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Mendel then crossed these dihybrids. If it is inevitable that round seeds must always be yellow and wrinkled seeds must be green, then he would have expected that this would produce a typical monohybrid cross: 75% round-yellow; 25% wrinkled-green. But, in fact, his mating generated seeds that showed all possible combinations of the color and texture traits.
- 9/16 of the offspring were round-yellow
- 3/16 were round-green
- 3/16 were wrinkled-yellow, and
- 1/16 were wrinkled-green
Rule of Independent Assortment
Finding in every case that each of his seven traits was inherited independently of the others, he formed his "second rule" the Rule of Independent Assortment:
The inheritance of one pair of factors (genes) is independent of the inheritance of the other pair.
Today we know that this rule holds only if two conditions are met:
- the genes are on separate chromosomes or
- the genes are widely separated on the same chromosome.
Mendel was lucky in that every pair of genes he studied met one requirement or the other. The table shows the chromosome assignments of the seven pairs of alleles that Mendel studied. Although all of these genes showed independent assortment, several were, in fact, syntenic with three loci occurring on chromosome 4 and two on chromosome 1. However, the distance separating the syntenic loci was sufficiently great that the genes were inherited as though they were on separate chromosomes.
Start with two different strains of corn (maize).
- one that is homozygous for two traits
- yellow kernels (C,C) which are filled with endosperm causing the kernels to be
- smooth (Sh,Sh).
- a second that is homozygous for
- colorless kernels (c,c) that are wrinkled because their endosperm is
- shrunken (sh,sh)
When the pollen of the first strain is dusted on the silks of the second (or vice versa), the kernels produced (F1) are all yellow and smooth. So the alleles for yellow color (C) and smoothness (Sh) are dominant over those for colorlessness (c) and shrunken endosperm (sh).
To simplify the analysis, mate the dihybrid with a homozygous recessive strain (ccshsh). Such a mating is called a test cross because it exposes the genotype of all the gametes of the strain being evaluated.
According to Mendel's second rule, the genes determining color of the endosperm should be inherited independently of the genes determining texture. The F1 should thus produce gametes in approximately equal numbers.
- CSh, as inherited from one parent.
- csh, as inherited from the other parent
- Csh, a recombinant
- cSh, the other recombinant.
In fact, the recombination frequency is 2.0%, telling us that the actual order of loci is
c — sh — bz.
Mapping by linkage analysis is best done with loci that are relatively close together; that is, within a few centimorgans of each other. Why? Because as the distance between two loci increases, the probability of a second crossover occurring between them also increases.
But a second crossover would undo the effect of the first and restore the parental combination of alleles. These would show up as nonrecombinants. Thus as the distance between two loci increases, the percentage of recombinants that forms understates the actual distance in centimorgans that separates them. And, in fact, that has happened in this example. Using a three-point cross reveals the existence of a small number of double recombinants and tells us that the actual distance c—bz is indeed 5 cM as we would expect by summing
- c—sh = 3 cM
- sh—bz = 2 cM
and not the 4.6 cM revealed by the dihybrid cross.
A three-point cross also tells us the gene order in a single cross rather than the three we needed here.
There are other problems with preparing genetic maps of chromosomes.
- The probability of a crossover is not uniform along the entire length of the chromosome.
- Crossing over is inhibited in some regions (e.g., near the centromere).
- Some regions are "hot spots" for recombination (for reasons that are not clear). Approximately 80% of genetic recombination in humans is confined to just one-quarter of our genome.
- In humans, the frequency of recombination of loci on most chromosomes is higher in females than in males. Therefore, genetic maps of female chromosomes are longer than those for males.
A genetic map of chromosome 9 (the one that carries the C, Sh, and bz loci) of the corn plant (Zea mays) is shown above. If one maps in small intervals from one end of a chromosome to the other, the total number of centimorgans often exceeds 100 (as you can see for chromosome 9). However, even for widely-separated loci, the maximum frequency of recombinants that can form is 50%. And this is also the frequency of recombinants that we see for genes independently assorting on separate chromosomes. So we cannot tell by simply counting recombinants whether a pair of gene loci is located far apart on the same chromosome or are on different chromosomes. As we saw above, several of Mendel's independently assorting traits are controlled by genes on the same chromosome but located so far apart that they are inherited as if they were located on different chromosomes.
Genes that are present on the same chromosome are called syntenic.
Construction of genetic linkage map and identification of a novel major locus for resistance to pine wood nematode in Japanese black pine (Pinus thunbergii)
Pine wilt disease (PWD), which is caused by the pine wood nematode (PWN) Bursaphelenchus xylophilus, is currently the greatest threat to pine forests in Europe and East Asian countries including Japan. Constructing a detailed linkage map of DNA markers and identifying PWD resistance genes/loci lead to improved resistance in Pinus thunbergii, as well as other Pinus species that are also susceptible to PWD.
A total F1 mapping population of 188 individuals derived from a cross between the PWD-resistant P. thunbergii varieties ‘Tanabe 54’ (resistant rank 2 to PWD) and ‘Tosashimizu 63’ (resistant rank 4 to PWD) was inoculated with PWN, and was evaluated for disease symptoms. To perform linkage analysis for PWN resistance, a set of three maps was constructed two parental maps generated using the integrated two-way pseudo-testcross method, and a consensus map with population-type cross-pollination. The linkage map of ‘Tanabe 54’ consisted of 167 loci, and covered 14 linkage groups (LGs), with a total genetic distance of 1214.6 cM. The linkage map of ‘Tosashimizu 63’ consisted of 252 loci, and covered 14 LGs, with a total genetic distance of 1422.1 cM. The integrated consensus map comprised 12 LGs with the basic chromosome number of P. thunbergii, and a total genetic distance of 1403.6 cM. Results from quantitative trait loci (QTL) analysis using phenotype data and linkage maps indicated that PWN resistance is controlled by a single dominant allele, which was derived from the ‘Tanabe 54’ female parent. This major QTL was located on linkage group 3 and was designated PWD1 for PINE WILT DISEASE 1.
The PWD1 locus is a major resistance QTL located on the Pinus consensus LG03 that acts in a dominant manner to confer pine wood nematode resistance. Information from the present study will be useful for P. thunbergii breeding programs to improve resistance to PWD, and also to help identify susceptibility genes in Pinus species.
Construction of high-density genetic linkage map of Pyropia yezoensis (Bangiales, Rhodophyta) and identification of red color trait QTLs in the thalli
Pyropia yezoensis is an important macroalga because of its extensive global distribution and economic importance. Color is an important trait in the thalli of P. yezoensis, it is also an effective marker to identify the hybridization in genetic breeding. In this study, a high-density genetic linkage map was constructed based on high-throughput single nucleotide polymorphism (SNP) markers, and used for analyzing the quantitative trait loci (QTLs) of red color trait in the thalli of P. yezoensis. The conchospore undergoes meiosis to develop into an ordered tetrad, and each cell has a haploid phenotype and can grow into a single individual. Based on this theory, F1 haploid population was used as the mapping population. The map included 531 SNP markers, 394.57 cM long on average distance of 0.74 cM. Collinear analysis of the genetic linkage map and the physical map indicated that the coverage between the two maps was 79.42%. Furthermore, QTL mapping identified six QTLs for the chromosomal regions associated with the red color trait of the thalli. The value of phenotypic variance explained (PVE) by an individual QTL ranged from 4.71%–63.11%. And QTL qRed-1-1, with a PVE of 63.11%, was considered the major QTL. Thus, these data may provide a platform for gene and QTL fine mapping, and marker-assisted breeding in P. yezoensis in the future.
Physical and Genetic Mapping of Genome: 3 Things to know About
This article throws light upon physical and genetic mapping of genome. The three things to know about are:
(1) Genetic Techniques Used for Cross-Breeding Experiment (2) Molecular Markers in Physical Mapping and (3) Restriction Mapping of DNA Fragments.
