Mapping a marker or a mutation to a well defined chromosomal region is an essential step in the genetic analysis of a plant and is also (unless the mutant is tagged) a prerequisite for molecular cloning of the corresponding gene. Determining the map position of a marker or a gene (as identified by its mutant phenotype) consists in testing linkage with a number of previously mapped markers.
Once linkage with a specific marker is detected, a refined mapping can be achieved by analysing linkage relations to more markers in that region.
Historically, mapping in plants primarily utilized morphological markers
such as mutants with an easily scorable phenotype and a defined map position. Typically, the mutant of interest is crossed to another mutant used as phenotypic marker, the resulting F1 double heterozygote is allowed to self, and segregation of the two phenotypes is analysed in the F2 population.
Once linkage with a specific marker is detected, a refined mapping can be achieved by analysing linkage relations to more markers in that region.Historically, mapping in plants primarily utilized morphological markers
such as mutants with an easily scorable phenotype and a defined map position. Typically, the mutant of interest is crossed to another mutant used as phenotypic marker, the resulting F1 double heterozygote is allowed to self, and segregation of the two phenotypes is analysed in the F2 population. The mutation used as marker should of course not interfere with the phenotype of the mutant to be mapped. The genetic distance is the number of meiotic recombination events that occur between the two loci in 100 chromosomes. The genetic distance is expressed in centiMorgans (cM), and can range from 0 cM (absolute linkage) to 50 cM (non-linked loci). It remains however difficult to score many different phenotypes in a single population. Hence, detailed mapping using morphological markers is tedious because it requires numerous crosses.
In contrast, a single cross can be used to analyse linkage with an essentially unlimited number of molecular markers. DNA markers were incorporated into mapping strategies once it was recognized that distantly related individuals differ in DNA sequence throughout their genome [Botstein, 1980]. Molecular markers exploit the natural differences between distinct lines. For instance, in Arabidopsis, it has been estimated that the widely used Landsberg erecta and Columbia ecotypes differ by approximately 0.5 to 1 % at the DNA sequence level [Chang, 1988; Hauser, 1998]. These local differences or polymorphisms of the DNA sequence are due to point mutations, insertion or deletions that randomly occurred in one ecotype and not in the other. These DNA polymorphism can be conveniently visualized by several methods.
In contrast, a single cross can be used to analyse linkage with an essentially unlimited number of molecular markers. DNA markers were incorporated into mapping strategies once it was recognized that distantly related individuals differ in DNA sequence throughout their genome [Botstein, 1980]. Molecular markers exploit the natural differences between distinct lines. For instance, in Arabidopsis, it has been estimated that the widely used Landsberg erecta and Columbia ecotypes differ by approximately 0.5 to 1 % at the DNA sequence level [Chang, 1988; Hauser, 1998]. These local differences or polymorphisms of the DNA sequence are due to point mutations, insertion or deletions that randomly occurred in one ecotype and not in the other. These DNA polymorphism can be conveniently visualized by several methods.
To map a novel mutation that was generated in line A, this mutant is crossed with a wild-type plant of a polymorphic line B, and the Fl progeny is allowed to self. The resulting F2 population can then be used to Self. The resulting between the mutation of interest and any DNA marker that distinguished line A and line B. As compared to Morphological markers, an additional advantage of molecular markers is that in most cases homozygous and heterozygous individuals can be readily distinguished.
In addition, data will be generated using M. truncatula Recombinant Imbred Lines (RKLs) in combination with the SSR ( Microsatellites) method. Data will be analyzed using the MAPMAKER program or the colormapping procedure.
DIFFERENT TYPE OF DNA MARKERS
· RFLP
· CAPS
· RAPD
· AFLP
· SSR
· SNPs
The types of markers that will used during the practical course are indicated with. However, other types of DNA markers that can be used for genetic mapping are also described. By definition we call "DNA marker" a DNA fragment which exhibit polymorphism between two lines.
