process involving small recombinant segments seems to occur between the two gene copies as a consequence of their location in the IRs. These and previous results support the hypothesis that the CPM protein is a component of the plastome mismatch repair (MMR) system, whose failure of the anti-recombination activity results in increased illegitimate recombination between the rpl23 gene and pseudogene.

Effects of ionizing radiation on Capsicum baccatum var. pendulum (Solanaceae).

Cytogenetic and somatic effects of various x-ray treatments were evaluated in pepper, Capsicum baccatum var. pendulum cv. "Cayenne", with the aim to assess optimal conditions for obtaining viable lines. The cytogenetic effects were quantified by counting chromosome aberrations. The level of DNA fragmentation was estimated with TUNEL test (terminal transferase mediated dUTP-fluorescein nick end labeling). Irradiation to 20 Gy with 16-h presoaking can be a suitable treatment of the selected pepper cultivar for a mutagenesis program.

A cytoplasmically inherited mutant controlling early chloroplast development in barley seedlings.

Cytoplasmic line 2 (CL2) has been previously reported as a cytoplasmically inherited chlorophyll-deficient mutant selected from a chloroplast-mutator genotype of barley. It was characterized by a localized effect on the upper part of the first-leaf blade. At emergence the CL2 seedlings-phenotype varied from a grainy light green to an albino color. They gradually greened during the following days, starting from the base of the blade and extending to cover most of its surface when it was fully grown. The present results, from both light microscopy and transmission electron microscopy (TEM), confirmed the previously described positional and time-dependent expression of the CL2 syndrome along the first-leaf blade. During the first days after emergence, light microscopy showed a normally developed chloroplast at the middle part of the CL2 first-leaf blade, meanwhile at the tip only small plastids were observed. TEM showed that the shapes and the internal structure of the small plastids were abnormal, presenting features of proplastids, amyloplasts and/or senescent gerontoplasts. Besides, they lack plastid ribosomes, contrasting with what was observed inside chloroplasts from normal tips, which presented abundant ribosomes. Phenotypic observations and spectrophotometric analysis of seedlings produced by mother plants that had been grown under different temperatures indicated that higher temperatures during seed formation were negatively associated with pigment content in CL2 seedlings. In contrast, higher temperatures during the growth of CL2 seedlings have been associated with increased pigment content. Aqueous solution with kanamycin and streptomycin, which are antibiotics known to interfere with plastid gene translation, were used for imbibition of wild-type and CL2 seeds. Antibiotic treatments differentially reduced the chlorophyll content in the upper part of the first-leaf blade in CL2, but not in wild-type seedlings. These results suggest that in the wild-type, plastid-gene proteins which are necessary for chloroplast development and chlorophyll synthesis in the upper part of the first-leaf blade are usually synthesized during embryogenesis. However, under certain circumstances, in CL2 seedlings, they would be synthesized after germination. In addition, a shortening of the sheath has been observed in association with pigment decrease suggesting the existence of plastid factors affecting the expression of some nuclear genes. We consider the CL2 mutant a unique experimental material useful to study biological phenomena and external factors regulating plastid, and nuclear gene expression during embryogenesis and early seedling development.

Isolation and molecular characterization of atrazine tolerant barley mutants.

Two atrazine-tolerant barley mutants were isolated from atrazine-selection experiments performed on barley chloroplast-mutator plants. Genetic analysis demonstrated that atrazine tolerance was maternally inherited. Molecular characterization of the mutants was performed by PCR amplification of an internal fragment of the chloroplast gene psbA. The BstXI restriction patterns of the amplified fragments showed two bands in both tolerant barley mutants and only one in the atrazine-sensitive control. The 277-bp amplified fragments from the parental line and both atrazine-tolerant mutants were cloned and sequenced. Sequence analysis showed a single nucleotide substitution in both barley atrazine-tolerant mutants, i.e. A to G at the +790 position of the psbA gene-coding sequence. This point mutation corresponds to an amino-acid change of serine- to -glycine and creates a BstXI restriction site. Our results confirmed the conservative variability involved in atrazine tolerance which was previously reported for several other species. To our knowledge this is the first report on the obtention of atrazine-tolerant barley. This finding provides support to the hypothesis that, in addition to a wide variety of chlorophyll deficiencies, the barley chloroplast mutator genotype induces variability in other traits, which could include agronomically valuable mutants.

A mutator nuclear gene inducing a wide spectrum of cytoplasmically inherited chlorophyll deficiences in barley.

Some striped plants were observed in plots of a long-grain mutant barley grown at a field nursery. All of the plants of these plots, which were naturally self pollinated, were individually harvested, and most of their progenies (92.5%) segregated seedlings carrying chlorophyll deficiencies (CD) as determined by greenhouse analysis. The majority of the mutant seedlings (84.3%) showed a pattern of longitudinal chlorophyll sectors. The spectrum of CD was wide among the solid mutant seedlings and consisted of three main types (albina, viridis and discontinuous). In association with some CD types morphological changes were frequently observed. Non-CD-associated morphological changes and diminished seed-set were scarce and, so far, none of them has proved to be inherited. Analysis of CD in reciprocal crosses and backcrosses proved that while CD were transmitted cytoplasmically their induction was controlled by a single nuclear mutator gene, active when homozygous. In addition once the CD were induced, they were expressed independently of the nuclear constitution. The results suggest that the mutator gene induces diverse mutational events on chloroplast (cp) DNA. In barley, as in other monocots, nuclear genes which are inductors of cytoplasmic genetic changes have been reported. However, all of them produced a narrower spectrum of CD and had a more rapid sorting-out of the cytoplasmic mutants than what we observed. On this basis a distinction between chloroplast and mitochondrial (mt) mutator genes is proposed. Accordingly, the chloroplast mutator here described would be the first one reported for monocots. Increased knowledge on this subject can play a fundamental role in elucidating organelle heredity and its interactions with the nuclear genome. Moreover, this material could be a valuable source of variability of the otherwise conservative genetic information encoded in the chloroplast.

Influence of Marker Genes on the Expression of Somatic Mutations in Barley.

Mutagenic agents have been evaluated in terms of their effects on germ cells. The study of induced somatic mutations is a faster, more economical, and less laborious method than the traditional ones. The induction of somatic sectors by x-rays, ethyl methanesulfonate, and sodium azide was studied in cultivated barley. Four stocks were used: a normal one, a balanced lethal genotype carrying two chlorophyll-deficiency genes of different expression (albina and golden), and two (semidominant) chlorophyll mutants of the same dominant gene, which in the progeny of selling segregate 1 normal green: 2 light green: 1 yellow seedlings. Treatments were applied to seeds, and the fourth, fifth, and sixth leaves of the main shoot were examined in the M1 generation for the presence of sectors showing conspicuous changes in chlorophyll content. Highly significant frequencies of dark green sectors were induced by all three mutagens in the (semidominant) heterozygous plants, whereas evidence of somatic crossing over or mitotic nondisjunction was very rare in this material and in the balanced lethal genotype. In all cases, the frequencies of induced sectors without chlorophyll were much higher in the heterozygotes than in the normal homozygotes, with each mutagen showing typical dose-response curves. A notable interaction between mutagen and genotype was found. The variation in the relative frequencies of sector types in the various genotypes points to the differential influence of the marker genes.