Biotehnologija u oplemenjivanju kukuruza

Maize is one of the most important economic crops and the best studied and most tractable genetic system among monocots. The development of biotechnology has led to a great increase in our knowledge of maize genetics and understanding of the structure and behaviour of maize genomes. Conventional breeding practices can now be complemented by a number of new and powerful techniques. Some of these often referred to as molecular methods, enable scientists to see the layout of the entire genome of any organism and to select plants with preferred characteristics by "reading" at the molecular level, saving precious time and resources. DNA markers have provided valuable tools in various analyses ranging from phylogenetic analysis to the positional cloning of genes. Application of molecular markers for genetic studies of maize include: assessment of genetic variability and characterization of germ plasm, identification and fingerprinting of genotypes, estimation of genetic distance, detection of monogamic and quantitative trait loci, marker assisted selection, identification of sequence of useful candidate genes, etc. The development of high-density molecular maps which has been facilitated by PCR-based markers, have made the mapping and tagging of almost any trait possible and serve as bases for marker assisted selection. Sequencing of maize genomes would help to elucidate gene function, gene regulation and their expression. Modern biotechnology also includes an array of tools for introducing or deieting a particular gene or genes to produce plants with novel traits. Development of informatics and biotechnology are resulted in bioinformatic as well as in expansion of microarrey technique. Modern biotechnologies could complement and improve the efficiency of traditional selection and breeding techniques to enhance agricultural productivity.Kukuruz je jedan od ekonomski najznačajnijih useva i model sistem za genetička ispitivanja kod monokotila. Razvoj biotehnologije je omogućio bolje razumevanje strukture i funkcije genoma kukuruza a konvencionalno oplemenjivanje je dopunjeno novim i moćnim tehnikama. Neke od njih omogućavaju naučnicima da sagledaju strukturu celog genoma i odaberu biljke s poželjnim svojstvima na molekularnom nivou, štedeći vreme i resurse. Primena molekularnih markera uključuje ispitivanje genetičke varijabilnosti i karakterizaciju germplazme; identifikaciju gena koji kontrolišu agronomski važne osobine; selekciju pomoću markera. Sekvencioniranje genoma kukuruza pomaže rasvetljavanju funkcije, regulacije i ekspresije gena. Moderna biotehnologija uključuje seriju tehnika koje omogućavaju prenos gena iz drugih organizama ili deaktivaciju postojećih gena i stvaranje genotipova sa novim osobinama. Razvoj informatike i biotehnologije rezultirao je u stvaranju bioinformatike i omogućio je širu primenu mikroarrey tehnike. Moderna biotehnologija može da dopuni i poboljša efikasnost klasičnog oplemenjivanja u cilju stvaranja visokorodnih genotipova kukuruza otpornih na bolesti i stres

Comparative genomics of Arabidopsis and maize: prospects and limitations

The completed Arabidopsis genome seems to be of limited value as a model for maize genomics. In addition to the expansion of repetitive sequences in maize and the lack of genomic micro-colinearity, maize-specific or highly-diverged proteins contribute to a predicted maize proteome of about 50,000 proteins, twice the size of that of Arabidopsis

FunnyBase: a systems level functional annotation of Fundulus ESTs for the analysis of gene expression

BACKGROUND: While studies of non-model organisms are critical for many research areas, such as evolution, development, and environmental biology, they present particular challenges for both experimental and computational genomic level research. Resources such as mass-produced microarrays and the computational tools linking these data to functional annotation at the system and pathway level are rarely available for non-model species. This type of "systems-level" analysis is critical to the understanding of patterns of gene expression that underlie biological processes. RESULTS: We describe a bioinformatics pipeline known as FunnyBase that has been used to store, annotate, and analyze 40,363 expressed sequence tags (ESTs) from the heart and liver of the fish, Fundulus heteroclitus. Primary annotations based on sequence similarity are linked to networks of systematic annotation in Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) and can be queried and computationally utilized in downstream analyses. Steps are taken to ensure that the annotation is self-consistent and that the structure of GO is used to identify higher level functions that may not be annotated directly. An integrated framework for cDNA library production, sequencing, quality control, expression data generation, and systems-level analysis is presented and utilized. In a case study, a set of genes, that had statistically significant regression between gene expression levels and environmental temperature along the Atlantic Coast, shows a statistically significant (P < 0.001) enrichment in genes associated with amine metabolism. CONCLUSION: The methods described have application for functional genomics studies, particularly among non-model organisms. The web interface for FunnyBase can be accessed at . Data and source code are available by request at [email protected]

