SNP (Single Nucleotide Polymorphism) molecular marker for identifying edgeworthia chrysantha germplasm edgeworthia

Abstract

The invention discloses an SNP (Single Nucleotide Polymorphism) molecular marker for identifying the edgeworthia chrysantha germplasm edgeworthia chrysantha performance and application, and belongs to the technical field of biology. The 11 SNP molecular markers related to the agilawood formation performance of the germplasm of the Machilus davidii are disclosed for the first time, the agilawood formation capability, namely the agilawood formation performance, of the germplasm of the Machilus davidii can be identified by utilizing the SNP molecular markers, workers are guided to selectively cultivate and produce high-quality agilawood, and identification and assistant breeding of the germplasm of the Machilus davidii are facilitated; the method is easy to operate, economical, practical, rapid and efficient, and is shorter in consumed time without waiting for agilawood formation compared with a traditional method.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an SNP molecular marker for identifying the resin formation performance of Qinan agarwood and its application. Background Technology

[0002] As a new chemical type of Aquilaria sinensis, Qinan agarwood is characterized by its "easy resin formation" and "high-quality resin formation," leading to the booming development of the artificial cultivation industry for Qinan agarwood. However, due to the different origins of the mother trees of various Qinan agarwood varieties, the quality of the agarwood produced varies, resulting in inconsistent quality of Qinan agarwood sold in the market. Traditional methods for judging the quality of resin formation in a tree species require inducing damage to mature trees for six months to a year or even several years to obtain results, which is time-consuming.

[0003] SNP molecular marker technology, as a novel molecular marker technology, has advantages such as fast detection speed and accurate results, and has been increasingly used in the field of plant germplasm identification in recent years, including in the identification of Qinan agarwood germplasm. For example, patent application No. 202411686912.9 discloses a method for identifying Qinan agarwood germplasm resources, which detects seven SNP sites, uses PCR amplification and primer effect testing, and digests the PCR products with enzymes to distinguish Qinan agarwood from Aquilaria sinensis, and / or distinguish different varieties of Qinan agarwood. This method has the technical advantages of simple operation and low cost. However, this method can only be used to identify different Qinan varieties and distinguish Qinan agarwood from Aquilaria sinensis; it cannot directly identify the resin-forming performance of Qinan. In the Qinan agarwood planting industry, the resin-forming ability of Qinan germplasm is of great concern because it directly affects the economic benefits of planting. Existing technologies are insufficient to meet this key need, resulting in a lack of effective technical support for growers. Therefore, there is an urgent need for a method that can rapidly and accurately identify the properties of Qinan agarwood formation using SNP molecular marker technology, in order to promote the further development of the Qinan agarwood planting industry. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the present invention discloses an SNP molecular marker for identifying the resin formation performance of Qinan agarwood and its application.

[0005] The technical solution of this invention mainly includes the following: An SNP molecular marker for identifying the resin formation performance of Qinan agarwood, wherein the molecular marker is: The FLAS-02-SNP1 located at position 830 of the nucleotide sequence shown in SEQ ID NO.1 has a T / G variation. The FLAS-04-SNP1 located at position 661 of the nucleotide sequence shown in SEQ ID NO.2 has an A / G variation. FLAS-06-SNP6, located at position 1397 of the nucleotide sequence shown in SEQ ID NO.2, has a G / T variation at this site; FLAS-06-SNP7, located at position 1522 of the nucleotide sequence shown in SEQ ID NO.2, has a G / A variation at this site; HMGR-01-SNP1, located at position 352 of the nucleotide sequence shown in SEQ ID NO.3, has an A / C variation at this site; The PKS7-01-SNP4 located at position 477 of the nucleotide sequence shown in SEQ ID NO.4 has a G / T variation. The PKS7-03-SNP5 located at position 1373 of the nucleotide sequence shown in SEQ ID NO.4 has an A / T variation. The TPS18-03-SNP1 located at position 1065 of the nucleotide sequence shown in SEQ ID NO.5 has a C / T variation. The TPS18-03-SNP4 located at position 1314 of the nucleotide sequence shown in SEQ ID NO.5 has a G / A variation. The TPS18-09-SNP4 nucleotide at position 4583 of the nucleotide sequence shown in SEQ ID NO.5 has a C / T variation. The TPS18-13-SNP5 located at position 6235 of the nucleotide sequence shown in SEQ ID NO.5 has a G / A variation.

