A molecular marker mboii‑33 for predicting and identifying the color of watermelon pulp and its application
A pulp and color technology, applied in the field of molecular biology, can solve the problems of small number of molecular markers, low polymorphism, narrow genetic distance of watermelon, etc., and achieve the effect of improving breeding efficiency and shortening the breeding period
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Embodiment 1
[0028] Example 1 Primer Design and Genomic DNA Extraction
[0029] The F 1 , F 2 The generation group was used as experimental material to verify the presence of lycopene in watermelon pulp by CAPS markers. The result is as figure 1 shown.
[0030] Using high-throughput sequencing technology to sequence the two watermelon lines COS and LSW-177, mining the base segment of the SNP mutation site in the sequencing data, analyzing the enzyme digestion information of the SNP site, and obtaining the sequence with the CAPS mutation. CAPS primers were designed on sequences where CAPS sites exist. The primer length is 18-26bp, the annealing temperature is 56-60°C, and the GC content is 40%-60%. Collect LSW-177, COS and the F obtained by crossing them 1 Generation, F 2 Genomic DNA was extracted from the young true leaves of the generation plants using the improved CTAB method.
Embodiment 2
[0031] The acquisition of embodiment 2 PCR product
[0032] The obtained CAPS primers and genomic DNA were used for PCR reaction, and the PCR reaction system was shown in Table 1 (20 μl):
[0033] Table 1. PCR reaction system
[0034]
[0035]Touchdown PCR (touchdown PCR, TD-PCR) was used for amplification, and the program was: 94°C pre-denaturation for 7 minutes, 94°C denaturation for 30 s, 60°C annealing for 30 s, 0.5°C lowering per cycle, 72°C extension for 40 s, 30 cycles; 94 Denaturation at ℃ for 30s, annealing at 45℃ for 30s, extension at 72℃ for 40s, 10 cycles; extension at 72℃ for 10min, storage at 4℃.
[0036] Utilize 1% agarose gel electrophoresis to detect the obtained PCR product, and the detection result is as follows figure 1 As shown, LSW-177, COS, F 1 and F 2 A fragment with a size of 524bp was amplified from each plant, which was consistent with the expected fragment size.
Embodiment 3
[0037] Embodiment 3 PCR product is carried out enzyme digestion verification
[0038] According to the restriction endonuclease operation guide of Thermo, use restriction endonuclease to check the enzyme digestion of the obtained PCR product. The enzyme digestion system is shown in Table 2: (15.3 μl)
[0039] Table 2. Enzyme digestion reaction system
[0040]
[0041] Enzyme digested overnight in a water bath at 37°C, and the digested products were detected by 1% agarose electrophoresis, and the detection results were as follows: figure 1 shown. In the tested 18 strains of F 2 Among the single plants of the present generation, 9 strains showed red phenotype in the field, and lycopene could be detected in the pulp tissue, and the remaining 9 strains were non-red, and lycopene could not be detected in the pulp tissue. The band is 524bp.
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