Term
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Definition
| A strong promoter in most cell types. Often used to over-express a gene experimentally. |
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Term
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Definition
| Three overlapping domains of genes are expressed in the flower to form floral organs. Sepals require Ap2, petals require Ap2 plus Ap3/Pi, stamens require Ap3/Pi plus Ag, and carpels require Ag. |
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Term
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Definition
| A family of transposable elements in corn. Ac is 4,563 bp, and is the autonomous element, encoding transposase. Ds is a non-autonomous element without Ac. At the site of insertion it prouces an 8 bp repeat that flanks the TE. It has an inverted repeat at termini of 11 bp, necessary for its excission. Usually jumps to adjacent regions on the same chromosome. It jumps into the pigment gene, producing white phenotype. It can jump in and out in development of a kernel, producing spotted phenotype. |
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Term
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Definition
| A phenolic compound released by wounded plants. It can protect plants, acting as a signalling molecule. Attracts Agrobacterium tumefaciens to the wounding site, and activates Vir A. |
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Term
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Definition
| A gene in the ABC model. Required for formation of stamens and carpels. It is repressed by Ap2. When mutated, stamens are replaced with petals, and carpels are replaced by sepals. In the mutant, there is Ap2 activity in whorls 3 and 4. |
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Term
| Agrobacterium tumefaciens |
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Definition
Rhizobium radiobacter
A method of introducing DNA into regenerable cells in plant genetic transformation. A free-living soil bacterium. Attracted to acetosyringone, which is released by wounded plants. Infects wounded plants and causes crown gall. Infects dicots regularly, but is not a disease of monocots, except for superstrains. Hijacks plant metabolism to produce a compound only the bacterium can use as food. Has a Ti plasmid, and inserts T-DNA into the plant host. |
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Term
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Definition
| A polyhaploid derived from an allopolyploid, such as durum wheat. |
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Term
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Definition
Alloploidy
Amphidiploidy
Results from interspecific and intergeneric hybridization, accompanied by sexual doubling of chromosomes. Played a major role in evolution of crops including wheat and oats. Enjoys benefits of hybridity as well as polyploidy, making it highly adaptable to diverse environments. |
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Term
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Definition
| A gene in the ABC model. Required for formation of sepals and petals. When mutated, sepals are replaced with carpels, and petals are replaced with stamens. The gene normally represses transcription of Ag in whorls 1 and 2. In the mutant, Ag activity occurs in these whorls. Its mRNA is present in all four whorls in wild-type, but its activity is suppressed in whorls 3 and 4 by miRNA 172. |
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Term
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Definition
| Plant transformation is simple, and does not requre a tissue culture system. The inflorescence is dipped in Agrobacterium tumefaciens, and seeds produced are transformed plants; very easy transformation. Its genome was sequenced in 2000, and it was discovered to have over 25,000 genes. In 2002, a huge project produced lines of T-DNA insertions using Agrobacterium tumefaciens. |
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Term
| Autonomous transposable element |
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Definition
| A DNA transposable element which can control its own movement. Can jump into a gene and knock out its expression. If an element excises late in development, chimeric tissues can be observed, producing phenotypes such as spotting in corn kernel aleurons. Encodes a transposase. Can allow non-autonomous transposable elements to move, revealing their presence when a line with autonomous elements is crossed with a line with non-autonomous elements. |
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Term
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Definition
| A polyhaploid derived from an autopolyploid, such as potato. |
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Term
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Definition
| A locus in corn which controls pigmentation of the stalk and husk. B-1 allele causes purple pigmentation, and can spontaneously mutate by an unknown cause, into B', which has the same DNA sequence, but causes no pigmentation (green). When B-1/B-1 is crossed with B'/B', the progeny, B'/B-1, have no pigment but when F1 is backcrossed to B-1/B-1, there is not the expected 1:1 segregation, instead all progeny have no pigment. This is paramutation; the B-1 allele became altered by B' when they were together in the heterozygote parent, becoming B-1*, which has the same DNA sequence, and the ability to silence other B-1 alleles. Used to identify mediator of paramutation 1. Both alleles have enhancer regions consisting of 7 copies of a 853 bp repeat, 100 kb away from the transcriptional start site. The enhancer is transcribed from both DNA strands, separately from the protein product, producing double-stranded RNA which is cleaved into siRNA by dicer. The siRNA serves as a primer, working with RNA-dependent RNA polymerase to create more double-stranded RNA, and more siRNA. A threshold is reached where DNA modifies the enhancer chromatin to inhibit protein product expression, with methylation, altering histones, and DNA folding. There are unknown factors that are necessary, apart from the siRNA. Tandem repeats are necessary for expression and silencing. |
