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Using CRISPR Cas9 and Marker Assisted Technologies for Cannabis Breeding

Using CRISPR-Cas9 and Marker Assisted Technologies for Cannabis Breeding What is CRISPR-Cas9? The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat) system evolved as an adaptive immune response in bacteria and archaea to defend against invading viruses. This allows the bacteria to essentially keep a genetic record of the invading viral DNA to speed up response time to render the virus inactive. This technology can be applied to plants breeding by allowing for specific gene loci to be targeted for gene alteration. Use for CRISPR Breeding processes that are assisted with CRISPR technologies may benefit from a reduction in the time to develop a new cultivar. What Are Marker Assisted Technologies? Isozyme variation RFLP PCR Microsatellites Reference Article Mandolino, Giuseppe & Carboni, Andrea. (2004). Potential of marker-assisted selection in hemp genetic improvement. Euphytica. 140. 107-120. 10.1007/s10681-004-4759-6. https://www.researchgate.net/publicat... Isozyme Variation Isozymes are protein markers. The technique is based on the principal that allelic variation exists from many different proteins. This uses the simple process of making a crude protein extract from some tissue sources, usually leaves that is separated by gel electrophoresis. Disadvantages: The number of isozyme loci that can be scored is limited. Different source tissues will provide different results *As a result, this is rarely used today in favor of RFLP’s RFLP (Restriction Fragment Length Polymorphism) RFLP: a molecular marker based on differences at the genomic DNA level. RFLP markers are defined by a specific enzyme-probe combination that identifies one single locus at a time and utilizes agarose gel separation. PCR (Polymerase Chain Reaction) The restriction digestion of genomic DNA is followed by a PCR amplification of the fragments obtained This uses labeled primers that anneal to the ends of the fragments Having a variable number of extra nucleotides randomly chosen, so to amplify only a subset of the total fragments obtained. The labeled amplification products are then run in polyacrilamide gels. Microsatellites Short sequences of two, three or more nucleotides that are repeated for a variable number of times in the genome In general, one single locus is identified by each PCR reaction, but the number of alleles that can be identified is very high, as the variability in the repeated motif number is high in the plant genomes Only recently identified and used for Cannabis Microsatellite markers were most useful in describing the hemp germplasm. Link to Lecture Slides: https://drive.google.com/file/d/18FTd... 420 Magazine Source Content by Slide Title: Title: Using CRISPR-Cas9 and Marker Assisted Technologies for Cannabis Breeding https://www.420magazine.com/community... Use for CRISPR https://www.420magazine.com/community... *Due to the description character limit the full work cited for "Using CRISPR Cas9 and Marker Assisted Technologies for Cannabis Breeding" can be viewed at... https://drive.google.com/file/d/1sQ6y...