[0006] The primers used to detect the SNP molecular marker are as follows: The primer sequences for detecting FLAS-02-SNP1 are shown in SEQ ID NO.6~SEQ ID NO.8; The primer sequences for detecting FLAS-04-SNP1 are shown in SEQ ID NO.9~SEQ ID NO.11; The primer sequences for detecting FLAS-06-SNP6 are shown in SEQ ID NO.12~SEQ ID NO.14; The primer sequences for detecting FLAS-06-SNP7 are shown in SEQ ID NO.15~SEQ ID NO.17; The primer sequences for detecting HMGR-01-SNP1 are shown in SEQ ID NO.18~SEQ ID NO.20; The primer sequences for detecting PKS7-01-SNP4 are shown in SEQ ID NO.21~SEQ ID NO.23; The primer sequences for detecting PKS7-03-SNP5 are shown in SEQ ID NO.24~SEQ ID NO.26; The primer sequences for detecting TPS18-03-SNP1 are shown in SEQ ID NO.27~SEQ ID NO.29; The primer sequences for detecting TPS18-03-SNP4 are shown in SEQ ID NO.30~SEQ ID NO.32; The primer sequences for detecting TPS18-09-SNP4 are shown in SEQ ID NO.33~SEQ ID NO.35; The primer sequences for detecting TPS18-13-SNP5 are shown in SEQ ID NO.36~SEQ ID NO.38.

[0007] The application of the SNP molecular marker or the reagent for detecting the SNP molecular marker in identifying the resin formation performance of Qinan germplasm.

[0008] Furthermore, the reagent includes the aforementioned primers.

[0009] Furthermore, this invention relates to a method for identifying the resin formation properties of Qinan agarwood, comprising the following steps: Using DNA from agarwood germplasm samples as a template, KASP genotyping was performed using the aforementioned primers; the identification method was as follows: (1) Preliminary classification: If the genotypes of the TPS18-03-SNP4, TPS18-13-SNP5, HMGR-01-SNP1, FLAs-04_SNP1 and TPS18-09-SNP4 loci of the sample are of the following X1 or X2 categories, then the sample is classified as category A, and other genotype combinations are classified as category B;

[0010] (2) Further precise screening is performed on the superior and inferior germplasm populations classified in step (1): In the A-type germplasm population, if the genotype of a sample at the TPS18-03-SNP1, FLAs-02-SNP1, FLAs-06-SNP6, FLAs-06-SNP7, PKS7-01-SNP4, and PKS7-03-SNP5 loci is at least one of the following Y1~Y3 categories, then the sample is classified as germplasm with poor resin formation performance; other genotype combinations are classified as germplasm with good resin formation performance.

[0011] In the B-type germplasm population, if the genotype of a sample at the TPS18-03-SNP1, FLAs-02-SNP1, FLAs-06-SNP6, FLAs-06-SNP7, PKS7-01-SNP4, and PKS7-03-SNP5 loci is at least one of the following Z1 to Z4 categories, then the sample is classified as a germplasm with good resin formation performance; other genotype combinations are classified as germplasm with poor resin formation performance.

[0012] / indicates that the locus is any genotype.

[0013] The beneficial effects of this invention are: This invention provides SNP molecular markers and their primers for identifying the resin-forming performance of Qinan agarwood. Molecular marker technology can be used to identify the ability of Qinan agarwood to produce high-quality agarwood, i.e., the quality of resin-forming performance, guiding researchers to selectively cultivate and produce high-quality agarwood, and aiding in germplasm identification and assisted breeding. This method is simple to operate, economical, practical, fast, and efficient; compared to traditional methods, it eliminates the need to wait for resin formation and is much faster.