Thing # 1. Genetic Techniques Used for Cross-Breeding Experiment:
Genetic mapping is based on the use of genetic techniques to construct maps showing the positions of genes and other sequences features on a genome. These genetic techniques include cross-breeding experiments or, in the case of humans, the examination of family histories.
Genetic mapping is based on the principles of inheritance as first described by Gregor Mendel in 1865 and genetic linkages.
Genetic maps are created to locate the genes or characters on the chromosome for their utilization in genetic studies. Physical maps are created to identify certain markers to detect or diagnose the specific character.
I. Genetic Linkage:
Genetic linkage occurs when particular genetic loci or alleles for genes are inherited jointly. Genetic linkage was first discovered by the British geneticists William Bateson and Reginald Punnett shortly after Mendel’s laws were rediscovered. Genetic loci on the same chromosome are physically connected and tend to stay together during meiosis, and are thus genetically linked. For example, in fruit flies the genes affecting eye color and wing length are inherited together because they appear on the same chromosome.
Alleles for genes on different chromosomes are usually not linked, due to independent assortment of chromosomes during meiosis. Because there is some crossing over of DNA when the chromosomes segregate, alleles on the same chromosome can be separated and go to different daughter cells. There is a greater probability of this happening if the alleles are far apart on the chromosome, as it is more likely that a cross-over will occur between them. The relative distance between two genes can be calculated using the offspring of an organism showing two linked genetic traits, and finding the percentage of the offspring where the two traits do not run together.
The higher the percentage of descendants that does not show both traits, the further apart on the chromosome they are. Among individuals of an experimental population or species, some phenotypes or traits occur randomly with respect to one another in a manner known as independent assortment.
Today scientists understand that independent assortment occurs when the genes affecting the phenotypes are found on different chromosomes or separated by a great enough distance on the same chromosome that recombination occurs at least half of the time. But in many cases, even genes on the same chromosome that are inherited together produce offspring with unexpected allele combinations. These results from a process called crossing over.
At the beginning of normal meiosis, a chromosome pair (made up of a chromosome from the mother and a chromosome from the father) intertwine and exchange sections or fragments of chromosome. The pair then breaks apart to form two chromosomes with a new combination of genes that differs from the combination supplied by the parents. Through this process of recombining genes, organisms can produce offspring with new combinations of maternal and paternal traits that may contribute to or enhance survival.
Ii. Genetic Map:
A genetic map is a linkage map of a species or experimental population that shows the position of its known genes and/or genetic markers relative to each other in terms of recombination frequency during crossover of homologous chromosomes. The greater the frequency of recombination (segregation) between two genetic markers, the farther apart they are assumed to be. Conversely, the lower the frequency of recombination between the markers, the smaller the physical distance between them.
Historically, the markers originally used were detectable phenotypes (enzyme production, color, shapes etc.) derived from coding DNA sequences. Now, non-coding DNA sequences such as microsatellites or those generating restriction fragment length polymorphisms (RFLPs) have been used. Genetic maps help researchers to locate other markers, such as other genes by testing for genetic linkage of the already known markers. A genetic map is not a physical map or gene map.
To be useful in genetic analysis, a gene must exist in at least two forms, or alleles each specifying a different phenotype. Earlier only those genes could be studied whose specifying phenotypes were distinguishable by visual observation. This approach soon became outdated as in many cases a single phenotypic character could be affected by more than one gene. For example, in 1922, 50 genes had been mapped onto the four fruit fly chromosomes, but nine of these genes were for eye color.
The observations by Thomas Hunt Morgan that the amount of crossing over between linked genes differs (partial linkage) led to the idea that crossover frequency might indicate the distance separating genes on the chromosome. Morgan’s student Alfred Sturtevant developed the first genetic map, also called a linkage map.
Iii. Recombination Frequency:
Sturtevant assumed that crossing over was a random event, there being an equal chance of it occurring at any position along a pair of lined-up chromatids. He proposed that the greater the distance between linked genes, the greater the chance that non-sister chromatids would cross over in the region between the genes. By working out the number of recombinants it is possible to obtain a measure for the distance between the genes. This distance is called a genetic map unit (m.u.), or a centimorgan and is defined as the distance between genes for which one product of meiosis in 100 is recombinant.
A recombinant frequency (RF) of 1% is equivalent to 1 m.u. A linkage map is created by finding the map distances between a numbers of traits that are present on the same chromosome, ideally avoiding having significant gaps between traits to avoid the inaccuracies that will occur due to the possibility of multiple recombination events.
Recombination frequency is the frequency that a chromosomal crossover will take place between two loci (or genes) during meiosis. Recombination frequency is a measure of genetic linkage and is used in the creation of a genetic linkage map. During meiosis, chromosomes assort randomly into gametes, such that the segregation of alleles of one gene is independent of alleles of another gene. This is stated in Mendel’s second law and is known as the law of independent assortment.
The law of independent assortment always holds true for genes that are located on different chromosomes, but for genes that are on the same chromosome, it does not always hold true. As an example of independent assortment, consider the crossing of the pure-bred homozygote parental strain with genotype AABB with a different pure-bred strain with genotype aabb. A and a and B and b represent the alleles of genes A and B. Crossing these homozygous parental strains will result in F1 generation offspring with genotype AaBb.
The F1 offspring AaBb produces gametes that are AB, Ab, aB, and ab with equal frequencies (25%) because the alleles of gene A assort independently of the alleles for gene B during meiosis. Note that 2 of the 4 gametes (50 %) Ab and aB-were not present in the parental generation. These gametes represent recombinant gametes. Recombinant gametes are those gametes that differ from both of the haploid gametes that made up the diploid cell. In this example, the recombination frequency is 50% since 2 of the 4 gametes were recombinant gametes.
The recombination frequency will be 50% when two genes are located on different chromosomes or when they are widely separated on the same chromosome. This is a consequence of independent assortment. When two genes are close together on the same chromosome, they do not assort independently and are said to be linked. Linked genes have a recombination frequency that is less than 50%.
As an example of linkage, consider the classic experiment by William Bateson and Reginald Punnett. They were interested in trait inheritance in the sweet pea and were studying two genes- the gene for flower color (P- purple and p- red) and the gene affecting the shape of pollen grains (L- long and I- round). They crossed the pure lines PPLL and ppll and then self-crossed the resulting PpLl lines.
According to Mendelian genetics, the expected phenotypes would occur in a 9:3:3:1 ratio of PL:P1:pL:p1. To their surprise, they observed an increased frequency of PL and pi and a decreased frequency of P1 and pL (Table 21.1).
Their experiment revealed linkage between the P and L alleles and the p and l alleles. The frequency of P occurring together with L and with p occurring together with I is greater than that of the recombinant PI and pL. The recombination frequency cannot be computed directly from this experiment, but it is less than 50%. The progeny in this case received two dominant alleles linked on one chromosome (referred to as coupling or cis arrangement).
However, after crossover, some progeny could have received one parental chromosome with a dominant allele for one trait (e.g., Purple) linked to a recessive allele for a second trait (eg round) with the opposite being true for the other parental chromosome (e.g., red and long). This is referred to as repulsion or a Trans-arrangement.
The phenotype here would still be purple and long but a test cross of this individual with the recessive parent would produce progeny with much greater proportion of the two crossover phenotypes. While such a problem may not seem likely from this example, unfavorable repulsion linkages do appear while breeding for disease resistance in some crops.
When two genes are located on the same chromosome, the chance of a crossover producing recombination between the genes is directly related to the distance between the two genes. Thus, the use of recombination frequencies has been used to develop linkage maps or genetic maps.
Iv. Genetic Mapping in Bacteria:
Bacteria are haploid organisms and do not undergo meiosis. So for creating their genetic maps geneticists made use of other methods to induce crossovers between homologous segments of bacterial DNA.
