A molecular marker combination for identifying pachouli germplasm types and application thereof

Abstract

The present application relates to a kind of molecular marker combination that can identify pachouli germplasm type and its application.The molecular marker combination includes first molecular marker, second molecular marker, third molecular marker and fourth molecular marker;The first molecular marker is located at 44138387bp of nuclear genome A02 chromosome, the second molecular marker is located at 25567854bp of nuclear genome A02 chromosome, the third molecular marker is located at 33983091bp of nuclear genome A03 chromosome, and the fourth molecular marker is located at 42533373bp of nuclear genome A05 chromosome.Compared with early molecular identification method, the present application can quickly and accurately identify different germplasm pachouli by molecular marker identification, and the developed molecular marker can be used for the commercial application of pachouli germplasm type identification, and can bring good economic benefit and good social value.

Description

Technical Field

[0001] This invention relates to the field of gene technology, specifically to a molecular marker combination for identifying patchouli germplasm types and its application. Background Technology

[0002] Patchouli is a famous southern Chinese medicinal herb. After nearly a thousand years of cultivation and domestication in my country, it has developed into several germplasm types, represented by Shipai patchouli, Zhaoqing patchouli, Hainan patchouli, and Zhanjiang patchouli. Among these, Hainan patchouli and Zhanjiang patchouli are actually of the same germplasm type. Based on chemical composition, patchouli can be divided into two categories: "alcoholic" and "ketoneic." Zhaoqing and Shipai patchouli are "ketoneic," while Hainan and Zhanjiang patchouli are "alcoholic." Due to the long-term reliance on cuttings for propagation, the problem of germplasm degeneration and mixing has become increasingly serious. In an attempt to overcome this problem, growers often blindly introduce patchouli germplasm from other producing areas, which not only fails to improve the germplasm degeneration problem but also leads to further mixing of germplasm. Traditionally, it is believed that the medicinal value of alcohol-scented and ketone-scented patchouli differs due to their different chemical compositions. Therefore, the problem of mixed patchouli germplasm not only affects the stability of patchouli medicinal material quality but also hinders the assurance of safe clinical application. Currently, researchers have conducted research on patchouli germplasm identification using macro / microscopic phenotypic traits, chemical fingerprinting, and conventional molecular markers, but a rapid and effective identification method has not yet been established.

[0003] Identifying patchouli through phenotypic traits largely relies on the experience and subjective judgment of the person identifying it. Morphological identification is affected by the environment and season, resulting in a large workload but low accuracy. Although chemical fingerprinting has achieved some success in the identification of patchouli, it still faces some challenges and limitations. For example, the sample processing is relatively complex and easily affected by various factors; at the same time, this method requires professional chemical analysis techniques and equipment, and places high demands on the technical skills of the operators.

[0004] Early molecular identification methods, such as amplified fragment length polymorphism (AFLP), random amplified polymorphism (RAPD), and restriction fragment length polymorphism (RFLP) markers, have been applied to the identification of patchouli germplasm, but the results are still unsatisfactory. With the rapid development of high-throughput sequencing technology, molecular markers developed based on genome-wide genetic variations, such as single nucleotide polymorphisms (SNPs) and insertions / deletions (InDels), are gradually playing an important role in germplasm identification. However, to date, there are no research reports on the use of genome-wide genetic variations to develop molecular markers for the identification of patchouli germplasm resources in my country. Therefore, this invention provides a combination of molecular markers for identifying patchouli germplasm types and their applications. Summary of the Invention

[0005] The technical problem to be solved by this invention is to provide a combination of molecular markers for identifying patchouli germplasm types and its application. The aim is to identify different patchouli germplasms using genome-wide genetic variation.

[0006] The technical solution of the present invention to solve the above-mentioned technical problems is as follows:

[0007] In a first aspect, a molecular marker combination for identifying patchouli germplasm types, the molecular marker combination comprising a first molecular marker, a second molecular marker, a third molecular marker, and a fourth molecular marker;

[0008] The first molecular marker is located at 44138387 bp on chromosome A02 of the nuclear genome, and the polymorphism is T or TTCT;

[0009] The second molecular marker is located at 25567854 bp on chromosome A02 of the nuclear genome, with a polymorphism of T or TGGAATTAGTGAGT;

[0010] The third molecular marker is located at 33983091 bp on chromosome A03 of the nuclear genome, and its polymorphism is A or AA;

[0011] The fourth molecular marker is located at 42533373 bp on chromosome A05 of the nuclear genome, and its polymorphism is A or GTA.

