Molecular markers related to the content of parthenolide in Magnolia biondii and its application

Abstract

The invention discloses a molecular marker related to the content of parthenolide in Magnolia biondii and application thereof. The molecular marker includes at least one of Mbi13_11039065 and Mbi13_11193126; Mbi13_11039065 is the 11039065th base of chromosome 13. When the genotyping of the site is homozygous G / G, the Magnolia bicolor does not contain parthenolide or the content is lower than 0.5%; when the genotyping of the site is homozygous A / A or heterozygous A / G, the content of parthenolide in the Magnolia bicolor is higher than 3.0%; Mbi13_11193126 is the 11193126th base of chromosome 13. When the genotyping of the site is homozygous C / C, the Magnolia bicolor does not contain parthenolide or the content is lower than 0.5%; when the genotyping of the site is homozygous T / T or heterozygous C / T, the content of parthenolide in the Magnolia bicolor is higher than 3.0%. The molecular markers Mbi13_11039065 and Mbi13_11193126 developed by the present invention can be used to identify the content of parthenolide in the root bark of Magnolia biondii at an early stage.

Description

Technical Field

[0001] The invention belongs to the field of plant molecular biology and relates to a molecular marker related to the content of parthenolide in Magnolia biondii and an application thereof. Background Art

[0002] In recent years, Chinese scholars have developed an original anti-glioma drug candidate ACT001 using parthenolide through chemical modification. The drug can enter the human brain and treat glioblastoma (Ding et al. 2020). For example, the world's medical problem - the survival time of patients with glioma has been extended from only 3 months to more than 3 years after monotherapy with this drug ( Figure 1 ). The Phase II clinical trial of ACT001 for the treatment of brain glioma was conducted at the University of Texas MD Anderson Cancer Center in the United States, and has been granted orphan drug status for the treatment of glioblastoma in the United States and the European Union. Currently, the China National Medical Products Administration has also quickly approved the clinical trial of the drug in combination with radiotherapy for the treatment of brain metastases of solid tumors. Once the drug is launched on the market, there will be a huge demand for the raw material parthenolide.

[0003] Parthenolide (PTL) was first extracted from chamomile flowers. Its molecular formula is C 15 H 20 O3, with a relative molecular mass of 248.3. Previous studies have shown that the content of parthenolide is highest in chamomile flowers, less in leaves, and almost undetectable in roots, but the content of parthenolide in chamomile flowers of different varieties and different ploidy is only 0.2% to 0.5% (Majdi et al., 2013). The development of synthetic chemistry has provided new ways for the production of many drugs, but the chemical synthesis of parthenolide requires 13 chemical reactions, which is complicated and costly (Freund & Arndt, 2016).

[0004] A large number of studies have shown that Michelia and Magnolia in the Magnoliaceae family also contain parthenolide (Xia Wei et al., 2014). Zhou Qian et al. measured the content of parthenolide in the leaves of eight species of Magnoliaceae plants, and the content of parthenolide in the leaves ranged from 0.002% to 0.35% (Zhou Qian et al., 2022). After years of research, the project team found that the content of parthenolide in the roots of Magnolia grandiflora was relatively high, and there was rich genetic variation in natural populations, ranging from 0.25% to 5.86%. The highest content of the individual was 18 times the highest content among the eight species of Magnolia plants measured by Zhou Qian et al. This provides an opportunity to breed new forest varieties with high content of parthenolide. However, the content of parthenolide in the roots of Magnolia grandiflora is a gradual accumulation process, which generally takes four years to reach stability. The high and low content of root parthenolide is difficult to distinguish by morphology. If the forestry production aims to extract parthenolide from the root bark of Magnolia bicolor, once the low-content asexual lines are mistakenly used, it will bring great losses to the producers. The phenotype of plants is controlled by genotype. The association analysis of phenotypic data and genotype is used to screen the key intervals that control the phenotypic shape. At the same time, the development of molecular markers with tightly linked phenotypes can predict the content of parthenolide in the root bark of Magnolia bicolor after cultivation through genotype identification at the seedling stage. However, there are currently no related genes and primers for early identification of parthenolide in the root system. Therefore, screening out primers associated with the content of parthenolide is the basis for early selection of asexual lines of Magnolia bicolor with high content of parthenolide and ensuring that all Magnolia bicolor planted are asexual lines with high content of parthenolide. Summary of the invention

[0005] Purpose of the invention: The first purpose of the present invention is to provide a molecular marker and its application that can be used to quickly identify the content of parthenolide in Magnolia bicolor at an early stage. The second purpose of the present invention is to provide a primer combination and a kit for detecting the above molecular marker. The third purpose of the present invention is to provide a method for identifying the content of parthenolide in Magnolia bicolor.

