Chap 9
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Chap. 9 Molecular Markers
1. 2. 3. 4. 5.
Purpose and expected outcomes Genetic markers (markers) are essentially landmarks on chromosomes, which are essential to finding out where genes are on a genetic map. Genes can be located by how close they are to the known landmarks or markers. To be useful, markers should be heritable and readily assayable or detected. A goal of the HGP is to identify markers that are linked to genetic diseases. In this chapter, you will learn: (a) The types of markers. (b) The types of molecular markers. (c) The properties of molecular markers (d) The importance of markers in biotechnology. Types of markers
1. Two categories of markers: (a) morphological markers i. are manifested on the outside of the organism as a product of the interaction of genes and the environment (i.e., an adult phenotype). (b) molecular markers i. are detected at the subcellular level and can be assayed before the adult stage in the life cycle of the organism. ii. Useful fir germplasm evaluation, map-based cloning of genes, and molecular breeding. 2. Markers are very useful for breeding quantitative traits and traits with low heritability. Molecular markers 1. Isozymes were the first true molecular markers to be discovered. 2. DNA markers that are currently widely used include: (a) RFLP (restriction fragment length polymorphism) (b) AFLP (amplified fragment length polymorphism) (c) PCR (polymerase chain reaction)-based (d) SNPs (single- nucleotide polymorphisms) (e) Microsatellites 3. Molecular markers may be grouped into two categories: (a) Single- locus, multi-allelic, codominant markers i. includes RFLPs and microsatellites (SSR). ii. Microsatellites are capable of detecting higher levels of polymorphisms than RFLPs and AFLPs.
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(b) Multi- locus, single-allelic, dominant markers i. includes AFLP and RAPD (random amplified polymorphic DNA) RFLPs: (a) They originate from natural variations in the genome that cause homologous chromosomes to produce different restriction products. (b) RFLPs are randomly distributed throughout the genome of an organism and may occur in both exons and introns. (c) A restriction enzyme is used to digest DNA, followed by electrophoresis, Southern blotting, and then probing to detect polymorphisms. (d) The DNA profiles or fingerprints produced are specific to the combination of restriction enzymes and probe. (e) Probes may be derived from a random genomic DNA library, cDNA library. (f) Fig. 9-1. RAPD (a) A PCR-based marker system. (b) The total genomic DNA is amplified using a single short (about 10 bp) random primer (Fig. 9-2). (c) The PCR product is submitted to gel electrophoresis. DNA amplification fingerprinting (DAF) (a) a variation of the RAPD methodology. (b) Uses very short (5-8 bp) random primers and consequently produces more variation than RAPDs. (c) More effective in distinguishing among closely related cultivars. Simple sequence repeats (SSRs) (a) occur in microsatellites in eukaryotic genomes. (b) Random tandem repeats of 2-5 nucleotides (e.g., GT, GACA) that are present in copy numbers that vary among individuals and are sources of polymorphism in plants and animals. (c) Also PCR-based AFLP (a) highly sensitive for DNA fingerprinting. (b) AFLP is simply RFLPs visualized by selective PCR amplification of DNA restriction fragments. (c) Fig. 9-3. Single nucleotide polymorphisms (SNPs) (a) SNP is a single base pair site in the genome that is different from one individual to another. (b) Most frequently occur ring genetic markers in humans, occurring at a rate of every 100 to 1,000 bp.
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Marker-assisted selection 1. Plant cultivar identification 2. Conventional breeding (and linkage mapping) 3. Markers for selection in transformation studies: (a) rDNA technology usually requires the use of a marker. (b) Cloning vectors have built- in selection marker systems. (c) Selection: a process by which host cells that have incorporated recombinant vectors are identified and isolated from untransformed cells. (d) Replica plating (Fig. 9-4). Key concepts Websites tutorial See p. 124 References 1. Barrett, B. and K.K. Kidwell. 1998. AFLP-based genetic diversity assessment among wheat cultivars from the Pacific Northwest. Crop Science, 38:1261-1271. 2. Davila, J.A., Y. Loarce, and E. Ferrer. 1999. Molecula r characterization and genetic mapping of random amplified microsatellite polymorphism in barley. Theoretical and Applied Genetics, 98:265-273. 3. Kwok, P.Y. and Z. Gu. 1999. SNP libraries: Why and how are we building them? Molecular Medicine Today, 12:538-543. 4. Rosenberg, M., M. Przybylska, and D. Strauss. 1994. RFLP subtraction: A method for making libraries of polymorphic markers. Proc. Nat. Acad. Sci. USA, 91:6113-6117. 5. Smigielski, E.M., K. Sirotkin, M. Ward, and S.T. Sherry. 2000. dbSNP: A database of single nucleotide polymorphisms. Nucleic Acid Research, 28(1):352-355.