[0012] Furthermore, when the base inserted at 44138387bp on chromosome A02 is TTCT, it is Zhanjiang patchouli - alcoholic type; when the base inserted at 25567854bp on chromosome A02 is TGGAATTAGTGAGT, it is ketone type - Zhaoqing patchouli; when the base inserted at 33983091bp on chromosome A03 is AA, it is ketone type - Shipai patchouli; when the base inserted at 42533373bp on chromosome A05 is GTA, it is intermediate type - patchouli with high alcohol and ketone content.

[0013] Secondly, a primer for amplifying a combination of molecular markers that can identify the germplasm type of patchouli includes a primer set for amplifying the molecular marker combination.

[0014] Furthermore, the primer set for the first molecular marker is shown in SEQ ID No:1-2;

[0015] The primer set for the second molecular marker is shown in SEQ ID No:3-4;

[0016] The primer set for the third molecular marker is shown in SEQ ID No:5-6;

[0017] The primer for the fourth molecular marker is shown in SEQ ID No:7.

[0018] Thirdly, a kit for amplifying molecular marker combinations that can identify patchouli germplasm types, comprising the primer set described above for amplifying molecular marker combinations that can identify patchouli germplasm types.

[0019] Fourthly, the application of a molecular marker combination for identifying patchouli germplasm types, wherein the molecular marker combination is used in the identification of patchouli germplasm types; or wherein the molecular marker combination is used in patchouli-assisted breeding.

[0020] Fifthly, the application of a primer set for amplifying molecular marker combinations that can identify patchouli germplasm types is characterized by the application of the primer set for the molecular marker combinations in the identification of patchouli germplasm types; or the application of the primer set for the molecular marker combinations in patchouli-assisted breeding.

[0021] Sixthly, the application of a kit for amplifying molecular marker combinations that can identify patchouli germplasm types, the application of the kit in the identification of patchouli germplasm types; or the application of the kit in patchouli-assisted breeding.

[0022] Seventhly, a method for identifying patchouli germplasm types includes the following steps:

[0023] Step 1: Extract DNA from the patchouli sample to be tested as a DNA template;

[0024] Step 2: Perform PCR amplification on the DNA template using the primer set or the kit described above to obtain the PCR amplification product;

[0025] Step 3: Sequencing the PCR amplification products and performing genotyping based on the sequencing results.

[0026] Furthermore, the PCR amplification reaction system in step 2 is as follows: 12.5 μl of 2X Pro Taq Master Mix (dye plus), 8.5 μL of ddH2O, 1.0 μL of 10 μmol / L forward and reverse primers, and 2.0 μl of 10–30 ng / μL template DNA;

[0027] The PCR amplification reaction program in step 2 is as follows: 94℃ pre-denaturation for 30s; 94℃ denaturation for 30s, 56℃ annealing for 30s, 72℃ extension for 1min, 35 cycles; 72℃ extension for 10min; 4℃ incubation.

[0028] The beneficial effects of this invention are as follows: This invention utilizes four molecular markers based on genome-wide genetic variation to identify different patchouli germplasms. Compared with earlier molecular identification methods, its advantage lies in its ability to rapidly and accurately identify patchouli germplasms of different types. Furthermore, this method is simple to operate, easy to perform in the laboratory, and readily applicable, possessing broad market application prospects. In addition, this technology can be effectively used for patchouli germplasm identification and protection, and the developed molecular markers can be used for commercial applications in patchouli germplasm type identification, bringing significant economic benefits and good social value. Attached Figure Description

[0029] Figure 1 The electrophoresis results of the amplified fragments of the first primer pair of this invention are as follows, from left to right: marker, Zhanjiang patchouli - alcohol type, ketone type - Shipai patchouli, ketone type - Zhaoqing patchouli, intermediate type - patchouli with high alcohol and ketone content;