Analysis of splicing related proteins to study characteristics of splicing mechanisms in plants

About 80% of plant genes are interrupted by introns. Splicing is the procedure to remove introns from pre-mRNA. Although the splicing machinery (splicesome) is generally conserved, the mechanism for splice site selection and splicing control may vary among eukaryotic organisms. Splicing mechanisms in plants are poorly understood due to the lack of an in vitro splicing system. In this study, both computational and experimental methods were used to study plant-splicing mechanisms. The Arabidopsis genome was searched by a three-round BLAST method using Drosophila and human splicing related proteins as query. In total, 262 splicing related proteins were identified and classified into four groups according to their functions and homologs. About 60% of splicing related genes were duplicated in Arabidopsis. The level of variation and duplication varied from snRNP to splicing regulators. snRNPs are the most conserved, splicing factors are less conserved. Splicing regulators display more variation and duplication than the other two groups. Proteins involved in splice site selection were studied in detail by experimental methods, with focus on U2AF1. Two copies of U2AF1 were found in both Arabidopsis (AUSa and AUSb) and maize (ZUSa and ZUSb). These homologs all have single intron in the 5\u27UTR region. RT-PCR revealed that both copies in Arabidopsis and maize were expressed in major tissues, and that the intron could be alternatively spliced. Promoter::GUS assay revealed subtle differences in the expression pattern between AUSa and AUSb. AUSa was expressed strongly in flower but weakly in roots, while AUSb was expressed strongly in roots. These facts indicate that the substrate of AUSa and AUSb may be different. Mutants with T-DNA inserted into the AUSa promoter showed late flowering phenotype, possibly due to the splicing pattern of some genes in flowering pathway were altered. Novel SR proteins in Arabidopsis and maize were also identified and compared. Both computational and experimental studies demonstrate that the general splicing mechanism is conserved in plants, but that the splicing regulation mechanism is more variable. This is reasonable for organisms to adapt to their environments. Plants may have developed special ways to control splicing during evolution

Discovery of induced point mutations in maize genes by TILLING

BACKGROUND: Going from a gene sequence to its function in the context of a whole organism requires a strategy for targeting mutations, referred to as reverse genetics. Reverse genetics is highly desirable in the modern genomics era; however, the most powerful methods are generally restricted to a few model organisms. Previously, we introduced a reverse-genetic strategy with the potential for general applicability to organisms that lack well-developed genetic tools. Our TILLING (Targeting Induced Local Lesions IN Genomes) method uses chemical mutagenesis followed by screening for single-base changes to discover induced mutations that alter protein function. TILLING was shown to be an effective reverse genetic strategy by the establishment of a high-throughput TILLING facility and the delivery of thousands of point mutations in hundreds of Arabidopsis genes to members of the plant biology community. RESULTS: We demonstrate that high-throughput TILLING is applicable to maize, an important crop plant with a large genome but with limited reverse-genetic resources currently available. We screened pools of DNA samples for mutations in 1-kb segments from 11 different genes, obtaining 17 independent induced mutations from a population of 750 pollen-mutagenized maize plants. One of the genes targeted was the DMT102 chromomethylase gene, for which we obtained an allelic series of three missense mutations that are predicted to be strongly deleterious. CONCLUSIONS: Our findings indicate that TILLING is a broadly applicable and efficient reverse-genetic strategy. We are establishing a public TILLING service for maize modeled on the existing Arabidopsis TILLING Project

