Set of chloroplast SNP and INDEL molecular marker combination for maternal traceability of maize
A molecular marker, chloroplast genome technology, applied in the field of crop molecular biology
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Embodiment 1
[0033] Example 1 Obtaining the combination of polymorphic sites in maize chloroplast genome
[0034] 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.
[0035] Sample preparation: The total DNA of 170 corn samples was extracted by CTAB method, and the RNA was removed. The quality of the extracted DNA was detected by ultraviolet spectrophotometer and agarose electrophoresis, and it was found that the extracted DNA met the relevant requirements for sequencing. Agarose electrophoresis showed a single DNA band without degradation; UV spectrophotometer detected that A260 / 280 was between 1.8-2.0 (DNA had no protein contamination and RNA content was low); A260 / 230 was between 1.8-2.0 (DNA Low salt...
Embodiment 2
[0041] Example 2 Construction of Chloroplast DNA Fingerprint Database of Maize Varieties Using Polymorphic Site Combinations
[0042] DNA extraction of corn varieties: The total DNA of all corn varieties used to construct the chloroplast DNA fingerprint database was extracted using the conventional CTAB method, and the DNA was diluted to form a working solution with a concentration of 20 ng / μL.
[0043] Design and synthesis of primers for 100 polymorphic sites: The polymorphic sites provided in this example are SNP and INDEL markers of biallelic genes, so primers can be designed for 100 sites based on the KASP detection platform technology, and the primers are common primers Does not contain fluorescence.
[0044] PCR amplification system: the total volume is 1 μL, add 1.5 μL to the PCR plate, dry, 0.5 μL deionized water, 0.5 μL MasterMix (UK LGC company).
[0045] The PCR amplification program was: 94°C for 15 min; 94°C for 20 s, 62°C for 1 min, 10 cycles (minus 0.8°C for ea...
Embodiment 3
[0047] Example 3 Maternal traceability analysis using maize chloroplast genome polymorphic sites
[0048] DNA is extracted from the maize hybrid A to be identified, PCR amplification is performed by using the chloroplast marker provided by the present invention, and the fluorescent signal is scanned to obtain fingerprint data. Concrete method is with embodiment 2. Based on the comparison of the DNA fingerprint data of sample A (Jingke 968) with the chloroplast genome fingerprint database of known maize inbred varieties, it was determined that the chloroplast fingerprint information of the sample to be tested was the same as that of the B inbred line (Jing 724), and it was speculated that the hybrid A ( The female parent of Jingke 968) is B (Jing 724).
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