EST-SSR (expressed sequence tag-simple sequence repeat) molecular marker primer group developed based on cerasus sikanensis transcriptome sequence and application

Abstract

The invention relates to the technical field of plant molecular markers, in particular to an EST-SSR (expressed sequence tag-simple sequence repeat) molecular marker primer group developed on the basis of a cerasus sikanensis transcriptome sequence and application. The EST-SSR molecular marker primer group comprises 19 pairs of primers, and the sequences of the primers are as shown in SEQ ID No.1-38. According to the EST-SSR molecular marker primer developed on the basis of cerasus sikanensis transcriptome data, the EST-SSR molecular marker has accuracy, stability and effectiveness compared with a traditional molecular marker, and the EST-SSR molecular marker is mostly distributed in a gene coding region or a region related to a regulatory sequence; a theoretical basis and an important technical means are provided for germplasm resource identification, genetic diversity analysis, molecular-assisted breeding and fingerprint construction of cerasus sikanensis, and the method has a good application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plant molecular markers, in particular to an EST-SSR molecular marker primer set developed based on the transcriptome sequence of Cerasus campanulata and application thereof. BACKGROUND

[0002] Cerasus campanulata is a high-quality cherry species in China, and is one of the four major ornamental species of cherry. It has high ornamental value, wide adaptability, strong stress resistance and other advantages. The variation coefficients of various traits among individuals of Cerasus campanulata are large, and it has the potential for selective breeding. In recent years, the breeding research and landscape application of Cerasus campanulata have developed rapidly in China, and many new varieties have been authorized. Therefore, it is particularly important to identify a large number of Cerasus campanulata varieties.

[0003] DNA fingerprint identification technology based on molecular markers has the advantages of accuracy, simplicity, rapidness and easy automation. Rapid identification of varieties through the construction of DNA fingerprint is an important technical means for variety identification and protection. SSR molecular markers have the advantages of large number, full genome coverage, high polymorphism, co-dominance and easy operation.

[0004] Publication No. CN117925898A, entitled "InDel marker primer combination for Cerasus campanulata germplasm resources and application thereof", discloses an InDel marker combination consisting of 5 pairs of primers, which can be used to identify 7 different geographical sources of Cerasus campanulata germplasm and construct a DNA fingerprint. However, this method only identifies limited germplasm resources from different geographical sources, and the number of samples and the breadth of genetic diversity covered are obviously insufficient. Moreover, the core polymorphism measurement indicators such as the number of alleles (Na), the number of effective alleles (Ne), and the polymorphic information content (PIC) analysis are not provided, and the actual polymorphism level and practical value of the primer set cannot be objectively evaluated. In addition, there is a lack of population genetics tests such as Hardy-Weinberg equilibrium (HWE) detection, which makes it difficult to assess potential problems such as whether the loci meet the expectations of population genetics. Compared with SSR markers, the InDel marker combination has inherent limitations in identification ability. SSR markers usually have multiple loci, high polymorphism and multiple alleles, and have stronger discrimination ability; while InDel markers are mostly di-allelic, with lower polymorphism, and have limited identification accuracy when distinguishing different varieties with close genetic relationship or from the same source. Although the selected InDel marker primer set can identify 7 geographical germplasms of Cerasus campanulata, there are still obvious deficiencies in the reliability of identification efficiency, the breadth of application range and the stability of primer polymorphism, which limits the efficiency and accuracy of Cerasus campanulata germplasm identification and genetic diversity analysis. SUMMARY

[0005] The present application is aimed at the problems existing in the prior art, and provides an EST-SSR molecular marker primer set developed based on a transcriptome sequence of Prunus triloba, which is used for constructing a Prunus triloba fingerprint and analyzing genetic diversity of germplasm resources.

[0006] To achieve the above object, the technical scheme adopted by the present application is that the EST-SSR molecular marker primer set developed based on the transcriptome sequence of Prunus triloba comprises 19 pairs of primer sets, including CCFSSR036, CCFSSR056, CCFSSR093, CCFSSR195, CCFSSR215, CCFSSR217, CCFSSR241, CCFSSR267, CCFSSR275, CCFSSR293, CCFSSR296, CCFSSR315, CCFSSR316, CCFSSR317, CCFSSR342, CCFSSR365, CCFSSR381, CCFSSR390 and CCFSSR398.

