Development and application of EST-SSR markers in deutzia gracilis based on transcriptome sequences

Abstract

The application discloses a set of EST-SSR marker primers of Deutzia developed based on transcriptome sequences and application thereof. The EST-SSR primer set has 16 pairs of primers, wherein the nucleotide sequences of the first to 16th pairs of primers are shown as SEQ ID No. 1 to SEQ ID No. 32. The SSR primer set obtained by the application has good polymorphism and universality; the DNA fingerprint map and the high-efficiency combination can realize rapid identification of Deutzia materials, and they will provide scientific tools for future researches on germplasm resource identification, origin evolution and germplasm resource innovation of Deutzia plants, and lay a foundation for popularization and application of the Deutzia plants.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to Deutzia EST-SSR marker primers developed based on transcriptome sequences and their applications. Background Technology

[0002] Ulva ( Deutzia *Deutzia* is an excellent summer ornamental shrub, prized for its beautiful shape, elegant flowers, and diverse phenotypes, making it highly valuable for development and application. Furthermore, the roots, leaves, and fruits of *Deutzia* can be used medicinally, possessing properties such as clearing heat and promoting diuresis, tonifying the kidneys, and treating malaria. *Deutzia* is also an important auxiliary nectar and pollen source in northern regions, giving it economic value. However, research on the *Deutzia* genus is extremely limited. Existing studies have primarily focused on morphology, physiology, and cytology, with little in-depth exploration at the genetic level. Due to limitations in research capabilities, *Deutzia* species currently face numerous challenges, including difficulties in identification, unclear classification, species mixing, and slow breeding progress, resulting in a large untapped germplasm resource. Currently, only a small number of SRAP and ISSR molecular markers have been successfully developed for *Deutzia*, insufficient to fully meet the needs of further research in this genus.

[0003] Expressed sequence tag microsatellites (EST-SSRs) are a novel type of molecular marker based on simple sequence repeats designed using expressed sequence tags. Compared to gSSRs developed using traditional methods, EST-SSR markers developed using RNA-Seq sequencing have a shorter development cycle, are highly correlated with phenotypic traits, and can be used in plants without a reference genome. They play a crucial role in discovering new genes, gene expression analysis, and germplasm identification, and have been developed and applied in various plant species in recent years.

[0004] Currently, there are no reports on the application of EST-SSR in Deutzia species. Relying solely on morphological markers for differentiation and identification of Deutzia species is extremely difficult, severely hindering the future development and utilization of Deutzia germplasm resources. Therefore, developing Deutzia SSR primers based on transcriptome sequences and constructing a Deutzia DNA fingerprint can help rapidly identify Deutzia germplasm, providing a scientific basis for future systematic classification and genetic breeding research of Deutzia. Summary of the Invention

[0005] The technical solution adopted in this invention is: a set of Deutzia EST-SSR molecular markers, which includes forward and reverse primers corresponding to the following 16 sites:

[0006]

[0007] The 16 primer pairs disclosed in this invention stably amplified a total of 395 bands in all Deutzia materials, of which 107 were polymorphic, with an average polymorphic band ratio of 27.40%. The polymorphism information content ranged from 0.51 to 0.86, all of which were highly polymorphic loci.

[0008] The method for developing the EST-SSR molecular marker of Deutzia spp. according to the present invention includes the following steps:

[0009] 1) Obtaining Deutzia spp. (small-flowered) via RNA-Seq Deutzia. parviflora Transcriptome data;

[0010] 2) Use MISA software to perform SSR locus search and feature analysis on the original gene database described in step 1);

[0011] 3) Primers were designed in batches using Primer3 software, and 100 pairs of primers were randomly selected and synthesized.

[0012] 4) The primers selected in 3) were used to amplify the urinary tract material, and the urinary tract EST-SSR markers were screened out after capillary electrophoresis.

[0013] 5) Based on the capillary electrophoresis results in 3), genetic cluster analysis was performed on the deutzia material according to the size of the amplification products, and a DNA fingerprint map was constructed.

