SSR primers for the endangered species *Taihang flower* and their applications

Abstract

This invention belongs to the field of Taihang flower research. It discloses 15 pairs of specific SSR primers for Taihang flower, a national second-class protected plant and endangered species, and their applications in the analysis of genetic diversity and genetic structure characteristics of the endangered species Taihang flower population. This can reveal the phylogenetic and evolutionary relationship of Taihang flower populations and has important theoretical significance for the research and protection of the endangered species Taihang flower.

Description

Technical Field

[0001] This invention relates to SSR primers, and more particularly to specific SSR primers for the endangered species *Taihangia taihanga*. The invention further relates to the application of these specific SSR primers in the analysis of genetic diversity and population structure characteristics of the endangered species *Taihangia taihanga*, belonging to the field of *Taihangia taihanga* research. Background Technology

[0002] Taihangia rupestris Yu et Li is a perennial herbaceous plant listed as endangered in the wild by the International Union for Conservation of Nature (IUCN) and also included in the China Species Red List. X. Sun et al. (Sun, X., Wang, YP, Liu, C., Huang, LF (2019). Molecular identification of Taihangia rupestris Yu et Li, an endangered species endemic to China. South African Journal of Botany, 124, 173-177.) developed a DNA barcode based on partial genes from chloroplasts and the nuclear genome (Rubisco large subunit, maturase K, psbA-trnH, internally transcribed spacer (ITS), and ITS2) to distinguish two species of the genus Taihangia (Taihangia rupestris and Taihangia rupestris var. ciliata). Hong-Wei Wang et al. (Wang, HW, Fang, XM, Ye, YZ, Cheng, YQ, Wang, ZS (2011). High genetic diversity in Taihangia rupestris Yu et Li, a rare cliff herb endemic to China, based on Inter-simple sequence repeat markers. Biochemical Systems and Ecology, 39(4-6), 553-561.) Using ISSR molecular markers, the genetic diversity and differentiation characteristics of 10 populations of *Aurea taihangensis* were analyzed, demonstrating high genetic diversity at the population level among 10 different populations from Henan, Hebei, and Shanxi provinces, indicating a significant positive correlation between geographical distance and genetic distance. A search revealed that no primers designed based on SSR markers developed from nuclear genomes have yet been used to study the genetic diversity and structure of *Aurea taihangensis*.

[0003] Simple sequence repeats (SSRs) are repetitive sequences consisting of 1 to 6 nucleotides. They exhibit high polymorphism and abundance and are commonly used genetic markers. SSR markers possess high polymorphism and co-dominance, making them significant for genetic research and the utilization of gene resources. Summary of the Invention

[0004] The technical problem to be solved by this invention is to provide specific SSR primers for *Taihangia spp.*, which can be used to study the genetic diversity of different populations of *Taihangia spp.* and analyze the genetic structural characteristics of different populations of *Taihangia spp.*. This has important theoretical and practical significance for the development, utilization and protection of this species.

[0005] The technical solution of this invention is: a specific SSR primer for the endangered species *Taihangia taihangensis*, comprising the following primer pairs:

[0006] Primer pair 1: Consists of two single-stranded DNA molecules as shown in SEQ ID No. 1 and SEQ ID No. 2;

[0007] Primer pair 2: consists of two single-stranded DNA molecules as shown in SEQ ID No. 3 and SEQ ID No. 4;

[0008] Primer pair 3: consists of two single-stranded DNA molecules as shown in SEQ ID No. 5 and SEQ ID No. 6;

[0009] Primer pair 4: consists of two single-stranded DNA molecules as shown in SEQ ID No. 7 and SEQ ID No. 8;

[0010] Primer pair 5: consists of two single-stranded DNA molecules as shown in SEQ ID No. 9 and SEQ ID No. 10;

[0011] Primer pair 6: consists of two single-stranded DNA molecules as shown in SEQ ID No. 11 and SEQ ID No. 12;

[0012] Primer pair 7: consists of two single-stranded DNA molecules as shown in SEQ ID No. 13 and SEQ ID No. 14;

[0013] Primer pair 8: consists of two single-stranded DNA molecules as shown in SEQ ID No. 15 and SEQ ID No. 16;

[0014] Primer pair 9: consists of two single-stranded DNA molecules as shown in SEQ ID No. 17 and SEQ ID No. 18;

[0015] Primer pair 10: consists of two single-stranded DNA molecules as shown in SEQ ID No. 19 and SEQ ID No. 20;

[0016] Primer pair 11: Consists of two single-stranded DNA molecules as shown in SEQ ID No. 21 and SEQ ID No. 22;

[0017] Primer pair 12: consists of two single-stranded DNA molecules as shown in SEQ ID No. 23 and SEQ ID No. 24;

[0018] Primer pair 13: consists of two single-stranded DNA molecules as shown in SEQ ID No. 25 and SEQ ID No. 26;

[0019] Primer pair 14: consists of two single-stranded DNA molecules as shown in SEQ ID No. 27 and SEQ ID No. 28;

[0020] Primer pair 15: consists of two single-stranded DNA molecules as shown in SEQ ID No. 29 and SEQ ID No. 30.

