MPB

Biological e.g. Agrobacterium mediated transformation

Mechanical e.g. Particle bombardment

Electroporation e. g. microinjection, Silica/carbon fibers, Laser mediated

Chemical e.g. Polyethylene glyco

Recent Examples of GM Biotech crops approved or commercialized in Brazil, Argentina, Canada, the United States:

Arctic® Apples that do not brown when sliced

Innate™ potatoes, with lower levels of acrylamide, a potential carcinogen, resistance to bruising, and late blight resistance

HB4 Transgenic wheat approved for cultivation:

Transcription factor HB4 from sunflower transferred into wheat leads to drought tolerance

Bacillus thuringinsis

Mechanism:

toxin protein ingested by insect when insect feeds on plant

toxin activated in insect gut -> alkaline conditions and specific proteases needed

inserts into gut epithelial cell membrane (specific receptors) and forms ion channel

Different codons are preferably used in different organisms. Before transfer of a sequence into another oranisms this has to be adapted to increase the expression rate in the organism.

Introduction of the resistance from an closely related species via wide cross or if present in the species by crossing

Mutation breeding (with e.g. radiation or chemical mutagenisis) and hoping for a resistance occure by mutation

Introduction via gene transfer from a plant within or outside of the species

“guide” RNA,

Editing nuclease: sequence optimized;

Selectable marker gene,

Promoter,

Nuclear targeting peptide,

terminator

Would be possible via creating mutations (random mutagenesis)

It is not GMO as it is defined in the regulations

The CRISPR/Cas9-system is a highly precise defence mechanism of bacteria against viruses. Thereby the bacteria stores a segement of an attaching but defeated virus. If it is again faced with the virus the sequence is recognised and cut.

We can use and modify the system by intruducing a target sequence into the system. If this sequence is recognised by the CRISPR/Cas9 system it is cut. This can be used to create knockouts or go further and introduce new segments.

They can be created so they are able to identify a sequence and cut at a specific site. This can be used to create knockouts or introduce a new segment at that loci.

Denaturation

Annealing

Extension/Elongation

During particle bombardment sequences are atteched to particles (as gold). Afterwards they are shot with high pressure on cells. Due to the high speed they are able to enter cells.

Oligonucleotide-directed mutagenesis (ODM) is a method by which a single mutation can be induced at a specific site in the genome. For this purpose, oligonucleotides - short pieces of DNA whose sequence matches the target sequence - are synthetically produced and introduced into the cell. As a result of a deliberately inserted faulty nucleotide, individual base pairs are exchanged or altered during the subsequent repair of the break. Among the random DNA changes at the break site, the one that corresponds to the respective breeding target must now be found out.

Canola (CIBUS):

Under the Falco™ brand, our first commercial product is a non-transgenic sulfonylurea herbicide tolerant canola (Raps) providing an alternative weed control option for canola farmers globally, including lower herbicide costs, non-GM premiums and a synergistic option for soybean rotations.

Cisgenics: gene from sexually compatible plant

Intragenics: Newly arranged sequence from compatible plant

Transgenics: original gene from sexual not compatible variety

CRISPER-Cas9 System (easy and cheap) design sgRNA, embed in Cas9 protein (cutting molecule), the complex binds to a PAM region and unwinds the double helix. When the correct sequence is found, Cas9 introduces a double strand break. Repairing the cut though is error-proned which often leads to mutations and therefore making CRISPR a great tool for knocking out genes

A guide RNA (gRNA) is a piece of RNA that functions as a guide for RNA- or DNA-targeting enzymes, with which it forms complexes. Very often these enzymes will delete, insert or otherwise alter the targeted RNA or DNA. They occur naturally, serving important functions, but can also be designed to be used for targeted editing, such as with CRISPR-Cas9

dsRNA is produced, in the cells transformed into siRNAs (small interfering RNA) are incorporated into a multicomponent nuclease complex. Afterwards it is cutting siRNA/mRNA with the complementary sequence. This enables plants to be resistant to viruses.

insertion of a coding sequence which produces dsRNA (hairpin construct) complementary to the sequence for the GBSSI enzyme.

Production of siRNA and incorporation into multicomponent nuclease complex.