They used the three methods of recombination that occurs in bacteria:
(a) Conjugation-Two bacteria come into physical contact and one bacterium (the donor) transfers DNA to the second bacterium (the recipient). The transferred DNA can be a copy of some or possibly the donor cell’s entire chromosome, or it could be a segment of chromosome DNA up to 1 mb in length integrated in a plasmid. The latter is called episome transfer,
(b) Transduction- It involves transfer of a small segment of DNA up to 50 kb or so, from donor to recipient via a bacteriophage,
(c) Transformation- The recipient cell takes up from its environment a fragment of DNA, rarely longer than 50 kb, released from a donor cell.
In bacteria, the phenotype studied are the biochemical characteristics like ability to synthesize tryptophan in the dominant or wild type strain and inability to synthesize tryptophan in other strain, which is the recessive allele. The gene transfer is usually set up between a donor strains that possesses dominant gene to the recipient strain that possesses recessive gene. The transfer into the recipient is monitored by looking for attainment of the biochemical function specified by the gene being studied. This can be understood by (Fig. 21.1). Here, the functional gene for tryptophan synthesis from a wild strain is being transferred to recipient that lacks the functional copy of that gene (trp – ).
This recipient is called as auxotroph (bacteria which can survive only if provided with tryptophan). The wild strain (trp + ) does not require tryptophan for its survival. After the transfer, two crossovers are needed to integrate the transferred gene into the recipient cell’s chromosome, converting the recipient from trp” to trp + .
The precise detail of the map depends on the method of gene transfer being used. During conjugation, DNA is transferred from donor to recipient in the same way that a string is pulled through a tube. The relative positions of markers on the DNA molecule can therefore be mapped by determining the times at which the markers appear in the recipient cell. For example in Fig. 21.2, markers A, B and C are transferred after 8, 20 and 30 minutes of beginning of conjugation. The entire E. coli DNA takes approx. 100 minutes to transfer.
In case of transformation and transformation mapping enable genes that are relatively close together to be mapped, because the transferred DNA segment is short (<50kb), so the probability of two genes being transferred together depends on how close together they are on the bacterial DNA (Fig. 21.3).
Elie Wollman and Francois Jacob (1950s) conducted first genetic mapping experiments in bacteria. They studied linear transfer of genes in conjugation experiments between Hfr (Hfr- High frequency of recombination) and F – (F – fertility factor) strains of E. coli. During the experiment they interrupted conjugation between bacteria at specific times termed as “Interrupted mating”. They noticed that the time it takes for genes to enter a recipient cell is directly related to their order along the chromosome.
This experiment derived them to give the hypothesis that (a) The chromosome of the Hfr donor is transferred in a linear manner to the F- recipient cell (b) The order of genes along the chromosome can be deduced by determining the time required for various genes to enter the recipient.
Conjugation studies have been used to map over 1,000 genes along the circular E. coli chromosome. The genetic maps are scaled in minutes e.g., E. coli chromosome is 100 minutes long, conjugative transfer of the complete chromosome takes approximately 100 minutes (Fig. 21.4).
V. Gene Mapping in Humans by Pedigree Analysis:
To map human chromosomes, obviously one cannot perform controlled mating experiments. However, it is possible to estimate map positions by examining linkage in several generations of relatives. This means that only limited data are available, and their interpretations is often difficult because a human marriage rarely results in a convenient test cross, and often the genotypes of one or more family members are unobtainable because those individuals are dead or unwilling to cooperate.
For example, blood samples from several large Mormon families in Utah, where all the members of at least three generations were alive to be sampled, have been collected and stored. These have already been used to establish genetic linkage relationships and will be available in the years ahead to study other human genes as they are identified.
Thing # 2. Molecular Markers in Physical Mapping:
Different types of molecular markers are used to understand and ascertain relationship in different organisms/individuals as well as to detect or diagnose character. These markers are to locate certain characteristics on the gel (banding pattern) which can be used to detect a specific character/defect in the genome. Unlike genetic mapping, physical mapping is not to locate the genes/characters on a genome, but to create a unique pattern by processing the genomic DNA.
There are several molecular markers available which are used depending upon the objective of the work and facilities available at the centre. Use of these markers to create maps (e.g., electrophoretic patterns) of an organism is known as ‘physical mapping’. Molecular markers used in physical mapping are described below. New technologies are also developed simultaneously to resolve biological problems and help legal proceedings.
Restriction Fragment Length Polymorphism (RFLP):
RFLP is a method used by molecular biologists to follow a particular sequence of DNA as it is passed on to other cells. RFLPs can be used in many different settings to accomplish different objectives. RFLPs can be used in paternity cases or criminal cases to determine the source of a DNA sample.
RFLPs can be used to determine the disease status of an individual. RFLPs can be used to measure recombination rates which can lead to a genetic map with the distance between RFLP loci measured in centiMorgans.
RFLP, as a molecular marker, is specific to a single clone/restriction enzyme combination. It is a difference in homologous DNA sequences that can be detected by the presence of fragments of different lengths after digestion of the DNA samples in question with specific restriction endonucleases. Most RFLP markers are co-dominant (both alleles in heterozygous sample will be detected) and highly locus-specific.
An RFLP probe is a labelled DNA sequence that hybridizes with one or more fragments of the digested DNA sample after they were separated by gel electrophoresis, thus revealing a unique blotting pattern characteristic to a specific genotype at a specific locus. Short, single- or low-copy genomic DNA or cDNA clones are typically used as RFLP probes. The RFLP probes are frequently used in genome mapping and in variation analysis (genotyping, forensics, paternity tests, hereditary disease diagnostics, etc.) (Fig. 21.5).
Usually, DNA from an individual specimen is first extracted and purified. Purified DNA may be amplified by polymerase chain reaction (PCR), The DNA is then cut into restriction fragments using suitable endonucleases, which only cut the DNA molecule where there are specific DNA sequences, termed recognition sequence or restriction sites that are recognized by the enzymes.
These sequences are specific to each enzyme, and may be either four, six, eight, ten or twelve base pairs in length. The more base pairs there are in the restriction site, the more specific it is and the lower the probability that it will find a place to be cut. The restriction fragments are then separated according to length by agarose gel electrophoresis. The resulting gel may be enhanced by Southern blotting. Alternatively, fragments may be visualized by pre-treatment or post-treatment of the agarose gel, using methods such as ethidium bromide staining or silver staining respectively.
RFLPs have provided valuable information in many areas of biology, including: screening human DNA for the presence of potentially deleterious genes (Fig. 21.6). Providing evidence to establish the innocence of or a probability of the guilt of, a crime suspect by DNA “fingerprinting”. The distance between the locations cut by restriction enzymes (the restriction sites) varies between individuals, due to insertions, deletions or trans-versions.
This causes the length of the fragments to vary, and the position of certain amplicons differs between individuals (thus polymorphism). This can be used to genetically tell individuals apart. It can also show the genetic relationship between individuals, because children inherit genetic elements from their parents. Mitochondrial DNA RFLP analyses can lead to the determination of maternal relationships.
Fragments may also be used to determine relationships among and between species by comparison of the resulting haplotypes (abridged for ‘haploid genotype’). RFLP is a technique used in marker assisted selection. Terminal Restriction Fragment Length Polymorphism (TRFLP or sometimes T-RFLP) is a molecular biology technique initially developed for characterizing bacterial communities in mixed-species samples. The technique has also been applied to other groups including soil fungi.
The technique works by PCR amplification of DNA using primer pairs that have been labelled with fluorescent tags. The PCR products are then digested using RFLP enzymes and the resulting patterns visualized using a DNA sequencer. The results are analyzed either by simply counting and comparing bands or peaks in the TRFLP profile, or by matching bands from one or more TRFLP runs to a database of known species.
ii. Measurement of distance between two RFLP loci:
To calculate the genetic distance between two loci, you need to be able to observe recombination. Traditionally, this was performed by observing phenotypes but with RFLP analysis, it is possible to measure the genetic distance between two RFLP loci whether they are a part of genes or not. Let’s look at a simple example in fruit flies. Two RFLP loci with two RFLP bands possible at each locus (Fig. 21.7).
