Primer for identifying authenticity of crape myrtle hybrid as well as application and method thereof

Abstract

The invention discloses a primer for identifying the authenticity of a crape myrtle hybrid as well as application and a method thereof. The primer molecular marker is high in resolution, can be used independently or in combination, and can be used for quickly and accurately identifying the authenticity of the crape myrtle hybrid and identifying the crape myrtle variety in the seedling stage. When the primer is used for identifying the authenticity of the lagerstroemia indica hybrid, the identification rate of hybrid offspring can reach 90%, and the primer is suitable for popularization and application in actual use. The method is simple to operate, convenient and fast, can directly utilize existing equipment, reagents and the like, and is convenient to popularize and use.

Description

Technical Field

[0001] This invention relates to the field of plant variety identification, specifically to a primer for identifying the authenticity of crape myrtle hybrids and its application and method. Background Technology

[0002] Crape myrtle (Lagerstroemia indica) is a deciduous shrub or small tree belonging to the genus Lagerstroemia in the family Lythraceae. With its vibrant flowers and long blooming period, it is an important woody flowering plant for summer. Since the 1960s, horticulturalists worldwide have cultivated numerous crape myrtle varieties through various breeding methods, including introduction and domestication, selective breeding, and hybridization. As of the end of September 2017, the United States alone had 94 patented crape myrtle varieties. Currently, China has over 300 crape myrtle varieties, while horticulturalists worldwide have cultivated over 100 more. Existing crape myrtle varieties can be classified by flower color into violet, red, silver, and multi-colored flower groups. Most are shrubs and ground cover types, with a few small trees and a lack of tree types. Therefore, the cultivation of new crape myrtle varieties urgently needs to be continued, especially the development of varieties with new flower colors and tree types.

[0003] There are approximately 56-80 species of Lagerstroemia worldwide, but most species are confined to tropical regions and lack cold resistance. Only a few species can be found in temperate regions, with China being the main distribution area. Among them, the Chinese endemic Lagerstroemia excelsa is a deciduous tree belonging to the same genus as Lagerstroemia in the family Lythraceae. It is a tall tree, reaching 20-30 meters in height, and is a rare tree species in the Lagerstroemia genus. It also has cold resistance and resistance to diseases and pests, but its flowers are small and white, making it less ornamental. Furthermore, there is a distant hybridization barrier between Lagerstroemia excelsa and Lagerstroemia excelsa (Wang Tian, ​​2022, Study on distant hybridization compatibility and seed germination characteristics of Lagerstroemia excelsa and Lagerstroemia excelsa, Master's thesis of Central South University of Forestry and Technology, p. 77; Li Xuelu, 2024, Preliminary study on the molecular mechanism of hybridization incompatibility between 'Ziyun' Lagerstroemia excelsa and Lagerstroemia excelsa, Master's thesis of Central South University of Forestry and Technology, p. 55). Hybridization between *Lagerstroemia indica* var. *chizophyllum* and different varieties of *Lagerstroemia* showed significant differences in seed set rate and the number of offspring seedlings. Reciprocal crosses between *Lagerstroemia indica* var. *chizophyllum* and 'Danhong Ziye' var. *chizophyllum* ... 0.79%, no hybrid seedlings were obtained, and the reciprocal cross seed setting rate was 9.24%, with only 2 hybrid seedlings obtained (Wang Tian, ​​2022, Study on the affinity and seed germination characteristics of distant hybridization between Lagerstroemia indica and Lagerstroemia indica var. chuanxiong, Master's thesis of Central South University of Forestry and Technology, p. 24). Although hybrid fruits were obtained, most of the embryos in the fruits were underdeveloped or missing, and the hybrid seeds could not germinate or had difficulty germinating. Therefore, it was impossible to obtain hybrid seedlings or only a small number of hybrid seedlings were obtained, and the authenticity of the hybrid species was not identified.