Complex degrades mRNA encoding for the GBSSI enzyme.

no transfer of the transgene locus instead transfer of the protein or construct (avoid selection cycle, maintain variety quality, since it is a vegetatively propagated variety)

contains sgRNA for the GBSSI enzyme (enzyme involved in starch synthesis) locus -> knockout of all copies of GBSSI

-> no elongation of amylose, almost exclusively amylopectin is created

Random mutagenesis (e.g. EMS) and afterwards TILLING

many other mutations as well -> decrease of quality

CRISPR/Cas9: targeted induced mutation that is knocking out the enyzme for the elongation of amylose

easier, fast, precise, but not allowed

transfer of the transgene locus encoding for the CRISPR/Cas9 construct (easier selection and higer transformation rate, but generative propagation needed)

or only transfer of the complex or vector (better if vegetative propagation and maintainance of the variety, hard selection)

Makes it easier to select the plant with the transformation

Easier to distinct between plants with and without the desired gene

a gene encoding an enzyme is added along with your gene

Common selectable marker genes:

Antibiotic resistance: NPTII - detoxifies kanamycin (antibiotic),

Herbicide resistance: bar - detoxifies glufosinate (herbicide),

positive selection marker: PMI Mannose-6-phosphat-isomerase: changes mannose to useable carbohydrate

directly produces parental lines for any heterozygous plant

generates perfectly complementing homozygous parental lines

Surpression of Meiotic Recombination

sRNA is either applied or produced by the plant after ingestion by the insect (and transformation into siRNA) is cutting siRNA/mRNA with the complementary sequence.

Use of sgRNA that enables cutting at a designated spot

Biological e.g. Agrobacterium mediated transformation

Mechanical e.g. particle bombardment

Electroporation e.g. Microinjection, Silica/carbon fibers, Laser mediated

Chemical e.g. polyethylene glyco

Plant breeding is the genetic improvement of plants for human benefit.

Short, easy and true.

The activity of plant breeding can be partitioned in three phases: (Modern biotechnology helps plant breeders)

1) Planning: the design/imagine a breeding target (the ‘ideal’ cultivar)

2) Generate (new) variants: e.g., crossing, mutagenesis, transgenics

Careful planning of new crosses, mutagenesis, transgenics

3) Find and maintain the ‘best’ new variants

selection (markers), testing of new candidates, check for purity

collection of techniques used to maintain or grow plant cells, tissues or organs under sterile conditions on a nutrient culture medium of a known composition

In-vitro-propagation (production of clones)

virus elimination

embryo rescue in wide crosses

tissue culture for doubled haploids (embryogenesis)

protoplast fusion (= somatic hybridisation allows fusion of different plant species)

single cell regeneration (e.g. after transformation)

Is a plant that has two identical chromosome sets

Homozygosity, diploid

can be produced via:

regeneration from haploid cells (gametes e.g., pollen or ovary), Chromosome elimination, Colchicine

Faster homozygosity in comparison to inbreeding

Labour intensive, working with toxic substances

Production from gametes (pollen or ovaries)

(Colchicine treatment or spontaneous diploidization)

Haploids can not sexually reproduce since during the meiosis the chromosome set can not be further split up

Homozygosity -> because this is needed for breeding

Inbreeding -> highest inbreeding -> selfing

Selfing takes place over several generations which is very time consuming

A gene is a molecular unit of heredity of a living organism

Gene for pea shape, flower colour

An allele is one of a number of alternative forms of the same gene or same genetic locus (generally a group of genes).

Allele for round or wrinkled pea shape

You can see them while the plant is growing or on the plant in general

->easy to notice

Some traits develop late in life, some are not easy detectable, there are just a few markers (a few dozen)

E.g., Spelt colour, flower colour, hypocotyl colour, hairy/naked pods etc.

White flowers and missing black spots on stipules are perfect markers for tannin free seeds

Biochemical markers are protein markers

They visualize polymorphisms in proteins, such as storage proteins or enzymatic proteins.

