Introduction to Molecular Markers and their Application in Biotechnology

Transcription

1 Introduction to Molecular Markers and their Application in Biotechnology

2 Molecular Markers 1. What are molecular markers? Introduction to technology Description of types of markers 2. Uses of molecular markers Application as a genetic tool for plant genotyping and gene mapping Application in agriculture Marker-assisted breeding Plant variety protection Assessing genetic diversity Applications in Human Health Association with genetic-based diseases Forensic studies

3 Molecular Markers 1. What are molecular markers? Heritable DNA sequence differences (polymorphisms) Phenotypically neutral, developmentally and environmentally stable Identified by techniques such as Southern hybridizations or PCR 2. Types of Markers Those detected by Southern Hybridizations RFLPs --Restriction Fragment Length Polymorphisms VNTRs -- variable number of tandem repeats (minisatellites) Those detected by PCR-based methods RAPD -- randomly amplified polymorphic DNA AFLP -- amplification fragment length polymorphism CAPS -- cleaved amplified polymorphic site SSR -- simple sequence repeats (microsatellites) SNP -- single nucleotide polymorphisms The best molecular markers are those that distinguish multiple alleles per locus (i.e. are highly polymorphic) and are co-dominant (each allele can be observed)

4 RFLP- a site in a genome where the distance between two restriction sites varies among different individuals. These sites are identified by restriction enzyme digests of chromosomal DNA, and the use of Southern blotting to identify the specific fragments. Requires a radioactive probe! CAPS-a site in the genome polymorphic for the presence or absence of a restriction enzyme site. Detected by PCR and restriction enzyme digests on gels. SSR-a site in the genome that contains many short tandem repeat sequences (microsatellites). These sites are usually in the size range of base pairs composed of dinucleotide and trinucleotide repeats. They are very polymorphic, scattered through out genomes. Genomes typically contain 1,000s of SSRs! They are detected by PCR using primers flanking the repeats and resolved on gels. SNP-a single nucleotide difference in the sequence of a gene or segment of the genome. There are typically tens of 1,000s of SNPs and a variety of methods for analyzing them, including highly automated/high throughput procedures with simultaneous scoring of many markers. Detection of SNPs can be done without gels.

5 RFLPs can arise through point mutations, deletions, insertions, etc. Allele A Allele B A deletion between two restriction enzyme sites probe polymorphism probe AA BB AB AA BB AB An insertion between two restriction enzyme sites transposon

6 RFLPs Restriction Fragment Length Polymorphism (RFLP) probe binding site 5Õ EcoRI EcoRI 3Õ CHR 1: NNNG AATTCNNN NNNG AATTCNNN NNNCTTAA GNNN NNNCTTAA GNNN 3Õ 5Õ probe binding site 5Õ EcoRI EcoRI 3Õ CHR 2: NNNG AATTCNNN NNNG AATTCNNN NNNCTTAA GNNN NNNCTTAA GNNN 3Õ 5Õ allele B allele A allele C Genomic DNA cut by EcoRI, DNA run on gel, blotted, and hybridized to probe Polymorphism: fragments of different lengths migrate differently in the gel

7 Allele A RFLPs Allele B Southern blot probe AA BB AB polymorphism probe Allele B (RFLP B ) appears associated with the phenotype! AB BB But not completely! #12 must be a recombinant between the phenotypic locus and the RFLP B * * * * * * * P1 P

8 Allele A RFLPs Allele B probe probe Southern blot AA BB AB Allele B * Allele A * * * * * * * P1 P

9 Review of Genetic Linkage and Recombination Genes that are close together on a chromosome tend to be inherited together. In the example shown at left, genes A and B would tend to be inherited together much more often than with gene C. Gene C would be inherited with B slightly more often than with A. Recombination & Crossing Over Exchange of alleles, but not gene order, between the two homologous chromosomes in diploids during meiosis. Frequency of crossing-over increases with distance between loci.

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11 Allele A RFLPs Allele B probe probe Southern blot AA BB AB Allele B * Allele A Allele B Allele A * * * * * * * P1 P * *

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15 CAPS (cleaved amplified polymorphic sequence) as a molecular marker. Allele A Allele B Sequence Specific Primers Amplify by PCR Digest with restriction enzyme Visualize on Agarose Gel polymorphism This procedure is much quicker than doing a Southern hybridization, yet yields the same information!! AA BB AB

16 SSR: simple sequence repeat (length polymorphism) PCR Primer (CA) (CA) n n+4 variation between strains in number of repeats at a given locus PCR yields products of different size:

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18 Simple Sequence Repeat (SSR) or Microsatellite Markers Polymorphism is based on the number of times a simple sequence of DNA, usually 2-3 base pairs, is repeated. The variant alleles are probably generated by stuttering of DNA polymerase or repair enzymes during DNA replication of repeated sequences. Male parent (red square) contains alleles 5 and 2 Female parent (black circle) contains alleles 6 and 3 Progeny segregate for alleles 2, 3, 5, and 6 One daughter is mated to a male containing another allele (4), resulting in offspring heterozygous for alleles 5 and 4.

19 Bottom line: interpretation of data for RFLP markers and microsatellites is the same, even though the data is generated by means of different techniques (Southern blotting for RFLP, PCR for microsatellite)

20 Molecular Markers 1. What are molecular markers? Introduction to technology Description of types of markers 2. Uses of molecular markers Application as a genetic tool for plant genotyping and gene mapping Application in agriculture Marker assisted breeding Plant variety protection Assessing genetic diversity Applications in Human Health Association with genetic-based diseases Forensic studies

21 Genotyping Progeny in a Standard Genetic Cross An example of a genetic cross wild type X teosinte branched mutant (+/+) X (tb1/tb1) F1 all wild type (+/tb1) F2 Normal : tb1 3 : 1 (+/+ or +/tb1) : (tb1/tb1) Normals are either +/+ or +/tb1, but one can not distinguish between the two genotypes because + is dominant to the recessive tb1! BUT, a molecular marker can easily distinguish between the genotypes! Prepare DNA from leaves of each type and use molecular marker to determine genotype!