[0014] The SNP molecular markers of this invention are closely related to the resin-forming properties of Qinan agarwood, and can accurately identify the resin-forming ability of Qinan agarwood at the molecular level. The detection results are relatively consistent with the actual resin-forming results, with an identification concordance rate of 93.2%, and the detection results are accurate and reliable. Attached Figure Description

[0015] Figure 1 Comparison of the resinous properties of samples used for development and labeling.

[0016] Figure 2 Flowchart for molecular marker identification of agarwood formation properties. Detailed Implementation

[0017] To better understand the technical content of this invention, the invention will be further described below with reference to specific embodiments and accompanying drawings.

[0018] Example 1: Development of SNP Molecular Markers 1. Experimental Methods (1) Screening of Qinan germplasm using traditional methods Thirty-six healthy, undamaged, smooth, straight lateral stems (3cm in diameter) of various agarwood varieties were selected. Three branches were selected from each treatment group, and several holes spaced 10cm apart were drilled vertically using a 4mm drill bit to induce resin formation. Samples were collected after 6 months. The stem was longitudinally dissected along the distance between two holes to observe the formation of agarwood and photographed.

[0019] After the resin formation period, the resin formation effect on the lateral branches of 36 agarwood species induced by mechanical damage was observed for 6 months. During the observation, without causing large-scale damage to the resin-containing wood, the bark and sapwood around the drill hole were peeled away until the boundary between the sapwood and the brown color was visible. The range of resin variation near the drill hole was observed, and the resin formation status around the drill hole on the agarwood lateral branch was recorded. The resin formation performance was evaluated based on the longitudinal resin diffusion length of the stem, with the furthest point of the dark brown resin diffusion defined as the actual diffusion edge. Six holes were selected for each species as three replicates, and the average resin diffusion length between two holes was measured and counted using a ruler. This evaluation will compare various types of samples. Samples with good resin formation performance are those where the resin overflows from both boreholes and diffuses outwards, exhibiting a clear resin formation trend and a unidirectional diffusion distance greater than 2 cm. Among these, samples where the resin connects the two boreholes are considered the best in terms of resin formation performance. Samples where the resin overflows from both boreholes and diffuses outwards, with a unidirectional diffusion distance less than 2 cm, are considered poor in terms of resin formation performance. Samples where the resin does not overflow from the boreholes and the resin formation trend is relatively insignificant are considered the worst in terms of resin formation performance.

[0020] (2) Preparation of plant samples Ten representative individuals with significant differences in resin-forming performance from Qinan germplasm were selected, including five germplasm groups with good resin-forming performance (QLX, TJ, YYZ, AS, and JSY) and five germplasm groups with poor resin-forming performance (HZZ, YGL, XS3, XS8, and DWG). Common Aquilaria sinensis germplasm (BMX) served as a control. Three trees were selected from each germplasm group, and several fresh, tender green leaves were picked and quickly placed in a self-sealing bag filled with color-changing silica gel. The bag was sealed immediately, and the silica gel was replaced regularly. The bags were stored at room temperature in a cool, dark place for later use. Genomic DNA was extracted from the leaves.

[0021] (3) Sequence polymorphism analysis The promoters and full-length sequences of five key genes were divided into 300-800 bp fragments, and primers were designed for each fragment using the primer design tool Primer Premier 6. PCR amplification was performed using DNA from 33 samples of 11 different varieties of Aquilaria sinensis (including a common Aquilaria agallocha control) as templates, and the PCR products were sequenced.

[0022] (4) Development of molecular markers SNP loci with genotypic differences among germplasms exhibiting superior and inferior resin formation performance were selected. KASP reaction primers were designed based on the SNP locus sequence information. KASP genotyping mixtures and DNA templates from 33 samples were added to 96-well or 384-well PCR plates. Each PCR plate required at least two negative controls (NTCs). The plates were sealed, centrifuged, and then sealed again with a fluorescent membrane. PCR was performed using a quantitative real-time PCR instrument, and fluorescence values ​​were read after the reaction.