E.g., Storage proteins (as glutenins and gliadins), Isozyme-Markers (Endopeptidase)

Point mutations (single nucleotide polymorphisms, SNP)

Insertion/Deletion (INDEL): a more or less large piece of DNA is missing or extra present

Use of restriction enzymes for generating DNA fragments of different size, their detection is based on DNA/DNA Hybridization -> RFLP

Markers based on PCR: microsatellites (SSR), CAPS, (RAPD, AFLP, S-SAP, ISSR, SRAP, STS, SCAR…)

Markers detection on high throughput DNA-chips, modern DNA sequencing methods or different variants of PCR:

SNP on chips, genotyping-by-sequencing

(DArT and numerous more)

Restriction-endonucleases are enzymes that cut DNA sequences at recognition sites

We need them for RFLP markers

Use of restriction enzymes for generating DNA fragments of different size, their detection is based on DNA/DNA Hybridization

Gel electrophoresis is a method for separation and analysis of biomacromolecules (DNA, RNA, proteins, etc.) and their fragments, based on their size and charge.

Nucleic acid molecules are separated by applying an electric field to move the negatively charged molecules through a matrix of agarose or other substances.

Gel, charged molecules ( e.g. DNA fragments), Buffer solution, electric field, colour for visualisation

Co-dominant markers

Are markers for which both alleles are expressed when co-occurring in an individual

Heterozygotes can be distinguished from homozygotes

SSR, STR and RFLP

Dominant markers

In dominant markers only one allele is visible and heterozygosity cannot be determined directly

ISSR, RAPD and AFLP

Polymerase Chain Reaction

a technique used to amplify a segment of DNA of interest

PCR enables you to produce millions of copies of a specific DNA sequence from an initially small sample      

Standard ingredients in the mixture are:

sequence of interest,

specific primers,

heat-resistant polymerase (Taq-polymerase),

dNTPs,

Buffer,

PCR machine

1. at approx. 90°C: Denaturing: the double stranded DNA becomes single stranded

2. at approx. 60°C: Annealing: primers bind to complementary sequences

3. at approx. 74°C: Elongation: the Taq-polymerase elongates the primers

Repeat steps 1-3 many times (…. 20-35 times)

Result after n cycles: Linear amplification of PCR product of not defined length: 2n

Exponential amplification of PCR product of defined length (defined by the primer ends): 2n - 2n – 2

Detection of SSR markers:

SSR markers are usually INDEL markers, detection needs therefore size determination

Basically there are TWO types of DNA polymorphisms that we assess using currently available DNA marker methods:

Point mutations (single nucleotide polymorphisms, SNP)

Insertion/Deletion (INDEL): a large piece of DNA is missing or extra present

SSR = Simple Sequence Repeats (=microsatellites)

-> SSRs are highly variable and evenly distributed throughout the genome.

This type of repeated DNA is common in eukaryotes

Gel based systems:

Agarose gel (low resolution),

high resolution agarose gel,

Native acrylamide gel,

sequencing gel (denaturing acrylamide gel)

Capillary electrophoresis:

Capillary electrophoresis systems (e.g., capillary sequencer, capillary fragment analyzer, …),

Chip-electrophoresis systems (miniaturized electrophoresis systems, …)

SSR are co-dominant markers

SSR loci are often multi-allelic

Allow typically (but not always) co-dominant scoring

For many crop plants, many SSR markers (primer pairs) are available in the public database, which can be used for research and breeding

Many markers are already mapped and publicly available

SNP-Markers detects Single Nucleotide Polymorphism,

Variations at specific nucleotide positions in the DNA of a species (base substitutions because of point mutations),

SNPs are the most abundant sequence-polymorphisms,

silent mutations because of the ‚degenerated’ code for amino acids and,

SNPs occur often outside the coding regions

Methods based on hybridization (e.g. microarrays)

Enzyme-based methods (CAPS, RFLP, primer-extension…)

Detection post PCR (e.g.. Single-stranded Conformation Polymorphism, Minisequencing, Kaspar System,…)

Uses allele specific PCR primers for amplification of SNPs combined with fluorescence detection

GBS is a method to discover SNPs and to perform a genotyping study.

For sequencing of large and complex genomes target enrichment or reduction of genome complexity must be performed to ensure sufficient overlap in sequence coverage for species with large genomes.

