International Journal of Molecular Evolution and Biodiversity 2024, Vol.14, No.2, 62-70 http://ecoevopublisher.com/index.php/ijmeb 69 3.3 SSR primer selection and PCR reaction In this experiment, the RM series SSR primers from the rice genome, synthesized by Shanghai Shenggong Company, were used, totaling 37 pairs (Table 3). Using the total DNA of common wild rice as the template, PCR amplification was performed with SSR primers distributed across 12 chromosomes. The total volume of the PCR reaction was 15 μL, consisting of 9.98 μL of sterile ultrapure water, 1.5 μL of 10× PCR Buffer (500 mmol/L KCl; 100 mmol/L Tris-HCl, pH 9.0; 1% Triton X-100), 1.0 μL of 20 mmol/L MgCl2, 0.4 μL of dNTPs (dATP, dCTP, dTTP, dGTP, each at 25 mmol/L), 0.5 μL of each forward and reverse primer at 4.5 μmol/L, 0.12 μL of 5U Taqase (provided by Promega), and 1.5 μL of 20 ng template DNA. The PCR reaction program was as follows: pre-denaturation at 94°C for 5 minutes; denaturation at 94°C for 45 seconds, annealing at 55°C for 45 seconds, and extension at 72°C for 1 minute, for 35 cycles; followed by a final extension at 72°C for 10 minutes. The PCR amplification of total DNA from common wild rice was performed using a PTC-100 PCR amplifier from MJ Research Inc. After completing the PCR amplification, the PCR products were subjected to non-denaturing polyacrylamide gel electrophoresis. Table 3 The 37 pairs of rice SSR primers used in the experiment Chromosome No. of rice SSR primer 1 RM5、RM140、RM237 2 RM18、RM48、RM166 3 RM114、RM143、RM293 4 RM127、RM131、RM185、RM280 5 RM26、RM163、RM169 6 RM3、RM111、RM162 7 RM2、RM11、RM295 8 RM42、RM80、RM126 9 RM160、RM242、RM321 10 RM171、RM222、RM239 11 RM202、RM206、RM229 12 RM17、RM19、RM20 3.3 Data recording In this study, the SSR primers used are codominant markers, with each pair of primers corresponding to a single locus. After PCR amplification using specific SSR primers, the resulting products are represented by bands on a non-denaturing polyacrylamide gel electrophoresis, which indicate different alleles. Each band position is marked by an Arabic numeral representing one allele, while another numeral represents another allele. DNA bands with the same migration rate, amplified using the same SSR primer set across all samples, are considered the same allele and are marked with the same Arabic numeral. This method allows for precise differentiation and recording of the genetic diversity at each locus in different individuals. 3.4 Statistical analysis When analyzing the genetic data for each SSR locus, allele frequency and polymorphism rate were calculated for each locus. Additionally, the heterozygosity at each SSR locus in the population was analyzed in detail, including observed heterozygosity, expected heterozygosity, and unbiased heterozygosity estimates. Unbiased heterozygosity is adjusted based on sample size and provides a more accurate reflection of actual genetic diversity. Moreover, using Wright’s FST method (Kitada et al., 2020), the degree of genetic differentiation between the two populations at different SSR loci was calculated using the appropriate formula T S T ST H H H F . Acknowledgments This research was conducted at the Hainan Provincial Key Laboratory of Molecular Breeding of Crops. We extend our gratitude to the two anonymous peer reviewers for their constructive comments on this manuscript.
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