In addition, data will be generated using M. truncatula Recombinant Imbred Lines (RKLs) in combination with the SSR ( Microsatellites) method. Data will be analyzed using the MAPMAKER program or the colormapping procedure.
DIFFERENT TYPE OF DNA MARKERS
· RFLP
· CAPS
· RAPD
· AFLP
· SSR
· SNPs
The types of markers that will used during the practical course are indicated with. However, other types of DNA markers that can be used for genetic mapping are also described. By definition we call "DNA marker" a DNA fragment which exhibit polymorphism between two lines.
Restriction Fragment Length Polymorphism (RFLP)
The first type of DNA markers that were used for genetic mapping were RFLPs. The DNA sequence differences between polymorphic lines may create
differences in the length of restriction fragments derived from genomic DNA. For instance a given restriction site may be present in one line and not in the other. This polymorphism can be revealed by genomic DNA blot hybridization (Southern) using as probe a DNA fragment corresponding to that region. The polymorphic bands can then be used as genetic markers to distinguish the two lines. Multiple RFLP markers can be identified and assembled into genetic maps.
An advantage of the RFLP mapping approach is that RFLP markers are co-dominant. Distinct patterns are indeed obtained for plants that are homozygous or heterozygous for the parental alleles. Hence, an the chromosomes of a given F2 population can be scored. In contrast, a main disadvantage is that RFLP mapping necessitates relatively large amounts of DNA because distinct RFLP markers may require digestion of genomic DNA samples with different diagnostic restriction enzymes.
The first type of DNA markers that were used for genetic mapping were RFLPs. The DNA sequence differences between polymorphic lines may create
differences in the length of restriction fragments derived from genomic DNA. For instance a given restriction site may be present in one line and not in the other. This polymorphism can be revealed by genomic DNA blot hybridization (Southern) using as probe a DNA fragment corresponding to that region. The polymorphic bands can then be used as genetic markers to distinguish the two lines. Multiple RFLP markers can be identified and assembled into genetic maps.An advantage of the RFLP mapping approach is that RFLP markers are co-dominant. Distinct patterns are indeed obtained for plants that are homozygous or heterozygous for the parental alleles. Hence, an the chromosomes of a given F2 population can be scored. In contrast, a main disadvantage is that RFLP mapping necessitates relatively large amounts of DNA because distinct RFLP markers may require digestion of genomic DNA samples with different diagnostic restriction enzymes.
Cleaved Amplified Polymorphism Sequences (CAPS)
The principle of CAPS markers is very similar to that of RFLP markers. The main difference is that PCR is used instead of DNA blot hybridisation to detect a restriction site polymorphism. A genomic DNA region is amplified by PCR using specific primers and those amplified fragments are then digested with diagnostic restriction enzymes to reveal the polymorphism. Hence, whereas RFLP probes can be anonymous colnes, CAPS markers require sequence information to design the specific PCR primers.
Like RFLPs, CAPS markers are co-dominant. CAPS markers are based on PCR for detection, and thus require only small quantities of genomic DNA. Typically a single leaf will provide enough DNA for analiysis with multiple CAPS markers.
Finally, CAPS markers can be easily assayed using standard agarose gel electrophoresis.
The principle of CAPS markers is very similar to that of RFLP markers. The main difference is that PCR is used instead of DNA blot hybridisation to detect a restriction site polymorphism. A genomic DNA region is amplified by PCR using specific primers and those amplified fragments are then digested with diagnostic restriction enzymes to reveal the polymorphism. Hence, whereas RFLP probes can be anonymous colnes, CAPS markers require sequence information to design the specific PCR primers.
Like RFLPs, CAPS markers are co-dominant. CAPS markers are based on PCR for detection, and thus require only small quantities of genomic DNA. Typically a single leaf will provide enough DNA for analiysis with multiple CAPS markers.
Finally, CAPS markers can be easily assayed using standard agarose gel electrophoresis.
