Pcr And Non Pcr Based Molecular Markers PdfBy Daila L. In and pdf 23.04.2021 at 01:27 9 min read
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- Molecular markers-types and applications
- Molecular marker
- Comparison of PCR-based molecular marker analyses of Musa breeding populations
- Molecular Markers and Marker-Assisted Breeding in Plants
Molecular markers-types and applications
A molecular marker is a DNA sequence in the genome which can be located and identified. As a result of genetic alterations mutations, insertions, deletions , the base composition at a particular location of the genome may be different in different plants. These differences, collectively called as polymorphisms can be mapped and identified. Plant breeders always prefer to detect the gene as the molecular marker, although this is not always possible. The alternative is to have markers which are closely associated with genes and inherited together.
The molecular markers are highly reliable and advantageous in plant breeding programmes:. Let us assume that there are two plants of the same species—one with disease sensitivity and the other with disease resistance. If there is DNA marker that can identify these two alleles, then the genome can be extracted, digested by restriction enzymes, and separated by gel electrophoresis. The DNA fragments can be detected by their separation. For instance, the disease resistant plant may have a shorter DNA fragment while the disease — sensitive plant may have a longer DNA fragment Fig.
Marker-based DNA hybridization is widely used. The major limitation of this approach is that it requires large quantities of DNA and the use of radioactivity labeled probes. RFLP is mainly based on the altered restriction enzyme sites, as a result of mutations and re-combinations of genomic DNA. The procedure basically involves the isolation of genomic DNA, its digestion by restriction enzymes, separation by electrophoresis, and finally hybridization by incubating with cloned and labeled probes Fig.
Based on the presence of restriction sites, DNA fragments of different lengths can be generated by using different restriction enzymes. In the Fig. In plant A, a mutations has occurred leading to the loss of restriction site that can be digested by EcoRI.
This results in a polymorphic pattern of separation. Locus non-specific markers e. Locus specific markers e. Single short oligonucleotide primers usually a base primer can be arbitrarily selected and used for the amplification DNA segments of the genome which may be in distributed throughout the genome.
The amplified products are separated on electrophoresis and identified. Based on the nucleotide alterations in the genome, the polymorphisms of amplified DNA sequences differ which can be identified as bends on gel electrophoresis. Genomic DNA from two different plants often results in different amplification patterns i. This is based on the fact that a particular fragment of DNA may be generated from one individual, and not from others. This represents polymorphism and can be used as a molecular marker of a particular species.
Thus, this technique combines the usefulness of restriction digestion and PCR. The DNA of the genome is extracted. It is subjected to restriction digestion by two enzymes a rare cutter e. Msel; a frequent cutter e. The cut ends on both sides are then ligated to known sequences of oligonucleotides Fig. PCR is now performed for the pre-selection of a fragment of DNA which has a single specific nucleotide.
By this approach of pre-selective amplification, the pool of fragments can be reduced from the original mixture. In the second round of amplification by PCR, three nucleotide sequences are amplified. Autoradiography can be performed for the detection of DNA fragments. Use of radiolabeled primers and fluorescently labeled fragments quickens AFLP. AFLP analysis is tedious and requires the involvement of skilled technical personnel.
Hence some people are not in favour of this technique. In recent years, commercial kits are made available for AFLP analysis. AFLP is very sensitive and reproducible. It does not require prior knowledge of sequence information. By AFLP, a large number of polymorphic bands can be produced and detected.
Sequence tagged sites represent unique simple copy segments of genomes, whose DNA sequences are known, and which can be amplified by using PCR.
STS markers are based on the polymorphism of simple nucleotide repeats e. STS have been recently developed in plants. STS loci have been analysed and studied in a number of plant species. Microsatellites are the tandemly repeated multi-copies of mono-, di-, tri- and tetra nucleotide motifs. In some instances, the flanking sequence of the repeat sequences may be unique.
Primers can be designed for such flanking sequences to detect the sequence tagged microsatellites STMS. This can be done by PCR. Selection of the desired traits and improvement of crops has been a part of the conventional breeding programmes.
This is predominantly based on the identification of phenotypes. It is now an accepted fact that the phenotypes do not necessarily represent the genotypes. Many a times the environment may mark the genotype. These traits include resistance to pathogens and insects, tolerance to abiotic stresses, and various other qualitative and quantitative traits. The advantage with a molecular marker is that a plant breeder can select a suitable marker for the desired trait which can be detected well in advance.
Accordingly, breeding programmes can be planned. The following are the major requirements for the molecular marked selection in plant breeding:. With rapid progress in molecular biology and genetic engineering, there is now a possibility of improving the crop plants with respect to yield and quality. The term molecular breeding is frequently used to represent the breeding methods that are coupled with genetic engineering techniques.
Improved agriculture to meet the food demands of the world is a high priority area. For several years, the conventional plant breeding programmes although time consuming have certainly helped to improve grain yield and cereal production. Many developments on the agriculture front are expected in the coming years as a result of molecular breeding. Linkage analysis basically deals with studies to correlate the link between the molecular marker and a desired trait.
This is an important aspect of molecular breeding programmes. Linkage analysis has to be carried out among the populations of several generations to establish the appropriate linkage. In the earlier years, linkage analysis was carried out by use of isoenzymes and the associated polymorphisms. Molecular markers are now being used.
The techniques employed for this purpose have already been described. These are many characteristics controlled by several genes in a complex manner. Some good examples are growth habit, yield, adaptability to environment, and disease resistance. These are referred to as quantitative traits.
