A method for identifying eucalypt male parent and variety by 15 heavy SSR markers

Abstract

The application discloses a 15-repetitive SSR marker detection method for identifying eucalyptus male parents and varieties. Through experiment screening, optimization and verification, 15 pairs of SSR marker primers are screened out, 15 SSR markers can be detected in one reaction system, and the reaction system is optimized, so that the 15-repetitive SSR marker detection method can be used for rapidly and efficiently identifying male parents of eucalyptus natural pollination offspring, determining whether the natural hybrid is natural or not, and detecting the variety attribution and authenticity of eucalyptus clones. The detection steps comprise the following steps: (1) DNA extraction and purification, (2) PCR amplification, (3) PCR product detection and SSR marker typing. The method provided by the application has the advantages of high experimental efficiency, accurate detection, convenient operation and suitability for different scales.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of molecular markers, and particularly relates to a 15-plex SSR marker detection method for identifying male parents and varieties of eucalyptus trees, which is suitable for rapidly and efficiently identifying male parents of eucalyptus trees naturally pollinated offspring and the variety attribution and authenticity of eucalyptus clones. BACKGROUND

[0002] Eucalyptus is a general term for tree species of the genus Eucalyptus, Angophora and Corymbia in the Myrtaceae family, which has the advantages of fast growth, high yield, strong stress resistance and high economic value, and is one of the three major types of afforestation trees in the world, and is an important raw material source for pulp and paper, wood-based panel, furniture, packaging, construction, eucalyptus oil and other industries. Eucalyptus is widely planted in South China, with an area of 82 million mu, producing nearly 25% of wood from less than 5% of artificial forest area in China, therefore, eucalyptus plays an important role in maintaining China's timber and ecological safety and promoting national economic development.

[0003] In the study of forest trees (including eucalyptus), full-sib offspring materials with the same maternal and paternal parents are often used, which are generally obtained by artificial controlled pollination between maternal and paternal parents. This is extremely inconvenient for forest trees that flower only after many years and have a large plant height, even if the plant height is reduced by grafting, there are still problems such as whether the flowering time and flowering amount meet the needs. Forest trees are generally cross-pollinated, and the pollen transmission distance is limited, so the pollen of maternal natural pollination mainly comes from the adjacent paternal parent, and the paternal identification can determine the full-sib offspring materials with the same paternal parent in the half-sib offspring of maternal natural pollination. Moreover, when the maternal parent and the identified paternal parent are different tree species, the corresponding offspring is an interspecific hybrid, thereby effectively identifying natural hybrids.

[0004] Simple sequence repeat (SSR, also known as microsatellite) marker is a kind of molecular marker based on the polymerase chain reaction (PCR) of specific primers, which is based on 2-6 base units and forms DNA fragment length polymorphism by repeating number variation. SSR has the advantages of co-dominance, wide distribution in genome, high polymorphism, good reproducibility and flexible detection method, and has been used in various studies assisted by molecular markers, such as paternal identification and full-sib offspring detection, variety fingerprint construction and authenticity identification, germplasm genetic diversity analysis, etc.

[0005] In addition, eucalyptus forest mainly uses tissue culture clone seedlings. Clone varieties may have name confusion and inferior products during long-term asexual propagation and promotion, and new clones produced by breeding work also need to be effectively distinguished from existing clones, which requires developing an effective method for identifying eucalyptus varieties. SUMMARY

[0006] In view of defects and deficiencies of the prior art, the present application aims to provide a 15-plex SSR marker detection method for identifying eucalyptus paternal and / or varieties.

[0007] The 15-plex fluorescence detection method is determined by a large number of experiments, screening and optimization of 132 SSR markers of eucalyptus developed in the early stage. The 15-plex SSR marker detection method obtained through experiment screening, optimization and verification can effectively amplify and clearly type random samples, and is suitable for quickly and efficiently identifying the paternal of eucalyptus natural pollination offspring and determining whether it is a natural hybrid, and detecting the variety attribution and authenticity of eucalyptus clones.