[0030] Figure 2 The electrophoresis results of the amplified fragments of the second primer pair of this invention are as follows, from left to right: marker, Zhanjiang patchouli - alcohol type, ketone type - Shipai patchouli, ketone type - Zhaoqing patchouli, intermediate type - patchouli with high alcohol and ketone content;

[0031] Figure 3 The electrophoresis results of the amplified fragments of the third primer pair of this invention are as follows, from left to right: marker, Zhanjiang patchouli - alcohol type, ketone type - Shipai patchouli, ketone type - Zhaoqing patchouli, intermediate type - patchouli with high alcohol and ketone content;

[0032] Figure 4The electrophoresis results of the amplified fragments of the fourth primer pair of this invention are as follows, from left to right: marker, Zhanjiang patchouli - alcohol type, ketone type - Shipai patchouli, ketone type - Zhaoqing patchouli, intermediate type - patchouli with high alcohol and ketone content;

[0033] Figure 5 The results of forward sequencing of the PCR products of the first primer pair of this invention are as follows: from top to bottom, they are Zhanjiang patchouli - alcohol type, ketone type - Shipai patchouli, ketone type - Zhaoqing patchouli, and intermediate type - patchouli with high alcohol and ketone content.

[0034] Figure 6 The reverse sequencing peak alignment results of the PCR products of the first primer pair of this invention are as follows: from top to bottom, they are Zhanjiang patchouli - alcohol type, ketone type - Shipai patchouli, ketone type - Zhaoqing patchouli, and intermediate type - patchouli with high alcohol and ketone content.

[0035] Figure 7 The results of forward sequencing of the PCR products of the second primer pair of this invention are as follows: from top to bottom, they are Zhanjiang patchouli - alcohol type, ketone type - Shipai patchouli, ketone type - Zhaoqing patchouli, and intermediate type - patchouli with high alcohol and ketone content.

[0036] Figure 8 The reverse sequencing peak alignment results of the PCR products of the second primer pair of this invention are as follows: from top to bottom, they are Zhanjiang patchouli - alcohol type, ketone type - Shipai patchouli, ketone type - Zhaoqing patchouli, and intermediate type - patchouli with high alcohol and ketone content.

[0037] Figure 9 The results of forward sequencing of the PCR products of the third primer pair of this invention are as follows: from top to bottom, they are Zhanjiang patchouli - alcohol type, ketone type - Shipai patchouli, ketone type - Zhaoqing patchouli, and intermediate type - patchouli with high alcohol and ketone content.

[0038] Figure 10 The reverse sequencing peak alignment results of the PCR products of the third primer pair of this invention are as follows: from top to bottom, they are Zhanjiang patchouli - alcohol type, ketone type - Shipai patchouli, ketone type - Zhaoqing patchouli, and intermediate type - patchouli with high alcohol and ketone content.

[0039] Figure 11 The results of forward sequencing of the PCR products of the fourth primer pair of this invention are as follows: from top to bottom, they are Zhanjiang patchouli - alcohol type, ketone type - Shipai patchouli, ketone type - Zhaoqing patchouli, and intermediate type - patchouli with high alcohol and ketone content.

[0040] Figure 12 This is a peak diagram of the forward primer sequencing of sample A in this invention;

[0041] Figure 13 This is a peak diagram of reverse primer sequencing of sample A in this invention;

[0042] Figure 14 This is a peak diagram of the forward primer sequencing of sample B in this invention;

[0043] Figure 15 This is a peak diagram of reverse primer sequencing for sample B in this invention;

[0044] Figure 16 This is a peak diagram of the forward primer sequencing of sample C in this invention;

[0045] Figure 17 This is a peak diagram of reverse primer sequencing for sample C in this invention;

[0046] Figure 18 This is a peak diagram of the forward primer sequencing of sample D in this invention. Detailed Implementation

[0047] The principles and features of this invention are described below. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they should be performed according to the techniques or conditions described in the literature in this field, or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0048] Example

[0049] 1. Development of germplasm-specific molecular markers for patchouli

[0050] The patchouli varieties of Shipai, Zhaoqing, and Zhanjiang, as well as the patchouli varieties with high alcohol and ketone content, of this invention were bred by Professor Ouyang Puyue of Guangdong Food and Drug Vocational College. Samples were taken from Liantang Town, Gaoyao District, Zhaoqing City, Guangdong Province on March 18, 2023. Whole-genome sequencing was performed on the Shipai, Zhaoqing, and Zhanjiang patchouli varieties, and germplasm-specific variations were found at chromosomes A02 (44138387 and 25567854), A03 (33983091), and A05 (42533373), respectively. Primers were designed to amplify these candidate loci.

