In this article we will discuss about:- 1. Meaning of Marker Assisted Selection 2. Features of Marker Assisted Selection 3. Procedure 4. Achievements 5. Merits and Demerits.
Meaning of Marker Assisted Selection:
Marker assisted selection refers to indirect selection for a desired plant phenotype based on the banding pattern of linked molecular (DNA) markers. It is also known as marker aided selection. Marker assisted selection is based on the concept that it is possible to infer the presence of a gene from the presence of a marker that is tightly linked to the gene.
If the marker and the gene are located far apart then the possibility that they will be transmitted together to the progeny individuals will be reduced due to double crossover recombination events.
Thus the following two things are prerequisite for marker assisted selection:
i. A tight linkage between marker and gene of interest.
ii. High heritability of the gene of interest.
Three types of breeding population’s viz.:
(1) Near isogenic lines (NILs),
(2) Introgression lines (ILs) and
(3) Bulk segregation population has been used to identify markers tightly linked to gene of interest.
These are briefly discussed below:
(1) Near Isogenic Lines (NILs):
NILs differ in the presence or absence of the target gene (gene of interest) and a small region flanking the target gene. If the sources of the gene and recurrent parent are sufficiently divergent, markers will reveal polymorphisms between the NILs and recurrent parent. Markers tightly linked to the pseudomonas resistance in tomato and the downy mildew resistance genes in lettuce have been identified using NILs. However, NILs are not available for many traits and generating NILs is time consuming.
(2) Introgression Lines (ILs):
These lines are nearly isogenic to the recipient parent except for the donor gene. These lines are useful in identifying markers linked to major genes and also for fine mapping of QTLs. This method was developed by Eshed and Zamir for mapping of yield associated QTLs in tomato.
(3) Bulk Segregation Analysis (BSA):
This method was developed by Michelmore et al. It makes use of the breeding population segregating for markers defining any target interval containing specific genes, QTLs or gaps in molecular maps. BSA has been used to identify tightly linked markers for pedicel abscission and fruit ripening in tomato, photoperiod-sensitive genie male sterility in rice and HI gene conferring resistance to potato cyst nematode.
Features of Marker Assisted Selection:
Improvement of crop plants for various economic characters using indirect selection for linked molecular markers is referred to as molecular breeding.
Main features of marker assisted selection (MAS) are given below:
1. Application:
Marker Assisted Selection (MAS) is applicable to all the three groups of crop plants, viz. self-pollinated, cross pollinated and asexually propagated species.
2. High Accuracy:
The molecular markers are correlated with conventional markers. Once this work is completed, molecular markers can be used with very high degree of accuracy. In other words, molecular markers have very high accuracy.
3. Rapid Method:
MAS is a rapid method of crop improvement. For example, in conventional breeding when we transfer a recessive character through backcross, one selfing is required after every backcross for identification of such character. MAS permits identification of recessive alleles even in heterozygous condition and thus speeds up the progress of crop improvement work.
4. Free from Environmental Effects:
DNA markers are not affected by the environmental conditions. They are free from environmental effects and indicate simply the presence or absence of specific base sequence. The expression of conventional markers, on the other hand, is largely influenced by environmental conditions. RFLP markers maintain their identification irrespective of genetic background.
5. Permits QTL Tagging:
The genes controlling polygenic or quantitative characters are scattered in different chromosomes. Hence mapping of such genes is not possible by conventional method. Such traits cannot be analysed by Mendelian genetics. Such characters are analysed using biometrical techniques.
DNA markers offer tagging or mapping of quantitative trait loci (QTL). However, for achieving this, first chromosome segments are correlated with quantitative characters. The DNA sequences controlling a particular quantitative trait are identified through RFLP technique.
6. Requires Sophisticated Laboratory:
DNA marker techniques require well equipped laboratory facilities. It requires expensive equipments, chemicals, glass-wares and trained manpower. Thus, this is an expensive method.
7. Health Hazards:
Some of the DNA marker techniques involve radioactive labelling resulting in health hazards of people working in such laboratories. Now non-radioactive labelling techniques are also available. Thus, from the above discussion it is very clear that conventional breeding differs from molecular breeding in many respects (Table 32.1).
Procedure of Marker Assisted Selection:
In the marker assisted selection, RFLP markers are widely used for genetic improvement of crop plants for various economic characters.
The marker assisted selection consists of five important steps, viz.:
(1) Selection of parents,
(2) Development of breeding population,
(3) Isolation of DNA from each plant,
(4) Scoring RFLPs, and
(5) Correlation with morphological traits.
These are briefly discussed below:
(1) Selection of Parents:
Selection of suitable parents is an important step in marker assisted selection. The parents should be such so that we can get usable level of polymorphism (variation) in the RFLP markers. In other words, parents with contrasting characters or divergent origin should be chosen.
This will help in identification of DNA of both the parents and also their segments in F2 generation in various recombination’s. For selection of parents, we have to screen germplasm and select parents with distinct DNA. The parents that are used for MAS should be pure (homozygous). In self-pollinated species, plants are usually homozygous. In cross-pollinated species, inbred lines are used as parents.
