InDel marker for identifying red/non-red properties of pulp of peaches, primer pair and application thereof

A primer pair and labeling technology, applied in the biological field, can solve the problems of cumbersome identification steps, red pulp, low accuracy, etc., and achieve low-cost effects.

Active Publication Date: 2020-05-01
ZHENGZHOU FRUIT RES INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, some of the existing linkage markers are far away from the red meat traits, and the accuracy rate in the early identification of the hybrid offspring is low; although some are closely linked to the red meat traits, the accuracy rate of the identification in the hybrid offspring is also relatively low. high, but it is not the main factor that causes the pulp to turn red, and they are all SSR type markers, and the identification steps are cumbersome. Therefore, it is necessary to develop molecular markers with high accuracy and convenient operation

Method used

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  • InDel marker for identifying red/non-red properties of pulp of peaches, primer pair and application thereof
  • InDel marker for identifying red/non-red properties of pulp of peaches, primer pair and application thereof
  • InDel marker for identifying red/non-red properties of pulp of peaches, primer pair and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Embodiment 1, the acquisition of indel marker (InDel)

[0027] In the present invention, two germplasms of peach germplasm resources "Tianjin Shuimi (red meat)" and "Baifeng (white meat)" are used as samples of the peach germplasm resources of Zhengzhou Institute of Fruit Trees, Chinese Academy of Agricultural Sciences. Two germplasms were resequenced to obtain 18Gb data, with an average sequencing depth of about 30×. According to the reads of 10kb-100kb obtained by sequencing, compare with peach reference genome v.2.0 (ftp: / / ftp.bioinfo.wsu.edu / species / Prunus_persica / Prunus_persica-genome.v2.0.a1 / ), identify 12692 large nucleotide indels were found, among which there was a 6647bp insertion sequence in "Tianjin Shuimi" at the promoter position of BL gene (between 707474bp and 707475bp of chromosome 5 of the peach genome). But 'Baifeng' did not. Afterwards, the upstream primers were designed according to the upstream reference sequence of the insertion site, and the do...

Embodiment 2

[0028] Embodiment 2 Utilizes the method for nucleotide insertion (InDel) marker identification peach pulp color

[0029] 1. DNA extraction

[0030] The modified CTAB method is used to extract the DNA of the sample tissue (leaf) to be tested, and the DNA sample volume is not less than 30 μl. Measure the OD value of the DNA sample at 260nm and 280nm with a UV spectrophotometer, and calculate the DNA content and OD 260 / 280 ratio. DNA sample purity OD 260 / 280 The value should be between 1.8-2.0, and the concentration should be diluted to around 10ng / μl.

[0031] 2. Primer design

[0032] Primers were designed according to the upstream reference sequence of the insertion site and the 5' end sequence of the insertion fragment (about 1680 bp in total), as shown in Table 1 below.

[0033] Table 1 Sequence information at both ends of the insertion site

[0034]

[0035]

[0036] Note: The nucleotide sequence in the gray bottom part is the reference sequence upstream of the ...

Embodiment 3

[0048] Example 3, 3 hybrid populations using the invented InDel markers to carry out performance blind test verification

[0049] 1. Selection of test materials

[0050] Using conventional peach varieties planted in the resource garden of Zhengzhou Fruit Tree Research Institute as experimental materials, 3 red-fleshed peach hybrid populations with investigated phenotypic traits were selected from them, with a total of 455 individual plants. See Table 5 for details.

[0051] Table 5 The name and population size information (unit: strain) of the hybrid population for testing

[0052]

[0053] Note: 07-4-28 and 09-1 Xi-28 are excellent red meat lines, Yangzhou 431 is a white meat local variety, Zhaohui is a white meat bred variety, Zhongyou 13 is a yellow meat bred variety.

[0054] 2. Utilize the identification method of InDel mark of the present invention

[0055] Using the InDel marker of the present invention, a total of 455 single-plant pulp colors of 3 red-flesh peach ...

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Abstract

The invention discloses an InDel marker for identifying red/non-red properties of pulp of peaches, a primer pair and an application thereof. The InDel marker is located between 707474bp and 707475bp of a fifth chromosome of a peach genome, a sequence of a corresponding forward primer BLproF is shown in SEQ ID No. 2, and a sequence of a corresponding reverse primer BLproR is shown in SEQ ID No. 3.By using that a forward primer of the primer pair is located on a known reference sequence, a reverse primer is located on an insertion sequence, and an amplification product is 1041bp, 455 single plants to be assayed of 3 hybrid colonies are subjected to molecular marker genotype accuracy verifying. It is proven that all single plants capable of amplifying about 1041bp bands are red-pulp peaches,single plants incapable of amplifying bands are non-red-pulp peaches, and the rate of accuracy of verifying reaches 100%. By identifying peach pulp colors by using the InDel marker disclosed by the invention, the InDel marker has the advantages of rapidness, convenience, low cost and the like and can achieve large-scale application in production.

Description

technical field [0001] The invention relates to an InDel marker for identifying the color (red / non-red) character of peach pulp, a pair of primers and an application thereof, belonging to the field of biotechnology. Background technique [0002] As the main agronomic trait of peach, pulp color plays an important sensory role in people's evaluation of peach fruit quality. According to the content of anthocyanins, peach pulp is divided into white meat, yellow meat, and red meat. The red meat is rich in anthocyanins, which not only has a strong visual impact, but also has the ability to remove free radicals in the body, resist tumors, and prevent heart disease. The effect of arteriosclerosis; in addition, anthocyanins in red meat, as a pigment additive, are more natural and healthy. Therefore, red meat peaches are favored by consumers. However, most of the red-flesh peaches currently used for planting are local varieties, which have defects in appearance, flavor and hardness....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12N15/11
CPCC12Q1/6895
Inventor 王力荣王蛟曹珂陈昌文王新卫方伟超朱更瑞王玲玲李勇吴金龙
Owner ZHENGZHOU FRUIT RES INST CHINESE ACADEMY OF AGRI SCI
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