[0023] 2. Results and Analysis (1) Sequence polymorphism analysis Based on the electrophoresis results of the PCR products, PCR products matching the target fragment size were sequenced. The sequencing results were then assembled, and the polymorphism of five genes in the sample sequences was analyzed. To screen for stable SNP sites, base changes below the peak threshold of 25% were ignored. After excluding fragments with special structures such as palindromes, GC structures, polymorphic structures, repetitive sequence structures, or primer-specific double peaks and fragments without sequencing results, 61 detectable SNP sites were successfully obtained.

[0024] (2) Development of molecular markers Based on the base variations at 61 SNP sites in 33 samples from 11 different species, 41 SNP sites with good biological repeatability and different genotypes among different species with varying resin-forming performance were selected as candidate molecular markers for KASP genotyping. The genotyping results were compared with the sequencing results, and sites with inconsistent results or those not detected were excluded. Eleven SNP sites that can be used to accurately identify the resin-forming ability of Qinan species were obtained (Table 1).

[0025] Table 1. SNP site-specific primer numbers and sequences

[0026] Table 2

[0027] (3) Construction of the identification process for the resin formation performance of Qinan germplasm Based on 11 accurately genotyped SNP loci, the genotype combinations at each locus were adjusted to construct a two-step process for identifying the resin-producing performance of Qinan germplasm. Figure 2 ): Preliminary classification step 1: If a sample simultaneously meets the genotyping criteria at the loci in Table 3, it is classified as population A; germplasm that does not meet the genotyping criteria is classified as population B. The second step is precise identification: In the A-type germplasm population, germplasm that simultaneously meets the genotyping criteria at the four loci in Table 4 is classified as germplasm with poor resin formation performance, while the rest are classified as germplasm with good resin formation performance. In the B-type germplasm population, germplasm that simultaneously meets the genotyping criteria at the five loci in Table 5 is classified as germplasm with good resin-forming performance, while the rest are classified as germplasm with poor resin-forming performance.

[0028] Table 3

[0029] Table 4

[0030] Table 5

[0031] Example 2: Verification and Application of the Constructed Qinan Germplasm Agarwood Formation Performance Identification Technology We collected 134 Qinan germplasms with different resin formation properties, extracted their leaf DNA, and performed KASP genotyping detection according to the above method to verify the accuracy of the constructed identification technology.

[0032] The results of the identification are shown in Table 6: Five loci (TPS18-03-SNP4, TPS18-13-SNP5, HMGR-01-SNP1, FLAs-04-SNP1, and TPS18-09-SNP4) were used to initially distinguish two germplasm populations with superior and inferior resin-forming performance. Then, six more loci (TPS18-03-SNP1, FLAs-02-SNP1, FLAs-06-SNP6, FLAs-06-SNP7, PKS7-01-SNP4, and PKS7-03-SNP5) were used to precisely identify individuals with different resin-forming performance. After combining 11 molecular markers, 45 germplasm samples with good resin-forming performance and 89 germplasm samples with poor resin-forming performance were identified, which was relatively consistent with the actual resin-forming results, with an identification concordance rate of 93.2%.

[0033] Table 6

[0034] The above description is only a part of the embodiments of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention shall fall within the protection scope of the present invention.