(2) Development of Breeding Populations:
This is the second important step for application of marker assisted selection. The selected parents are crossed to obtain F1 plants. F1 plants between two pure-lines or inbred lines are homogeneous (alike phenotypically) but are heterozygous for all the RFLPs of two parents involved in the F1. The F2 progeny is required for the study of segregation pattern of RFLPs. Generally 50-100 F2 plants are sufficient for the study of segregation of RFLP markers.
(3) Isolation of DNA:
The third important step is isolation of DNA from breeding population. The main advantage of MAS is that DNA can be isolated even from the seedlings and we need not to wait for flowering or seed development stage. The DNA is isolated from each plant of F2 population. Standard procedures are available for DNA isolation.
The isolated DNA is digested with specific restriction enzyme to obtain fragments of DNA. The DNA fragments of different sizes are separated by subjecting the digested DNA to agarose gel electrophoresis. The gel is stained with ethidium bromide and the variation in DNA fragments can be viewed in the ultraviolet light.
The DNA of chloroplasts, when digested with specific enzyme, produces about 40 fragments of different sizes. The nuclear DNA of higher plants, when digested with specific restriction enzymes, produces millions of fragments in a continuous range of sizes. It is a tedious job to identify individual DNA fragment in such cases.
(4) Scoring RFLPs:
The polymorphism in RFLPs between the parents and their involvement in the recombinants in F2 population is determined by using DNA probes. The labelled probes are used to find out the fragments having similarity. The probe will hybridize only with those segments which are complementary in nature. Generally 32P is used for radioactive labelling of DNA probe. Now non-radioactive probe labelling techniques are also available. In this way RFLPs are determined.
(5) Correlation with Morphological Traits:
The DNA marker (say RFLPs) are correlated with morphological markers and the indirect selection through molecular markers is confirmed. Once the correlation of molecular markers is established with morphological markers, MAS can be effectively used for genetic improvement of various economic traits.
Achievements of Marker Assisted Selection:
The marker assisted selection (MAS) has been used in genetic improvement of different field crops such as barley, maize, potato, rice, tomato etc. for various economic characters. In these crops, RFLP markers have been used for marker aided selection. The numbers of enzymes used to detect polymorphism in RFLPs differ from species to species depending upon the variation present in the parental lines used in the hybridization.
For example, in inbred lines of maize 1 or 2 enzymes are required to detect the polymorphism. In rice, on the other hand, 11 enzymes were used to detect polymorphism in RFLPs.
The marker aided selection can be used as a tool in the crop improvement programme. It can speed up the progress of breeding programmes. It can cut short the time required for development of new varieties. However, this technique cannot be used as a substitute for conventional breeding approaches. Marker aided selection has its own limitations.
Merits and Demerits of Marker Assisted Selection:
Merits:
1. It permits early screening of traits that are expressed late in the life of plant. For example characters such as grain or fruit quality, flower colour, male sterility, photoperiod sensitivity that express late in the life of a plant can be screened in the seedling stage. In other words, DNA isolated at seedling stage can throw light about the traits which are expressed later on.
2. It permits screening of traits that are extremely difficult, expensive or time consuming to score phenotypically. For example, screening for traits such as root morphology, resistance to biotic (insects, diseases) and abiotic stresses (drought, salinity, mineral deficiencies or toxicity) is very easy through marker aided selection.
3. It helps in distinguishing the homozygous versus heterozygous condition of many loci in a single generation without the need of progeny testing, because molecular markers are co-dominant.
4. DNA markers permits marker aided selection for several characters at one time. In some cases, simultaneous marker aided selection can offer an opportunity to screen for a character or characters that could not be included in the programme because of cost or difficulty in traditional screening procedures.
5. RFLP markers help in indirect selection of recessive allele, in the backcross programme in the heterozygous condition without selfing or progeny testing. Thus this is a rapid method of crop improvement. In other words, it speeds up the progress of crop improvement work.
6. The accuracy of marker aided selection is very high, molecular markers are not affected by environmental conditions.
Demerits:
Marker assisted selection or molecular breeding has many advantages as discussed above.
It has some demerits also which are briefly presented below:
1. It requires a sophisticated and well equipped laboratory to initiate the work on DNA marker assisted selection.
2. The molecular breeding techniques are very expensive, because these techniques require very costly equipments, glass-wares and chemicals.
3. It requires well trained manpower for handling of equipments, isolation of DNA molecule and study of DNA markers.
4. The detection of various DNA markers (RFLP, AFLP, and RAPD) is a laborious and time consuming work.
5. For RFLP markers, a huge breeding population has to be screened to get meaningful results which are a very combursome job. This limits the use of RFLP in plant breeding. However, short cut methods are expected to be developed in the future.
6. Molecular breeding/marker assisted selection involves use of radioactive isotopes in labeling of DNA, which may lead to serious health hazards. This is a major disadvantage of RFLP based DNA markers. The polymerase Chain Reaction (PCR) is a cheaper, easier and safer method than RFLPs which can be used for marker aided selection with large plant populations.
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