Comparative characteristics of oat doubled haploids and oat × maize addition lines: Anatomical features of the leaves, chlorophyll a fluorescence and yield parameters.

As a result of oat (Avena sativa L.) × maize (Zea mays L.) crossing, maize chromosomes may not be completely eliminated at the early stages of embryogenesis, leading to the oat × maize addition (OMA) lines development. Introgression of maize chromosomes into oat genome can cause morphological and physiological modifications. The aim of the research was to evaluate the leaves' anatomy, chlorophyll a fluorescence, and yield parameter of oat doubled haploid (DH) and OMA lines obtained by oat × maize crossing. The present study examined two DH and two disomic OMA lines and revealed that they differ significantly in the majority of studied traits, apart from: the number of cells of the outer bundle sheath; light energy absorption; excitation energy trapped in PSII reaction centers; and energy dissipated from PSII. The OMA II line was characterized by larger size of single cells in the outer bundle sheath and greater number of seeds per plant among tested lines.

Effect of Soil Drought Stress on Selected Biochemical Parameters and Yield of Oat × Maize Addition (OMA) Lines.

Plant growth and the process of yield formation in crops are moderated by surrounding conditions, as well as the interaction of the genetic background of plants and the environment. In the last two decades, significant climatic changes have been observed, generating unfavorable and harmful impacts on plant development. Drought stress can be considered one of the most dangerous environmental factors affecting the life cycle of plants, reducing biomass production and, finally, the yield. Plants can respond to water deficit in a wide range, which depends on the species, genetic variability within the species, the plant's ontogenesis stage, the intensity of the stress, and other potential stress factors. In plants, it is possible to observe hybrids between different taxa that certain traits adopted to tolerate stress conditions better than the parent plants. Oat × maize addition (OMA) plants are good examples of hybrids generated via wide crossing. They can exhibit morphological, physiological, and biochemical variations implemented by the occurrence of extra chromosomes of maize, as well as the interaction of maize and oat chromatin. The initial goal of the study was to identify OMA lines among plants produced by wide crossing with maize. The main goal was to investigate differences in OMA lines according to the Excised Leaf Water Loss (ELWL) test and to identify specific biochemical changes and agronomic traits under optimal water conditions and soil drought. Additionally, detection of any potential alterations that are stable in F2 and F3 generations. The aforementioned outcomes were the basis for the selection of OMA lines that tolerate growth in an environment with limited water availability. The molecular analysis indicated 12.5% OMA lines among all tested descendants of wide oat-maize crossing. The OMA lines significantly differ according to ELWL test results, which implies some anatomical and physiological adaptation to water loss from tissues. On the first day of drought, plants possessed 34% more soluble sugars compared to control plants. On the fourteen day of drought, the amount of soluble sugars was reduced by 41.2%. A significant increase of phenolic compounds was observed in the fourteen day of drought, an average of 6%, even up to 57% in line 9. Soil drought substantially reduced stem biomass, grains number, and mass per plant. Lower water loss revealed by results of the ELWL test correlated with the high yield of OMA lines. Phenolic compound content might be used as a biochemical indicator of plant drought tolerance since there was a significant correlation with the high yield of plants subjected to soil drought.

Genetic melting pot and importance of long-distance dispersal indicated in the Gladiolus imbricatus L. populations in the Polish Carpathians.

The genetic diversity in 11 populations of Gladiolus imbricatus in five mountain ranges, including the Tatra, Pieniny, Gorce, Beskid Niski (Western Carpathians) and Bieszczady Mts (Eastern Carpathians), was studied with inter-simple sequence repeat (ISSR) markers. The species is a perennial plant occurring in open and semi-open sites of anthropogenic origin (meadows and forest margins). We checked a hypothesis on the microrefugial character of the plant populations in the Pieniny Mts, a small calcareous Carpathian range of complicated relief that has never been glaciated. Plant populations in the Tatra and Pieniny Mts had the highest genetic diversity indices, pointing to their long-term persistence. The refugial vs. the non-refugial mountain ranges accounted for a relatively high value of total genetic variation [analysis of molecular variance (AMOVA), 14.12%, p = 0.003]. One of the Pieniny populations was of hybridogenous origin and shared genetic stock with the Tatra population, indicating there is a local genetic melting pot. A weak genetic structuring of populations among particular regions was found (AMOVA, 4.5%, p > 0.05). This could be an effect of the frequent short-distance and sporadic long-distance gene flow. The dispersal of diaspores between the remote populations in the Western Carpathians and Eastern Carpathians could be affected by the historical transportation of flocks of sheep from the Tatra to Bieszczady Mts.

Functioning of the Photosynthetic Apparatus in Response to Drought Stress in Oat × Maize Addition Lines.

