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MNP core primer combination for molecular identification of kiwi fruit varieties and application of MNP core primer combination

A technology of molecular identification and primer combination of varieties, applied in the field of molecular biology, to ensure production safety and speed up the breeding cycle

Active Publication Date: 2022-05-10
WUHAN BOTANICAL GARDEN CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no standard for the identification of varieties by MNP marker fingerprints in kiwifruit

Method used

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  • MNP core primer combination for molecular identification of kiwi fruit varieties and application of MNP core primer combination
  • MNP core primer combination for molecular identification of kiwi fruit varieties and application of MNP core primer combination
  • MNP core primer combination for molecular identification of kiwi fruit varieties and application of MNP core primer combination

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Screening and Validation of MNP Core Marker Primer Combinations for Molecular Identification of Kiwifruit DNA Varieties

[0024] (1) Screening of kiwifruit MNP primer marker combinations

[0025] Taking the publicly released kiwifruit genome as a reference (http: / / eggplant.kazusa.or.jp / ), using the resequencing data of 76 kiwifruit genomes carried out by our team for comparative analysis, 347 MNP marker sites were screened ( figure 1 ), design primers (see Table 1 for primer details).

[0026] (2) Correctness analysis of kiwifruit MNP primer marker combination

[0027] In order to test the accuracy of the kiwifruit MNP marker method, 8 samples were collected from the national kiwifruit resource garden, and a reproducibility experiment was carried out. The same DNA of each variety was constructed twice, and the two libraries were sequenced twice at different times. When comparing the first and second sequencing of different batches of libraries, the number of differenc...

Embodiment 2

[0033] Construction of standard DNA fingerprint of kiwifruit with MNP core marker primer combination for molecular identification of kiwifruit DNA varieties

[0034]1. DNA extraction and quality assessment

[0035] Genomic DNA of all kiwifruit varieties used to construct the kiwifruit fingerprint database was extracted by conventional CTAB method, and the quality of DNA was detected by agarose gel and ultraviolet spectrophotometer after RNase treatment. If the DNA band is single, there is no obvious degradation, the UV spectrophotometer detects that the A260 / 280 is between 1.8-2.0, and the A260 / 230 is between 1.8-2.0, there is no obvious light absorption at 270nm, and the DNA concentration is greater than 50ng / ul, it means Genomic DNA meets the relevant quality requirements and can be used for subsequent experiments.

[0036] 2. MNP marker detection

[0037] Using 347 synthetic kiwifruit MNP-labeled primers, multiplex amplification, next-generation high-throughput sequencing...

Embodiment 3

[0041] Authenticity of kiwifruit varieties detected by MNP-labeled primer combination for molecular identification of kiwifruit DNA varieties

[0042] 1. Determination of the discrimination degree of kiwifruit MNP primer marker varieties and the determination of the authenticity test results of kiwifruit varieties

[0043] The 91 kiwifruit samples in Example 2 were pairwise compared to analyze the differences of their MNP marker sites, and a total of 4095 pairs of comparison results were obtained, the average number of MNP marker differences between any varieties was 89.22, and the genetic distance difference of 4095 pairs of samples Between 0.30% and 82.26%, the average difference is 47.85%, and no difference is 0 in pairwise comparison. Using the developed marker detection method, 100% of kiwifruit varieties could be distinguished ( figure 2 ), indicating that the developed kiwifruit MNP markers have high polymorphism and strong variety discrimination. Therefore, the 347 ...

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Abstract

The invention belongs to the technical field of molecular biology, and particularly discloses an MNP core primer combination for molecular identification of kiwi fruit varieties and application of the MNP core primer combination. The primer combination formed by 347 MNP core primers provided by the invention can be compatible with a next-generation sequencing platform, has the characteristics of high efficiency, correctness, high reproducibility and the like, and meets the requirements of standard fingerprint data construction of kiwi fruits; the authenticity of a kiwi fruit sample to be detected and a control variety can be accurately identified by identifying the kiwi fruit sample to be detected and the control variety; the MNP site has high polymorphism and repeatability, can distinguish different kiwi fruit groups, and has a wide application range.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a combination of MNP core primers for molecular identification of kiwifruit DNA varieties and an application thereof. [0002] technical background [0003] Kiwifruit (Actinidia chinensis) is an economic crop of the genus Actinidia. The fruit is rich in nutrition and is popular all over the world. China is the origin of Actinidia plants and the original origin center of Actinidia genetic resources. [0004] With the introduction of new varieties of kiwifruit, the identification of new varieties is a major problem to be solved. The identification of kiwifruit varieties is only carried out by traditional methods such as tree vigor, leaf shape, flowering period, flower shape, and fruit appearance, which are inevitably affected by environmental and other human factors. Uncertainty about kiwifruit varieties restricts the healthy and sustainable development of th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12N15/11
CPCC12Q1/6895C12Q2600/156
Inventor 张琼钟彩虹郑浩
Owner WUHAN BOTANICAL GARDEN CHINESE ACAD OF SCI
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