SSR primer pair for genetic diversity evaluation of betula microphylla and application of SSR primer pair

Abstract

The invention discloses an SSR primer pair for genetic diversity evaluation of betula microphylla and application of the SSR primer pair, and relates to the technical field of biology. The SSR primer pair comprises a primer pair BM-11 and a primer pair BM-14, wherein the primer pair BM-11 is a single nucleotide sequence; the primer pair BM-11 comprises an upstream primer of which the nucleotide sequence is as shown in SEQ ID NO.1 and a downstream primer of which the nucleotide sequence is as shown in SEQ ID NO.2; the primer pair BM-14 comprises an upstream primer of which the nucleotide sequence is shown as SEQ ID NO.3 and a downstream primer of which the nucleotide sequence is shown as SEQ ID NO.4. The primer pair BM-14 has the advantages that the nucleotide sequence is shown as SEQ ID NO.3; the SSR primer pair can be used for genetic diversity analysis of the betula microphylla, so that the classification status of the betula microphylla can be better illuminated. The method can provide important scientific values for species classification status determination, high-quality provenance conservation and regional specific wild forest resource utilization of the birch microphylla.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to an SSR primer pair for evaluating the genetic diversity of Betula microphylla and an application thereof. Background Art

[0002] Betula microphylla Bge., a member of the genus Betula (Betula L.) in the family Betulaceae, is a clumping or trunk-bearing tree with a shallow root system and bark ranging from grayish-white to brownish-red. Its leaves range from rhombus-shaped, rhombus-elliptical, to rhombus-obovate. Its catkins are unisexual and hermaphroditic. Its fruit is leathery, and its nutlets are covered with membranous wings. Betula microphylla has significant development value for improving wetland environments and enhancing landscape optimization. Through detailed observation of key morphological characteristics of the small-leaved birch, including leaves, bracts, and bark shedding, researchers have identified five subspecific variants at the subspecific level: broad-bract small-leaved birch (B. microphylla Bunge var. latibracteata), Kanas small-leaved birch (B. microphylla Bunge var. harasiica), swamp small-leaved birch (B. microphylla Bunge var. paludosa), Tumant small-leaved birch (B. microphylla Bunge var. tumantica), and Lake Ebinurica small-leaved birch (B. microphylla Bunge var. ebinurica). The habitat and phenotypic diversity of this species, resulting in these five variants, clearly indicate that its classification remains controversial. Furthermore, field surveys have revealed trends of premature aging and increased mortality. Clarifying the taxonomic status and genetic diversity of this unique wild plant resource is urgent, in order to provide guidance for the efficient utilization and adequate protection of this unique wild plant resource.

[0003] SSR (Simple Sequence Repeat) and ISSR (Inter-Simple Sequence Repeat) are two commonly used molecular marker technologies in molecular biology, both of which rely on microsatellite (SSR) sequences in the genome. Microsatellites are short tandem repeat sequences composed of 1-6 nucleotides. Unlike some marker SNPs, ISSR and SSR markers do not require complete genomic sequence information to be developed. In comparison, SSR is more accurate, highly reproducible and site-specific, but ISSR has low reproducibility due to the loose primer annealing conditions, which may produce non-specific bands. Previous studies on the taxonomic status of the Ebinur Lake small-leaved birch population were also conducted using universal ISSR primers. In recent years, researchers have successively developed SSR primers for white birch (Betula. platyphylla) and weeping birch (Betula. pendula), but no SSR primers have been developed for small-leaved birch. Summary of the Invention

[0004] The present invention aims to provide an SSR primer pair for evaluating the genetic diversity of Betula microphylla and its application to solve the problems existing in the above-mentioned prior art. The SSR primer pair can be used for genetic diversity analysis of Betula microphylla, thereby better clarifying the taxonomic status of Betula microphylla.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides an SSR primer pair for evaluating the genetic diversity of Betula microphylla, comprising a primer pair BM-11 and a primer pair BM-14;

[0007] The primer pair BM-11 includes an upstream primer having a nucleotide sequence as shown in SEQ ID NO.1 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO.2;

[0008] The primer pair BM-14 includes an upstream primer whose nucleotide sequence is shown as SEQ ID NO.3 and a downstream primer whose nucleotide sequence is shown as SEQ ID NO.4.

[0009] The present invention also provides a primer pair combination for evaluating the genetic diversity of Betula microphylla, comprising 6 ISSR primers and the above-mentioned SSR primer pair;

[0010] The nucleotide sequences of the six ISSR primers are shown in SEQ ID NOs. 5-10, respectively.

