Method for early identification of gender of date palm plant

Abstract

The application discloses a method for early identification of the gender of Phoenix dactylifera L. plant, and belongs to the field of plant molecular biology and agricultural biotechnology. The method uses genomic DNA of Phoenix dactylifera L. as a template, and adopts specific primer pairs shown in SEQ ID NO. 5-6 to perform PCR amplification. The amplification procedure is as follows: 95 DEG C pre-denaturation for 2 min, 98 DEG C denaturation for 10 s, 61 DEG C annealing for 15 s, 68 DEG C extension for 1 min, a total of 35 cycles, and final extension at 68 DEG C for 2 min. The amplification product is detected by agarose gel electrophoresis, if a specific band of 320 bp is presented, it is determined as a female plant, and if a specific band of 420 bp is presented, it is determined as a male plant. The method has high specificity and good stability (the coincidence degree of repeated verification reaches 98.3%), can complete gender identification at the seedling stage of Phoenix dactylifera L., is 4-8 years earlier than traditional morphological identification, is simple and easy to operate, low in cost, and suitable for large-scale popularization and application in breeding units and production lines of Phoenix dactylifera L.

Description

Technical Field

[0001] This invention belongs to the fields of plant molecular biology and agricultural biotechnology, and in particular relates to a method for early identification of the sex of date palm plants. Background Technology

[0002] Dates ( Phoenix dactylifera Date palms (L.) are dioecious plants belonging to the genus *L.* of the palm family (Arecaceae). Their fruits have extremely high economic and nutritional value, earning them the nickname "desert bread." In commercial date palm cultivation, female plants are responsible for fruit production, while male plants only provide pollen; therefore, the economic value of female plants is far higher than that of male plants. However, the sex of date palm plants cannot be distinguished by morphological characteristics during the seedling stage (vegetative growth period). Traditional methods require waiting until the plants reach flowering stage (usually 4-8 years) for sex determination, leading to an imbalanced sex ratio, making it impossible to rationally allocate pollinator trees, and resulting in a significant waste of land, water, fertilizer, and management costs.

[0003] While existing molecular marker technologies have been used for sex identification in crops such as chestnuts, the sex determination mechanism of dates is complex. Existing primer combinations often exhibit problems such as band diffusion, severe tailing, and poor specificity under different experimental conditions, making it impossible to reliably distinguish the sex of dates. Therefore, establishing a highly specific, stable, and easy-to-operate method for early sex identification of dates is of great significance for the efficient breeding and cultivation management of the date industry. Summary of the Invention

[0004] To address the above technical problems, this invention provides a method for early sex identification of date palm plants, the method comprising the following steps: (1) Using date palm genomic DNA as a template, PCR amplification was performed using specific primer pairs, the nucleotide sequences of which are shown in SEQ ID NO.5-6; The PCR amplification reaction system consisted of the following components in a total volume of 30 μL: 1.00 μL GXL DNA polymerase, 6.00 μL 5×GXL Buffer, 5.00 μL dNTPs, 1.00 μL upstream primer, 1.00 μL downstream primer, 1.00 μL template DNA, with the remainder being sterile water to a final volume of 30 μL. The concentration of GXL DNA polymerase was 1.25 U / μL; the concentration of each dNTP was 2.5 mM; and the concentrations of both upstream and downstream primers were 10 pmol / μL. The PCR amplification program was as follows: 95℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, 61℃ annealing for 15 s, 68℃ extension for 1 min, for a total of 35 cycles; 68℃ final extension for 2 min. (2) Detect the PCR amplification products in step (1) and determine the sex of the date palm plant to be tested based on the size of the amplification product bands: If the amplification product shows a specific band of 320 bp, it is determined to be female; If the amplification product shows a specific band of 420 bp, it is determined to be male.

[0005] Preferably, the step (1) includes the extraction of date palm genomic DNA.

[0006] More preferably, the date palm genomic DNA is derived from the leaves, inflorescences, or stems of the date palm.

[0007] More preferably, the PCR amplification products in step (2) are detected by agarose gel electrophoresis.

[0008] More preferably, the judgment criteria in step (3) also include: if there is no obvious band or the band signal is weak, it is judged as unknown and DNA needs to be extracted again or the reaction conditions optimized for retesting.

[0009] The present invention also provides a kit for early sex identification of date palm plants, the kit comprising the aforementioned primers.

[0010] Preferably, it also contains GXL DNA polymerase, 5×GXL Buffer, dNTPs, and sterile water.

[0011] Compared with the prior art, the present invention has the following beneficial effects: (1) High specificity: After systematic screening of multiple candidate primer pairs and optimization of PCR system, the primer pairs mentioned in this patent are finally determined to be able to specifically amplify sex-related gene fragments in the date palm genome, with clear product bands, no impurities, and no non-specific amplification.

[0012] (2) High discrimination: The amplification products of female and male plants have obvious differences in band position (about 320 bp for females and about 420 bp for males), which can be easily distinguished and interpreted in agarose gel electrophoresis without the need for sequencing verification.

[0013] (3) Good stability: The optimized 30μL PCR reaction system has been repeatedly verified and its stability is over 98%; the identification results of 150 samples of unknown sex are compared with known results and the consistency is 98.3%.

[0014] (4) Early identification: This method can complete the sex identification during the seedling stage (vegetative growth period) of date palms, without waiting until the flowering period, which is 4-8 years earlier than the traditional morphological identification, providing technical support for early breeding selection and cultivation management.

[0015] (5) Simple operation: It can be achieved using conventional PCR technology and agarose gel electrophoresis. No expensive equipment or complicated operation is required, making it easy to promote and apply in breeding units and production lines. Attached Figure Description

[0016] Figure 1 Electrophoretic images showing the sex determination of dates at different annealing temperatures in Example 1, where A is annealing temperature 55℃; B is annealing temperature 58℃; and C is annealing temperature 61℃. C and H represent female plants; J and P represent male plants.