[0008] The 15-plex SSR marker detection method for identifying eucalyptus paternal and / or varieties of the present application is that 15 pairs of SSR marker detection primers are mixed in the same PCR system to complete the PCR reaction at the same time. The specific content is as follows:

[0009] 1. The 15-plex SSR marker detection primer set in the application is composed of the following 15 pairs of SSR marker detection primers:

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0026] The 5' end of the primer pair of EUCeSSR0568, EUCeSSR536, EUCeSSR0957, EUCeSSR046, EUCeSSR1007 is modified with red light-emitting substance Rox;

[0027] The 5' end of the primer pair of EUCeSSR537, EUCeSSR509, EUCeSSR241, EUCeSSR668, EUCeSSR1098 is modified with black fluorescent substance Tamara;

[0028] The 5' end of the primer pair of EUCeSSR683, EUCeSSR0056, EUCeSSR1127, EUCeSSR349, EUCeSSR1018 is modified with green fluorescent substance Hex.

[0029] 3. The SSR marker detection primer set described in the invention is used for identifying the father of eucalyptus, and the steps are as follows:

[0030] S1. Extracting the DNA of the eucalyptus to be identified, the mother eucalyptus and the candidate father eucalyptus;

[0031] S2. Using the 15-fold SSR marker detection primer set to perform PCR reaction with the DNA extracted in step S1 as the corresponding template DNA, and obtaining the PCR product;

[0032] S3. Detecting and SSR marker typing the PCR product;

[0033] S4. Comparing the SSR alleles of the eucalyptus to be identified with the SSR alleles of the mother eucalyptus and the candidate father eucalyptus, and determining the father of the eucalyptus to be identified.

[0034] 4. The SSR marker detection primer set described in the invention is used for identifying the variety of eucalyptus, and the steps are as follows:

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

[0036] S2. Using the 15-fold SSR marker detection primer set to perform PCR reaction with the DNA extracted in step S1 as the corresponding template DNA, and obtaining the PCR product;

[0037] S3. Detecting and SSR marker typing the PCR product;

[0038] S4. Comparing the SSR alleles of the eucalyptus to be identified with the SSR alleles of the related candidate eucalyptus variety, and determining the variety attribution and the authenticity of the eucalyptus to be identified.

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

[0040] Total volume 25 μL, including 12.5 μL PCR buffer, 7.375 μL labeled detection primer group mixture, 10 U Taq DNA polymerase, 40 ng template DNA, and the rest is ddH2O; the PCR buffer contains dNTP and Mg 2+ etc.

[0041] 6. The detection primer group mixture for identifying the male parent of Eucalyptus and identifying the Eucalyptus variety, wherein the final concentrations of different primer pairs in the PCR system are as follows:

[0042] The final concentration of the primer pair of EUCeSSR0568, EUCeSSR509 and EUCeSSR1098 is 0.20 μM;

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

[0044] The final concentration of the primer pair of EUCeSSR0957, EUCeSSR1007 and EUCeSSR668 is 0.40 μM;

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

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

[0047] 7. The PCR reaction procedure for identifying the male parent of Eucalyptus and identifying the Eucalyptus variety is as follows:

[0048] 94 ℃ pre-denaturation for 4 min; 94 ℃ denaturation for 30 s, 60 ℃ annealing for 30 s, 72 ℃ extension for 50 s, 35 cycles; 72 ℃ extension for 5 min.

[0049] The beneficial effects of the present application are as follows:

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

[0051] (2) By using the method provided by the present application, the male parent of 3007 Eucalyptus natural pollination offspring, the authenticity of 12 different source clone varieties, and the source of 4 Eucalyptus varieties are successfully identified. The method is suitable for different sample sizes, is helpful for quickly constructing the full sibling offspring materials required by the research, and guarantees the rights and interests of the breeders and users.

[0052] (3) The 15-plex SSR marker detection method of the application can not only quickly and efficiently identify the male parent of the natural pollination offspring of eucalyptus and determine whether the offspring is a natural hybrid, but also detect the variety attribution and authenticity of eucalyptus clones, which is a key technology for eucalyptus excellent germplasm breeding and variety ownership maintenance, and has great application potential in the sustainable development and industrialization promotion of eucalyptus plantation resources. BRIEF DESCRIPTION OF DRAWINGS

[0053] Figure 1 is a peak shape diagram obtained by randomly detecting one eucalyptus sample based on the 15-plex SSR marker primer setting different annealing temperatures; wherein the annealing temperatures from top to bottom are 62℃ and 60℃.