[0011] The present invention also provides the use of the above-mentioned SSR primer pair in preparing a product for evaluating the genetic diversity of Betula microphylla.

[0012] The present invention also provides use of the primer pair combination in preparing a product for evaluating the genetic diversity of Betula microphylla.

[0013] Furthermore, the product is a kit.

[0014] The present invention also provides a product for evaluating the genetic diversity of Betula microphylla, comprising the above-mentioned SSR primer pair or primer pair combination.

[0015] Furthermore, the product is a kit.

[0016] The present invention also provides application of the above-mentioned SSR primer pair in evaluating the genetic diversity of Betula microphylla.

[0017] The present invention also provides application of the primer pair combination in evaluating the genetic diversity of Betula microphylla.

[0018] The present invention also provides application of the above product in evaluating the genetic diversity of Betula microphylla.

[0019] The present invention discloses the following technical effects:

[0020] The present invention has developed SSR primers that can be used for genetic diversity analysis of Betula microphylla, which can better clarify the taxonomic status of Betula microphylla. It has been verified that the effective number of alleles (Ne) revealed by PCR amplification results of SSR primers is 1.5657, and that of ISSR is 1.3844, indicating that the alleles detected by SSR primers are richer and more polymorphic; Nei's gene diversity index (H) is greater in SSR (0.3293) than in ISSR (0.2472), indicating that the population genetic diversity detected by SSR primers is higher. Shannon diversity index (I) is greater in SSR (0.4973) than in ISSR (0.3960), further supporting that SSR can detect richer genetic variation than ISSR.

[0021] The present invention further uses SSR primers and ISSR to jointly calculate the genetic distance and evaluate the genetic diversity of Betula microphylla populations, which not only clarifies the taxonomic status of Betula microphylla, but also provides a scientific basis for the rational utilization and protection of germplasm resources in the future.

[0022] The present invention can provide important scientific value for determining the taxonomic status of Betula microphylla species, conserving high-quality provenance, and utilizing regional unique wild forest resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0024] Figure 1 Agarose gel electrophoresis results of PCR amplification products screened by multiple primers;

[0025] Figure 2 Agarose gel electrophoresis results of PCR amplification products screened by single primers;

[0026] Figure 3 Agarose gel electrophoresis results of PCR amplification products of Betula microphylla DNA samples JH-1-10, SW-1-10, WS-1-10, YM-1-10, TL-1-10, XY-1-9, GL-1-10 and MGH-1-7 using the screened SSR primers;

[0027] Figure 4 Agarose gel electrophoresis results of PCR amplification products of Betula microphylla DNA samples ZS-1-10, KC-1-10, NLK-1-10, HC-1-6, MNS-1-7, TKS-1-6 and YC-1-10 using the screened SSR primers;

[0028] Figure 5 Agarose gel electrophoresis results of PCR amplification products of Betula microphylla DNA samples TCG-1-18, HBH-1-5, HF-1-9, WQ-1-10, EM-1-10, JMN-1-7 and BSW-1-2 using the screened SSR primers;

[0029] Figure 6 Agarose gel electrophoresis results of PCR amplification products of Betula microphylla DNA samples JH-1-10, SW-1-10, WS-1-10, YM-1-10, TL-1-10, XY-1-9 and GL-1-10 using 6 pairs of ISSR primers;

[0030] Figure 7 Agarose gel electrophoresis results of PCR amplification products of Betula microphylla DNA samples TCG-1-18, HBH-1-5, HF-1-9, WQ-1-10, EM-1-10, JMN-1-7 and BSW-1-2 using 6 pairs of ISSR primers;

[0031] Figure 8Agarose gel electrophoresis results of PCR amplification products of Betula microphylla DNA samples ZS-1-10, KC-1-10, NLK-1-10, HC-1-6, MNS-1-7, TKS-1-6, YC-1-10 and MGH-1-7 using 6 pairs of ISSR primers;

[0032] Figure 9 The figure shows the polymorphism comparison of 2 pairs of SSR primers and 6 pairs of ISSR primers; A is the result of primer genetic diversity analysis; B is the result of primer population heterozygosity, gene flow and genetic differentiation analysis;

[0033] Figure 10 This is a phylogenetic tree based on the Nei's genetic distance calculated comprehensively. DETAILED DESCRIPTION

[0034] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0035] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0036] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0037] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0038] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0039] Example 1