[0006] Technical solution: The molecular markers related to the content of parthenolide in Magnolia biondii described in the present invention include at least one of Mbi13_11039065 and Mbi13_11193126; Mbi13_11039065 is the 11039065th base of chromosome 13, and there are three genotypes at this site, namely G / G, A / A and A / G; Mbi13_11193126 is the 11193126th base of chromosome 13, and there are three genotypes at this site, namely C / C, T / T and C / T.

[0007] Among them, when the genotyping of the Mbi13_11039065 site is homozygous G / G, the Magnolia bicolor does not contain parthenolide or the content is lower than 0.5%; when the genotyping of the Mbi13_11039065 site is homozygous A / A or heterozygous A / G, the content of parthenolide in the Magnolia bicolor is higher than 3.0%; when the genotyping of the Mbi13_11193126 site is homozygous C / C, the Magnolia bicolor does not contain parthenolide or the content is lower than 0.5%; when the genotyping of the Mbi13_11193126 site is homozygous T / T or heterozygous C / T, the content of parthenolide in the Magnolia bicolor is higher than 3.0%.

[0008] The fluorescent quantitative PCR detection primer combination described in the present invention comprises a primer pair for detecting Mbi13_11039065 and / or Mbi13_11193126 molecular markers; the primer pair for detecting the Mbi13_11039065 molecular marker comprises a forward primer having a nucleotide sequence as shown in SEQ ID NO.1 to 2, and a universal reverse primer having a nucleotide sequence as shown in SEQ ID NO.3; the primer pair for detecting the Mbi13_11193126 molecular marker comprises a forward primer having a nucleotide sequence as shown in SEQID NO.4 to 5, and a universal reverse primer having a nucleotide sequence as shown in SEQ ID NO.6.

[0009] The kit of the present invention contains the above-mentioned fluorescent quantitative PCR detection primer combination.

[0010] The kit also contains 2×castPCR Genotyping Master Mix and ddH2O.

[0011] The invention discloses an application of the molecular marker, primer combination and kit in identifying the content of parthenolide in Magnolia biondii.

[0012] The method for identifying the content of parthenolide in Magnolia bicolor of the present invention comprises the following steps: extracting genomic DNA of Magnolia bicolor to be tested; using the extracted genomic DNA of Magnolia bicolor as a template, performing fluorescence quantitative PCR amplification using the primer combination pair of the above-mentioned SNP molecular markers Mbi13_11039065 and Mbi13_11193126, and If the amplification product of bi13_11039065 contains a homozygous G / G site, the Magnolia bicolor does not contain parthenolide or the content is lower than 0.5%; if the amplification product of Mbi13_11039065 contains a homozygous A / A or heterozygous A / G site, the content of parthenolide in the Magnolia bicolor to be tested is higher than 3.0%; if the amplification product of Mbi13_11193126 contains a homozygous C / C site, the Magnolia bicolor does not contain parthenolide or the content is lower than 0.5%; if the amplification product of Mbi13_11193126 contains a homozygous T / T or heterozygous C / T site, the content of parthenolide in the Magnolia bicolor to be tested is higher than 3.0%.

[0013] The reaction system of the fluorescent quantitative PCR includes 2×castPCR Genotyping Master Mix, a primer combination and ddH2O.

[0014] Specifically, the PCR reaction system is as follows: in every 20 μL system, 2×castPCR Genotyping MasterMix 10.0 μL, 30 ng / μL DNA template 2 μL, 10 μM Allele1 and Allele2 primers 0.3 μL each, 10 μM universal reverse primer 0.8 μL, and ddH2O 6.6 μL.

