GPB (521)Molecular Breeding PYQ

• Questions Asked:

  1. Fill the blanks. (2.0 Marks)

     a. VIGS is...

     b. ZFN is...

     c. TALEN is...

     d. PEG is...

  2. Write about Cisgenesis and Intragenesis. (4.0 Marks)

  3. What is Linkage Disequilibrium? (1.0 Marks)

  4. Write difference between selectable and scorable markers. (2.0 Marks)

  5. Write traits to increase potential and to remove constraints in crop plants. (3.0 Marks)

  6. What is biopharming? (1.0 Marks)

  7. Compare genetic engineering and conventional breeding. (3.0 Marks)

  8. Write about biosafety measures in view of transgenics. (2.0 Marks)

  9. What are factors important for efficiency of genetic transformation? (2.0 Marks)

  10. Compare chemical and biological methods of transformation. (2.0 Marks)

  11. Explain physical methods of genetic transformation. (2.0 Marks)

  12. Write about phenotyping and genotyping. (2.0 Marks)

  13. Write steps in PCR and profile for RAPD markers. (2.0 Marks)

  14. What is reverse breeding? How is it useful in crop improvement. (2.0 Marks)

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Solutions and Explanations

Here are the correct answers and detailed explanations for the questions on the paper.

1. Fill the blanks.

• a. VIGS is Virus-Induced Gene Silencing. (A method used to study gene function by temporarily suppressing a target gene's expression using a modified plant virus).

• b. ZFN is Zinc-Finger Nuclease. (A class of engineered DNA-binding proteins used for targeted genome editing).

• c. TALEN is Transcription Activator-Like Effector Nuclease. (Another class of genome editing enzymes, similar to ZFNs, known for their high specificity).

• d. PEG is Polyethylene Glycol. (A polymer used in molecular biology to induce cell fusion or make cell membranes permeable for DNA uptake, as in protoplast transformation).

2. Cisgenesis and Intragenesis

Cisgenesis and Intragenesis are genetic modification techniques that use genes from the species itself or a closely related, crossable species. This distinguishes them from transgenesis, where genes from unrelated species are used.

• Cisgenesis: This involves inserting a natural, complete gene (including its own promoter and terminator sequences) from a crossable plant (same species or a sexually compatible relative) into a recipient plant. The resulting plant is considered a "cisgenic" plant and contains no foreign DNA.

• Intragenesis: This is similar but allows for more modification. It uses genetic elements (like promoters, coding sequences, and terminators) that are sourced from the same species or a crossable relative, but they can be combined in new, synthetic arrangements. For example, the promoter of one gene could be fused to the coding sequence of another gene from the same plant.

The key difference is that cisgenesis uses an exact, unmodified copy of a gene, while intragenesis can use new combinations of gene fragments.

3. What is Linkage Disequilibrium (LD)?

Linkage Disequilibrium (LD) is the non-random association of alleles at two or more different loci in a population.

In simple terms, if two alleles from different genes are inherited together more or less often than would be expected by chance, they are in LD. LD is primarily caused by a low recombination frequency between the loci, which is often due to their close physical proximity on the same chromosome. Other factors influencing LD include selection, mutation, genetic drift, and population structure.

The student's handwritten answer is good: "Non-random occurrence of alleles on the chromosome is called as linkage disequilibrium. Linkage Disequilibrium occurs because of selection constraint and close proximity of 2/more alleles on the same chromosome."

4. Selectable vs. Scorable Markers

Both are used in genetic transformation to identify cells that have successfully incorporated the new gene(s).

Feature	Selectable Markers	Scorable (Screenable) Markers
Function	They confer a trait (e.g., resistance) that allows only the transformed cells to survive under specific conditions.	They produce a visible signal (e.g., color, fluorescence) that allows researchers to identify or screen for transformed cells.
Mechanism	Kills or inhibits the growth of non-transformed cells.	Does not kill non-transformed cells; it just makes the transformed ones visually distinct.
Examples	Antibiotic resistance genes (nptII for kanamycin resistance), herbicide resistance genes (bar gene).	GUS gene (produces a blue color with X-Gluc substrate), GFP (Green Fluorescent Protein), LUC (Luciferase).

