15-fold SSR (Simple Sequence Repeat) marker detection method for identifying male parent and variety of eucalyptus

Abstract

The invention discloses a 15-fold SSR (Simple Sequence Repeat) marker detection method for identifying male parents and varieties of eucalyptus. Through experimental screening, optimization and verification, 15 pairs of SSR marker primers are screened out, 15 SSR markers can be detected in a reaction system, the reaction system is optimized, and the method can be used for rapidly and efficiently identifying male parents of natural pollination offspring of eucalyptus, determining whether natural hybrids exist or not and detecting variety attribution and authenticity of clone of eucalyptus. The detection method comprises the following steps: (1) DNA extraction and purification, (2) PCR amplification, and (3) PCR product detection and SSR marker typing. The method has the advantages of being high in experiment efficiency, accurate in detection, convenient to operate, suitable for different scales and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular markers, and specifically relates to a 15-fold SSR marker detection method for identifying eucalyptus male parents and varieties, which is suitable for quickly and efficiently identifying the male parent of eucalyptus natural pollination progeny and the variety affiliation and authenticity of eucalyptus asexual lines. Background Art

[0002] Eucalyptus is a general term for species of Eucalyptus, Angophora and Corymbia in the Myrtaceae family. It has the advantages of fast growth and high yield, tolerance to barrenness, strong resistance to adversity and high economic value. It is one of the three major types of afforestation species in the world and an important source of raw materials for industries such as pulp and paper, wood-based panels, furniture, packaging, construction, and eucalyptus oil. Eucalyptus is widely planted in South China, covering an area of ​​82 million mu. With less than 5% of my country's artificial forest area, it produces nearly 25% of the wood. Therefore, eucalyptus plays an important role in maintaining my country's wood and ecological security and promoting national economic development.

[0003] In the study of forest trees (including eucalyptus), full-sib progeny materials with the same maternal and paternal parents are often used. The acquisition of these materials generally requires artificial controlled pollination between the maternal and paternal parents, which is extremely inconvenient for tall trees that take many years to bloom. Even if they are dwarfed by grafting, there are still problems such as whether the flowering time and flowering amount meet the needs. Forest trees are generally outcrossed, and the pollen propagation distance is limited. The pollen of the maternal natural pollination mainly comes from the adjacent paternal parent. The full-sib progeny materials with the same paternal parent in the half-sib progeny of the natural pollination of a maternal parent can be determined through the identification of the paternal parent. In addition, when the maternal parent and the identified paternal parent are different tree species, the corresponding progeny are interspecific hybrids, thereby effectively identifying natural hybrids.

[0004] Simple sequence repeat (SSR, also known as microsatellite) marker is a molecular marker based on polymerase chain reaction (PCR) with specific primers. It is based on 2 to 6 base units and forms polymorphism of DNA fragment length through variation of the number of repeats. SSR has the advantages of co-dominance, wide distribution in the genome, high polymorphism, good repeatability and flexible detection methods. It has been used in many types of research assisted by molecular markers, such as paternal identification and full-sib progeny detection, variety fingerprint map construction and authenticity identification, germplasm genetic diversity analysis, etc.

[0005] In addition, eucalyptus afforestation mainly uses tissue cultured clones. In the long-term asexual propagation and promotion of asexual varieties, there will be confusion in the names and inferior products will be sold as good ones. In addition, the new asexual lines produced by breeding work must also be effectively distinguished from the existing asexual lines, so it is necessary to develop effective methods for eucalyptus variety identification. Summary of the invention

[0006] In view of the defects and shortcomings of the prior art, the present invention aims to provide a 15-fold SSR marker detection method for identifying Eucalyptus male parents and / or varieties.

[0007] The 132 SSR markers of eucalyptus developed in the early stage were subjected to multiple combination and typing tests, and the 15-fold fluorescence detection method in the present invention was determined through a large number of experimental screening and optimization. The 15-fold SSR marker detection method obtained through experimental screening, optimization and verification can effectively amplify and clearly type random samples, and is suitable for quickly and efficiently identifying the father of the natural pollination progeny of eucalyptus, determining whether it is a natural hybrid, and detecting the variety ownership and authenticity of the eucalyptus asexual line.