[0015] The amplification program of the fluorescent quantitative PCR is as follows: pre-denaturation at 94°C for 15 min; denaturation at 94°C for 20 s, annealing-extension at 62°C for 60 s, for a total of 10 cycles, with the annealing temperature reduced by 0.6°C after each cycle; denaturation at 94°C for 20 s, annealing-extension at 56°C for 60 s, for a total of 30 cycles; and 37°C for 1 min.

[0016] The invention discloses an application of the molecular markers in the breeding of Magnolia biondii with a high content of parthenolide.

[0017] Beneficial effects: Compared with the prior art, the present invention has the following outstanding significant advantages: the molecular markers Mbi13_11039065 and Mbi13_11193126 developed by the present invention can be used to identify the content of parthenolide in the root bark of Magnolia biondii at an early stage, and can be applied to the breeding of Magnolia biondii with high content of parthenolide and the selection of excellent seedlings for afforestation. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1The clinical results of ACT001 in treating brain glioma;

[0019] Figure 2 is the genome-wide association site for parthenolide content;

[0020] Figure 3 This is the agarose gel test result of KASP amplification product;

[0021] Figure 4 This is the screening result of the primers for parthenolide that are tightly linked to KASP;

[0022] Figure 5 The results of testing 70 individuals in a natural population using the screened KASP primers. DETAILED DESCRIPTION

[0023] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0024] Example 1 Development of molecular markers and primers

[0025] The invention conducts association analysis on the content of parthenolide in the root bark and the genotype through the natural population material of Magnolia biondii, and obtains the key site for the synthesis of parthenolide.

[0026] First, referring to the determination method of parthenolide in the root bark of Magnolia grandiflora by Zhu Xinghua et al. (ZL201710854730.1), the content of parthenolide in the root bark of 200 Magnolia grandiflora samples from Nanzhao County, Henan Province was determined. Twenty-four individuals with content below 0.5% and above 3% were selected for whole genome resequencing, with an average sequencing depth of 30×. FastQC and Trimmomatic were used to filter the low-quality sequences and adapter sequences of the sequencing. Then, BWA (Li et al., 2010) was used to align the filtered sequences to the Magnolia grandiflora reference genome (https: / / doi.org / 10.5061 / dryad.s4mw6m947). Samtools (Li et al., 2009) was used to sort the alignment results and remove duplicates. GATK software was used for local realignment and base quality correction to ensure the accuracy of the obtained SNPs and Indels. The missing genotype information was inferred using the ProbABEL R package, and the genotype was associated with the phenotypic traits using the GEMMA software. Based on the degree of significant association, the key sites controlling the synthesis of parthenolide were identified. A total of 24,051,833 SNP sites were obtained through resequencing data analysis of 48 individuals. Through association analysis with the content of parthenolide in the root bark, it was found that 17 SNP sites located in the interval of 11,039,065 to 11,193,126 bp on chromosome 13 were closely associated with the content of parthenolide (the results of the genome-wide association analysis (GWAS) of parthenolide in Magnolia officinalis are shown in Figure 2). Figure 2 As shown in the figure, the parthenolide-related sequence signals are all distributed in the proximal telomeric region of chromosome 13).

[0027] KASP primers were developed based on the SNP loci in the 11,039,065-11,193,126 bp interval of chromosome 13 of Magnolia bicolor. PCR amplification was performed on natural population materials of Magnolia bicolor. Two SNP loci (Mbi13_11039065 located at base 11,039,065 of chromosome 13 and Mbi13_11193126 located at base 11,193,126 of chromosome 13) with the contents of parthenolide in the root bark of Magnolia bicolor higher than 3% and lower than 0.5% were screened out. Two pairs of specific KASP universal primers were designed, namely Mbi13_11039065 (Mbi13_11039065F-A forward primer: 5′-gaaggtgaccaagttcatgctTCAATGTTAGAGTATTTTATGTGCCA-3′; Mbi13_11039065F-G forward primer: 5′-gaaggtcggagtcaacggattTCAATGTTAGAGTATTTTATGTGCCG-3′; Mbi13_11039065R universal reverse primer: 5′-TGGGGTCTCCTTTATGTGGA-3′) and Mbi13_11193126 (Mbi13_11193126F-T forward primer: 5′-gaaggtgaccaagttcatgctTTCTCACCTTACATG GTATCAGAGAT-3′; Mbi13_11193126F-C forward primer: 5′-gaaggtcggagtcaacggattTTCTC ACCTTACATGGTATCAGAGAC-3′; Mbi13_11193126R universal reverse primer: 5′-GGGACA CTAGATTTCTGCGCT-3′).