[0051] Specifically, a molecular marker combination for identifying patchouli germplasm types includes a first molecular marker, a second molecular marker, a third molecular marker, and a fourth molecular marker; the first molecular marker is located at 44138387 bp on chromosome A02, with a polymorphism of T or TTCT; the second molecular marker is located at 25567854 bp on chromosome A02 of the nuclear genome, with a polymorphism of T or TGGAATTAGTGAGT; the third molecular marker is located at 33983091 bp on chromosome A03 of the nuclear genome, with a polymorphism of A or AA; and the fourth molecular marker is located at 42533373 bp on chromosome A05 of the nuclear genome, with a polymorphism of A or GTA.

[0052] When the base inserted at 44138387bp on chromosome A02 is TTCT, it is Zhanjiang patchouli - alcoholic type; when the base inserted at 25567854bp on chromosome A02 is TGGAATTAGTGAGT, it is ketone type - Zhaoqing patchouli; when the base inserted at 33983091bp on chromosome A03 is AA, it is ketone type - Shipai patchouli; when the base inserted at 42533373bp on chromosome A05 is GTA, it is intermediate type - patchouli with high alcohol and ketone content.

[0053] 2. Primer design for molecular markers

[0054] Primer design was performed using Primer3 software, with the following parameter settings:

[0055] PRIMER_PICK_LEFT_PRIMER = 1,

[0056] PRIMER_PICK_INTERNAL_OLIGO=0,

[0057] PRIMER_PICK_RIGHT_PRIMER = 1,

[0058] PRIMER_OPT_SIZE = 20,

[0059] PRIMER_MIN_SIZE = 18,

[0060] PRIMER_MAX_SIZE = 22,

[0061] PRIMER_PRODUCT_SIZE_RANGE=80-150, PRIMER_EXPLAIN_FLAG=1.

[0062] First primer pair of the first molecule marker (Zhanjiang patchouli - alcoholic type):

[0063] Forward primer: 5'-AGGTGAGGGAAGGCATTGTT-3' (SEQ ID No: 1),

[0064] Reverse primer: 5'-TCAACCACCATCAACCACACT-3' (SEQ ID No:2).

[0065] The second primer pair for the second molecular marker (ketone aroma type - Zhaoqing patchouli):

[0066] Forward primer: 5'-AGGAGCCTTTAGGTGTTGCT-3' (SEQ ID No: 3),

[0067] Reverse primer 2: 5'-ACACTATGGAAAAGGGAGGCC-3' (SEQ ID No:4).

[0068] The third primer pair with the third molecule marker (ketone aroma type - Shipai patchouli):

[0069] Forward primer: 5'-CCCTGAAAAGAAAAGGCTACC-3' (SEQ ID No: 5),

[0070] Reverse primer: 5'-CCCACCATAGTAAAAAAGTTGTAAG-3' (SEQ ID No: 6).

[0071] The fourth primer with the fourth molecule label (patchouli with high content of both intermediate-type alcohol and ketone):

[0072] Primer: 5'-TGGTGCACTTGAACAATCCT-3' (SEQ ID No: 7).

[0073] 3. Validation of the primer set for molecular markers

[0074] A method for identifying patchouli germplasm types includes the following steps:

[0075] Step 1: Extract DNA from the patchouli sample as a DNA template. DNA extraction was performed using the Tiangen Plant Genomics Kit (DP305) method, as described in the manufacturer's instructions. The purity and concentration of the DNA were determined using a Nanodrop 2000c (Thermo Scientific, USA). DNA samples with an OD260 / 280 range of 1.8–2.0 were stored at -20°C for later use.

[0076] Step 2: Use the primer set described above to perform PCR amplification on the DNA template to obtain the PCR amplification product;

[0077] The PCR reaction system consisted of: 12.5 μl of 2X Pro Taq Master Mix (dye plus), 8.5 μl of ddH2O, 1.0 μl of 10 μmol / L forward and reverse primers, and 2.0 μl of 10–30 ng / μL template DNA.