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Term
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Definition
1902 - 1992
Discovered DNA transposable elements. Published articles on transposable elements in 1953, however there were only two reprint requests, and most of the scientific community did not understand her work. It was not mainstream genetics, and many people did want to see that genes could move themselves. She applied to study plant breeding at Cornell, but was rejected because she was a woman! It took a long time for her to be recognized for her work. She received the Nobel Prize in medicine in 1983 at age 81. Genetics 136: 1 - 10 is an interesting biography of her life. |
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Term
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Definition
| It can take 16 - 40 weeks to produce a transformed plant. Leaf disk transformation doesn't work. A pollinated inflorescence must be dissected for immature embryos, which are treated with Agrobacterium tumefaciens superstrains. |
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Term
| Bacillus thuringiensis (Bt) |
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Definition
| A bacteria with an endotoxin that is inserted as a transgene into plants to confer insect resistance. Kills European corn borer by causing problems in their gut. |
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Term
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Definition
| Produces only random chromosome assortment, regardless of where the gene is on the chromosome. |
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Term
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Definition
Undifferentiated cells
Can be derived from an explant, with intermdiate ratios of cytokinins to auxin. May be transformed. It can form an embryo, or shoots and roots in tissue culture medium. |
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Term
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Definition
| An enzyme used to cut the mismatched DNA strands near SNPs during tilling. |
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Term
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Definition
| Frequency of codons used differs between plants and bacteria, resulting in low protein levels in transgenes that come from bacteria (including Bacillus thuringiensis). Certain degenerate codons are used at higher frequencies than others, and tRNAs are produced at corresponding levels to biased codon usage. Reduced expression of transgenes is caused by insufficient quantities of corresponding tRNA to make proteins. To solve the problem, mutate the transgene to have codons that match those in high frequency in the plant, guaranteeing sufficient tRNA. |
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Term
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Definition
Maize
It cannot be transformed with a floral dip. A tissue culture sytem with embryos is necessary to generate transformed plants. It is impractical to use T-DNA insertions to generate large populations of insertion knockouts for reverse genetics. The genome is 85% transposable elements. A model plant. During development of kernels, stripes can occur due to the way cells divide in columns. |
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Term
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Definition
| A system that can cause a double-stranded break. Two possible outcomes: gene knockout, or gene insertion by homologous recombination. Can be used to create knockout mutations in any gene. An old gene may be spliced out and replaced, or a new gene inserted to modify an old one. Could be used to insert a transgene into an area of the genome with high expression, as a solution to position effects and chromatin structure. It can be switched out for another gene, or as an insertion nearby, where it will likely be in active chromatin. Before its discovery, plant scientists had no method of knocking out specific genes. Technology has advanced, and is a new hot topic. Became widely applicable in 2015, and is now used routinely. If there is no mutant for a gene of interest available in genetic resources it is the best approach. Delivery of CRISPR/Cas9 constructs into plants is evolving such that development of stable transgenic lines through Agrobacterium tumefaciens transformation may not be necessary to insert a gene or cause mutation. Transient expression of one or both components of the system by various methods may be sufficient to achieve a desirable result. Delivery systems include infiltration of Agrobacterium into leaf cells, viral vector delivery, particle gun bombardment of both constructs, gRNA into a plant with stable Cas9 transformation, delivery of ribonucleotide complex containing Cas9 protein and gRNA, and direct delivery of RNA for the Cas9 and gRNA scaffold. |
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Term
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Definition
| A disease in plants caused by Agrobacterium tumefaciens. The bacteria infect wounded plants, and cause abnormal tumour-like growths. Gall tissuses can grow in culture without hormones added, because they produce their own hormones. Gall tissues produce unusual amino acids, opine, nopaline, and octapine, which the plant cannot use, but are catabolized by the bacteria. |
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Term
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Definition
| Used to identify trisomic lines. Rings can occur when there are two crossover events. It is a dying art, but can be useful. |
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Term
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Definition