The oat × maize chromosome addition (OMA) lines, as hybrids between C3 and C4 plants, can potentially help us understand the process of C4 photosynthesis. However, photosynthesis is often affected by adverse environmental conditions, including drought stress. Therefore, to assess the functioning of the photosynthetic apparatus in OMA lines under drought stress, the chlorophyll content and chlorophyll a fluorescence (CF) parameters were investigated. With optimal hydration, most of the tested OMA lines, compared to oat cv. Bingo, showed higher pigment content, and some of them were characterized by increased values of selected CF parameters. Although 14 days of drought caused a decrease of chlorophylls and carotenoids, only slight changes in CF parameters were observed, which can indicate proper photosynthetic efficiency in most of examined OMA lines compared to oat cv. Bingo. The obtained data revealed that expected changes in hybrid functioning depend more on the specific maize chromosome and its interaction with the oat genome rather than the number of retained chromosomes. OMA lines not only constitute a powerful tool for maize genomics but also are a source of valuable variation in plant breeding, and can help us to understand plant susceptibility to drought. Our research confirms more efficient functioning of hybrid photosynthetic apparatus than oat cv. Bingo, therefore contributes to raising new questions in the fields of plant physiology and biochemistry. Due to the fact that the oat genome is not fully sequenced yet, the mechanism of enhanced photosynthetic efficiency in OMA lines requires further research.

3-D Nucleus Architecture in Oat × Maize Addition Lines.

The nucleus architecture of hybrid crop plants is not a well-researched topic, yet it can have important implications for their genetic stability and usefulness in the successful expression of agronomically desired traits. In this work we studied the spatial distribution of introgressed maize chromatin in oat × maize addition lines with the number of added maize chromosomes varying from one to four. The number of chromosome additions was confirmed by genomic in situ hybridization (GISH). Maize chromosome-specific simple sequence repeat (SSR) markers were used to identify the added chromosomes. GISH on 3-D root and leaf nuclei was performed to assess the number, volume, and position of the maize-chromatin occupied regions. We revealed that the maize chromosome territory (CT) associations of varying degree prevailed in the double disomic lines, while CT separation was the most common distribution pattern in the double monosomic line. In all analyzed lines, the regions occupied by maize CTs were located preferentially at the nuclear periphery. A comparison between the tissues showed that the maize CTs in the leaf nuclei are positioned closer to the center of the nucleus than in the root nuclei. These findings shed more light on the processes that shape the nucleus architecture in hybrids.

Chlorophyll a Fluorescence Parameters of Hulled and Hull-less Barley (Hordeum vulgare L.) DH Lines Inoculated with Fusarium culmorum.

Barley worldwide is affected seriously by Fusarium seedling blight (FSB) and Fusarium head blight (FHB) diseases caused by the Fusarium species. The objective of this study was to facilitate the resistance of hulled and hull-less barley at different growth stages to F. culmorum according to direct parameters: disease rating (DR), fresh weight of leaves and roots, kernel weight per spike, kernel number per spike, plump kernels, and indirect parameters - chlorophyll a fluorescence (CF). Plate assay, greenhouse and field tests were performed on 30 spring barley doubled haploid (DH) lines and their parents infected with Fusarium culmorum. Direct parameters proved that hulled genotypes show less symptoms. Most studied chlorophyll a fluorescence (CF) parameters (apart from DIo/CS - amount of energy dissipated from PSII for laboratory test, TRo/CS - amount of excitation energy trapped in PSII reaction centers, ETo/CS - amount of energy used for electron transport and RC/CS - number of active reaction centres in the state of fully reduced PSII reaction center in field experiment) were significantly affected by F. culmorum infection. In all experiments, hulled genotypes had higher values of CF parameters compared to hull-less ones. Significant correlations were detected between direct and indirect parameters and also between various environments. It was revealed that ABS/CS, TRo/CS, and RC/CS have significant positive correlation in greenhouse test and field experiment. Significant correlations suggest the possibility of applying the CF parameters in selection of barley DH lines resistant to F. culmorum infection.

Genotype by environment interaction using AMMI model and estimation of additive and epistasis gene effects for 1000-kernel weight in spring barley (Hordeum vulgare L.).

