Lecture 6: fungus aspergillus Flashcards
key of the lecture
Key methodologies such as backcrossing, multiple mutants in one complementation group, segregation analysis, and bulk segregant analysis (BSA) are employed to reduce non-relevant mutations and identify critical genetic changes.
Parasexual Cycle in A. niger
Since A. niger lacks a known sexual cycle, researchers use the parasexual cycle to create genetic crosses. This involves forcing the formation of diploids using auxotrphic color mutants and then inducing haploidization using chemicals like benomyl, which destabilize the diploid genome.
Sexual Cycle: The natural reproductive cycle involving the fusion of two haploid (single set of chromosomes) gametes to form a diploid organism, which typically leads to genetic variation in offspring.
Parasexual Cycle: is an asexual method of genetic recombination used in organisms that lack a natural sexual cycle.
Diploids: Organisms or cells with two complete sets of chromosomes, one from each parent. In the parasexual cycle, diploids are artificially created from haploid cells.
Auxotrophic: A condition where an organism cannot synthesize a certain compound and must obtain it from the environment.
Color Mutants: Mutations that affect pigmentation in organisms, commonly used as markers in genetic experiments to track inheritance.
Haploidization: The process of reducing the chromosome number of a diploid organism to a haploid state (one set of chromosomes).
Genome sequencing
Genome sequencing involves comparing the genome of a wild-type Aspergillus niger strain with that of a mutant strain displaying a specific phenotype (e.g., UV-induced mutations). This approach identifies differences, such as single nucleotide polymorphisms (SNPs) or insertions/deletions (indels), that may be responsible for the observed phenotype.
reduce the effort to identify the responsible mutation:
Backcrossing
a genetic technique where a mutant organism, which carries the trait of interest, is repeatedly crossed with a wild-type organism. Each cross removes about half of the irrelevant mutations, as half of the genetic material comes from the wild-type strain. Over successive backcrosses, the proportion of non-relevant mutations decreases, while the relevant mutation remains. By the final backcross, only the mutation responsible for the phenotype should remain, making it easier to identify the cause of the phenotype.
Cross your mutant strain with the parental strain (make a diploid), then make a haploid again. Half of the yeast strain will be from mutant, and other half will be parental. Then do this multiple times, selecting for the strains that still show the phenotype. Eventually you will find which gene is responsible for the phenotype.
reduce the effort to identify the responsible mutation:
Complementation analysis
helps identify whether different mutants with the same phenotype are affected by mutations in the same gene or different genes. It allows researchers to group mutants with overlapping genetic defects, narrowing down the location of the mutations and identifying key genes involved in a biological pathway.
Recessive Mutations: CA is typically done with recessive mutants because the phenotype only appears when both copies of the gene are mutated (homozygous recessive). In heterozygous conditions, the wild-type allele can compensate for the defective one.
Complementation: When two mutant strains are crossed, if their offspring show the wild-type phenotype (i.e., they are not red), this means the mutations are in different genes. The two mutated genes “complement” each other, as each mutant provides a functional copy of the gene that the other lacks.
No Complementation: If the offspring still show the mutant phenotype (e.g., red), it indicates that both mutants have mutations in the same gene. In this case, the mutations do not complement each other, as neither mutant provides a functional copy of the gene.
You have two red mutants, each with a mutation causing the red color.
Crossing Mutant 1 × Mutant 2:
If the offspring are wild-type color (not red), it suggests that the two mutants have mutations in different genes, and the wild-type copy from each mutant complements the other.
If the offspring are red, it suggests the mutations are in the same gene, and they cannot complement each other.
reduce the effort to identify the responsible mutation:
Isolation: Selecting many mutants with the same phenotype to identify shared SNPs
If you have a lot of mutants, you can sequence them all and increase the likelihood of identifying mutations responsible for the phenotype.
reduce the effort to identify the responsible mutation:
Segregation analysis
is used to study how a mutant phenotype is inherited over generations.
The process involves:
Crossing a mutant strain with a wild-type strain to create a diploid organism. This allows for the mixing of genetic material from both parents.
Haploidization: The diploid is then forced to lose one set of chromosomes (haploidization) using chemicals like benomyl, which generate haploid progeny (segregants).
Segregant Analysis: The offspring (segregants) are analyzed for their phenotypes to see how the traits are inherited. .
By comparing the genomes of segregants that show different phenotypes, researchers can narrow down the region of the genome associated with the trait of interest.
In summary, segregation analysis helps track how a trait segregates across generations and allows for identification of the genetic variation responsible for the phenotype by comparing segregants from a mutant × wild-type cross.
Bulk Segregant Analysis (BSA)
is an advanced method used to identify phenotype-associated mutations by analyzing large pools of segregants, typically from a cross between a mutant and wild-type strain.
**Create Two Pools: **After performing the cross and generating segregants, two pools of offspring are created: one with the desired phenotype (e.g., a specific color or resistance) and one without.
The genomes of both pools are sequenced in-depth, and Single Nucleotide Polymorphisms (SNPs) between the two pools are compared.
If a mutation is associated with the phenotype, it will be present in all individuals of the phenotype pool but not in the non-phenotype pool. This shows a consistent genetic association with the trait. Non-linked mutations will appear in both pools, as the segregation of those SNPs is not tied to the phenotype.
SNPs in phenotype-associated pool: These are the mutations that are more likely linked to the desired trait, as they are found exclusively or predominantly in individuals with the trait.
Difference BSA and seggregrant anlysis
- Segregation Analysis focuses on studying the inheritance pattern of a phenotype over generations and uses phenotypic segregation to map the genes responsible for a trait.
- BSA involves pooling offspring based on phenotype, sequencing their genomes, and comparing SNPs to identify mutations that are statistically linked to the phenotype.
Both methods complement each other, with segregation analysis helping track inheritance and BSA providing a high-throughput way to identify the genetic causes of the phenotype.