| An enzyme which recognizes and cuts double stranded miRNA hairpins, from replicating viruses or in PTGS. Cuts dsRNA into 21 - 25 nucleotide fragments, siRNA, which can then bind to RISC complexes. Can come from replication of plant viruses. Includes many types, for different pathways. |
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Term
| DNA transposable element (TE) |
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Definition
DNA transposon
Discovered by Barbara McClintock. A piece of DNA that can move within the nuclear genome. Not to be confused with a retrotransposon. Includes autonomous and non-autonomous transposable elements. A source of mutations when inserted into genes. Can excise from a gene and cause reversions, seen as spots or stripes in corn kernels. Reversion events at different times in development are associated with different magnitudes of reversion. Includes Ac/Ds and Spm. |
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Term
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Definition
Triticum turgidum
Macaroni wheat
Important cereal for human consumption. Several commercial cultivars are produced by hybridization with corn, forming disomic tetraploid plants. Includes emmer wheat, a tetraploid from the cross of T. urartu and Aegilops speltoides. |
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Term
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Definition
| A piece of differentiated plant tissue. A stem segment, hypocotyl, or leaf disk. It is manipulated on artificial medium to yield a new plant, either by forming a callus or embryo. Response of cells is manipulated by the ratio of cytokinin to auxin. A high ratio produces shoots, and a low ratio produces roots. Intermediate ratios form callus. No hormone causes no growth. |
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Term
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Definition
| First a mutation is caused, and then the genotype and gene are found. |
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Term
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Definition
| DNA sequencing projects can predict genes present in an organism. Substantial numbers of genes have no predicted function. Computers can't define function of genes with high confidence using similarities from previously studied genes; experimental confirmation is needed in most cases. Indirect information on cellular or developmental function can be obtained from spatial and temporal expression patterns. The most precise way to determine gene function is to knock out expression and observe phenotype. |
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Term
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Definition
| Sometimes there is no mutant phenotype unless all genes in a family are mutated. In this case, it is nearly impossible to randomly mutate members of a gene family simultaneously in one M2 plant. Instead, T-DNA tagged lines may be crossed to produce single, double, or triple mutants in forward genetic screens. |
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Term
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Definition
Create a binary system with two plasmids. Remove oncogenes for overproduction of hormones, and manipulate hormones in culture medium. Add selectable marker. Genes need eukaryotic promoter and 3' regions to terminate transcription and add poly-A tails. Plasmids include:
1. Disarmed Ti plasmid, with missing T-DNA.
2. Plasmid with T-DNA, gene of interest, and selectable marker. |
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Term
| Genome in situ hybridization (GISH) |
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Definition
| DNA is labelled with a fluorescent probe. |
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Term
| Global analysis of gene expression |
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Definition
| Measurement of gene expression of all genes, under different conditions. Allows for identification of genes which may be involved in particular biochemical or physiological pathways important for traits. Could lead to candidates for knockouts and advanced genetic analysis. |
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Term
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Definition
2n = x
Monohaploid
Monoploid
A plant with half the chromosome number, derived from a diploid species. Sporophytes are still denoted by 2n, despite having one set of chromosomes. Because there is only one copy and allele of each gene, it makes studying mutations easy. Used in practical breeding. |
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Term
| Herbicide resistance marker |
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Definition
| A selectable marker that is herbicide resistance. A treatment of herbicide will kill all non-transofrmed cells. |
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Term
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Definition
| A reactive oxygen species. A possible treatment that could affect gene expression of some genes. |
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Term
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Definition
| A method of marker free transgenics. Independent assortment of the gene of interest from the selectable marker. A plasmid is constructed with two independent T-DNA regions, one containing the gene of interest and the other with the selectable marker. The two genes are not linked when integrating into the DNA, and they can insert on different chromosomes, or at different loci in the same chromosome. After a plant is selected with both the gene of interest and selectable marker, the two genes are separated through independent assortment, or recombination if they are linked. Recovers a plant with only the gene of interest. |
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Term
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Definition
Shotgun approach