Ascorbate content of wheat leaves is not determined by maximal L-galactono-1,4-lactone dehydrogenase (GalLDH) activity under drought stress

Although ascorbic acid (AA) is a high-abundance metabolite, relatively little is known about the factors controlling its accumulation in leaves. To address this issue, we examined the role of l-galactono-1,4-lactone dehydrogenase (GalLDH), the enzyme which catalyses the last step of this pathway, in the control of AA content under optimal and stress conditions. In a range of species, no clear relationship between AA content and leaf GalLDH protein and activity was found under optimal growth conditions. To explore the effect of drought stress on GalLDH activity and protein content, wheat (Triticum aestivum L.) was selected for detailed analysis, using two cultivars that differ in their constitutive AA level. In well-watered plants, the AA content of cv Buck Chambergo (BCH) was over twice that of cv Cooperativa Maipún (CM) but dehydroascorbic acid content was similar in both cv. In agreement with this, dehydroascorbate reductase and glutathione reductase activities were higher in cv BCH than in cv CM, indicating a higher capacity for AA regeneration. Neither leaf DHA content nor activities of AA regenerating enzymes were modified by drought. Although drought caused a substantial increase in GalLDH protein and activity in the low AA cv CM, this treatment had no effect on these parameters in cv BCH. Notably, leaf AA content was unaffected by drought in either cv. These results suggest that GalLDH protein and activity cannot be used as an indicator for changes in the capacity for ascorbate biosynthesis and that AA biosynthesis is constrained by other factors under stress. This can be explained by the importance of regeneration in maintaining AA levels and possibly also by redox regulation of GalLDH.Instituto de Fisiología Vegeta

Conserved Subgroups and Developmental Regulation in the Monocot rop Gene Family

Rop small GTPases are plant-specific signaling proteins with roles in pollen and vegetative cell growth, abscisic acid signal transduction, stress responses, and pathogen resistance. We have characterized the rop family in the monocots maize (Zea mays) and rice (Oryza sativa). The maize genome contains at least nine expressed rops, and the fully sequenced rice genome has seven. Based on phylogenetic analyses of all available Rops, the family can be subdivided into four groups that predate the divergence of monocots and dicots; at least three have been maintained in both lineages. However, the Rop family has evolved differently in the two lineages, with each exhibiting apparent expansion in different groups. These analyses, together with genetic mapping and identification of conserved non-coding sequences, predict orthology for specific rice and maize rops. We also identified consensus protein sequence elements specific to each Rop group. A survey of ROP-mRNA expression in maize, based on multiplex reverse transcriptase-polymerase chain reaction and a massively parallel signature sequencing database, showed significant spatial and temporal overlap of the nine transcripts, with high levels of all nine in tissues in which cells are actively dividing and expanding. However, only a subset of rops was highly expressed in mature leaves and pollen. Intriguingly, the grouping of maize rops based on hierarchical clustering of expression profiles was remarkably similar to that obtained by phylogenetic analysis. We hypothesize that the Rop groups represent classes with distinct functions, which are specified by the unique protein sequence elements in each group and by their distinct expression patterns

Genome-wide mutagenesis of Zea mays L. using RescueMu transposons

Derived from the maize Mu1 transposon, RescueMu provides strategies for maize gene discovery and mutant phenotypic analysis. 9.92 Mb of gene-enriched sequences next to RescueMu insertion sites were co-assembled with expressed sequence tags and analyzed. Multiple plasmid recoveries identified probable germinal insertions and screening of RescueMu plasmid libraries identified plants containing probable germinal insertions. Although frequently recovered parental insertions and insertion hotspots reduce the efficiency of gene discovery per plasmid, RescueMu targets a large variety of genes and produces knockout mutants