[0004] To obtain a superior crape myrtle variety that combines the genotypes of both Lagerstroemia indica and Lagerstroemia speciosa, the inventors mixed wild Lagerstroemia speciosa with several other crape myrtle varieties and continuously selected them through open pollination over eight years. This resulted in the natural hybrid arborescent white-flowered crape myrtle variety 'Qianchao Xingbai' (variety right number: 20250992). 'Qianchao Xingbai' exhibits significantly different morphological characteristics from its parent, Lagerstroemia speciosa, and also from any other hybrid crape myrtle variety. 'Qianchao Xingbai' has 18-24 stamens, including 6 long stamens, with a flower diameter of 1.20-1.50 cm and purple petal claws (RHS 75C); while the parent Lagerstroemia speciosa has only 6 stamens, a flower diameter of 0.5-0.6 cm, and white petal claws; furthermore, both 'Qianchao Xingbai' and the parent Lagerstroemia speciosa have white flowers. (See...) Figure 1 and Figure 2'Qianchao Xingbai', a tall crape myrtle variety containing the genotype of Sichuan and Guizhou crape myrtle, is an ideal breeding parent for obtaining arborescent crape myrtle. Although its flower diameter is twice that of the parent plant, it is still a very small type, and its flower color is white. Its ornamental characteristics still need to be improved, and its market acceptance is low. Therefore, by hybridizing 'Qianchao Xingbai' with red-flowered and crape myrtle-colored crape myrtle varieties, it is hoped that a tall arborescent non-white-flowered crape myrtle variety can be obtained.

[0005] The key to obtaining superior traits in hybrid breeding lies in identifying the authenticity of hybrid offspring. Traditional phenotypic identification based on organs such as flowers, stems, leaves, and fruits is time-consuming and unreliable, especially in the early stages. SSR molecular markers, due to their co-dominance, high polymorphism, large information content, good repeatability, and high reliability, are widely used in germplasm resource identification and kinship determination. Therefore, if SSR molecular markers can be used to identify the authenticity of Lagerstroemia indica hybrid offspring, it is expected to improve breeding efficiency, shorten the breeding cycle, and thus obtain new and superior flower colors and tall, tree-type flowering Lagerstroemia indica varieties. Summary of the Invention

[0006] In order to identify the authenticity of 'Qianchao Xingbai' crape myrtle hybrids with red-flowered and crape myrtle-colored crape myrtle varieties, this invention provides primers for identifying the authenticity of crape myrtle hybrids and crape myrtle varieties, as well as their applications and methods, which can quickly and accurately identify the authenticity of crape myrtle hybrids in the seedling stage.

[0007] A primer for identifying the authenticity of crape myrtle hybrids and for identifying crape myrtle varieties, wherein the primer is one or more of primers S04, S17, and S20;

[0008] The primer S04:

[0009] Forward primer: 5'-ATGTTCATGTGGCTCGGTCA-3' (SEQ ID NO: 1)

[0010] Reverse primer: 5'-CAGATGATGGCTCCAGCGAT-3' (SEQ ID NO: 2);

[0011] Primer S17:

[0012] Forward primer: 5'-GTCTCTCAGATACACCGCCG-3' (SEQ ID NO: 3)

[0013] Reverse primer: 5'-GACCAGACAGACAGACAGGC-3' (SEQ ID NO: 4);

[0014] The primer S20:

[0015] Forward primer: 5'-GGTGGAGATGCTAACAAGCAAG-3' (SEQ ID NO: 5)

[0016] Reverse primer: 5'-GGATTTTTGCTGTAGGGTGATT-3' (SEQ ID NO: 6).

[0017] Optionally, the primers can be designed as fluorescently labeled primers by incorporating fluorescent dyes, fluorescent probes, or other materials for fluorescent labeling, to adapt to various fluorescence detection or reading devices. Generally, the forward primer is fluorescently labeled, for example, by modifying the forward primer with FAM (blue) or HEX (green) biofluorescent genes.

[0018] The primers described above for identifying the authenticity of crape myrtle hybrids can be used to identify both the authenticity of the hybrids and the crape myrtle varieties. The primers can be used directly to identify the authenticity of crape myrtle hybrids and the crape myrtle varieties; alternatively, reagents or kits containing the primers can be used to identify the authenticity of crape myrtle hybrids and the crape myrtle varieties. The crape myrtle varieties are one or more of 'Qianchao Xingbai', 'Hongsheng', and 'Ewei No. 3'.