A method of knocking out specific genes which was used in the early 2000s, before discovery of CRISPR. Large populations are generated with the hope of producing mutants for every gene. Mutant lines were catalogued, identifying mutated genes by sequencing. Scientists could order genetic stocks for genes of interest. Includes T-DNA tagged lines, natural transposons, and tilling. |
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Term
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Definition
| Crossed tetraploid wheat (AABB) with corn (CC) to get ABC. They then eliminated C to get AB, an allopolyhaploid. The C genome chromosomes formed a micronucleus which degraded in the cell. The plants were mostly sterile, but produced small amounts of seeds. |
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Term
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Definition
| A locus which encodes a micro RNA that is constitutively expressed from the 35S promoter. When mutated it results in altered leaf development (jaw-D). Has homology to TCP genes which are important for cell division in developing leaves. After cell division the miRNA is expressed in some cells to repress TCP to stop further divisions so that differentiation can take place. When it is overexpressed, there are crinkled leaves, resulting from altered cell divisions. |
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Term
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Definition
| Knocking out single genes to determine function. Can be done using CRISPR, insertion collections, or tilling. |
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Term
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Definition
| Includes N, P, and K. Needed in tissue culture mixture. |
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Term
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Definition
| A method of producing transgenic plants without selectable markers. Strategies consider eliminating markers after transformation. Includes independent assortment and recombination with the T-DNA. |
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Term
| Matrix attachment site (MARS) |
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Definition
| Specific scaffold attachment regions which define domains in the genome. Transgenes may be placed between MARS sites to insulate them from the environment, as a solution to position effects and chromatin structure. In some cases it causes less variability among transgenics, and in somec cases causes overall increase in expression levels. Can sometimes work to prevent position effect. |
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Term
| Mediator of paramutation (Mop-1) |
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Definition
| A large F1 population of B'/B-1 was created by crossing B'/B' with active Mu transposons with B-1/B-1. Over 500 F1 plants were selfed, producing 500 F2 families, 25 of which were segergating 3 no pigment:1 pigment, and the rest of which were all no pigment. The segergating families are caused by a Mu transposon insertion into a locus which was mapped with molecular markers and named mop-1. This locus allows for paramutation and gene silencing of the B-1 allele. Prevents paramutation when a Mu transposon is inserted, making it null. It was found to be syntenic with a region of the rice genome that encoded RNA-dependent RNA polymerase. |
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Term
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Definition
| Meiosis I reduces chromosome number. Meiosis II maintains chromosome number. |
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Term
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Definition
| A selectable marker that is an enzyme, such as phosphor-mannose isomerase (PMI). If you add mannose-6-phosphate to the growth medium as a carbon source, cells will not grow unless PMI is also present to convert it into fructose-6-phosphate, which is usable by the plant cell. Better than an herbicide marker. |
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Term
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Definition
| Associated with chromatin folding and inactivation of gene expression. siRNA from RNA polymerase V leads to methylation and silencing of retrotransposons. When DNA is methylated it leads to histone methylation, leading to inactive loops. Silences thousands of transposons in the genome. |
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Term
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Definition
| Non-coding, endogenous small RNAs. A gene with a promoter encodes RNA over several hundred nucleotides long, which can structurally form a hairpin loop. The transcript does not encode protein, but it has homology to a sector of a known, functional gene that encodes protein product, often a transcription factor. The hairpin loop forms double-stranded RNA which is recognized by dicer to produce a miRNA duplex. One miRNA can bind to the RISC complex to cleave the mRNA of the homologous gene. Processes regulated this way include leaf, floral, shoot, root, and vascular development, phase change in meristems from vegetative to floral, and phosphate accumulation. Species vary with the number of miRNAs, but most plants have over 1,000. Can be grouped into families based on sequence similarities. Can be classified as non-transposable element-like or transposable element-like. |
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Term
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Definition
| A microRNA in Arabidopsis. Has homology to Ap2 locus. It is expressed in whorls 3 and 4. Represses Ap2 through siRNA, rather than cleavage and degradation. Support for it regulating Ap2 gene expression: when it is overexpressed in all four whorls with a 35S promoter, ap2 mutant phenotype is observed because it eliminates Ap2 in all whorls; and when mutated miRNA 172 is expressed with a 35S promoter, ag mutant phenotype is observed, because Ap2 is not repressed and will repress Ag activity. |