These loci are located on the same chromosome for the female (left) and the male (right). The upper locus can produce two different bands called 1 and 3. The lower locus can produce bands called 2 or 4. The male is homozygous for band 1 at the upper locus and 2 for the lower locus. The female is heterozygous at both loci. Their RFLP banding patterns can be seen on the Southern blot below (Fig. 21.8).
The male can only produce one type of gamete (1 and 2) but the female can produce four different gametes. Two of the possible four are called parental because they carry both RFLP bands from the same chromosome 1 and 2 from the left chromosome or 3 and 4 from the right chromosome. The other two chromosomes are recombinant because recombination has occurred between the two loci and thus the RFLP bands are mixed so that 1 is now linked to 4 and 3 is linked to 2.
When these two flies mate, the frequency of the four possible progeny can be measured and from this information, the genetic distance between the two RFLP loci (upper and lower) can be determined (Fig. 21.9).
In this example, 70% of the progeny were produce from parental genotype eggs and 30% were produced by recombinant genotype eggs. Therefore, these two RFLP loci are 30 centiMorgans apart from each other.
Isolation of sufficient DNA for RFLP analysis is time consuming and labor intensive. However, PCR can be used to amplify very small amounts of DNA, usually in 2-3 hours, to the levels required for RFLP analysis. Therefore, more samples can be analyzed in a shorter time. An alternative name for the technique is Cleaved Amplified Polymorphic Sequence (CAPS) assay.
RFLP is a multistep procedure involving restriction enzymatic cleavage, electrophoresis, southern blotting and detection of specific sequences. It is a time consuming process.
Random Amplified Polymorphic DNA (RAPD):
This technique can be used to determine taxonomic identity, assess kinship relationships, detect inter-specific gene flow, analyze hybrid speciation, and create specific probes. Advantages of RAPDs include suitability for work on anonymous genomes, applicability to work where limited DNA is available, efficiency and low expense. It is also useful in distinguishing individuals, cultivars or accessions. RAPDs also have applications in the identification of asexually reproduced plant varieties for forensic or agricultural purposes, as well as ecological ones.
In RAPD by using different primers, molecular characters can be generated that are diagnostic at different taxonomic levels. This is really a stripped-down version of PCR but uses a single sequence in the design of the primer (i.e., two primers are still needed for PCR: the same primer is used at either end).
The primer may be designed specifically, but could be chosen randomly and is used to amplify a series of samples which will include both the material of interest as well as other control samples with which the experimental material needs to be compared. Choice of primer length will be critical to the determination of band complexity in the resulting amplification pattern. Eventually a particular probe will be found that is able to distinguish between the sample of interest and those that are different.
Unlike traditional PCR analysis, RAPD (pronounced ‘rapid’) does not require any specific knowledge of the DNA sequence of the target organism: the identical 10-mer primers will or will not amplify a segment of DNA, depending on positions that are complementary to the primers’ sequence. For example, no fragment is produced if primers annealed too far apart or 3′ ends of the primers are not facing each other.
Therefore, if a mutation has occurred in the template DNA at the site that was previously complementary to the primer, a PCR product will not be produced, resulting in a different pattern of amplified DNA segments on the gel (Fig. 21.10). RAPD is an inexpensive yet powerful typing method for many bacterial species
RAPD amplification products can be either variable (polymorphic) or constant (non- polymorphic). In a RAPD analysis of several individuals within a species, and species within a genus, constant fragments diagnostic for a genus may be identified, as well as fragments which are polymorphic between species of the genus. RAPDs can be applied to analyze fusion of genotypes at different taxonomic levels. At the level of the individual, RAPD markers can be applied to parentage analysis, while at the population level, RAPD can detect hybrid populations, species or subspecies.
The detection of genotype hybrids relies on the identification of diagnostic RAPD markers for the parental genotypes under investigation. However RAPD markers tend to underestimate genetic distances between distantly related individuals, for example in inter-specific comparisons.
It is wise to be cautious when using RAPD for taxonomic studies above the species level. Conventional RFLP techniques are ill-suited for the analysis of paternity and estimation of reproductive success in species with large offspring clutches, because of the need to determine paternity for each individual offspring. RAPD fingerprinting provides a ready alternative for such cases.
Synthetic offspring may be produced by mixing equal amounts of the DNA of the mother and the potential father. The amplification products from the synthetic offspring should ideally contain the full complement of bands that appear in any single offspring of these parents (Table 21.2).
ii. Limitations of RAPD:
1. Nearly all RAPD markers are dominant, i.e., it is not possible to distinguish whether a DNA segment is amplified from a locus that is heterozygous (1 copy) or homozygous (2 copies). Co-dominant RAPD markers, observed as different-sized DNA segments amplified from the same locus, are detected only rarely.
2. PCR is an enzymatic reaction, therefore the quality and concentration of template DNA, concentrations of PCR components, and the PCR cycling conditions may greatly influence the outcome. Thus, the RAPD technique is notoriously laboratory dependent and needs carefully developed laboratory protocols to be reproducible.
3. Mismatches between the primer and the template may result in the total absence of PCR product as well as in a merely decreased amount of the product. Thus, the RAPD results can be difficult to interpret.
Amplification Fragment Length Polymorphism (AFLP):
Amplified Fragment Length Polymorphism (AFLP) is a polymerase chain reaction (PCR) based genetic fingerprinting technique that was developed in the early 1990’s by Keygene. AFLP can be used in the fingerprinting of genomic DNA of varying origins and complexities. The amplification reaction is rigorous, versatile and robust, and appears to be quantitative.
While AFLP is capable of producing very complex fingerprints (100 bands where RAPD produces 20), it is a technique that requires DNA of reasonable quality and is more experimentally demanding. AFLP uses restriction enzymes to cut genomic DNA, followed by ligation of complementary double stranded adaptors to the ends of the restriction fragments.
A subset of the restriction fragments are then amplified using 2 primers complementary to the adaptor and restriction site fragments. The fragments are visualized on denaturing polyacrylamide gels either through auto-radiographic or fluorescence methodologies.
AFLP-PCR is a highly sensitive method for detecting polymorphisms in DNA. The technique was originally described by Vos and Zabeau in 1993. The procedure of this technique is divided into three steps (Fig. 21.11):
1. Digestion of total cellular DNA with one or more restriction enzymes that cuts frequently (Msel, 4 bp recognition sequences) and one that cuts less frequently (EcoRI, 6 bp recognition sequence). The resulting fragments are ligated to end-specific adaptor molecules.
2. Selective amplification of some of these fragments with two PCR primers that have corresponding adaptor and restriction site specific sequences.
3. Electrophoretic separation of amplicons on a gel matrix, followed by visualisation of the band pattern.
In a second, “selective”, PCR, using the products of the first as template, primers containing two further additional bases, chosen by the user, are used. The EcoRI-adaptor specific primer used bears a label (fluorescent or radioactive). Gel electro-phoretic analysis reveals a pattern (fingerprint) of fragments representing about 1/4000th of the EcoRl-Msel fragments.
AFLP’s, can be co-dominant markers, like RFLP’s. Co-dominance results when the polymorphism is due to sequences within the amplified region. Yet, because of the number of bands seen at one time, additional evidence is needed to establish that a set of bands result from different alleles at the same locus.
If, however, the polymorphism is due to presence/absence of a priming site, the relationship is dominance. The non-priming allele will not be detected as a band. Compared to RAPD, fewer primers should be needed to screen all possible sites. AFLP can be used for mapping, fingerprinting and genetic distance calculation between genotypes. The advantage of AFLP is its high multiplexity and therefore the possibility of generating high marker densities.