[0078] The PCR reaction program was as follows: 94℃ pre-denaturation for 30s; 94℃ denaturation for 30s, 56℃ annealing for 30s, 72℃ extension for 1min, 35 cycles; 72℃ extension for 10min; storage at 4℃.

[0079] Step 3: Sequencing the PCR amplification products and performing genotyping based on the sequencing results.

[0080] (1) Electrophoretic detection

[0081] The PCR products were detected by 0.7% agarose gel electrophoresis. The electrophoresis conditions were 80V and 50min. The results are as follows: Figures 1-4 As shown in the image, the imaging results indicate that all four primer pairs successfully amplified clear bands for the four different patchouli samples. However, the band length of the PCR amplification product of the same primer did not differ significantly among the four patchouli samples. This result suggests that, on the gel imaging layer, we cannot effectively distinguish between Zhanjiang patchouli (alcoholic type), Zhaoqing patchouli (ketone type), Shipai patchouli (ketone type), and the intermediate type (patchouli with high alcohol and ketone content).

[0082] (2) Sanger sequencing and sequence alignment

[0083] PCR products and their corresponding forward and reverse primers were sent to Sangon Biotech (Shanghai) Co., Ltd. for bidirectional sequencing. The sequencing results for each sample included both forward and reverse sequences. The sequencing results of the forward and reverse primers and the corresponding reference sequences were imported into SnapGene software for sequence alignment. The sequence alignment results are shown below. Figures 5-12 As shown. Comparing the T base at position 44138387 on chromosome A02 of the nuclear genome, when the base is inserted at TTCT, it is Zhanjiang patchouli - alcoholic type; comparing the T base at position 25567854 on chromosome A02 of the nuclear genome, when the base is inserted at TGGAATTAGTGAGT, it is ketone-type - Zhaoqing patchouli; comparing the A base sequence at position 33983091 on chromosome A03 of the nuclear genome, when an additional A is inserted at this position, it is ketone-type - Shipai patchouli; comparing the A base at position 42533373 on chromosome A05 of the nuclear genome, when the base is inserted at GTA, it is intermediate type - patchouli with high alcohol and ketone content.

[0084] 4. Application of primer sets with 4 marker sites in the practical identification of patchouli germplasm types

[0085] A single-blind experimental design was adopted, in which the designers numbered the various germplasms, and then the testers conducted tests on samples with different numbers. To verify the detection efficacy of the developed molecular markers, different germplasm types of patchouli were randomly numbered (A: ketone-type - Shipai patchouli, B: Zhanjiang patchouli - alcohol-type, C: ketone-type - Zhaoqing patchouli, D: intermediate type - patchouli with high alcohol and ketone content) (Table 1). Operators unaware of the germplasm type performed DNA extraction, PCR amplification, and first-generation sequencing. The identification steps were the same as described in Part 3 above, directly using primers for PCR amplification, and the sequencing data was sent out for germplasm identification based on the sequence composition.

[0086] Table 1 Sample Source and Number

[0087]

[0088] Depend on Figure 12-18 It can be seen that the identification results are consistent with the actual situation, and the germplasm types of all samples can be successfully detected.

[0089] In summary, this invention utilizes four molecular markers based on genome-wide genetic variation to identify different patchouli germplasms. Compared to earlier molecular identification methods, its advantages lie in its ability to rapidly and accurately identify patchouli germplasms of different types. Furthermore, this method is simple to operate, easy to perform in the laboratory, and readily applicable, possessing broad market application prospects. In addition, this technology can be effectively used for patchouli germplasm identification and conservation, and the developed molecular markers can be used for commercial applications in patchouli germplasm type identification, bringing significant economic benefits and good social value.

