A fingerprint map of violet based on SSR markers, construction method and application thereof

Abstract

The present invention provides a violet fingerprint map based on SSR markers, a construction method and its application, and relates to the technical field of plant variety identification. The present invention provides a method for constructing a violet variety fingerprint map, using 8 pairs of SSR marker primers obtained by screening as core primers for constructing the violet fingerprint map, and using "1" and "0" to mark the presence or absence of amplified fragments at the same migration position, respectively, to construct fingerprint maps of 37 violet varieties. The specific band combination of the 8 pairs of primers is used as the DNA fingerprint of a variety, which can be used to identify different varieties. The violet fingerprint map provided by the present invention provides a scientific basis for screening valuable breeding materials from domestic and foreign violet variety germplasm resources, reduces breeding blindness, improves violet breeding efficiency, and provides a basis for the protection of new variety intellectual property rights, variety identification, and innovative violet breeding.

Description

Technical Field

[0001] The present invention relates to the technical field of plant variety identification, and in particular to a violet fingerprint map based on SSR markers, a construction method and applications thereof. Background Art

[0002] SSRs (simple sequence repeats), also known as microsatellites, are tandemly repeated DNA motifs occurring in coding and non-coding regions. Research has found that SSRs are ubiquitous in the genome, with repeat units ranging from 1 to 6 nt. SSR molecular markers offer advantages such as high abundance, high polymorphism, extensive allelic variation, codominance, low cost, and ease of PCR detection. They are widely used in genetic analysis, such as population structure and genetic diversity analysis, QTL mapping, marker-assisted selection breeding, and DNA fingerprinting.

[0003] Violet (Matthiola incana (L.) R.Br.), a member of the genus Viola in the family Cruciferae, is native to the Mediterranean coast of continental Europe and is considered one of Europe's most famous flowers. Violet boasts a rich variety of colors, abundant flowers, a long flowering period, and diverse plant shapes, making it suitable for both outdoor planting in courtyards and along roadsides, as well as for a variety of indoor potted ornamental applications. Its excellent cold tolerance makes it one of the few ornamental plants that can bloom in cool seasons. Currently, hundreds of different types of horticultural cultivars have been bred abroad, focusing on traits such as plant shape (tall, medium, and short), flower color (white, yellow, orange, pink, red, and purple), and flower shape (single, double, and overlapping petals). Domestic violet breeding is limited, with high-end varieties relying on imports. my country has introduced dozens of violet varieties, but there is no unified, standardized standard for variety identification, leading to widespread substandard products and confusion among varieties. To promote the selection, identification, and promotion of varieties that meet market demand, it is imperative to establish appropriate violet variety identification standards. Therefore, violet variety identification is of great significance for germplasm resource evaluation, innovative germplasm utilization, quality supervision of commercial flowers, and market regulation. Currently, there are no reports in China on the identification of violet varieties using SSR molecular markers. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a violet fingerprint map based on SSR markers and a construction method, which realizes the identification of violet varieties for the first time and provides a scientific basis for screening valuable breeding materials from violet variety germplasm resources at home and abroad.

[0005] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0006] The present invention provides an SSR marker primer set for identifying violet. The primer set comprises 8 pairs of specific primer pairs. The nucleotide sequences of the primer pairs are shown as SEQ ID NO.1 to SEQ ID NO.16 respectively.

[0007] The present invention provides a method for constructing a fingerprint of violet varieties, the method comprising the following steps:

[0008] The DNA of each violet variety is amplified by PCR using a primer set, and the resulting product is subjected to electrophoresis detection to obtain the fingerprint of the violet variety.

[0009] Preferably, the primer set includes 8 pairs of specific primers, and the nucleotide sequences of the primer pairs are shown as SEQ ID NO.1 to SEQ ID NO.16 respectively.

[0010] Preferably, the violet varieties include: Harmonious Deep Rose Red, Harmonious Light Rose, Harmonious Light Purple, Harmonious Purple, Christmas Apricot, Christmas Purple, Christmas Deep Red, Christmas White, and Noble White whose provenance is Takii, Japan; Brilliant White, Brilliant Pink, Brilliant Peach Pink, Brilliant Rose Red, Brilliant Purple, Brilliant Light Purple, Maimeng Light Pink, Maimeng White, Maimeng Purple, Maimeng Rose Red, and Maimeng Red whose provenance is Sakata, Japan; Katz Apricot, Katz Ruby, Katz White, Iron Blue, Iron Apricot, Iron Light Yellow Primrose, Iron Palace Purple, Fine Wine White, Fine Wine Rose Red, Fine Wine Purple, and Rainbow Sugar Cube whose provenance is Pan American, United States; Strawberry Sorbet, Violet Purple, Violet Deep Rose Red, Violet Rose Red, Violet Peach Red, and Violet Milky White whose provenance is United States.

