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Restriction
Fragment
Length
Polymorphism
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RFLP -
Use of Cloned fragments of chromosomal DNA as genetic markers is usually termed RFLP.
This technique is dependent on natural variation in DNA base sequence and digestion of DNA with restriction enzyme.
Homologous restriction fragments of DNA that differs in size (length) can be used as genetic markers to follow chromosomal segments through genetic crosses.
Using this technique, RFLP linkage maps are prepared.
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These molecular maps and markers provide a direct method for selecting desirable genes for, say, disease resistance, variety identification and so on. Extremely high saturation of RFLP markers around genes of interest can be achieved with near isogenic lines.
The basic advantage of this technique is that a probe can be identified as linked to a gene just by comparison of the RFLP patterns of the donor parent, the recurrent parent and one or more lines isogenic for that trait of interest.
Continuous variation for most of the traits in nature resulted from the concept that quantitative traits can arise from segregation of multiple genes, modified by environmental effects. Linkage and accurate systematic mapping of quantitative trait loci were not possible because the inheritance of an entire genome could not be studied with genetic markers.
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Use of RFLPs has increased the efficiency of mapping quantitative trait loci because a greater number of markers can be scored in a single population relative to other markers, such as isozymes or morphological markers.
It is also possible to examine the effects of environment on expression of individual gene loci involved in a complex trait and to determine functional genotype environment interaction.
In agriculture, quantitative trait loci analysis could help in achieving species resistant to diseases and pests and tolerant to drought, heat, cold and other diverse conditions. Efficient use of resources and nutritional quality would also be augmented.
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Since 1985, restriction fragment length polymorphism has emerged as a very powerful tool for genetic analysis in plants. RFLP, like many other phenomena, was first identified in microbes, then utilized in humans and later flourished in plants.
The most exciting feature about RFLP is the possible use of a cloned DNA fragment as a genetic marker.
This ability to detect variation directly at the DNA level makes RFLP a very important tool in plant genetics.
Secondly, RFLPs have an extremely nigh resolution power for detection of genetic variation compared to other existing technologies; hence they can be directly integrated into the existing breeding technologies with considerable ease.
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RFLP-assisted plant breeding has opened entirely new and exciting vistas.
To date, 14 major crop species are under study using RFLP for development of suitable breeding technologies to overcome such present problems as biotic and abiotic stresses, yield etc.
For example, identification of specific genes in the latter problems is difficult and also affects the specific trait of interest.
The crop species under study using RFLPs include:
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MONOCOTS:
(i) Allium cepa and
(ii) Triticale sp.;
DICOTS:
(i) Lycopersicon spp.,
(ii) Glycine max,
(iii) Solanum spp.,
(iv) Brassica spp.,
(v) Arabidopsis thaliana,
(vi) Lactuca sativa,
(vii) Lens culinaris,
(viii) Vicia spp., and
(ix) Beta vulgaris.
The most studied aspect using RFLPs, in the above species is the genetic diversity feasibility, followed by the construction of RFLP maps and their use in breeding. Availability of good genetic background has aided RFLP work in rice, maize and tomato, for which a good correlation has been obtained between morphological and isozyme data with RFLP data. Molecular mapping of economically important genes is one of the immediate applications of an RFLP map for a species.
Basically, any gene of interest can be tagged using the same method for both monogenic and polygenic traits. The genetic materials may be either nearly isogenic lines (NILs) or F2 segregating populations as long as two parents are diverse enough in background. Tightly linked DNA markers serve as "Tags" for these genes of interest. Such tags may facilitate early selection for these genes in breeding programs and ultimately lead to cloning them via chromosome walking.
RFLP analysis in rice has been the focus of a major research thrust under the Rice Biotechnology program of the Rockefeller Foundation. Currently around 400 markers, distributed at an average spacing of 20 cm have been located on the genome. This number is anticipated to increase to around 500 in the next year. With the advent of RAPD technology, new markers specific to any interval can be obtained with ease.
Secondary trisomics representing all the 12 rice chromosomes have been identified. These trisomics are being used to locate centromeres on the RFLP maps to study the orientation of linkage groups and to delimit the area locations of the markers.
The available RFLP information has been used to tag 11 genes of economic importance, including those for bacterial blight (x a 21), blast (pi-1, pi-2, pi-4), resistance to the white-backed plant hopper (wph-1), grain aroma (f g r), photoperiodic flowering response (se-1), and semidwarf habit {sd-1). Molecular DNA probes have also been used for analysis of the blast pathogen in order to investigate genetic relationships between isolates that infect rice and those that infect other grasses.
A repeat sequence, MGR, was found to be present in a relatively high copy number in isolates infecting rice but in few copies in isolates not infecting rice.
RFLP techniques have been exploited in Indian rice and rape mustard programs to enhance resistance to bacterial leaf blight (Xanthomonas campestris), blast (Pyricularia oryzae), and aroma in rice. Indica rices and brassicas have been used to develop embryogenic cell suspension cultures, protoplast regeneration, leaf-based mesophyll cells, and somatic hybrids.
Some 15 RFLP/RAPD have been in routine use in rice in the resistance development program against rice bacterial leaf blight, blast and aroma. More RFLP/RAPDs are being developed in rice (Oryza sativa), rape mustard (Brassica nap us, Brassica campestris), and chickpea (Cicer arietinum) through the Indian crop biotechnology network program.
Two genes of rice, Pi-2 (t) and Pi-4, confer complete resistance to the blast fungal pathogen Pyricularia oryzae. DNA markers (RFLP) closely linked to these genes have been identified. Among them, Pi-Z (t) confers resistance to several blast fungal races.
This gene is closely linked to a single-copy DNA clone on chromosome 6. Pi-1 (t) and Pi-4 (t) are each linked to two DNA clones. Pi-i (t) is located on chromosome 11 and Pi-4 (t) on chromosome 12.
In addition, two other blast resistant genes have been mapped on chromosome 12. Efforts have been undertaken to isolate more DNA markers around the regions of these genes for both marker-aided selection and map-based cloning. Different blast-resistant genes on the same chromosomal regions are presently under comparison.
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