Chromosome inversion co-separated from peach fruit shape traits and application thereof
A technology of co-segregation and chromosome, which is applied in the field of plant molecular biology, can solve the problems of unclear genetic basis of peach flat fruit shape, and achieve the effects of improving breeding efficiency, saving breeding time, and rapid detection
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Embodiment 1
[0051] Example 1, Illumina next-generation resequencing library construction and data processing
[0052] Library preparation: First, high-quality genomic DNA was extracted from leaves using the CTAB method (Porebski et al. 1997). After passing the test, it was implemented according to the Illumina standard library construction protocol, including sample quality inspection, library construction, library quality inspection, and library sequencing. After the genomic DNA of the sample is qualified, the DNA is fragmented by mechanical fragmentation (ultrasonic wave), and then the fragmented DNA is purified, repaired, added A at the 3′ end, connected to the sequencing adapter, and then analyzed by agarose gel. Electrophoresis was used to select the size of the fragment, and PCR amplification was performed to form a sequencing library. The built library was first subjected to library quality inspection, and the library that passed the quality inspection was sequenced by Illumina. Th...
Embodiment 2
[0054] Example 2, Pacbio three-generation resequencing library construction and data processing
[0055] Library construction: Qualified DNA samples are randomly broken into fragments by the Covaris ultrasonic breaker; large fragments of DNA are enriched and purified by magnetic beads; fragmented DNA is repaired, damage repaired, and end repaired; stem-loops are connected at both ends of the DNA fragments The adapters were sequenced and exonucleases were used to remove fragments that failed to ligate. The constructed library was sequenced by Pacbio Sequel.
[0056] Data processing: Using MaSuRCA software, Illumina next-generation sequencing data was used to correct the PacBio third-generation data. Valid high-quality sequencing data were aligned to the reference genome by ngmlr software (parameter: -t 4). Use SAMTOOLs (mpileup-m 2-F 0.002-d 1000) to detect small fragment insertions and deletions (InDels) with a length less than 50 bp, and then use ANNOVAR software to annotat...
Embodiment 3
[0057] Example 3, Development of Molecular Markers for Chromosomal Inversions at S Site
[0058]According to the information of chromosomal inversion breakpoints at the S site, we designed three pairs of primers, P1F / P1R, P2F / P2R and P3F / P3R, for screening and identifying flat peach traits, which can amplify the regions of RefPB, PanPB and PanDB, respectively. The primer sequences are shown in SEQ ID NO.5, 6...10.
[0059] The PCR enzyme was selected from Qingke Biological Gold Medal Mix (green), the annealing temperature in the cycle was 58°C, and the extension time was 30s, a total of 35 cycles. After PCR is finished, use 1% agarose gel electrophoresis.
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