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CN-122012794-A - Primer probe set, kit and application for identifying cassava varieties south planting 199 based on MNP (MNP) markers

CN122012794ACN 122012794 ACN122012794 ACN 122012794ACN-122012794-A

Abstract

The invention discloses a primer probe set, a kit and application for identifying a cassava variety south planting 199 based on MNP (molecular weight distribution) markers. The primer probe set comprises a first primer pair, a first probe, a second primer pair, a second probe, a third primer pair and a third probe, wherein each primer pair comprises an upstream primer and a downstream primer, the kit comprises the primer probe set, the primer probe set for identifying the cassava varieties south planting 199 based on MNP marks, the kit and the application are used, and SNP differences among varieties are analyzed based on the detection result of multiple TaqMan probes qPCR, so that the rapid and accurate identification of the cassava varieties south planting 199 is realized.

Inventors

  • ZHANG BAOLONG
  • LI QIONG
  • PENG HAI
  • HE YUN
  • FANG ZHIWEI
  • LIN YAOYAO
  • LI TIANTIAN
  • CAI DIWEI
  • HUANG QIONG

Assignees

  • 江汉大学
  • 中国热带农业科学院热带作物品种资源研究所
  • 三亚崖州湾创新发展中心有限公司

Dates

Publication Date
20260512
Application Date
20260327

Claims (6)

  1. 1. The primer probe group for identifying the cassava varieties south planting 199 based on MNP (molecular weight distribution) markers is characterized by comprising a first primer pair, a first probe, a second primer pair, a second probe, a third primer pair and a third probe, wherein each primer pair comprises an upstream primer and a downstream primer, the nucleotide sequence of the upstream primer of the first primer pair is shown as SEQ ID NO. 1 in a sequence table, the nucleotide sequence of the downstream primer of the first primer pair is shown as SEQ ID NO. 2 in the sequence table, the nucleotide sequence of the upstream primer of the first probe is shown as SEQ ID NO. 3 in the sequence table, the nucleotide sequence of the downstream primer of the second primer pair is shown as SEQ ID NO. 4 in the sequence table, the nucleotide sequence of the downstream primer of the second primer pair is shown as SEQ ID NO. 5 in the sequence table, the nucleotide sequence of the upstream primer of the third primer pair is shown as SEQ ID NO. 6 in the sequence table, and the nucleotide sequence of the downstream primer of the third primer pair is shown as SEQ ID NO. 8 in the sequence table.
  2. 2. A kit for identifying a cassava variety south-planting 199 based on MNP markers, comprising the primer probe set of claim 1.
  3. 3. The application of the primer probe group for identifying the south planting 199 of the cassava varieties based on MNP marks is characterized in that the application comprises the steps of adopting the primer probe group in claim 1 to perform qPCR amplification, and then analyzing SNP differences among the cassava varieties according to the amplification result to judge whether the cassava varieties are the south planting 199.
  4. 4. The method of claim 3, wherein the step of amplifying the third probe to obtain a Ct value of 32 or less is performed, and the step of amplifying the first probe and the second probe to obtain a Ct value of 35 or less is performed, wherein the sample to be tested is the south plant 199.
  5. 5. The use according to claim 3, wherein the thermal cycling program of qPCR amplification comprises 95℃2 min and 40 cycles of 95℃15 s and 60℃60 s.
  6. 6. The method according to claim 3, wherein each 20. Mu.L of the qPCR amplification procedure comprises 2X QPCR MASTER Mix 10. Mu.L of each of the upstream primer and the downstream primer of the first primer pair, the second primer pair and the third primer pair of 300 to nM, each of the first probe, the second probe and the third probe of 150 to nM, and the template DNA of the sample to be tested is 2. Mu.L, which is supplemented with RNase-free water.