[0017] Figure 2 Electrophoresis images of date palms sexed using different primers in Example 2: A. Primers 1 and 2; B. Primers 3 and 4; Primers 5 and 6. C and H represent female plants; J and P represent male plants.

[0018] Figure 3 The image shows a representative electrophoresis diagram of date palms sexed using primers 5 and 6 in Example 3. 4-1 to 4-8 are 1-year-old date palm plants whose sex is to be determined. C and H are control female plants; J and P are control male plants.

[0019] Figure 1-3 The markers in the image are Takara DL2000 DNA markers, and from bottom to top they are 100bp, 250bp, 500bp, 750bp, 1000bp, and 2000bp. Detailed Implementation

[0020] Example 1 Optimization of PCR reaction system Using leaf DNA from date palm plants of known sex as templates, single-factor gradient optimization was performed on the concentrations of GXL DNA polymerase, dNTPs, primers, and annealing temperature. The results showed that the amplification products exhibited the clearest bands, no nonspecific amplification, and the best repeatability when the GXL DNA polymerase (1.25 U / μL) concentration was 1 μL / 30 μL, the dNTP concentration was 5 μL, the forward and reverse primers (10 pmol / μL) were 1 μL each, and the annealing temperature was 61℃. (See [link to relevant documentation]). Figure 1 ).

[0021] Table 1

[0022] The PCR amplification procedure is as follows: Pre-denaturation at 95℃ for 2 min; denaturation at 98℃ for 10 s; annealing at 61℃ for 15 s; extension at 68℃ for 1 min, for a total of 35 cycles; final extension at 68℃ for 2 min; storage at 4℃.

[0023] Example 2 Primer screening and specificity verification Three pairs of candidate primers designed to target sex-related genes in dates were selected for validation. The results showed that Primer-1 / Primer-2 and Primer-3 / Primer-4 could not produce clear and interpretable specific bands under the experimental conditions. Khayri MJ et al. (Al-Khayri, JM, Jain, SM, Johnson, DV (Eds.) (2017). DatePalm Biotechnology Protocols Volume II: Germplasm Conservation and Molecular Breeding. Methods in Molecular Biology, vol. 1638. Humana Press, SpringerScience+Business Media LLC, New York. ISBN 978-1-4939-7158-9 (Print), 978-1-4939-7159-6 (eBook). DOI: 10.1007 / 978-1-4939-7159-6.) used some primer combinations to amplify products that were diffuse and contained many extraneous bands, making it impossible to determine the sex of the date palm plants. Finally, Primer-5 (1F) / Primer-6 (1R) amplification was selected from the primers of Khayri MJ et al. (2017), yielding clear and highly specific amplification bands. The bands in male individuals were positioned higher (approximately 420 bp), while those in female individuals were positioned lower (approximately 320 bp), showing a significant difference (see [link to study]). Figure 2 ).

[0024] Table 2 Candidate primers for date palm plant sex identification

[0025] Example 3: Gender identification of unknown samples Using an optimized PCR system and Primer-5 / Primer-6 primers, DNA samples from leaf leaves of 150 date palm seedlings of unknown sex were analyzed. Of these, 100 seedlings (66.67%) were female, 23 were male (15.33%), and 27 could not be identified (18.00%). The results for identifying female and male seedlings were completely consistent with the actual findings. Figure 3 Representative electrophoresis images for identifying the sex of dates using Primer-5 and Primer-6.

[0026] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for early sex identification of date palm plants, characterized in that, The method includes the following steps: (1) Using date palm genomic DNA as a template, PCR amplification was performed using specific primer pairs, the nucleotide sequences of which are shown in SEQ ID NO.5-6; The PCR amplification reaction system consisted of the following components in a total volume of 30 μL: 1.00 μL GXL DNA polymerase, 6.00 μL 5×GXL Buffer, 5.00 μL dNTPs, 1.00 μL upstream primer, 1.00 μL downstream primer, and 1.00 μL template DNA. The remaining volume was made up to 30 μL with sterile water. The concentration of GXL DNA polymerase was 1.25 U / μL. The concentration of each dNTP was 2.5 mM. The concentration of both upstream and downstream primers was 10 pmol / μL. The PCR amplification program was as follows: 95℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, 61℃ annealing for 15 s, 68℃ extension for 1 min, for a total of 35 cycles; 68℃ final extension for 2 min. (2) Detect the PCR amplification products in step (1) and determine the sex of the date palm plant to be tested based on the size of the amplification product bands: If the amplification product shows a specific band of 320 bp, it is determined to be female; If the amplification product shows a specific band of 420 bp, it is determined to be male.

2. The method for early sex identification of date palm plants according to claim 1, characterized in that, The step (1) is preceded by the extraction of date palm genomic DNA.

3. The method for early sex identification of date palm plants according to claim 2, characterized in that, The date palm genomic DNA was derived from the leaves, inflorescences, or stems of the date palm.

4. The method for early sex identification of date palm plants according to claim 3, characterized in that, The PCR amplification products in step (2) are detected by agarose gel electrophoresis.

5. The method for early sex identification of date palm plants according to claim 4, characterized in that, The judgment criteria in step (3) also include: if there is no obvious band or the band signal is weak, it is judged as unknown and DNA needs to be extracted again or the reaction conditions optimized for retesting.

6. A kit for early sex identification of date palm plants, characterized in that, The kit includes the primers described in claim 1.

7. The reagent kit according to claim 6, characterized in that, It also contains GXL DNA polymerase, 5×GXL Buffer, dNTPs, and sterile water.

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