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(Agricultural College, Yanbian University, Longjing, Jilin 133400, China)
Application and Prospect of RFLP Technology in Crop Breeding
Zhang Baoguo Liu Wei
Abstract: RFLP is a molecular marker technology developed along with several fields of biotechnology. It can be used to make RFLP maps of plants and apply them to plant genetics and breeding research. This paper briefly introduces the basic principles, technical steps, advantages and disadvantages of RFLP technology.
Key words: RFLP; crop research; application
CLC number: S5.032 Document code: A Article ID: 1008-7508( 2007)03-0105-03
First, the basic principle
Re Restriction end nuclease recognizes a specific base sequence on the DNA duplex and can cleave it. Different restriction endonucleases have their own specific recognition sequences. In the corresponding DNA segment of the homologous chromosome, digestion with a restriction endonuclease, due to different base composition, will produce fragments of different lengths, and then the corresponding DNA probes labeled with these fragments Hybridization, the displayed map can reflect whether there is a difference in the composition of the genomic DNA base sequence. The basis of this technique is to reveal the similarities and differences of DNA base sequence composition by restriction endonuclease fragment length polymorphism, which is called Restriction Fragment Length Polymorp Hism (RFLP). technology.
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Second, the basic steps of RFLP technology
 This technology mainly consists of three major steps:
1. Preparation of target DNA First, extract the genomic DNA, select appropriate restriction endonuclease to digest the genomic DNA, and separate the DNA fragments of various lengths by agarose gel electrophoresis. According to the length of the fragments, the DNA fragment was denatured and transferred to a nitrocellulose membrane or a nylon membrane, and the blot was transferred (Southernb1ot), and baked at 80 ° C or irradiated with long-wave ultraviolet rays to fix the DNA on the membrane. 
2. Labeling of nucleic acid probes Purification of DNA fragments prepared as probes (these DNA fragments may be a fragment of genomic DNA, or cDNA, or synthetic oligonucleotides), using radioactive elements (such as α- 32p) or non-radioactive elements (such as Dig-dUTP, etc.) are labeled and purified before use. 
3. Hybridization shows that the labeled probe is hybridized with a single-stranded nucleic acid on a nitrocellulose membrane or a nylon membrane, and after washing the membrane to remove the unhybridized labeled probe, the autoradiograph or the substrate to which the enzyme is added is subjected to a color reaction. The displayed bands are analyzed.
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Third, the advantages of RFLP technology and attention to problems 
 1, the advantages of RFLP technology 
First, the number of markers can be infinite: RFLP reveals a natural variation in DNA levels, the number of which is almost limitless. 
Second, most markers are codominant: the sites that express RFLP are generally single sequences, and each site usually has two alleles (dominant). Following Mendelian inheritance, RFLP markers can also be constructed using traditional gene mapping methods. 
Third, any growth period is predictable and unaffected by the environment: DNA molecule level markers have no developmental stage or organ specificity and are not affected by environmental conditions and genetic interactions. 
Fourth, high variability: Each plant will have a large number of polymorphisms. Usually, as long as there is sexual hybridization, a mapping population can construct a rich RFLP map. 
2. When doing RFLP analysis techniques, there are several issues that require close attention:
First, the DNA molecule to be detected must maintain a large molecule, and avoid artificially cutting the DNA molecule into small fragments of DNA during the extraction of DNA. Otherwise, the final displayed RFLP pattern may be an illusion;
Second, when digesting macromolecular DNA with restriction endonucleases, the DNA is completely digested, otherwise the results obtained are not reliable;
Third, the concentration of DNA to be digested cannot be too high;
Fourth, use low-pressure electrophoresis when electrophoresis;
Fifth, the probe must be fully denatured before hybridization;
Sixth, the conditions of hybridization and membrane washing should be mastered according to the condition of the probe labeling and the degree of complementarity between the probe and the target DNA and the content of G and C;
Seventh, in the case of autoradiography, the exposure time is determined according to factors such as radioactivity on the film after hybridization.
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IV. Application of RFLP in Crop Genetics and Breeding
 1. Select the target trait at the molecular level
The RFLP map itself is not directly useful for plant breeding, but only when it is combined with the classical marker, the originally located gene, when determining which RFLP marker is synergistically separated from the target trait, ie, the gene of interest is linked to RFLP. This makes it easy to select the desired recombinant form, selects the target trait at the molecular level, and is not affected by the genetic background of the environmental conditions. The RFLP linkage map provides breeders with an efficient means of selection in potato, corn, and pepper. There have been reports of RFLP mapping on cotton, soybean, lettuce, sweet potato, tomato and wheat crops. As long as the breeder selects one or more RFLP markers that are tightly linked to the gene of interest, the gene of interest is selected because the RFLP marker linked to the gene of interest is readily detectable. This indirect is particularly effective for those who have difficulty or are too time consuming, laborious, and the choice of recessive traits in separate populations. The genes identified in tomato that are linked to the RFLP marker include tomato mosaic virus, Fusarium, bacterial plaque and root-knot nematode resistance genes, and major genes controlling plant growth habits (SP) and fruit ripening characteristics. The RFLP marker was also established in the maize dwarf mosaic virus resistance gene and the lettuce downy mildew resistance gene. 
