A set of maize chloroplast indel molecular markers suitable for capillary electrophoresis detection platform
A molecular marker and molecular marker-assisted technology, applied in the field of crop molecular biology, can solve problems such as undocumented chloroplast genome polymorphisms
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Embodiment 1
[0037] Example 1 Obtaining of InDel polymorphic sites and primers in maize chloroplast genome
[0038] Sample selection: 170 widely representative maize inbred lines were selected for whole genome sequencing. These 170 samples include common corn, waxy corn, sweet corn, popcorn and other corn types; including all heterosis groups in my country, Tang Sipingtou, Lvda Honggu, Reid, Lanka, improved Reid, improved Lanka, Group P and landraces.
[0039] Sample preparation: 170 corn samples were germinated in the incubator for 5 days, the first 3 days were under no light conditions, and the next 2 days were under light conditions (that is, sufficient light was given after emergence). For each sample, leaves were selected from 30 green seedlings, mixed, and fully ground under liquid nitrogen conditions. Total DNA was extracted by CTAB method, and RNA was removed. The quality of the extracted DNA was detected by UV spectrophotometer and agarose electrophoresis respectively. Agarose ...
Embodiment 2
[0048] Example 2 Using the InDel polymorphic primers provided by the present invention to construct a maize variety chloroplast DNA-InDel fingerprint database
[0049] DNA extraction of corn varieties: Each sample was seeded with seeds, given light, and formed green shoots. The method of DNA extraction was mixed plants, and the green leaves of 30 individual plants were mixed. The specific steps of DNA extraction were carried out in accordance with the molecular identification standard of maize DNA (Wang Fengge et al., 2014). Dilute the DNA to form a working solution with a concentration of 20ng / μL.
[0050] Primer synthesis: According to the verified 6 pairs of primer sequences provided in Table 2, that is, the 6 pairs of primer nucleotide sequences determined in Example 1 are SEQ ID NO.13-14, SEQ ID NO.15-16, SEQ ID NO .17-18, SEQ ID NO.19-20, SEQ ID NO.21-22, SEQ ID NO.23-24 synthetic primers; one of each pair of primers is labeled with a fluorescent group at the 5' end.
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Embodiment 3
[0053] Example 3 Using the Maize Chloroplast Genome InDel Polymorphic Primer Provided by the Invention to Carry Out Maternal Tracing Analysis
[0054]For the maize hybrid A to be identified, DNA was extracted from the green leaves of the seeds, and the 6 pairs of InDel polymorphic primers identified in Example 1 of the present invention were used for PCR amplification and fluorescent capillary electrophoresis to obtain fingerprint data. Concrete method is with embodiment 2. Based on the comparison of InDel fingerprint data of sample A (Jingke 968) with the chloroplast InDel marker fingerprint database of known maize inbred varieties, it was confirmed that the chloroplast fingerprint information of the sample to be tested was the same as that of inbred line B (Jing 724), and it was speculated that hybrid A The female parent of (Jingke 968) is B (Jing 724).
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