[0014] Furthermore, in step 2), the number of repeats of the di, tri, tetra, penta, and hexanucleotides at the SSR site is at least 5, 4, 3, 3, and 3 times, respectively.

[0015] Furthermore, in step 3), the EST-SSR primer design adopts the following parameter settings: primer length is 18-27 bp; GC content is 40%~60%; annealing temperature is 45~65℃; expected product fragment size is 100-300 bp; and the difference in Tm value between upstream and downstream primers is less than 5℃.

[0016] Furthermore, in step 3), a 16-base sequence of a universal tag (CAGTCGGGCGTCATCA) is added to the 5' end of all forward primers F during primer synthesis; four types of fluorescent labels are used: FAM (blue), HEX (green), TAMRA (yellow) and ROX (red).

[0017] Furthermore, the Deutzia materials in step 4) include: Deutzia macrocarpa, Deutzia heterochroma, Deutzia serrata, Deutzia macrocarpa, Deutzia xerophyta, Deutzia szechuanensis, 'Edelweiss', 'Snow Cherry Blossom', 'Clock', 'Alice', 'Tourbillon Jewel', 'Strawberry Field', 'Rose Pearl', 'Banli', 'Rochester's Glory', 'Pink Bell', Deutzia coarse-toothed, and 'Double-petaled Deutzia'.

[0018] Furthermore, the PCR amplification reaction system in step 4) is: 17 μl 1.1×T3 Super PCR Mix, 1 μl forward primer (10 μM), 1 μl reverse primer (10 μM) and 1 μl template DNA.

[0019] Furthermore, the PCR amplification program in step 4) is as follows: pre-denaturation at 98℃ for 2 min; denaturation at 98℃ for 10 s, annealing at 60℃ for 10 s, extension at 72℃ for 10 s, for a total of 35 cycles; and final extension at 72℃ for 5 min.

[0020] Further, in step 5), the method for constructing the DNA plant map is as follows: Based on the STR typing data, the amplification band types of 16 pairs of screening primers in the Deutzia material are compiled and recorded in the form of bp values; the bp values ​​are arranged in ascending order, corresponding to the numbers "1~9" and the letters "A~Z" sequentially, to obtain the amplification bp value coding table. The lengths of the amplification products of 18 Deutzia materials are matched with the bp value coding table, and the codes are concatenated one by one to obtain the SSR band fingerprint code of the Deutzia material. The QR code of the Deutzia SSR fingerprint map is constructed using the QR CodeGenerator v1.210 QR code generation tool.

[0021] This invention discloses EST-SSR primers for Deutzia and their applications, which have at least the following advantages: the 16 selected SSR primer pairs can be stably amplified in Deutzia materials, exhibiting good polymorphism and versatility; the constructed DNA fingerprint and efficient combination can achieve rapid identification of Deutzia materials. These will provide scientific tools for future research on the identification, origin, evolution, and innovation of Deutzia germplasm resources, laying the foundation for the application and promotion of Deutzia plants. Attached Figure Description

[0022] Figure 1 Several common STR electrophoresis patterns in SSR primer screening; Figures A and B are both STR electrophoresis patterns of 54 primer pairs; Figure C is a STR electrophoresis pattern of 23 primer pairs; the expected product length of primer P025 in Figure C is 267bp; Figure D is a STR electrophoresis pattern of 16 primer pairs.

[0023] Figure 2 UPGMA clustering diagram of 18 desiccated materials. Detailed Implementation

[0024] The present invention will be further described below with reference to specific embodiments.

[0025] Example 1

[0026] 1. Materials and Methods

[0027] 1.1 Test Materials

[0028] Eighteen wild species and varieties of Deutzia were obtained through collection and purchase, and were planted as test materials at the National Experimental Teaching Demonstration Center for Landscape Architecture of Nanjing Forestry University (Table 1). Young leaves were collected as samples in the spring of 2021, flash-frozen in liquid nitrogen, transferred, and stored at -80℃ for later use.