[0040] Leaves of the tested Betula microphylla were collected and subjected to simplified genome sequencing using the SLAF-seq (Specific-Locus Amplified Fragment Sequencing) technology independently developed by Beijing Biomarker Biotechnology Co., Ltd. SLAF markers were developed and SNP information was collected. SSR loci were identified and primers were designed based on their flanking sequences. The designed SSR primers were screened. 193 leaf DNA samples from 22 Betula microphylla populations (Table 1) were amplified by polymerase chain reaction (PCR) using the screened primers. A "0 / 1" matrix was created for the amplification results, and the genetic diversity correlation index was calculated using software, as detailed below:

[0041] Table 1 Betula microphylla populations

[0042] serial number Population Abbreviation Altitude / m Habitat 1 Wusu City WS 315 wetlands 2 Mushroom Lake in Shihezi City MGH 350 plain wetland 3 Shawan City SW 1500 Mountain Valley 4 Manas County MNS 1127 Mountain Valley 5 Wenquan County WQ 1680 Mountain 6 Jinghe County JH 240 swamp wetlands 7 Yumin County YM 1960 Mountain grassland 8 Habahe County HBH 384 swamp wetlands 9 Beishawo area of ​​Jimunai County BSW 874 plain wetland 10 Emin County EM 672 Mountain meadow 11 Jimunai County JNM 962 swamp wetlands 12 Tiechanggou Town, Tuoli County TCG 656 wetlands 13 Tori County TL 850 plain wetland 14 Hefeng County HF 1167 wetlands 15 Xinyuan County XY 1430 Mountain Valley 16 Zhaosu County ZS 1400 meadow 17 Huocheng County HC 1500 River Valley 18 Nileke County NLK 1009 River Valley 19 Tekes County TKS 1143 River Valley 20 Gongliu County GL 1676 meadow 21 Yecheng County YC 2600 Mountain Valley 22 Kuqa City KC 2064 Mountain Valley

[0043] DNA extraction from the collected leaves specifically included the following steps: 0.1-0.3 g of frozen leaves were taken and ground into a fine powder in a mortar; the powder was transferred to preheated CTAB extraction buffer (containing 2% CTAB, 1.4 M NaCl, 20 mM EDTA, 100 mM Tris-HCl pH 8.0, and 1% PVP) and incubated at 65°C in a water bath for 60 min; β-mercaptoethanol (final concentration 0.2-2%) was added to reduce disulfide bonds and prevent polyphenol oxidation; after cooling, an equal volume of chloroform:isoamyl alcohol (24:1) was added, the mixture was gently mixed, and centrifuged at 12,000 rpm for 15 min; the supernatant was extracted 1-2 times until no precipitate was present at the interface; 0.6 times the volume of isopropanol was added to the supernatant, the mixture was gently inverted and allowed to stand at -20°C for 30 min; the white flocculent precipitate was collected by centrifugation and washed twice with 70% ethanol; the precipitate was dried and then washed with TE buffer (10 mM Tris-HCl, 1 mM EDTA) to dissolve DNA; NanoDrop concentration meter to measure A 260 / A 280 (1.8-2.0 is optimal; below 1.8 may indicate protein / phenol contamination); agarose gel electrophoresis shows clear main bands without tailing, indicating that the extracted DNA is of good quality.

[0044] Simplified genome sequencing was performed using the SLAF-seq (Specific-Locus Amplified Fragment Sequencing) technology independently developed by Beijing Biomarker Biotechnology Co., Ltd. The specific process includes: electronic enzyme digestion prediction of the reference genome of closely related species, and determination of the restriction endonuclease combination as RsaⅠ+HaeⅢ based on the enzyme digestion scheme selection principle; sequencing quality value distribution check: To ensure the quality of project analysis, a read length of 126bp×2 was used as the subsequent data evaluation and analysis data, and the sequencing quality value (Q) was calculated to evaluate the single-base error rate of high-throughput sequencing. The higher the sequencing quality value, the lower the corresponding base sequencing error rate. The corresponding formula for the base sequencing error rate P and the sequencing quality value Q is: Q -score = -10×log 10 P; Base Distribution Check: SLAF-seq sequencing reads are enzyme-digested fragments of genomic DNA. Their base distribution is affected by the enzyme cleavage site and PCR amplification. The first two bases of a sequencing read will show a base separation consistent with the enzyme cleavage site, and the subsequent base distribution will fluctuate to varying degrees. Sequencing Data Output and Quality Statistics: Sequencing data for each sample was statistically analyzed, including read number, Q30, and GC content. Based on the above steps, the sequencing data summary shows that a total of 387 Mb of reads were obtained in this project, with an average sequencing Q30 of 95.47% and an average GC content of 42.95%.