[0021] In addition, this invention also provides the application of the specific SSR primers described above in the analysis of genetic diversity and population genetic structure of *Taihangia* populations.

[0022] Furthermore, the application includes the following steps:

[0023] (1) Extraction of Taihang flower genomic DNA

[0024] (2) PCR amplification was performed using the specific SSR primers described above.

[0025] (3) Detection of amplification products using polyacrylamide gel electrophoresis.

[0026] (4) Perform genetic diversity and population genetic structure analysis on the results of step (3).

[0027] The PCR reaction system for the aforementioned application is as follows:

[0028]

[0029] The PCR amplification procedure for the aforementioned application is as follows:

[0030]

[0031] Furthermore, the application involves conducting genetic diversity analysis on the Taihang flower population, using the CRVUS (version 3.0) software to calculate its genetic diversity indicators, which include the number of alleles, observed heterozygosity, expected heterozygosity, polymorphism information content, Hardy-Weinberg equilibrium, and null allele frequency of the Taihang flower population.

[0032] Furthermore, the application involves genetic structure analysis of the Taihang flower population, using Gene AlEx software to perform principal coordinate analysis based on genetic distance data.

[0033] The present invention has the following beneficial effects:

[0034] (1) This invention provides specific SSR primers for *Taihangia taihanga* to be used in the study of the genetic diversity of different populations of *Taihangia taihanga* and to analyze the genetic structural characteristics of different populations. The SSR primers developed in this invention are of great significance for the study of the phylogenetic evolution of the endangered plant *Taihangia taihanga*.

[0035] (2) Compared with other molecular markers such as RFLP and RAPD, microsatellite DNA molecular markers (SSR) have the characteristics of good specificity, high polymorphism and co-dominance, which provide important theoretical support for the scientific protection of the endangered species Taihang flower. Attached Figure Description

[0036] Figure 1 Polyacrylamide gel electrophoresis images of 45 individuals of *Taihangia* using 15 primer pairs (TH-1 to TH-15).

[0037] Figure 2 PCoA analysis based on genetic distance for 45 individuals of *Taihangia* (pop 1: population 1) and (pop 2: population 2). Detailed Implementation Plan

[0038] The present invention will be further illustrated below through a detailed description of specific embodiments, but this is not intended to limit the invention and is merely an illustrative example.

[0039] Example 1: Design and Screening of Specific SSR Primers

[0040] 1. Find sequences containing microsatellite repeat units.

[0041] (1) Obtaining the original sequence of Taihang flower: The nuclear genome DNA of Taihang flower was provided. This sequencing was performed using the Illumina Novaseq 6000 platform and de novo sequencing was performed to obtain the original sequence of the nuclear genome of Taihang flower.

[0042] (2) Raw data preprocessing and quality assessment: The raw data obtained from high-throughput sequencing in step (1) above were processed by adapter cutting, low-quality sequence removal, and short fragment (<35bp) removal to filter out low-quality data. Blast+ was used to compare 2000 randomly selected sequences from the raw data to determine whether the raw sequences were contaminated.

[0043] (3) Use Misa (version 1.0) software to find SSRs. The parameters are as follows: minimum SSR spacing 200bp, selection criteria are shown in Table 1:

[0044] Table 1 SSR cutoff

[0045]

[0046] 2. SSR primer design and screening

[0047] After obtaining the SSR sequence information, primers were designed and organized for these SSR sequences using Primer3 (version 2.3.5).

[0048] A total of 72,768 SSR loci were identified, and three different primer pairs were designed for each SSR locus. One primer pair was selected for each locus. Among these primer pairs, 100 primer pairs that met the criteria were selected using primer screening principles (annealing temperature around 60℃; GC content of forward and reverse primers between 45% and 55%; base repetition more than 5 times).

[0049] Preliminary experiments were conducted using these 100 primer pairs. Ten *Taihangia* species from different populations were randomly selected, and their genomic DNA was extracted using the CTAB method. Amplification reactions were performed according to the PCR reaction systems shown in Tables 2 and 3 below, followed by detection using polyacrylamide gel electrophoresis. Based on the electrophoresis band results, 20 pairs of SSR markers with good stability and high polymorphism were selected from the 100 primer pairs. Software was used to verify the SSR markers for invalid alleles or other read errors, ultimately selecting 15 valid SSR marker pairs (Table 4).

[0050] Table 2 PCR reaction system

[0051]

[0052] Table 3 PCR Amplification Procedure

[0053]

[0054] Table 4 shows the 15 pairs of SSR primers selected from *Taihangia*.