Description

Primer probe set, kit and application for identifying cassava varieties south planting 199 based on MNP (MNP) markers Technical Field The invention relates to the fields of molecular biology and genetic breeding, in particular to a primer probe set, a kit and application for identifying a cassava variety south planting 199 based on MNP (molecular weight distribution) markers. Background The south planting 199 of the cassava variety is not only used as an important raw material for starch processing and industrial ethanol production, but also can directly promote the development of related industries, can help the grower to increase the yield and income, and has remarkable social and economic benefits. In recent years, along with the acceleration of the expansion of the cultivation area of the south planting 199 and the improvement of varieties, the variety authenticity identification has significantly improved importance in the aspects of production, seedling supervision, quality control, intellectual property protection and the like. Conventional variety identification is based on multiple morphological features or on simple molecular markers, but these methods suffer from slow speed, low sensitivity to doped samples, and difficulty in rapid identification on site. Therefore, there is a need for a method of identifying samples that are fast and sensitive to doped samples. BRIEF SUMMARY OF THE PRESENT DISCLOSURE In order to solve the problems that the existing identification method is low in speed, low in sensitivity to doped samples and difficult to identify rapidly on site, the embodiment of the disclosure provides a primer probe set, a kit and application for identifying the cassava varieties south planting 199 based on MNP (molecular weight distribution) markers. The technical scheme is as follows: In one aspect, the disclosure provides a primer probe set for identifying a cassava variety south plant 199 based on MNP (molecular weight distribution) markers, wherein the primer probe set comprises a first primer pair, a first probe, a second primer pair, a second probe, a third primer pair and a third probe, each primer pair comprises an upstream primer and a downstream primer, the nucleotide sequence of the upstream primer of the first primer pair is shown as SEQ ID NO. 1 in a sequence table, the nucleotide sequence of the downstream primer of the first primer pair is shown as SEQ ID NO. 2 in the sequence table, the nucleotide sequence of the first probe is shown as SEQ ID NO. 3 in the sequence table, the nucleotide sequence of the upstream primer of the second primer pair is shown as SEQ ID NO. 4 in the sequence table, the nucleotide sequence of the downstream primer of the second primer pair is shown as SEQ ID NO. 5 in the sequence table, the nucleotide sequence of the second primer pair is shown as SEQ ID NO. 6 in the sequence table, the nucleotide sequence of the downstream primer of the third primer pair is shown as SEQ ID NO. 8 in the sequence table. In another aspect, the present disclosure provides a kit for identifying a cassava variety, south-planting 199, based on MNP markers, comprising the above primer probe set. In yet another aspect, the disclosure provides an application of a primer probe set for identifying a cassava variety south planting 199 based on MNP markers, wherein the application comprises the steps of analyzing SNP differences among cassava varieties according to amplification results after qPCR amplification by using the primer probe set, and judging whether the cassava variety is the south planting 199. Specifically, the thermal cycling program of qPCR amplification comprises 95 ℃ 2 min and 40 more cycles, each cycle comprising 95 ℃ 15s and 60 ℃ 60 s. Specifically, each 20 mu L of the qPCR amplification procedure comprises 2X QPCR MASTER Mix 10 mu L of each of the upstream primer and the downstream primer of the first primer pair, the second primer pair and the third primer pair, 300 nM of each of the upstream primer and the downstream primer of the first primer pair, the second probe and the third probe, 150 nM of each of the first probe, the second probe and the third probe, and 2 mu L of template DNA of the sample to be detected, which is supplemented with RNase-free water. The technical scheme provided by the embodiment of the disclosure has the beneficial effects that the embodiment of the invention provides the primer probe group, the kit and the application for identifying the south planting 199 of the cassava varieties based on MNP marks, and the primer probe group can realize accurate variety distinguishing based on qPCR results, so that the identification of the south planting 199 of the cassava varieties can be efficiently, quickly and high-throughput completed. Specifically, to meet the requirements of rapid, sensitive and industrialized production of variety identification, the embodiment of the disclosure utilizes a cassava polynucleotide polymorphism (Multiple