PLNT3140 Introductory Cytogenetics

I. MAPPING A PHENOTYPE 

A. A molecular definition of "allele"

B. RFLPs are molecular alleles.

1. Restriction sites can be assayed for polymorphism within a population by Southern hybridization.

2. Restriction sites can reveal polymorphism between genotypes.

3. RFLPs behave like any other Mendelian trait.

I. MAPPING A PHENOTYPE

Why do we still need genetic mapping? Can't we just sequence the genome?

No matter how sophisticated and rapid DNA sequencing and other genomics technologies become, there is still no getting around the fact that what we're really interested in, in practical terms, is phenotypes. We want to somehow get at traits that are responsible for diseases in humans, or for agronomically useful characteristcs in crop plants and livestock. Examples of the latter would include disease resistance, yeild, protein quality, oil quality, or uniform height or flowering time. Many of these traits are quantitative traits, governed by multiple loci.

In many cases, the only thing we know at the beginning is the phenotype.

Consequently, Mendelian genetics is more relevant than ever, as the primary way to go from a trait we can see or measure, to a specific chromosomal locus and gene product.

The field of genetics was built upon the mapping of individual genes to nearest neighbors using 3 point crosses. Until molecular markers, there were no specific projects to map entire genomes.
Genetics will never be the same:
- greater precision (< 0.5cM)
- fewer crosses
- use of F₂ data
- fewer breeding generations
- more reliance on maps, mapping kits
- "everything has already been mapped"

A.  A molecular definition of "allele"

Modern genetic mapping using molecular markers still relies upon the basic principles of Mendellian genetics. We observe genetic segregation based on the segregation of alleles in genetic crosses. But for molecular markers, we need a broader molecular definition of what an allele is.


What is an allele?

Can be defined as:
• variant of a phenotype (phenotypic)
• variant of a DNA sequence (molecular)
Do silent mutations count as alleles?
Do they give us different phenotypes?

An allele defined by molecular means should have exactly the same genetic properties as a phenotypically-defined allele.

Infinite alleles model

1. Mutations within protein coding sequences define molecular allelism at many levels and degrees of severity.

• Identical sequences - the baseline against which mutations are measured
• Silent mutation - mutation in "degenerate" position in a codon, so that no amino acid replacement occurs (see Genetic Code below)
  
• Missense mutation - mutation leading to an amino acid substitution. Many, and perhaps most missense mutations have little or no effect on the function of the protein. Evidence for this comes from the fact that comparison of amino acid sequences from homologous proteins from many species often shows that some regions of a protein are highly conserved, while other regions appear to tolerate a great deal of amino acid substitution.
• Nonsense mutation - mutation which converts a codon into a stop codon. Occurrence of a stop codon within a protein coding sequence results in a truncated protein.
• Insertions/Deletions/Rearrangements - Insertions and deletions within a protein coding sequence can interrupt protein function. As well, insertions or deletions whose length is not a multiple of 3 will cause a frameshift mutation, meaning that incorrect amino acids will be inserted downstream from the mutation. Rearrangements in a protein coding sequence will probably result in loss of function.

Divergence of protein sequences during plant evolution. Amino acids from the N-terminal region of thionin proteins from several plant species are shown. Where amino acids have been inserted or deleted during the course of evolution, gap (-) characters have been inserted to optimize the alignment of homologous positions within the proteins.

-- but, mutations within genes are only a small percentage of the total number of mutations occurring in the genome!


2. Because only a small percentage of eukaryotic genomes codes for proteins, most mutations will occur within non-coding DNA, and will usualy be selectively neutral.

In most eukaryotic genomes, the chromosome is a sea of non-coding DNA punctuated by genes. This is illustrated in an approximately 72 kb region taken from Arabidopsis thaliana chromosome 4:



Click here to go to the Genome Data Viewer at NCBI

Additionally, genes are composed of both exons and introns. In comparison to exons, introns appear to mutate very rapidly, suggesting that there is little selective pressure against intronic mutations. In many higher eukaryotes, especially in animals, introns may be very long compred to exons. (Note that this appears not to be the case in plants, in which introns tend to be short.).