Random amplified polymorphic DNA (RAPD)
RAPD markers are another type of PCR-based markers that have been used for genetic mapping [Wiliams, 1993]. This approach is based on the amplification of random DNA segments with single primers of arbitrary nucleotide sequence. The oligonucleotide (around 10bp long) is used for
PCR at low annealing temperatures. When the oligonucleotide hybridises to both DNA strands at sites within an appropriate distance from each other, the DNA region delimited by these two sites will be amplified. Small nucleotide changes (polymorphism) at one of the two sites may prevent hybridization of the oligonucleotide and hence also prevent DNA amplification [Williams, 1990). Typically a RAPD primer will amplify a given fragment from Line A and not from line B. It will impossible to distinguish an homozygous individual AA from an heterozygous individuals AB. In other words, RAPDs are dominant markers and are thus less efficient than co-dominant markers in extracting information from a given F2 population. Another limitation of RAPD marker is that because of the low annealing temperatures used, the amplification of a given polymorphic band seems to be highly sensitive to PCR conditions and hence less consistently reproducible in different laboratories.
Amplified restriction Fragment Length Polymorphism (AFLP)
AFLP TM is a patented technology developed by Key Gene, Wageningen, the Netherlands [Vos et al.,1995]. In this
procedure, the genomic DNA is digested by two different restriction enzymes, a rare cutter and frequent cutter. Double-stranded adapters are then ligated to the ends of the restriction fragments. The fragments are then amplified by PCR using primers that correspond to the adapter and restriction site sequences. These primers have additional nucleotides at the 31 en
ds extending into the restriction fragments, in order to limit the number of fragments that will be amplified. The AFLP products are detected by labeling one of two primers, and lablled DNA fragments are separated by electrophoresis in denaturing polyacrylamide gels (similar to sequencing gels). Typically, 50 to 100 amplification products are detected in a single lane. polymorphic bands can be identified by comparing the amplification products derived from two lines. Like RAPDs, AFLPs are typically dominant markers.
Simple sequence repeats (SSR)
Like other eukaryotic genomes, the plant genome contains tandem repeats of one- two or three-nucleotide mot
ifs. These microsatellite repeat sequences are usually polymorphic in different lines because of variations in the number of repeat units. These polymorphisms are called SSR, and can be conveniently used as co-dominant genetic markers. Specific primers are used to PCR amplify a small genomic region (150 to 250 bp) that contains a polymorphic microsatellite sequence. The size of the amplified fragment will vary depending on the number of repeats present in a given line. These polymorphic fragments can be separated and visualized by electrophoresis in agarose or polyacrylamide gels.
As compared to CAPS markers, SSR offer the additional advantage that they do not involve the use of restriction endonucleases and thus avoid the problems associated with partial digestions.
Single Nucleotide Polymorphisms(SNPs)
The most common class of DNA polymorphisms present both in natural lines and after induced mutagenesis is single nucleotide polymorphisms (SNPs). The RFLP and CAPS methods can detect only the SNPs which alter a recognition site for a restriction enzyme. The RAPD and AFLP methodologies can in principle detect any type of SNPs, however these two techniques are not very convenient to target a selected genomic region. In contrast, plant
genome sequencing is generating a wealth of sequence information which provides a starting point for the development of PCR-based markers. In other words, once the sequence of a region is known, primers can be synthesized to amplify alleles of interest which can then be analyzed to find allele-specific polymorphisms in that region.
Although they have not been extensively used in plants thus far, a large number of techniques have been developed to scan a defined region of DNA for SNPs reviewed by Cotton, 1997. In particular, single-strand conformation polymorphism (SSCP) is based on the fact that a strand of single-stranded DNA folds differently from another if it differs by a single base, which leads to different motilities of these two strands in non-denaturing gel electrophoresis. Heteroduplex analysis is based on the different motilities of homo- and heteroduplexes in non-denaturing gel electrophoresis, or in slightly denaturing high-performance liquid chromatography. It should be noted that methods to detect SNPs can also be extremely useful in the step of positional cloning of a mutant locus, namely to locate the mutation within the DNA region has been delimited by mapping.
