MicroRNA-Based Markers in Plant Genome Response to Abiotic Stress and Their Application in Plant Genotyping

The high conservation of miRNA sequences provided an opportunity to develop an effective type of markers that is useful not only for genetic diversity study but also as potential biomarkers in plant stress responses. The fundamental potential of miRNA-based markers relies on the primer design based on the sequences of mature miRNAs, which are part of the step-loop structures. The advantages of this marker system include high polymorphism, reproducibility, and transferability across species. The abundance of mature miRNAs, which is linked to the expression of MIRNA genes, varies greatly among miRNAs, tissue types, or developmental stages, indicating the spatially and temporally regulated expression patterns of plant miRNAs. The results confirm the significance, reliability, and the position of miRNA-based markers as stress-sensitive biomarkers, indicating their potential in a wide range of applications of agricultural research

Lunasin detection in coloured wheat genotype

Lunasin is a biologically active protein, composed of 43 amino acid residues. There has been proven many health-promoting effects of lunasin peptide. The most important health benefits include: anti-hypertension, antioxidant activity, cancer prevention or therapy. It was also demonstrated anti-inflammation, hypocholesterolemic activity, anti-obesity and immunomodulation. The focus of our research is to summarize the discovery, characterization and biological activities of lunasin, which will provide a reference for the future development and utilization of lunasin, and a basis for exploring the underlying mechanisms of these health-beneficial functions. Lunasin was first isolated in 1987 at Niigata University School of Medicine in Japan, during the screening of protease inhibitors from soybean seeds. It was subsequently found in other beans, grains and herbal plants, including wheat, barley, rye, triticale. Concentration of lunasin is ranging from 0.013 to 70.5 mg protein lunasin/g of protein. Big step forward in the understanding of the lunasine operating mechanism in the fight against cancer has arisen after study on cloning of the soybean lunasin gene and subsequent transfection into mammalian cells which led to the discovery that the lunasin gene can disrupt mitosis and induce chromosome breakage, ultimately leading to cell apoptosis. The main goal of our work was to evaluate collection of wheat with unusual grain colour for presence of lunasin gene. DNA was extracted by commercial kit and lunasin gene was detected by PCR reaction. Our results showed presence of lunasin gene detected by 3 combinations of 2 sets of primer pair and indicated lunasin peptide presence in cereal grains. These findings are necessary to confirmed by proteome analysis

Comparison of American and European maize (Zea mays L.) protein profiles

Maize is an important crop with great use in agriculture, food and other industries. The aim of this work was to compare 30 varieties of Zea mays L., that were grown in the USA and Europe in terms of nutritional quality and to genetically distinguish them by creating a dendrogram of relationship. The content of total nitrogen, crude protein content, fractional composition of proteins and the calculated coefficient of nutritional quality were all determined. Compared to American varieties, higher nutritional quality was shown by the varieties of European origin with the coefficient of nutritional quality higher about 17.55%. However, the highest value was achieved by the American variety Howling Mob (331.91%). Storage proteins - glutelins were divided according to molecular weight into HMW-GS (high molecular weight glutelin subunits), LMW-GS (low molecular weight glutelin subunits) plus zeins and residual albumins and globulins by electrophoretic separation. The biggest difference was recorded among HMW-GS content - 5.77% (American varieties) and 7.35% (European varieties), while the LMW-GS plus zeins and residual albumins and globulins content was similar. 20 protein bands (15 polymorphic and 5 monomorphic) in American varieties and 18 protein bands (10 polymorphic and 8 monomorphic) in European varieties were evaluated in the electrophoretograms. Based on the presence of individual proteins, a dendrogram was constructed, using the UPGMA (Unweighted Pair-Group Method Using Arithmetic Averages) method and the Jaccard coefficient of similarity. In the dendrogram, 2 clusters of varieties with the dissimilarity 0.481 were formed. In the subgroups, from the point of glutelin polymorphism, the varieties Illinois Hulles and Queen Anna were separated from the others. The European varieties were segregated within the second cluster and created the common subgroup of 2 varieties - Madarska cukrova (Hungary) and Zlota Handlowa (Poland)

Polymorfizmus bielkovín vo vybraných slovenských odrodách pšenice letnej použitím SDS-PAGE

