Genetic diversity is a historical product of organisms formed in the course of long-term evolution and is an important component of biological diversity. The size of a species' genetic diversity determines the species' adaptability to environmental changes and its evolutionary potential. The study of species genetic diversity can reveal the evolutionary history of a species or population, and also provide an important scientific basis for further analysis of its evolutionary potential and future fate, especially for exploring the mechanism of species rarity or endangerment. In recent years, various molecular markers have been rapidly applied to the study of plant genetic diversity, including restriction fragment length polymorphism (RFLP), simple repeat sequence interval polymorphism (ISSR), amplified fragment length polymorphism (AFLP) and simple sequence repeat polymorphism (SSR). Among them, SSR molecular markers have been widely used in plant genetic diversity, phylogeny, germplasm identification, genetic map construction and gene localization due to their abundance, high reliability, good repeatability and co-dominant inheritance.
Operation method
Detection of plant genetic diversity
Materials and Instruments
Material: Move The basic process of plant genetic diversity detection can be divided into the following steps: SSR marker technology mainly includes steps such as DNA extraction, PCR amplification, electrophoretic detection, result statistics, and data analysis.
Young leaves of three groups of plants of Codonopsis pilosula [Codonopsis pilosula (Fr.) Nannf], dried and preserved in discolored silica gel.
Apparatus:
① Tabletop high-speed freezing centrifuge
② 9700 PCR amplifier
③ Centrifuge tubes
④ Pipette guns of various types ⑤ Horizontal electrophoresis apparatus
⑤ Horizontal electrophoresis apparatus
⑥ Vertical electrophoresis instrument
⑦ Gel Imager
⑧ microwave oven
⑨ Autoclave
⑩ Refrigerator
Reagents:
① PCR amplification reagent dNTP
Taq DNA polymerase
③ Primers were purchased from Sangong Bioengineering (Shanghai) Co., Ltd. and other chemical reagents were imported or domestic analytically pure reagents.
The primers were designed according to the sequences published by the National Center for Biotechnology Information (NCBI) and synthesized by Shanghai Sangon Company. Three samples were selected from each population of Codonopsis pilosula, and eight primers with good amplification and polymorphism were chosen for SSR-PCR reaction.
2. PCR systemThe total system was 20 μL, including 2.0 μL of 10 × PCR Buffer, 3 mmol/L of each dNTP, 50 ng of template DNA, 1 unit of Taq DNA polymerase, 1.0 μL each of forward and reverse primers, and 16.4 μL of sterile water. The PCR program was as follows: pre-denaturation at 94 ℃ for 4 min, 35 cycles of denaturation at 94 ℃ for 30 s, and an annealing temperature of 50~56 ℃. （The PCR program consisted of pre-denaturation at 94 ℃ for 4 min, 35 cycles of denaturation at 94 ℃ for 30 s, annealing at 50-56 ℃ for 30 s, extension at 72 ℃ for 40 s, and final extension at 72 ℃ for 4 min. After completion of the reaction, the PCR products were detected by agarose gel electrophoresis with 1% agarose gel. 3.
3. Polyacrylamide gel electrophoresis(1) Electrophoresis procedure
(1) Gel filling: Clean large and small glass plates are assembled into the gel preparation rack, paying attention to the correct installation of the glass plates and keeping the bottom of the glass plates on a flat surface to prevent leakage of liquid during gel filling. Use an ImL pipette to carefully add prepared gel solution between two glass plates to the upper part of the plate, carefully insert a comb to prevent air bubbles, and coagulate for 0.5 h or so.
(2) Electrophoresis: Remove the gel from the gel-making rack, rinse off the excess residue outside under tap water, install it correctly on the vertical electrophoresis apparatus, take an appropriate amount of 1 × TBE (diluted with 10 × TBE) and add it to the positive and negative tanks, pull out the comb carefully, pay attention to not deform the gel holes, blow the gel holes with a 1 mL pipette to remove the impurities inside, add 9 μL of denatured DNA samples into each sampling hole, and leave one hole for each gel to be added with DNA. Add 9 μL of denatured DNA samples to each sample well, add 8 μL of 50 bp Ladder Marker (3 μL of the original solution + 5 μL of 3 × Loading Buffer) into one well of each piece of glue, electrophoresis at 18 mA for 40-60 min, and wait for the Desuphrine Blue to reach the lower edge of the glass plate, and then the Desuphrine Blue will be run out of the glass plate if the target fragments are larger.
2) Silver staining
(1) Fixation: Separate the two glass plates carefully, place the gel in a 14 cm Petri dish, add appropriate amount of 25% ethanol, and fix it on shaking table for about 5 min, at this time, the gel has been completely detached from the glass plate.
(2) Rinsing: Place the gel in appropriate amount of distilled water and rinse for 1 min on a shaker.
(3) Oxidization: Put the gel in 1% HNO3, rinse it in shaker for 4~6 min, and rinse it in distilled water for 2~3 times.
(4) Staining: place the gel in appropriate amount of newly prepared 2% AgNO3 solution, stain for 20~30 min, rinse with distilled water once.
(5) Developing: Add 100 mL of freshly prepared color development solution and shake gently until the bands appear completely.
[Color development solution: 3 g of sodium carbonate ( Na2CO3 ) was added to 100 mL of water, and 200 μL of formaldehyde and 10 μL of sodium thiosulfate (NazSzCh, 16 mg/mL) were added to it].
(6) Fixing: Place the gel with bands in appropriate amount of 10% acetic acid, shake gently for about 1 min, rinse off the residual acetic acid with tap water, save it, take pictures, and analyze the bands.
4. Data processingThe electrophoretic bands were analyzed by UVI Photo MV software, which showed that the 8 pairs of primers had obvious DNA length polymorphisms among the populations, and then the primer bands with obvious polymorphisms were analyzed by GENEPOP version 3.4. Specific calculations were performed to determine the number of alleles (Aa), the expected heterozygosity (He), the observed heterozygosity (Ho), the number of observed alleles (Na), the number of effective alleles (Ne), the polymorphic locus information content (PIC), the Shannon information index (I), and the gene differentiation coefficient ( Fst ), etc. The results were then analyzed using NtsysPC version 2.2.2. UPGMA cluster analysis was then performed using NtsysPC 2.10.
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