Steps for SSR labeling experiments

Summary

SSR simple sequence repeat markers can be used to: design pairs of primers with a specific sequence of microsatellite regions, and polymorphism of SSR loci among different individuals can be shown by PCR, by polyacrylamide gel electrophoresis.

Operation method

Steps of SSR labeling experiment

Principle

Compared with other molecular markers, SSR markers have the following advantages: (1) they are abundant, covering the whole genome and revealing high polymorphism; (2) they are multi-allelic and provide high information; (3) they are inherited in Mendelian manner and are co-dominant; and (4) each locus is determined by the sequence of primers designed, which makes it easy for different laboratories to communicate and cooperate with each other to develop primers. Therefore, this technique has been widely used in the construction of genetic maps [11, 12, 18, 19, 33], the calibration of target genes [8, 9, 21, 22, 26], fingerprinting [22] and other studies. However, it should be noted that the establishment of SSR markers first requires basic research on microsatellite flanking sequences such as cloning, sequencing, artificial design of synthetic primers, as well as localization and mapping of the markers, and thus their development costs are quite high, and various laboratories must collaborate to develop more markers. Due to the greater application value and species specificity of SSR markers, SSR marker research in some of the major crops is now collaborating on the development of STMS primers.

Materials and Instruments

Plant leaf samples
Liquid Nitrogen CTAB Chloroform Isoamyl Alcohol Isopropyl Alcohol TE Anhydrous Ethanol
Centrifuge tubes Refrigerator Water bath Centrifuge

Move

Taking leaves → grinding samples → extracting DNA → PCR amplification → electrophoretic detection → staining → reading bands for labeling.

1. DNA extraction

The procedure was carried out according to Doyle and Dickson's (1987) CTAB method (`Cetyl triethyl ammonium bromide) with slight improvement as follows:

① Take leaves at maturity with liquid nitrogen and grind them into powder, transfer to 1.5 ml centrifuge tube and store in the refrigerator at -20℃;

② Add 600 μl of 2 × CTAB at 65 ℃ to mix and place in a 65 ℃ water bath for 30-60 minutes;

③ Remove, cool, shake up and down and add 600 μl of 24:1 chloroform isoamyl alcohol;

④ Centrifuge at 12000 rpm for 10 min and take the supernatant in another 1.5 ml centrifuge tube;

⑤ Add isopropanol and centrifuge at 12000 rpm for 10 minutes, pour off the supernatant;

⑤ Add isopropanol, centrifuge at 12000 rpm for 10 minutes, pour off the supernatant; ⑥ Wash with 100% spermicide after drying, and add appropriate amount of TE after drying. 2. PCR amplification

The concentration of template DNA was about 25ng/μl, and the amplification reaction system was 20μl, as follows:

Sterile ddH2O 11μl

PCR Buffer 3μl

dNTP-mix 0.5μl

Primer1 1μl

Primer2 1μl

Taq polymerase 0.5ul (2U/μl)

DNA 3μl

PCR amplification program is: (2h30min)

① Pre-denaturation: 94℃, 5 minutes;

② Denaturation: 94℃, 40 sec;

① Pre-denaturation: 94℃, 5 minutes; ② Denaturation: 94℃, 40 seconds; ③ Annealing: 55℃, 40 seconds

③ Annealing: 55℃ for 40 seconds; ④ Extension: 72℃ for 1 minute;

⑤ Cycle: 38 cycles from 2 to 4;

⑥ Final extension at 72 ℃ for 5 min; 4 ℃ for 5 min, the amplification product was stored in the refrigerator at 4 ℃. 3. Electrophoretic separation of amplification products

Gluing→filling→sampling→electrophoresis.

The amplification products were separated by electrophoresis with 8% polyacrylamide denaturing gel, and the steps were as follows:

① Prepare electrode solution (1×TBE);

② Set out the comb and quickly rinse the gel surface with electrode solution;

③ No pre-electrophoresis;

④ Spot sample and electrophoresis 220V;

⑤ Remove the plate and wash the inner plate, side strips and comb in time.

4、Gel staining

① Fixing: 10% acetic acid, 5 minutes;

② Staining: 10 minutes;

③ Rinsing: dH2O, 5-10 seconds;

④ Color development: formaldehyde-NaOH until satisfactory;

⑤ Rinsing: dH2O, 2 minutes.

5、Self-sealing tape sealing, reading tape marking, digital photography and video, air-drying at room temperature.

Common Problems

1, Advantages:

(1) The markers are abundant, have a high number of allelic variants, and are widely distributed on all chromosomes;

(2) It is a co-dominant marker, showing Mendelian inheritance;

(3) Good technical reproducibility, easy to operate, and reliable results.

2, Disadvantages:

The development of such markers requires prior knowledge of the sequence information at both ends of the marker, and the cost of primer synthesis is high.

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