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
What is the mutation frequncy?
Mutation frequency = total length of DNA screened (kbp) / number of mutations
(must times by two if there are x number of diploid plants)
What is the arabidopsis genome size?
137Mbp
What is the process of backcrossing following EMS mutagenesis that is used to remove high mutation load?
- Design molecular markers and backcross to recurrent parent (elite line for crops)
- Once do one backcross 50% progeny will be elite parent (recurrent parent) and 50% will be mutant
- If backcross F2 generation again this will be further halved
- Go through multiple backcrosses all the time selecting for individuals carrying genes of interest
- Using molecular markers to identify scewed individuals so can get away with 1 or 2 backcrosses
What is the latin name of the opium poppy and why was it named so?
Papaver somniferum L.
The milky juice (latex) was seen to be sleep inducing which inspired linnaeus
Morpheus greek god of sleep - morphine
What were the features of morphine when it was first isolated?
- Isolated by Fredrich Serturner, 21 yr old pharmacists apprentice
- 1804
- first plant derived drug to be isolated and prepared in pure form
- commercially sold by merk in 1827
- can’t be produced industrially because of the centre structure
What are the main five alkoids produced in opium poppy?
- Morphine (analgesic)
- Codeine (analgesic, cough supressant)
- Oripavine
- Thebaine
- Noscapine (cough supressant, potential anticancer agent)
Through what pathway are Sanguinarine, Noscapine, Codeine and Morphine thought to be produced?
Benzylisoquinoline alkaloid metabolism in opium poppy
Looking to use TILLING on these steps to improve C flux through morphine production
How were ten genes identified that were thought to be involved in noscapine synthesis?
- Through transcriptome analysis in P. somniferum cultivar
- Had a high morphine variety, high thebaine variety and a high noscopine variety. Sequences and isolated DNA from capsules (where most of the drug is produced) and identified a block of genes encoding enzymes that were only expressed in the variety making noscopine
- Hypothesised that these genes were responsible for production of noscopine
What allowed the development of PCR based dominant moleulcar markers for genes related to noscapine?
The ten genes exclusively expressed in HN1 are absent in HM1 and HT1
-identified by PCR on genomic DNA
What did the development of PCR based dominant moleulcar markers for genes related to noscapine allow?
Follow the segregation of each of the genes in F2 mapping populations and investigate their link with the noscapine trait
What evidence suggested that the 10 genes related to noscapine existed as a gene cluster?
Tight linkage in F2 (when HN1 crossed when HM1 and F1 plants selfed)
Co-segregation with the noscapine trait
What level of noscapine is produced when the gene cluster is in a heterozygous state?
Produced at a low level
How was it discovered that all of the 10 noscapine genes have evolved together and become clustered in the genome?
Made a library of genominc DNA from HN plants, cloned DNA into bacterial articificial chromosomes (BACs)
Allows the cloning of massive fragments (around 200kbs) into a single plasmid and express it in E.coli
Make a BACV library and sequenced
How was the pathway of noscapine expression identified?
Used virus induced gene silencing to intoroduce antisense versions of the genes into plants
Knock down genes and then able to construct the pathway
(functional genetics)
What did sequencing reveal about the noscapine gene cluster and how>?
- screening of an HN1 BAC library using part of a CYP82X2 as screening probe yeilded 6 overlapping clones
- sequencing relvealed that the ten genes span a region of 221 kb
- for some genes duplication and neofunctions appear to have occured at the cluster site
- For others gene dupliation appears to have been at a remote site followed by relocation
What was the impact on breeding opum poppy varieties with novel alkoid profiles?
- accumulation of nomally low abundance intermediates in BIGS-plants (e.g. narcotoline)
- raises the prospect of obtaining novel varieties with stable high levels of these compounds through mutation breeding
- the complex biochemical trait of noscapine biosynthesis segregating like a single medelian genetic locus mean that it can be introgressed with relative ease into opium poppy cultivars optimised for other traits (e.g. agricultural performance, specific alkoid profiles)
What does artemenisin do?