[0019] The method for identifying the authenticity of crape myrtle hybrids includes the following steps:

[0020] (1) Extract DNA from fresh leaves of each sample of 'Qianchao Xingbai', 'Hongsheng', 'Ewei No. 3', and the crape myrtle hybrid to be tested;

[0021] (2) Using the DNA extracted in step (1) as DNA templates, PCR amplification was performed using the primers used to identify the authenticity of the crape myrtle hybrid to obtain the PCR amplification products of each primer.

[0022] (3) The PCR amplification products of each primer in step (2) are detected by capillary electrophoresis to obtain the capillary electrophoresis pattern of each sample. The authenticity of the crape myrtle hybrid is identified based on the capillary electrophoresis pattern of the crape myrtle hybrid to be tested.

[0023] Optionally, the criteria for determining the authenticity of a crape myrtle hybrid based on its capillary electrophoresis pattern are as follows: if the capillary electrophoresis pattern of the crape myrtle hybrid shows both parental characteristic peaks or only the paternal characteristic peak, it is determined to be a genuine hybrid; otherwise, it is determined to be a false hybrid.

[0024] Optionally, the crape myrtle hybrid is one or more of the following: 'Qianchao Xingbai' × 'Hongsheng' hybrid, 'Hongsheng' × 'Qianchao Xingbai' hybrid, and 'Qianchao Xingbai' × 'Ewei No. 3' hybrid. The 'Qianchao Xingbai' × 'Hongsheng' hybrid is a hybrid with 'Qianchao Xingbai' as the female parent and 'Hongsheng' as the male parent. The 'Hongsheng' × 'Qianchao Xingbai' hybrid is a hybrid with 'Hongsheng' as the female parent and 'Qianchao Xingbai' as the male parent. The 'Qianchao Xingbai' × 'Ewei No. 3' hybrid is a hybrid with 'Qianchao Xingbai' as the female parent and 'Ewei No. 3' as the male parent.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] This invention provides primers for identifying the authenticity of crape myrtle hybrids and crape myrtle varieties. These primers have high molecular marker resolution and can be used alone or in combination. They can quickly and accurately identify the authenticity of crape myrtle hybrids in the seedling stage, and can also quickly and accurately identify the crape myrtle varieties 'Qianchao Xingbai', 'Hongsheng', and 'Ewei No. 3' in the seedling stage.

[0027] The primers used in this invention to identify the authenticity of crape myrtle hybrids can achieve an identification rate of up to 90% for hybrid offspring, making them suitable for widespread application in practical use.

[0028] The method of this invention is simple to operate, convenient and quick, and can directly utilize existing equipment and reagents, making it easy to promote and use. Attached Figure Description

[0029] Figure 1 These are photos of a single flower, among which... Figure 1 Photo a shows a single flower of Lagerstroemia indica from Sichuan and Guizhou. Figure 1 b is a photo of a single flower of the 'Qian Chaoxing Bai' variety;

[0030] Figure 2 This is a photo of the inflorescence, in which... Figure 2 Photo 'a' shows the inflorescence of Lagerstroemia indica from Sichuan and Guizhou. Figure 2 b is a photo of the inflorescence of 'Qianchao Xingbai';

[0031] Figure 3 Comparison of single flowers of hybrid Z1-8, hybrid Z1-16, female parent 'Qianchao Xingbai', and male parent 'Hongsheng';

[0032] Figure 4 Comparison of single flowers of hybrid Z2-6, hybrid Z2-9, female parent 'Qianchao Xingbai', and male parent 'Ewei No. 3';

[0033] Figure 5 Capillary electrophoresis image for the identification of 'Qianchao Xingbai' using primer S04;

[0034] Figure 6Capillary electrophoresis image of primer S04 for the identification of 'Hongsheng';

[0035] Figure 7 Capillary electrophoresis image of primer S04 for the identification of 'Ewei No. 3';

[0036] Figure 8 Capillary electrophoresis image of primer S04 for the identification of 'Qianchao Xingbai' × 'Hongsheng' hybrid Z1-8;

