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

Active Publication Date: 2017-04-26
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Marker loci and molecular identification applications f...

Method used

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  • Set of chloroplast SNP and INDEL molecular marker combination for maternal traceability of maize
  • Set of chloroplast SNP and INDEL molecular marker combination for maternal traceability of maize
  • Set of chloroplast SNP and INDEL molecular marker combination for maternal traceability of maize

Examples

Experimental program
Comparison scheme
Effect test

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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Abstract

The invention relates to a set of chloroplast SNP and INDEL molecular marker combination for maternal traceability of maize. On the basis of high quality resequencing data, the invention develops a set of maize chloroplast genome polymorphic locus combination, the polymorphic loci are CPM001-CPM100, which include 96 SNP loci and 4 IDNEL loci. The 100 chloroplast loci can be applied to construction of a maize chloroplast DNA fingerprint database, parental traceability, and reciprocal cross identification. Through application of the chloroplast polymorphic loci, the range of available marker loci for maize on the genomic level is expanded; and a new idea and method are provided for maize varieties, germplasm resource identification, genetic relationship evaluation, cytoplasmic genetic characteristics and other studies.

Description

technical field [0001] The invention belongs to the technical field of crop molecular biology, and in particular relates to a set of chloroplast SNP and INDEL molecular marker combinations for maize maternity tracing. Background technique [0002] Chloroplast is the organelle of photosynthesis in green plants, and has its own complete set of genome, called chloroplast genome. The structure of the chloroplast genome is very conservative, and the DNA is generally a double-stranded circular molecule. The size of the chloroplast genome in higher plants is generally 120-160kb. The double-stranded circular DNA consists of four basic parts, namely, the large single copy region (Largesingle copy region, LSC), the small single copy region (small single copy region, SSC), and two inverted repeat regions (Inverted Repeats, IRs) . [0003] Due to the following advantages, chloroplast genome information is widely used in the research and application of plant varieties, germplasm resour...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6895C12Q2600/156
Inventor 王凤格赵久然田红丽董文攀许理文
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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