[0029] Table 1. Germplasm resource collection status of the genus Deutzia

[0030]

[0031]

[0032] 1.2 Deutzia transcriptome sequencing and analysis

[0033] Deutzia simonii, with its relatively small chromosome number and wide distribution in my country, was selected as the sequencing material. Newly acquired young Deutzia simonii leaf samples were sent to Guangzhou Gediao Biotechnology Co., Ltd. for Illumina HiSeq high-throughput sequencing. A total of 60,707 unigene sequences were obtained, ranging in length from 201 to 14,679 bp, with an average length of 791 bp, for a total length of 48,053,766 bp, approximately 48 MB. More than half of the unigenes were between 200 and 500 bp in length, with the 200-300 bp unigenes being the most numerous, accounting for 35.78% of the total. The average GC content was approximately 41.88%, and the N50 number was 1,502 bp in length, with an average length of 791 bp.

[0034] 1.3 Development of EST-SSR Molecular Markers for Deutzia

[0035] 1.3.1 Obtaining Deutzia genomic DNA

[0036] Genomic DNA was extracted using a modified CTAB plant genomic DNA rapid extraction kit (Adley Biotechnology, Beijing, model DN-14 centrifuge column). DNA quality was assessed using a micro-volume nucleic acid and protein analyzer (Quawell, USA). Extracted DNA was labeled and stored at 4°C (short-term) or -20°C (long-term).

[0037] 1.3.2 Design and Synthesis of SSR Primers

[0038] Based on transcriptome sequencing results, all EST-SSR loci in the transcriptome were searched using MISA software. The search criteria were: dinucleotide repeats greater than 5; trinucleotide repeats greater than 4; and tetranucleotide, pentanucleotide, and hexanucleotide repeats greater than 3. The selected SSR loci were characterized using Microsoft Excel 2019 software.

[0039] Based on EST-SSR search results, primers were designed in batches using Primer3 software. The main parameters set during primer design were: primer length 18-27 bp; GC content 40%-60%; annealing temperature 45-65℃; expected product fragment size 100-300 bp; and a Tm difference of less than 5℃ between upstream and downstream primers. One hundred pairs of initial screening primers were randomly selected using Excel software and sent to Nanjing Qingke Biotechnology Co., Ltd. for primer synthesis. During synthesis, a 16-base primer sequence with a universal tag (CAGTCGGGCGTCATCA) was added to the 5' end of all forward primers (F). Four types of fluorescent labels were used: FAM (blue), HEX (green), TAMRA (yellow), and ROX (red).

[0040] 1.3.3 Screening of polymorphic SSR primers

[0041] One hundred pairs of random primers were used for PCR amplification on 18 deutzia materials. The total PCR amplification reaction volume was 20 μl, including 17 μl of 1.1×T3 Super PCR Mix, 1 μl of forward primer (10 μM), 1 μl of reverse primer (10 μM), and 1 μl of template DNA. The amplification program was as follows: pre-denaturation at 98℃ for 2 min; denaturation at 98℃ for 10 s, annealing at 60℃ for 10 s, extension at 72℃ for 10 s, for a total of 35 cycles; secondary extension at 72℃ for 5 min; PCR products were stored at 4℃. For the second amplification, fluorescently modified adapter primers and reverse primers were used together, and the amplification program was the same as above. The amplified PCR products were sent to Nanjing Qingke Biotechnology Co., Ltd. for capillary electrophoresis to obtain the STR electrophoresis patterns and genotyping information of the primers. Statistical analysis of STR genotyping data was performed using Excel 2019 software. Primers with good polymorphism were selected as usable Deutzia SSR molecular markers based on the number and length of amplified bands of each pair of SSR primers.

[0042] 1.4 Analysis of genetic diversity in Deutzia

[0043] The genetic coefficients of 16 pairs of SSR marker primers across different samples were calculated using PopGene32 and Powermarker software, including the average number of alleles. Na ), number of effective alleles ( Ne ), observed heterozygosity ( Ho ), expected heterozygosity ( He ), polymorphic information content ( PIC ), Shannon Diversity Index ( I ) and Nei's average expected heterozygosity ( Nei The genetic similarity coefficients of each sample were calculated using NTsys 2.10e software, and a genetic clustering diagram was constructed based on the UPGMA clustering method.