[0028] Among them, the amplification product of primer Mbi13_11039065 in Magnolia bicolor individuals with a parthenolide content higher than 3% is a homozygous A / A or heterozygous A / G site; the amplification product of primer Mbi13_11039065 in Magnolia bicolor individuals without parthenolide or with a content lower than 0.5% is a homozygous G / G site. The amplification product of primer Mbi13_11039065 in Magnolia bicolor individuals with a parthenolide content higher than 3% is a homozygous T / T or heterozygous C / T site, and the amplification product of primer Mbi13_11039065 in Magnolia bicolor individuals without parthenolide or with a content lower than 0.5% is a homozygous C / C site.

[0029] Example 2 Validation test of primers Mbi13_11039065 and Mbi13_11193126

[0030] (1) Conventional PCR test

[0031] Primers Mbi13_11039065 and Mbi13_11193126 were used to perform PCR amplification on Magnolia grandiflora, using a common PCR instrument. Four Magnolia grandiflora with a content of 0% to 0.5% and four with a content of more than 3% in natural populations were amplified and tested. The genomic DNA of Magnolia grandiflora root bark was extracted using Tiangen New Plant Genome Extraction Kit (DP320), and the extracted DNA was diluted to 30 ng / μL and stored at -20℃ for later use. The PCR reaction system was as follows: in every 20 μL system, 10.0 μL of 2×castPCRGenotyping Master Mix, 2 μL of 30 ng / μL DNA template, 0.3 μL each of 10 μM Allele1 primer (Mbi13_11039065F-A or Mbi13_11193126F-T) and Allele2 primer (M bi13_11039065F-G or Mbi13_11193126F-C), 0.8 μL of 10 μM universal reverse primer, and 6.6 μL of d HO. The PCR amplification program was as follows: pre-denaturation at 94°C for 15 min; denaturation at 94°C for 20 s, annealing-extension at 62°C for 60 s, for a total of 10 cycles (annealing temperature was reduced by 0.6°C after each cycle); denaturation at 94°C for 20 s, annealing-extension at 56°C for 60 s, for a total of 30 cycles; and 37°C for 1 min.

[0032] The results are as follows Figure 3 As shown, 1 to 4 in the figure are individuals with a content of 0% to 0.5% of parthenolide, and 5 to 8 are individuals with a content of more than 3%; the resequencing results show that in individuals with a content of 0% to 0.5%, the site is homozygous G / G, while in individuals with a content of more than 3%, the site is homozygous A / A or heterozygous A / G; Figure 3 In A, since the last base at the 3' end of the Mbi13_11193126F-A primer is A, and individuals with a content of 0% to 0.5% of parthenolide are homozygous G / G, it cannot be successfully amplified in individuals with a content of 0% to 0.5%, while individuals with a high content of more than 3% have a homozygous A / A or heterozygous A / G at this site, so it can be successfully amplified in these individuals; Figure 3 In B, since the last base at the 3' end of the Mbi13_11193126F-G primer is G, and individuals with a parthenolide content of 0% to 0.5% are homozygous G / G, and individuals with a high content of more than 3% are homozygous A / A or heterozygous A / G at this site, this primer can successfully amplify in these individuals; therefore, ordinary PCR instruments cannot accurately distinguish heterozygous sites.