[0045] The process for SLAF marker development and SNP information statistics is as follows: Reads generated by sequencing are derived from enzyme-digested fragments of the same length produced by the same restriction endonuclease on different samples. Reads from each sample are clustered based on sequence similarity, and reads clustered together are derived from a SLAF fragment (SLAF tag). The sequence similarity between different samples for the same SLAF tag was much higher than the similarity between different SLAF tags. To ensure the reliability and practicality of the final SLAF tags, sequences with enzyme-digested fragment lengths between 264 and 344 bp were defined as SLAF tags. A SLAF tag with sequence differences between different samples (i.e., polymorphism) was defined as a polymorphic SLAF tag. SNP marker development used the highest-depth sequence type within each SLAF tag as a reference. Clean reads from all sample sequencing were aligned to the reference genome using BWA (0.7.10-r789). SNPs were developed across all samples using GATK (v3.8) and SALMTools (v1.9). The intersection of these two data sets yielded the final reliable SNP marker dataset. Combining the above processes, a total of 311,670 SLAF tags were developed, with an average sequencing depth of 11.99x, resulting in 334,223 SNP markers.

[0046] The contents and basis for designing primers based on the flanking sequences of SSR sites include: the polymorphism of SLAF tags is mainly due to the presence of SNP (Single Nucleotide Polymorphism) sites. SSR sites are searched on sequences with SNP sites ≥10, and SSR primers are designed based on the flanking sequences of SSR sites. The design of SSR primers is based on (1) the number of base motif repetitions ≥5; (2) the maximum temperature difference between forward and reverse primers is guaranteed to be 2°C; (3) the primer itself does not have 4 consecutive bases complementary to avoid the self-cyclization phenomenon of the primer when it is not bound to the template; (4) the G+C content is generally within 40%-60%. The Tm value is the melting temperature of the oligonucleotide chain. If the GC content of the upstream and downstream primers is very different, it will affect the Tm value; (5) the four bases of the primer are randomly distributed. And there is no polypurine or polypyrimidine, especially no more than three consecutive G or C bases at the 3' end, otherwise the primer will make mistakes in the G+C rich sequence region; (6) The length of the amplified product is ≥120bp, and a suitable DNA marker can be selected and easily distinguished from the primer dimer band to avoid band reading errors.

[0047] Screening of designed SSR primers using multiple primers and single primers:

[0048] A total of 30 designed primer pairs were divided into 10 groups, and three pairs of primers were added simultaneously to a PCR amplification system. Five DNA samples belonging to five populations were selected. 25 μL of multi-primer screening PCR system was added with 9.2 μL of ddH2O, 1.6 × 3 μL of primers (each at a concentration of 10 μmol / L), 1 μL of template DNA (25-50 ng / μL), and 10 μL of 2× Taq PCR premix II.

[0049] The 25 μL single primer screening PCR system includes 12.4 μL of ddH2O, 1.6 μL of primers (each primer concentration is 10 μmol / L), 1 μL of template DNA (25-50 ng / μL), and 10 μL of 2×Taq PCR premix reagent II.

[0050] Agarose gel electrophoresis results of PCR amplification products screened by multiple primers are shown in Figure 1 The agarose gel electrophoresis results of the PCR amplification products screened by single primers are shown in Figure 2 .

[0051] The selected SSR primers (Table 2) were used to amplify the leaf DNA materials of 22 Betula microphylla populations. The PCR amplification system was the same as the single primer screening PCR system mentioned above. A "0 / 1" matrix was established for the amplification results, and the genetic diversity correlation index was calculated using software: the amplified products were subjected to agarose gel electrophoresis ( Figure 3-Figure 5 ), observe and photograph with an ultraviolet imager and select the manual band reading method to analyze the bands with high clarity and good repeatability. The bands with bands are assigned "1" and the bands without bands are assigned "0". The original "0 / 1" numerical matrix is ​​obtained and input into POPGEN32 to calculate the mean number of alleles Na, the effective number of alleles Ne, Nei's gene diversity index H, and the Shannon information index i.

[0052] Table 2 Screened SSR primers

[0053]

[0054] Example 2

[0055] Six ISSR primers (see Table 3) were used to amplify the leaf DNA materials of 22 Betula microphylla populations (same as in Example 1), a "0 / 1" matrix was established for the amplification results, and the genetic diversity correlation index was calculated using software.