[0044] 1.5 Construction of Deutzia DNA fingerprinting

[0045] Based on the amplification performance of selected SSR marker sites on the *Deutzia* material, an SSR band fingerprint of *Deutzia* was constructed. QR codes for the *Deutzia* SSR fingerprint were constructed using the QR Code Generator v1.210 tool.

[0046] 2 Results and Analysis

[0047] 2.1 Search and Characterization of SSR Loci in the Deutzia Transcriptome

[0048] A total of 15,465 SSR loci matching the criteria were found, distributed across 10,767 different Unigenes. The occurrence frequency (number of Unigenes with SSR loci / total number of Unigenes) was 17.74%, and the distribution frequency (total number of SSR loci / total number of Unigenes) was 25.47%. The average distribution distance of the SSRs was 3.11 kb, and the average length was 31.99 bp.

[0049] Statistical analysis of repeat types at *Deutzia macrophylla* EST-SSR loci (Table 2) shows that dinucleotide repeats are the most common type, with 11,222 repeats, accounting for 72.26% of the total SSR loci. Trinucleotide repeats are the second most common, with 3,169 repeats, accounting for 20.49%. Tetranucleotide, pentanucleotide, and hexanucleotide repeats are less common, accounting for 2.11%, 1.51%, and 3.33% of the total SSR loci, respectively. Statistical analysis of the number of repeats for different repeat types (Table 3) shows that repeats of 5-10 times are the most common, accounting for 67.47% of the total. SSRs with 4 or more repeats are less common, both below 5%. The most common repeat type is 6-times repeats, with 2,929 repeats, accounting for 18.94%. In terms of repeat types, trinucleotide repeats have the widest coverage, appearing in all repeats with ≥5 repetitions; followed by dinucleotide repeats, appearing in all repeats with ≥6 repetitions; when the number of repetitions is ≥12, the SSR repeat types are only dinucleotide and trinucleotide. Statistical results of repeat motifs show that there are 13 main repeat motifs in Deutzia SSRs, of which 3 are dinucleotide repeat motifs, with AG / CT accounting for the highest proportion (57.61%), followed by AC / GT and AT / AT accounting for 9.71% and 4.49%, respectively; among the trinucleotide repeat motifs, AAT / CTT has the highest proportion (6.49%), ranking third among all motifs.

[0050] Table 2. Repeat frequencies of different types of SSRs in Deutzia Unigenes

[0051]

[0052] Table 3. Statistical analysis of the number of repetitions of different repeating types of SSRs in Deutzia spp.

[0053]

[0054] 2.2 Development of EST-SSR primers for Deutzia spp.

[0055] Statistical analysis of the amplification results showed that out of 100 pairs of SSR primers, 25 pairs could amplify the target product, with an effective amplification rate of 25%; 54 pairs of primers failed to identify the specific peak. Figure 1 -A) or the presence of many heterogeneous peaks ( Figure 1 -B); the amplification products of 23 primer pairs did not match the expected product extent ( Figure 1 -C). After further screening to remove low-polymorphic primers with less than 3 polymorphic bands after amplification, 16 pairs of SSR primers with excellent polymorphism and good universality were finally selected. Figure 1-D). The 16 primer pairs stably amplified a total of 395 bands, of which 107 were polymorphic, with an average polymorphism rate of 27.40%. Among them, P002 had the highest polymorphism, with a polymorphic band rate of 50.00%; P017 had the lowest polymorphism, with a polymorphic band rate of 15.38%.