One limitation of the AFLP technique is that fingerprints may share few common fragments when genome sequence homology is less than 90%. Therefore, AFLP cannot be used in comparative genomic analysis with hybridization-based probes or when comparing genomes that are evolving rapidly such as those of some microbes. Conversely, very homogeneous genomes may not be suitable for AFLP analysis.
A study on the genetic diversity of an endangered alpine plant (Eryngium alpinum L. (Apiaceae) demonstrated that AFLP markers enable a quick and reliable assessment of intraspecific genetic variability in conservation genetics. The study showed that although the endangered plant occurred in small isolated populations, these populations contained a high genetic diversity, a good indication that recovery of the species was possible.
ii. Limitations of AFLP:
1. Proprietary technology is needed to score heterozygotes and homozygotes. Otherwise, AFLP must be dominantly scored.
2. Developing locus-specific markers from individual fragments can be difficult.
3. Need to use different kits adapted to the size of the genome being analyzed.
Microsatellites can be used to determine genetic diversity within a species, as well as being able to distinguish varieties and even individuals, as well as parentage. The distribution of genetic variability is commonly used to verify species, subspecies or population division. Monitoring change in diversity may also be useful for predicting populations in peril as the persistence of a population partially depends on maintaining its evolutionary significance which requires genetic variation.
Microsatellites have been used to estimate demographic bottlenecks in some species. A bottleneck, when it severely and temporarily reduces population size, can also drastically reduce the genetic diversity of a population. A common theme in conservation genetics is the use of genetic variation to identify populations that have experienced bottlenecks, as numerous threatened or endangered species and populations have been found to have low levels of genetic variation.
Inter-Simple Sequence Repeats (ISSR):
ISSRs can be used to assess hybridization in natural populations of plants, as a study on Penstemon (Scrophulariaceae) did. Eight ISSR primers were used to examine patterns of hybridization and hybrid speciation in a hybrid complex involving four species, as well as allowing examination of pollen-mediated gene flow. Previous studies using allozymes, restriction-site variation of nuclear rDNA and chloroplast DNA failed to determine whether gene flow occurs among species other than P. cenranthifolius.
The previous studies also failed to provide support for hypotheses of diploid hybrid speciation. ISSR proved to be a much more successful technique in this study, allowing all species and all DNA accessions to be differentiated. ISSR has also been used to detect varieties and diversity in rice, revealing much more data than RFLPs. The technique allowed for dissection below the subspecies level and this gives it a good level of applicability in the study of rare or endangered plants.
ISSRs have been used in conjunction with RAPD data to determine the colonization history of Olea europaea in Macronesia, along with lineages in the species complex. The two techniques have also been utilized in examining the historical biogeography of Sea rocket (Cakile maritima) and Sea Holly (Eryngium maritimum), comparing different and only distantly related taxa of broadly similar extant distribution. The trees generated by the different methods were largely similar topologically. Using the result, dispersal routes of the species along a linear coast line could be construed.
Joint use of RAPD and ISSR has also been used to examine clonal diversity in Calamagrostis porteri ssp. insperata (Poaceae), a rare grass that has little or no sexual reproduction, and spreads by vegetative reproduction. The relative advantages and disadvantages of various molecular markers in physical mapping are summarized in Table 21.3. This information suggests that RFLP, SSR and AFLP markers are most effective in detecting polymorphism.
However, given the large amount of DNA required for RFLP detection and the difficulties in automating RFLP analysis, AFLP and SSR are currently most popular markers.
The main uses of these markers include:
1. Assessment of genetic variability and characterization of germplasm.
2. Identification and fingerprinting of genotypes.
3. Estimation of genetic distances between population, inbreeds and breeding material.
4. Detection of monogenic and qualitative trait loci.
5. Marker assisted selection.
6. Identification of sequences of useful candidate genes.
Thing # 3. Restriction Mapping of DNA Fragments:
Genetic mapping using RFLPs as DNA markers can locate the positions of polymorphic restriction sites within a genome, but very few of the restriction sites in a genome are polymorphic, so many sites are not mapped by this technique.
We increase the marker density on a genome map by using an alternative method to locate the positions of some of the non polymorphic restriction sites. This is what restriction mapping achieves, although in practice the technique has limitations that means it is applicable only to relatively small DNA molecules.
Methodology for Restriction Mapping:
The simplest way to construct a restriction map is to compare the fragment sizes produced when a DNA molecule is digested with two different restriction enzymes that recognize different target sequences. An example using the restriction enzymes EcoRI and BamHI is shown in figure. 21.12. First, the DNA molecule is digested with just one of the enzymes and the sizes of the resulting fragments measured by agarose gel electrophoresis. Next, the molecule is digested with the second enzyme and the resulting fragments again sized in an agarose gel.
The results of subsequent use of two enzymes give clear picture about restriction sites creating a large number of fragments but this method do not allow their relative positions to be determined. Additional information is therefore obtained by cutting the DNA molecule with both enzymes together. In the example shown in Figure 21.12, the double restriction enables three of the sites to be mapped. However, a problem arises with the larger EcoRI fragment because this contains two BamHI sites and there are two alternative possibilities for the map location of the outer one of these.
The problem dissolved by going back to the original DNA molecule and treating it again with BamHI on its own, but this time preventing the digestion from going to completion by, for example, incubating the reaction for only a short time rousing a suboptimal incubation temperature. This is called a partial restriction and leads to a more complex set of products. The complete restriction products now being supplemented with partially restricted fragments that still contain one or more uncut BamHI sites.
In the example shown in Figure 21.12, the size of one of the partial restriction fragments is diagnostic and the correct map can be identified. A partial restriction usually gives the information needed to complete a map, but if there are many restriction sites then this type of analysis becomes bulky, simply because there are many different fragments to consider. An alternative strategy is simpler because it enables the majority of the fragments to be ignored. This is achieved by attaching a radioactive or other type of marker to each end of the starting DNA molecule before carrying out the partial digestion.
The result is that many of the partial restriction products become “invisible” because they do not contain an end-fragment and so do not show up when the agarose gel is screened for labeled products (Fig. 21.12). The sizes of the partial restriction products that are visible enable unmapped sites to be positioned relative to the ends of the starting molecule.
The scale of restriction mapping is limited by the sizes of the restriction fragments. Restriction maps are easy to generate if there are relatively few cut sites for the enzymes being used. However, as the number of cut sites increases, so also do the numbers of single, double and partial-restriction products whose sizes must be determined and compared in order for the map to be constructed. Computer analysis can be brought into play but problems still eventually arise.
A stage will be reached when a digest contains so many fragments that individual bands merge on the agarose gel, increasing the chances of one or more fragments being measured incorrectly or missed out entirely. If several fragments have similar sizes then even if they can all be identified, it may not be possible to assemble them into a clear map. Restriction mapping is therefore more applicable to small rather than large molecules, with the upper limit for the technique depending on the frequency of the restriction sites in the molecule being mapped.
In practice, if a DNA molecule is less than 50 kb in length it is usually possible to construct a clear restriction map for a selection of enzymes with six nucleotide recognition sequences. Restriction maps are equally useful after bacterial or eukaryotic genomic DNA has been cloned, if the cloned fragments are less than 50kb in length, because a detailed restriction map can then be built up as a preliminary to sequencing the cloned region. This is an important application of restriction mapping in projects sequencing large genomes.
Restriction mapping can be used for mapping of entire genomes larger than 50kb by slightly eliminating the limitations of restriction mapping by choosing enzymes expected to have infrequent cut sites in the target DNA molecule.
These “rare cutters” fall into two categories:
1. A few restriction enzymes cut at seven- or eight-nucleotide recognition sequences. Examples are Sapl (5′-GCTCTTC-3′) and SgfI (5′-GCGATCGC-3′). The enzymes with seven-nucleotide recognition sequences would be expected, on average, to cut a DNA molecule with GC content of 50% once every 47 = 16,384 bp.