[0037] Figure 9 Capillary electrophoresis image for the identification of 'Qianchao Xingbai' using primer S17;

[0038] Figure 10 Capillary electrophoresis image for the identification of 'Hongsheng' using primer S17;

[0039] Figure 11 Capillary electrophoresis image of primer S17 for the identification of 'Ewei No. 3';

[0040] Figure 12 Capillary electrophoresis image of primer S17 for identification of 'Qianchao Xingbai' × 'Hongsheng' hybrid Z1-8;

[0041] Figure 13 Capillary electrophoresis image of primer S17 for the identification of 'Qianchao Xingbai' × 'Hongsheng' hybrid Z1-16;

[0042] Figure 14 Capillary electrophoresis image of primer S17 for identifying the hybrid Z2-9 of 'Qianchao Xingbai' × 'Ewei 3';

[0043] Figure 15 Capillary electrophoresis image for the identification of 'Qianchao Xingbai' using primer S20;

[0044] Figure 16 Capillary electrophoresis image for the identification of 'Hongsheng' using primer S20;

[0045] Figure 17 Capillary electrophoresis image of primer S20 for the identification of 'Ewei No. 3';

[0046] Figure 18 Capillary electrophoresis image of primer S20 for identification of 'Qianchao Xingbai' × 'Hongsheng' hybrid Z1-8;

[0047] Figure 19 Capillary electrophoresis image of Z2-6, a hybrid of 'Qianchao Xingbai' × 'Ewei 3', identified with primer S20;

[0048] Figure 20 Capillary electrophoresis image of Z2-9, a hybrid of 'Qianchao Xingbai' × 'Ewei 3', identified with primer S20;

[0049] In the capillary electrophoresis diagram, the horizontal axis is bp. Detailed Implementation

[0050] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments, so that those skilled in the art can better understand the technical solution of the present invention.

[0051] To further improve its ornamental traits, 'Qianchao Xingbai' was crossed with the red-flowered crape myrtle variety 'Hongsheng' and the purple-flowered crape myrtle variety 'Ewei No. 3' in reciprocal crosses, with the aim of obtaining a tall, tree-type crape myrtle variety with vibrant colors and large flower diameters.

[0052] The hybrids of 'Qianchao Xingbai' and 'Hongsheng' crape myrtle, and the hybrids of 'Qianchao Xingbai' and 'Ewei No. 3' crape myrtle, usually require flowering before observation to determine if they are true hybrids, and the accuracy of the observation results is low. If they do not flower, it cannot be determined whether they are true hybrids, and it is impossible to select superior arbor trees from them. Therefore, all bred hybrids need to be retained, resulting in a long breeding cycle, low efficiency, and high management costs. If hybrid identification could be performed on one-year-old seedlings, and only true hybrids could be retained in later selection, breeding efficiency could be improved and seedling costs reduced.

[0053] Example 1

[0054] 1. Test Methods

[0055] 1.1 Hybrid pollination and hybrid seedling cultivation

[0056] In August 2022, during the peak flowering period of three crape myrtle varieties—'Qianchao Xingbai,' 'Hongsheng,' and 'Ewei No. 3'—freshly opened flowers were collected between 7:00 and 8:00 AM and brought back to the laboratory. The anthers were peeled onto tracing paper, and the flowers were allowed to naturally disperse pollen at room temperature. They were then wrapped in tracing paper and placed in a sealed bag with silicone sealant, stored at 4°C. The following morning, between 5:00 and 8:00 AM, the flowers of the three crape myrtle varieties were artificially emasculated and covered with tracing paper bags. After complete emasculation, artificial pollination was performed using pollen collected the day before, and the bags were re-covered. Two reciprocal cross experiments were conducted for two hybridization combinations (see Table 1). Three days after pollination, the tracing paper bags were removed, and newly emerging flower buds were periodically observed and removed. Three months after pollination, when the capsules turned brownish-black, the fruits were harvested promptly, the number of fruits was counted, the fruits were opened, and the seeds were extracted and stored at 4°C.

