Marker site, primer pair and detection kit used for identifying disease resistance/disease susceptibility character of peach trees to meloidogyne incognita and application

A technique for marking sites of root-knot nematode incognita, applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve problems such as unclear key genes and achieve low-cost effects

Active Publication Date: 2018-11-23
ZHENGZHOU FRUIT RES INST CHINESE ACADEMY OF AGRI SCI
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  • Abstract
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AI Technical Summary

Problems solved by technology

The inventor verified the SSR markers with high repeatability among the above markers, and found that the accuracy rate of identification in the hybrid offspring was only about 90%, and the key genes were not clear, so it is necessary to further develop corresponding more accurate markers

Method used

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  • Marker site, primer pair and detection kit used for identifying disease resistance/disease susceptibility character of peach trees to meloidogyne incognita and application
  • Marker site, primer pair and detection kit used for identifying disease resistance/disease susceptibility character of peach trees to meloidogyne incognita and application
  • Marker site, primer pair and detection kit used for identifying disease resistance/disease susceptibility character of peach trees to meloidogyne incognita and application

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Experimental program
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Effect test

Embodiment 1I

[0026] The acquisition of embodiment 1 Indel site

[0027] The present invention uses the hybrid progeny F of 'Honggen Gansu Tao No. 1' and 'Beilei' constructed from the peach germplasm resource nursery of Zhengzhou Institute of Pomology, Chinese Academy of Agricultural Sciences. 1 F 2 The germplasm of 250 selfing populations was used as samples, which were inoculated with Meloidogyne incognita and the disease resistance index was investigated. Then 20 disease-resistant and 20 high-susceptibility germplasms were selected to construct mixed gene pools, and the sample DNA was extracted by conventional CTAB method, and the parental 'Honggengansutao 1' and 'Beilei' were sequenced by Illumina HiSeq 2000 sequencer. , resistant mixed pool, susceptible mixed pool and F 1 A total of 5 samples of DNA were sequenced, and 47.58Gb of data was obtained, covering an average of 95.50%-96.49% of the peach genome, and the average sequencing depth was between 38.04× and 49.18×. According to t...

Embodiment 2

[0028] Example 2 The method for identifying peach tree resistance to root-knot nematode incognita using the PCR marker developed by the polymorphic Indel site

[0029] 1. DNA extraction

[0030] The DNA of the peach sample tissue to be tested was extracted by the conventional CTAB method, and the RNA was removed. The total volume of the DNA sample was not less than 15 μl. Measure the OD value of the DNA sample at 260nm and 280nm with a UV photometer, and calculate the DNA content and OD 260 / 280 ratio. DNA sample purity OD 260 / 280 The value should be between 1.8 and 2.0, and the concentration should be diluted to 50ng / μl.

[0031] 2. Design primers

[0032] The primers were designed according to the 200 bp sequence upstream and downstream of the 35 bp deletion sequence site at the 4,467,346-4,467,380 position of chromosome 2 of the peach genome (see Table 1 for the specific nucleotide sequence).

[0033] Table 1 35bp Indel flanking sequence information

[0034]

[0035...

Embodiment 3

[0047] Example 3 Verification of the phenotypic traits of peach germplasm using the PCR markers developed at the Indel locus of peach tree resistance to root-knot nematode incognita

[0048] 1. Selection of experimental materials

[0049] The hybrid progeny F of 'Honggen Gansu Tao No. 1 F 2 250 self-bred population germplasms were used as experimental materials.

[0050] 2. An identification method for phenotypic prediction using markers developed at Indel loci associated with peach tree resistance to root-knot nematode incognita

[0051] Using a pair of PCR primers developed from the 200 bp upstream and downstream sequences of the 2nd chromosome of the peach genome of the present invention, respectively, to identify the disease resistance / susceptibility traits of 250 self-bred population test materials, the specific identification method refers to The method of embodiment 2.

[0052] 3. Prediction of phenotype by typing results in selfing populations

[0053] Table 4 and...

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Abstract

The invention discloses a polymorphism marker site, primer pair and detection kit used for identifying a disease resistance / disease susceptibility character of peach trees to meloidogyne incognita andapplication. The polymorphism marker site is a site with a sequence, on the positions 4, 467, 346-4, 467 and 380 bp with 35 bp deletion, of the second chromosome of a peach genome. The PCR primers designed according to upstream and downstream 200 bp sequences of the site can be used for identifying or assisting in identifying the disease resistance / disease susceptibility character of the peach trees to the meloidogyne incognita and has high accuracy when used for identifying the disease resistance / disease susceptibility character of the peach trees to the meloidogyne incognita. Verification is conducted on the PCR marking accuracy of 250 to-be-detected peach single plants of an F2 group cross-bred by prunus kansuensis and prunus persica, and a result shows that when phenotypic character prediction of a hybrid group is conducted, the accuracy can reach 100.00%; therefore, the PCR primers developed by means of the marker site have the advantages of being simple, fast and low in cost when used for detection, and large-scale application in production can be achieved.

Description

technical field [0001] The invention relates to a marker site, a primer pair, a kit and an application for identifying the disease resistance / susceptibility traits of peach trees to root-knot nematode incognita, and belongs to the field of biotechnology. Background technique [0002] Progeny screening is one of the most important links in breeding. It refers to the selection of genotypes that meet the expected breeding goals in a cross-breeding population for subsequent re-selection and regional testing, thereby completing the breeding process of new varieties. In traditional conventional breeding, since it is difficult to know the genotype of the offspring, the basis for selection is usually based on the phenotype of a single plant. This selection method is generally effective for qualitative traits, but for quantitative traits, because the phenotype is obviously affected by the environment; and quantitative traits are controlled by multiple loci, and there is no clear corr...

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 曹珂王力荣王新卫张倩刘扩展朱更瑞方伟超陈昌文
Owner ZHENGZHOU FRUIT RES INST CHINESE ACADEMY OF AGRI SCI
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