2. RFLP genetic markers and crop quantitative traits gene mapping
There are many economic traits in crops that are quantitative traits that are controlled by a single gene effect and are susceptible to environmental influences. For many years, genetic studies of such traits have mainly adopted quantitative genetic methods, through appropriate experimental design and The statistical model estimates the genetic parameters such as mean, variance, covariance, heritability, and combining ability. Using these parameters to describe the genetic characteristics of the population, it has a certain guiding role in breeding research, but the number of genes controlling quantitative traits, the number of quantitative trait loci (Quantitative Trait Loci referred to as QTL) on the chromosome, QTL gene locus Questions such as the contribution of quantitative traits and their intergenic relationships require RFLP detection techniques. At present, molecular markers on high-density RFLP maps are used as important quantitative traits, such as yield, plant height, fruit and seed component content, etc. It is expected to introduce the number of multiple genes controlling quantitative traits and determine the location of QTLs on chromosomes. Determine the effect of a single gene. The application of molecular markers to locate QTL is to first detect and screen the parents and construct the genetic population, construct a molecular marker linkage map, and then achieve the purpose of locating the QTL according to the linkage relationship between the statistical model and the method and the QTL on the chromosome. In recent years, the application of RFLP markers in QTL mapping of tomato, maize, rice and other crops has achieved gratifying results, such as rice blast resistance multi-gene localization, tomato fruit weight, soluble solid content, pH QTL mapping research, control QTL analysis of maize maturity. 
3. Determination of genetic purity of varieties or strains
An understanding of the RFLP maps at many linkage loci in the entire genome allows for genotypic mapping of a variety or line to represent species-specificity of this line, so some minor variations in the genome can be altered from RFLP maps. It is monitored; RFLP mapping technology can also be used in seed patent registration to resolve infringement disputes, which have been used by some commercial breeders in Europe to protect their breed rights. 
4, improved backcross breeding technology
Sexual crossbreeding is the hybridization of the traits with the traits to be improved, and the chromosomes of the isolated progeny are inlaid by the chromosomal fragments of the parents, with the desired parental traits and undesired parental traits. . After the introduction of the donor gene by the backcross breeding technology, the method for restoring the original excellent genotype of the parent can be genetically and receptively through multiple backcross selection or backcrossing and selfing selection (the target gene is recessive). Parents are similar, but plants with the gene of interest are added. The limitation of backcross breeding lies in the long time, the target gene is effective and easy to detect. The emergence of RFLP technology is expected to overcome the limitations of backcross breeding. If the gene to be transferred is closely linked with the RFLP marker, then the seedling stage, ie the trait has not yet been The presence of the gene of interest can be detected prior to expression, and backcross breeding can also be performed on quantitative trait genes using RFLP technology. 
5. Establish genetic relationships between incompatible crops and determine genomic homology between crops
In taxonomy, some sexually incompatible crops have certain genetic relationships. For example, broccoli, radish, and cabbage are all of the genus Brassica. Both corn and sorghum belong to the family Poaceae, and potatoes, tomatoes, and peppers belong to the family Solanaceae. Little is known about the homology of their genomes. If the gene sequence of a chromosome component is highly conserved among relatives, it is possible to replace a single chromosome or chromosome fragment by means of somatic hybridization or primal DNA introduction. , combining the excellent traits of the crop with those genes that are not available within the normal hybridization range of one species. Using the same set of probes for RFLP mapping, the chromosome composition and gene sequence conservation of tomato, potato and pepper have been determined, and a comparative linkage map between the three is established. By comparing the highly conserved relationship between tomato and potato, the chromosome has Partial homology, it is possible to exchange chromosomes or fragments, while peppers have poor homology.
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V. Problems and prospects
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RFLP technology is incorporated into routine breeding programs to enable people to acquire, transfer and combine genes quickly and accurately. Due to the direct impact of RFLP on breeding methodologies, the application of RFLP spectroscopy will likely be one of the first biotechnologies to be included in commercial or government breeding programs, and will be the first biotechnologies to influence agricultural production. However, the degree of availability of the RFLP map depends on its saturation level, ie the number of fragments and the level of distribution of these fragments in the staining group and the level of polymorphism exhibited, and also requires a certain polymorphic feature and a desired phenotype. Linked. One of the goals of the RFLP mapping program is to create a "saturated map". All chromosomes have RFLP markers every 10 to 20 exchange units (cM). A saturated graph should contain 150 or so uniformly spaced markers. With hundreds of explorations, it is possible to reach saturation levels. Therefore, the cost of this technology and the problem of radioisotope labeling are the limitations of the current application of RFLP technology. However, in the future, a simple method of development, using biotin or enzyme-labeled DNA probes, will reduce costs. 
References: 
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