Claims

1. A SNP molecular marker for identifying the resin formation performance of Qinan agarwood, characterized in that, The molecular marker is: The FLAS-02-SNP1 located at position 830 of the nucleotide sequence shown in SEQ ID NO.1 has a T / G variation. The FLAS-04-SNP1 located at position 661 of the nucleotide sequence shown in SEQ ID NO.2 has an A / G variation. FLAS-06-SNP6, located at position 1397 of the nucleotide sequence shown in SEQ ID NO.2, has a G / T variation at this site; FLAS-06-SNP7, located at position 1522 of the nucleotide sequence shown in SEQ ID NO.2, has a G / A variation at this site; HMGR-01-SNP1, located at position 352 of the nucleotide sequence shown in SEQ ID NO.3, has an A / C variation at this site; The PKS7-01-SNP4 located at position 477 of the nucleotide sequence shown in SEQ ID NO.4 has a G / T variation. The PKS7-03-SNP5 located at position 1373 of the nucleotide sequence shown in SEQ ID NO.4 has an A / T variation. The TPS18-03-SNP1 located at position 1065 of the nucleotide sequence shown in SEQ ID NO.5 has a C / T variation. The TPS18-03-SNP4 located at position 1314 of the nucleotide sequence shown in SEQ ID NO.5 has a G / A variation. The TPS18-09-SNP4 nucleotide at position 4583 of the nucleotide sequence shown in SEQ ID NO.5 has a C / T variation. The TPS18-13-SNP5 located at position 6235 of the nucleotide sequence shown in SEQ ID NO.5 has a G / A variation.

2. Primers for detecting the SNP molecular marker of claim 1, characterized in that, The primer sequences for detecting FLAS-02-SNP1 are shown in SEQ ID NO.6~SEQ ID NO.8; The primer sequences for detecting FLAS-04-SNP1 are shown in SEQ ID NO.9~SEQ ID NO.11; The primer sequences for detecting FLAS-06-SNP6 are shown in SEQ ID NO.12~SEQ ID NO.14; The primer sequences for detecting FLAS-06-SNP7 are shown in SEQ ID NO.15~SEQ ID NO.17; The primer sequences for detecting HMGR-01-SNP1 are shown in SEQ ID NO.18~SEQ ID NO.20; The primer sequences for detecting PKS7-01-SNP4 are shown in SEQ ID NO.21~SEQ ID NO.23; The primer sequences for detecting PKS7-03-SNP5 are shown in SEQ ID NO.24~SEQ ID NO.26; The primer sequences for detecting TPS18-03-SNP1 are shown in SEQ ID NO.27~SEQ ID NO.29; The primer sequences for detecting TPS18-03-SNP4 are shown in SEQ ID NO.30~SEQ ID NO.32; The primer sequences for detecting TPS18-09-SNP4 are shown in SEQ ID NO.33~SEQ ID NO.35; The primer sequences for detecting TPS18-13-SNP5 are shown in SEQ ID NO.36~SEQ ID NO.

38.

3. The application of the SNP molecular marker of claim 1 or the application of the reagent for detecting the SNP molecular marker of claim 1, characterized in that, Used to identify the resin formation properties of Qinan agarwood.

4. The application according to claim 3, characterized in that, The reagent includes the primers as described in claim 2.

5. A method for identifying the resin formation properties of Qinan agarwood, characterized in that, Includes the following steps: Using DNA from agarwood germplasm samples as a template, KASP genotyping was performed using the primers described in claim 2; the identification method was as follows: (1) Preliminary classification: If the genotypes of the TPS18-03-SNP4, TPS18-13-SNP5, HMGR-01-SNP1, FLAs-04_SNP1 and TPS18-09-SNP4 loci of the sample are of the following X1 or X2 categories, then the sample is classified as category A, and other genotype combinations are classified as category B; (2) Precisely filter within the groups categorized in step (1): In the A-type germplasm population, if the genotype of a sample at the TPS18-03-SNP1, FLAs-02-SNP1, FLAs-06-SNP6, FLAs-06-SNP7, PKS7-01-SNP4, and PKS7-03-SNP5 loci is at least one of the following Y1~Y3 categories, then the sample is classified as germplasm with poor resin formation performance; other genotype combinations are classified as germplasm with good resin formation performance. In the B-type germplasm population, if the genotype of a sample at the TPS18-03-SNP1, FLAs-02-SNP1, FLAs-06-SNP6, FLAs-06-SNP7, PKS7-01-SNP4, and PKS7-03-SNP5 loci is at least one of the following Z1 to Z4 categories, then the sample is classified as a germplasm with good resin formation performance; other genotype combinations are classified as germplasm with poor resin formation performance. / indicates that the locus represents any genotype.

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