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MNP core primer combination for molecular identification of DNA varieties of nelumbo nucifera L. and application of MNP core primer combination

A technology of core primers and primer pairs, applied in DNA/RNA fragments, recombinant DNA technology, genomics, etc.

Pending Publication Date: 2021-12-24
WUHAN ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] At present, there are no widely used DNA fingerprints and variety identification standards in lotus, but the development, screening and application of MNP markers have a good application basis in plants.

Method used

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  • MNP core primer combination for molecular identification of DNA varieties of nelumbo nucifera L. and application of MNP core primer combination
  • MNP core primer combination for molecular identification of DNA varieties of nelumbo nucifera L. and application of MNP core primer combination
  • MNP core primer combination for molecular identification of DNA varieties of nelumbo nucifera L. and application of MNP core primer combination

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Screening and validation of MNP core marker primer combinations for molecular identification of lotus DNA varieties (1) Screening of lotus MNP primer marker combinations

[0028] Taking the lotus root genome version GCA_003033685.1 as the reference genome, the sequence download address is: https: / / www.ncbi.nlm.nih.gov / assembly / GCA_003033685.1, the total length of the sequence is 817,267,751bp, including 8 chromosomes and multiple unmapped contig,; use the genome resequencing data of 296 germplasm resources provided by Wuhan Academy of Agricultural Sciences (link https: / / db.cngb.org / search / project / CNP0001227 / ), compare and analyze the sequencing data, and screen out Design primers for 525 MNP marker sites (see Table 1 for primer details).

[0029] Table 1 Sequence information table of 525 pairs of MNP marker amplification primers

[0030]

[0031]

[0032]

[0033]

[0034]

[0035]

[0036]

[0037]

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[0039]

[0040] ...

Embodiment 2

[0052] Example 2 The combination of MNP core marker primers for the molecular identification of lotus DNA varieties is applied to the construction of lotus standard DNA fingerprints

[0053] 1. DNA extraction and quality assessment

[0054] Genomic DNA of all lotus varieties used to construct the lotus 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.

[0055] 2. MNP marker detection

[0056] Using the 525 pairs of lotus MNP marker primer combinations screened in Example 1, multiple amplific...

Embodiment 3

[0060] Example 3 The combination of MNP core marker primers for the molecular identification of lotus DNA varieties is applied to detect the authenticity of lotus varieties 1, the determination of the differentiation degree of lotus MNP primer marker varieties and the determination of the judgment criteria for the authenticity of lotus varieties

[0061] The 56 lotus samples in Example 2 were compared in pairs to analyze the differences of their MNP marker loci, and a total of 1540 pairs of comparison results were obtained, the average number of MNP marker differences between any varieties was 212.65, and the genetic distance difference of 1540 pairs of samples Located between 0%-89.5%, the average difference is 44.15%, of which 1 pair has a difference of 0, accounting for 0.06% of the total number of sample pairs. Using the developed marker detection method, 99.93% of lotus species could be distinguished ( figure 2 ), indicating that the lotus MNP marker provided by the pres...

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Abstract

The invention discloses an MNP core primer combination for molecular identification of DNA varieties of nelumbo nucifera L. and an application of the MNP core primer combination. On the basis of re-sequencing data of the nelumbo nucifera L., 525 pairs of MNP core primer combinations for detecting polymorphic MNP marker sites of the nelumbo nucifera L. are screened by combining a high-throughput sequencing technology, and the nucleotide sequences of the combinations are as shown in SEQ ID No.1-SEQ ID No.1050. The 525 pairs of MNP site detection primers provided by the invention can be compatible with a next-generation sequencing platform, have the characteristics of high efficiency, accuracy, high reproducibility and the like, and meet the requirements of standard fingerprint data construction of the nelumbo nucifera L. The primer combination can be used for identifying a to-be-detected nelumbo nucifera L.sample and a control variety, can accurately identify the authenticity of the to-be-detected nelumbo nucifera L. sample, can be applied to the aspects of constructing a nelumbo nucifera L. DNA fingerprint database, detecting the authenticity of the nelumbo nucifera L. variety, analyzing genetic diversity of nelumbo nucifera L. germplasm resources or breeding nelumbo nucifera L.molecules and the like, and provides technical support for breeding nelumbo nucifera L. molecules and analyzing seed resources. The breeding period of the new nelumbo nucifera L.variety is accelerated.

Description

technical field [0001] The invention relates to a primer combination for molecular identification of plant varieties, in particular to an MNP core primer combination for molecular identification of lotus DNA varieties and its application in the molecular identification of lotus DNA varieties, and belongs to the field of molecular identification of lotus DNA varieties. Background technique [0002] Lotus (Nelumbo nucifera L.) is a vegetable crop of the genus Lily in the family Nymphaeaceae, the most characteristic aquatic horticultural plant in China. (Lotus) is one of China's top ten famous flowers, and lotus is also one of the horticultural plants with the most processed products. The cultivation area of ​​lotus root in China is about 400,000 hm 2 , the cultivation area of ​​seed lotus is about 100,000 hm 2 , mainly distributed in the Yangtze River Basin, Pearl River Basin and Yellow River Basin, among which the middle and lower reaches of the Yangtze River have the large...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/6858C12N15/11G16B40/30G16B30/10G16B20/20
CPCC12Q1/6895C12Q1/6858G16B40/30G16B30/10G16B20/20C12Q2600/156C12Q2600/16C12Q2537/143C12Q2531/113C12Q2535/122
Inventor 孙亚林王直新柯卫东朱红莲刘正位李明华李双梅李峰匡晶
Owner WUHAN ACADEMY OF AGRI SCI
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