[0008] In the 15-fold SSR marker detection method for identifying the male parent and / or variety of eucalyptus invented this time, 15 pairs of SSR marker detection primer sets are mixed in the same PCR system to complete the PCR reaction simultaneously. The specific contents are as follows:

[0009] 1. The 15-plex SSR marker detection primer set described in the invention consists of the following 15 pairs of SSR marker detection primers:

[0010] The primer pair of EUCeSSR0568, the sequence of the forward primer is shown in SEQ ID NO.1, and the sequence of the backward primer is shown in SEQ ID NO.2;

[0011] The primer pair of EUCeSSR536, the sequence of the forward primer is shown in SEQ ID NO.3, and the sequence of the backward primer is shown in SEQ ID NO.4;

[0012] The primer pair of EUCeSSR0957, the sequence of the forward primer is shown in SEQ ID NO.5, and the sequence of the backward primer is shown in SEQ ID NO.6;

[0013] The primer pair of EUCeSSR046, the sequence of the forward primer is shown in SEQ ID NO.7, and the sequence of the backward primer is shown in SEQ ID NO.8;

[0014] The primer pair of EUCeSSR1007, the sequence of the forward primer is shown in SEQ ID NO.9, and the sequence of the backward primer is shown in SEQ ID NO.10;

[0015] The primer pair of EUCeSSR537, the sequence of the forward primer is shown in SEQ ID NO.11, and the sequence of the backward primer is shown in SEQ ID NO.12;

[0016] The primer pair of EUCeSSR509, the sequence of the forward primer is shown in SEQ ID NO.13, and the sequence of the backward primer is shown in SEQ ID NO.14;

[0017] The primer pair of EUCeSSR241, the sequence of the forward primer is shown in SEQ ID NO.15, and the sequence of the backward primer is shown in SEQ ID NO.16;

[0018] The primer pair of EUCeSSR668, the sequence of the forward primer is shown in SEQ ID NO.17, and the sequence of the backward primer is shown in SEQ ID NO.18;

[0019] The primer pair of EUCeSSR1098, the sequence of the forward primer is shown in SEQ ID NO.19, and the sequence of the backward primer is shown in SEQ ID NO.20;

[0020] The primer pair of EUCeSSR683, the sequence of the forward primer is shown in SEQ ID NO.21, and the sequence of the backward primer is shown in SEQ ID NO.22;

[0021] The primer pair of EUCeSSR0056, the sequence of the forward primer is shown in SEQ ID NO.23, and the sequence of the backward primer is shown in SEQ ID NO.24;

[0022] The primer pair of EUCeSSR1127, the sequence of the forward primer is shown in SEQ ID NO.25, and the sequence of the backward primer is shown in SEQ ID NO.26;

[0023] The primer pair of EUCeSSR349, the sequence of the forward primer is shown in SEQ ID NO.27, and the sequence of the backward primer is shown in SEQ ID NO.28;

[0024] The primer pair of EUCeSSR1018, the sequence of the forward primer is shown in SEQ ID NO.29, and the sequence of the backward primer is shown in SEQ ID NO.30.

[0025] 2. The SSR marker detection primer set described in the invention, the fluorescent modification of the 5' end of the 15 primer pairs is as follows:

[0026] The 5′ ends of the primer pairs of EUCeSSR0568, EUCeSSR536, EUCeSSR0957, EUCeSSR046, and EUCeSSR1007 were modified with the red luminescent substance Rox;

[0027] The 5′ ends of the primer pairs of EUCeSSR537, EUCeSSR509, EUCeSSR241, EUCeSSR668, and EUCeSSR1098 were modified with the black fluorescent substance Tamara;

[0028] The 5' ends of the primer pairs of EUCeSSR683, EUCeSSR0056, EUCeSSR1127, EUCeSSR349, and EUCeSSR1018 were modified with the green fluorescent substance Hex.

[0029] 3. The SSR marker detection primer set described in the invention is used to identify the male parent of Eucalyptus, and the steps are as follows:

[0030] S1. Extracting DNA from the eucalyptus progeny to be identified, the maternal eucalyptus and the candidate paternal eucalyptus;

[0031] S2. Using the DNA extracted in step S1 as the corresponding template DNA, a PCR reaction is performed using the 15-plex SSR marker detection primer set to obtain a PCR product;

[0032] S3. Detection of PCR products and SSR marker typing;

[0033] S4. Compare the SSR alleles of the offspring eucalyptus to be identified with the SSR alleles of the maternal eucalyptus and the candidate paternal eucalyptus to determine the paternal parent of the offspring eucalyptus to be identified.

[0034] 4. The SSR marker detection primer set described in the invention is used to identify eucalyptus varieties, and the steps are as follows:

[0035] S1. Extracting DNA of the eucalyptus to be identified and related candidate eucalyptus varieties;

[0036] S2. Using the DNA extracted in step S1 as the corresponding template DNA, a PCR reaction is performed using the 15-plex SSR marker detection primer set to obtain a PCR product;

[0037] S3. Detection of PCR products and SSR marker typing;

[0038] S4. Compare the SSR alleles of the eucalyptus to be identified with the SSR alleles of related candidate eucalyptus varieties to determine the variety affiliation and variety authenticity of the eucalyptus to be identified.