The objective of this study was to assess genotype by environment interaction for 1000-kernel weight in spring barley lines grown in South Poland by the additive main effects and multiplicative interaction model. The study comprised of 32 spring barley (Hordeum vulgare L.) genotypes (two parental genotypes-breeding line 1 N86 and doubled haploid (DH) line RK63/1, and 30 DH lines derived from F1 hybrids), evaluated at six locations in a randomized complete block design, with three replicates. 1000-kernel weight ranged from 24.35 g (for R63N/42 in 2011) to 61.46 g (for R63N/18 in 2008), with an average of 44.80 g. AMMI analyses revealed significant genotype and environmental effects as well as GE interaction with respect to 1000-kernel weight. In the analysis of variance, 16.86% of the total 1000-kernel weight variation was explained by environment, 32.18% by differences between genotypes, and 24.50% by GE interaction. The lines R63N/61, R63N/22, and R63N/1 are recommended for further inclusion in the breeding program because their stability and the highest averages of 1000-kernel weight. The total additive effect of all genes controlling the trait and the total epistasis effect of 1000-kernel weight were estimated. Additive gene action effects based on DH lines were always larger that this parameter estimated on the basis of parental lines. Estimates of additive gene action effects based on the all DH lines were significantly larger than zero in each year of study. Epistasis effects based on all DH lines were statistically significant in 2011 and 2013.

Complex characterization of oat (Avena sativa L.) lines obtained by wide crossing with maize (Zea mays L.).

BACKGROUND: The oat × maize addition (OMA) lines are used for mapping of the maize genome, the studies of centromere-specific histone (CENH3), gene expression, meiotic chromosome behavior and also for introducing maize C4 photosynthetic system to oat. The aim of our study was the identification and molecular-cytogenetic characterization of oat × maize hybrids. METHODS: Oat DH lines and oat × maize hybrids were obtained using the wide crossing of Avena sativa L. with Zea mays L. The plants identified as having a Grande-1 retrotransposon fragment, which produced seeds, were used for genomic in situ hybridization (GISH). RESULTS: A total of 138 oat lines obtained by crossing of 2,314 oat plants from 80 genotypes with maize cv. Waza were tested for the presence of maize chromosomes. The presence of maize chromatin was indicated in 66 lines by amplification of the PCR product (500 bp) generated using primers specific for the maize retrotransposon Grande-1. Genomic in situ hybridization (GISH) detected whole maize chromosomes in eight lines (40%). All of the analyzed plants possessed full complement of oat chromosomes. The number of maize chromosomes differed between the OMA lines. Four OMA lines possessed two maize chromosomes similar in size, three OMA-one maize chromosome, and one OMA-four maize chromosomes. In most of the lines, the detected chromosomes were labeled uniformly. The presence of six 45S rDNA loci was detected in oat chromosomes, but none of the added maize chromosomes in any of the lines carried 45S rDNA locus. Twenty of the analyzed lines did not possess whole maize chromosomes, but the introgression of maize chromatin in the oat chromosomes. Five of 66 hybrids were shorter in height, grassy type without panicles. Twenty-seven OMA lines were fertile and produced seeds ranging in number from 1-102 (in total 613). Sixty-three fertile DH lines, out of 72 which did not have an addition of maize chromosomes or chromatin, produced seeds in the range of 1-343 (in total 3,758). Obtained DH and OMA lines were fertile and produced seeds. DISCUSSION: In wide hybridization of oat with maize, the complete or incomplete chromosomes elimination of maize occur. Hybrids of oat and maize had a complete set of oat chromosomes without maize chromosomes, and a complete set of oat chromosomes with one to four retained maize chromosomes.

Evolution of meiotic recombination genes in maize and teosinte.

BACKGROUND: Meiotic recombination is a major source of genetic variation in eukaryotes. The role of recombination in evolution is recognized but little is known about how evolutionary forces affect the recombination pathway itself. Although the recombination pathway is fundamentally conserved across different species, genetic variation in recombination components and outcomes has been observed. Theoretical predictions and empirical studies suggest that changes in the recombination pathway are likely to provide adaptive abilities to populations experiencing directional or strong selection pressures, such as those occurring during species domestication. We hypothesized that adaptive changes in recombination may be associated with adaptive evolution patterns of genes involved in meiotic recombination. RESULTS: To examine how maize evolution and domestication affected meiotic recombination genes, we studied patterns of sequence polymorphism and divergence in eleven genes controlling key steps in the meiotic recombination pathway in a diverse set of maize inbred lines and several accessions of teosinte, the wild ancestor of maize. We discovered that, even though the recombination genes generally exhibited high sequence conservation expected in a pathway controlling a key cellular process, they showed substantial levels and diverse patterns of sequence polymorphism. Among others, we found differences in sequence polymorphism patterns between tropical and temperate maize germplasms. Several recombination genes displayed patterns of polymorphism indicative of adaptive evolution. CONCLUSIONS: Despite their ancient origin and overall sequence conservation, meiotic recombination genes can exhibit extensive and complex patterns of molecular evolution. Changes in these genes could affect the functioning of the recombination pathway, and may have contributed to the successful domestication of maize and its expansion to new cultivation areas.

Conversion of oat (Avena sativa L.) haploid embryos into plants in relation to embryo developmental stage and regeneration media.