[0007] The sequence of the CCRSSR036 primer set is shown as SEQ ID No. 1-2;

[0008] The sequence of the CCRSSR056 primer set is shown as SEQ ID No. 3-4;

[0009] The sequence of the CCRSSR093 primer set is shown as SEQ ID No. 5-6;

[0010] The sequence of the CCRSSR195 primer set is shown as SEQ ID No. 7-8;

[0011] The sequence of the CCRSSR215 primer set is shown as SEQ ID No. 9-10;

[0012] The sequence of the CCRSSR217 primer set is shown as SEQ ID No. 11-12;

[0013] The sequence of the CCRSSR241 primer set is shown as SEQ ID No. 13-14;

[0014] The sequence of the CCRSSR267 primer set is shown as SEQ ID No. 14-15;

[0015] The sequence of the CCRSSR275 primer set is shown as SEQ ID No. 17-18;

[0016] The sequence of the CCRSSR293 primer set is shown as SEQ ID No. 19-20;

[0017] The CCRSR296 primer set sequence is shown as SEQ ID No. 21-22;

[0018] The CCRSR315 primer set sequence is shown as SEQ ID No. 23-24;

[0019] The CCRSR316 primer set sequence is shown as SEQ ID No. 25-26;

[0020] The CCRSR317 primer set sequence is shown as SEQ ID No. 27-28;

[0021] The CCRSR342 primer set sequence is shown as SEQ ID No. 29-30;

[0022] The CCRSR365 primer set sequence is shown as SEQ ID No. 31-32;

[0023] The CCRSR381 primer set sequence is shown as SEQ ID No. 33-34;

[0024] The CCRSR390 primer set sequence is shown as SEQ ID No. 35-36;

[0025] The CCRSR398 primer set sequence is shown as SEQ ID No. 37-38.

[0026] The second object of the present application is to provide a kit for analyzing the genetic diversity of or constructing a fingerprint of a Prunus serrulata germplasm resource, wherein the kit contains the primer sets described in SEQ ID No. 1-38.

[0027] The third object of the present application is to provide the application of the sequences of SEQ ID No. 1-38 or the above-mentioned kit in the analysis of the genetic diversity of a Prunus serrulata germplasm resource.

[0028] The fourth object of the present application is to provide the application of the sequences of SEQ ID No. 1-38 or the above-mentioned kit in the construction of a Prunus serrulata fingerprint.

[0029] The beneficial technical effect of the present application is that the EST-SSR molecular marker primer developed based on the transcriptome data of Prunus serrulata is accurate, stable and effective compared with traditional molecular markers, and the EST-SSR marker is mainly distributed in the region related to the gene coding region or the regulatory sequence, which provides a theoretical basis and an important technical means for Prunus serrulata germplasm identification, genetic diversity analysis, molecular assisted breeding and fingerprint construction, and has a good application prospect. The present application has the characteristics of high efficiency and low cost, the developed molecular marker sites are multiple and the information is comprehensive, and the developed molecular marker sites are multiple and the information is comprehensive. BRIEF DESCRIPTION OF DRAWINGS

[0030] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0031] Figure 1 The agarose gel electrophoresis diagram of the PCR products after amplification of the 8 Prunus serrulata germplasm materials in Example 1 using CCFSSR315-CCFSSR320 molecular marker primers. In the figure, SSR 315, SSR 316, SSR 317, SSR 318, SSR 319, SSR 320 represent primer group numbers; C20, C22, C25, C29, C31, C37, F91, F101 represent Prunus serrulata sample numbers; and M is Marker.

[0032] Figure 2 The capillary fluorescence detection diagram of the PCR products after amplification of the 8 Prunus serrulata germplasm materials in Example 1 using CCFSSR275 primers.