[0054] Figure 2 is a peak shape diagram obtained by randomly detecting one eucalyptus sample based on the 15-plex SSR marker primer adding different DNA sample amounts; wherein the DNA sample amounts from top to bottom are 20 ng, 40 ng, 60 ng and 80 ng.

[0055] Figure 3 is a peak shape diagram obtained by detecting four random samples based on two different 15-plex SSR primer combinations MIX1 and MIX2; from top to bottom, the first sub-diagram to the fourth sub-diagram are the peak shape diagrams obtained by detecting the selected four random samples using MIX1, and the fifth sub-diagram to the eighth sub-diagram are the peak shape diagrams obtained by detecting the selected four random samples using MIX2.

[0056] Figure 4 is a peak shape diagram obtained by randomly detecting one eucalyptus sample using the optimized 15-plex SSR marker detection method; wherein the second to fourth sub-diagrams are different fluorescence extracted from the first sub-diagram, corresponding to HEX green fluorescence, TAMARA black fluorescence and ROX red fluorescence, respectively.

[0057] Figure 5 is a peak shape diagram obtained by randomly detecting one eucalyptus sample using the optimized 15-plex SSR marker detection method; wherein the second to fourth sub-diagrams are different fluorescence extracted from the first sub-diagram, corresponding to HEX green fluorescence, TAMARA black fluorescence and ROX red fluorescence, respectively.

[0058] Figure 6 is a peak shape diagram obtained by detecting 12 Rut45-33 clones of different sources using the optimized 15-plex SSR marker detection method; wherein the peak shape diagrams of Rut45-33 clones 4533a-4533l from top to bottom are shown, and the peak shape diagram of 4533a is taken as a control.

[0059] Figure 7is the peak shape chart of 4 eucalyptus samples detected by the optimized 15-plex SSR marker detection method; wherein, from top to bottom, the peak shape charts of No. 1, No. 2, No. 3 and No. 4 of Eucalyptus camaldulensis are in turn.

[0060] Figure 8 is the comparison of the peak shape charts of No. 1 of Eucalyptus camaldulensis and DH32-28 detected by the optimized 15-plex SSR marker detection method; wherein, from top to bottom, the peak shape charts of No. 1 of Eucalyptus camaldulensis and DH32-28 are in turn.

[0061] Figure 9 is the comparison of the peak shape charts of No. 2, No. 4 of Eucalyptus camaldulensis and Guanglin 9 detected by the optimized 15-plex SSR marker detection method; wherein, from top to bottom, the peak shape charts of No. 2, No. 4 of Eucalyptus camaldulensis and Guanglin 9 are in turn.

[0062] Figure 10 is the comparison of the peak shape charts of No. 3 of Eucalyptus camaldulensis and Q9 detected by the optimized 15-plex SSR marker detection method; wherein, from top to bottom, the peak shape charts of No. 3 of Eucalyptus camaldulensis and Q9 are in turn. DETAILED DESCRIPTION

[0063] The following examples are further illustrations of the present application and are not intended to limit the present application.

[0064] The 15-plex SSR marker detection method obtained through experiment screening, optimization and verification is suitable for rapidly and efficiently identifying the paternal of eucalyptus natural pollination offspring and determining whether it is a natural hybrid and detecting the variety attribution and authenticity of eucalyptus clones.

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

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

[0067]

[0068]

[0069] In the following examples, the PCR buffer (containing dNTP and Mg 2+ , hereinafter referred to as buffer) and Taq DNA polymerase kit added in the PCR system are purchased from Nanjing Nuowei Biological Technology Co., Ltd., and the product number is PM201-01; the internal standard GeneScan TM 500LIZ TMDye fragment standard, purchased from Applied Biosystems, USA, item number 4322682.

