Marker Assisted Selection (MAS): Meaning, Steps and Application

In this article we will discuss about:- 1. Meaning of Marker Assisted Selection 2. Features of Marker Assisted Selection 3. Steps 4. Applications 5. Achievements 6. Advantages7. Limitations 8. Future Outlook.

Contents:

1. Meaning of Marker Assisted Selection
2. Features of Marker Assisted Selection
3. Steps of Marker Assisted Selection
4. Applications of Marker Assisted Selection
5. Achievements of Marker Assisted Selection
6. Advantages of Marker Assisted Selection
7. Limitations of Marker Assisted Selection
8. Future Outlook of Marker Assisted Selection

---

1. Meaning of Marker Assisted Selection (MAS):

Marker Assisted Selection [MAS] refers to indirect selection for a desired plant phenotype based on the banding pattern of linked molecular (DNA) markers. MAS is based on the concept that it is possible to infer the presence of a gene from the presence of a marker which is tightly linked to the gene of interest.

If the marker and the gene are located far apart then the possibility of their transmission together to the progeny individuals will be reduced due to double crossover recombination events.

2. Features of Marker Assisted Selection (MAS):

The main features of MAS are briefly presented below:

i. Other Terms Used:

Marker assisted selection (MAS) is also termed as marker aided selection and marker assisted breeding (MAB). It differs from gene assisted selection (GAS) which refers to the selection which is based on QTLs (quantitative trait locus or loci).

ii. Pre-Requisites:

There are two pre-requisites for marker assisted selection. These are: (i) a tight linkage between molecular marker and gene of interest, and (ii) high heritability of the gene of interest.

iii. Application:

MAS is applicable for genetic improvement of plants as well as animals. In plants, it is equally applicable in both self-pollinated and cross pollinated species.

iv. Markers Used:

MAS makes use of various types of molecular markers. The most commonly used molecular markers include amplified fragment length polymorphisms (AFLP), restriction fragment length polymorphisms (RFLP), random amplified polymorphic DNA (RAPD), simple sequence repeats (SSR) or micro satellites, single nucleotide polymorphisms (SNP), etc. The use of molecular markers differs from species to species also.

v. Efficiency:

The relative efficiency of MAS is greatest for characters with low heritability, if a large fraction of the additive genetic variance is associated with the marker loci. In other words, MAS is useful when the heritability of the trait is low. Moreover, MAS is more efficient than purely phenotypic selection in quite large populations.

It has been found by some workers that MAS may become less efficient than phenotypic selection in the long term. This is because the rate of fixation of unfavourable alleles at QTLs with small effects is higher under MAS than under phenotypic selection. It may be a consequence of the strong selection applied to QTLs with large effects under MAS in early generation. However, such problem comes after a long period.

vi. Accuracy:

Molecular markers have very high accuracy. They are not affected by environmental conditions. MAS is a new breeding tool which is available to make more accurate and useful selections in breeding populations. MAS allows heritable traits to be linked to the DNA which is responsible for controlling that trait.

vii. Speed of Progress:

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 recessive character. MAS permits identification of recessive alleles even in heterozygous condition and thus speeds up the progress of crop improvement work.

viii. Traits Improved:

MAS can be used for improvement of both oligogenic and polygenic traits. In the past, MAS has been mostly used for the genetic improvement of oligogenic traits and little progress has been made with polygenic traits.

ix. Material Developed:

MAS leads to development of non-transgenic genotypes or cultivars. In other words, MAS is used for development of non-transgenic cultivars. The transgenic cultivars face public resistance. On the other hand, cultivars developed by MAS are acceptable by consumers.

x. Cost:

MAS is very costly as compared to phenotypic selection. In MAS, the costly items include equipment’s, consumables, infrastructure, labour and DNA extraction process. MAS requires sophisticated and well equipped laboratory.

3. Steps in Marker Assisted Selection (MAS):

In the marker aided selection, RFLP markers are widely used for genetic improvement of crop plants for various economic characters.

The marker aided selection consists of five important steps, viz:

(i) Selection of parents,

(ii) Development of breeding population,

(iii) Isolation of DNA from each plant,

(iv) Scoring RFLPs, and

(v) Correlation with morphological traits.