[0033] (2) Fluorescence quantitative PCR detection

[0034] Primers Mbi13_11039065 and Mbi13_11193126 were used to perform fluorescence quantitative PCR amplification on Magnolia grandiflora, and six Magnolia grandiflora with a content of 0% to 0.5% and six with a content of more than 3% in natural populations were amplified and detected. Genomic DNA from Magnolia grandiflora leaves was extracted using Tiangen New Plant Genome Extraction Kit (DP320), and the extracted DNA was diluted to 30 ng / μL and stored at -20℃ for later use. The PCR reaction system was as follows: in every 20 μL system, 10.0 μL of 2×castPCRGenotyping Master Mix, 2 μL of 30 ng / μL DNA template, 0.3 μL each of 10 μM Allele1 primer (Mbi13_11039065F-A or Mbi13_11193126F-T) and Allele2 primer (Mbi13_11039065F-G or Mbi13_11193126F-C), 0.8 μL of 10 μM universal reverse primer, and 6.6 μL of ddH2O. The PCR amplification program was as follows: pre-denaturation at 94°C for 15 min; denaturation at 94°C for 20 s, annealing-extension at 62°C for 60 s, for a total of 10 cycles (annealing temperature was reduced by 0.6°C after each cycle); denaturation at 94°C for 20 s, annealing-extension at 56°C for 60 s, for a total of 30 cycles; and fluorescence signal collection at 37°C for 1 min.

[0035] The results are as follows Figure 4 As shown, Figure 4 A is the band of 12 natural populations of Magnolia biondii amplified by primer Mbi13_11039065. Each sample was repeated twice. The individual genotype was homozygous A / A ( Figure 4 A blue dot) or heterozygous A / G ( Figure 4 A green dot) locus, and the individual genotype with a content of 0% to 0.5% is homozygous G / G ( Figure 4 A red dot) site. Figure 4 B is the spectrum of 12 natural populations of Magnolia biondii amplified by primer Mbi13_11193126. Each sample was repeated twice. The content was higher than 3% and the individual genotype was homozygous T / T ( Figure 4 B blue dots) or heterozygous T / C ( Figure 4 B green dots) locus, and the individual genotype with a content of 0% to 0.5% is homozygous C / C ( Figure 4 B red dots) sites. H2O was also tested as a negative control (3 replicates), which is shown as × in the figure. The specific results are shown in Table 1. The results show that the use of fluorescent quantitative PCR amplification can accurately distinguish between homozygous and heterozygous sites.

[0036] Table 1 The content of parthenolide (PTL) in the root bark of 12 individuals of Magnolia biondii natural population amplified by primers Mbi13_11039065 and Mbi13_11193126 and the genotypes detected by the corresponding primers

[0037]

[0038]

[0039] Note: NOAMP means the sample under test was not amplified.

[0040] Example 3 Actual detection of primers Mbi13_11039065 and Mbi13_11193126

[0041] Primers Mbi13_11039065 and Mbi13_11193126 were used to perform fluorescence quantitative PCR amplification on Magnolia grandiflora. The natural populations of Magnolia grandiflora were collected from two different locations, Nanzhao, Henan (individuals 1 to 51 in Table 2) and Xiangyang, Hubei (individuals 52 to 70 in Table 2), with a total of 70 individuals. The Tiangen New Plant Genome Extraction Kit (DP320) was used to extract genomic DNA from Magnolia grandiflora leaves, and the extracted DNA was diluted to 30 ng / μL and stored at -20℃ for later use. The PCR reaction was a 20μL system: 2×castPCR Genotyping Master Mix 10.0μL, 30ng / μL DNA template 2μL, 10μM Allele1 and Allele2 primers 0.3μL each, 10μM universal reverse primer 0.8μL, ddH2O6.6μL. The PCR amplification program was as follows: pre-denaturation at 94°C for 15 min; denaturation at 94°C for 20 s, annealing-extension at 62°C for 60 s, for a total of 10 cycles (annealing temperature was reduced by 0.6°C after each cycle); denaturation at 94°C for 20 s, annealing-extension at 56°C for 60 s, for a total of 30 cycles; and fluorescence signal collection at 37°C for 1 min.

