Clubroot-resistant Chinese cabbage gene CRb closely-linked molecular markers, primers and selection method of clubroot-resistant plant

A technology of clubroot resistance and molecular markers, applied in biochemical equipment and methods, microbe measurement/inspection, DNA/RNA fragments, etc., can solve problems such as long genetic distances, shorten the breeding process, and improve breeding efficiency Effect

Inactive Publication Date: 2013-09-04
SHENYANG AGRI UNIV
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

Two of the closest flanking markers, TCR05 and TCR09, are described in (Piao et al., SCAR and CAPS mapping of CRb, agene conferring resistance to Plasmodiophora brassicae in Chinese cabbage (Brassicarapassp.pekinensis), TheorApplGenet, 108:1458-1465), but these markers are genetically distant from CRb

Method used

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  • Clubroot-resistant Chinese cabbage gene CRb closely-linked molecular markers, primers and selection method of clubroot-resistant plant
  • Clubroot-resistant Chinese cabbage gene CRb closely-linked molecular markers, primers and selection method of clubroot-resistant plant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Example 1: Acquisition of Chinese cabbage clubroot resistance gene CRb tightly linked marker

[0075] 1. Construction of Separation Groups

[0076] F 1 , the obtained F 1 All the offspring plants showed resistance to the disease. The above two parents 'CRShinkiDH' and 'QG501' are preserved in Shenyang Agricultural University. Select individual plant F 1 selfing build F 2 There are 2,896 groups in the mapping group. All 2896 F in seeded 2 In the group, it was planted in the greenhouse of Baicao Garden of Shenyang Agricultural University in early August 2012, and inoculated with Plasmodium ten days later. The identification of disease resistance was carried out in September of the same year, and 2896 F 2 In the population, 2203 were resistant and 693 were susceptible, and the chi-square test met the segregation ratio of 2:1 (χ 2 =1.672 0.05 =3.84). Select 31 disease-resistant individuals to self-cross to obtain F 2:3 Pedigrees for disease resistance identifica...

Embodiment 2

[0089] Example 2: Acquisition and identification of Chinese cabbage clubroot resistance gene CRb-linked markers

[0090] 1. Screening of polymorphic primers

[0091] According to the two flanking markers TCR05 and TCR09 sequences of the clubroot resistance CRb gene published by piao et al. in 2004 (Piao et al., SCAR and CAPS mapping of CRb, agene conferring resistance to Plasmodiophora brassicae in Chinese abbage (Brassicara passsp. pekinensis), TheorApplGenet, 108:1458-1465), it was found that the gene is located in Brassica Species A3 chromosome 205kb interval. The sequence was downloaded from the BrassicaDatabase website (http: / / brassicadb.org), and the analysis found that there were 19 genes and 30 SSRs in this segment sequence. Among them, 5 genes and 25 SSRs were randomly selected, and primers were designed using Primer5.0 software, and a total of 35 pairs of primers were designed.

[0092] Using the designed 35 pairs of primers to amplify the DNA of the parental 'CRSh...

Embodiment 3

[0123] Example 3: Application of 5 Molecular Markers Linked to Chinese Cabbage Clubroot Resistance Gene CRb in Assisted Selection of Chinese Cabbage Clubroot Plants

[0124] 1. Genomic DNA extraction

[0125] Carry out the extraction of genomic DNA by the method in embodiment 1

[0126] 2. PCR amplification:

[0127] a. Reaction system: 10μL system, the content of each component is 15ngtemplate; 5pmol L -1 primer; 1.0mmol·L -1 dNTP;1×PCRBuffer;0.5UTaqDNApolymerase;ddH 2 Make up 10 μL of O, mix well, and centrifuge;

[0128] b. Amplification program: pre-denaturation at 94°C / 5min; 94°C / 30s; 60°C / 45s; 72°C / 30s; after 35 cycles; extension at 72°C for 10 minutes; and finally storage at 4°C.

[0129] 3. Electrophoresis:

[0130] TCR25, TCR30, TCR74: Mix the amplified product with an equal volume of loading buffer (98% formamide, 10mM EDTA, 0.001% xylenecyanol and bromphenolblue). Denatured at 94°C for 6 minutes, then electrophoresed on a denaturing polyacrylamide gel (6%) on...

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Abstract

The invention provides 5 clubroot-resistant Chinese cabbage gene CRb closely-linked molecular markers, primers and a selection method of a clubroot CRb-resistant plant. The 5 clubroot-resistant Chinese cabbage gene CRb closely-linked molecular markers comprise TCR25, TCR108, TCR116, TCR30 and TCR74. Distances of the gene CRb respectively to the TCR25, the TCR108, the TCR116, the TCR30 and the TCR74 are 0.07cM, 0.04cM, 0.08cM, 0.19cM and 0.27cM. The 5 clubroot-resistant Chinese cabbage gene CRb closely-linked molecular markers can be used for molecular marker-assistant selection of clubroot-resistant genes of brassica such as Chinese cabbage and Shanghai Bak choi. In assistant selection, the 5 clubroot-resistant Chinese cabbage gene CRb closely-linked molecular markers are used simultaneously so that theoretical selection accuracy is 100%. The 5 clubroot-resistant Chinese cabbage gene CRb closely-linked molecular markers have good repeatability, high reliability and a low detection cost and saves time and labor.

Description

technical field [0001] The invention relates to a molecular marker closely linked with the Chinese cabbage clubroot disease-resistant gene CRb, a primer sequence and a method for screening Chinese cabbage clubroot disease-resistant plants. Background technique [0002] In 2002, our research group obtained a Chinese cabbage double haploid line 'CRShinkiDH' resistant to brassica clubroot, and the research showed that the disease-resistant material was resistant to Physiological races 2, 4 and 8 of Phylophyllum brassicae (Piao et al., Conversion of AFLP marker linked to club root resistance gene into SCAR marker. 2002, JKorSocHortSci43:653–665). Further studies have proved that the clubroot resistance gene is a dominant single gene and named CRb (Piao et al., SCAR and CAPS mapping of CRb, agene conferring resistance to Plasmodiophora brassicae in Chinese cabbage (Brassicarapassp.pekinensis), TheorApplGenet, 2004, 108:1458-1465). [0003] During the transformation of brassica s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/68
CPCC12Q1/6895
Inventor 朴钟云张腾赵卓陈慧慧陈静静张椿雨李宏博李海燕李艳辉
Owner SHENYANG AGRI UNIV
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