[0056] The ISSR primers were amplified using a 20 μL PCR system consisting of 7.2 μL of ddH2O, 0.8 μL of primers (10 μmol / L), 1 μL of template DNA (25-50 ng / μL), and 11 μL of 2×Taq PCR premix II.

[0057] A "0 / 1" matrix was established for the amplification results, and the genetic diversity related index was calculated using software: the amplified products were subjected to agarose gel electrophoresis ( Figure 6-Figure 8 ), observe and photograph using an ultraviolet imager and select the manual band reading method to analyze the bands with high clarity and good repeatability. The bands with bands are assigned "1" and the bands without bands are assigned "0". The original "0 / 1" numerical matrix is ​​obtained and input into POPG EN32 to calculate the mean number of alleles Na, the effective number of alleles Ne, Nei's gene diversity index H, and the Shannon information index i.

[0058] Table 3 ISSR primer sequences

[0059] ISSR primers Nucleotide sequence (5'-3') Sequence number UBC807 AGAGAGAGAGAGAGAGT SEQ ID NO.5 UBC811 GAGAGAGAGAGAGAGAC SEQ ID NO.6 UBC814 CTCTCTCTCTCTCTCTA SEQ ID NO.7 UBC827 ACACACACACACACACACG SEQ ID NO.8 UBC844 CTCTCTCTCTCTCTCTRC SEQ ID NO.9 UBC845 CTCTCTCTCTCTCTCTRG SEQ ID NO.10

[0060] The average number of alleles Na, effective number of alleles Ne, Nei's gene diversity index H, Shannon information index i, Nei's genetic distance D, genetic consistency and genetic distance were calculated based on the SSR primers and ISSR primers to construct the phylogenetic tree of 22 Betula microphylla populations. The main operations are: open the "ITOL" evolutionary tree production website, import the "0 / 1" matrix into the "tree files" generated by POPGEN32, and the results can be generated. Figure 10 The 22 Betula microphylla populations were divided into two major groups. Group I included seven populations, namely KC, MNS, HC, TKS, NLK, ZS, and YC, while another group consisted of 15 populations, namely GL, XY, TCG, WQ, HF, TL, WS, MGH, JH, SW, YM, EM, JMN, HBH, and BSW.

[0061] like Figure 9 As shown, comparing Example 1 and Example 2, the effective allele number (Ne) is higher in SSR (1.5657) than in ISSR (1.3844), indicating that the alleles detected by the SSR primers are richer and more polymorphic; and the closer the effective allele number is to 2, the stronger the polymorphism of the SSR primers (theoretical maximum value = 2). The Nei's gene diversity index (H) is higher in SSR (0.3293) than in ISSR (0.2472), indicating that the population genetic diversity detected by the SSR primers is higher.

[0062] The Shannon diversity index (I) was higher in the range of SSR (0.4973) than ISSR (0.3960), further supporting that SSR can detect richer genetic variations than ISSR.

[0063] In summary, the two pairs of SSR primers developed in the present invention have higher polymorphism and higher resolution than the six ISSR primers, and can be applied to the population genetic analysis of Betula microphylla.

[0064] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. An SSR primer pair for evaluating genetic diversity of Betula microphylla, characterized in that: Includes primer pair BM-11 and primer pair BM-14; The primer pair BM-11 includes an upstream primer having a nucleotide sequence as shown in SEQ ID NO.1 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO.2; The primer pair BM-14 includes an upstream primer having a nucleotide sequence as shown in SEQ ID NO.3 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO.

4.

2. A primer pair combination for evaluating the genetic diversity of Betula microphylla, characterized in that: comprising 6 ISSR primers and the SSR primer pair according to claim 1; The nucleotide sequences of the six ISSR primers are shown in SEQ ID NOs. 5-10, respectively.

3. Use of the SSR primer pair according to claim 1 in preparing a product for evaluating the genetic diversity of Betula microphylla.

4. Use of the primer pair combination according to claim 2 in preparing a product for evaluating the genetic diversity of Betula microphylla.

5. The use according to claim 3 or 4, characterized in that The product is a test kit.

6. A product for evaluating the genetic diversity of Betula microphylla, characterized in that: It includes the SSR primer pair described in claim 1 or the primer pair combination described in claim 2.

7. The product according to claim 6, characterized in that The product is a test kit.

8. Use of the SSR primer pair according to claim 1 in evaluating the genetic diversity of Betula microphylla.

9. Use of the primer pair combination according to claim 2 in evaluating the genetic diversity of Betula microphylla.

10. Use of the product according to claim 6 or 7 in evaluating the genetic diversity of Betula microphylla.

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