SSR molecular marker primer based on ixora chinensis transcriptome sequence and application of SSR molecular marker primer

Abstract

The invention discloses an SSR molecular marker primer based on a Chinese ixora transcriptome sequence and application of the SSR molecular marker primer, and belongs to the technical field of molecular markers. The SSR molecular marker primer is developed based on a transcriptome sequence of the ixora chinensis, on the basis of transcriptome sequencing, batch screening is conducted on a large amount of sequencing data, SSR sequences are obtained, SSR marker primer design is conducted, and the effectiveness of the SSR molecular marker primer is verified through different varieties of the ixora chinensis. The invention provides a valuable molecular marker for the research of genetic diversity analysis, genetic relationship analysis and the like of the existing ixora germplasm resources.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular markers and relates to an SSR molecular marker primer based on a transcriptome sequence of Ixora ilicifolia and an application thereof. Background Art

[0002] Ixora chinensis Lam. is a plant of the Rubiaceae family. It is also known as water hydrangea, white day red, fairy dan flower, mountain dan flower, etc. It is native to China and distributed in Guangxi Zhuang Autonomous Region, Hainan, Fujian, Guangdong, Hainan and other places. The genus Ixora is rich in plant resources. There are more than 400 species in the world, and about 19 species are distributed in my country, most of which are distributed in tropical regions. As a traditional Chinese medicinal material, the roots, stems, leaves and flowers of Ixora chinensis Lam. can be used as medicine, with the effects of clearing heat and cooling blood, dispersing blood stasis and relieving pain. Chinese Ixora chinensis is rich in chemical components. Modern pharmacological studies have shown that it has analgesic, anti-inflammatory, antioxidant, anti-tumor and other pharmacological effects. It is also a plant with ornamental value and is widely used in garden beautification and other aspects. Studies on genetic diversity analysis, variety identification and genetic map construction of Ixora chinensis are of great significance for its resource protection and variety breeding. However, there is currently a lack of effective molecular marker methods for Ixora, especially simple sequence repeat markers (SSR molecular markers) developed based on transcriptome information. Traditional molecular marker methods may have problems such as insufficient polymorphism and low specificity, which limits in-depth genetic research on Ixora. Summary of the invention

[0003] The purpose of the present invention is to provide an SSR molecular marker primer based on the transcriptome sequence of Ixora sibiricum and its application. In order to solve the problem of confusing naming of Ixora sibiricum varieties and unknown kinship, the present invention discovered a total of 15262 SSR sites based on the transcriptome sequencing data of Ixora sibiricum, and screened out 12 pairs of polymorphic SSR molecular marker primers, which can be used for genetic diversity detection and kinship analysis of Ixora sibiricum populations.

[0004] To achieve the above object, the present invention adopts the following technical solutions:

[0005] The present invention, based on transcriptome sequencing of mixed samples of Ixora sibiricum, deeply excavates the SSR sites of Ixora sibiricum and designs specific primers, and screens out 12 pairs of polymorphic SSR molecular marker primers that can be used for genetic diversity analysis and kinship analysis of Ixora sibiricum, which are: LC-16, LC-17, LC-26, LC-33, LC-34, LC-38, LC-41, LC-42, LC-43, LC-57, LC-58, LC-61. Genomic DNA of 55 Ixora sibiricum is extracted, and the genomic DNA is used as a template, and each pair of primers in the SSR marker primer set is used to perform PCR amplification reaction respectively, and then the PCR amplification products are subjected to polyacrylamide gel electrophoresis, and the band detection results are statistically analyzed, and the genetic diversity analysis and kinship analysis of Ixora sibiricum are performed using MEGA software.

[0006] The screening method of SSR molecular marker primers based on the transcriptome sequence of Ixora ilicifolia comprises the following steps:

[0007] (1) Total RNA was extracted from I. coccinea 'Apric Gold' and Ixora paruiflora, and the integrity and purity of the RNA were tested to prepare RNA libraries.