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Term
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Definition
| Used in a system designed to take advantage of autonomous and non-autonomous transposons, to prevent transposons from jumping out of genes of interest, producing stable insertions throughout the genome. Can be very effective because they transpose into low-copy number, non-repeated regions (gene rich), and can jump across chromosomes. A genetic stock with many non-autonomous Mu transposons is crossed to a line with autonomous Mu transposons, activating non-autonomous elements in F1. F1 is then selfed to produce F2 and F3 families where lines with only non-autonomous Mu elements are selected through independent assortment. These lines have stable Mu insertions in new locations. A large number of plants with Mu insertions across the genome can be produced. Flanked sequences around Mu elements in each family could be high-throughput sequenced and entered in a database so researchers can order genetic lines with an insertion in a gene of interest. Because there would be 40 - 50 Mu insertions in the genome, the researcher would need to backcross with a tester to produce a line with a Mu insertion only in the gene of interest, to properly observe knockout phenotype. Used to identify mediator of paramutation 1. |
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Term
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Definition
Transposon tagging
An insertion collection technique for producing knockouts used in corn. Includes engineered transposon systems such as Mu transposon. |
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Term
| Non-autonomous transposable element |
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Definition
| A DNA transposable element which relies upon the presence of autonomous elements for movement. Stable, and do not move without the presence of an autonomous element, which provides transposase. |
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Term
| Non-transposable element-like |
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Definition
| Micro RNAs which arise from duplication of protein-coding genes. |
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Term
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Definition
| A mutation where gene expression is completely absent. Can be produced from T-DNA insertions. The plant may not tolerate complete absence of a functional protein if it is critical for metabolism, growth, or development. Lethal null phenotypes are not informative; there are no seeds, just empty ovules. |
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Term
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Definition
| In paramutation, the allele with normal expression until it is in the same plant with a paramutagenic allele. |
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Term
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Definition
| In paramutation, the allele with no expression or altered expression. It alters the expression of the paramutable allele. |
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Term
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Definition
| A process where one allele influences the expression of another when two are present in a heterozygote. A paramutagenic allele alters the expression of a paramutable allele, without altering DNA sequence. Includes R and B alleles. |
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Term
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Definition
A method of introducing DNA to regenerable cells in plant genetic transformation. Often used in monocot species in days before superstrains of Agrobacterium tumefaciens were developed. DNA is precipitated onto small gold particles which are then shot by a puff of air into plant cells on a screen. Takes a lot of time to form a callus. Location of DNA insertion is erratic. Commonly there are inverted repeats in the transgene that form hairpins as mRNA and are identified as dsRNA, leading to methylation of the DNA.
1. Bombard cells
2. Callus growth without selection
3. Callus growth with selection
4. Regeneration of transgenic plant |
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Term
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Definition
| It would not be a good gene to use as a selectable marker, because it may have issues with expression, and can lead to false negatives. |
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Term
| Plant genetic transformation |
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Definition
| Introduction of a DNA fragment into a plant cell, such that it is inherited in a stable fashion. Can be in nuclear or cytoplasmic DNA. First done by Monsanto in tobacco in 1984, now done in over 100 species in over 35 families. When it was first discovered it was thought that it was the end of plant breeding, and plants could be designed to be perfect. It is not this simple, and is not always consistent. Often thousands of plants are transformed before a good one is found. For transformation, target tissue needs to be available, including competent cells for plant regeneration. A method of introducing DNA to the regenerable cell is needed: common methods include Agrobacterium tumefaciens and particle gun bombardment. Needs a procedure to select and regenerate transformed plants at a satisfactory frequency. In most plants an in vitro technique is required, and complexity of protocols can vary from transforming a leaf disk (common in Solanaceae) to transforming immature embryos dissected from developing seeds. A system of plant tissue culture is often required, where the explant is manipulated on artificial medium to yield a new plant. DNA can be introduced to an explant or callus derived from an explant. Results is a plant with foreign DNA that is sexually transmissible to progeny. Problems with transgenics include low levels of expression caused by position effects and chromatin structure, codon usage, and transgene silencing. Never assume that a transgene is incorporated without testing it. |