5. Traits for Crop Improvement

These traits can be categorized into two groups:

• Traits to Increase Genetic Potential:

  • Higher Yield: Increasing grain number, grain size, or biomass.

  • Improved Quality: Enhancing nutritional value (e.g., Golden Rice with Vitamin A), oil content, protein content, or shelf life.

  • Enhanced Photosynthetic Efficiency: Improving the plant's ability to convert light into energy.

• Traits to Remove Constraints (Stress Tolerance):

  • Biotic Stress Resistance: Resistance to pests (e.g., Bt cotton against bollworms) and diseases (fungal, bacterial, or viral resistance).

  • Abiotic Stress Tolerance: Tolerance to environmental challenges like drought, high salinity, extreme temperatures (heat/cold), and waterlogging.

6. What is Biopharming?

Biopharming (or molecular pharming) is the technology of using genetically engineered plants or animals to produce valuable pharmaceutical compounds, such as vaccines, antibodies, therapeutic proteins, and hormones. Instead of being synthesized in industrial bioreactors, these molecules are grown in crops. For example, research has focused on producing insulin in safflower and edible vaccines in bananas or potatoes.

7. Genetic Engineering vs. Conventional Breeding

Feature	Conventional Breeding	Genetic Engineering (Transgenesis)
Gene Source	Limited to the same species or sexually compatible wild relatives.	Can use genes from any living organism (plant, animal, bacteria, virus).
Precision	Imprecise. Involves crossing whole genomes, leading to the transfer of thousands of desired and undesired genes (linkage drag).	Precise. Allows for the transfer of a single or a few known genes.
Speed	Slow. Requires multiple generations of crossing and selection (often >10 years).	Fast. Can develop a new trait in a shorter timeframe.
Outcome	Shuffles existing genetic variation within a species.	Can introduce completely novel traits not present in the species' gene pool.

8. Biosafety Measures for Transgenics

Biosafety measures are protocols and regulations designed to assess and manage the potential risks associated with genetically modified organisms (GMOs). Key measures include:

1. Contained Use: Conducting initial experiments in secure labs and greenhouses to prevent environmental release.

2. Field Trial Regulations: Requiring isolation distances for field trials to prevent gene flow (cross-pollination) to non-GM crops or wild relatives.

3. Food & Feed Safety Assessment: Rigorous testing for potential toxicity, allergenicity, and unintended changes in nutritional composition before commercial approval.

4. Environmental Impact Assessment: Evaluating potential effects on non-target organisms (e.g., monarch butterflies and Bt corn pollen) and the risk of creating "superweeds."

5. Regulatory Oversight: National and international bodies (like GEAC in India, EFSA in Europe) review all data before approving a GMO for cultivation or consumption.

9. Factors for Efficient Genetic Transformation

The success and efficiency of genetic transformation depend on several critical factors:

• Transformation Method: The choice of method (Agrobacterium-mediated, biolistics, etc.) is crucial. Agrobacterium is often preferred for its efficiency and cleaner integrations.

• Plant Genotype: Some species and even cultivars within a species are highly "recalcitrant" (resistant) to transformation and regeneration.

• Explant Type: The type of tissue used (e.g., embryos, leaf discs, calli) and its physiological state significantly impact success.

• Vector Design: The construction of the plasmid vector, including the choice of promoters, markers, and terminators, is vital for proper gene expression.

• Tissue Culture Conditions: The composition of the growth media, including hormones and selection agents, must be optimized for cell growth, selection, and regeneration.

10. Chemical vs. Biological Methods of Transformation

• Biological Methods: This primarily refers to Agrobacterium-mediated transformation. It uses the natural ability of the bacterium Agrobacterium tumefaciens to transfer a piece of its plasmid DNA (the T-DNA) into the plant genome. It is highly efficient, precise, and typically results in low-copy-number integrations.