The locations on the chromosomes for these genes are regarded as quantitative trait loci QTL. The major problem, the plant breeder faces is how to improve the a complex character controlled by many genes. It is not an easy job to manipulate multiple genes in genetic engineering. Therefore, it is a very difficult and time consuming process. For instance, development of Golden Rice with enriched pro-vitamin A involving the insertion of just three genes took about seven years.
The terms arid zone is used to refer to harsh environmental conditions with extreme heat and cold. The fields have limited water and minerals. It is different task to grow plants and achieve good crop yield in arid zones. Semi-arid regions are characterized by unpredictable weather, inconsistent rainfall, long dry seasons, and poor nutrients in the soil. Most parts of India and many other developing countries Africa, Latin America, and Southeast Asia have semi- arid regions.
Crops like sorghum, millet, groundnut and cowpea are mostly grown in semi-arid tropics. Besides unpredictable weather, biotic and abiotic stresses contribute to crop loss in these areas. The biotechnological approaches for the breeding programmes in the semi-arid regions should cover the following areas:.
Micro-propagation techniques to spread economically important plants which can withstand harsh environmental conditions. Some success has been achieved in improving sorghum, millet and legume crops that are grown in semi-arid regions. Genetic transformation in sorghum was possible by using micro projectile method. Greenhouse literally means a building made up of glass to grow plants. Green houses are required to grow regenerated plants for further propagation and for growing plants to maturity.
Greenhouses are the intermediary stages involving the transitional step between the plant cultures and plant fields. The purpose of greenhouses is to acclimatize and test the plants before they are released into the natural environment.
The plants are grown in greenhouse to develop adequate root systems and leaves so as to withstand the field environment.
The greenhouses are normally equipped with cooling systems to control temperature. Greenhouses have chambers fitted with artificial lights. It is possible to subject the plants to different lighting profiles. In recent years many improvements have been made in the development of more suitable greenhouses. These include the parameters such as soil, and humidity.
The major limitation of greenhouse technology is an increase in CO 2 production that in turn increases temperature.
A molecular marker is a DNA sequence in the genome which can be located and identified. As a result of genetic alterations mutations, insertions, deletions , the base composition at a particular location of the genome may be different in different plants. These differences, collectively called as polymorphisms can be mapped and identified. Plant breeders always prefer to detect the gene as the molecular marker, although this is not always possible. The alternative is to have markers which are closely associated with genes and inherited together. The molecular markers are highly reliable and advantageous in plant breeding programmes:.
Molecular markers! Combines restriction disgestion with PCR amplification. • Selectively amplifies a Mostly based on differences in number of di- or tri nucleotide repeats at a Repeats less frequent in coding than non-coding regioins.
Comparison of PCR-based molecular marker analyses of Musa breeding populations
A molecular marker is a molecule contained within a sample taken from an organism biological markers or other matter. It can be used to reveal certain characteristics about the respective source. DNA , for example, is a molecular marker containing information about genetic disorders and the evolutionary history of life.
Molecular Markers and Marker-Assisted Breeding in Plants
Public genomic databases have provided new directions for molecular marker development and initiated a shift in the types of PCR-based techniques commonly used in plant science. Alongside commonly used arbitrarily amplified DNA markers, other methods have been developed. Targeted fingerprinting marker techniques are based on the well-established practices of arbitrarily amplified DNA methods, but employ novel methodological innovations such as the incorporation of gene or promoter elements in the primers.
was conducted to compare different PCR-based marker systems (RAPD, VNTR and AFLP) Molecular markers based on PCR are the most ap- The frequency of polymorphic non-parental alleles White RP: Genstat 5 Reference Manual.
A genetic marker is a known location on a chromosome used for identification of individuals among and between species. Thus from the definition, we can say that broadly, the Genetic markers are used,. In the present article, we will understand some common markers used in the molecular genetic technique along with its applications. The genetic marker is a known DNA sequence or gene located on the chromosome which can be applied in the identification of individual species or organism or we can use it in the identification of other genes or DNA sequences. Before that let me give you a brief idea about how different genetic markers are developed. If we mark some sentences or paragraphs, we can use it every time in exams, assignments and other activities. Similarly, by marking a specific sequence of DNA, we can use it in different types of genetic studies.
A molecular marker is a DNA sequence in the genome which can be located and identified. As a result of genetic alterations mutations, insertions, deletions , the base composition at a particular location of the genome may be different in different plants. These differences, collectively called as polymorphisms can be mapped and identified. Plant breeders always prefer to detect the gene as the molecular marker, although this is not always possible. The alternative is to have markers which are closely associated with genes and inherited together. The molecular markers are highly reliable and advantageous in plant breeding programmes:. Let us assume that there are two plants of the same species—one with disease sensitivity and the other with disease resistance.
Progress in the breeding of plantain and banana has been restricted by the complex genetic structure and behaviour of cultivated polyploid Musa. Genetic improvement has been hindered due to the large amount of space required for growth and maintenance of plant populations, in addition to the long growth cycle and the low levels of fertility and seed viability characteristic of cultivated genotypes. Molecular marker assisted breeding has the potential to dramatically enhance the pace and efficiency of genetic improvement in Musa. All three assays detected a high level of polymorphism between parental genotypes and within progeny populations. AFLP analysis of a full-sib tetraploid hybrid population confirmed previous reports based on VNTR analysis, of a high frequency of recombination during 2n 3x gamete formation by a triploid plantain landrace.