Winter wheat is especially used for bread-making. The specific composition of the grain storage proteins and the representation of individual subunits determines the baking quality of wheat. The aim of this study was to analyze 15 slovak varieties of the winter wheat (Triticum aestivum L.) based on protein polymorphism and to predict their technological quality. SDS-PAGE method by ISTA was used to separate glutenin protein subunits. Glutenins were separated into HMW-GS (15.13%) and LMW-GS (65.89%) on the basis of molecular weight in SDS-PAGE. At the locus Glu- A1 was found allele Null (53% of genotypes) and allele 1 (47% of genotypes). The locus Glu-B1 was represented by the HMW-GS subunits 6+8 (33% of genotypes), 7+8 (27% of genotypes), 7+9 (40% of genotypes). At the locus Glu-D1 were detected two subunits, 2+12 (33% of genotypes) and 5+10 (67% of genotypes) which is correlated with good bread-making properties. The Glu – score was ranged from 4 (genotype Viglanka) to 10 (genotypes Viola, Vladarka). According to the representation of individual glutenin subunits in samples, the dendrogram of genetic similarity was constructed. By the prediction of quality the results showed that the best technological quality was significant in the varieties Viola and Vladarka which are suitable for use in food processing.Pšenica letná, forma ozimná sa využíva najmä na výrobu chleba. Špecifické zloženie zásobných bielkovín zrna a zastúpenie individuálnych podjednotiek určuje pekársku kvalitu pšenice. Cieľom tejto práce bolo analyzovať 15 slovenských odrôd pšenice letnej, formy ozimnej (Triticum aestivum L.) z hľadiska polymorfizmu bielkovín a predigovať ich technologickú kvalitu. Na separáciu glutenínových podjednotiek bola použitá metóda ISTA SDS-PAGE. Gluteníny boli rozdelené v SDS - PAGE na základe molekulovej hmotnosti na HMW-GS (15,13%) a LMW-GS (65,89%). Na lokuse Glu-A1 bola zistená prítomnosť alely 0 (53% genotypov) a alely 1 (47% genotypov). Lokus Glu-B1 bol reprezentovaný HMW-GS podjednotkami 6+8 (33% genotypov), 7+8 (27% genotypov), 7+9 (40% genotypov). Na lokuse Glu-D1 boli detegované dve podjednotky, a to 2+12 (33% genotypov) a 5+10 (67% genotypov), ktorá je spájaná s dobrými pekárskymi vlastnosťami. Hodnota Glu-skóre sa pohybovala od 4 (odroda Viglanka) do 10 (odrody Viola, Vladarka). Na základe zastúpenia jednotlivých glutenínových podjednotiek vo vzorkách bol zostrojený dendrogram genetickej príbuznosti. Predikciou kvality z výsledkov vyplýva, že najlepšiu technologickú kvalitu vykazovali odrody Viola a Vladarka, ktoré sú vhodné na využitie v potravinárskom priemysle

Start codon targeted (scot) polymorphism reveals genetic diversity in european old maize (Zea mays L.) Genotypes

Maize (Zea mays L.) is one of the world's most important crop plants following wheat and rice, which provides staple food to large number of human population in the world. It is cultivated in a wider range of environments than wheat and rice because of its greater adaptability. Molecular characterization is frequently used by maize breeders as an alternative method for selecting more promising genotypes and reducing the cost and time needed to develop hybrid combinations. In the present investigation 40 genotypes of maize from Czechoslovakia, Hungary, Poland, Union of Soviet Socialist Republics, Slovakia and Yugoslavia were analysed using 20 Start codon targeted (SCoT) markers. These primers produced total 114 fragments across 40 maize genotypes, of which 86 (76.43%) were polymorphic with an average of 4.30 polymorphic fragments per primer and number of amplified fragments ranged from 2 (SCoT 45) to 8 (SCoT 28 and SCoT 63). The polymorphic information content (PIC) value ranged from 0.374 (ScoT 45) to 0.846 (SCoT 28) with an average of 0.739. The dendrogram based on hierarchical cluster analysis using UPGMA algorithm was prepared. The hierarchical cluster analysis showed that the maize genotypes were divided into two main clusters. Unique maize genotype (cluster 1), Zuta Brzica, originating from Yugoslavia separated from others. Cluster 2 was divided into two main clusters (2a and 2b). Subcluster 2a contained one Yugoslavian genotype Juhoslavanska and subcluster 2b was divided in two subclusters 2ba and 2bb. The present study shows effectiveness of employing SCoT markers in analysis of maize, and would be useful for further studies in population genetics, conservation genetics and genotypes improvement

Detection genetic variability of secale cereale L. by scot markers

Rye (Secale cereale L.) is our traditional cereal used for baking. The genetic variability of grown rye has been reduced by modern agronomic practices, which subsequently prompted the importance of search for species that could be useful as a gene pool for the improving of flour quality for human consumption or for other industrial uses. Therefore, the aim of this study was to detect genetic variability among the set of 45 rye genotypes using 8 SCoT markers. Amplification of genomic DNA of 45 genotypes, using SCoT analysis, yielded 114 fragments, with an average of 14.25 polymorphic fragments per primer. The most polymorphic primer was SCoT 36, where 21 polymorphic amplification products were detected. In contract the lowest polymorphic primer was SCoT 45 with 5 polymorphic products. Genetic polymorphism was characterized based on diversity index (DI), probability of identity (PI) and polymorphic information content (PIC). The hierarchical cluster analysis showed that the rye genotypes were divided into 2 main clusters. One rye genotype Motto, origin from Poland formed a separate subcluster (1b). Subscluster 2a included only genotype Valtické (CSK). In this experiment, SCoT proved to be a rapid, reliable and practicable method for revealing of polymorphism in the rye cultivars