[0057] In March 2023, the seeds were removed, soaked in a 37℃ water bath for 2 hours, and then sown in seedling trays containing substrate (peat:perlite = 5:1, by volume). At the end of May, when the seedlings grew to 10cm in height, they were transplanted into non-woven bags containing the same substrate ratio, and watered and fertilized as needed.

[0058] Table 1. Hybrid combination design and hybridization seed setting rate

[0059]

[0060] 1.2 Identification of SSR molecular markers in hybrid offspring

[0061] Once the seedlings have grown 8-10 leaves, collect fresh leaves from each hybrid offspring as samples and extract total DNA from each sample using a DNA extraction kit.

[0062] During the flowering period of 'Jianminhong' crape myrtle, its petals were collected and stored in liquid nitrogen. Transcriptome sequencing was performed by Illumina to obtain crape myrtle petal transcriptome data. Based on this crape myrtle petal transcriptome data, SSR analysis was performed on the selected single sequences larger than 1kb using the MISA tool (MicroStellite identification tool). SSR molecular marker primers were designed using Primer 5.0 software. 45 pairs of SSR primers were screened from the designed SSR molecular marker primers, and 15 pairs of SSR primers were screened from recent published literature, for a total of 60 pairs of SSR primers. Three primer pairs that can effectively distinguish 'Qianchaoxingbai', 'Hongsheng', and 'Ewei No. 3' were selected from the 60 SSR primer pairs for capillary electrophoresis. Two primer pairs (S04 and S17) were developed independently, and one primer pair (S20) was obtained from the literature (Wang, Xinwang, Dean, Deborah, et al. Development of Microsatellite Markers from Crape Myrtle (Lagerstroemia L.). HortScience, 2010, 45(5): 842-844). Primer sequence information is shown in Table 2.

[0063] The total volume of the PCR amplification reaction system was 15 μL: 1 μL of 50 ng / μL DNA template, 7.5 μL of 2×PCRMix, 0.3 μL of 10 mmol / L upstream primer (i.e., forward primer), 0.3 μL of 10 mmol / L downstream primer (i.e., reverse primer), and 5.9 μL of deionized water.

[0064] PCR amplification procedure: First, pre-denaturation was performed at 98°C for 3 min. Then, 35 cycles were performed, each cycle including a denaturation step at 94°C for 20 s, an annealing step at 55°C to 60°C for 20 s, and an extension step at 72°C for 40 s. After all cycles were completed, a final extension was performed at 72°C for 5 min.

[0065] PCR amplification product detection: Before capillary electrophoresis, 1 μL of PCR amplification product was added to 9 μL of highly deionized formamide (HiDi), denatured at 95°C for 3 min, immediately incubated in an ice-water bath, and then loaded for capillary electrophoresis. Capillary electrophoresis was performed using an ABI 3730XL sequencer.

[0066] Table 2 Primer sequence information

[0067]

[0068] The criteria for determining the authenticity of hybrids are as follows: if the capillary electrophoresis results of the hybrid offspring show both parental characteristic peaks or only the paternal characteristic peak, then it is determined to be a true hybrid (Zhao, et al., Hybrid identification and genetic variation of Elymus sibiricus hybrid populations using EST-SSR markers, Hereditas, 2017, Vol. 154, No. 15, p. 3).

[0069] 2. Research Results

[0070] 2.1 Hybridization efficiency

[0071] The fruit set rates of the two hybrid combinations ranged from 1.19% to 17.02% (see Table 1). Specifically, the fruit set rate of 'Qianchao Xingbai' × 'Hongsheng' was 4.20%, yielding 17 hybrid offspring; the fruit set rate of 'Hongsheng' × 'Qianchao Xingbai' was 17.02%, but due to external factors after sowing and transplanting (seedling damage caused by stray animals entering the seedling area after transplanting), only one hybrid offspring was ultimately obtained; the fruit set rate of 'Qianchao Xingbai' × 'Ewei No. 3' was 6.17%, yielding 12 hybrid offspring; and the fruit set rate of 'Ewei No. 3' × 'Qianchao Xingbai' was 1.19%, yielding no hybrid offspring.