[0055]

[0056]

[0057] Note: TH-1 to TH-15 represent 1 to 15 pairs of specific SSR primers for *Taihangia*.

[0058] Example 2: Genetic diversity analysis of Taihang flower populations

[0059] Genetic diversity indicators mainly include the number of alleles (Na), the number of effective alleles (Ne), observed heterozygosity (Ho), expected heterozygosity (He), and inbreeding coefficient (FIS). We selected two populations from Guanshan, Xinxiang, Henan Province: population 1 (35°33'46"N, 113°31'31"E, n=24) and population 2 (35°33'54"N, 113°31'40"E, n=21). We used cervus (version 3.0) software to calculate the various genetic diversity indicators of the two populations, and the results are shown in Table 5.

[0060] Table 5. Genetic characteristics analysis of 15 pairs of SSR markers from 45 individuals of *Taihangia*.

[0061]

[0062] Note: TH-1 to TH-15 represent the 1st to 15th pairs of specific SSR primers for *Taihangia*. HW: Hardy-Weinberg equilibrium.

[0063] Example 3: Genetic Structure Analysis of Taihang Flower Population

[0064] We selected two populations from Guanshan, Xinxiang, Henan Province: population 1 (35°33'46"N, 113°31'31"E, n=24) and population 2 (35°33'54"N, 113°31'40"E, n=21). We used GenAlEx (version 6.5) software to perform principal coordinate analysis (PCoA) based on genetic distance data. The results of the principal coordinate analysis based on genetic distance are shown below. Figure 2 .

Claims

1. Specific SSR primers for the endangered species *Taihangia*, characterized in that, Including the following 15 primer pairs: Primer pair 1: Consists of two single-stranded DNA molecules as shown in SEQ ID No. 1 and SEQ ID No. 2; Primer pair 2: consists of two single-stranded DNA molecules as shown in SEQ ID No. 3 and SEQ ID No. 4; Primer pair 3: consists of two single-stranded DNA molecules as shown in SEQ ID No. 5 and SEQ ID No. 6; Primer pair 4: consists of two single-stranded DNA molecules as shown in SEQ ID No. 7 and SEQ ID No. 8; Primer pair 5: consists of two single-stranded DNA molecules as shown in SEQ ID No. 9 and SEQ ID No. 10; Primer pair 6: consists of two single-stranded DNA molecules as shown in SEQ ID No. 11 and SEQ ID No. 12; Primer pair 7: consists of two single-stranded DNA molecules as shown in SEQ ID No. 13 and SEQ ID No. 14; Primer pair 8: consists of two single-stranded DNA molecules as shown in SEQ ID No. 15 and SEQ ID No. 16; Primer pair 9: consists of two single-stranded DNA molecules as shown in SEQ ID No. 17 and SEQ ID No. 18; Primer pair 10: consists of two single-stranded DNA molecules as shown in SEQ ID No. 19 and SEQ ID No. 20; Primer pair 11: Consists of two single-stranded DNA molecules as shown in SEQ ID No. 21 and SEQ ID No. 22; Primer pair 12: consists of two single-stranded DNA molecules as shown in SEQ ID No. 23 and SEQ ID No. 24; Primer pair 13: consists of two single-stranded DNA molecules as shown in SEQ ID No. 25 and SEQ ID No. 26; Primer pair 14: consists of two single-stranded DNA molecules as shown in SEQ ID No. 27 and SEQ ID No. 28; Primer pair 15: consists of two single-stranded DNA molecules as shown in SEQ ID No. 29 and SEQ ID No.

30.

2. The application of the specific SSR primers described in claim 1 in the analysis of genetic diversity and genetic structure characteristics of Taihang flower populations.

3. The application according to claim 2, characterized in that... Includes the following steps: (1) Extracting genomic DNA from *Taihangia stenoptera*; (2) PCR amplification using the specific SSR primers described in claim 1; (3) The amplification products were detected by polyacrylamide gel electrophoresis; (4) Use the results of (3) above to analyze the genetic diversity and genetic structure characteristics of the Taihang flower population.

4. The application as described in claim 3, characterized in that, The PCR reaction system is as follows: 。 5. The application as described in claim 3, characterized in that, The PCR amplification procedure is as follows: 。 6. The application as described in claim 3, characterized in that: The application described herein is for the analysis of genetic diversity of the Taihang flower population. The Cervus version 3.0 software is used to calculate the genetic diversity indicators of the Taihang flower population. The genetic diversity indicators include the number of alleles, observed heterozygosity, expected heterozygosity, polymorphism information content, Hardy-Weinberg equilibrium, and null allele frequency.

7. The application as described in claim 3, characterized in that: The application described is to perform genetic structure analysis on the Taihang flower population, using Gen AlEx version 6.5 software to perform principal coordinate analysis based on genetic distance data.

Images