Many agriculturally important traits like yield, disease resistance, insect resistance and quality traits are governed by quantitative trait loci. Although significant improvement has been made for agronomically important traits, considerable difficulties are encountered because of genotype- environment interactions. A major breakthrough in the characterization of quantitative traits was initiated by the development of DNA markers in 1980’s.

Marker assisted selection refers to selection of plants which carries the genomic regions that are involved in expression of trait of interest through molecular markers. Or it is method where phenotype is selected based on the genotype of the marker.

With the development and availability array of molecular markers and dense molecular maps it has become possible to transfer the traits governed by both major and minor genes into the elite genotype.

• Salient requirements of MAS:

  • A genetic map with an adequate number of uniformly spaced polymorphic markers to accurately locate the desired QTLs or major genes.

  • Close linkage between the major gene or QTL and the adjacent markers

  • Adequate recombination between the marker and the rest of the genome

  • An ability to analyze large number of plants in time and cost-effective manner

  The success of MAS depends upon the location of the marker with respect to the gene of interest. Three kinds of relations between the marker and with the respective gene of interest could be distinguished

  • The molecular marker is located within the gene of interest which is ideally referred to as gene based marker

  • The marker is in linkage disequilibrium with the gene of interest called as LD-MAS

  • The marker is in linkage equilibrium with the gene of interest- most difficult and challenging situation for applying MAS

• Application of MAS is to use DNA based markers for three basic purposes:

  • Tracing the favorable allele (dominant or recessive) across generations; in order to accumulate favorable alleles

  •  Identifying the most suitable individuals among segregating generations, based on the allelic composition of a part or of the entire genome

  • Breaking the possible linkage of favorable alleles with undesirable loci

• The advantages of MAS:

  • The time saving from the substitution of complex field trials (that need to be conducted at a particular times of year or at specific locations, or are technically complicated) with molecular tests

  • Elimination of unreliable phenotypic evaluations associated with field trials due to environmental effects

  • Selection of genotypes at seedling stage

  • Gene pyramiding or combining multiple genes simultaneously

  • Avoid the transfer of undesirable or deleterious genes (‘linkage drag’)

  • Selecting for traits with low heritability

  • Testing for specific traits where phenotypic evaluation is not feasible

• Marker assisted foreground selection:

  Marker assisted foreground selection was proposed by Tanksley (1983). This is used to trace the presence of target genes. It is used to trace the traits which are difficult to identify using phenotypic screening methods. Also used to identify the plants at the seedling stage, allowing the best plants to be selected and used for backcrossing. Recessive alleles can be selected, which are very difficult to identify using conventional breeding methods.

• Marker assisted background selection:

  This was originally proposed by Young and Tanksley (1989a) and later termed background selection by Hospital and Charcosset (1997). The objective of the background selection is to accelerate the return to recipient recurrent parent genome outside the target genome, so as to

  • To reduce the length of the intact chromosomal segment of the donor parent dragged around the target gene on the carrier chromosome.

  • Reduce donor genome on the non carrier chromosome to the maximum extent

  • If the phenotype of the desired gene is not easily assayed, BC progeny possessing a marker allele from the donor parent at a locus near/within the target gene can be selected

• Main considerations for the use of DNA markers in MAS:

  • Reliability: Markers should be tightly linked to the target loci, preferably less than 5 centi morgan genetic distance. The use of flanking markers or intragenic markers will greatly increase the reliability of the markers to predict the phenotype.

  • DNA quality and quantity: Some marker techniques require large amounts and high quality of DNA, which may sometimes be difficult to obtain in practice, and this adds to the cost of the procedures.

  • Technical procedures: The level of simplicity and the time required for the technique are critical considerations. High throughput simple and quick methods are highly desirable

  • Level of polymorphism: Ideally, the marker should be highly polymorphic in breeding material. (i.e. it should discriminate between the different genotypes)

  • Cost: The marker assay must be cost effective in order for MAS to be useful

   

• Applications of MAS in plant breeding:

  (a) Marker assisted evaluation of breeding material:

  Marker assisted evaluation of breeding material: Prior to crossing and line development, there are several applications in which DNA marker data mat be useful be for breeding, such as

  • A cultivar identity
  • Assessment of genetic diversity
  • Parental selection
  • Confirmation of hybrids

  (1) Cultivar identity / assessment of cultivar: Seed of different strains is often mixed due to the difficulties of handling large numbers of seed samples used within and between crop breeding programmes. The maintenance of high levels of genetic purity is essential in cereal hybrid production in order to exploit heterosis.

