Plant Genetics: Final – Flashcards

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Transposon tagging

An insertion collection technique for producing knockouts used in corn. Includes engineered transposon systems such as Mu transposon.

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

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.

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.

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.