[0056] 2.3 Genetic diversity analysis of Deutzia

[0057] 2.3.1 Genetic diversity analysis of SSR markers

[0058] A total of 107 alleles were detected in 18 Deutzia materials, with an average number of alleles ( ). Na The average value is 6.69 (Table 4). Ne The value is 3.70, and Na The values ​​vary to some extent, with the Deutzia population exhibiting a higher degree of genetic variation. Shannon diversity index (… I The range was 0.92 to 2.11, with an average of I The value is 1.46; observed heterozygosity ( Ho The value ranged from 0.12 to 0.48, with an average of 0.12 to 0.4 Ho The value is 0.29. Expected heterozygosity ( He The range is 0.51 to 0.86, with an average of He The value is 0.71, and all SSR loci... He If the values ​​are all greater than 0.5 and close to 1, then the selected SSR primers have good genetic diversity. Nei Values ​​ranged from 0.51 to 0.86, with an average of 0.69. Polymorphism information content ( PIC The values ​​ranged from 0.51 to 0.86, with an average of 0.69; 16 pairs of SSR primers PIC All values ​​are greater than 0.5, indicating that they are all highly polymorphic loci with excellent polymorphism and universality. Based on the above heritability indices, primer P088 exhibits the best polymorphism and universality, while primer P008 shows relatively weaker polymorphism and universality.

[0059] Table 4. Genetic diversity indicators of SSR loci

[0060]

[0061] 2.3.2 SSR-based cluster analysis

[0062] Based on the UPGMA clustering graph results ( Figure 2It can be seen that at a similarity coefficient of 0.72, the 18 Deutzia materials can be divided into 5 groups according to their kinship: (I) Deutzia macrocarpa (D06); (II) Deutzia heterocarpa (D17) and Deutzia davidii (D12); (III) Deutzia macrocarpa (D15) and Deutzia xerophyta (D16); (IV) Deutzia szechuanensis (D10), 'Edelweiss' (D13), 'Snow Cherry' (D14) and 'Clock' (D18); (V) 'Alice' (D01), 'Tourbillon Jewel' (D11), 'Strawberry Field' (D05), 'Rose Pearl' (D09), 'Variegated' (D02), 'Rochester's Glory' (D08), 'Pink Bell' (D07), Deutzia coarse-toothed (D04), and 'Double-flowered Deutzia' (D03).

[0063] 2.4 Construction of Deutzia DNA fingerprinting

[0064] 2.4.1 Construction of Deutzia SSR Strip Fingerprint Code

[0065] Based on STR genotyping data, the amplification band types of 16 pairs of screening primers in the deutzia material were compiled and recorded in bp value form. The bp values ​​were arranged in ascending order and numbered sequentially according to the numbers "1~9" and the letters "A~Z" to obtain the amplification bp value coding table (Table 5). The coding table shows that a total of 115 band patterns were amplified from the 16 SSR loci in the deutzia material. Based on the correspondence between the amplification product length of the deutzia material and the bp value coding table, the codes were concatenated to obtain the SSR band pattern fingerprint code of the deutzia material (Table 6).

[0066] Of the 18 Deutzia materials, 6 did not have specific genotypes; the remaining 12 materials had a total of 60 specific genotypes, which could be quickly identified using only one pair of SSR primers: Sichuan Deutzia, Coarse-toothed Deutzia, Heterochromatic Deutzia, Small-flowered Deutzia, Large-flowered Deutzia, Large-calyxed Deutzia, Xerophytic Deutzia, 'Edelweiss', 'Snow Cherry Blossom', 'Variegated', 'Double-flowered Deutzia', and 'Zhong'.

[0067] Based on the primer combinations, the following SSR primer combinations were found to enable rapid identification of 18 Deutzia materials: (1) P002+ P008+ P026+ P050; (2) P008+ P012+ P026+ P050; (3) P008+ P022+ P026+ P050; (4) P008+ P026+ P033+ P050; (5) P008+ P026+ P050+ P077; (6) P008+ P026+ P050+ P085; (7) P008+ P026+ P050+ P088; (8) P008+ P026+ P050+ P096.

[0068] 2.4.2 QR Encoding of Ureaplasma purpureus SSR Fingerprint

[0069] Based on the previously constructed SSR banding fingerprint codes of Deutzia materials, QR code generation software was used to construct QR codes for the SSR fingerprint spectrum of all Deutzia materials in this study. After scanning the constructed QR codes, relevant information about the Deutzia material can be obtained online, including its Latin name, Chinese scientific name or trade name, and the SSR banding fingerprint code. For example, scanning Sichuan Deutzia yields the following information:

[0070] (1) Material name: Deutzia setchuenensis

[0071] (2) Chinese scientific name: Sichuan deutzia

[0072] (3) SSR fingerprint codes: 524231926A354665.