SNP genotyping on microarrays

Disadvantages of SNP ARRAYS:

development costs, therefore available for ‚important‘ species,

only those SNP that are represented on the array can be measured (may have a bias)

Advantages:

easily comparable and highly reproducible, robust,

High number can be detected at relatively low costs

GBS – Genotyping by Sequencing:

Advantages:

open platform, not restricted to pre-selected SNPs,

can also be used for minor crops/less economically important species

Recombination:

Interchromosomal and Intrachromosomal

Have ideally two homozygous parent (lines) that are distinct from each other in two or multiple traits/loci -> creation of a hybrid/cross (F1) -> selfing (or backcrossing) (alternatively development of homozygous progeny) -> measuring of the traits in the F2/homozygous progeny -> comparison of the ratio parental phenotype/traits and the newly combined phenotype/traits

Best to have >100

Parents that differ in one trait, high and low starch content

Make a cross F1

Selfing or backcross with one parent -> segregating population

Recombinant inbreed line or DH -> we want immortal populations -> DH or RIL with a genotype with a lot of markers and phenotype for the trait (with very few percentage of heterozygosity)

Data analysis

QTL validation

F2: fast but not many recombinant events, low homozygosity

RIL: needs a long time but reproducible and more recombination events

DH lines: fast, labour intensive, 100% homozygosity, less recombinations than RIL

BC1F1: fast, only one recombination event

Yes it makes a difference, it is not possible to distinguish between the two alleles

Co-dominant because it has a finer resolution (we want to distinguish between homozygotes and heterozygotes)

a. False

I can combine all markers; it does not matter which type of marker

There is no need to differ between DNA and morphological marker, for the mapping it does not matter, as long as we can differ between A1A1, A1A2, A2A2

b. True, for more see a.

c. False, for more see a.

Populations in which the individuals have a high degree of homozygosity are called ‚immortal populations ‘, each line can be multiplied easily and remains genetically constant

examples: DH populations, RIL populations

Mapping populations with a high degree of heterozygosity (example BC1F1 or F2) are ‚temporary populations ‘

Yes

DH have only one recombination event

RIL has double the amount of recombination events so the resolution is higher

it will increase

-> more possibilities for crossover

I) false

II) true

III) false

The more recombination events the higher the resolution

The resolution of any linkage map depends on the number of effective meiotic events that took place in the population

a. No

b. Yes

c. Yes

d. No

e. Yes

…. 1% per meiosis event, which means ONE crossover per 100 independent meiosis events

recombination [in cM] = number of recombined gametes/numbers of all gametes * 100

Recombination frequency (r) is not a good measure of genetic distance since the average recombination frequency cannot exceed 0.50 or 50%.

Meioses that contain multiple chrossovers are counted on average as containing only one crossover so r is biased downwards, especially when r > 0.20

A cross-over event usually reduces the chance for another crossover event nearby.

-> for these problems we need a mapping function

Recombination rate is the number of recombined gametes / number of all gametes,

Genetic distance is a distance that is generated from the recombination frequency and can be used to generate a genetic map.

No it is not uniform

There are less recombinations in the centromeric region

a. 1000

b. Yes

c. 500 RILs, double as informativ as DH

Genotypes that often have marker alleles in common are closer related to each other than genotypes that possess often different marker alleles.

Jaccarde distance: number of common bands in both genotypes divided by all bands,

shared band index: number of shared bands/ number of bands of genotype i and j,

Helpful for selecting the parents

To know where to search for possible advantageous alleles

relatedness is important for differentiation between cultivars of different breeders

detect duplicates in your popultaion

Cluster-Analysis-Methods

Principle Component Analysis: Data reduction to visualize principal structure in the data

Have two distinct parents for the trait -> create an F1 and F2 -> do phenotyping and genotyping (high marker density every 5-10 cM one Marker) -> See which marker fits to the distribution of the trait

Qualitative: discrete variations (e.g. flower color in peas, hypocotyl colour in soy, qualitative resistances, …)

Quantitative: continuous variation (e.g. grain yield, plant height, protein contents, …)

The environment is modulating the trait

Multiple genes contribute to the trait variation

Yes, it does. Only crosses are going to lead to new combinations, which are relevant for selection

 -> use molecular markers as quality control

quantitative trait locus/loci a locus in the genome, that influences a quantitative trait

or a region on a chromosome, which is significantly associated with variation of a quantitative trait

Where are genes located that influence a trait,

how much of the variation can be explained by the locus,

How many (significant) QTL govern a certain trait in a certain situation?