The enzymes with eight nucleotide recognition sequences should cut once every 48 = 65,536 bp. These figures compare with 46 = 4096 bp for enzymes with six-nucleotide recognition sequences, such as BamHI and EcoRI.
Cutters with seven-or eight-nucleotide recognition sequences are often used in restriction mapping of large molecules, but the approach is not as useful as it might by simply because not many of these enzymes are known.
2. Enzymes can be used whose recognition sequences contain motifs that are rare in the target DNA. Genomic DNA molecules do not have random sequences and some molecules are significantly deficient in certain motifs. For example, the sequence 5′-CG- 3′ is rare in the genomes of vertebrates because vertebrate cells possess an enzyme that adds a methyl group to carbon 5 of the C nucleotide in this sequence.
Domination of the resulting 5-methylcytosine gives thymine. The consequence is that during vertebrate evolution, many of the 5′-CG sequences that were originally in these genomes have become converted to 5′-TG-3′.
Restriction enzymes that recognize a site containing 5′- CG-3′ therefore cut vertebrate DNA relatively infrequently. Examples are Smal (5′-CCCGGG-3′), which cuts human DNA once every 78 kb on average, and BssHII (5′- GCGCGC′), which cuts once every 390 kb.
The potential of restriction mapping is therefore increased by using rare cutters. It is still not possible to construct restriction maps of the genomes of animals and plants, but it is feasible to use the technique with large cloned fragments, and with the smaller DNA molecules of prokaryotes and lower eukaryotes such as yeast and fungi.
If a rare cutter is used then it may be necessary to employ a special type of agarose gel electrophoresis to study the resulting restriction fragments. This is because the relationship between the length of DNA molecule and its migration rate in an electrophoresis gel is not linear, the resolution decreasing as the molecules get longer (Fig. 21.13A). This means that it is not possible to separate molecules more than about 50 kb in length, because all of these longer molecules run as a single, slowly migrating band in a standard agarose gel.
To separate them it is necessary to replace the linear electric field used in conventional gel electrophoresis with a more complex field. An example is provided by orthogonal field alternation gel electrophoresis (OFAGE), in which the electric field alternates between two pairs of electrodes, each positioned at an angle of 45″ to the length of the gel (Fig. 21.13B).
The DNA molecules still move down through the gel, but each change in the field forces the molecules to realign. Shorter molecules realign more quickly than longer ones and so migrate more rapidly through the gel. The overall result is that molecules much longer than those separated by conventional gel electrophoresis can be resolved. Related techniques include CHEF (contour clamped homogeneous electric fields) and FIGE (field inversion gel electrophoresis).
Genetic versus Physical Maps
Chromosome mapping by counting recombinant phenotypes produces a genetic map of the chromosome. But all the genes on the chromosome are incorporated in a single molecule of DNA. Genes are simply portions of the molecule (open reading frames or ORFs) encoding products that create the observed trait (phenotype). The rapid progress in DNA sequencing has produced complete genomes for hundreds of microbes and several eukaryotes.
Having the complete sequence makes it possible to determine directly the order and spacing of the genes. Maps drawn in this way are called physical maps.
What is the relationship between the genetic map and the physical map of a chromosome? As a very rough rule of thumb, 1 cM on a chromosome encompasses 1 megabase (1 Mb = 10 6 bp) of DNA. But for the reasons mentioned above, this relationship is only approximate. Although the genetic maps of human females average 90% longer than the same maps in males, their chromosomes contain the same number of base pairs. So their physical maps are identical.
There are two distinctive types of "Maps" used in the field of genome mapping: genetic maps and physical maps. While both maps are a collection of genetic markers and gene loci,  genetic maps' distances are based on the genetic linkage information, while physical maps use actual physical distances usually measured in number of base pairs. While the physical map could be a more "accurate" representation of the genome, genetic maps often offer insights into the nature of different regions of the chromosome, e.g. the genetic distance to physical distance ratio varies greatly at different genomic regions which reflects different recombination rates, and such rate is often indicative of euchromatic (usually gene-rich) vs heterochromatic (usually gene poor) regions of the genome.
Gene mapping Edit
Researchers begin a genetic map by collecting samples of blood, saliva, or tissue from family members that carry a prominent disease or trait and family members that don't. The most common sample used in gene mapping, especially in personal genomic tests is saliva. Scientists then isolate DNA from the samples and closely examine it, looking for unique patterns in the DNA of the family members who do carry the disease that the DNA of those who don't carry the disease don't have. These unique molecular patterns in the DNA are referred to as polymorphisms, or markers. 
The first steps of building a genetic map are the development of genetic markers and a mapping population. The closer two markers are on the chromosome, the more likely they are to be passed on to the next generation together. Therefore, the "co-segregation" patterns of all markers can be used to reconstruct their order. With this in mind, the genotypes of each genetic marker are recorded for both parents and each individual in the following generations. The quality of the genetic maps is largely dependent upon these factors: the number of genetic markers on the map and the size of the mapping population. The two factors are interlinked, as a larger mapping population could increase the "resolution" of the map and prevent the map from being "saturated".
In gene mapping, any sequence feature that can be faithfully distinguished from the two parents can be used as a genetic marker. Genes, in this regard, are represented by "traits" that can be faithfully distinguished between two parents. Their linkage with other genetic markers is calculated in the same way as if they are common markers and the actual gene loci are then bracketed in a region between the two nearest neighboring markers. The entire process is then repeated by looking at more markers that target that region to map the gene neighborhood to a higher resolution until a specific causative locus can be identified. This process is often referred to as "positional cloning", and it is used extensively in the study of plant species. One plant species, in particular in which positional cloning is utilized is in maize.  The great advantage of genetic mapping is that it can identify the relative position of genes based solely on their phenotypic effect.
Genetic mapping is a way to identify exactly which chromosome has which gene and exactly pinpointing where that gene lies on that particular chromosome. Mapping also acts as a method in determining which gene is most likely recombine based on the distance between two genes. The distance between two genes is measured in units known as centimorgan. A centimorgan is a distance between genes for which one product of meiosis in one hundred is recombinant. The further two genes are from each other, the more likely they are going to recombine. If it were closer, the opposite would occur. [ citation needed ]
Physical mapping Edit
Since actual base-pair distances are generally hard or impossible to directly measure, physical maps are actually constructed by first shattering the genome into hierarchically smaller pieces. By characterizing each single piece and assembling back together, the overlapping path or "tiling path" of these small fragments would allow researchers to infer physical distances between genomic features. The fragmentation of the genome can be achieved by restriction enzyme cutting or by physically shattering the genome by processes like sonication. Once cut, the DNA fragments are separated by electrophoresis.  The resulting pattern of DNA migration (i.e. its genetic fingerprint) is used to identify what stretch of DNA is in the clone. By analyzing the fingerprints, contigs are assembled by automated (FPC) or manual means (pathfinders) into overlapping DNA stretches. Now a good choice of clones can be made to efficiently sequence the clones to determine the DNA sequence of the organism under study.
In physical mapping, there are no direct ways of marking up a specific gene since the mapping does not include any information that concerns traits and functions. Genetic markers can be linked to a physical map by processes like in situ hybridization. By this approach, physical map contigs can be "anchored" onto a genetic map. The clones used in the physical map contigs can then be sequenced on a local scale to help new genetic marker design and identification of the causative loci.
Macrorestriction is a type of physical mapping wherein the high molecular weight DNA is digested with a restriction enzyme having a low number of restriction sites.
There are alternative ways to determine how DNA in a group of clones overlaps without completely sequencing the clones. Once the map is determined, the clones can be used as a resource to efficiently contain large stretches of the genome. This type of mapping is more accurate than genetic maps.