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Term
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Definition
| Infects plants systemically, spreading throughout the whole plant. Most are single-stranded mRNA viruses, which replicate using a dsRNA intermediate, requiring RNA-dependent RNA polymerase. Transcript levels are very high. Plant cells can often clear out a virus. |
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Term
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Definition
| A mutation that causes an amino acid substitution or premature stop codon that is not lethal, but may result in a weakened protein that provides an identifiable phenotype to study gene function. One can easily generate hundreds of families with point mutations in different genes. Correct family with a mutated gene of interest must be identified from the population. |
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Term
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Definition
| A haploid plant derived from a polyploid species. Includes allopolyhaploids and autopolyhaploids. |
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Term
| Position effects & chromatin structure |
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Definition
| A problem with transgenics, causing low levels of expression. Different domains are present in the genome, based on loops in scaffolding. Domains have different levels of gene activity based on DNA folding, which can change during development, altering gene expression. A transgene may not be expressed in a folded inactive domain, or expression may vary through development. Ideally a transgene would be in a domain that is active throughout the life cycle. One may need to screen thousands of transformants to find one with stable, high expression. Possible solutions include: place the transgene between matrix attachment sites, to insulate the transgene from the environment; find a plant with an appropriately expressed transgene, and use it as the foundation for new lines by switching the original transgene with another, or adding one nearby; and CRIPSR/Cas9. |
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Term
| Post-transcriptional gene silencing (PTGS) |
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Definition
| Transgene silencing where there is transcription, but no accumulation of mRNA, and no protein or gene expression. Related to natural mechanisms of virus resistance. Plant RNA-dependent RNA polymerase (RdRP) makes RNAs into dsRNA, such as those with high copy number (including viral mRNA, transgene mRNA, or mRNA with defencts). Transgene mRNA is often expressed with strong constitutive promoters, and can be aberrant, prematurely terminated, or miss-spliced. The dsRNA is cut by dicer, and the siRNA produced is used to cleave all homologous mRNA with a RISC complex. A virus infects the plant and spreads systemically, with viral mRNA at high levels in the cell. Viral ssRNA replicates using RdRP, with a dsRNA intermediate; this allows PTGS system to recognize and cleave viral mRNA. |
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Term
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Definition
| Regulation of traits can be through changes in protein levels or structure, independent of gene transcription levels. Changes which affect gene expression will not be detectable by global analysis of gene expression, unless proteins were measured. |
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Term
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Definition
| A locus in corn which controls pigmentation in the seed aleurone. When the paramutable allele, R-r/R-r (full pigment) is crossed with the null allele, r/r (no pigment), the progeny, R-r/r, have full pigment. When the paramutagenic allele, R-st/R-st (stippled) is crossed to R-r/R-r, the progeny, R-r/R-st, have full pigment. When this heterozygote, R-r/R-st is crossed to r/r, instead of the expected 1 stippled:1 full pigment segregation, there is 1 stippled:1 no pigment. This is paramutation: the R-r allele is altered by the R-st allele when they are together in the heterozygote parent, such that it becomes non-functional, however the DNA sequence of R-r is unchanged. |
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Term
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Definition
| A method of marker free transgenics. Within the left and right borders of the T-DNA, insert a gene of interest plus a region that will be spliced out through homologous recombination. Within this region, place the selectable marker and reombinase gene, controlled by a chemically inducible promoter such as ethanol. The transformed plant is identified by the selectable marker, and is then treated with ethanol that activates the recombinase, leading to excision of all genetic material between recombination sequences, leaving only the transgene of interest. |
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Term
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Definition
| Before the internet, if you wanted to read a scientific article, you could request that a personal copy be sent to you from the owner of the original. |
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Term
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Definition
| Produces RNA which is reverse-transcribed into double-stranded DNA that is incorporated into the genome. Not to be confused with a DNA transposon. |
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Term
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Definition
| First a gene sequence is obtained, then the function is determined by knocking it out to look for mutant phenotype. It does not miss genes which would be invisible using forward screening, such as cauliflower mutant in Arabidopsis. |