[0070] Example 1: Verification experiment of 15 SSR marker detection method

[0071] 1. Verification of annealing temperature

[0072] (1) PCR system

[0073] 25 μL total volume, consisting of 12.5 μL buffer, 7.375 μL 15 marker primer mixture (the final concentration of each marker primer pair in the reaction system is shown in Table 1), 10 U Taq DNA polymerase, 40 ng DNA, and the rest is ddH2O.

[0074] (2) PCR program

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

[0076] (3) PCR product detection

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

[0078] The experimental results show that, under the same conditions, randomly selecting one eucalyptus sample DNA as a template, 60°C annealing temperature is obviously better than 62°C, and under the condition of 60°C, more and better SSR alleles can be amplified, see Figure 1 .

[0079] 2. Verification of template DNA addition amount

[0080] (1) PCR system

[0081] 25 μL total volume, consisting of 12.5 μL buffer, 7.375 μL 15 marker primer mixture (the final concentration of each marker 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 rest is ddH2O.

[0082] (2) PCR program

[0083] 94°C pre-denaturation 4 min; 35 cycles: 94°C denaturation 30 s, 60°C annealing 30 s, 72°C extension 50 s; final 72°C extension 5 min.

[0084] (3) PCR product detection

[0085] PCR product 0.5 μL was added to 9.34 μL ultrapure formamide and 0.16 μL GeneScan 500LIZ internal standard dilution, 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 showed that randomly selecting one eucalyptus sample DNA as a template, too little or too much template DNA would seriously affect the acquisition of target PCR products. Among the four template DNA amounts, 40 ng was the best, and details were shown in Table 2. Figure 2 .

[0087] 3.15 Verification of 15-primer group

[0088] (1) PCR system

[0089] 25 μL total volume, consisting of: 12.5 μL buffer, 7.375 μL different 15-SSR marker primer mixtures (MIX1 or MIX2), 10 U Taq DNA polymerase, 40 ng DNA, and the rest was ddH2O.

[0090] (2) PCR program

[0091] 94°C pre-denaturation 4 min; 35 cycles: 94°C denaturation 30 s, 60°C annealing 30 s, 72°C extension 50 s; final 72°C extension 5 min.

[0092] (3) PCR product detection

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

[0094] (4) Explanation of MIX1 and MIX2

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

[0096] In MIX2, the primer pair of EUCeSSR181 (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 are the same as those of the primer pair of EUCeSSR349 in MIX1) is used to replace the primer pair of EUCeSSR349 in MIX1, and the other 14 primer pairs and their final concentrations in the reaction system are the same as those in MIX1.

[0097] The amplification of four random samples by using the primer combinations of MIX1 or MIX2 respectively shows that the amplification effect of the primer combination of MIX1 is obviously better than that of the primer combination of MIX2, which is shown in detail in Figure 3 .

[0098] 4.15 Optimal experimental system of detection method of heavy SSR markers

[0099] (1) Optimal PCR system

[0100] 25 μL total volume, consisting of 12.5 μL buffer, 7.375 μL 15 marker primer mixture (the final concentration of each marker primer pair in the reaction system is shown in Table 1), 10 U Taq DNA polymerase, 40 ng DNA, and the rest is ddH2O.

[0101] (2) Optimal PCR program

[0102] 94℃ pre-denaturation for 4 min; 35 cycles of 94℃ denaturation for 30 s, 60℃ annealing for 30 s, and 72℃ extension for 50 s; and finally 72℃ extension for 5 min.

[0103] A random eucalyptus sample DNA is selected as the template DNA, and after amplification by using the optimal PCR system and PCR program described above, the peak shape diagram of the detection and typing of the PCR product is shown in Figure 4 .

[0104] Example 2: Identification of the father of the eucalyptus naturally pollinated offspring and determination of the natural hybrid

[0105] The task comes from a foreign-funded company in Nanning, Guangxi, and the implementation time is from March 2023 to December 2023. The father of the offspring seedlings of 7 mother clones is identified, and the natural hybrid in the offspring is determined. The seeds are collected in late March, and the leaves of 10 candidate father eucalyptus trees within 100 m around the mother tree are collected in early April. The seeds are sown in early April, and the tender leaves of the offspring seedlings are collected in early September. The DNA is extracted, and the optimal scheme obtained by screening in Example 1 is used to carry out detection experiments of 15 heavy SSR markers. The sources of the eucalyptus clone naturally pollinated seeds and the number of samples of the participating offspring seedlings are shown in Table 2.

