The polymorphism in RFLPs between the parents and their involvement in the recombinants in F 2 population is determined by using DNA probes. It is a tedious job to identify individual DNA fragment in such cases. The nuclear DNA of higher plants, when digested with specific restriction enzymes, produces millions of fragments in a continuous range of sizes. The DNA of chloroplasts, when digested with specific enzyme, produces about 40 fragments of different sizes. The gel is stained with ethidium bromide and the variation in DNA fragments can be viewed in the ultraviolet light. The DNA fragments of different sizes are separated by subjecting the digested DNA to agarose gel electrophoresis. The isolated DNA is digested with specific restriction enzyme to obtain fragments of DNA. Standard procedures are available for DNA isolation. The DNA is isolated from each plant of F 2 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 third important step is isolation of DNA from breeding population.
Generally 50-100 F 2 plants are sufficient for the study of segregation of RFLP markers. The F 2 progeny is required for the study of segregation pattern of RFLPs.
F 1 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 F 1. The selected parents are crossed to obtain F 1 plants. This is the second important step for application of marker aided selection. In cross-pollinated species, inbred lines are used as parents. In self- pollinated species, plants are usually homozygous. The parents that are used for MAS should be pure (homozygous). This will help in identification of DNA of both the parents and also their segments in F 2 generation in various recombination’s.įor selection of parents, we have to screen germplasm and select parents with distinct DNA. In other words, parents with contrasting characters or divergent origin should be chosen. The parents should be such so that we can get usable level of polymorphism (variation) in the RFLP markers. Selection of suitable parents is an important step in marker aided selection. Moreover, MAS is more efficient than purely phenotypic selection in quite large populations. In other words, MAS is useful when the heritability of the trait is low. 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. The use of molecular markers differs from species to species also. 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. MAS makes use of various types of molecular markers. In plants, it is equally applicable in both self-pollinated and cross pollinated species. MAS is applicable for genetic improvement of plants as well as animals.
These are: (i) a tight linkage between molecular marker and gene of interest, and (ii) high heritability of the gene of interest. There are two pre-requisites for marker assisted selection. It differs from gene assisted selection (GAS) which refers to the selection which is based on QTLs (quantitative trait locus or loci). Marker assisted selection (MAS) is also termed as marker aided selection and marker assisted breeding (MAB).
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