[0008] (2) After enriching eukaryotic mRNA with a polyA tail using magnetic beads with Oligo(dT), the mRNA was sheared using ultrasound. The fragmented mRNA was used as a template and random oligonucleotides as primers to synthesize the first cDNA chain in the M-MuLV reverse transcriptase system. The RNA chain was then degraded with RNaseH, and the second cDNA chain was synthesized using dNTPs as raw materials in the DNA polymerase I system. The purified double-stranded cDNA was end-repaired, A-tailed, and connected to sequencing adapters. AMPure XP beads were used to screen cDNA of about 200 bp, PCR amplification was performed, and the PCR product was purified again using AMPure XP beads to finally obtain the library.

[0009] (3) The transcriptome sequencing library of Illumina was sequenced by high-throughput sequencing to obtain raw reads.

[0010] (3) The raw reads were quality controlled using fastp to filter low-quality data and obtain clean reads. The clean reads were assembled and spliced ​​using Trinity software, and all assembled Unigenes were compared with four major databases, including Nr, KEGG, KOG, and SwissProt (evalue < 0.00001), to obtain the Unigenes database;

[0011] (4) The Unigenes database was searched to find SSRs in Unigene, and primers were designed on both sides of the SSR sequence using primer3 (version 1.1.4) based on the SSR information found.

[0012] (6) 100 pairs of primers were randomly selected and PCR amplification was performed using 55 samples of Ixora aurea DNA as templates. The products were detected by polyacrylamide gel electrophoresis to observe the specificity and band clarity of the amplified products, and primers with good amplification effect and strong specificity were screened out.

[0013] The above screening method is as follows:

[0014] 1) Collect fresh leaves of Ixora from different sources, quickly freeze them in liquid nitrogen, and then store them in a -80°C refrigerator. At the same time, prepare relevant reagents and instruments for RNA extraction, reverse transcription, PCR amplification, etc., such as RNA extraction kit, reverse transcriptase, DNA polymerase, dNTPs, primer synthesis reagents, centrifuge, PCR instrument, electrophoresis instrument, etc.

[0015] 2) RNA extraction and quality testing

[0016] Total RNA was extracted from the leaves of Ixora australis using a suitable RNA extraction kit. The concentration and purity of RNA were tested by a nucleic acid analyzer (OD260 / OD280 ratio between 1.8-2.0), and the integrity of RNA was checked by agarose gel electrophoresis, requiring that the 28S and 18S rRNA bands were clear and the brightness of the 28S rRNA band was about twice that of the 18S rRNA.

[0017] 3) cDNA synthesis

[0018] Use a high-quality reverse transcription kit to synthesize cDNA using the extracted RNA as a template. Prepare the reaction system according to the kit instructions, including an appropriate amount of RNA, reverse transcription primers, dNTPs, reverse transcriptase, and buffer, and complete the reverse transcription reaction under appropriate temperature and time conditions.

[0019] 4) Transcriptome sequencing and analysis

[0020] The synthesized cDNA was sequenced using the Illumina sequencing platform, and the quality of the obtained raw sequencing data was controlled. After removing low-quality reads, the transcript sequence was assembled using the professional transcriptome assembly software Trinity. SSR sites were found in the transcripts through a special SSR search algorithm and software, and sequence regions where the number of repetitions of repeating units (such as dinucleotides, trinucleotides, etc.) reached a certain threshold (such as dinucleotide repeats ≥ 6 times) were identified, and the location, repeat type, and length of the SSR site were recorded.

[0021] 5) SSR primer design

[0022] According to the conserved sequences on both sides of the SSR site searched, primers are designed using primer design software (such as Primer3). The following principles are followed when designing primers: primer length 18-22bp, GC content 40%-60%, primer annealing temperature difference no more than 5°C, avoid the formation of primer secondary structure and primer dimer.

[0023] 6) PCR amplification and detection

[0024] Using Ixora dasyphylla genomic DNA (obtained by conventional plant genomic DNA extraction methods) as a template, PCR amplification was performed using designed primers. The PCR reaction system includes template DNA, primers, dNTPs, DNA polymerase, and buffer, etc., and the reaction conditions (such as annealing temperature, number of cycles, etc.) are optimized. The amplified product is detected by agarose gel electrophoresis to observe whether there are clear and specific amplified bands.

[0025] 7) Polymorphism analysis and marker verification

[0026] For SSR primers with amplified products, amplification was performed in more Ixora individuals to analyze the polymorphism of amplified products between different individuals, such as by comparing the size of amplified fragments, the presence or absence of amplified fragments, etc. The polymorphism of SSR markers was determined. The selected polymorphic SSR markers were verified by repeated experiments and amplification on different batches of DNA samples to ensure the stability and reliability of the markers.