[0072] 2.2 SSR identification of hybrid offspring

[0073] Seventeen hybrid offspring from the orthogonal cross (Z1) 'Qianchaoxingbai' (♀) × 'Hongsheng' (♂) were analyzed by capillary electrophoresis with primer S04. The maternal characteristic peak was 266 / 269, and the paternal characteristic peak was 263 / 272. Compared with the parental characteristic peaks, except for offspring Z1-4, Z1-15, and Z1-16, the remaining 14 offspring all possessed the characteristic peaks of both parents, thus identifying them as true hybrids. After capillary electrophoresis with primer S17, the maternal characteristic peak was 308 / 312, and the paternal characteristic peak was 263 / 272. The characteristic peak is 324 / 328. Compared with the parental characteristic peaks, except for the two offspring plants Z1-4 and Z1-15, the remaining 15 offspring plants all possess the characteristic peaks of both parents, and are identified as true hybrids. After capillary electrophoresis with primer S20, the maternal characteristic peak is 129 / 172, and the paternal characteristic peak is 133 / 140. Compared with the parental characteristic peaks, except for the three offspring plants Z1-4, Z1-15, and Z1-16, the remaining 14 offspring plants all possess the characteristic peaks of both parents, and are identified as true hybrids. That is, among the 17 hybrid offspring plants from the orthogonal cross of hybrid combination 1 (Z1), 14 plants can be identified as true hybrid offspring plants by primer S04 or primer S20, and 15 plants can be identified as true hybrid offspring plants by primer S17. The identification results showed that Z1-4 and Z1-15 were not true hybrids of 'Qianchao Xingbai' (♀) × 'Hongsheng' (♂), while the remaining 15 plants were true hybrids of 'Qianchao Xingbai' (♀) × 'Hongsheng' (♂).

[0074] The sole offspring of the reciprocal cross (Z1n) 'Hongsheng' (♀) × 'Qianchao Xingbai' (♂) was identified as a true hybrid offspring by capillary electrophoresis with primers S04, S17, and S20, and compared with the characteristic peaks of the parents. The identification results show that Z1n-1 is a true hybrid of 'Hongsheng' (♀) × 'Qianchao Xingbai' (♂).

[0075] Twelve offspring from the orthogonal cross Z2, 'Qianchaoxingbai' (♀) × 'Ewei No. 3' (♂), were analyzed by capillary electrophoresis with primer S04. The maternal genotype was 266 / 269, and the paternal genotype was 266 / 278, meaning the maternal characteristic peak was 269 and the paternal characteristic peak was 278. Comparison with the parental characteristic peaks revealed that five offspring, Z2-1, Z2-2, Z2-5, Z2-7, and Z2-8, possessed characteristic peaks from both parents, thus identifying them as true hybrids. Further analysis with capillary electrophoresis with primer S17... The maternal phenotypic peak was 308 / 312, and the paternal phenotypic peak was 328 / 328. Comparison with the parental phenotypic peaks revealed that five offspring plants (Z2-2, Z2-5, Z2-7, Z2-9, and Z2-10) possessed phenotypic peaks from both parents, thus identifying them as true hybrids. After capillary electrophoresis with primer S20, the maternal phenotypic peak was 129 / 172, and the paternal phenotypic peak was 125 / 125. Comparison with the parental phenotypic peaks revealed that, except for Z2-1 and Z2-3, the remaining ten offspring plants possessed phenotypic peaks from both parents, thus identifying them as true hybrids. In other words, among the 12 hybrid offspring from the orthogonal cross of hybrid combination 2 (Z2), five plants could be identified as true hybrids by primers S04 or S17, and ten plants could be identified as true hybrids by primer S20. The identification results showed that Z2-3 was not a true hybrid of 'Qianchao Xingbai' (♀) × 'Ewei No. 3' (♂), while the other 11 plants were true hybrids of 'Qianchao Xingbai' (♀) × 'Ewei No. 3' (♂).

