Molecular marker MboII-37 for indicating and identifying watermelon pulp color and application
A technology of mboii-37 and molecular markers, applied in the field of molecular biology, can solve the problems of low polymorphism, small number of molecular markers, narrow genetic distance of watermelon, etc., and achieve the effect of shortening the breeding period and improving the efficiency of breeding
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Embodiment 1
[0036]Example 1 Primer Design and Genomic DNA Extraction
[0037] 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.
[0038] Two watermelon lines, COS and LSW-177, were sequenced using high-throughput sequencing technology, and the cds region sequence of the watermelon lycopene β-cyclase gene was obtained through the NCBI website, and compared with the sequenced material, the obtained The chromosomal interval where the cds region sequence of the watermelon lycopene β-cyclase gene exists, within this interval, the base segment with the SNP mutation site in the sequencing data is mined, and the enzyme digestion information of the SNP site is analyzed to obtain the existence For the sequence of CAPS mutation, CAPS primers were designed on the sequence with CAPS site. The primer length is 18-26bp, the annealing temperature is 56-60°C, and the GC content i...
Embodiment 2
[0039] The acquisition of embodiment 2PCR product
[0040] The obtained CAPS primers and genomic DNA were used for PCR reaction, and the PCR reaction system was shown in Table 1 (20 μl):
[0041] Table 1. PCR reaction system
[0042]
[0043] 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℃.
[0044] 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 787bp was amplified from each plant, which was consistent with the expected fragment size.
Embodiment 3
[0045] Embodiment 3 PCR product is carried out enzyme digestion verification
[0046] 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)
[0047] Table 2. Enzyme digestion reaction system
[0048]
[0049] 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. LSW-177 has enzyme cleavage sites at 488bp, 491bp, 644bp and 871bp, so the fragment sizes obtained after digestion are 339bp, 227bp, 153bp, 64bp and 3bp, and COS only has enzymes at 644bp and 871bp The cleavage site, the size of the fragment obtained after digestion is 495bp, 227bp and 64bp. f 1 Representatives exhibiting co-dominant features include both LSW-177 restriction fragments and COS restriction restriction fragments. ...
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