Where in the genome are they located?

Which markers are closest to the QTL and how close are they?

What is the contribution of the individual QTL to the overall trait variation?

What is the gene action of the QTLs (additive, dominant, epistatic)?

Parent 1 x Parent 2 (are differing from each other in the trait of interest) -> creation of a segregating population -> genotyping (with a lot of markers approximately every 5-10 cM) and phenotyping -> Data analysis -> QTL validation

Populations in which the individuals have a high degree of homozygosity are called ‚immortal populations‘, each line can be multiplied easily and remains genetically constant, examples: DH populations, RIL populations

Mapping populations with a high degree of heterozygosity (example BC1F1 or F2) are ‚temporary populations‘

Yes, A for seed color and B for yield.

Because B is a nearly immortal population yield is a trait influenced by a lot of loci so it makes sense to have a population with which replications are performable.

Also, it is possible to do finer mapping with population B because of more recombination events

We want to find out which QTL markers are associated with our trait

Single marker-trait analysis -> looks at each individual marker and calculates the chance that it is associated with the trait

Simple interval mapping -> calculates the possible position in the genome and returns the likelihood that it is in a given interval

No, I don’t know the gene and its function we only have an interval and the sequence there -> synteny analysis or knockout experiments with the genes in the interval

a. False

b. True

Even though you don’t know the specific gene relevant for the resistance you can use the information for MAS e.g. in a marker-assisted backcross or simply in a cross to see if the region and thus the resistance is in the genome.

Cross and multiple rounds of backcrossing.

Creation of sister lines with small segments of donor and QTL mapping.

Useful for transferring QTL alleles from a non-adapted line or wild relative into an adapted cultivar and mapping of QTL at the same time.

Introgression library is a series of introgression lines which are almost identical and have small segements from the donor lines.

Association mapping or linkage disequilibrium (LD) mapping is an alternative approach for mapping quantitative traits in populations.

LD mapping is based on genetic variation in ‚natural‘ populations for high resolution mapping of desired genes.

The non-random association between two markers or two genes (QTL) or between a marker and a gene (QTL)

LD mapping is based on genetic variation in ‚natural‘ populations for high resolution mapping of desired genes.

An alternative approach for mapping quantitative traits in populations

Marker assisted selection

MAS is a variant of indirect selection

It combines phenotypic and marker selection (in contrast to marker based selection)

Use of markers that are closely linked to the trait and selecting of those markers instead of the trait

Multi-Parent Advanced Generation Intercrosses

Advantage: population with LARGE genetic diversity:

* more parents leads to more diversity

* potentially segregating for multiple alleles at each locus

Bottlenecks:

* needs longer to develop,

* must have many lines (~100 per founder)

Phenotypic selection is labour intensiv, expensive or time consuming

Phenotypic selection is only possible in advanced generations

needs much input (many environments, expensive testing methods, …)

The breeder wants to combine alleles which cannot be distinguished on the phenotype

In a back-crossing program when a breeder wants to transfer only a few ‘positive’ alleles from a donor line into a well adapted productive line. Especially in cases where the donor line is a wild relative or a landrace with poor agronomic performance

(B) False

we just need markers and need to know if they are linked to our trait of interest and how much the loci influences the trait.

In case of GS we need many markers, a phenotyped and genotyped training group to vreat a prediction model for the performance of a breeding population.

form of marker-assisted selection which uses a training population that is phenotyped and genotyped and applies a model using all markers applied to the genotype.

This model is afterwards applied to a breeding population, where the performance is predicted.

Early selection possible, reduces the high costs for phenotyping.

Lower costs compared to phenotypic tests

Genomic predictions of breeding values can be obtained earlier in the breeding program than phenotypic predictions.

