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Molecular markers of maize chloroplast genome and its application in cultivar identification

A chloroplast genome and molecular marker technology, applied in the field of crop molecular biology

Active Publication Date: 2018-09-04
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report on the development of marker loci based on the maize chloroplast genome and the establishment of its high-throughput detection technology

Method used

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  • Molecular markers of maize chloroplast genome and its application in cultivar identification
  • Molecular markers of maize chloroplast genome and its application in cultivar identification
  • Molecular markers of maize chloroplast genome and its application in cultivar identification

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 Obtaining a set of polymorphic primers suitable for KASP sample high-throughput detection platform of maize chloroplast genome

[0043] 1. 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.

[0044] 2. 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 unearthed). 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 spectrophotom...

Embodiment 2

[0051] Example 2 Using the present invention to obtain 63 pairs of polymorphic primer combinations to construct a corn variety chloroplast DNA fingerprint database

[0052] 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. The 63 pairs of primers in Table 1 of the present invention were used to construct chloroplast DNA fingerprints (based on SNP and InDel markers) based on the KASP platform.

[0053]PCR amplification: PCR reaction system: DNA template 1.5 μL, KASP ROX standard reaction mix (Kbiosciences, Herts UK) 0.5 μL, KASP Primer mix 0.014 μL, ddH 2 O 0.5 μL; the concentration of each prim...

Embodiment 3

[0055] Example 3 Maternal traceability analysis using maize chloroplast genome polymorphic sites

[0056] For maize hybrid A to be identified, DNA was extracted from the green leaves of the seedlings, PCR amplification was performed using 63 pairs of polymorphic primers provided in Table 3 of the present invention, and the fluorescent signal was scanned to obtain fingerprint data. Concrete method is with embodiment 1. Based on the comparison of the DNA fingerprint data of sample A (Jingke 968) with the chloroplast SNP and InDel marker 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 the hybridization was speculated The female parent of species A (Jingke 968) is B (Jing 724).

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Abstract

The invention provides molecular markerd of a maize chloroplast genome and its application in cultivar identification. The molecular markerd comprise 57 SNP markers and 6 InDel markers. The 63 molecular markers and primers developed according to the markers can be used in any one of the following: (1) constructing a corn variety chloroplast DNA-SNP and InDel fingerprint database, (2) carrying outcorn maternal line traceability analysis, and (3) performing reciprocal cross identification on corn samples. Through the application of the chloroplast polymorphic primer composition, the range of available marker sites of the corn is expanded at the genome level and the new ideas and technical means are provided for the study of maize variety and germplasm resource identification, genetic relationship evaluation and cytoplasmic genetic characteristics.

Description

technical field [0001] The invention belongs to the technical field of crop molecular biology, and in particular relates to molecular markers of maize chloroplast genome and its application in variety identification. 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 resourc...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q1/6895C12Q2600/13C12Q2600/156C12Q2531/113
Inventor 王凤格杨扬易红梅田红丽赵久然许理文王蕊刘亚维
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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