Chap. 9 Molecular Markers
1. 2. 3. 4. 5.
Purpose and expected outcomes Genetic markers (markers) are essentially landmarks on chromosomes, which are essential to finding out where genes are on a genetic map. Genes can be located by how close they are to the known landmarks or markers. To be useful, markers should be heritable and readily assayable or detected. A goal of the HGP is to identify markers that are linked to genetic diseases. In this chapter, you will learn: (a) The types of markers. (b) The types of molecular markers. (c) The properties of molecular markers (d) The importance of markers in biotechnology. Types of markers
1. Two categories of markers: (a) morphological markers i. are manifested on the outside of the organism as a product of the interaction of genes and the environment (i.e., an adult phenotype). (b) molecular markers i. are detected at the subcellular level and can be assayed before the adult stage in the life cycle of the organism. ii. Useful fir germplasm evaluation, map-based cloning of genes, and molecular breeding. 2. Markers are very useful for breeding quantitative traits and traits with low heritability. Molecular markers 1. Isozymes were the first true molecular markers to be discovered. 2. DNA markers that are currently widely used include: (a) RFLP (restriction fragment length polymorphism) (b) AFLP (amplified fragment length polymorphism) (c) PCR (polymerase chain reaction)-based (d) SNPs (single- nucleotide polymorphisms) (e) Microsatellites 3. Molecular markers may be grouped into two categories: (a) Single- locus, multi-allelic, codominant markers i. includes RFLPs and microsatellites (SSR). ii. Microsatellites are capable of detecting higher levels of polymorphisms than RFLPs and AFLPs.
2
4.
5.
6.
7.
8.
9.
(b) Multi- locus, single-allelic, dominant markers i. includes AFLP and RAPD (random amplified polymorphic DNA) RFLPs: (a) They originate from natural variations in the genome that cause homologous chromosomes to produce different restriction products. (b) RFLPs are randomly distributed throughout the genome of an organism and may occur in both exons and introns. (c) A restriction enzyme is used to digest DNA, followed by electrophoresis, Southern blotting, and then probing to detect polymorphisms. (d) The DNA profiles or fingerprints produced are specific to the combination of restriction enzymes and probe. (e) Probes may be derived from a random genomic DNA library, cDNA library. (f) Fig. 9-1. RAPD (a) A PCR-based marker system. (b) The total genomic DNA is amplified using a single short (about 10 bp) random primer (Fig. 9-2). (c) The PCR product is submitted to gel electrophoresis. DNA amplification fingerprinting (DAF) (a) a variation of the RAPD methodology. (b) Uses very short (5-8 bp) random primers and consequently produces more variation than RAPDs. (c) More effective in distinguishing among closely related cultivars. Simple sequence repeats (SSRs) (a) occur in microsatellites in eukaryotic genomes. (b) Random tandem repeats of 2-5 nucleotides (e.g., GT, GACA) that are present in copy numbers that vary among individuals and are sources of polymorphism in plants and animals. (c) Also PCR-based AFLP (a) highly sensitive for DNA fingerprinting. (b) AFLP is simply RFLPs visualized by selective PCR amplification of DNA restriction fragments. (c) Fig. 9-3. Single nucleotide polymorphisms (SNPs) (a) SNP is a single base pair site in the genome that is different from one individual to another. (b) Most frequently occur ring genetic markers in humans, occurring at a rate of every 100 to 1,000 bp.
3
Marker-assisted selection 1. Plant cultivar identification 2. Conventional breeding (and linkage mapping) 3. Markers for selection in transformation studies: (a) rDNA technology usually requires the use of a marker. (b) Cloning vectors have built- in selection marker systems. (c) Selection: a process by which host cells that have incorporated recombinant vectors are identified and isolated from untransformed cells. (d) Replica plating (Fig. 9-4). Key concepts Websites tutorial See p. 124 References 1. Barrett, B. and K.K. Kidwell. 1998. AFLP-based genetic diversity assessment among wheat cultivars from the Pacific Northwest. Crop Science, 38:1261-1271. 2. Davila, J.A., Y. Loarce, and E. Ferrer. 1999. Molecula r characterization and genetic mapping of random amplified microsatellite polymorphism in barley. Theoretical and Applied Genetics, 98:265-273. 3. Kwok, P.Y. and Z. Gu. 1999. SNP libraries: Why and how are we building them? Molecular Medicine Today, 12:538-543. 4. Rosenberg, M., M. Przybylska, and D. Strauss. 1994. RFLP subtraction: A method for making libraries of polymorphic markers. Proc. Nat. Acad. Sci. USA, 91:6113-6117. 5. Smigielski, E.M., K. Sirotkin, M. Ward, and S.T. Sherry. 2000. dbSNP: A database of single nucleotide polymorphisms. Nucleic Acid Research, 28(1):352-355.