Obtaining oat DH lines is only effective via wide crossing with maize. Seven hundred haploid embryos from 21 single F1 progeny obtained from wide crosses with maize were isolated, divided into four groups according to their size (<0.5 mm, 0.5-0.9 mm, 1.0-1.4 mm, and ≥1.5 mm), and transferred into 190-2 regeneration medium with different growth regulators: 0.5 mg L-1 kinetin (KIN) and 0.5 mg L-1 1-naphthaleneacetic acid (NAA); 1 mg L-1 zeatin (ZEA) and 0.5 mg L-1 NAA; or 1 mg L-1 dicamba (DIC), 1 mg L-1 picloram (PIC), and 0.5 mg L-1 kinetin (KIN). Among all isolated embryos, approximately 46.1% were between 1.0-1.4 mm, while the smallest group of embryos (7.1%) were those <0.5 mm. The ability of haploid embryos to germinate varied depending on oat genotypes and the size of embryos. Haploid embryos <0.5 mm were globular and did not germinate, whereas embryos ≥1.5 mm had clearly visible coleoptiles, radicles, and scutella, and were able to germinate. Germination of oat haploid embryos varied depending on growth regulators in the regeneration medium. Most haploid embryos germinated on medium with 0.5 mg L-1 NAA and 0.5 mg L-1 KIN, while the fewest germinated on medium with 1 mg L-1 DIC, 1 mg L-1 PIC, and 0.5 mg L-1 KIN. One hundred thirty germinated haploid embryos converted into haploid plants. Fifty oat DH lines were obtained after colchicine treatment.

In silico and in vivo studies of molecular structures and mechanisms of AtPCS1 protein involved in binding arsenite and/or cadmium in plant cells.

This paper reports a continuation of our previous research on the phytochelatin synthase1 (PCS1) gene involved in binding and sequestration of heavy metals or metalloids in plant cells. Construction of a 3D structure of the Arabidopsis thaliana PCS1 protein and prediction of gene function by employing iterative implementation of the threading assembly refinement (I-TASSER) revealed that PC ligands (3GC-gamma-glutamylcysteine) and Gln50, Pro53, Ala54, Tyr55, Cys56, Ile102, Gly161, His162, Phe163, Asp204 and Arg211 residues are essential for formation of chelating complex with cadmium (Cd²âº) or arsenite (AsIII). This finding suggests that the PCS1 protein might be involved in the production of the enzyme phytochelatin synthase, which might in turn bind, localize, store or sequester heavy metals in plant cells. For validation of the in silico results, we included a T-DNA tagged mutant of Arabidopsis thaliana, SAIL_650_C12, (mutation in AtPCS1 gene) in our investigation. Furthermore, using reverse transcriptase PCR we confirmed that the mutant does not express the AtPCS1 gene. Mutant plants of SAIL_650_C12 were exposed to various amounts of cadmium (Cd²âº) and arsenite (AsIII) and the accumulation of these toxic metals in the plant cells was quantified spectrophotometrically. The levels of Cd²âº and AsIII accumulation in the mutant were approximately 2.8 and 1.6 times higher, respectively, than that observed in the wild-type controlled plants. We confirmed that the results obtained in in silico analyses complement those obtained in in vivo experiments.

In silico and in vivo studies of an Arabidopsis thaliana gene, ACR2, putatively involved in arsenic accumulation in plants.

Previously, our in silico analyses identified four candidate genes that might be involved in uptake and/or accumulation of arsenics in plants: arsenate reductase 2 (ACR2), phytochelatin synthase 1 (PCS1) and two multi-drug resistant proteins (MRP1 and MRP2) [Lund et al. (2010) J Biol Syst 18:223-224]. We also postulated that one of these four genes, ACR2, seems to play a central role in this process. To investigate further, we have constructed a 3D structure of the Arabidopsis thaliana ACR2 protein using the iterative implementation of the threading assembly refinement (I-TASSER) server. These analyses revealed that, for catalytic metabolism of arsenate, the arsenate binding-loop (AB-loop) and residues Phe-53, Phe-54, Cys-134, Cys-136, Cys-141, Cys-145, and Lys-135 are essential for reducing arsenate to arsenic intermediates (arsenylated enzyme-substrate intermediates) and arsenite in plants. Thus, functional predictions suggest that the ACR2 protein is involved in the conversion of arsenate to arsenite in plant cells. To validate the in silico results, we exposed a transfer-DNA (T-DNA)-tagged mutant of A. thaliana (mutation in the ACR2 gene) to various amounts of arsenic. Reverse transcriptase PCR revealed that the mutant exhibits significantly reduced expression of the ACR2 gene. Spectrophotometric analyses revealed that the amount of accumulated arsenic compounds in this mutant was approximately six times higher than that observed in control plants. The results obtained from in silico analyses are in complete agreement with those obtained in laboratory experiments.