[0039] 5. The PCR system for identifying the male parent of eucalyptus and the eucalyptus variety is as follows:

[0040] The total volume is 25 μL, including 12.5 μL PCR buffer, 7.375 μL marker detection primer set mixture, 10 U Taq DNA polymerase, 40 ng template DNA, and the balance is ddH2O; the PCR buffer contains dNTP and Mg 2+ wait

[0041] 6. A mixture of detection primer sets for identifying eucalyptus male parents and eucalyptus varieties, wherein the final concentrations of different primer pairs in the PCR system are as follows:

[0042] The final concentration of the primer pairs for EUCeSSR0568, EUCeSSR509, and EUCeSSR1098 was 0.20 μM;

[0043] The final concentration of the primer pairs for EUCeSSR536, EUCeSSR046, EUCeSSR537, EUCeSSR241, EUCeSSR683, EUCeSSR0056, and EUCeSSR1018 was 0.10 μM;

[0044] The final concentration of the primer pairs for EUCeSSR0957, EUCeSSR1007, and EUCeSSR668 was 0.40 μM;

[0045] The final concentration of the primer pair for EUCeSSR1127 was 0.15 μM;

[0046] The final concentration of the primer pair for EUCeSSR349 was 0.30 μM.

[0047] 7. The PCR reaction procedures for identifying the male parent of eucalyptus and the eucalyptus variety are as follows:

[0048] Pre-denaturation at 94°C for 4 min; denaturation at 94°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 50 s, 35 cycles; extension at 72°C for 5 min.

[0049] Beneficial effects of the present invention:

[0050] (1) The method provided by the present invention has the advantages of high efficiency, accurate detection, convenient operation, and cost saving.

[0051] (2) Using the method provided by the present invention, the male parents of 3007 naturally pollinated progeny of eucalyptus, the authenticity of 12 clones from different sources, and the origins of 4 eucalyptus varieties were successfully identified. This method is applicable to samples of different scales, which helps to quickly construct full-sib progeny materials required for research and protect the rights and interests of variety breeders and users.

[0052] (3) The 15-fold SSR marker detection method of the present invention can not only quickly and efficiently identify the father of the naturally pollinated progeny of eucalyptus and determine whether the progeny are natural hybrids, but also detect the variety ownership and authenticity of the eucalyptus asexual lines. It is a key technology for the selection and breeding of excellent eucalyptus germplasm and the maintenance of variety rights, and has great application potential in the sustainable development and industrial promotion of eucalyptus plantation resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 This is a peak graph obtained by setting different annealing temperatures based on 15-plex SSR marker primers and randomly testing a eucalyptus sample; among them, the annealing temperatures from top to bottom are 62℃ and 60℃ respectively.

[0054] Figure 2 This is a peak graph obtained by adding different DNA sample amounts to 15-plex SSR marker primers and randomly testing one eucalyptus sample; among them, the DNA sample amounts from top to bottom are 20ng, 40ng, 60ng and 80ng respectively.

[0055] Figure 3 It is a peak graph based on the detection of 4 random samples using two different 15-plex SSR primer combinations, MIX1 and MIX2; from top to bottom, the 1st to 4th sub-graphs are peak graphs of the detection of 4 selected random samples using MIX1, and the 5th to 8th sub-graphs are peak graphs of the detection of 4 selected random samples using MIX2.

[0056] Figure 4 It is a peak graph of a eucalyptus sample randomly detected using the optimized 15-plex SSR marker detection method; among them, the 2nd to 4th sub-graphs are different fluorescence extracted from the 1st sub-graph, corresponding to HEX green fluorescence, TAMARA black fluorescence, and ROX red fluorescence, respectively.

[0057] Figure 5 This is a peak diagram of the male parent (top), female parent (middle) and offspring (bottom) of a eucalyptus sample randomly detected using the optimized 15-plex SSR marker detection method; from top to bottom are the male parent DH32-29, the female parent DH33-27, and the hybrid offspring.

[0058] Figure 6 The peak graphs of 12 Rut45-33 asexual lines from different sources were detected using the optimized 15-plex SSR marker detection method; among them, from top to bottom are the peak graphs of Rut45-33 asexual lines 4533a-4533l, with the peak graph of 4533a as a control.

[0059] Figure 7It is the peak graph of 4 eucalyptus samples detected by the optimized 15-plex SSR marker detection method; among them, from top to bottom are the peak graphs of Zhong Eucalyptus No. 1, Zhong Eucalyptus No. 2, Zhong Eucalyptus No. 3 and Zhong Eucalyptus No. 4.