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Term
| RNA-dependent RNA polymerase (RdRP) |
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Definition
| Synthesizes RNA from an RNA template. Used by plant viruses to replicate ssRNA at high copy numbers, with a dsRNA intermediate. Also found in plants, where it makes RNAs with high copy number or defects double-stranded, and they are cut by dicer. Allows plant cells to clear viral mRNA infections in some cases. A mutation causes plants to be hypersensitive to viral infections, with more intense symptoms. |
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Term
| RNA-induced sliencing complex (RISC) |
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Definition
| A protein complex to which the siRNA binds. siRNA is made by dicer. Causes cleavage events in mRNA of the functional gene homologous to the siRNA. Results in cleavage and degradation of the mRNA. A defence against viruses. Occurs most frequently in plants. |
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Term
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Definition
| Used for transcription of genes. |
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Term
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Definition
| Transcribes heterochromatic regions with transposons, producing single-stranded RNA that is involved in generation of siRNA through dicer. |
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Term
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Definition
| Transcribes heterochromatic regions with transposons, producing single stranded RNA that is invovled in the silencing complex of DNA. The siRNA binds to RNA as it is synthesized, and methylates the DNA in that region of the chromosome. |
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Term
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Definition
RNA seq
Uses high-throughput DNA sequencing technologies to sequence cDNA of RNA samples from different genotypes or treatments. Frequency of any transcript in a treatment is estimated, and treatments are compared to see response in gene expression to treatment. Deep-sequencing of a large number of reads can identify transcripts of very low abundance in a sample. All sequencing projects produce a large amount of data; bioinformatics is necessary to process the information. Once genes are identified they can be classified into functional groups, and promoters can be examined to see if common and specific enhancer sequences are activated. One could obtain knockouts of specific genes and determine how phenotype is affected, or repeat RNA analysis in wild-type and knockouts to see what genes are downstream of the knockout. Relative expression between treatments can be plotted to look for interesting genes; genes expressed over 1.5 are considered up-regulated. Not the ultimate measure of gene activity, because mRNAs may not be translated into protein, or proteins may be regulated post-translationally by phosphorylation. |
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Term
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Definition
| Antibiotic resistance, herbicide resistance, or metabolic markers. Used in genetic engineering. Allows you to kill all non-transformed plants, leaving only transformed plants. There is concern that they may be released into the environment. On selective media, some un-transformed cells inside a cluster may be randomly unharmed, and not die. |
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Term
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Definition
| A highly successful way that allopolyploids arise in nature. Far more efficient than somatic chromosome doubling. Effected by functioning of unreduced 2n gametes in parental species or their hybrids. Unreduced gametes of both parents give rise to fertile allopolyploids. Does not require colchicine treatments. Instrumental in the evolution of grain, fibre, and oilseed crops. |
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Term
| Short interfering RNA (siRNA) |
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Definition
| Small RNA fragments, 21 - 25 nucleotides long, formed from dsRNA by dicer. Becomes associated with a RISC complex, which cleaves all homologous mRNA, which is then degraded. |
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Term
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Definition
| 21 - 24 nucleotides long. Can bind to mRNA to block translation. Occurs frequently in animals. |
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Term
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Definition
| A family of transposable elements. 8,287 bp. Produces 3 bp repeats at the site of insertion. The TE has inverted repeat at termini of 13 bp, necessary for its excision. |
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Term
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Definition
| Strains of Agrobacterium tumefaciens with an extra Vir gene that enables them to infect monocots. Before they were identified, particle bombardment was used to transform monocots. |
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Term
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Definition
| An insertion collection technique for producing knockouts used in Arabidopsis. T-DNA can be mutagenic when inserted into a gene. A large population, over 100,000 of independent Agrobacterium-derived transformants are developed using floral dip transformation. Plants are selfed to recover homozygotes of the mutations, and sequences flanking T-DNA are obtained for each transformant. A database is developed for insertion lines, and genes into which T-DNA has been inserted. May be used to identify and study the phenotype of new genes from genome sequencing projects. For duplicate genes one can find knockouts for each, and make single, double, or triple mutants. This is important for forward genetic screens where you can miss genes that occur in gene families. |