[0042] The results are as follows Figure 5 The results of the two sets of primers for the detection of Magnolia bicolor leaves and the detection of parthenolide content in the root bark were exactly the same. The genotype amplified by primer Mbi13_11039065 in individuals with a content higher than 3% was homozygous A / A ( Figure 5 A blue dot) or heterozygous A / G ( Figure 5 A green dot) locus, and the individual genotype with a content of 0% to 0.5% is homozygous G / G ( Figure 5 A red dot) site. Figure 5 B is the band of 70 natural populations of Magnolia biondii amplified by primer Mbi13_11193126, and the genotype of individuals with a content of more than 3% is homozygous T / T ( Figure 5B blue dots) or heterozygous T / C ( Figure 5 B green dots) locus, and the individual genotype with a content of 0% to 0.5% is homozygous C / C ( Figure 5 B red dots) site. H2O was also detected as a negative control, shown as yellow dots in the figure. The specific results are shown in Table 2. Table 2 Primers Mbi13_11039065 and Mbi13_11193126 amplified the content of parthenolide (PTL) in the root bark of 70 individuals in the natural population of Magnolia bicolor and the genotypes detected by the corresponding primers

[0043]

[0044]

[0045]

[0046]

[0047] The results showed that the identification results of the two primers were consistent and both can be used for the early identification of the content of parthenolide in the root bark of Magnolia biondii, which is of great significance in the molecular marker-assisted selection breeding of Magnolia biondii with high content of parthenolide and the selection of excellent seedlings for afforestation.

Claims

1. A fluorescent quantitative PCR detection primer combination, characterized in that: The primer combination includes a primer pair for detecting the Mbi13_11039065 and / or Mbi13_11193126 molecular marker; Mbi13_11039065 is the 11039065th base of chromosome 13, and there are three genotypes at this site, namely G / G, A / A and A / G; Mbi13_11193126 is the 11193126th base of chromosome 13, and there are three genotypes at this site, namely C / C, T / T and C / T; the primer pair for detecting the Mbi13_11039065 molecular marker includes a forward primer whose nucleotide sequence is shown in SEQ ID NO.1~2, and a universal reverse primer whose nucleotide sequence is shown in SEQ ID NO.3; the primer pair for detecting the Mbi13_11193126 molecular marker includes a forward primer whose nucleotide sequence is shown in SEQ ID NO.4~5, and a universal reverse primer whose nucleotide sequence is shown in SEQ ID NO. .6 shows the universal reverse primer.

2. A kit, characterized in that: The kit contains the fluorescent quantitative PCR detection primer combination according to claim 1.

3. The kit for identifying the content of parthenolide in Magnolia biondii according to claim 2, characterized in that: The kit also includes 2×castPCR Genotyping Master Mix and ddH2O.

4. Use of the primer combination according to claim 1 and the kit according to any one of claims 2 to 3 in identifying the content of parthenolide in Magnolia biondii.

5. A method for identifying the content of parthenolide in Magnolia biondii, characterized in that: The following steps are involved: Extract the genomic DNA of Magnolia bicolor to be tested; use the extracted genomic DNA of Magnolia bicolor as a template, and use the primer combination of SNP molecular markers Mbi13_11039065 and Mbi13_11193126 in claim 3 to perform fluorescence quantitative PCR amplification; if the amplified product of Mbi13_11039065 is a homozygous G / G site, the Magnolia bicolor does not contain parthenolide or the content is less than 0.5%; if Mbi13_110 If the 39065 amplification product contains a homozygous A / A or heterozygous A / G site, the content of parthenolide in the tested Magnolia bicolor is higher than 3.0%; if the Mbi13_11193126 amplification product contains a homozygous C / C site, the Magnolia bicolor does not contain parthenolide or the content is lower than 0.5%; if the Mbi13_11193126 amplification product contains a homozygous T / T or heterozygous C / T site, the content of parthenolide in the tested Magnolia bicolor is higher than 3.0%.

6. The method for identifying the content of parthenolide in Magnolia biondii according to claim 5, characterized in that: The reaction system of fluorescence quantitative PCR includes 2×castPCR Genotyping Master Mix, primer combination and ddH2O.

7. The method for identifying the content of parthenolide in Magnolia biondii according to claim 5, characterized in that: The amplification program of fluorescence quantitative PCR was as follows: pre-denaturation at 94 °C for 15 min; denaturation at 94 °C for 20 s, annealing-extension at 62 °C for 60 s, for a total of 10 cycles, with the annealing temperature lowered by 0.6 °C after each cycle; denaturation at 94 °C for 20 s, annealing-extension at 56 °C for 60 s, for a total of 30 cycles; and incubation at 37 °C for 1 min.

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