[0076] In summary, three pairs of SSR primers were used to identify the hybrids of 30 offspring from the two hybrid combinations obtained in this invention. Of the 17 offspring from the reciprocal cross (Z1) of hybrid combination 1, 15 were identified as true hybrids using the primers of this invention; the only offspring from the reciprocal cross (Z1n) of hybrid combination 1 was identified as a true hybrid using all three pairs of primers; and of the 12 offspring from hybrid combination 2, 11 were identified as true hybrids using the primers of this invention. The results are shown in Table 3. The identification rate of hybrid offspring using the three pairs of primers reached 90%.

[0077] Table 3. Capillary electrophoresis results of hybrid parents and offspring

[0078]

[0079] In the table, " / " indicates that the peak value of the total DNA amplification result of the sample is not ideal, and "-" indicates that no judgment is made.

[0080] 3. Identification of hybrid representative types

[0081] To further verify the reliability of the SSR marker, in the summer of 2024, 4 out of 30 hybrid progeny plants flowered. Among them, 2 were the offspring of combination 1 (Z1) 'Qianchao Xingbai' (♀) × 'Hongsheng' (♂) (Z1-8, Z1-16), and 2 were the offspring of combination 2 (Z2) 'Qianchao Xingbai' (♀) × 'Ewei No. 3' (♂) (Z2-6, Z2-9). The flower diameter and flower color of these 4 flowering progeny plants were investigated in accordance with the "Guidelines for Testing the Distinctiveness, Uniformity and Stability of New Plant Varieties - Crape Myrtle", with 5 replicates.

[0082] Among them, the offspring Z1-8 and Z1-16 of the orthogonal cross Z1 'Qianchao Xingbai' (♀) × 'Hongsheng' (♂) exhibited flower diameter and color between the parents (Table 4), showing hybrid characteristics. The flower diameter of the maternal parent 'Qianchao Xingbai' was significantly smaller than that of the paternal parent 'Hongsheng'. The flower diameters of offspring Z1-8 and Z1-16 were significantly larger than those of the maternal parent and significantly smaller than those of the paternal parent, but there was no significant difference between the two offspring. The flower color of offspring Z1-8 and Z1-16 was light purple, corresponding to color card numbers RHS 75A and RHS 75B, respectively. The maternal parent 'Qianchao Xingbai' had white flowers (RHS N155A), and the paternal parent 'Hongsheng' had deep pinkish-purple flowers (RHS N57C). It can be seen that the flower color of the hybrid offspring was also between that of the parents. Phenotypic identification showed that Z1-8 and Z1-16 are true hybrids of 'Qianchao Xingbai' (♀) × 'Hongsheng' (♂). This is consistent with the SSR identification results. Specifically, progeny Z1-8 was identified as a true hybrid by all three primer pairs, and Z1-16 was identified as a true hybrid by primer S17. Individual flowers of hybrids Z1-8 and Z1-16 are shown... Figure 3 The SSR was identified as a hybrid, and its phenotype also supported this, demonstrating the reliability of the selected molecular marker.

[0083] Table 4. Results of phenotypic determination of parents and offspring in orthogonal cross of combination 1

[0084]

[0085] In Table 4, a, b, and c represent the differences in the analysis of variance. Different letters indicate significant differences, while the same letter indicates no significant differences.

[0086] The offspring Z2-6 and Z2-9 of the orthogonal cross (Z2) 'Qianchao Xingbai' (♀) × 'Ewei 3' (♂) exhibited flower diameter and color between their parents (Table 5), showing hybrid characteristics. The maternal parent 'Qianchao Xingbai' had a significantly smaller flower diameter than the paternal parent 'Ewei 3', while the offspring Z2-6 and Z2-9 had significantly larger flower diameters than both the maternal and paternal parents, with significant differences between the two offspring. The flower color of the hybrid offspring Z2-6 and Z2-9 was light purple (RHS 75C), while the maternal parent 'Qianchao Xingbai' had white flowers (RHSN155A), and the paternal parent 'Ewei 3' had dark purple flowers (RHS N72B), indicating that the flower color of the hybrid offspring was also between that of their parents. Phenotypic identification showed that offspring Z2-6 and Z2-9 were true hybrids of 'Qianchao Xingbai' (♀) × 'Ewei 3' (♂). This is consistent with the identification results of SSR. Among them, progeny Z2-6 can be identified as a true hybrid by primer S20, and Z2-9 can be identified as a true hybrid by both primers S17 and S20. Individual flowers of hybrids Z2-6 and Z2-9 are shown... Figure 4 The SSR was identified as a hybrid, and its phenotype also supported this, demonstrating the reliability of the selected molecular marker.