[0060] Figure 8 This is a comparison of the peak graphs obtained by detecting Zhong'an No. 1 and DH32-28 using the optimized 15-plex SSR markers; among them, from top to bottom are the peak graphs of Zhong'an No. 1 and DH32-28.

[0061] Fig. 9 This is a comparison of the peak graphs obtained by detecting Zhong'an No. 2, Zhong'an No. 4 and Guanglin 9 using the optimized 15-plex SSR markers; among them, from top to bottom are the peak graphs of Zhong'an No. 2, Zhong'an No. 4 and Guanglin 9.

[0062] Fig.10 This is a comparison of the peak graphs obtained by detecting Zhong'an No. 3 and Q9 using the optimized 15-plex SSR markers; among them, from top to bottom are the peak graphs of Zhong'an No. 3 and Q9. DETAILED DESCRIPTION

[0063] The following examples are provided to further illustrate the present invention, rather than to limit the present invention.

[0064] The 15-fold SSR marker detection method obtained through experimental screening, optimization and verification is suitable for quickly and efficiently identifying the paternal parent of the naturally pollinated progeny of eucalyptus, determining whether it is a natural hybrid, and detecting the variety affiliation and authenticity of eucalyptus asexual lines.

[0065] The sequences of the primer pairs of 15 SSR markers used in the 15-plex SSR marker detection method, modified fluorescence, final primer concentrations in the PCR system, and reference fragment ranges of PCR products are detailed in Table 1.

[0066] Table 1 Primer sequences, modified fluorescence, final concentration of PCR system and reference fragment range of PCR products of 15 SSR markers

[0067]

[0068]

[0069] In the following examples, the PCR buffer (containing dNTP and Mg) added in the PCR system 2+ etc., hereinafter referred to as buffer) and Taq DNA polymerase kit were purchased from Nanjing Novozyme Biotechnology Co., Ltd., with the catalog number PM201-01; the internal standard GeneScan TM 500LIZ TMDye fragment standards were purchased from Applied Biosystems, USA, with the catalog number 4322682.

[0070] Example 1: Experimental system verification experiment of 15-plex SSR marker detection method

[0071] 1. Verification of annealing temperature

[0072] (1) PCR system

[0073] The total volume was 25 μL, and the composition was as follows: 12.5 μL buffer, 7.375 μL 15 labeled primer mixtures (the final concentration of each labeled primer pair in the reaction system is shown in Table 1), 10 U Taq DNA polymerase, 40 ng DNA, and the balance was ddH2O.

[0074] (2) PCR procedure

[0075] Pre-denaturation at 94°C for 4 min; 35 cycles: denaturation at 94°C for 30 s, annealing at different annealing temperatures (62°C and 60°C) for 30 s, extension at 72°C for 50 s; and finally extension at 72°C for 5 min.

[0076] (3) PCR product detection

[0077] 0.5 μL of the PCR product was diluted with 9.34 μL ultrapure formamide and 0.16 μL GeneScan 500LIZ internal standard, denatured at 95°C for 5 min, quickly cooled on ice, and SSR markers were typed on an ABI 3130xl genetic analyzer.

[0078] The experimental results show that, under the same other conditions, when randomly selecting a eucalyptus sample DNA as a template, the annealing temperature of 60℃ is significantly better than 62℃. Under the condition of 60℃, more and better SSR allele fragments can be amplified. Figure 1 .

[0079] 2. Verification of the amount of template DNA added

[0080] (1) PCR system

[0081] The total volume was 25 μL, and the composition was as follows: 12.5 μL buffer, 7.375 μL 15 labeled primer mixtures (the final concentration of each labeled primer pair in the reaction system is shown in Table 1), 10 U Taq DNA polymerase, different amounts of template DNA (20 ng, 40 ng, 60 ng and 80 ng, respectively), and the balance was ddH2O.

[0082] (2) PCR procedure

[0083] Pre-denaturation at 94°C for 4 min; 35 cycles: denaturation at 94°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 50 s; and finally extension at 72°C for 5 min.

[0084] (3) PCR product detection

[0085] 0.5 μL of the PCR product was diluted with 9.34 μL ultrapure formamide and 0.16 μL GeneScan 500LIZ internal standard, denatured at 95°C for 5 min, quickly cooled on ice, and SSR markers were typed on an ABI 3130xl genetic analyzer.

[0086] The experimental results show that when a eucalyptus sample DNA is randomly selected as a template, too little or too much template DNA will seriously affect the acquisition of the target PCR product. Among the four template DNA amounts in the experiment, 40ng is the optimal amount. Figure 2 .

