Wheat is an annual grass plant. It is restricted due to the scarcity of salt-tolerant germplasm resources. In particular, the salt-tolerance variation of wheat is very limited and the genetic basis is narrow, which seriously restricts the research and utilization of salt-tolerance in wheat. Therefore, conducting research on the genetic diversity of wheat salt tolerance, broadening the genetic basis, and screening new salt-tolerant genes are of great theoretical value and practical significance for developing and utilizing salinized soil, expanding wheat planting area, and increasing unit yield. Randomly amplified DNA (RAPD) molecular markers can detect genetic differences across the entire genome at the molecular level. They have many marker sites and are not affected by environmental conditions. Therefore, they have been widely used in many aspects.
Any biological species has a specific sequence and structure of genetic material - DNA. Due to different selectivities in the process of biological evolution, different regions of biological genome DNA show highly conserved or highly variable phenomena, and have different genetic diversity.
RAPD labeling technology is a technology that diagnoses the laws of intrinsic gene arrangement and external trait expression in organisms by analyzing the diversity of DNA amplified by PCR technology. RAPD uses PCR technology to analyze polymorphisms from amplified DNA fragments. Since the fragment is selectively amplified by the primer, the amplified fragment can be clearly displayed on the gel, so that the polymorphism of the amplified band with the same primer can reflect the polymorphism of the template. RAPD requires only one primer, about 10 nucleotides in length, and the primer order is random. Therefore, the genome of the subject can be analyzed without any molecular biological information.
Single-primer amplification is achieved by random pairing of a primer on two complementary strands of DNA, but there may be long or short spaced inverted repeat sequences in the genomic DNA molecule. Then there is a primer binding site on each of the two single strands, forming a template molecule for single-primer PCR amplification. If the nucleotide sequence of the primer is very short and the annealing temperature is low, the chance of the primer and DNA template inverting the repeated sequence increases, producing several single-primer PCR amplification products, forming a specific pattern of the primer. The number of inverted repeat sequences and the length of the intervals in different DNAs are different, and the amplified bands are different, that is, polymorphism occurs. When the primers are short, many adjacent and opposite primer sites on the chromosome exist in the genome. PCR technology scans the genome containing these inverted repeats and amplifies inserted DNA fragments of varying lengths. In practice, amplified DNA fragments with a length of 400-2,000 bp appear as a band on an agarose gel. Generally, DNA fragments shorter than 400 bp or longer than 2,000 bp do not appear on agarose gels.
For DNA extraction and purification methods, please refer to extraction and purification of rice genomic DNA.
Run 50 or 100 PCR reactions with slightly different DNA samples or primers in each tube. It is necessary to first prepare a mixed reaction solution of all reagents with different amounts of components, and then aliquot them into each tube. Since agarose gel is required for verification, the final volume of the reaction is 20 µL.
a. Prepare 100 PCR reaction mixtures for different DNA on ice. The recipe is shown in Table 1. Prepare a mixture of 100 PCR reactions containing different primers. The recipe is shown in Table 2.
b. Dispense equal amounts of the above reaction mixture into each reaction tube.
c. Add DNA or primers.
d. Add two drops of mineral oil to each tube.
e. Tighten the lid, place it on the PCR machine, and perform the PCR reaction.
f. The three-step procedure of RAPD-PCR reaction is:
The first step, 1 cycle. 94°C, 300 s.
The second step is 45 cycles. 94°C, 60 s; 36°C, 60 s; 72°C, 90 s.
The third step, 72°C, 300 s.
Table 1. Recipe of PCR reaction (100 times) mixture for different DNAs
| Composition | Volume |
|---|---|
| Water | 1,355 µL |
| 10x buffer | 200 µL |
| 25 mmol/L MgCl2 | 120 µL |
| 10x dNTPS | 200 µL |
| Primer (20 µmol/L) | 20 µL |
| DNA polymerase | 5 µL |
| Final volume | 1,900 µL |
| DNA (10-100ng) | 1 µL per tube |
Table 2. Recipe for PCR reaction (100 times) mixture containing different primers
| Composition | Volume |
|---|---|
| DNA (appropriate concentration) | 100 µL |
| Water | 1,355 µL |
| 10x buffer | 200 µL |
| 25 mmol/L MgCl2 | 120 µL |
| 10x dNTPS | 200 µL |
| DNA polymerase | 5 µL |
| Final volume | 1,980 µL |
| Primer (20 µmol/L) | 0.2 µL per tube |
a. Use TAE buffer to prepare a 1.5%-2% agarose gel.
b. After PCR amplification, add 4 µL loading buffer to each sample.
c. Load 20 µL of sample into each lane and select the appropriate relative molecular mass marker.
d. Electrophoresis for an appropriate period of time.
e. Immerse the gel in ethidium bromide solution for 30-60 minutes.
f. Wash the gel with water.
g. Observe on the ultraviolet transilluminator and take pictures.
Analyze experimental data using appropriate statistical software. On the electrophoresis gel plate, the presence of bands at the same position is scored as 1, the absence of a band is scored as 0, and the failure or missing band is scored as 9. Enter the amplification results of all RAPD primers into the data table and use statistical software for statistical analysis.
Changes in PCR conditions, the number of primers, and the interpretation of electrophoresis results will affect the credibility of RAPD results. Interpretation of electrophoresis results mainly involves the selection of weak bands. The selection of weak bands mainly depends on the probability of their recurrence. Repeated experiments can be performed, and electrophoretic bands appearing at weak band positions that are difficult to choose can also be excluded from statistics. This may lose some polymorphic information sites and has little impact on the pedigree relationship of the research materials, but it is not advisable when studying molecular markers or constructing gene linkage maps.
