Lecture 9 - Molecular breeding of medicinal crops Flashcards
Summarise the different approaches in molecular breeding
Must begin with genetic variation
- Natural variation from wild ancestors
- Induced variation from mutagenesis
- Engineered variation from transgenics
Then use next generation sequencing for DNA markers and gene discovery
- Parent selection MAS
- Forward (trait) screens
- Reverse (genetic) screens/TILLING/NGS
- Transgenic event selection MAS
Identify indiviual with improved genetics/traits
- Quality control using DNA markers
- Stacking of traits from the same or different surces of variation using MAS
Field trial potential new varieties
What is the process of 454 pyrosequencing?
- A pool of small DNA fragments are generated from genomic or cDNA sources
- A single strand is bound to a bead, which is then amplified in a water-in-oil microreactor resulting in 10 million copies of a unique DNA template per bead
- A single clonally amplified bead is depositied into a well in the PicoTiterPlate device with 400,000 wells
- Bases (TACG) are flowed sequentially across the picotiterplate device during a sequencing run
- when a nucleotide is incorperated, pyrophosphotase is released and this is linked to a luciferase based light emission which is recorded by the CCD camera
- The signal strength is proportional to the numeber of nucleotides incorperated
- The current FLX system generates 400 000 quality reads with an average read length of 230 bases
Give a disadvantage of 454 pyrosequencing
Hard to quantify the number of repeated bases
What is the time period for 454 pyrosequencing?
DNA library preparation and titration - between 4.5 and 10.5 hours
emPCR - 8 hours
sequencing - 7.5 hours
Why is 454 pyrosequencing important?
Important in the manner which it is used
- Interactions of soil microorganisms
- Gut microbiome
- Plant natural variation
What can RNA sequencing be used for?
- Gene discovery
- Establishing expression levels of an expressed gene
- Identifying polymorphisms within genes
What is the method of RNA sequencing to establish the levels of expression within genes?
- Isolate mRNA
- Convert to cope DNA (cDNA) using reverse transcriptase
- Sequence cDNA (electronic northern blot - hybrididsation based method for quantifying mRNA instead of radioactively labelling probe which gives a signal when it binds to mRNA)
- If the sequence occurs at a higher proportion to others (e.g. the small subunit of RuBISCO would expect a high band of mRNA) whereas low levels of expression e.g. TF not see bright band
What is the process of marker assisted breeding?
- Identify molecular markers based on natural/induced variation as a result of the polyplodism of DNA
- Single nucleotide polymorphisms (SNPs)
- Short sequence repeats (SSRs)
- Amplified FRagment Length polymorphisms (often come from using specific primers to amplify DNA, if there is a polymorphism where the primer normally binds it won’t work)
- Construct a linkage (genetic) map based on the segregation between hundreds of markers
- Identify quantitative trait loci (QTLs) based on the cosegragation of traits with molecular markers. This can be achieved by genotyping and phenotyping F1 or F2 mapping populations
- Use markers that associate with positive QTLs for tracking traits and selecting individuals with good genetic background for plant breeding
How can SNPs and SSRs be identified from 454 sequence data?>
If take individuals from populations and sequence data the majority are going to be identical but some polymorphisms. Mix up DNA and sequence it in ‘pool’. Computer algorith will spot where there are consistantly gene alterations
Define forward genetics
Screen for mutatns with the phenotype of your dream, characterise the mutant and backcross to the recurring parent
Finding out which gene you have mutated to give the phenotype of interest is challenging. The manner by which the gene is identified depends on how the mutant was created.
What are the pros of forward genetics?
No prior knowledge of relevent genes required
What are the cons of forward genetics?
Screens can be labourious/impractical
Small gene families/gene homologues can mean single gene mutations do not have a phenotype
Define reverse genetics
Start with the gene sequence, identify a mutation in that gene and see what the effect is on the plant
Loss-of-function mutants are normally the most informative
How can loss of function mutants be produced?
Can be produced by antisense technology, insetional mutatgenesis or by heteroduplex mapping. (HDM = TILLING) in a mutagenised population
What are the pros of reverse genetics?
For HDM/TILLING mutations can be detected in the heterozygous state and crosses and selfing be used to produce homozygous single and double mutants
What are the cons of reverse genetics?
- Extensive knowledge of the candidate genes required
- Predominantly find loss of function mutations
- Not very good for increasing the activity of an enzyme
What is the CNAP fast track breeding system?
- Take seeds
- Treat with a chemical (EMS)
- Caues mutations in germline cells that go onto form seed
- Grow up plants and collect seed
- Grow lots of M2 individuals and isolate DNA (from around 5000 individuals)
- Use TILLING method to identify mutations in individual genes
What is the TILLING method?
- Isolate DNA from lots of individuals on gene A
- Want to find mutations in gene A so have lots of copies of gene A
- Use primers to amplify gene A (already know sequence)
- Put into pools of individuals and amplify with PCR
- During melting and reanealing if the first individual carrys a SNP mutation get an area of heteroduplex mismatch
- Enxyme Cel1 nicks areas with a heteroduplex in DNA
- When this is ran on polyacrylamide gel, can spot samples that have been cut as labelled the DNA
What does EMS mutatgensis result in ?
High mutation frequency in each individual
A backcross breeding strategy is used to remove mutation load
What is the mode of action of EMS?
Guanine to O-6-ethylguanine which can pair with thymine