[0087] 3.15 Validation of primer set

[0088] (1) PCR system

[0089] The total volume was 25 μL, and the composition was as follows: 12.5 μL buffer, 7.375 μL different 15-plex SSR marker primer mix (MIX1 or MIX2), 10 U Taq DNA polymerase, 40 ng DNA, and the balance was ddH2O.

[0090] (2) PCR procedure

[0091] Pre-denaturation at 94°C for 4 min; 35 cycles: denaturation at 94°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 50 s; and finally extension at 72°C for 5 min.

[0092] (3) PCR product detection

[0093] 0.5 μL of the PCR product was diluted with 9.34 μL ultrapure formamide and 0.16 μL GeneScan 500LIZ internal standard, denatured at 95°C for 5 min, quickly cooled on ice, and SSR markers were typed on an ABI 3130xl genetic analyzer.

[0094] (4) Description of MIX1 and MIX2

[0095] The 15 pairs of labeled primers in MIX1 and their final concentrations in the reaction system are shown in Table 1.

[0096] In MIX2, the EUCeSSR349 primer pair in MIX1 was replaced by the EUCeSSR181 primer pair (F: GCCCGCTGAAGTGTTTGT, SEQ ID NO.31; R: TGTGGTAGGAGGGTTTGG, SEQ ID NO.32; the fluorescent modification at the 5' end and the final concentration in the reaction system were the same as the EUCeSSR349 primer pair in MIX1), and the remaining 14 primer pairs and their final concentrations in the reaction system were the same as in MIX1.

[0097] Four random samples were amplified using MIX1 or MIX2 primer combinations. The results showed that the amplification effect of MIX1 primer combination was significantly better than that of MIX2 primer combination. Figure 3 .

[0098] 4.15 Optimal experimental system for the detection method of multiple SSR markers

[0099] (1) Optimal PCR system

[0100] The total volume was 25 μL, and the composition was as follows: 12.5 μL buffer, 7.375 μL 15 labeled primer mixtures (the final concentration of each labeled primer pair in the reaction system is shown in Table 1), 10 U Taq DNA polymerase, 40 ng DNA, and the balance was ddH2O.

[0101] (2) Optimal PCR program

[0102] Pre-denaturation at 94°C for 4 min; 35 cycles: denaturation at 94°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 50 s; and finally extension at 72°C for 5 min.

[0103] A eucalyptus sample DNA was randomly selected as the template DNA, and after amplification using the above optimal PCR system and PCR program, the peak shape of the PCR product was detected and typed as shown in the figure. Figure 4 .

[0104] Example 2: Identification of the male parent of eucalyptus natural pollination progeny and determination of natural hybrids

[0105] The task comes from a foreign-funded company in Nanning, Guangxi. The implementation time is from March 2023 to December 2023. The father parent of the offspring seedlings collected from 7 asexual mothers is identified, and the natural hybrids in the offspring are determined. Seeds were collected in late March, and leaf samples of the seed mother tree and 10 candidate male eucalyptus trees within 100m around the mother tree were collected. Sowing was done in early April, and tender leaves of the offspring seedlings were collected in early September. DNA was extracted and the optimal scheme obtained by screening in Example 1 was used to carry out the detection experiment of 15-fold SSR markers. The source of natural pollination seeds of the participating eucalyptus asexual lines and the number of participating offspring seedling samples are detailed in Table 2.

[0106] Table 2 Sources of natural pollination seeds of the eucalyptus clones and the number of progeny seedlings in the test

[0107]

[0108]

[0109] The specific implementation steps and experimental procedures are as follows:

[0110] 1. DNA extraction of eucalyptus offspring, maternal eucalyptus and candidate paternal eucalyptus

[0111] (a) About 300 mg of leaf sample was weighed and placed in a 2 mL centrifuge tube. Four 3 mm diameter steel balls were placed in the tube and cooled in liquid nitrogen. The tube was placed in a cryo-grinder with the frequency set to 55 Hz and the oscillation time set to 3 min.

[0112] (b) Add 1 mL of CTAB lysis buffer to the sample tube, vortex and incubate at 65°C in a water bath for 60 min, shaking every 10 min.

[0113] (c) After the water bath, the sample was centrifuged at 10,000 rpm for 10 min and the supernatant was collected.

[0114] (d) Add a mixed solution of chloroform and isoamyl alcohol in an equal volume to the supernatant (calculated by volume ratio, chloroform: isoamyl alcohol = 24:1), shake well, centrifuge at 10,000 rpm for 10 min, and collect the supernatant.

