AFLP Analysis of Genetic Diversity and Genetic Relationships of Spinach Germplasm

Spinach belongs to the genus Spinacia of the Amaranthaceae and subfamily Chenopodioideae and is an annual herbaceous plant. Randomly amplified DNA (RAPD) and other technologies have been reported in the genetic diversity analysis and germplasm identification of spinach germplasm resources. Certain results have also been achieved in the identification of disease resistance and cold tolerance of spinach germplasm. However, basic research on spinach is relatively weak. Further research on the genetic diversity evaluation and genetic relationships of spinach germplasm resources is of great significance for the innovative utilization of spinach germplasm and the breeding of new varieties.

Principle

The principle of amplified fragment length polymorphism (AFLP) labeling is based on double enzyme digestion of plant genomic DNA, followed by PCR amplification to select restriction fragments. When using AFLP labeling technology for research, due to the different sizes of genomic DNA in different species, the relative molecular masses of the restriction fragments generated after the genomic DNA is digested with restriction enzymes are different. Use a specific double-stranded adapter to connect to the enzyme-digested DNA fragment as a template for the amplification reaction, and use primers containing selective bases to amplify the template DNA. The type, number and sequence of selective bases determine the specificity of the amplified fragment. Therefore, only those restriction fragments whose nucleotides flanking the restriction site match the selective bases of the primer can be amplified. The amplified products are labeled with radioisotopes and separated by polyacrylamide gel electrophoresis, and then polymorphisms are detected based on the presence or absence of DNA fingerprints on the gel.

When analyzing genomes using AFLP, two restriction enzymes are generally used to digest genomic DNA in an appropriate buffer system. One is a low-frequency cleavage enzyme (the recognition site is a rare cutter with six bases), and the other is a high-frequency cleavage enzyme (the recognition site is a four-base frequent cutter). EcoRI, Hind III, Pst I, Sac I, and Apa I are all low-frequency cleavage enzymes with six base recognition sites, but EcoRI is more commonly used. Mse I and Taq I are high-frequency cleavage enzymes with four-base recognition sites, but the former is often used to cut A-rich eukaryotic genomic DNA strands to produce smaller restriction fragments, and is more commonly used in AFLP fingerprint analysis. The latter produces an unequal distribution of amplified fragments, often appearing in the upper part of the gel.

The enzyme combination commonly used in AFLP analysis is EcoRI and Mse I. In the experiment, the digested fragments were first connected to artificial adapters containing common sticky ends, and the connected sticky end sequences and adapters were used as primer binding sites for subsequent PCR reactions. The primer mentioned here includes three parts: a core sequence (CORE) at the 5' end that is complementary to the artificial linker sequence, a restriction endonuclease specific sequence (ENZ), and the sticky end with selective bases at the 3' end (selective extension, EXT). Among them, the design of connectors, including CORE and ENZ, is key. They are generally double-stranded DNA with a length of 14-18 nucleotides. The base sequence of the adapter and the enzyme fragment adjacent to the adapter is the binding site of the primer. The synthesized oligonucleotide linker was denatured at 94°C for 2 min, annealed at 37°C for 10 min, and stored at 4°C for later use.

In addition, selective amplification can be achieved by selecting different primers that add 1-3 selective nucleotides at the ends. These selective nucleotides enable the primers to selectively recognize and bind to endonuclease fragments with specific matching sequences to achieve specific amplification. The number of these selective nucleotides is mainly determined by the genome size of the DNA of the sample to be tested. Theoretically, for each additional selective base, only 1/4 of the restriction fragment will be amplified, and with three selective bases on both primers, only 1/4,096 of the fragment will be obtained. Therefore, selective bases are a precise and efficient method to select specific restriction fragments for amplification. The greater the number of selective nucleotides, the stronger the selectivity and the fewer the amplification products. On the contrary, if its number is small, the selectivity will be poor and the amplification products will be more. Therefore, using double enzyme digestion can produce a better amplification reaction and produce fragments of appropriate size suitable for separation on the gel. Different endonuclease combinations and the number and type of selective bases can flexibly adjust the number of fragments, thereby producing different AFLP fingerprints.