22 Physical Maps Molecular markers can be mapped relative to one another This leads to a physical map that illustrates the linkage relationships between physical markers on each of the chromosomes Now, one can map a new mutation (morphological marker) relative to the molecular markers by following linkage between the mutant phenotype and polymorphisms in molecular markers.

23 Each chromosome is divided into bins These bins are about 20 cm Bins are defined by molecular markers

24 Lets use molecular markers to find the map location of a newly isolated mutant!

25 Map your mutant by comparing its inheritance relative to the inheritance of mapped molecular markers First, make a mapping population: New mutant: tb1-like. This mutant was found in an EMS screen for new mutants in maize inbred B73 tb tb In a B73 background x Mo17 B1 B2 tb B3 B4 B1 B2 tb B3 B4 x M1 M2 TB M3 M4 M1 M2 TB M3 M4 F1 B1 B2 tb B3 B4 M1 M2 TB M3 M4

26 Generate Mapping Population F1 B1 B2 tb B3 B4 B1 B2 TB B3 B4 F2 B1 B2 TB B3 B4 B1 B2 TB B3 B4 B1 B2 tb B3 B4 B1 B2 TB B3 B4 B1 B2 tb B3 B4 B1 B2 tb B3 B4 WT WT tb mutant This population will segregate 3:1 for wild type and tb-like plants. You will isolate DNA from some number of mutant (tb) plants. Every chromosome transmitted is potentially recombinant. Every F2 mutant can be scored for 0/2, 1/2, or 2/2 recombinant chromosomes. For each molecular marker to be tested for linkage with tb mutant, identify a polymorphism between the B73 and MO17. Then score inheritance of the polymorphism. You look to see if the polymorphism is inherited with (linked to) your mutant.

27 If a molecular marker is very closely linked to the mutation (tb), then those progeny homozygous for the mutation will also be homozygous for the B73 polymorphism B73 Mo17 tb 1 tb 2 tb 3 tb 4 tb 5 tb 6 tb 7 tb 8 tb 9 tb 10 tb 11 tb 12 tb 13 tb 14 tb 15 tb 16 tb 17 tb 18 tb 19 tb 20

28 If a molecular marker is unlinked to the mutation (tb), then those progeny homozygous for the mutation are just as likely to inherit the MO17 polymorphism as the B73 polymorphism B73 Mo17 tb 1 tb 2 tb 3 tb 4 tb 5 tb 6 tb 7 tb 8 tb 9 tb 10 tb 11 tb 12 tb 13 tb 14 tb 15 tb 16 tb 17 tb 18 tb 19 tb recombinant chromosomes out of a total of 40 scored This marker is unlinked to the mutation!

29 If a molecular marker is linked to the mutation (tb), then those progeny homozygous for the mutation are more likely to inherit the B73 polymorphism then the MO17 polymorphism B73 Mo17 tb 1 tb 2 tb 3 tb 4 tb 5 tb 6 tb 7 tb 8 tb 9 tb 10 tb 11 tb 12 tb 13 tb 14 tb 15 tb 16 tb 17 tb 18 tb 19 tb 20 8 recombinant chromosomes out of a total of 40 scored 8/40 = 0.2 or 20% recombination or 20 map units (20 cm between this marker and the mutation). You have found a linked molecular marker!! B1 B2 tb B3 B4

30 If a molecular marker is linked to the mutation (tb), then those progeny homozygous for the mutation are more likely to inherit the B73 polymorphism then the MO17 polymorphism B73 Mo17 tb 1 tb 2 tb 3 tb 4 tb 5 tb 6 tb 7 tb 8 tb 9 tb 10 tb 11 tb 12 tb 13 tb 14 tb 15 tb 16 tb 17 tb 18 tb 19 tb 20 8 recombinant chromosomes out of a total of 40 scored 8/40 = 0.2 or 20% recombination or 20 map units (20 cm between this marker and the mutation). You have found a linked molecular marker!! B1 B2 tb B3 B4

31 Bulked Segregant Analysis B73 Mo17 mutants If a molecular marker is unlinked to the mutation B73 Mo17 Pooled (bulked) mutants B73 Mo17 mutants If a molecular marker is linked to the mutation F1 Pooled (bulked) normals Pooled (bulked) mutants B73 Mo17 Pooled (bulked) mutants

32 SNP: single nucleotide polymorphisms Outgrowth of sequencing projects Detection of SNPs can be done without gels: highly automated/high throughput and/or highly parallel (simultaneous scoring of MANY markers)

33 Allele-Specific Extension & Identification in CE: Minisequencing (ABI SNaPShot TM )

34 Degree of Multiplexing Depends on Resolution dr6g dr110 ABI SNaPshot on 3130xl

35 Genotyping by SBE and Mass Spectrometry PCR Amplification SAP Treatment Single Base Extension Spot on 384-place Chips MALDI-TOF Mass Spec

36 Other uses for Molecular Markers Plant Variety Protection Verify varietal identity, purity and stability Marker-Assisted Breeding For accelerated trait/transgene introgressions For introgression of quantitative traits Estimation of Genetic Variation For phylogenetic analysis For preservation of rare plant and animal species For management of wild species Plant Pathogen Identification

37 Molecular Markers in Human Health Identification of human genetic diseases Sickel cell anemia Huntingtons disease Tay Sachs disease Cystic Fibrosis (and many more) Forensic Science Paternity Determinations

38 Introduction to Molecular Markers and their Application in Biotechnology