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Definition
Tumour-inducing plasmid
Found in Agrobacterium tumefaciens. A portion of this plasmid, containing genes for opines and hormone synthesis, is incorporated into plant DNA: T-DNA. There is also a Vir region. The only sequences necessary for integration into the plant chromosome are terminal repeats, and the information between them can be replaced, and the Ti plasmid can be used as a vector for transformation and genetic engineering. |
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Targeted Induced Local Lesions IN Genomes
Used to identify knockout mutants with T-DNA insertions when there is no insertion in the gene of interest in any lines, or when T-DNA insertion causes a lethal mutation. When M1 is selfed, half of progeny are Aa, segregating 3:1 (1 AA:2 Aa:1 aa) and half are AA, segregating 1:0 (all AA), overall M2 is segregating 7:1. Fluorescently labelled PCR primers are designed to amplify the DNA sequence for the gene we want to knock out. DNA is extracted from each family and pooled, and genes are amplified with primers. For families segregating 3:1, half the DNA contains the mutant allele, and when the DNA is made single-stranded, half of reannealed strands will be heteroduplexes, where there are mismatched nucleotides, A/C and G/T. Families segregating 1:0 will not have any mismatches because all the DNA has the wild-type allele. Cel1 is used to cutmismatched DNA strands, producing different length strands of DNA, assuming the mutation did not occur in the centre of the fragment. When separating the DNA on gel, it is easy to distinguish samples which contained the mutation and samples which did not: there are three or one bands, respectively. Each family is pooled and superpooled into rows and columns, and PCR is done on each group. This method allows you to find a mutated family in a group of 100,000 families with only 210 PCR reactions, by narrowing down the search strategically by row and column. |
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Definition
| Contains salts with macro- and micronutrients: N, P, K, S, Ca, Mg, Mb, B, Cl, Cu, and Mb. There is a carbon source, sucrose, and an organic nitrogen source, amino acids. The sucrose is an energy source for young cells which may not have photosynthesis. There are vitamins, and a balance of the phytohormones cytokinin and auxin. The solid support is agar. The exact mixture and conditions needed is different for every plant species. |
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| An oncogenic region of T-DNA. Has 8 open reading frames encoding genes. Genes are transferred to the plant, and affect hormone synthesis and opine production. |
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| The first plant to be genetically transformed, in 1984. It is a model plant that is easy to transform. Work done in the 1960s showed that it had good response to tissue culture. |
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| A region of T-DNA. Genes code for additional opines. |
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| Transcriptional silencing |
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Definition
| Transgene silencing where transcription is inhibited by methylation or chromatin folding. Related to natural mechanisms to silence retrotransposons. In heterochromatic regions with transposons, RNA polymerase IV and V transcribe single stranded RNA. Transgenes may be inserted as an inverted repeat sequence, which produces double-stranded hairpins as RNA, which may be recognized as dsRNA and cleaved by dicer. |
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Definition
| A region of the Ti plasmid in Agrobacterium tumefaciens which is transferred into the plant. Has TL and TR regions. Flanked by 24 bp direct repeats; the right-hand border repeat is essential for transfer, but not the left-hand border. The genes have eukaryotic promtoers and poly-A addition signals. ChvA and ChvB are necessary on the bacterial chromosome for attachment to the plant cell wall. |
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Definition
| Includes transcriptional and post-transcriptional silencing. |
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| Transposable element-like |
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Definition
| Micro RNAs which are derived from transposable elements which normally have inverted repeats flanking a DNA sequence. |
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Definition
| Generated by crossing a tetraploid and a diploid. |
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| A gene in the Vir region of the Ti plasmid of Agrobacterium tumefaciens. Activated by acetosyringone by autophosphorylation. This is a signal transduction pathway leading to a cellular response. |
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Definition
| A gene in the Vir region of the Ti plasmid of Agrobacterium tumefaciens. Encodes export apparatus. |
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Definition
| A gene in the Vir region of the Ti plasmid of Agrobacterium tumefaciens. Vir D 1, 2, and 3 cause a single-stranded nick in the T-DNA, and Vir D 2 covalently links to the 5 end of the displaced single stranded DNA molecule. DNA repair replaces one strand of DNA. |
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Definition
| A gene in the Vir region of the Ti plasmid of Agrobacterium tumefaciens. Single stranded DNA formed by Vir D becomes bound by Vir E 2 proteins. |
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| A region on the Ti plasmid containing virulence genes responsible for transfer of T-DNA into the plant cell nucleus. 24 genes in a 40 kb region. Vir A and Vir G genes are produced in free-living Agrobacterium tumefaciens. Also includes Vir B and E. Forms single-stranded T-DNA and encodes export appratus. Acts in trans; does not have to be on the same plasmid with T-DNA in order to work. |
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