Kills the malaria parasite in the blood
What is the time line for artemisinin discovery?
1964 - malaria causes huge numbers of casulties
1967 - initiaition of progect 523 in may 1967
1971-2 - discovery of atriminsin extracts effective at curing malaria, inspired by an ancient recipe from traditional chinese medicene. Arteminisin identified as the active ingredient
1977 - first publication of the checmical structure of arteminisin
2001 - WHO recommended the use of artemenisin based combination therapies (ACTs) in global fight against malaria
What are the targets for the development of new varieties of A annua with increased yield of arteminsin per hectare?
- Increase amount of artemenisin in the trichome metabolite pathways
- Increase the number of tichome/lead: trichome density/laf area
- Increase the number of leaves on a plant: braching, total biomass, leaf:stem ratio, delayed flowering
How was it discovered that arteminisin is made in the trichomes?
- Isolated RNA from trichomes
- Sequenced RNA
- Discvovered genes for arteminisin production
- Used a number of strategies to try and make plants that made more ateminisisin
How were individuals with high arteminisin yield produced?
Induced variation -> reverse genetic screens
Natural variation -> marker assisted breeding
- > identfied individuals with high artemininsin yield
- > developed robust new varieties
What traits were looked at for high levels of arteminisin production and how were these measured?
Flowering times
- Time to flower
- number of leaves at flowering
- number of branches at flowering
Biomass
- Number of branches
- Height
- Average branch length
- Conformation by wet and dry weight
Trichome density
- Trichome reflection
- Haririness
- confirming through microscopy
Arteminisin content
- High-throughput ELISA screening
- Medium-throughput TLC screening
- Confirmation by HPLC
What were the features of the forward (trait) screen for high arteminisin levels?
High through-put screen of M2 (selfed) plants for arteminisin yield
- chloroform dip
- UPLC MS with 2.5 min run time
Identify high yielding individuals and confirm trait in the field
1000 plants screened every two weeks
What were the results from the forward screen on arteminisin levels?
- Screened 21000/25000 plants
- identified 230 high yeilding individuals
- (between 1.5 and 3 times higher)
What were the actions taken following the forward screen of arteminisin levels?
- SNP genotyping in artemis
- Linkage map of Artemisia annua (mapping - matebolites, archtechure, leaf traits, tichomes)
- 1536 SNPs were genotyped for 1152 samples using illumina
- 275 (breeding population) F1 artimis mapping population (to produce genetic linkage map)
- 2 artemis parental lines (C3 and C4) per plate as controls
- segregation data on each SNP provided the genotype data for mapping
- 1536 SNPs were genotyped for 1152 samples using illumina
- Screened thousands of markers against locus of interest
*
What QTLs were identified for atemenisin based on co segragagation of traits with molecular markers?
- Metabolites
- arteminism
- Trichome
- number
- density
- Leaves
- leaf area
- leaf to stem ration
- Biomass
- leaf dry weight
- whole plant dry weight
- Flowering time
- Plant architechure
- height
- branching
- number of nodes
What was found of the hybrids generated from selected parents for high artemisin levels?
Number of positive QTLs in best hybrids better than the number of positive QTLs in lower hybrids
What is Hyb8001 “Zenith”?
- A fast growing, high biomass hybrid suited to china uganda and madagascar
- Trialled at 13 independent sites across chine, india, madagascar and uganda
- Artemisinin yeild: Maximum concentration 1.44% (madagascar); Maxiumu leaf dry weight 4488kg/ha (china); Maxiumum yield; 54.6kg/ha (madagascar)
What have been the impacts of the artemisinin study?
- Molecular breeding platform - linkage and QTL maps
- Growing seed sales of CNAP hybrids into Africa over the last 3 years sufficient to provide up to 200 million ACT treatments for malaria sufferes in the developing world
- registration of a lead hybrid Hyb8001r in china in 2015