[0033] Figure 3 UPGMA clustering result of the 47 Prunus serrulata germplasm materials in Example 2. DETAILED DESCRIPTION

[0034] The technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only some embodiments of the present application, not all embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0035] Example 1

[0036] Development and screening of Prunus serrulata polymorphic primers

[0037] 1. Transcriptome sequencing

[0038] Chung-hua cherry samples were collected from Xiamen, Fujian Province. Healthy young leaves were selected for RNA extraction. After high-throughput transcriptome sequencing, 75582 Unigenes were obtained for SSR site search, with an average length of 1.69 Kb.

[0039] 2. SSR site identification

[0040] MISA was used to detect Unigene and obtain EST-SSR site information. A total of 30654 SSR sites were detected in 21217 Unigenes.

[0041] 3. Primer design

[0042] EST-SSR primers were designed using Primer 3 software, and 16692 pairs of primers were designed in batches. The main parameters set during primer design were: primer length between 20-23 bp; annealing temperature around 60℃; base repeat number ≤4; no 2 consecutive A / T bases at 5' and 3' ends. The designed primers were evaluated using Oligo 7.0 software. Finally, 400 pairs were selected for marker development and verification.

[0043] 4. Genomic DNA extraction and detection

[0044] Eight cherry germplasm materials with large differences in ornamental traits (Table 1) were selected for healthy young leaves, and TSINGKE plant DNA extraction kit (universal type) was used to extract genomic DNA. After PCR amplification, the DNA concentration and purity were detected by 1% agarose gel electrophoresis and ND-1000 spectrophotometer (NanoDrop, wilmington, DE, USA). After detection, the DNA extraction solution was diluted to 25 ng / μl and stored in a refrigerator at -20℃ for standby.

[0045] Table 1 Eight cherry germplasm materials for testing

[0046]

[0047] 5. PCR amplification reaction

[0048] The PCR amplification system and amplification program are as follows (Table 2, Table 3).

[0049] Table 2 PCR amplification reaction system

[0050]

[0051] Table 3 PCR amplification program

[0052]

[0053] Note: The first amplification annealing 62℃, the second amplification annealing 55℃ with fluorescent modified linker primer.

[0054] 6. Polymorphic primer screening

[0055] The amplified PCR products were detected by 1% agarose gel electrophoresis (2 μl sample + 6 μl bromophenol blue) to obtain the identification gel map. When 'Shuangqi' was used as the DNA template, 303 pairs of primers were successfully amplified, and 303 sets of effective image data were obtained, all of which showed high consistency. Figure 1 The most representative CCFSSR315-CCFSSR320 group was selected for display. Subsequently, the template concentration was determined by gel map, diluted with water, and further screened by capillary electrophoresis. The results showed that 186 pairs of primers were successfully amplified in 8 samples as shown in Figure 2 , accounting for 46.5% of the synthesized primers. After further analysis, 169 pairs of primers with high polymorphism in 8 cherry varieties were finally selected.

[0056] Using these 169 pairs of clock cherry EST-SSR polymorphic primers as candidate primers, 19 pairs of primers (Table 4) with high polymorphic information content and stable amplification bands were finally selected as core primers for clock cherry genetic diversity analysis and fingerprint construction.

[0057] Table 4 Information of 19 pairs of EST-SSR core primers selected from clock cherry transcriptome

[0058]

[0059] Example 2 Application of EST-SSR molecular marker primer set to clock cherry genetic diversity analysis

[0060] 1. Germplasm resource sample source

[0061] Among the 47 test materials (Table 5), 38 were clock cherry (i.e. Fujian mountain cherry) strains, 3 were C. dielsiana strains, 2 were C. speciosa strains, 2 were C. yedoensis strains, 1 was C. yunnanensis strain, and 1 was C. cerasoides.