[0011] Preferably, the PCR amplification reaction system includes: 2 μL of 50 ng / μL DNA template, 0.5 μL of 10 ng / μL upstream primer, 0.5 μL of 10 ng / μL downstream primer, 5 μL of PCRMasterMix, and ddH2O added to 10 μL.

[0012] Preferably, the reaction procedure of the PCR amplification is: 94°C for 5 min; 94°C for 30 s, 60°C for 30 s, 72°C for 30 s, 9 cycles, with the annealing temperature decreasing by 0.5°C in each cycle; 94°C for 30 s, 55°C for 30 s, 72°C for 30 s, 30 cycles; 72°C for 10 min.

[0013] Preferably, after the electrophoresis detection, the SSR amplified bands are counted, and at the same migration position, the bands are recorded as "1" and the bands without are recorded as "0", forming respective data matrices, which are the fingerprint maps of the violet varieties.

[0014] The present invention also provides the application of the primer set or the construction method in identifying violet varieties.

[0015] The present invention also provides a method for identifying violet varieties, the method comprising the following steps:

[0016] Extracting genomic DNA from the violet to be tested, performing PCR amplification on the genomic DNA using the above primer set, and performing electrophoresis detection on the obtained PCR amplification products;

[0017] The obtained electrophoresis detection results are compared with the fingerprint map constructed by the above construction method to realize the variety identification of the violet to be tested.

[0018] Preferably, the electrophoresis detection result is: after electrophoresis detection, the SSR amplification bands are counted, and at the same migration position, the bands are recorded as "1" and the bands without are recorded as "0" to form a data matrix.

[0019] Beneficial effects of the present invention:

[0020] The present invention provides a method for constructing a fingerprint map of violet varieties. Eight pairs of SSR marker primers obtained by screening are used as core primers for constructing the fingerprint map of violet varieties. At the same migration position, "1" and "0" are used to mark the presence or absence of amplified fragments, respectively, to construct fingerprint maps of 37 violet varieties. The specific band combination of the eight pairs of primers is used as the DNA fingerprint of a variety, which can be used to identify different varieties. The violet fingerprint map provided by the present invention provides a scientific basis for screening valuable breeding materials from domestic and foreign violet variety germplasm resources, reduces breeding blindness, improves violet breeding efficiency, and provides a basis for the protection of new variety intellectual property rights, variety identification, and innovative violet breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the electrophoresis result of the 22 pairs of polymorphic primers selected. DETAILED DESCRIPTION

[0022] The present invention provides an SSR marker primer set for identifying violet. The primer set comprises 8 pairs of specific primer pairs. The nucleotide sequences of the primer pairs are shown as SEQ ID NO.1 to SEQ ID NO.16 respectively.

[0023] In the present invention, the SSR marker primer set is derived from the sequence of the developed violet whole genome SSR primer; the primer set is preferably synthesized by Guangzhou Tianyi Huiyuan Gene Technology Co., Ltd. The eight primer sets screened in the present invention have clear amplification bands and high polymorphism. The specific sequence information of the primer sets is shown in Table 1 below:

[0024] Table 1. Primer sets for SSR markers of violet

[0025]

[0026] The present invention utilizes 300 SSR primers to amplify 6 violet DNAs, screens out a series of polymorphic primers, and selects 22 pairs of SSR primers with clear bands and high polymorphism located on chromosome 7 for analysis of all 37 violet materials. Analysis of the obtained polymorphic loci reveals that the above 8 pairs of primers can distinguish 37 types of violet materials. Therefore, the 8 pairs of SSR marker primers screened out are used as core primers for constructing a fingerprint map. The specific band combination of the 8 pairs of primers is used as the DNA fingerprint of a variety, which can be used to identify different varieties.