Bioinformatic approach in the identification of arabidopsis gene homologous in amaranthus

Bioinfomatics offers an efficient tool for molecular genetics applications and sequence homology search algorithms became an inevitable part for many different research strategies. Appropriate managing of known data that are stored in public available databases can be used in many ways in the research. Here, we report the identification of RmlC-like cupins superfamily protein DNA sequence than is known in Arabidopsis genome for the Amaranthus - plant specie where this sequence was still not sequenced. A BLAST based approach was used to identify the homologous sequences in the nucleotide database and to find suitable parts of the Arabidopsis sequence were primers can be designed. In total, 64 hits were found in nucleotide database for Arabidopsis RmlC-like cupins sequence. A query cover ranged from 10% up to the 100% among RmlC-like cupins nucleotides and its homologues that are actually stored in public nucleotide databases. The most conserved region was identified for matches that posses nucleotides in the range of 1506 up to the 1925 bp of RmlC-like cupins DNA sequence stored in the database. The in silico approach was subsequently used in PCR analysis where the specifity of designed primers was approved. A unique, 250 bp long fragment was obtained for Amaranthus cruentus and a hybride Amaranthus hypochondriacus x hybridus in our analysis. Bioinformatic based analysis of unknown parts of the plant genomes as showed in this study is a very good additional tool in PCR based analysis of plant variability. This approach is suitable in the case for plants, where concrete genomic data are still missing for the appropriate genes, as was demonstrated for Amaranthus.&nbsp

Molecular characterization of rye cultivars

The results of molecular analysis of 45 rye taxa (Secale cereale L.) represented by agricultural varieties originated from Central Europe and the Union of Soviet Socialist Republics (SUN) are presented. The genetic diversity of rye cultivars by 6 SSR markers was evaluated. Six specific microsatellite primer pairs produced 58 polymorphic alleles with an average of 9.7 alleles per locus. The number of alleles ranged from 6 (SCM2) to 14 (SCM86). Genetic polymorphism was characterized based on diversity index (DI), probability of identity (PI) and polymorphic information content (PIC). The diversity index (DI) of SSR markers ranged from 0.5478 (SCM2) to 0.887 (SCM86) with an average of 0.778. The lowest value of polymorphic information content was recorded for SCM2 (0.484) and the highest value for SCM86 (0.885) of PIC was detected in SCM86 with an average of 0.760.The dendrogram of genetic similarity was constructed, based on UPGMA algorithm. The hierarchical cluster analysis divided rye genotypes into 4 main clusters. The first cluster of 14 genotypes was subdivided in two subclusters (1a and 1b) where 50% of genotypes were Czechoslovak origin. The second cluster contained four genotypes were three (75%) of them had Czech or Czechoslovak origin. In the third subcluster separated three rye genotypes of different origin. The rest (24) of rye genotypes in the fourth cluster were divided into two subclusters (4a and 4b) where clearly separated group of Polish (4aa) and Czech and Czechoslovak (4ab) genotypes. Two genotypes of 4aa subcluster (Wojcieszyckie and Dankowskie Nowe) from Poland were genetically the closest. In the dendrogram alle genotypes were differentiated and clustering partially reflects geographic origin of studied rye genotypes. In this experiment, SSRs markers proved to be a high informative and usefull tool in genetic diversity research for the distinguishing and characterization of close related varieties

Molecular variability of oat based on gene specific markers

Oat (Avena sativa L.) is a grass planted as a cereal crop. Cultivation of oat is increasing in the recent years because of its good nutrition value. The aim of our study was to analyze genetic variability of oat accessions based on SCoT markers. Eighteen primers were used to study polymorfism of 8 oat genotypes. All 18 primers produced polymorphic and reproducible data. Altogether 153 different fragments were amplified of which 67 were polymorphic with an average number of 3.72 polymorphic fragments per genotype. The number of polymorphic fragments ranged from one (SCoT9, SCoT62) to nine (SCoT40). The percentage of polymorphic bands ranged from 14.29% (SCoT9) to 60% (SCoT59) with an average of 41.62%. Genetic polymorphism was characterized based on diversity index (DI), probability of identity (PI) and polymorphic information content (PIC). The diversity index of the tested SCoT markers ranged from 0 (SCoT9, SCoT62) to 0.878 (SCoT40) with an average of 0.574. The polymorphic information content ranged from 0 (SCoT9, SCoT62) to 0.876 (SCoT40) with an average of 0.524. Dendrogram based on hierarchical cluster analysis using UPGMA algorithm grouped genotypes into two main clusters. Two genotypes, Taiko and Vok were genetically the closest. Results showed the utility of SCoT markers for estimation of genetic diversity of oat genotypes leading to genotype identification