[0062] Table 5 Basic information of 47 cherry germplasm resources for testing

[0063]

[0064] 2. Genomic DNA extraction

[0065] DNA of 47 germplasm resources in Table 5 was extracted using TSINGKE Plant DNA Extraction Kit (Universal Type). After extraction, the DNA concentration and purity were detected by 1% agarose gel electrophoresis and ND-1000 spectrophotometer. After passing the detection, the DNA extraction solution was diluted to 25 ng / μl and stored in a refrigerator at -20°C for standby use.

[0066] 3. PCR amplification reaction

[0067] PCR amplification was performed using the above DNA as a template and 19 pairs of polymorphic primers selected in Table 4. The PCR reaction system and procedure were the same as those in Table 2 and Table 3.

[0068] 4. Genetic diversity analysis

[0069] The PCR amplification products were detected and analyzed by capillary electrophoresis. The genetic diversity of Chonghua cherry was analyzed by comparing the amplified polymorphic bands. Specifically, in PopGen 32 and GenALEx version 6.501 software, the genetic diversity indexes of SSR loci and populations were calculated, including observed alleles (Na), effective alleles (Ne), Shannon index (I), polymorphic information index (PIC), observed heterozygosity (Ho), expected heterozygosity (He), major allele frequency (MP), and inbreeding coefficient (Fis).

[0070] Table 6 Polymorphism of 19 pairs of EST-SSR primers

[0071]

[0072] Note: Na: observed alleles, Ne: effective alleles, I: Shannon index, PIC: polymorphic information index, Ho: observed heterozygosity, He: expected heterozygosity, PHWE: Hardy-Weinberg equilibrium. *: P<0.05, **: P<0.01, ***: P<0.001, ns: P>0.05.

[0073] The results showed that the 19 primer pairs detected a total of 139 alleles in 47 samples (Table 6). The number of alleles ranged from 4 to 15, with an average of 7.3 per locus. The total number of effective alleles was 49.7590, ranging from 1.3867 to 7.6568, with an average of 2.6189 per locus. The Shannon index (I) ranged from 0.5554 to 2.2745, with an average of 1.1335. The polymorphic information content (PIC) ranged from 0.2579 to 0.8563, with an average of 0.4968. These results indicate that the 19 EST-SSR primer pairs have high polymorphism. The observed heterozygosity (Ho) and expected heterozygosity (He) ranged from 0.1277 to 0.7234 and 0.2819 to 0.8787, respectively, with means of 0.3577 and 0.5416. The average inbreeding coefficient was 0.3324. The Hardy-Weinberg equilibrium test revealed that 18 of the 19 SSR loci deviated from Hardy-Weinberg equilibrium at P < 0.01, likely due to excess heterozygosity.

[0074] 5. Cluster Analysis

[0075] Based on Nei's genetic distance, a UPGMA tree (set 1000 replicates) was constructed in POPULATIONS 1.2.30 software, and cluster analysis was performed on the 47 test materials. The cluster tree was beautified and edited in FigTree version 1.4.2 software. Clustering results ( Figure 3 ) shows that C36 Gaopen cherry is a separate branch, while the three tail-leaf cherry varieties C2, C5, and C22 are clustered together, and C25 Showa cherry is also separated from the other varieties. Within the bell-flower cherry lineage, phenotypically and closely related varieties, such as the red double-petaled 'Mudan Ying,' 'Jiangshan Meiren,' 'Wangdi,' 'Fugui,' and 'Xia Meiren,' are clustered together. Meanwhile, pink, open-petaled varieties, such as 'Hongfen Jiaren,' 'Guangzhou Ying,' and 'Fuzhou Feihan,' are clustered together. This demonstrates that the EST-SSR clustering results can well reflect the genetic relationships among the tested cherry varieties and provide a reference for tracing pedigrees between varieties.

[0076] Example 3 Application of EST-SSR Molecular Marker Primer Sets in the Construction of DNA Fingerprints of Different Germplasms of Prunus campanulata

[0077] Capillary electrophoresis was performed on the amplified bands of the 19 EST-SSR primer pairs in 47 germplasm samples. Based on the test results, fingerprint analysis and genotyping were performed. The allelic marker configuration at each SSR locus was recorded for each sample. The 19 SSR primer pairs were sequentially encoded, and the encoded information was combined to form the SSR fingerprint of the Prunus campanulata samples.