[0027] The present invention provides a method for constructing the fingerprint of the above-mentioned violet variety, comprising the following steps:

[0028] The above primer sets are used to perform PCR amplification on the DNA of various violet varieties, and the resulting products are subjected to electrophoresis detection to obtain the fingerprint of the violet varieties.

[0029] In the present invention, the primer set includes 8 pairs of specific primers, and the nucleotide sequences of the primer pairs are shown in SEQ ID NO. 1 to SEQ ID NO. 16. In the present invention, the violet varieties and sources are shown in Table 2 below:

[0030] Table 2 Violet varieties and sources

[0031]

[0032] In the present invention, the reaction system of the PCR amplification preferably includes: 2 μL of 50 ng / μL DNA template, 0.5 μL of 10 ng / μL upstream primer, 0.5 μL of 10 ng / μL downstream primer, 5 μL of PCR MasterMix, and ddH2O added to 10 μL. In the present invention, the reaction procedure of the PCR amplification is preferably: 94°C for 5 min; 94°C for 30 s, 60°C for 30 s, 72°C for 30 s, 9 cycles with the annealing temperature decreasing by 0.5°C for each cycle; 94°C for 30 s, 55°C for 30 s, 72°C for 30 s, 30 cycles; 72°C for 10 min. In the present invention, the SSR amplification bands are counted after the electrophoresis detection, and the bands at the same migration position are recorded as "1" and the bands without bands are recorded as "0". The data matrices formed are the fingerprint maps of the violet varieties. The results of the violet variety plant map constructed by the above construction method are shown in Table 6.

[0033] The present invention also provides the application of the primer set or the construction method in identifying violet varieties.

[0034] The present invention also provides a method for identifying violet varieties, the method comprising the following steps:

[0035] Extracting genomic DNA from the violet to be tested, performing PCR amplification on the genomic DNA using the above primer set, and performing electrophoresis detection on the obtained PCR amplification products;

[0036] The obtained electrophoresis detection results are compared with the fingerprint map constructed by the above construction method to realize the variety identification of the violet to be tested.

[0037] In the present invention, the electrophoresis detection result is: after electrophoresis detection, the SSR amplification bands are counted, and at the same migration position, the bands are recorded as "1" and the bands without are recorded as "0" to form a data matrix.

[0038] The present invention does not particularly limit the method for extracting DNA from the violet sample, and conventional DNA extraction methods in the art can be used.

[0039] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0040] In the following examples, unless otherwise specified, all methods are conventional.

[0041] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0042] Example 1

[0043] 1. Screening of SSR primers and PAGE electrophoresis

[0044] The SSR primers used were derived from the sequences of the developed violet whole genome SSR primers.

[0045] 300 SSR primer pairs were amplified and screened using DNA (CTAB extraction) from 6 samples of violet (Table 3). A series of polymorphic primers were screened, from which 22 pairs of SSR primers with clear bands and high polymorphism were selected ( Figure 1 The amplification system (10 μL) consisted of 2 μL DNA template, 0.5 μL each of upstream and downstream primers (10 ng / μL), 5 μL of PCRMasterMix, and ddH2O to 10 μL. PCR products were run on a 6% polyacrylamide gel, silver-stained, developed, and photographed before reading the bands.

[0046] Table 3 Violet variety names and sources

[0047]

[0048] Table 4 Primer sequence information

[0049]

[0050]

[0051] 2. SSR fingerprint construction

[0052] According to the electrophoresis results, the SSR amplification bands were counted, and the bands at the same migration position were recorded as "1" and the bands without bands were recorded as "0", and their respective data matrices were formed. The number of alleles (Na), effective number of alleles (Ne), allele frequency and other information of each primer pair were calculated using POPGENE software, and the polymorphism information content (PIC) of the primers was calculated as follows: PIC = 1-∑(P i ) 2 , P i represents the frequency of the ith allele.

[0053] Table 5 Polymorphism information of primers

[0054]

[0055] Based on the polymorphic information content (PIC) of the primers, we screened the primer combinations that could distinguish all 37 accessions from each other, ranked from highest to lowest. After screening, comparison, and verification, we ultimately identified eight primer pairs (No. 2, No. 5, No. 6, No. 8, No. 12, No. 19, No. 20, and No. 21; Table 1) that could distinguish all 37 accessions and served as core primers for fingerprint construction. At the same migration position, the presence or absence of the amplified fragment was marked with a "1" or a "0," respectively. Fingerprints for the 37 violet varieties were constructed, as shown in Table 6.