[0115] (e) Repeat step (d).

[0116] (f) Add 2 / 3 volume of refrigerated isopropanol to the supernatant, shake gently to mix, and place at -20°C for more than 2 h.

[0117] (g) Centrifuge at 10,000 rpm for 10 min, discard the supernatant, add 1 mL of 75% and 95% alcohol respectively for washing, dry, add 100 μL of 1×TE solution, soak the precipitate for 10 min, and flick to fully dissolve the DNA.

[0118] (h) Centrifuge briefly, add 1 μL of 10 mg / mL RNase A solution, and incubate at 37°C for 30 min.

[0119] 2. PCR process

[0120] (a) PCR system

[0121] The total volume was 25 μL, and the composition was as follows: 12.5 μL buffer, 7.375 μL of a mixture of 15 SSR marker primers (the final concentrations of different marker primer pairs in the reaction system are shown in Table 1 ), 10 U Taq DNA polymerase and 40 ng DNA, with the remainder being ddH O.

[0122] (b) PCR procedure

[0123] Pre-denaturation at 94°C for 4 min; 35 cycles: denaturation at 94°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 50 s; and final extension at 72°C for 5 min.

[0124] 3. PCR product detection

[0125] 0.5 μL of PCR product was diluted with 9.34 μL ultrapure formamide and 0.16 μL GeneScan 500LIZ internal standard, denatured at 95°C for 5 min, and quickly cooled on ice. Automatic detection by capillary electrophoresis was performed on an ABI 3130xl genetic analyzer, and data were collected using GeneMapper 4.1 software according to the sequencer operation manual.

[0126] 4. SSR allele comparison analysis

[0127] Eucalyptus is basically diploid, and there are generally two alleles at one SSR locus, with the same length (homozygous) or different lengths (heterozygous). If the two alleles are the same length, they will be superimposed into one peak. The peak graph of the sample mainly depends on whether the position of the peak on the horizontal axis is the same. If the position of the peak between samples is the same, it means that the alleles of the length corresponding to the peak are the same; the height of the peak (vertical axis) is not very important, it just indicates the difference in the amount of PCR amplification product.

[0128] 5. Identification results

[0129] The results of SSR allele comparison analysis of offspring seedlings, maternal parents and candidate paternal parents showed that 1766 of the 3007 asexually pollinated offspring seedlings involved in the identification were confirmed to have a paternal variety that was different from the maternal asexual variety, and were natural hybrids (Table 3). Among them, based on the detection of a sample of eucalyptus offspring seedlings based on 15-fold SSR markers, the peak graphs of the offspring, paternal parent and maternal parent are shown in Figure 5 .from Figure 5 It can be seen that the female parent is DH33-27, and the male parent is DH32-29, so the offspring seedlings are natural hybrids.

[0130] Table 3 Identification results of the male parent of the natural pollination progeny of Eucalyptus and the natural hybrid progeny of the determined male parent

[0131]

[0132] Example 3: Authenticity Identification of the E.urophylla × E.tereticornis Clone Rut45-33 (Rut45-33 for short)

[0133] The task originated from a private company in Nanning, Guangxi, and was implemented in December 2023. The 15-fold SSR marker detection method was used to analyze the authenticity of 12 strains of Rut45-33 (see Table 4) from different sources.

[0134] Table 4 Sources of 12 strains of Rut45-33

[0135]

[0136]

[0137] The implementation steps and experimental procedures are the same as those in "Example 2".

[0138] The implementation results show that among the 12 Rut45-33 samples from different sources tested this time, except for 4533f, the SSR allele lengths of the remaining samples (the combination of which is called molecular fingerprint) are consistent with the original plant 4533a of Rut45-33 seedlings from the earliest experimental forest in Zhangzhou, Fujian.

[0139] The identification results showed that the 4533f provided by a company in Beihai, Guangxi was not the genuine Rut45-33 asexual line. It was possible that it was confused with other asexual lines that were being propagated simultaneously during tissue culture rapid propagation and promotion. The remaining asexual line samples were all genuine Rut45-33 asexual lines.

[0140] The lengths of allele fragments of 12 Rut45-33 samples from different sources at 15 SSR markers are shown in Table 5; the peak shape diagram of 15-plex SSR marker detection of 12 samples is shown in Figure 6 .