[0090] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A primer set for amplifying molecular marker combinations that can identify the germplasm type of patchouli, characterized in that, The molecular marker combination comprises a first molecular marker, a second molecular marker, a third molecular marker, and a fourth molecular marker; the nucleotide sequence of the first molecular marker is shown in SEQ ID No:8, and an insertion / deletion as follows is present at 35 bp of the nucleotide sequence shown in SEQ ID No:8: TTCT; the nucleotide sequence of the second molecular marker is shown in SEQ ID No:9, and an insertion / deletion as follows is present at 39 bp of the nucleotide sequence shown in SEQ ID No:9: TGGAATTAGTGAGT; the nucleotide sequence of the third molecular marker is shown in SEQ ID No:10, and an insertion / deletion as follows is present at 47 bp of the nucleotide sequence shown in SEQ ID No:10: A; the nucleotide sequence of the fourth molecular marker is shown in SEQ ID No:11, and an insertion / deletion as follows is present at 31 bp of the nucleotide sequence shown in SEQ ID No:11: GTA; The primer set is as follows: The primer set for the first molecular marker is shown in SEQ ID No:1-2; The primer set for the second molecular marker is shown in SEQ ID No:3-4; The primer set for the third molecular marker is shown in SEQ ID No:5-6; The primers for the fourth molecular marker are shown in SEQ ID No:7; When the first molecular marker inserts a base of TTCT, it is Zhanjiang patchouli - alcoholic type; when the second molecular marker inserts a base of TGGAATTAGTGAGT, it is ketone type - Zhaoqing patchouli; when the third molecular marker inserts a base of A, it is ketone type - Shipai patchouli; when the fourth molecular marker inserts a base of GTA, it is intermediate type - patchouli with high alcohol and ketone content.

2. A kit for amplifying molecular marker combinations that can identify the germplasm type of patchouli, characterized in that, The primer set includes the molecular marker combination for amplifying the germplasm type of patchouli as described in claim 1.

3. The application of a primer set for amplifying molecular marker combinations that can identify patchouli germplasm types, characterized in that, The application of the primer set described in claim 1 in the identification of patchouli germplasm types: when the base inserted by the first molecular marker is TTCT, it is Zhanjiang patchouli - alcoholic type; when the base inserted by the second molecular marker is TGGAATTAGTGAGT, it is ketone type - Zhaoqing patchouli; when the base inserted by the third molecular marker is A, it is ketone type - Shipai patchouli; when the base inserted by the fourth molecular marker is GTA, it is intermediate type - patchouli with high alcohol and ketone content.

4. The application of a kit for amplifying molecular marker combinations that can identify patchouli germplasm types, characterized in that, The application of the kit described in claim 2 in the identification of patchouli germplasm types: when the base inserted by the first molecular marker is TTCT, it is Zhanjiang patchouli - alcoholic type; when the base inserted by the second molecular marker is TGGAATTAGTGAGT, it is ketone type - Zhaoqing patchouli; when the base inserted by the third molecular marker is A, it is ketone type - Shipai patchouli; when the base inserted by the fourth molecular marker is GTA, it is intermediate type - patchouli with high alcohol and ketone content.

5. A method for identifying the germplasm type of patchouli, characterized in that, Includes the following steps: Step 1: Extract DNA from the patchouli sample to be tested as a DNA template; Step 2: Perform PCR amplification on the DNA template using the primer set described in claim 1 or the kit described in claim 4 to obtain the PCR amplification product; Step 3: Sequencing the PCR amplification products and performing genotyping based on the sequencing results; When the first molecule marker inserts a base of TTCT, it is Zhanjiang patchouli – alcoholic type; when the second molecule marker inserts a base of TGGAATTAGTGAGT, it is ketone type – Zhaoqing patchouli; when the third molecule marker inserts a base of A, it is ketone type – Shipai patchouli; when the fourth molecule marker inserts a base of GTA, it is intermediate type – patchouli with high alcohol and ketone content.

6. The method for identifying patchouli germplasm types according to claim 5, characterized in that, The reaction system for PCR amplification in step 2 is as follows: 12.5 μl of 2X Pro Taq Master Mix (dye plus), 8.5 μl of ddH2O, 1.0 μl of 10 μmol / L primer set, and 2.0 μl of 10~30 ng / μL DNA template; The PCR amplification reaction program in step 2 is as follows: 94℃ pre-denaturation for 30s; 94℃ denaturation for 30s, 56℃ annealing for 30s, 72℃ extension for 1min, 35 cycles; 72℃ extension for 10min; 4℃ incubation.

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