Mapping of mutational sites within a gene Edit
In the early 1950s the prevailing view was that the genes in a chromosome are discrete entities, indivisible by genetic recombination and arranged like beads on a string. During 1955 to 1959, Benzer performed genetic recombination experiments using rII mutants of bacteriophage T4. He found that, on the basis of recombination tests, the sites of mutation could be mapped in a linear order.   This result provided evidence for the key idea that the gene has a linear structure equivalent to a length of DNA with many sites that can independently mutate.
In 1961, Francis Crick, Leslie Barnett, Sydney Brenner and Richard Watts-Tobin performed genetic experiments that demonstrated the basic nature of the genetic code for proteins.  These experiments, involving mapping of mutational sites within the rIIB gene of bacteriophage T4, demonstrated that three sequential nucleobases of the gene's DNA specify each successive amino acid of its encoded protein. Thus the genetic code was shown to be a triplet code, where each triplet (called a codon) specifies a particular amino acid. They also obtained evidence that the codons do not overlap with each other in the DNA sequence encoding a protein, and that such a sequence is read from a fixed starting point.
Edgar et al.  performed mapping experiments with r mutants of bacteriophage T4 showing that recombination frequencies between rII mutants are not strictly additive. The recombination frequency from a cross of two rII mutants (a x d) is usually less than the sum of recombination frequencies for adjacent internal sub-intervals (a x b) + (b x c) + (c x d). Although not strictly additive, a systematic relationship was demonstrated  that likely reflects the underlying molecular mechanism of genetic recombination.
Genome sequencing Edit
Genome sequencing is sometimes mistakenly referred to as "genome mapping" by non-biologists. The process of "shotgun sequencing"  resembles the process of physical mapping: it shatters the genome into small fragments, characterizes each fragment, then puts them back together (more recent sequencing technologies are drastically different). While the scope, purpose and process are totally different, a genome assembly can be viewed as the "ultimate" form of physical map, in that it provides in a much better way all the information that a traditional physical map can offer.
Identification of genes is usually the first step in understanding a genome of a species mapping of the gene is usually the first step of identification of the gene. Gene mapping is usually the starting point of many important downstream studies.
Disease association Edit
The process to identify a genetic element that is responsible for a disease is also referred to as "mapping". If the locus in which the search is performed is already considerably constrained, the search is called the fine mapping of a gene. This information is derived from the investigation of disease manifestations in large families (genetic linkage) or from populations-based genetic association studies.
8.4: Genetic linkage and Genetic Maps - Biology
By the end of this section, you will be able to do the following:
- Define genomics
- Describe genetic and physical maps
- Describe genomic mapping methods
Genomics is the study of entire genomes, including the complete set of genes, their nucleotide sequence and organization, and their interactions within a species and with other species. Genome mapping is the process of finding the locations of genes on each chromosome. The maps that genome mapping create are comparable to the maps that we use to navigate streets. A genetic map is an illustration that lists genes and their location on a chromosome. Genetic maps provide the big picture (similar to an interstate highway map) and use genetic markers (similar to landmarks). A genetic marker is a gene or sequence on a chromosome that co-segregates (shows genetic linkage) with a specific trait. Early geneticists called this linkage analysis. Physical maps present the intimate details of smaller chromosome regions (similar to a detailed road map). A physical map is a representation of the physical distance, in nucleotides, between genes or genetic markers. Both genetic linkage maps and physical maps are required to build a genome’s complete picture. Having a complete genome map of the genome makes it easier for researchers to study individual genes. Human genome maps help researchers in their efforts to identify human disease-causing genes related to illnesses like cancer, heart disease, and cystic fibrosis. We can use genome mapping in a variety of other applications, such as using live microbes to clean up pollutants or even prevent pollution. Research involving plant genome mapping may lead to producing higher crop yields or developing plants that better adapt to climate change.
The study of genetic maps begins with linkage analysis, a procedure that analyzes the recombination frequency between genes to determine if they are linked or show independent assortment. Scientists used the term linkage before the discovery of DNA. Early geneticists relied on observing phenotypic changes to understand an organism’s genotype. Shortly after Gregor Mendel (the father of modern genetics) proposed that traits were determined by what we now call genes, other researchers observed that different traits were often inherited together, and thereby deduced that the genes were physically linked by their location on the same chromosome. Gene mapping relative to each other based on linkage analysis led to developing the first genetic maps.
Observations that certain traits were always linked and certain others were not linked came from studying the offspring of crosses between parents with different traits. For example, in garden pea experiments, researchers discovered, that the flower’s color and plant pollen’s shape were linked traits, and therefore the genes encoding these traits were in close proximity on the same chromosome. We call exchanging DNA between homologous chromosome pairs genetic recombination, which occurs by crossing over DNA between homologous DNA strands, such as nonsister chromatids. Linkage analysis involves studying the recombination frequency between any two genes. The greater the distance between two genes, the higher the chance that a recombination event will occur between them, and the higher the recombination frequency between them. (Figure) shows two possibilities for recombination between two nonsister chromatids during meiosis. If the recombination frequency between two genes is less than 50 percent, they are linked.
Figure 1. Crossover may occur at different locations on the chromosome. Recombination between genes A and B is more frequent than recombination between genes B and C because genes A and B are farther apart. Therefore, a crossover is more likely to occur between them.
The generation of genetic maps requires markers, just as a road map requires landmarks (such as rivers and mountains). Scientists based early genetic maps on using known genes as markers. Scientists now use more sophisticated markers, including those based on non-coding DNA, to compare individuals’ genomes in a population. Although individuals of a given species are genetically similar, they are not identical. Every individual has a unique set of traits. These minor differences in the genome between individuals in a population are useful for genetic mapping purposes. In general, a good genetic marker is a region on the chromosome that shows variability or polymorphism (multiple forms) in the population.
Some genetic markers that scientists use in generating genetic maps are restriction fragment length polymorphisms (RFLP), variable number of tandem repeats (VNTRs), microsatellite polymorphisms, and the single nucleotide polymorphisms (SNPs). We can detect RFLPs (sometimes pronounced “rif-lips”) when the DNA of an individual is cut with a restriction endonuclease that recognizes specific sequences in the DNA to generate a series of DNA fragments, which we can then analyze using gel electrophoresis. Every individual’s DNA will give rise to a unique pattern of bands when cut with a particular set of restriction endonucleases. Scientists sometimes refer to this as an individual’s DNA “fingerprint.” Certain chromosome regions that are subject to polymorphism will lead to generating the unique banding pattern. VNTRs are repeated sets of nucleotides present in DNA’s non-coding regions. Non-coding, or “junk,” DNA has no known biological function however, research shows that much of this DNA is actually transcribed. While its function is uncertain, it is certainly active, and it may be involved in regulating coding genes. The number of repeats may vary in a population’s individual organisms. Microsatellite polymorphisms are similar to VNTRs, but the repeat unit is very small. SNPs are variations in a single nucleotide.
Because genetic maps rely completely on the natural process of recombination, natural increases or decreases in the recombination level given genome area affects mapping. Some parts of the genome are recombination hotspots whereas, others do not show a propensity for recombination. For this reason, it is important to look at mapping information developed by multiple methods.
A physical map provides detail of the actual physical distance between genetic markers, as well as the number of nucleotides. There are three methods scientists use to create a physical map: cytogenetic mapping, radiation hybrid mapping, and sequence mapping. Cytogenetic mapping uses information from microscopic analysis of stained chromosome sections ((Figure)). It is possible to determine the approximate distance between genetic markers using cytogenetic mapping, but not the exact distance (number of base pairs). Radiation hybrid mapping uses radiation, such as x-rays, to break the DNA into fragments. We can adjust the radiation amount to create smaller or larger fragments. This technique overcomes the limitation of genetic mapping, and we can adjust the radiation so that increased or decreased recombination frequency does not affect it. Sequence mapping resulted from DNA sequencing technology that allowed for creating detailed physical maps with distances measured in terms of the number of base pairs. Creating genomic libraries and complementary DNA (cDNA) libraries (collections of cloned sequences or all DNA from a genome) has sped the physical mapping process. A genetic site that scientists use to generate a physical map with sequencing technology (a sequence-tagged site, or STS) is a unique sequence in the genome with a known exact chromosomal location. An expressed sequence tag (EST) and a single sequence length polymorphism (SSLP) are common STSs. An EST is a short STS that we can identify with cDNA libraries, while we obtain SSLPs from known genetic markers, which provide a link between genetic and physical maps.