[0078] Any single pair of primers cannot completely distinguish 47 materials, according to the polymorphism information index of primers, first select CCRSSR342 and CCRSSR316 two pairs of primers for variety identification, according to the results, then add a pair of primers for combination identification in turn. When selecting CCRSSR342, CCRSSR316 and CCFSSR317 three pairs of primers for combination identification, C11, C15, C17, C18, C20 and C28 still cannot be distinguished. Analyzing the remaining primers found that CCRSSR215 can distinguish C17, C20 and C28, CCRSSR267 and CCRSSR036 two pairs of primers can distinguish C11, C15 and C18. Therefore, finally selected CCRSSR342, CCRSSR316, CCFSSR317, CCRSSR267, CCRSSR036 and CCRSSR215 six pairs of primers (Table 7) to construct the DNA fingerprint of 47 cherry germplasm materials (Table 8).

[0079] Table 7 six pairs of EST-SSR primers used for fingerprint construction

[0080]

[0081] Table 8 EST-SSR fingerprint code of 47 cherry varieties

[0082]

[0083] Finally, it should be noted that: the above examples are only used to illustrate but not limit the technical solutions of the present application, although the present application is described in detail with reference to the above examples, those skilled in the art should understand that: the present application can still be modified or replaced, without departing from the spirit and scope of the present application, any modification or partial replacement, which should be covered in the scope of the claims of the present application.

Claims

1. An EST-SSR molecular marker primer set developed based on the transcriptome sequence of Prunus campanulata, characterized by: The EST-SSR molecular marker primer set comprises 19 pairs of primer sets, including CCFSSR036, CCFSSR056, CCFSSR093, CCFSSR195, CCFSSR215, CCFSSR217, CCFSSR241, CCFSSR267, CCFSSR275, CCFSSR293, CCFSSR296, CCFSSR315, CCFSSR316, CCFSSR317, CCFSSR342, CCFSSR365, CCFSSR381, CCFSSR390 and CCFSSR398; The CCRSSR036 primer set sequence is shown in SEQ ID No. 1-2; The CCRSSR056 primer set sequence is shown in SEQ ID No. 3-4; The CCRSSR093 primer set sequence is shown in SEQ ID No. 5-6; The CCRSSR195 primer set sequence is shown in SEQ ID No. 7-8; The CCRSSR215 primer set sequence is shown in SEQ ID No. 9-10; The CCRSSR217 primer set sequence is shown in SEQ ID No. 11-12; The CCRSSR241 primer set sequence is shown in SEQ ID No. 13-14; The CCRSSR267 primer set sequence is shown in SEQ ID No. 14-15; The CCRSSR275 primer set sequence is shown in SEQ ID No. 17-18; The CCRSSR293 primer set sequence is shown in SEQ ID No. 19-20; The CCRSSR296 primer set sequence is shown in SEQ ID No. 21-22; The CCRSSR315 primer set sequence is shown in SEQ ID No. 23-24; The CCRSSR316 primer set sequence is shown in SEQ ID No. 25-26; The CCRSSR317 primer set sequence is shown in SEQ ID No. 27-28; The CCRSSR342 primer set sequence is shown in SEQ ID No. 29-30; The CCRSSR365 primer set sequence is shown in SEQ ID No. 31-32; The CCRSSR381 primer set sequence is shown in SEQ ID No. 33-34; The CCRSSR390 primer set sequence is shown in SEQ ID No. 35-36; The sequences of the CCRSSR398 primer set are shown in SEQ ID Nos. 37 to 38.

2. A kit for analyzing the genetic diversity of Cherry Blossom germplasm resources or constructing fingerprints, characterized in that: The kit contains the primer set according to claim 1.

3. Use of the primer set according to claim 1 or the kit according to claim 2 in the genetic diversity analysis of Cerasus chinensis germplasm resources.

4. Use of the primer set according to claim 1 or the kit according to claim 2 in the construction of a fingerprint of Sakura chinensis.

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