[0056] Table 6 Fingerprints of 37 violet resources constructed based on 8 pairs of SSR primers

[0057]

[0058]

[0059] Example 2

[0060] Identification of unknown violet varieties

[0061] 1. Amplification of SSR primer set and PAGE electrophoresis

[0062] DNA from three unknown violet varieties (A, B, and C) (commercially unavailable violet varieties) was extracted using the CTAB method. PCR amplification was performed using the primer sets shown in Table 1. The amplification system consisted of 10 μL of the following: 2 μL of DNA template, 0.5 μL of each upstream and downstream primer (10 ng / μL), 5 μL of PCR MasterMix, and ddH2O added to 10 μL. The reaction procedure was as follows: 1 cycle at 94°C for 5 min; 9 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s, with the annealing temperature decreasing by 0.5°C each cycle; 30 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s; and 72°C for 10 min. The PCR products were subjected to 6% polyacrylamide gel electrophoresis, silver-stained, developed, and photographed before band reading.

[0063] 2. SSR fingerprint construction

[0064] According to the electrophoresis results, the SSR amplified bands were counted, and the bands at the same migration position were recorded as "1" and the bands without bands were recorded as "0". The data matrices were formed to obtain the fingerprints of the three unknown varieties as shown in Table 7.

[0065] Table 7 SSR fingerprint results of test materials

[0066]

[0067] Comparing Table 7 with the fingerprints of the 37 varieties provided by this invention (i.e., Table 6) reveals that variety A is identical to M8 (Brilliant Rose Red), B is identical to M16 (Maimeng Light Pink), and C is not identical to any other variety. Therefore, based on the fingerprint identification results, it can be determined that A is variety 'Brilliant Rose Red', B is variety 'Maimeng Light Pink', and C is a new variety not included in the 37 varieties.

[0068] The fingerprint of the present invention can distinguish 37 violet varieties in Table 2, and realizes the identification of violet varieties based on SSR markers.

[0069] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. An SSR marker primer set for identifying violet, characterized in that: The primer set includes 8 pairs of specific primers, and the nucleotide sequences of the primer pairs are shown as SEQ ID NO.1 to SEQ ID NO.16 respectively; The violet varieties include: Harmonious Deep Rose Red, Harmonious Light Rose, Harmonious Light Purple, Harmonious Purple, Christmas Apricot, Christmas Purple, Christmas Deep Red, Christmas White, and Noble White whose sources are from Takii, Japan; Brilliant White, Brilliant Pink, Brilliant Peach Pink, Brilliant Rose Red, Brilliant Purple, Brilliant Light Purple, Maimeng Light Pink, Maimeng White, Maimeng Purple, Maimeng Rose Red, and Maimeng Red whose sources are from Sakata, Japan; Katz Apricot, Katz Ruby, Katz White, Iron Blue, Iron Apricot, Iron Light Yellow Primrose, Iron Palace Purple, Fine Wine White, Fine Wine Rose Red, Fine Wine Purple, and Rainbow Sugar Cube whose sources are from Pan American, United States; Strawberry Sorbet, Violet Purple, Violet Deep Rose Red, Violet Rose Red, Violet Peach Red, and Violet Milky White whose sources are from the United States.

2. A method for constructing a fingerprint of violet varieties, characterized in that: The construction method comprises the following steps: The DNA of each violet variety is amplified by PCR using the primer set, and the resulting product is subjected to electrophoresis detection to obtain the fingerprint of the violet variety; The violet varieties include: Harmonious Deep Rose Red, Harmonious Light Rose, Harmonious Light Purple, Harmonious Purple, Christmas Apricot, Christmas Purple, Christmas Deep Red, Christmas White, and Noble White, all of which are from Takii, Japan; Brilliant White, Brilliant Pink, Brilliant Peach Pink, Brilliant Rose Red, Brilliant Purple, Brilliant Light Purple, Maimeng Light Pink, Maimeng White, Maimeng Purple, Maimeng Rose Red, and Maimeng Red, all of which are from Sakata, Japan; Katz Apricot, Katz Ruby, Katz White, Iron Blue, Iron Apricot, Iron Light Yellow Primrose, Iron Palace Purple, Fine Wine White, Fine Wine Rose Red, Fine Wine Purple, and Rainbow Sugar Cube, all of which are from Pan American, USA; and Strawberry Sorbet, Violet Purple, Violet Deep Rose Red, Violet Rose Red, Violet Peach Red, and Violet Milky White, all of which are from the USA. The primer set includes 8 pairs of specific primers, and the nucleotide sequences of the primer pairs are shown as SEQ ID NO.1 to SEQ ID NO.16 respectively.

