SSR molecular marker in linkage with radish clubroot-resisting QTL and applications of SSR molecular marker

A molecular marker and anti-clubroot technology, applied in the field of molecular biology, can solve the problems of low throughput, high development cost, and limited application of SNP markers

Inactive Publication Date: 2019-01-29
INST OF ECONOMIC CROP HUBEI ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traditional SNP marker development methods have low throughput and high development costs, which greatly limit the application of SNP markers in high-density genetic maps

Method used

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  • SSR molecular marker in linkage with radish clubroot-resisting QTL and applications of SSR molecular marker
  • SSR molecular marker in linkage with radish clubroot-resisting QTL and applications of SSR molecular marker
  • SSR molecular marker in linkage with radish clubroot-resisting QTL and applications of SSR molecular marker

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Simplified Genome Sequencing

[0033] Take 130 F 2 Genomic DNA of radish was extracted by CTAB method from the young leaves of individual plants and parents. Genomic DNA was digested with restriction endonuclease EcorI and digested at 37°C for 15 minutes. After digestion and random interruption, 300bp-500bp DNA was recovered after electrophoresis on 1% agarose gel, and A was added after the end was repaired: Add Solexa Adapter P2Adapter (the P2 adapter is a bifurcated Y-shaped adapter, which can prevent the amplification of fragments not connected to the P1 adapter), and purify it, take 5 μL for PCR amplification, purify and recover 350-550bp DNA on 1% agarose gel, and then carry out the library Detection and sequencing on the machine, the sequencing instrument is Illumina Hiseq 4000, F 2The average information collection depth of offspring is 0.5X, and the average information collection volume is not less than 0.25G. The parental resequencing depth is 30X,...

Embodiment 2

[0037] Embodiment 2: Obtaining of SNP genetic markers

[0038] SNP markers ( figure 1 ).

[0039] All the filtered data of each sample obtained after filtering were compared with the BGI assembly reference genome sequence R.sativus.sequence.v130428.chr.fa (394M) using SOAP2 software, and the comparison information was calculated.

[0040] Using the consensus sequence, the polymorphic sites compared with the reference sequence are screened out and filtered to obtain the SNP site information of each sample. SNPs and consensus sequences are obtained from parents and offspring, and all individual SNPs are integrated to obtain high-quality genotype markers for the entire population. When performing SNP integration, if the quality value of the genotype is less than 20, the copy number is greater than 1.5, the depth of the parental genotype is less than 5 or greater than 300, and the depth of the offspring's genotype is less than 3 or greater than 300, record such a genotype For m...

Embodiment 3

[0051] Example 3: Radish F 3 Phenotypic Identification of Clubroot Resistance in Families

[0052] Will F 2:3 The seeds of each numbered phenotype were sown in 50-hole trays. 3 days after the emergence of the seedlings, the root irrigation method was adopted (the concentration of the clubroot spores was 1 × 10 7 ·mL －1 ), the resistance identification of 25 plants was carried out with No. 4 physiological race respectively. The soil should be kept moist and the temperature should not be lower than 10°C. After 6 weeks of inoculation, wash the root soil, and investigate the incidence of individual plants according to the 0-3 standard. The grading criteria are: grade 0, no tumor in the root; grade 1, small tumor in the lateral root; grade 2, large tumor in the lateral root or nodular tumor in the main root; grade 3, obvious tumor in the main lateral root. Level 0 is disease-resistant, and others are susceptible. The identification of disease resistance was carried out in Hu...

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Abstract

The invention discloses an SSR molecular marker in linkage with radish clubroot-resisting QTL. The radish clubroot-resisting QTL has 5 sites, respectively RsCr1, RsCr2, RsCr3, RsCr4 and RsCr5. The sequences of a forward primer and a reverse primer of the molecular marker are SEQ ID NO.1 and 2. The invention further discloses an acquiring method of the SSR molecular marker. The method comprises thefollowing steps: simplifying genomic sequencing, constructing a radish high-density SNP genetic map, carrying out phenotype identification on the clubroot resistance of the radish F3 family, carryingout radish clubroot-resisting QTL analysis, and developing and screening the SSR molecular marker. The SSR molecular marker provided by the invention can be directly utilized for identifying the clubroot resistance of radish and molecular marker assistant breeding, and has great significance in prevention and treatment research of radish clubroot.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to an SSR molecular marker linked with a radish clubroot resistance QTL and application thereof. Background technique [0002] In recent years, clubroot disease (Clubroot disease) has been found in Northeast China, Southwest China, and the middle and upper reaches of the Yangtze River, and the incidence area has a tendency to further expand. Most of the cruciferous crop producing areas in my country are harmed by clubroot. Clubroot has become one of the main diseases of Brassica crops in my country. Clubroot damage causes Brassica crops to lose as much as 10%-15% of their annual yield around the world (Dixon, 2009). The incidence of radish clubroot in my country is becoming more and more serious. The prevention and treatment of radish clubroot is urgent. [0003] Summary The control of clubroot at home and abroad generally includes crop rotation, cultivation of disease-resistant...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12N15/11
CPCC12Q1/6895C12Q2600/13C12Q2600/156
Inventor 甘彩霞邓晓辉崔磊袁伟玲於校青
Owner INST OF ECONOMIC CROP HUBEI ACADEMY OF AGRI SCI
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