GS therefore generally should increase the selection gain per unit time.

a. classical MAS: for qualitative traits or traits influenced only by a few loci, possible to create QTL ideotype

b. GS: highly quantitative trait influenced by a lot of minor loci and a trait that is hard to screen for large breeding populations (costs for phenotypisation high and selection on that way slow)

c. 

MAS: having closely linked markers to a few loci of interest that are screened for

GS: using many marker to predict the performance of an individual based on a training population

training population that is genotyped and phenotyped,

good prediction model,

breeding population that is only genotyped

higher reliability if you use 2 markers simultaneously,

using one marker a crossover could happen destroying the function, but two crossovers are rare -> increases the selection precision

Increase the frequency of desired alleles by removing unwanted individuals (= those which possess the un-desired alleles for at least one QTL homozygous)

If multiple Genes (QTL) are wanted in a genotype F2 enrichment is decreasing the number of lines needed

we have more markers to estimate than phenotyped individuals

solved by treating markers as random effects -> using estimation methods such as ridge regression BLUP (best linear unbiased prediction, RR-BLUP) or G-BLUP and several further approaches

True F1s are 9, 11, 13 and 15, since they have the allele from both parents all others are selfings from the mother plant

There are multiple mechanisms in plants against viruses the main mechanism is gene silencing -> whereby foreign genetic material is cut up 

A genetic marker is a gene or DNA sequence with a known location on a chromosome that can be used to identify individuals or species.

De novo sequencing is the sequencing of a genome, where no reference sequence is available.

In contrast for genome resequencing a reference genome exists, to which the sequences can and are alligned.

The PacBio sequencing is single molecule real time sequencing (SMRT) and creates long reads. It records the DNA synthesis in real time.

Advantages: Long reads

Disadvantages: high error rate (but unbiased errors)

50% of the assembly is within contigs or scaffolds of 1 mb or larger

Likelihood of wrong base in final assembly

Missing parts between contigs will be annotated with N

Proportion of base N in the assembly- 5-10% N is normal

Multiple reasons possible:

Biology -> natural variation

technology -> e.g. sequencing errors, biased sequencing errors, errors within reference sequence

Completeness evaluated based on genes e.g. Benchmarking universal single-copy othologes (BUSCO) or others

The key difference between contig and scaffold is that a contig does not have gaps while a scaffold consists of contigs and gaps.

Variety that is well known and widely used

Additionally material of young plants that have a lot of DNA and not jet accumulated many mutations

homozygous genotypes -> Inbred lines (e.g. from selfing or backcross)

Heterozygosity causes assembly fragmentation

DH genotypes (from plant biotechnology)

Parasite, symbionts, accidental contamination

Organisms colonized by microbes

Sequence database curators perform contamination screens

Attaching information to genome

e.g. genes, transposable elements, variants

Sequencing of a population

decide one genome as "reference"

Search for variants, differ from your reference

Variants should be called on both strands

Variants called from just one strand may be artefacts due to context-dependent biases

Multiple reasons possible:

technology -> e.g. sequencing errors, biased sequencing errors, errors within reference sequence

hierarchical sequencing: in hierarchical shotgun sequencing, the genome is broken into larger fragments prior to sequencing

in whole genome shotgun sequencing, the entire genome is broken into small fragments for sequencing

In contrast in whole genome sequencing there is no BAC library created

N50 of 1000bp reports that 50% of the assembly is in units of 1000bp or bigger

There will always be contigs that are too short to be of any use to anyone

If the number is very small it might not be a good assembly

Advantage:

Such near isogenic sister lines allow a very precise estimation of the effects of genes on the donor segments on the traits of interest.

Disadvantage:

Generation of a good introgression library is time and resource demanding.

One needs to backcross several times and has to analyze the progeny with appropriate markers after each backcross

Cannot answer:

which gene is exactly influencing the trait,

are the loci that were found the only loci influencing the trait,

We do not know if we found ALL QTL for this trait

uses existing populations due to many recombination events during their (breeding) history.

After many recombination events only very tightly linked markers should still be associated (be in LD) with the allele of interest

in contrast classical bi-parantal crossing populations and classical QTL mapping has relatively few recombination events