3. The construction method according to claim 2, characterized in that The PCR amplification reaction system includes: 2 μL of 50 ng / μL DNA template, 0.5 μL of 10 ng / μL upstream primer, 0.5 μL of 10 ng / μL downstream primer, 5 μL of PCR Master Mix, and ddH2O added to 10 μL.

4. The construction method according to claim 2, characterized in that The reaction program of the PCR amplification is: 94°C for 5 min; 94°C for 30 s, 60°C for 30 s, 72°C for 30 s, 9 cycles, with the annealing temperature decreasing by 0.5°C in each cycle; 94°C for 30 s, 55°C for 30 s, 72°C for 30 s, 30 cycles; 72°C for 10 min.

5. The construction method according to claim 2, characterized in that After the electrophoresis detection, the SSR amplified bands are counted, and at the same migration position, the bands are recorded as "1" and the bands without are recorded as "0", forming their respective data matrices, which are the fingerprint maps of the violet varieties.

6. Use of the primer set according to claim 1 or the construction method according to any one of claims 2 to 5 in identifying violet varieties; the violet varieties include: The source varieties are Harmonious Deep Rose Red, Harmonious Light Rose, Harmonious Light Purple, Harmonious Purple, Christmas Apricot, Christmas Purple, Christmas Deep Red, Christmas White, and Noble White from Takii, Japan; the source varieties are Brilliant White, Brilliant Pink, Brilliant Peach Pink, Brilliant Rose Red, Brilliant Purple, Brilliant Light Purple, Maimeng Light Pink, Maimeng White, Maimeng Purple, Maimeng Rose Red, and Maimeng Red from Sakata, Japan; the source varieties are Katz Apricot, Katz Ruby, Katz White, Iron Blue, Iron Apricot, Iron Light Yellow Primrose, Iron Palace Purple, Fine Wine White, Fine Wine Rose Red, Fine Wine Purple, and Rainbow Sugar from Pan American, United States; the source varieties are Strawberry Sorbet, Violet Purple, Violet Deep Rose Red, Violet Rose Red, Violet Peach Red, and Violet Milky White from the United States.

7. A method for identifying violet varieties, characterized in that: The identification method comprises the following steps: Extracting genomic DNA from the violet to be tested, performing PCR amplification on the genomic DNA using the primer set of claim 1, and detecting the obtained PCR amplification product by electrophoresis; The obtained electrophoresis detection result is compared with the fingerprint spectrum constructed by the construction method according to any one of claims 2 to 5 to realize the identification of the variety of the violet to be tested; The violet varieties include: Harmonious Deep Rose Red, Harmonious Light Rose, Harmonious Light Purple, Harmonious Purple, Christmas Apricot, Christmas Purple, Christmas Deep Red, Christmas White, and Noble White whose sources are from Takii, Japan; Brilliant White, Brilliant Pink, Brilliant Peach Pink, Brilliant Rose Red, Brilliant Purple, Brilliant Light Purple, Maimeng Light Pink, Maimeng White, Maimeng Purple, Maimeng Rose Red, and Maimeng Red whose sources are from Sakata, Japan; Katz Apricot, Katz Ruby, Katz White, Iron Blue, Iron Apricot, Iron Light Yellow Primrose, Iron Palace Purple, Fine Wine White, Fine Wine Rose Red, Fine Wine Purple, and Rainbow Sugar Cube whose sources are from Pan American, United States; Strawberry Sorbet, Violet Purple, Violet Deep Rose Red, Violet Rose Red, Violet Peach Red, and Violet Milky White whose sources are from the United States.

8. The identification method according to claim 7, characterized in that The electrophoresis detection result is: after electrophoresis detection, the SSR amplification bands are counted, and at the same migration position, the bands are recorded as "1" and the bands without bands are recorded as "0" to form a data matrix.

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