These are briefly discussed below:

i. Selection of Parents:

Selection of suitable parents is an important step in marker aided 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.

ii. Development of Breeding Populations:

This is the second important step for application of marker aided 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.

iii. 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.

iv. 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.

v. 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.

4. Applications of Marker Assisted Selection (MAS):

In crop improvement programmes MAS can be used in various ways. In other words, MAS has several useful applications in plant breeding.

Important applications of MAS in plant breeding are briefly presented below:

i. MAS is very effective, efficient and rapid method of transferring resistance to biotic and abiotic stresses in crop plants.

ii. It is useful in gene pyramiding for disease and insect resistance.

iii. It is being used for transfer of male sterility and photo period insensitivity into cultivated genotypes from different sources.

iv. MAS is being used for improvement of quality characters in different crops such as for protein quality in maize, fatty acid (linolenic acid) content in soybean and storage quality in vegetables and fruit crops.

v. MAS can be successfully used for transferring desirable transgene (such as Bt gene) from one cultivar to another.

vi. MAS is very effective in introgression of desirable genes from wild into cultivated genotypes.

vii. MAS is equally effective in genetic improvement of plants and animals.

viii. MAS is useful in genetic improvement of tree species where fruiting takes very long time (say 20 years) because for application of phenotypic selection we have to wait for such a long time.

ix. MAS has wide application for genetic improvement of oligogenic traits as compared to polygenic traits.

5. Achievements of Marker Assisted Selection (MAS):

MAS has been used for genetic improvement of different field crops such as maize, barley, rice, wheat, sorghum, soybean, chickpea, pea, sunflower, tomato, potato and some fruit crops for various economic characters. MAS has been mainly used for developing disease resistant cultivars in different crops (Table 34.2).

Some notable examples of the use of MAS are given below:

i. Rice:

In rice MAS has been successfully used for developing cultivars resistant to bacterial blight and blast. For bacterial blight resistance four genes (Xa₄, Xa₅, Xa₁₃ and Xa₂₁) have been pyramided using STS (sequence tagged site) markers.

The pyramided lines showed higher level of resistance to bacterial blight pathogen. In Indonesia, two bacterial blight resistant varieties of rice viz Angke and Conde have been released through MAS. For blast resistance, three genes (Pil, Piz5 and Pita) have been pyramided in a susceptible rice variety Co 39 using RFLP and PCR based markers.

ii. Maize:

In maize, normal lines have been converted into quality protein maize (QPM) lines through MAS using opaque 2 recessive allele. This work has been done at CIMMYT (international centre for wheat and maize improvement, Mexico).

Three SSR markers (Umc 1066, Phi 057 and Phi 112) present within opaque 2 gene have been used for this purpose. The MAS used for conversion of normal maize lines into QPM is simple, rapid and accurate.

iii. Soybean:

In soybean cyst nematodes pose serious problem and most of the varieties are susceptible to this parasite. The resistant gene (rhg 1) is available. In soybean, nematode resistant lines have been developed through MAS using SSR marker (Sat 309).

MAS has been used for genetic improvement of various characters in different crops. Important characters which have been improved through MAS in different crops include disease resistance, insect resistance, salinity resistance, shattering resistance.

It has also been used for transfer of various characters such as male sterility, photoperiod insensitivity, earliness, and improvement of protein contents in some crop plants (Table 34.2).

In MAS various types of DNA markers have been used in different crop plants. Molecular markers which have been widely used in MAS in different crops include Restriction fragment length polymorphisms (RFLP), random amplified polymorphic DNA (FAPD) and simple sequence repeats (SSR) or microsatellite.

Other markers which have been used in some crops include amplified fragment length polymorphism (AFLP), sequence tagged site (STS), expressed sequence tags (EST), and SCAR markers. Single nucleotide polymorphism (SNP) is also used. SNPs have been identified for all the major cereal crops.

MAS is also being used for genetic improvement of forage and fruit crops. Among fruit crops MAS has been extensively used in pomegranate, apple and pear. In fruit crops MAS is based on RFLP, RAPD, SSR and AFLP markers. In these fruit crops MAS is being used for higher fruit production, better keeping quality for storage and disease resistance.

In vegetable crops MAS is being used in tomato and potato based on RFLP, RAPD and AFLP markers mainly for disease resistance. MAS has been found useful for genetic improvement of tree crops such as coconut and rubber.

In majority of field crops genes and linked markers for various important traits have been identified which are being used for MAS.

6. Advantages of Marker Assisted Selection (MAS):

MAS has several advantages over phenotypic selection and other breeding techniques.

Some important advantages of MAS are briefly discussed below:

i. Accuracy:

The accuracy of MAS, is very high because molecular markers are not affected by environmental conditions. It is very effective even with the characters having low heritability.

ii. Rapid Method:

MAS is a rapid method of crop improvement. It takes 3-5 years for developing a new cultivar against 10-15 years taken by the conventional method of breeding.

iii. Non-transgenic Product:

MAS leads to development of non-transgenic cultivars which are acceptable to everybody. In other words, it does not involve transgene. Hence there is no question of gene silencing.

iv. Identification of Recessive Alleles:

MAS permits identification of recessive alleles even in heterozygous condition and thus speeds up the progress of crop improvement programmes. In other words, it is equally effective for the genetic improvement of recessive characters.

v. Early Detection of Traits:

MAS permits early detection 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 tested at seedling stage can through light about the trait which are expressed later on.

vi. Screening of Difficult Traits:

MAS permits screening traits that are extremely difficult expressive and time consuming to score phenotypically. For example, screening for traits such as root morphology and resistance to biotic (insects and diseases) and abiotic stresses (drought, salinity, heat, frost etc.) is very easy through MAS.

vii. Gene Pyramiding:

MAS is very effective method in accumulating multiple genes for resistance to specific pathogens and pests within the same cultivar. This process is called gene pyramiding. Maker assisted backcrossing is routinely applied in breeding programmes for gene introgression. MAS can provide an effective and efficient breeding tool for detecting, tracking, retaining, combining and pyramiding genes for disease resistance.

viii. Small Sample for Testing:

MAS requires only a small amount of plant tissue for DNA testing. In other words, MAS can be carried out with small breeding populations. Moreover, MAS can be applied at any stage of plant growth.

ix. Permits QTL Mapping:

MAS permits mapping or tagging of quantitative trait loci (QTL) which is not possible by conventional method.

x. Highly Reproducible:

The MAS is based on DNA fingerprinting technique and the results of DNA fingerprinting pattern are highly reliable and reproducible.

7. Limitations of Marker Assisted Selection (MAS):

MAS has several advantages as discussed.

However, it has some limitations or drawbacks which are briefly below:

i. MAS is a costly method. It requires well equipped laboratory viz. expensive equipment’s, glassware and chemicals.

ii. MAS requires well trained manpower for handling of sophisticated equipments, isolation of DNA molecule and study of DNA markers.

iii. The detection of various linked DNA markers (AFLP, RFLP, RAPD, SSR, SNP etc.) is a difficult, laborious and time consuming task.

iv. MAS sometimes involves use of radioactive isotopes in labelling of DNA, which may lead to serious health hazards. This is a major disadvantage of RFLP based markers. The PCR, markers are safe in this regard.

v. It has been reported that MAS may become less efficient than phenotypic selection in the long term.

vi. The use of MAS is more difficult for QTL because they have minor cumulative effects and are greatly influenced by environmental conditions and genetic background.

8. Future Outlook of Marker Assisted Selection (MAS):

It is believed that MAS is a potential tool for crop improvement and hence it should be an integral part of plant breeding techniques. It has been reported that MAS has been used mainly by developed countries because of high cost of the technology in terms of infrastructure, equipment’s, chemicals and glassware.

The high cost of MAS technology can not be afforded by many developing countries. To make this technology usable by all developing and under developed world, the following ways and means may be helpful in the spread of this technology:

i. The costly technology like MAS should be supported by Consultative Group on International Agricultural Research (CGIAR) that promotes collaborative research and training on global bases. It will help in rapid spread of technology (MAS) in developing countries.

ii. The FAO and Rockefeller Foundation may play key role in making MAS technology available to resource poor countries.

iii. The private Industries should support MAS technology for the benefit of common man and in the global interest.

iv. To take advantage of such technology, there is need for International collaboration of research Institutes engaged in MAS programmes.

v. There is need for collaborative approach between public and private organizations to provide access to MAS technology in developing countries.

vi. International organizations such as FAO and CGIAR should organize training programme on regional level for capacity building in MAS technology.

vii. 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.