[0027] The present invention obtains an SSR molecular marker primer based on the transcriptome sequence of Ixora australis through the above screening method, including the following primer pairs:

[0028]

[0029]

[0030] Beneficial effects of the present invention:

[0031] The invention develops SSR molecular marker primers based on the transcriptome of Ixora sibiricum, which is highly targeted and can more accurately reflect the genetic information related to the gene expression of Ixora sibiricum. The developed SSR molecular markers have high polymorphism and can be effectively used for genetic diversity analysis, variety identification and genetic map construction of Ixora sibiricum, providing strong support for genetic research and breeding practice of Ixora sibiricum. The method has clear steps and strong operability, and the quality and reliability of molecular markers are guaranteed through strict quality control and verification steps. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1It is a statistical chart of the proportion of SSRs with different tandem repeat unit types in the total SSRs.

[0033] Figure 2 There are 55 germplasm resources of Ixora.

[0034] Figure 3 This is the result of LC-57 amplification.

[0035] Figure 4 This is a dendrogram of the phylogenetic relationships of 55 Ixora germplasm resources. DETAILED DESCRIPTION

[0036] The present invention is further described below by means of specific examples.

[0037] 1. Transcriptome data sources

[0038] The stems, buds, leaves, stamens, flowers and other parts of I. coccinea'Apric Gold' and Ixora paruiflora in different growth states were collected from the nursery of the Botanical Garden of Minnan Normal University, RNA was extracted from the mixed samples for transcriptome sequencing, and 71349 Unigenes were obtained by sequencing on the Illumina platform and assembled using Trinity software. 31676 Unigenes were annotated, and 7734 simple repeat sequences SSRs were screened. The present invention uses the 31676 Unigenes to search and analyze SSR sites.

[0039] 2. Search for SSR sites in the transcriptome of Ixora australis

[0040] Using MISA software to search 31676 Unigenes in the transcriptome of Ixora australis, 9432 SSR sites that meet the conditions were found, with an occurrence frequency (the ratio of the number of Unigenes containing SSR to the total number of Unigenes) of 10.84%. 9432 SSR sites that meet the conditions were identified, with an occurrence probability (the ratio of the number of SSRs to the total number of Unigenes) of 13.22%. The total length of the sequence was 74427.112 kb, and an average of one SSR site appeared every 8.39 kb. This shows that the transcriptome of Ixora australis has abundant SSR sites.

[0041] 3. Distribution characteristics of SSR loci in the transcriptome of Ixora australis

[0042] The SSR types of Ixora australis are rich and widely distributed. As shown in Table 1, it contains 4628 dinucleotides (49.07%), 3289 trinucleotides (34.87%), 908 tetranucleotides (9.63%), 330 pentanucleotides (3.5%), and 288 hexanucleotides (2.94%). Among them, the sequence containing two SSR sites is the largest, with a number of 4628, accounting for 49.07%. The sequence containing six SSR sites is the least, with 288, accounting for 2.94%; among various repeat types, the SSR site with the most repeats is 6 times, reaching 2287, accounting for 24.25%, including di-, tri-, tetra-, penta-, and hexanucleotide types, among which the number of dinucleotides is the largest, with 1375. The SSR site with the least repeats is 13 times, which is 150, accounting for 1.27%, and only includes dinucleotide types and trinucleotide types.

[0043] Table 1 Number of SSRs with different motif lengths and repeats in the transcriptome of Ixora lanceolata

[0044]

[0045]

[0046] 4. Repeating unit types and probabilistic characteristics in Ixora

[0047] like Figure 1 As shown in the figure, a statistical analysis was performed on all the nucleotide repeating unit types of Ixorax ilicifolia, among which the AG / CT motif types were the largest in the dinucleotide motif types, accounting for 27.8%; the AAG / CTT motif types were the largest in the trinucleotide motif types, accounting for 8.58%; the AAAT / ATTT motif types were the largest in the tetranucleotide motif types, accounting for 3.66%; the AAAAG / CTTTT motif types were the largest in the pentanucleotide motif types, accounting for 1.16%.