[0141] Table 5 Allelic fragment lengths of 12 Rut45-33 strains from different sources detected by 15-plex SSR markers

[0142]

[0143]

[0144] Example 4: Species Identification of Eucalyptus Samples

[0145] The task came from the Forestry Bureau of a county in Liuzhou, Guangxi, to identify the varieties of 4 eucalyptus samples. The implementation time was April 2024. A total of 4 eucalyptus samples, namely, Zhong Eucalyptus 1, Zhong Eucalyptus 2, Zhong Eucalyptus 3 and Zhong Eucalyptus 4, were identified and differentiated. The SSR allele length (the combination of which is called molecular fingerprint) of the samples was analyzed using the 15-fold SSR marker detection method.

[0146] The implementation steps and experimental procedures are the same as those of "Example 2" and "Example 3".

[0147] The results showed that Zhongan 2 and Zhongan 4 are the same variety, and are different from Zhongan 1 and Zhongan 3. Compared with the SSR molecular fingerprints of 14 major domestically cultivated varieties, Zhongan 1 has the same SSR molecular fingerprint as DH32-28, Zhongan 2 and Zhongan 4 have the same SSR molecular fingerprint as Guanglin 9, and Zhongan 3 has the same molecular fingerprints as Q9 except for two sites with poor amplification effect. Therefore, it is determined that Zhongan 1 should be DH32-28, Zhongan 2 and Zhongan 4 should be Guanglin 9, and Zhongan 3 is suspected to be Q9.

[0148] The detection peak graphs of the four samples are shown in Figure 7 ; Figure 8 , Fig. 9 and Fig.10 The peak shape comparison of each sample and the determined varieties is shown respectively.

[0149] The fragment lengths of the four eucalyptus samples and related varieties DH32-28, Guanglin 9 and Q9 at 15 SSR markers are shown in Table 6.

[0150] Table 6 Allele lengths of 15 SSR markers in four eucalyptus samples and related varieties

[0151]

[0152]

Claims

1. A 15-fold SSR marker detection primer set for identifying eucalyptus male parents and / or varieties, characterized in that: It consists of the following 15 pairs of primers: The primer pair of EUCeSSR0568, the sequence of the forward primer is shown in SEQ ID NO.1, and the sequence of the backward primer is shown in SEQ ID NO.2; The primer pair of EUCeSSR536, the sequence of the forward primer is shown in SEQ ID NO.3, and the sequence of the backward primer is shown in SEQ ID NO.4; The primer pair of EUCeSSR0957, the sequence of the forward primer is shown in SEQ ID NO.5, and the sequence of the backward primer is shown in SEQ ID NO.6; The primer pair of EUCeSSR046, the sequence of the forward primer is shown in SEQ ID NO.7, and the sequence of the backward primer is shown in SEQ ID NO.8; The primer pair of EUCeSSR1007, the sequence of the forward primer is shown in SEQ ID NO.9, and the sequence of the backward primer is shown in SEQ ID NO.10; The primer pair of EUCeSSR537, the sequence of the forward primer is shown in SEQ ID NO.11, and the sequence of the backward primer is shown in SEQ ID NO.12; The primer pair of EUCeSSR509, the sequence of the forward primer is shown in SEQ ID NO.13, and the sequence of the backward primer is shown in SEQ ID NO.14; The primer pair of EUCeSSR241, the sequence of the forward primer is shown in SEQ ID NO.15, and the sequence of the backward primer is shown in SEQ ID NO.16; The primer pair of EUCeSSR668, the sequence of the forward primer is shown in SEQ ID NO.17, and the sequence of the backward primer is shown in SEQ ID NO.18; The primer pair of EUCeSSR1098, the sequence of the forward primer is shown in SEQ ID NO.19, and the sequence of the backward primer is shown in SEQ ID NO.20; The primer pair of EUCeSSR683, the sequence of the forward primer is shown in SEQ ID NO.21, and the sequence of the backward primer is shown in SEQ ID NO.22; The primer pair of EUCeSSR0056, the sequence of the forward primer is shown in SEQ ID NO.23, and the sequence of the backward primer is shown in SEQ ID NO.24; The primer pair of EUCeSSR1127, the sequence of the forward primer is shown in SEQ ID NO.25, and the sequence of the backward primer is shown in SEQ ID NO.26; The primer pair of EUCeSSR349, the sequence of the forward primer is shown in SEQ ID NO.27, and the sequence of the backward primer is shown in SEQ ID NO.28; The primer pair of EUCeSSR1018, the sequence of the forward primer is shown in SEQ ID NO.29, and the sequence of the backward primer is shown in SEQ ID NO.

30.