[0106] Table 2. Eucalyptus clones tested, source of seeds and number of progeny seedlings

[0107]

[0108]

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

[0110] 1. DNA extraction of Eucalyptus progeny, maternal Eucalyptus and candidate paternal Eucalyptus

[0111] (a) Weigh about 300 mg of leaf sample into a 2 mL centrifuge tube, add 4 steel balls with a diameter of 3 mm, cool in liquid nitrogen, and place in a frozen grinder with a frequency setting of 55 Hz for 3 min of oscillation.

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

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

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

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

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

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

[0118] (h) After slight centrifugation, 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 concentration of different marker primer pairs in the reaction system is shown in Table 1), 10 U Taq DNA polymerase, and 40 ng DNA, with the balance being ddH2O.

[0122] (b) PCR procedure

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

[0124] 3. PCR product detection

[0125] 0.5 μL of the PCR product was added to 9.34 μL of ultrapure formamide and 0.16 μL of GeneScan 500LIZ internal standard dilution, denatured at 95°C for 5 min, and rapidly cooled on ice. Capillary electrophoresis automatic detection was performed on an ABI 3130xl genetic analyzer, and data collection was performed using software GeneMapper 4.1 according to the operation manual of the sequencer.

[0126] 4. SSR allelic fragment comparison analysis

[0127] Eucalyptus is basically diploid, and generally has two allelic fragments at one SSR site, which are the same length (homozygous) or different lengths (heterozygous), and two allelic fragments of the same length are superimposed as one peak. The peak graph of a sample is mainly observed for whether the positions of the peaks on the horizontal coordinate are the same, and the positions of the peaks between samples are the same, which indicates that the allelic fragments of the same length are the same; and the height of the peak (vertical coordinate) is not very important, and only indicates the difference in the amount of PCR amplification product.

[0128] 5. Identification results

[0129] The SSR allelic fragment comparison analysis results of the progeny seedlings, the maternal parent, and the candidate paternal parent show that, among the 3007 seedlings of the progeny of the clones that are naturally pollinated and are identified, 1766 seedlings have a paternal parent that is a clone different from the maternal parent, and are natural hybrids (Table 3). Among them, based on the detection of a sample of a progeny seedling of Eucalyptus by 15 SSR markers, the peak shape graph of the sample, the parent, and the maternal parent is shown in Figure 5 From Figure 5 , it can be known that the progeny seedling of the maternal parent DH33-27 has a paternal parent DH32-29, and thus the progeny seedling is a natural hybrid.

[0130] Table 3 Identification results of the paternal parent of the naturally pollinated progeny of Eucalyptus and the natural hybrid progeny whose paternal parent is determined

[0131]

[0132] Example 3: Authenticity identification of E. urophylla x E. tereticornis clone Rut45-33 (abbreviation Rut45-33)

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

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

[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 fragment lengths (their combination is called molecular fingerprint) of the remaining samples are consistent with those of the original 4533a seedling of Rut45-33 in the earliest test forest in Zhangzhou, Fujian.

[0139] The identification results show that 4533f provided by a company in Beihai, Guangxi, is not a true Rut45-33 clone, and it is possible that it was confused with other clones during tissue culture and rapid propagation and promotion; the remaining clone samples are true Rut45-33 clones.

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

[0141] Table 5 SSR marker allele fragment lengths of 12 Rut45-33 samples from different sources based on 15-plex SSR marker detection

[0142]

[0143]

[0144] Example 4: Variety identification of eucalyptus samples

[0145] The task comes from a county forestry bureau in Liuzhou, Guangxi, to identify the varieties of 4 eucalyptus samples. The implementation time is April 2024, and 4 eucalyptus samples, including No. 1, No. 2, No. 3 and No. 4, are identified and distinguished. The SSR allelic fragment length (its combination is called molecular fingerprint) of the samples is analyzed by using the 15-SSR marker detection method.