Procedures

1. DNA extraction and purification

For DNA extraction and purification methods, please refer to extraction and purification of rice genomic DNA.

2. Restriction enzyme digestion and ligation

Add the reagent ingredients in Table 1 to a 0.5 mL centrifuge tube and mix well. Centrifuge for a few seconds, incubate at 37°C for 5 h, 8°C for 4 h, and 4°C overnight.

Table 1. Reagents required for enzyme digestion and ligation in AFLP

3. Pre-amplification reaction

Add the following ingredients to a 0.5 mL or 0.2 mL centrifuge tube. Template DNA (2 µL), pre-amplification primers (1 µL), dNTPs (0.5 µL), 10× PCR buffer (2.5 µL), TaqDNA polymerase (2 U/µL, 0.5 µL), and water (18.5 µL). After mixing, centrifuge for a few seconds and perform 30 cycles of PCR amplification according to the following parameters: 94°C, 30 s; 56°C, 30 s; 72°C, 80 s.

Dilute the sample 1:20 with TE buffer.

4. Selective PCR amplification

In a 0.5 mL or 0.2 mL centrifuge tube, add the following reagents (25 µL in total):

Pre-amplification diluted sample (2 µL), 10×PCR buffer (2.5 µL), dNTPs (0.5 µL), EcoRI primer (1 µL, 8 types in total, choose one as needed), Mse I primer (1 µL, 8 types in total, choose one as needed), Taq DNA polymerase (1 µL) and water (17 µL). Mix well, centrifuge for a few seconds, and set aside.

Perform PCR amplification according to the following parameter settings. One round of amplification parameters: 94°C, 30 s; 65°C, 30 s; 72°C, 80 s. Decrease the temperature by 0.7°C in each subsequent cycle and amplify for 12 cycles; or decrease the temperature by 1°C in each subsequent cycle and amplify for 10 cycles. Then amplify for 23 cycles according to the following parameters: 94°C, 30 s; 55°C, 30 s; 72°C, 80 s.

5. Gel analysis

Mix the PCR amplification product and formamide sample solution at a ratio of 8:3, denature at 95°C for 5-8 min, and then immediately bathe in ice. Prepare a denaturing gel with a mass fraction of 5%-6%, and a thickness of 0.4 mm to facilitate silver staining. The constant power of pre-electrophoresis is 50-60 W, about 30 min. Load 5-7 µL of sample, run at constant power 40-50 W and electrophorese until the bromophenol blue indicator line approaches the bottom of the gel, then stop electrophoresis. Silver stain the gel and observe the results. The steps for silver staining are as follows: fix with 10% acetic acid for 20-30 min, or overnight; rinse with distilled water 2-3 times; 0.1% silver nitrate, 0.56% formaldehyde, stain for 30 min, rinse lightly with distilled water for a few seconds; 0.28 mol/L sodium carbonate, 0.56% formaldehyde, 200 µL sodium thiosulfate (10 mg/mL), develop color at low temperature for 5-10 min. When the contrast between the band and the background is clearest, stop the color development with 10% acetic acid and rinse with distilled water for 2 minutes.

6. Data processing

For a certain strip, "yes" is recorded as "1" and "no" is recorded as "0". Only record the clearly identifiable amplification bands, and input the DNA amplification band data of all selective amplification primer products into the data matrix. The genetic similarity between samples can be calculated using Jaccard similarity coefficient method to obtain a similarity matrix. The similarity matrix was used to perform SAHN cluster analysis using the unweighted pair group method with arithmetic mean (UPGMA). Use AFLP marker polymorphism data to analyze DNA nucleotide polymorphisms, calculate and draw evolutionary trees, and conduct analysis.

Note

When combined with commonly used PCR instruments for selective amplification of AFLP, the reaction conditions of different PCR instruments are also different.

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