Lab 1: Genotyping & Phenotyping Flashcards
To improve fruit quality
What is the primary objective of fruit breeding programs?
Fruit quality
Driven by consumer demand
Outline the scientific process of developing new fruit varieties with improved traits.
Done by selecting and cross-breeding existing varieties
What is a molecular marker?
A molecular marker (DNA marker) is a fragment of DNA that is associated with a certain location within the genome.
Describe the mapping process.
Marker development process
Define: phenotype
Consequence of the interaction between the plant genetic background (i.e., genotype) and the biotic and abiotic conditions experienced by the plant in its growing environment.
Define: phenotyping.
The process of characterizing traits in detail.
Define: genotype
The plant genetic background. Also used to refer to individuals with distinct genetic background.
Define: genotyping
The analysis of variations in genomes between individual organisms
Describe the evolution of molecular markers. [5]
- Restriction fragment length polymorphism
- Random amplified polymorphic DNA
- Amplified fragment length polymorphism
- Simple sequence repeat (a.k.a. microsatellites)
- Single nucleotide polymorphism
What is a SNP marker?
- A single nucleotide polymorphism
- A variation at a single position in a DNA sequence among individuals
- Occurs in more than one percent of the general population
Describe the process from DNA extraction to SNP calling.
Define: genetic mapping
Statistical methods that link certain complex phenotypes to specific regions of chromosomes.
Using genome-wide association mapping.
Describe how to interpret a Manhattan plot for a genome-wide association study. [5]
- Scatter plot
- The dots are SNPs
- x axis shows chromosomes
- y axis shows negative log of p value
- Green line is the threshold for significance
Provide the background for raspberries as a crop to focus on for improvement.
- Red raspberry is a high value crop
- Diploid, 2n = 14
- Native to North America and Eurasia
- Mexico, California, and the Pacific Northwest are leading producers worldwide
- In Canada, BC accounts for over half of the marketed production, with a farm-gate value of ~$14 million per year
Raspberries display a broad range of colours (red-yellow) and intensities (dark-light).
What do the requirements of fruit colour depend on, and what are the major pigments found in raspberries?
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Colour requirements:
- Fresh: bright medium red
- Processing: dark red
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Major pigments:
- Anthocyanins (red)
- Carotenoids (yellow)
Describe the structure of anthocyanins.
Anthocyanins are glycosylated anthocyanidins
Describe the structure of carotenoids.
Describe the history of known raspberry colour genetics.
What are Claudia Baldassi’s (PhD candidate) goals regarding raspberries?
- Research gap: the genetic control of different red intensities remains elusive, hindering breeding for desired colour
- Objective 1: Map the regions underlying different red intensities of raspberry fruit through a genome-wide association study
- Objective 2: Develop markers to enable marker assisted breeding for specific fruit colour intensities
What is the pH differential method?
Widely used method for the determination of total anthocyanins which leverages the absorbance difference of anthocyanins at different pHs.
How does the pH differential method work?
- pH 1
- Anthocyanins absorb light in the visible spectrum; coloured form (flavylium cation)
- pH 4.5
- Anthocyanins don’t absorb light in the visible spectrum; colourless form
- Difference in absorbance between pH 1 and pH 4.5
- Removes background noise (non-anthocyanin absorbance)
- The differential absorbance is proportional to the total anthocyanin amount.
Describe sample preparation for anthocyanin extraction.
- The first 3 steps involve weighing out a specific amount
- The following 6 steps involve preparing a supernatant for analysis.
Describe the total anthocyanin measurement process.
- Involves measuring at 2 different wavelengths and 2 different pH
- By measuring the absorbance at two different wavelengths, you can capture the full spectral behavior of the anthocyanins.
- One wavelength detects the absorbance specific to the colored form of anthocyanins, and the other helps confirm the absence of absorbance in the colorless form, ensuring that the anthocyanin measurement is accurate by compensating for any non-anthocyanin compounds that might also absorb light.
Why do we measure at 2 different wavelengths and 2 different pHs in anthocyanin determination?
- Two pH levels differentiate the colored and colorless forms of anthocyanins.
- Two wavelengths help ensure accurate readings by both capturing the absorbance of the anthocyanins (at the first wavelength) and correcting for any background noise (at the second wavelength).
Why are the absorbance values at 700 nm subtracted from the 510 nm values?
To correct for haze, i.e., remove background noise
Why are the absorbance values at pH 4.5 subtracted from the absorbance values at pH 1?
To remove the absorbance of non-anthocyanin compounds that absorb at both pH levels.
Absorbance at 510 nm is subtracted from absorbance at 700 nm.
True or False?
False.
Absorbance at 700 nm is subtracted from absorbance at 510 nm.
To correct for haze, i.e., remove the background noise.
Absorbance at 700 nm is subtracted from absorbance at 510 nm.
True or False?
True.
To correct for haze, i.e., remove the background noise.
Absorbance values at pH 1 are subtracted from absorbance values at pH 4.5.
True or False?
False.
Absorbance values at pH 4.5 are subtracted from absorbance values at pH 1.
To remove the absorbance of non-anthocyanin compounds that absorb at both pH levels.
Absorbance values at pH 4.5 are subtracted from absorbance values at pH 1.
True or False?
True.
Why is concentration given in milligrams of cyanidin-3-O-glucoside per 100 grams of fresh weight?
- To use units commonly found in the literature to facilitate comparisons
What is the formula to calculate total anthocyanins (mg/100g FW)?
- This calculation accounts for weight of sample and extraction volume (volume used to extract anthocyanins from each sample)
How can we use the calibration curve to calculate total anthocyanin content?
Solve for x (concentration in ppm)
y = absorbance
How is a calibration curve prepared?
- By preparing dilutions of the anthocyanin standard cyanidin-3-O-glucoside (C3G), a common anthocyanin found in raspberries.
- Concentrations are all expressed as equivalents of C3G.
What is a boxplot?
Describe its ‘anatomy’.
- A great tool for data inspection
- Boxplot anatomy
- Min and max
- Interquartile range
- Median
- Potential outliers
What is the link between anthocyanin content and colour?
Increased anthocyanin content = darker colour
What is the link between anthocyanin content and colour?
Increased anthocyanin content = darker colour
What is a violin plot?
- Add info about data density at each value
- The diamond symbol stands for the mean of the distribution
- The dotted line is a global average
What is a histogram?
- Frequency
- The more the samples, the more the distribution approximates a gaussian distribution
Describe how histograms can be used to determine SNP distribution.
How is a Manhattan plot used to conduct a genome wide association study?
Describe how molecular markers for improving fruit quality can be found. [7]