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

[0147] The implementation results show that No. 2 and No. 4 of the eucalyptus are the same variety, and are different from No. 1 and No. 3. Compared with the SSR molecular fingerprints of 14 domestic main varieties, the SSR molecular fingerprint of No. 1 is consistent with that of DH32-28, the SSR molecular fingerprints of No. 2 and No. 4 are consistent with that of Guanglin 9, and the molecular fingerprints of the other 13 SSR markers of No. 3 are consistent with that of Q9 except for two sites with poor amplification effect. Therefore, it is determined that No. 1 should be DH32-28, No. 2 and No. 4 should be Guanglin 9, and No. 3 is suspected to be Q9.

[0148] The detection peak shape chart of the 4 samples is shown in Figure 7 ; Figure 8 , Figure 9 and Figure 10 , respectively, showing the peak shape comparison of each sample with the determined variety.

[0149] The fragment length of the 4 eucalyptus samples and related varieties DH32-28, Guanglin 9 and Q9 at 15 SSR markers is shown in Table 6.

[0150] Table 6 Allelic fragment length of 4 eucalyptus samples and related varieties at 15 SSR markers

[0151]

[0152]

Claims

1. A method for identifying the male parent of Eucalyptus using 15 SSR markers, characterized in that, Comprising the following steps: S1. Extracting DNA of the to-be-identified offspring eucalyptus, the female parent eucalyptus and the candidate male parent eucalyptus; S2. Using the 15-repeated SSR marker detection primer group to perform PCR reaction with the DNA extracted in step S1 as the corresponding template DNA, and obtaining PCR products; S3. Detecting and performing SSR marker typing on the PCR products; S4. Comparing the SSR alleles of the to-be-identified offspring eucalyptus with the SSR alleles of the female parent eucalyptus and the candidate male parent eucalyptus, and determining the male parent of the to-be-identified offspring eucalyptus; The 15-repeated SSR marker detection primer group is a primer group composed of the following 15 pairs of primers: The primer pair of EUCeSSR0568, wherein the sequence of the forward primer is shown as SEQ ID NO. 1, and the sequence of the reverse primer is shown as SEQ ID NO. 2; The primer pair of EUCeSSR536, wherein the sequence of the forward primer is shown as SEQ ID NO. 3, and the sequence of the reverse primer is shown as SEQ ID NO. 4; The primer pair of EUCeSSR0957, wherein the sequence of the forward primer is shown as SEQ ID NO. 5, and the sequence of the reverse primer is shown as SEQ ID NO. 6; The primer pair of EUCeSSR046, wherein the sequence of the forward primer is shown as SEQ ID NO. 7, and the sequence of the reverse primer is shown as SEQ ID NO. 8; The primer pair of EUCeSSR1007, wherein the sequence of the forward primer is shown as SEQ ID NO. 9, and the sequence of the reverse primer is shown as SEQ ID NO. 10; The primer pair of EUCeSSR537, wherein the sequence of the forward primer is shown as SEQ ID NO. 11, and the sequence of the reverse primer is shown as SEQ ID NO. 12; The primer pair of EUCeSSR509, wherein the sequence of the forward primer is shown as SEQ ID NO. 13, and the sequence of the reverse primer is shown as SEQ ID NO. 14; The primer pair of EUCeSSR241, wherein the sequence of the forward primer is shown as SEQ ID NO. 15, and the sequence of the reverse primer is shown as SEQ ID NO. 16; The primer pair of EUCeSSR668, wherein the sequence of the forward primer is shown as SEQ ID NO. 17, and the sequence of the reverse primer is shown as SEQ ID NO. 18; The primer pair of EUCeSSR1098, wherein the sequence of the forward primer is shown as SEQ ID NO. 19, and the sequence of the reverse primer is shown as SEQ ID NO. 20; The primer pair of EUCeSSR683, wherein the sequence of the forward primer is shown as SEQ ID NO. 21, and the sequence of the reverse primer is shown as SEQ ID NO. 22; The primer pair of EUCeSSR0056, wherein the sequence of the forward primer is shown as SEQ ID NO. 23, and the sequence of the reverse primer is shown as SEQ ID NO. 24; The primer pair of EUCeSSR1127, wherein the sequence of the forward primer is shown as SEQ ID NO. 25, and the sequence of the reverse primer is shown as SEQ ID NO. 26; a primer pair of EUCeSSR349, the sequence of the forward primer of which is shown as SEQ ID NO. 27, and the sequence of the reverse primer of which is shown as SEQ ID NO. 28; a primer pair of EUCeSSR1018, the sequence of the forward primer of which is shown as SEQ ID NO. 29, and the sequence of the reverse primer of which is shown as SEQ ID NO. 30; the total volume of the PCR reaction system is 25 μL, including 12.5 μL of PCR buffer, 7.375 μL of the mixture of the 15-plex SSR marker detection primer set, 10 U of Taq DNA polymerase, 40 ng of template DNA, and the rest of ddH2O; the final concentrations of 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 pair of EUCeSSR0568, EUCeSSR509 and EUCeSSR1098 is 0.20 μM; the final concentration of the primer pair of EUCeSSR536, EUCeSSR046, EUCeSSR537, EUCeSSR241, EUCeSSR683, EUCeSSR0056 and EUCeSSR1018 is 0.10 μM; the final concentration of the primer pair of EUCeSSR0957, EUCeSSR1007 and EUCeSSR668 is 0.40 μM; the final concentration of the primer pair of EUCeSSR1127 is 0.15 μM; the final concentration of the primer pair of EUCeSSR349 is 0.30 μM.

