Rice is an annual grass plant. Whether it is conventional breeding or hybrid rice breeding, the selection of parents is very important. It requires the parents to carry the target traits or genes and expand the genetic differences between the parents. Although it is effective for breeders to judge the genetic differences between varieties or strains based on the agronomic traits and pedigree relationships of the parents, it is limited by time, space and experience, and is difficult to quantify. The development of molecular marker technology provides an effective tool for detecting genetic differences between varieties and strains at the DNA level. Because RFLP markers have a large number of sites and are not affected by spatiotemporal restrictions on gene expression and environmental conditions, they are widely used in species classification, detection and evaluation of genetic diversity, and gene mapping research.
Restriction fragment length polymorphism (RFLP) labeling uses restriction enzymes to digest sample DNA to produce a large number of restriction fragments, and the DNA fragments are separated according to their respective lengths through gel electrophoresis. When there are a large number of enzymatic fragments, such as plant genomic DNA, although they are separated according to fragment length after electrophoresis, they still form a continuous band. In order to detect polymorphic fragments, the DNA in the gel needs to be denatured. Transfer to a support membrane such as nitrocellulose filter membrane or nylon membrane through Southern blotting to firmly combine the DNA single strand with the support membrane. The isotope or digoxigenin-labeled probe is then used to hybridize with the enzyme-digested fragment on the membrane, and the hybridization band is displayed by autoradiography, that is, the restriction fragment length polymorphism is detected.
When using the same restriction endonuclease to digest different varieties or different individuals of the same variety, since the target DNA has both homology and variation, the digested fragments will be different, and the positions of the hybridization bands displayed by different materials will also be different. This difference is restriction fragment polymorphism. This difference at the DNA molecular level may be due to changes in the endonuclease recognition sequence, or may involve partially determined deletions, insertions, translocations, inversions, etc. RFLP actually reflects differences at the DNA molecular level, and this variation is heritable. RFLP labeling technology requires a sufficient number of copies of the target sequence in the sample DNA. The quantity and purity of the target sequence in the sample determines the method of RFLP labeling technology.
The current research methods of RFLP labeling technology can be divided into two categories: standard RFLP labeling technology and PCR-RFLP labeling technology. Standard RFLP labeling technology is suitable for analyzing samples with a relatively high content of target sequences, such as mitochondrial DNA, ribosomal DNA or PCR amplification products. However, the use of standard RFLP labeling technology is limited due to the need to prepare probes, screen restriction enzymes, and cumbersome operations. PCR-RFLP labeling technology uses PCR technology to amplify the DNA region that determines allele specificity. The amplified product is digested with restriction enzymes and then separated by electrophoresis. Based on the obtained restriction fragment length polymorphism pattern, the specificity of different alleles was judged. PCR-RFLP labeling technology overcomes the shortcoming of standard RFLP labeling technology that requires a large amount of DNA, and can study trace amounts of DNA samples. Moreover, the products amplified by PCR can be directly detected by EB staining after digestion with restriction enzymes, which is quick and easy. The only disadvantage is that the primers for amplifying the sequence need to be selected and designed in advance.
The first step is to extract high-quality genomic DNA. Compared with other molecular marker experiments, RFLP labeling technology has high requirements on the quality of DNA and must ensure a certain quantity.
a. Add 5 µL of genomic DNA into a 1.5 mL centrifuge tube, add 5 µL of 10× digestion buffer and 20 units of restriction enzyme, and add ddH2O to 50 µL. In a 50 µL reaction system, perform the enzyme digestion reaction.
b. Shake slightly, centrifuge, and react at 37°C overnight. Take it out and store it in the refrigerator at 4°C for later use.
c. Take 5 µL of the reaction solution and conduct 0.8% agarose electrophoresis to observe whether the enzyme digestion is complete. At this time, there should be no obvious bands larger than 30 kb.
a. The enzymatically digested DNA is subjected to 0.8% agarose gel electrophoresis (can be electrophoresed at 18V overnight) and then observed by EB staining.
b. Immerse the gel piece in 0.25 mol/L HCI for depurination for 10 min.
c. Take out the gel piece, rinse with distilled water, transfer to denaturing solution for denaturation for 45 min, rinse with distilled water, then transfer to neutralizing solution for neutralization for 30 min.
d. Soak the nylon membrane and filter paper in water in advance, and then immerse them in 10×SSC. Place a glass plate rack in a basin and lay a layer of filter paper, then invert the gel piece, cover it with a nylon membrane, and cover it with two layers of filter paper. Then cover with absorbent paper, press with a 0.5 kg weight, and blot with 10× SSC salt solution for 18-24 h. Electric transfer or vacuum transfer can also be used.
e. Remove the nylon membrane, soak it in 0.4 mol/L NaOH solution for 30 s, quickly transfer to 0.2 mol/L Tris-HCI (pH 7.5) and 2× SSC solution, and rinse for 5 min.
f. Sandwich the membrane between two layers of filter paper and vacuum dry at 80°C for 2 h.
g. Autoradiography: Label the probe (the amount of DNA probe per lane is 5 ng, the amount of 32P-dCTP is 1 µCi) and then perform molecular hybridization. The probe and nylon membrane were insulated at 65°C and rotated slowly for 16-18 h. Wash the membrane and press it into a piece, take out the hybridization membrane, and wash away the unhybridized probe using SSC method and SDS method. Wrap the nylon film with plastic wrap, press the X-ray film, and perform autoradiography at -70°C for 5-15 d. After the film is washed and developed, take it out, fix it, dry it and store it.
The amount of data obtained using the RFLP method is large and requires computer processing. The data matrix is generated based on the presence or absence of restriction enzyme sites on PCR amplification products of different taxa and the variation in length. According to the presence or absence of a certain position in the PCR product, it is expressed as 1 or 0, and analysis software is used to perform various statistical analysis and cluster analysis.
Undigested DNA must be prevented from being degraded, and the enzyme digestion reaction must be thorough. Special attention should be paid to the following aspects when preparing samples for RFLP analysis.