[0073] Table 5. Correspondence between amplification bp values ​​and codes for 16 pairs of SSR primers.

[0074]

[0075] Table 6. SSR banding fingerprint codes of 18 hydrophobic materials

[0076]

Claims

1. A Deutzia EST-SSR marker primer developed based on transcriptome sequences, characterized in that, The EST-SSR primer set includes the following 16 pairs of primers: Primer P001-F has the nucleotide sequence shown in SEQ ID No. 1 of the sequence listing. Primer P001-R having the nucleotide sequence shown in SEQ ID No. 2 in the sequence listing; Primer P002-F has the nucleotide sequence shown in SEQ ID No. 3 in the sequence listing. Primer P002-R having the nucleotide sequence shown in SEQ ID No. 4 in the sequence listing; Primer P008-F has the nucleotide sequence shown in SEQ ID No. 5 of the sequence listing. Primer P008-R having the nucleotide sequence shown in SEQ ID No. 6 of the sequence listing; Primer P012-F has the nucleotide sequence shown in SEQ ID No. 7 of the sequence listing. Primer P012-R having the nucleotide sequence shown in SEQ ID No. 8 in the sequence listing; Primer P017-F has the nucleotide sequence shown in SEQ ID No. 9 of the sequence listing. Primer P017-R having the nucleotide sequence shown in SEQ ID No. 10 of the sequence listing; Primer P021-F has the nucleotide sequence shown in SEQ ID No. 11 of the sequence listing. Primer P021-R having the nucleotide sequence shown in SEQ ID No. 12 in the sequence listing; Primer P022-F has the nucleotide sequence shown in SEQ ID No. 13 of the sequence listing. Primer P022-R having the nucleotide sequence shown in SEQ ID No. 14 in the sequence listing; Primer P026-F has the nucleotide sequence shown in SEQ ID No. 15 of the sequence listing. Primer P026-R having the nucleotide sequence shown in SEQ ID No. 16 of the sequence listing; Primer P033-F has the nucleotide sequence shown in SEQ ID No. 17 of the sequence listing. Primer P033-R has the nucleotide sequence shown in SEQ ID No. 18 in the sequence listing; Primer P050-F has the nucleotide sequence shown in SEQ ID No. 19 of the sequence listing. Primer P050-R having the nucleotide sequence shown in SEQ ID No. 20 of the sequence listing; Primer P054-F has the nucleotide sequence shown in SEQ ID No. 21 of the sequence listing. Primer P054-R has the nucleotide sequence shown in SEQ ID No. 22 in the sequence listing; Primer P077-F has the nucleotide sequence shown in SEQ ID No. 23 in the sequence listing. Primer P077-R has the nucleotide sequence shown in SEQ ID No. 24 in the sequence listing; Primer P078-F has the nucleotide sequence shown in SEQ ID No. 25 of the sequence listing. Primer P078-R has the nucleotide sequence shown in SEQ ID No. 26 in the sequence listing; Primer P085-F has the nucleotide sequence shown in SEQ ID No. 27 of the sequence listing. Primer P085-R having the nucleotide sequence shown in SEQ ID No. 28 in the sequence listing; Primer P088-F has the nucleotide sequence shown in SEQ ID No. 29 of the sequence listing. Primer P088-R has the nucleotide sequence shown in SEQ ID No. 30 in the sequence listing; Primer P096-F has the nucleotide sequence shown in SEQ ID No. 31 of the sequence listing. Primer P096-R has the nucleotide sequence shown in SEQ ID No. 32 in the sequence listing.

2. The application of the Deutzia EST-SSR marker primers developed based on transcriptome sequences as described in claim 1 in identifying the phylogenetic relationship of Deutzia spp.

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