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KASP marker primer for authenticating types of cytoplasts Cam and Pol of brassica crops and application of KASP marker primer

A technology for labeling primers and Brassica, which is applied in biochemical equipment and methods, microbial determination/inspection, recombinant DNA technology, etc., can solve the problems of high economic cost and time cost, and achieve accurate and reliable detection results and high throughput. , the effect of simple operation

Active Publication Date: 2020-05-15
INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Using SNP sites for genotyping only needs to detect part of the SNP sites in the sample sequence, while Sanger sequencing or gene chip technology has relatively high economic and time costs

Method used

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  • KASP marker primer for authenticating types of cytoplasts Cam and Pol of brassica crops and application of KASP marker primer
  • KASP marker primer for authenticating types of cytoplasts Cam and Pol of brassica crops and application of KASP marker primer
  • KASP marker primer for authenticating types of cytoplasts Cam and Pol of brassica crops and application of KASP marker primer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Development of KASP-tagged primers for identification of Cam and Pol cytoplasmic types in Brassica crops:

[0074] (1) Sample selection: 54 representative Brassica experimental materials were selected (see Table 1 for specific information), including Nap, Cam and Pol cytoplasmic types;

[0075] (2) Sample preparation: select 10 plants for each sample and take its fresh and young plant leaf tissue, avoid selecting diseases, insect pests and aging leaves, wash 3 times with clean water, and dry it with filter paper; fully grind it with liquid nitrogen and quickly put it into In the homogenizer, add pretreatment buffer (0.5M Sucrose+1mM EDTA+70mM KH2PO4+0.6%PVP(v / v)+0.8%BSA(v / v)+0.1%β-mercaptoethanol(v / v); PH=7.5), after mixing, homogenize in a low temperature environment; gradient centrifugation in a low temperature environment, extract cytoplasm, and then use CTAB method to extract DNA and remove RNA;

[0076] (3) High-throughput sequencing of cytoplasmic DNA: The cytopl...

Embodiment 2

[0141] Utilize the KASP primer that the present invention develops to carry out KASP experiment verification to the material of known Cam and Pol cytoplasmic type:

[0142] (1) Cam cytoplasmic material: Shengli rape, Zhongshuang 2, Shan 2B; Pol cytoplasmic material: Jianyang rape, Xiang 5A, 20A;

[0143] (2) PCR amplification program: 94°C for 15 minutes; use falling PCR method, 94°C for 20s, 60°C for 1min, (60°C drops to 48°C, each cycle decreases by 1.2°C) cycle 10 times; 94°C for 20s, 55°C 1min, cycle 30 times; 1min at 37°C, collect fluorescence signal in the last 1s, the results are as follows: figure 1 ;

[0144] (3) figure 1 The aggregation points close to the Y axis are the materials of Cam cytoplasm, in which figure 1 The aggregation point near the Y axis in a indicates that the detected polymorphic site is C, and the aggregation point near the X axis indicates that the detected polymorphic site is A; the aggregation point near the Y axis in b indicates that the det...

Embodiment 3

[0148] Identification of cytoplasmic types of 482 Brassica napus inbred lines:

[0149] (1) DNA sample extraction of Brassica napus to be identified: using the CTAB method to extract the total plant DNA by using the CTAB method to extract DNA from each sample of 482 Brassica napus;

[0150] (2) Select 4 pairs of primers Cc3, Cm1, Pc1 and Pm2 from Example 1, and carry out KASP test on 482 copies of Brassica napus DNA: PCR reaction system is 5 μL, including 1 μL DNA template, 2.5 μL 2×Master Mix, 0.7 μL PrimerMix, 0.8μL ddH2O; PCR reaction program: 94°C for 15min; use falling PCR method, 94°C for 20s, 60°C for 1min, 60°C to 48°C, each cycle lowering 1.2°C, cycle 10 times; 94°C for 20s , 55°C for 1min, cycle 30 times; 37°C for 1min, collect fluorescence signal in the last 1s;

[0151] (3) Judgment method of cytoplasmic type: judge based on the alleles of each site listed in Table 2 in Brassica napus Cam and Pol cytoplasmic types, if it is the same as the alleles described in the...

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Abstract

The invention discloses a KASP marker primer for authenticating the types of cytoplasts Cam and Pol of brassica crops and application of the KASP marker primer. A competitive allelic specific PCR (KASP) primer is developed aiming at representative single nucleotide polymorphism (SNP) sites among the types of cytoplasts distributed in all genome ranges of chloroplasts and mitochondria or insertion / deletion (short InDel) polymorphism of small fragments based on high-throughput sequencing data of large-scale group samples. The typing of cytoplast genomes of genetic resources or breeding materialsCam and Pol of the brassica crops can be rapidly realized based on the set of KASP markers, a detection result is accurate and reliable, the operation is simple, the throughput is high, and the costis low; and compared with a traditional molecular marker, the range of usable marker sites is expanded at a genome level, and new knowledge information, methods and tools are provided for groundwork,genetic innovation, breeding of breeding materials and the like of the genetic resources of the brassica.

Description

technical field [0001] The invention belongs to the technical field of molecular breeding of Brassica crops, and in particular relates to KASP marker primers designed based on multi-type Brassica napus cytoplasmic genomes for detecting Cam and Pol cytoplasmic types of Brassica crops and applications thereof. Background technique [0002] Brassica cultivars are involved in many aspects such as vegetables, oil crops, fertilizer crops, ornamental plants, etc., mainly including three diploids and three allotetraploids evolved from them as parents. Diploids include cabbage (Brassica.rapa, 2n=20, AA), black mustard (B.nigra, 2n=16, BB) and cabbage (B.oleracea, 2n=18, CC), allotetraploids include Brassica napus (B. napus, 2n=38, AACC), Brassica napus (B. juncea, 2n=36, AABB) and Ethiopian mustard (B. carinata, 2n=34, BBCC). Among them, three allotetraploids are formed by two-two hybridization of three diploids and natural doubling. This relationship is called "Yu's triangle" (UN, ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12N15/11
CPCC12Q1/6895C12Q2600/13C12Q2600/156
Inventor 张效君乔江伟伍晓明陈碧云
Owner INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
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