Plant Genomics Flashcards
What is functional genomics and how can it be done?
The goal of functional genomics is to identify functions for all of the genes in an organism.
Take one gene, knock it out and see what is missing.
What is forward genetics?
Look at phenotype and determine the genotype.
Select a phenotype (e.g. flowering, height).
Generate mutants.
Screen the plants for the trait.
Map & clone the mutated genes by genetic and
molecular approaches.
(takes a lot of time, but rewarding since it is massively parallel- many genes at once.)
What is reverse genetics?
Look at genotype and determine the phenotype.
Select a set of genes (or the whole genome).
Generate a pool of mutants.
Screen mutants for altered gene sequence.
Select mutant in target gene.
Study phenotypes of mutants.
How are BACs used in forward genetics?
What are some problems?
Often uses naturally occurring phenotypes – or radiation-induced.
Uses conventional mapping and map based cloning approaches.
Map the trait in a mapping population (gives you a genetic map position).
Screen a BAC library with markers linked to trait.
Sequence BAC clone(s) by NGS.
Identify candidate genes from sequence data.
Test them by introducing the undamaged gene into a mutant plant (i.e. by genetic complementation).
It can take years to map a trait accurately enough to narrow down the search region to a few BACs – and if there’s more than a few BACs then you’ve probably got too many candidate genes to have a good chance of confirming one as the target you are after.
AND It’s effectively impossible to complement a QTL trait with sufficient accuracy that you can be sure of the gene, because a QTL is QUANTITATIVE! Quantitative traits can have many variants, and many genes can be responsible for the phenotype, but each gene may only contribute a small amount to the phenotype.
How does positional cloning work?
Genetic mapping population. QTL localised at approx. 10-30 cM. Narrow down the search region. NILs cross. Fine genetic mapping. QTL localised at
What is OVATE?
Plant growth supressor. It was a previously uncharacterized class of genes. Modulation of OVATE gene function can drive dramatic morphological changes.
One of the genes responsible for tomato fruit shape.
Cloned more than 10 years ago.
What is OVATE?
Plant growth supressor. It was a previously uncharacterized class of genes. Modulation of OVATE gene function can drive dramatic morphological changes.
One of the genes responsible for tomato fruit shape.
Cloned more than 10 years ago.
There were 17 candidate genes in the BAC19 insert.
Pear shaped fruit trait in tomato is determined by a major QTL on chromosome 2.
What is RNA mediated gene silencing?
Way of knocking out a gene.
Attack RNA that is encoded by the gene.
Silencing- way of knocking out RNA by inducing defence mechanism in cell that destroys double stranded RNA.
dsRNA is processed by a Dicer conatining complex. Generate siRNAs. Endonuclease-containing complex (RISC) is guided by the antisense strand of siRNA to cleave mRNAs.
What is virus induced gene silencing?
Another way of silencing- works in the same way as RNA silencing.
Clone gene fragments into a plant viral vector.
infect plants with the recombinant virus.
Virus induces RISC.
The cloned fragment is transcribed and this leads to degradation of the genuine mRNA of the corresponding gene.
Eg. phyotene synthase silencing induces a photo-bleaching phenotype.
What is mutagenesis?
Way of making mutants.
Uses plant transposons and other mobile DNA sequences to mutate genes in plant genomes.
End to make point mutations and hunt for them.
What is tilling and how does it work?
Targetted induced local lesions.
Make a mutant population (typically using a mutagen like EMS which generates point mutations)-Tens of thousands of seeds that have been mutagenised.
Isolate DNA from thousands of plants and pool the DNAs in a way that allows you to find the individual sample with a few assays.
Select a gene- design PCR primers.
Amplify DNA pools by PCR.
Screen pooled DNAs for mutation.
Find mutant plants and analyse corresponding phenotypes.
How does tilling pooling work?
We need to pool thousands of DNAs in a way that allows you to find an individual sample containing a rare mutation in your gene of interest with a few assays.
Imagine ten 384-well plates = 3840 wells, each with a different plant DNA sample.
Generate 6 different types of pooled DNAs.
Assays on these 6 pools can identify a single well containing a ‘positive’ sample for the assay, provided ‘positives’ are rare!
Groupings of plates- there are different ways of grouping them. Pool last row of each of them together.
Have different slices for each group.
Do PCR on these slices.
Can cross reference samples found in PCR to slice and row.
What is the CEL1 assay for SNP mutation?
CEL1 is from celery.
CEL1 nuclease cuts specifically at bubbles (mismatched duplexes/base pairs). Cleaves PCR product into two pieces.
Can identify a single mutation.
Take DNA pool, pull apart the strands and let them come back together, get mismatches which are heterozygosity.
How is WAVE dHPLC used to screen tilling populations?
Detects the products of cleavage with high sensitivity against a huge background of uncleaved DNA. Mutations are rare.
High performance liquid chromatography.
Detect tiny amounts of cleaved products.
Can detect multiples.
Reverse genetics can happen once a “hit” has been found.
How is tilling by sequencing used to screen tilling populations?
Uses pooling.
More modern method.