Finally, an efficient method has been described that allows to create a PCR-base marker for any known point mutation. This technique is called derived cleaved amplified polymorphic sequence (dCAPS), and has been recently applied to Arabidopsis (Michaels 1998; Neff, 1998). The dCAPS method is primarily used when the point mutation of interest does not alter an existing restriction site. In this case, the dCAPS technique consists in designing a primer with one or two mismatches which, together with the mutation, will create a unique restriction site in one only of the two alleles. A second primer (usually without mismatch) is used to PCR amplify the region, and the amplification products are digested with the appropriate restriction enzyme, exactly as for CAPS markers. dCAPS are useful for genetic mapping, and to follow known mutations in segregating populations.
When selecting DNA markers on genetic maps, or later in interpreting linkage data to these markers, it is important to bear in mind how these reference genetic maps were constructed. A genetic map only displays the relative genetic distances between the particularly set of markers that were analysed in a given mapping population. Hence, a given marker will typically have different map positions in genetic maps that were constructed independently, or in successive versions of the same map as new markers are incorporated. In order to construct a better reference map, in particular with respect to the relative order of markers, it is essential that all markers are mapped in the same population. Even with the development of PCR-based markers which require at smaller amount of genomic DNA than RFLP markers, only a limited number of markers and phenotypic traits can be mapped in a given F2 population. This provided the impetus for the generation of populations of recombinant inbred (RI) lines for mapping.
To construct an RI population, individual F2 plants are selfed, and for each F3 family a single F3 plant is selected at random and allowed to self. This process, called single-seed descent, is repeated to the F7. At each generation, the average level of heterozygocity is reduced by 50%. Hence, F7 lines are over 98% homozygous. These RI lines thus constitute permanent mapping populations because they are near-homozygous and can therefore be multiplied indefinitely, enabling multiple laboratories to use the same mapping population.
RAPD markers are another type of PCR-based markers that have been used for genetic mapping [Wiliams, 1993]. This approach is based on the amplification of random DNA segments with single primers of arbitrary nucleotide sequence. The oligonucleotide (around 10bp long) is used for
PCR at low annealing temperatures. When the oligonucleotide hybridises to both DNA strands at sites within an appropriate distance from each other, the DNA region delimited by these two sites will be amplified. Small nucleotide changes (polymorphism) at one of the two sites may prevent hybridization of the oligonucleotide and hence also prevent DNA amplification [Williams, 1990). Typically a RAPD primer will amplify a given fragment from Line A and not from line B. It will impossible to distinguish an homozygous individual AA from an heterozygous individuals AB. In other words, RAPDs are dominant markers and are thus less efficient than co-dominant markers in extracting information from a given F2 population. Another limitation of RAPD marker is that because of the low annealing temperatures used, the amplification of a given polymorphic band seems to be highly sensitive to PCR conditions and hence less consistently reproducible in different laboratories.Amplified restriction Fragment Length Polymorphism (AFLP)
AFLP TM is a patented technology developed by Key Gene, Wageningen, the Netherlands [Vos et al.,1995]. In this
procedure, the genomic DNA is digested by two different restriction enzymes, a rare cutter and frequent cutter. Double-stranded adapters are then ligated to the ends of the restriction fragments. The fragments are then amplified by PCR using primers that correspond to the adapter and restriction site sequences. These primers have additional nucleotides at the 31 en
ds extending into the restriction fragments, in order to limit the number of fragments that will be amplified. The AFLP products are detected by labeling one of two primers, and lablled DNA fragments are separated by electrophoresis in denaturing polyacrylamide gels (similar to sequencing gels). Typically, 50 to 100 amplification products are detected in a single lane. polymorphic bands can be identified by comparing the amplification products derived from two lines. Like RAPDs, AFLPs are typically dominant markers.Simple sequence repeats (SSR)
Like other eukaryotic genomes, the plant genome contains tandem repeats of one- two or three-nucleotide mot
ifs. These microsatellite repeat sequences are usually polymorphic in different lines because of variations in the number of repeat units. These polymorphisms are called SSR, and can be conveniently used as co-dominant genetic markers. Specific primers are used to PCR amplify a small genomic region (150 to 250 bp) that contains a polymorphic microsatellite sequence. The size of the amplified fragment will vary depending on the number of repeats present in a given line. These polymorphic fragments can be separated and visualized by electrophoresis in agarose or polyacrylamide gels.As compared to CAPS markers, SSR offer the additional advantage that they do not involve the use of restriction endonucleases and thus avoid the problems associated with partial digestions.