Summary:

i) Molecular alleles should segregate by the same Mendelian Principles as phenotypic alleles.
ii) In most cases, molecular alleles are selectively neutral.
iii) All of the rules of population genetics apply to molecular alleles.
iv) Practically speaking, there are an infinite number of possible molecular alleles. A given phenotypic allele observed in the population could, in principle, consist of a set of different molecularly-defined alleles.
 
Practical consequence: Because most mutations are selectively neutral and can occur anywhere in the genome, we can use molecular alleles as markers for chromosome mapping.

B. RFLP's (Restriction Fragment Polymorphisms) are molecular alleles

1. Restriction sites can be assayed for polymorphism within a population by Southern hybridization.

Assuming that individuals 1 & 2 are homozygous at the locus detected by these probes: 




2. Restriction sites can reveal polymorphism between genotypes.

DNA sequences are mutating all the time. As two populations diverge over time, they each accumulate different mutations at different sites. When restriction sites in the vicinity of a given gene are compared from one genotype to another, one genotype may have the site, and the other will not. This is referred to as a polymorphism. High polymorphism between two genotypes is evidence of genetic divergence. Mike Freeling and coworkers have compared restriction sites within the maize Alcohol dehydrogenase 1 gene in several maize lines. They found that although the restriction maps are identical within the Adh1 gene for all varieties tested, very few of the restriction sites outside of the coding region are conserved.

Johns, M. A., Strommer, J.N. and Freeling, M. (1983) Exceptionally high levels of restriction site polymorphism in DNA near the maize Adh1 gene. Genetics 105:733-743.
 

Maize genomic DNA was digested usng HindIII and the bands analyzed by Southern blot hybridization. DNA from seven maize lines was compared. The blot was hybridized using the pZmL84 probe, whose location on with respect to the AdhI gene is shown in the figure below. Note that this probe overlaps the 3' half of the adh1 transcript. Since the HindIII site is internal to the probe region, the probe detects two bands in all digests. In all maize lines, a conserved 2.5 kb fragment is seen, as well as a second band. The size of the second band is determined by how far away the next HindIII site is, downstream from the conserved HindIII site. Because numerous mutations have accumulated in the downstream region, in the different maize lines over time, the location of the downstream HindIII site differs between lines.


 
 
 

A restriction map of the corresponding region of the adh1 locus is shown below.

i) in all lines the 5' fragment detected by the pZmL84 probe is 2.5kb. The map shows that the two Hind3 sites that define this fragment are conserved.
ii) The 3' fragment is different in each line, because the next distal Hind3 site occurrs at a different location in each line.

Restriction maps of the seven Adh 1 chromosomal regions. The boundaries of the transcription units are denoted by the vertical dashed lines, and the region that hybridized with the ADH1-cDNA probe, pZML84, is also shown.

 
 

Now that hundreds of thousands of genes have been sequenced from thousands of species, we can generalize that protein coding regions are typically slow to diverge, because there is usually selective pressure to retain a given amino acid sequence. Outside of the protein coding region, and in non-coding DNA flanking genes, there is little selection against random mutations. Hence mutations are allowed to accumulate quickly in non-coding regions.

3. RFLPs behave like any other Mendelian trait.

Each band seen in a Southern blot indicates the presence of one or more restriction sites in a sequence. The sequence containing a restriction site is one allele, while the corresponding sequence missing the restriction site is the other allele. The "phenotypes" of these alleles are the differences in banding patterns, due to presence or absence of bands. The following exercises are designed to illustrate that the rules of Mendelian genetics apply for RFLPs exactly they do for any convential genetic trait.

IN-CLASS EXERCISES

EXERCISE 1 - Segregation in the F₁ generation
EXERCISE 2 - Measuring Recombination in a Test Cross
EXERCISE 3 - F₂ phenotypes

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