Figure 2. A cytogenetic map shows the appearance of a chromosome after scientists stain and exam it under a microscope. (credit: National Human Genome Research Institute)
Genetic and Physical Maps Integration
Genetic maps provide the outline and physical maps provide the details. It is easy to understand why both genome mapping technique types are important to show the big picture. Scientists use information from each technique in combination to study the genome. Scientists are using genomic mapping with different model organisms for research. Genome mapping is still an ongoing process, and as researchers develop more advanced techniques, they expect more breakthroughs. Genome mapping is similar to completing a complicated puzzle using every piece of available data. Mapping information generated in laboratories all over the world goes into central databases, such as GenBank at the National Center for Biotechnology Information (NCBI). Researchers are making efforts for the information to be more easily accessible to other researchers and the general public. Just as we use global positioning systems instead of paper maps to navigate through roadways, NCBI has created a genome viewer tool to simplify the data-mining process.
Scientific Method Connection
How to Use a Genome Map Viewer
Problem statement: Do the human, macaque, and mouse genomes contain common DNA sequences?
To test the hypothesis, click this link.
In Search box on the left panel, type any gene name or phenotypic characteristic, such as iris pigmentation (eye color). Select the species you want to study, and then press Enter. The genome map viewer will indicate which chromosome encodes the gene in your search. Click each hit in the genome viewer for more detailed information. This type of search is the most basic use of the genome viewer. You can also use it to compare sequences between species, as well as many other complicated tasks.
Is the hypothesis correct? Why or why not?
Link to Learning
Online Mendelian Inheritance in Man (OMIM) is a searchable online catalog of human genes and genetic disorders. This website shows genome mapping information, and also details the history and research of each trait and disorder. Click this link to search for traits (such as handedness) and genetic disorders (such as diabetes).
Genome mapping is similar to solving a big, complicated puzzle with pieces of information coming from laboratories all over the world. Genetic maps provide an outline for locating genes within a genome, and they estimate the distance between genes and genetic markers on the basis of recombination frequencies during meiosis. Physical maps provide detailed information about the physical distance between the genes. The most detailed information is available through sequence mapping. Researchers combine information from all mapping and sequencing sources to study an entire genome.
What is Physical Mapping
Physical mapping, on the other hand, is the technique used to indicate the physical distance of two genes. Generally, the low resolution of genetic maps due to fewer crossovers as well as their limited accuracy make physical mapping important. Also, it gives the actual distance between markers by means of the number of nucleotides. Basically, the most important forms of physical mapping techniques include restriction mapping, FISH (fluorescent in situ hybridization), and STS (sequence-tagged sites) mapping.
In restriction mapping, restriction sites serve as DNA markers. Of these, polymorphic restriction sites used are a few, but non-polymorphic restriction sites used are numerous. Generally, the simplest way to construct a restriction map is to compare the fragment sizes produced by the digestion of a DNA molecule with two different restriction enzymes, having different target sequences. However, restriction mapping is more applicable to short DNA fragments with relatively few cut sites. Still, there is a possibility to analyze entire genomes larger than 50 kb by using rare cutters with infrequent cut sites. Additionally, optical mapping i s another technique for constructing ordered, genome-wide, high-resolution restriction maps called “optical maps” from single, stained molecules of DNA.
Figure 4: Optical Mapping
Fluorescent in situ hybridization permits the direct visualization of the position of the marker on the chromosome. For that, it uses the hybridization of either radioactive or fluorescent probes. Also, it uses metaphase chromosomes, which are highly condensed. However, this leads to low-resolution mapping. Hence, the use of mechanically-stretched metaphase chromosomes or non- metaphase chromosomes would increase the resolution.
Figure 5: FISH
Sequence tagged mapping is the high resolution, rapid, and less-technically demanding mapping procedure. Therefore, it is the most powerful p hysical mapping technique and the one that has been responsible for the generation of the most detailed maps of large genomes. Normally, an STS or a sequence-tagged site is a short DNA s equence, between 100 and 500 bp in length and it is easily recognizable and occurs only once in a specific chromosome or genome. Therefore, an STS map can be generated by using a collection of overlapping DNA fragments from a single chromosome.
8.4: Genetic linkage and Genetic Maps - Biology
Genetic Mapping: Linkage and Recombination
Genetic mapping is a key tool for classical genetics. When a phenotype or a disease is observed, it is always desirable to determine where the gene is located.
Linkage means when gametes form, genes are not assorted independently, instead, they are linked together. This is because these genes are located close to each other on the same chromosome. Linkage can be tested by chi square.
Genes located close enough can crossover and then recombination occurs. The recombination frequency can be used to measure the genetic distance between two markers: the longer distance, the more recombination events, the higher recombination frequency.
Genetic map is a chromosome map of a species or experimental population that shows the position of its known genes and/or markers relative to each other. It is based on the frequencies of recombination between markers. Genetic mapping can be carried out by two-point or three-point testcrosses.
A genetic map has all the known genes or genetic markers arranged on chromosome according to their genetic distance. The map is drawn according to the recombination frequencies of the linked genes. Genes on different chromosomes belong to different linkage group. Genetic mapping can also be achieved by advanced techniques such as tetrad analysis, somatic cell hybridization and genome project.
- External concept map to describe the relationship of Mendelian genetics and other disciplines in biology
- Internal concept map to reveal the relationships among the content within this tutorial
- Step by step analysis of Mendel’s original experiments
- Brief explanation of Mendelian genetics at chromosome level
- Flow chart on calculating chi square
- Diagrams and symbols for human pedigree reading
- Discovery of linkage
- Linkage theory development
- Linkage and recombination
- Genetic distance
- Two-point testcross
- Genetic map
- Three-point testcross
- Coefficient of coincidence
Advanced mapping technique
See all 24 lessons in Genetics, including concept tutorials, problem drills and cheat sheets:
Teach Yourself Genetics Visually in 24 Hours
An Update of Congenital Adrenal Hyperplasia1
Disorders of steroidogenesis transmitted by an autosomal recessive gene have been investigated by hormonal and immunogenetic techniques.
Genetic linkage between the genes for HLA and steroid 21-hydroxylase has been demonstrated by family studies indicating that the gene for 21-hydroxylase is on the sixth chromosome close to the HLA-B locus. Various phenotypic forms of 21-hydroxylase deficiency have been described which may represent allelic variants at the 21-hydroxylase locus. Genetic linkage disequilibrium between HLA-Bw47 and the more severe 21-hydroxylase deficiency has been demonstrated. The cryptic and late onset 21-hydroxylase defects appear to be in nonrandom gametic association with HLA-B14 and DR1. Hormonal tests have confirmed the prediction of heterozygosity by HLA genotyping.
There is no genetic linkage between HLA and 11β-hydroxylase deficiency and neither HLA genotyping nor hormonal studies are useful in detecting heterozygosity for this disorder.
Prenatal diagnosis of the 21-hydroxylase deficiency by combined hormonal testing and HLA genotyping is possible, whereas only hormonal studies are useful in prenatal diagnosis of 11β-hydroxylase deficiency.
Further hormonal and immunogenetic studies should prove helpful in elucidating the biological basis for the variable expression of these disorders of adrenal steroidogenesis.