• Chemical Methods: These are direct DNA delivery methods that use chemicals to make the protoplast (plant cell without a cell wall) membrane permeable to DNA. The most common is PEG (Polyethylene Glycol)-mediated transformation, where PEG facilitates the uptake of plasmid DNA into protoplasts. This method is simpler but often less efficient and can lead to complex integration patterns.

11. Physical Methods of Genetic Transformation

These methods use physical force to breach the cell wall and membrane to deliver DNA directly.

• Biolistics (Gene Gun): Microscopic particles of gold or tungsten are coated with DNA and "shot" at high velocity into plant cells or tissues.

• Electroporation: A high-voltage electric pulse is applied to cells, creating temporary pores in the cell membrane through which DNA can enter.

• Microinjection: A fine glass micropipette is used to inject DNA directly into the nucleus of a single cell.

• Silicon Carbide Whiskers: DNA and plant cells are vortexed with tiny, sharp silicon carbide fibers. The fibers pierce the cells, allowing DNA to enter.

12. Phenotyping and Genotyping

• Genotyping: The process of determining the specific genetic makeup (genotype) of an individual. This involves analyzing its DNA to identify alleles, mutations, or variations, often using techniques like PCR, DNA sequencing, or molecular markers (e.g., SSRs, SNPs).

• Phenotyping: The process of measuring and analyzing an individual's observable traits (its phenotype), such as height, yield, disease resistance, flower color, etc. The phenotype is a result of the interaction between the genotype and the environment ($P=G+E$).

13. Steps in PCR and Profile for RAPD Markers

Steps in a PCR (Polymerase Chain Reaction) Cycle:

PCR is a technique to amplify a specific segment of DNA. A typical cycle consists of three steps:

1. Denaturation (~95°C): The reaction is heated to separate the double-stranded DNA template into two single strands.

2. Annealing (~50-65°C): The temperature is lowered to allow short DNA primers to bind (anneal) to their complementary sequences on the single-stranded template DNA.

3. Extension (~72°C): The temperature is raised to the optimal temperature for the Taq DNA polymerase enzyme, which synthesizes a new DNA strand complementary to the template, starting from the primer.

   These three steps are repeated for 25-35 cycles, leading to an exponential amplification of the target DNA sequence.

Profile for RAPD Markers:

RAPD (Random Amplified Polymorphic DNA) is a type of PCR-based marker.

• It uses a single, short (usually 10 bases), arbitrary (random sequence) primer.

• This single primer anneals to multiple random locations in the genome where a complementary sequence exists on both strands in an inverted orientation and within an amplifiable distance.

• The resulting PCR products are separated by size using agarose gel electrophoresis.

• The "profile" is the banding pattern seen on the gel. If there is a genetic difference (polymorphism) between two individuals (e.g., a mutation at a primer binding site), it will alter the banding pattern. The presence or absence of a specific band is the marker.

14. Reverse Breeding

Reverse breeding is a modern plant breeding method that allows for the rapid creation of homozygous parental lines that, when crossed, can exactly recreate a superior but complex heterozygous hybrid.

How it works:

1. Start with an elite heterozygous hybrid plant.

2. Use genetic modification to suppress meiotic recombination in this plant (e.g., by knocking out key recombination genes).

3. Generate haploid cells (gametes) from this non-recombining plant via tissue culture. Since there was no recombination, these gametes carry non-recombined parental chromosomes.

4. Use chromosome doubling techniques (e.g., with colchicine) to create a collection of Doubled Haploid (DH) lines. Each DH line is perfectly homozygous.

5. This collection of homozygous lines can then be screened and crossed to identify the pair that reconstitutes the original elite hybrid.

Usefulness: It drastically speeds up the process of creating parental lines for hybrid production, bypassing the many generations of selfing and selection required in conventional breeding. It is particularly useful for crops where creating inbred lines is difficult or for fixing complex traits governed by multiple genes.