[0087] Table 5. Results of phenotypic determination of parents and offspring in orthogonal crosses of combination 2

[0088]

[0089] In Table 5, a, b, c, and d represent the differences in the analysis of variance. Different letters indicate significant differences, while the same letter indicates no significant differences.

Claims

1. The application of a primer for identifying the authenticity of crape myrtle hybrids in identifying the authenticity of crape myrtle hybrids and crape myrtle varieties, characterized in that, The primers are one or more of primers S04, S17, and S20; The primer S04: Forward primer: 5'-ATGTTCATGTGGCTCGGTCA-3', Reverse primer: 5'-CAGATGATGGCTCCAGCGAT-3'; Primer S17: Forward primer: 5'-GTCTCTCAGATACACCGCCG-3', Reverse primer: 5'-GACCAGACAGACAGACAGGC-3'; The primer S20: Forward primer: 5'-GGTGGAGATGCTAACAAGCAAG-3', Reverse primer: 5'-GGATTTTTGCTGTAGGGTGATT-3'.

2. The application according to claim 1, characterized in that, The crape myrtle hybrid is one or more of the following: 'Qianchao Xingbai' × 'Hongsheng' hybrid, 'Hongsheng' × 'Qianchao Xingbai' hybrid, and 'Qianchao Xingbai' × 'Ewei No. 3' hybrid.

3. The application according to claim 1, characterized in that, The crape myrtle varieties mentioned are one or more of 'Qianchao Xingbai', 'Hongsheng', and 'Ewei No. 3'.

4. The application according to claim 1, characterized in that, The primers are fluorescently labeled primers.

5. The application according to claim 1, characterized in that, The application uses reagents or kits containing primers for identifying the authenticity of crape myrtle hybrids and for identifying crape myrtle varieties.

6. A method for identifying the authenticity of crape myrtle hybrids, characterized in that, Including the following steps: (1) Extract DNA from fresh leaves of each sample of 'Qianchao Xingbai', 'Hongsheng', 'Ewei No. 3', and the crape myrtle hybrid to be tested; (2) Using the DNA extracted in step (1) as DNA templates, PCR amplification was performed using the primers described in claim 1 or 4 to identify the authenticity of the crape myrtle hybrid, and the PCR amplification products of each primer were obtained. (3) The PCR amplification products of each primer in step (2) are detected by capillary electrophoresis to obtain the capillary electrophoresis pattern of each sample. The authenticity of the crape myrtle hybrid is identified based on the capillary electrophoresis pattern of the crape myrtle hybrid to be tested.

7. The method according to claim 6, characterized in that, The criteria for determining the authenticity of a crape myrtle hybrid based on its capillary electrophoresis pattern are as follows: if the capillary electrophoresis pattern of the crape myrtle hybrid shows both parental characteristic peaks or only the paternal characteristic peak, it is determined to be a genuine hybrid. Conversely, it is determined to be a false hybrid.

8. The method according to claim 6, characterized in that, The crape myrtle hybrid is one or more of the following: 'Qianchao Xingbai' × 'Hongsheng' hybrid, 'Hongsheng' × 'Qianchao Xingbai' hybrid, and 'Qianchao Xingbai' × 'Ewei No. 3' hybrid.

9. A primer for identifying the authenticity of crape myrtle hybrids, characterized in that, The primer is one or both of primer S04 and primer S17; The primer S04: Forward primer: 5'-ATGTTCATGTGGCTCGGTCA-3', Reverse primer: 5'-CAGATGATGGCTCCAGCGAT-3'; Primer S17: Forward primer: 5'-GTCTCTCAGATACACCGCCG-3', Reverse primer: 5'-GACCAGACAGACAGACAGGC-3'.

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