  (2) Assessment of genetic diversity and parental selection: Breeding programmes depend upon on high level of genetic diversity for achieving progress from selection. Broadening the genetic base of core breeding material requires the identification of diverse strains for hybridization with elite cultivars.

  (3) Study of heterosis:DNA markers have been used to define heterotic groups that can be used to exploit heterosis. The development of inbred lines for use in producing superior hybrids is very time consuming and expensive procedure.

  (b)Marker Assisted Backcrossing (MAB):

  Backcrossing is a plant breeding method which is used since from centuries to transfer the useful characters into the elite variety. Elite variety is the one which has very useful genes but deficient in one or two characters.

  Use of DNA markers in backcrossing increase the efficiency of selection. There are three levels in marker assisted backcrossing.

  (i) Foreground selection (already explained above)

  (ii) Recombinant selection

  (iii) Background selection (explained above)

  Recombinant selection: The purpose of recombinant selection is to reduce the donor chromosome segment. Selection of BC progeny with the target locus and recombination between the target locus and linked flanking markers. Because if the donor segment contains undesirable genes which are linked to the target locus it will affect the crop performance. It is mainly carried out to minimize the linkage drag.

  (c) Marker assisted pyramiding:

  Pyramiding is the process of combining the two or more genes into a single genotype. Using the conventional methods it is very difficult to identify the plants with one or two genes. A conventional method involves destructive type bioassays. But molecular markers assays are non destructive type. With linked molecular markers, one can determine the number of genes present in a plant.

  The motive of marker assisted pyramiding is to develop durable or stable resistance (5 years or more than years) since pathogens frequently overcome the single resistance gene because of emergence of new pathogen races.

  Ex: In rice for blast resistance genes (major gene) are pyramided using SSR markers.

  For bacterial blight disease three gene genes, Xa21, xa 5, Xa4, xa 13 (QTL’s) have been transferred using molecular markers.

  (d) Early generation marker assisted selection:

  It is often very difficult to select the plants at early stages for some of the characters like yield because of their quantitative nature which are highly influenced by the environment. In such cases MAS plays a very important role in early generations because most of the undesirable plants can be eliminated which increases the efficiency of selection. So that it reduces the burden to carry the large number of genotypes for the next generation.

  Ex: In self pollinated crops the aim is to fix the alleles in homozygous state. In single seed descent and bulk method the selection is carried out in F5 or F6 generations so that plants have attained homozygous condition. Using the codominant markers, it is possible to fix the alleles in homozygous form as early as in F2 generations but it requires the large population.

   

The markers which are identified in the preliminary study are seldom used for the MAS programme. They require further testing and development. The markers which are not tested are not reliable for Phenotyping.

The steps involved in further development of the marker that are useful in MAS are:

• High resolution mapping of QTL’s:In preliminary QTL mapping the aim is to cover all chromosomes evenly in order to find the markers flanking those QTL’s that governs the trait of interest. Sometimes even the closest marker flanking a QTL may not be tightly linked to the trait of interest. Hence by using large number of population and greater number of molecular markers one can identify the marker which is tightly linked to the QTL (<1 centi morgan) which governs the trait of interest which is known as ‘high resolution mapping’.

• Validation of markers:Validation of markers means testing of marker in determining target phenotype in independent population and in different environments. It is very important step because there is o guarantee that the marker identified in one genetic background will be useful in different population, especially when the population originate s from distantly related population. The marker should reveal sufficient polymorphism in different population derived from different parental genotypes.

• Marker conversion:Marker conversion is needed in two instances: when there are problem of reproducibility and marker technique is complex, time consuming and costly. Such problems can be overcome by developing the markers which are highly reproducible and easy to carry out. Ex: SCAR markers can be developed by the RAPD markers which have a low reproducibility. The PCR based markers can be utilized in MAS which are technically easy, less time consuming and cheaper.

• General Scenario for MAS in potato breeding includes five steps:

  • To cross progenitors and to produce segregating progenies. The genetic value and specific characteristics inherited by the progenitors will be known.

  • To grow seedling plants

  • To collect samples and to isolate DNA from each of the progenies. This can be done through:

    • Direct PCR (Polymerase chain reaction) that will be preferred as no additional isolation steps are required,

    •  One -step DNA isolation,

    • Multi-step DNA isolation, without liquid nitrogen,

    • Multi-step DNA isolation using liquid nitrogen

  • To apply molecular markers to the DNA samples. This could be done by:

    • One-step marker application (e.g. via real-time PCR), or

    • Multi-step, including amplification, separation of amplicons and visualization

  • To select those seedlings that possesses most of the specific alleles as detected by the markers.