2. The method of claim 1, wherein, the reaction procedure of the PCR reaction is as follows: pre-denaturation at 94 ℃ for 4 min, denaturation at 94 ℃ for 30 s, annealing at 60 ℃ for 30 s, extension at 72 ℃ for 50 s, 35 cycles, and extension at 72 ℃ for 5 min.

3. The method of claim 1, wherein, the fluorescence modification conditions of the 5' ends of the 15 primer pairs in the 15-plex SSR marker detection primer set are as follows: the 5' ends of the primer pairs of EUCeSSR0568, EUCeSSR536, EUCeSSR0957, EUCeSSR046 and EUCeSSR1007 are modified with a red light-emitting substance Rox; the 5' ends of the primer pairs of EUCeSSR537, EUCeSSR509, EUCeSSR241, EUCeSSR668 and EUCeSSR1098 are modified with a black fluorescent substance Tamara; the 5' ends of the primer pairs of EUCeSSR683, EUCeSSR0056, EUCeSSR1127, EUCeSSR349 and EUCeSSR1018 are modified with a green fluorescent substance Hex.

4. A method for identifying eucalypt varieties using 15 SSR markers, characterised by, comprising the following steps: S1. extracting DNA of the eucalyptus to be identified and related candidate eucalyptus varieties; S2. using the 15-plex SSR marker detection primer set to perform PCR reaction with the DNA extracted in step S1 as the corresponding template DNA to obtain PCR products; S3. detecting and performing SSR marker typing on the PCR products; S4. Comparing the SSR allelic fragments of the to-be-identified eucalyptus with the SSR allelic fragments of the related candidate eucalyptus varieties, and determining the variety attribution and authenticity of the to-be-identified eucalyptus; The 15 heavy SSR marker detection primer groups are primer groups composed of the following 15 pairs of primers: The primer pair of EUCeSSR0568, wherein the sequence of the forward primer is shown as SEQ ID NO. 1, and the sequence of the reverse primer is shown as SEQ ID NO. 2; The primer pair of EUCeSSR536, wherein the sequence of the forward primer is shown as SEQ ID NO. 3, and the sequence of the reverse primer is shown as SEQ ID NO. 4; The primer pair of EUCeSSR0957, wherein the sequence of the forward primer is shown as SEQ ID NO. 5, and the sequence of the reverse primer is shown as SEQ ID NO. 6; The primer pair of EUCeSSR046, wherein the sequence of the forward primer is shown as SEQ ID NO. 7, and the sequence of the reverse primer is shown as SEQ ID NO. 8; The primer pair of EUCeSSR1007, wherein the sequence of the forward primer is shown as SEQ ID NO. 9, and the sequence of the reverse primer is shown as SEQ ID NO. 10; The primer pair of EUCeSSR537, wherein the sequence of the forward primer is shown as SEQ ID NO. 11, and the sequence of the reverse primer is shown as SEQ ID NO. 12; The primer pair of EUCeSSR509, wherein the sequence of the forward primer is shown as SEQ ID NO. 13, and the sequence of the reverse primer is shown as SEQ ID NO. 14; The primer pair of EUCeSSR241, wherein the sequence of the forward primer is shown as SEQ ID NO. 15, and the sequence of the reverse primer is shown as SEQ ID NO. 16; The primer pair of EUCeSSR668, wherein the sequence of the forward primer is shown as SEQ ID NO. 17, and the sequence of the reverse primer is shown as SEQ ID NO. 18; The primer pair of EUCeSSR1098, wherein the sequence of the forward primer is shown as SEQ ID NO. 19, and the sequence of the reverse primer is shown as SEQ ID NO. 20; The primer pair of EUCeSSR683, wherein the sequence of the forward primer is shown as SEQ ID NO. 21, and the sequence of the reverse primer is shown as SEQ ID NO. 22; The primer pair of EUCeSSR0056, wherein the sequence of the forward primer is shown as SEQ ID NO. 23, and the sequence of the reverse primer is shown as SEQ ID NO. 24; The primer pair of EUCeSSR1127, wherein the sequence of the forward primer is shown as SEQ ID NO. 25, and the sequence of the reverse primer is shown as SEQ ID NO. 26; The primer pair of EUCeSSR349, wherein the sequence of the forward primer is shown as SEQ ID NO. 27, and the sequence of the reverse primer is shown as SEQ ID NO. 28; a primer pair of EUCeSSR1018, wherein the sequence of the forward primer is shown as SEQ ID NO. 29, and the sequence of the reverse primer is shown as SEQ ID NO. 30; The total volume of the PCR reaction system is 25 μL, including 12.5 μL of PCR buffer, 7.375 μL of the mixture of the 15-plex SSR marker detection primer set, 10 U of Taq DNA polymerase, 40 ng of template DNA, and the balance of ddH2O; The final concentrations of 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 concentrations of primer pairs of EUCeSSR0568, EUCeSSR509 and EUCeSSR1098 are 0.20 μM; The final concentrations of primer pairs of EUCeSSR536, EUCeSSR046, EUCeSSR537, EUCeSSR241, EUCeSSR683, EUCeSSR0056 and EUCeSSR1018 are 0.10 μM; The final concentrations of primer pairs of EUCeSSR0957, EUCeSSR1007 and EUCeSSR668 are 0.40 μM; The final concentration of primer pairs of EUCeSSR1127 is 0.15 μM; The final concentration of primer pairs of EUCeSSR349 is 0.30 μM.

5. The method of claim 4, wherein, The reaction program of the PCR reaction is as follows: pre-denaturation at 94 ℃ for 4 min; denaturation at 94 ℃ for 30 s, annealing at 60 ℃ for 30 s, extension at 72 ℃ for 50 s, 35 cycles; and extension at 72 ℃ for 5 min.

6. The method of claim 4, wherein, The fluorescence modification conditions of the 5' ends of the 15 primer pairs in the 15-plex SSR marker detection primer set are as follows: The 5' ends of primer pairs of EUCeSSR0568, EUCeSSR536, EUCeSSR0957, EUCeSSR046 and EUCeSSR1007 are modified with a red light-emitting substance Rox; The 5' ends of primer pairs of EUCeSSR537, EUCeSSR509, EUCeSSR241, EUCeSSR668 and EUCeSSR1098 are modified with a black fluorescent substance Tamara; The 5' ends of primer pairs of EUCeSSR683, EUCeSSR0056, EUCeSSR1127, EUCeSSR349 and EUCeSSR1018 are modified with a green fluorescent substance Hex.

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