Single Nucleotide Polymorphisms(SNPs)
The most common class of DNA polymorphisms present both in natural lines and after induced mutagenesis is single nucleotide polymorphisms (SNPs). The RFLP and CAPS methods can detect only the SNPs which alter a recognition site for a restriction enzyme. The RAPD and AFLP methodologies can in principle detect any type of SNPs, however these two techniques are not very convenient to target a selected genomic region. In contrast, plant
genome sequencing is generating a wealth of sequence information which provides a starting point for the development of PCR-based markers. In other words, once the sequence of a region is known, primers can be synthesized to amplify alleles of interest which can then be analyzed to find allele-specific polymorphisms in that region.Although they have not been extensively used in plants thus far, a large number of techniques have been developed to scan a defined region of DNA for SNPs reviewed by Cotton, 1997. In particular, single-strand conformation polymorphism (SSCP) is based on the fact that a strand of single-stranded DNA folds differently from another if it differs by a single base, which leads to different motilities of these two strands in non-denaturing gel electrophoresis. Heteroduplex analysis is based on the different motilities of homo- and heteroduplexes in non-denaturing gel electrophoresis, or in slightly denaturing high-performance liquid chromatography. It should be noted that methods to detect SNPs can also be extremely useful in the step of positional cloning of a mutant locus, namely to locate the mutation within the DNA region has been delimited by mapping.
Finally, an efficient method has been described that allows to create a PCR-base marker for any known point mutation. This technique is called derived cleaved amplified polymorphic sequence (dCAPS), and has been recently applied to Arabidopsis (Michaels 1998; Neff, 1998). The dCAPS method is primarily used when the point mutation of interest does not alter an existing restriction site. In this case, the dCAPS technique consists in designing a primer with one or two mismatches which, together with the mutation, will create a unique restriction site in one only of the two alleles. A second primer (usually without mismatch) is used to PCR amplify the region, and the amplification products are digested with the appropriate restriction enzyme, exactly as for CAPS markers. dCAPS are useful for genetic mapping, and to follow known mutations in segregating populations.
When selecting DNA markers on genetic maps, or later in interpreting linkage data to these markers, it is important to bear in mind how these reference genetic maps were constructed. A genetic map only displays the relative genetic distances between the particularly set of markers that were analysed in a given mapping population. Hence, a given marker will typically have different map positions in genetic maps that were constructed independently, or in successive versions of the same map as new markers are incorporated. In order to construct a better reference map, in particular with respect to the relative order of markers, it is essential that all markers are mapped in the same population. Even with the development of PCR-based markers which require at smaller amount of genomic DNA than RFLP markers, only a limited number of markers and phenotypic traits can be mapped in a given F2 population. This provided the impetus for the generation of populations of recombinant inbred (RI) lines for mapping.
To construct an RI population, individual F2 plants are selfed, and for each F3 family a single F3 plant is selected at random and allowed to self. This process, called single-seed descent, is repeated to the F7. At each generation, the average level of heterozygocity is reduced by 50%. Hence, F7 lines are over 98% homozygous. These RI lines thus constitute permanent mapping populations because they are near-homozygous and can therefore be multiplied indefinitely, enabling multiple laboratories to use the same mapping population.
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