[0048] 5. Design and verification of SSR primers for Ixora australis

[0049] MISA was used to search all Unigenes in the transcriptome to find SSRs in Unigenes. Primers were designed on both sides of the sequence using primer3 (version 1.1.4) for the SSR information found. A total of 23,286 pairs of SSR primers were designed, from which 100 pairs of SSR primers were randomly selected. Effective amplification was verified in Ixora chinensis, Ixora leucophylla, and Ixora chinensis. 36 pairs of primers were screened to amplify bands. 55 Ixora chinensis germplasm resources were collected to perform polymorphism analysis and evaluation on the 36 pairs of SSR primers ( Figure 2 ) The results showed that 12 pairs of SSR primers were polymorphic, as shown in Table 2.

[0050] Table 2 Information on 12 pairs of polymorphic SSR primers of Ixora fasciata

[0051]

[0052]

[0053] 6. PCR amplification and polyacrylamide electrophoresis of SSR primers of Ixora cyrtonema

[0054] PCR amplification was performed using 55 copies of Ixora sibiricum DNA as templates. The PCR reaction system was 10 μL: 1 μL DNA template, 5 μL 2xTag PCR Super Mix, 0.15 μL upstream and downstream primers, and ddHzO was added to 10 μL. The PCR reaction conditions were: 95°C pre-denaturation for 5 min; 95°C denaturation for 30 s, 56°C annealing for 30 s, 72°C extension for 45 s, 35 cycles, and finally 72°C extension for 5 min. The product was stored at 4°C. The PCR amplification product was taken out and added to 5 ul loading buffer (0.1 g xylene cyanol, 0.1 g bromophenol blue, 0.5 M EDTA ph8.0 solution added to 1 ml), and then denatured at 95 degrees for 5 min for later use. Prepare 5.7% polyacrylamide gel, wait for the gel to solidify, insert the electrophoresis comb, add 2.5ul of sample, 80W power, and perform electrophoresis for 50 minutes. After completion, separate the glass plates, keep the glass plate with the gel adhered to it horizontally with the gel surface facing upward, and place it in a container for the following operations. During the process, the liquid surface must be kept above the gel surface: (1) Rinse the gel plate once with deionized water for 30 seconds. (2) Soak in silver nitrate solution for 10 minutes (0.01% silver nitrate solution). (3) Rinse the gel plate once with deionized water for 30 seconds. (4) Soak in developer solution for 10 minutes (1.65% NaOH, 0.05% Na 2 CO 3 , 1% HCHO) until clear electrophoresis bands appear. (5) Rinse with tap water for 5 minutes. (6) Dry the gel plate at room temperature and store it or place it under an X-ray film observation lamp for observation and photography. The amplification results of primer LC-57 are as follows Figure 3 shown.

[0055] 7. SSR result analysis

[0056] Through band analysis, the genetic similarity of 55 Ixora species was analyzed based on the polymorphic sites amplified by SSR markers, and the neighbor-joining method in MEGA5 was used to construct a phylogenetic map, such as Figure 4As shown. The green part is subgroup Ⅰ, the red part is subgroup Ⅱ, the blue part is subgroup Ⅲ, and the yellow part is subgroup Ⅳ. The green part is subgroup Ⅰ, the red part is subgroup Ⅱ, the blue part is subgroup Ⅲ, and the yellow part is subgroup Ⅳ. Subgroup Ⅰ includes 20 germplasms, subgroup Ⅱ includes 15 germplasms, subgroup Ⅲ includes 7 germplasms, and subgroup Ⅳ includes 13 germplasms. The results show that the development of SSR markers based on transcriptomes provides valuable molecular markers for the genetic diversity analysis and kinship analysis of existing Ixora germplasm resources.

Claims

1. A SSR molecular marker primer based on the transcriptome sequence of Ixora ilicifolia, characterized in that: It includes the following 12 primer pairs:

2. The SSR molecular marker primer based on the transcriptome sequence of Ixora australis according to claim 1, characterized in that: The size of the PCR product is 175-280 bp.

3. The SSR molecular marker primer based on the transcriptome sequence of Ixora australis according to claim 1, characterized in that: The annealing temperature of the primer pair is:

4. Use of the SSR molecular marker primer according to any one of claims 1 to 3 in genetic diversity analysis, variety identification or genetic map construction of Ixora.

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