2. The primer set according to claim 1, characterized in that The fluorescent modification of the 5' ends of the 15 pairs of primers in the 15-plex SSR marker detection primer set is as follows: The 5′ ends of the primer pairs of EUCeSSR0568, EUCeSSR536, EUCeSSR0957, EUCeSSR046, and EUCeSSR1007 were modified with the red luminescent substance Rox; The 5′ ends of the primer pairs of EUCeSSR537, EUCeSSR509, EUCeSSR241, EUCeSSR668, and EUCeSSR1098 were modified with the black fluorescent substance Tamara; The 5' ends of the primer pairs of EUCeSSR683, EUCeSSR0056, EUCeSSR1127, EUCeSSR349, and EUCeSSR1018 were modified with the green fluorescent substance Hex.

3. A 15-fold SSR marker detection method for identifying the male parent of Eucalyptus, characterized in that: The following steps are involved: S1. Extracting DNA from the offspring eucalyptus to be identified, the mother eucalyptus and the candidate father eucalyptus; S2. Using the DNA extracted in step S1 as the corresponding template DNA, a PCR reaction is performed using the 15-plex SSR marker detection primer set according to claim 2 to obtain a PCR product; S3. Detection of PCR products and SSR marker typing; S4. Compare the SSR alleles of the offspring eucalyptus to be identified with the SSR alleles of the maternal eucalyptus and the candidate paternal eucalyptus to determine the paternal parent of the offspring eucalyptus to be identified.

4. The method according to claim 3, characterized in that The total volume of the PCR reaction system is 25 μL, including 12.5 μL PCR buffer, 7.375 μL of the mixture of the 15-plex SSR marker detection primer set described in claim 2, 10 U Taq DNA polymerase, 40 ng template DNA, and the balance is ddH2O.

5. The method according to claim 4, characterized in that The final concentrations of the different primer pairs in the mixture of the 15-plex SSR marker detection primer set in the PCR reaction system are as follows: The final concentration of the primer pairs for EUCeSSR0568, EUCeSSR509, and EUCeSSR1098 was 0.20 μM; The final concentration of the primer pairs for EUCeSSR536, EUCeSSR046, EUCeSSR537, EUCeSSR241, EUCeSSR683, EUCeSSR0056, and EUCeSSR1018 was 0.10 μM; The final concentration of the primer pairs for EUCeSSR0957, EUCeSSR1007, and EUCeSSR668 was 0.40 μM; The final concentration of the primer pair for EUCeSSR1127 was 0.15 μM; The final concentration of the primer pair for EUCeSSR349 was 0.30 μM.

6. The method according to claim 3, characterized in that The reaction procedure of the PCR reaction is: pre-denaturation at 94°C for 4 minutes; denaturation at 94°C for 30 seconds, annealing at 60°C for 30 seconds, extension at 72°C for 50 seconds, 35 cycles; extension at 72°C for 5 minutes.

7. A 15-fold SSR marker detection method for identifying eucalyptus varieties, characterized in that: The following steps are involved: S1. Extracting DNA of the eucalyptus to be identified and related candidate eucalyptus species; S2. Using the DNA extracted in step S1 as the corresponding template DNA, a PCR reaction is performed using the 15-plex SSR marker detection primer set according to claim 2 to obtain a PCR product; S3. Detection of PCR products and SSR marker typing; S4. Compare the SSR alleles of the eucalyptus to be identified with the SSR alleles of related candidate eucalyptus varieties to determine the variety affiliation and variety authenticity of the eucalyptus to be identified.

8. The method according to claim 7, characterized in that The total volume of the PCR reaction system is 25 μL, including 12.5 μL PCR buffer, 7.375 μL of the mixture of the 15-plex SSR marker detection primer set described in claim 2, 10 U Taq DNA polymerase, 40 ng template DNA, and the balance is ddH2O.

9. The method according to claim 8, characterized in that The final concentrations of the different primer pairs in the mixture of the 15-plex SSR marker detection primer set in the PCR reaction system are as follows: The final concentration of the primer pairs for EUCeSSR0568, EUCeSSR509, and EUCeSSR1098 was 0.20 μM; The final concentration of the primer pairs for EUCeSSR536, EUCeSSR046, EUCeSSR537, EUCeSSR241, EUCeSSR683, EUCeSSR0056, and EUCeSSR1018 was 0.10 μM; The final concentration of the primer pairs for EUCeSSR0957, EUCeSSR1007, and EUCeSSR668 was 0.40 μM; The final concentration of the primer pair for EUCeSSR1127 was 0.15 μM; The final concentration of the primer pair for EUCeSSR349 was 0.30 μM.

10. The method according to claim 7, characterized in that The reaction procedure of the PCR reaction is: pre-denaturation at 94°C for 4 minutes; denaturation at 94°C for 30 seconds, annealing at 60°C for 30 seconds, extension at 72°C for 50 seconds, 35 cycles; extension at 72°C for 5 minutes.

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