Primer combination of molecular marker for early identification of gender of bird-attracting plant ilex asprella and application thereof

Abstract

The present application relates to a primer combination of a molecular marker for early identification of the gender of bird-attracting plant iron camellia and application thereof, and belongs to the technical field of molecular biology. The primer combination of the molecular marker for early identification of the gender of bird-attracting plant iron camellia comprises an upstream primer and a downstream primer; the nucleotide sequence of the upstream primer is shown as SEQ ID No: 7; and the nucleotide sequence of the downstream primer is shown as SEQ ID No: 8. The present application identifies restriction associated DNA tag fragments (RAD-tags) associated with gender by performing population-level simplified genome deep sequencing on female and male plants of different sources, designs corresponding polymerase chain reaction (PCR) primers, and realizes rapid identification of the gender of iron camellia at an early stage (seedling stage).

Description

Technical Field

[0001] This invention relates to the field of molecular biology, and in particular to primer combinations of molecular markers for early identification of the sex of the bird-attracting plant Ilex chinensis and their applications. Background Technology

[0002] Iron holly (Ilex rotunda Thunb.) is an evergreen shrub or tree belonging to the genus Ilex in the family Aquifoliaceae. It is a common native tree species in southern my country, valued for its evergreen nature, beautiful tree shape, and especially its abundant red berries in autumn, resembling cinnabar beads. Furthermore, its berries attract various wild birds, creating micro-habitats within urban green spaces and effectively enhancing the ecological benefits of green areas. It is widely used in landscaping and gardening.

[0003] Ilex chinensis is dioecious. In production, propagation from seedlings is often hampered by the inability to determine sex, leading to insufficient female plants and fruit production, thus impacting landscape design and ecological benefits. However, traditionally, plants are transplanted after maturity and flowering, increasing costs and reducing efficiency. Therefore, developing sex-specific molecular markers for Ilex chinensis to enable early sex identification of seedlings allows for precise allocation of male and female plants, improving both landscape design and ecological benefits.

[0004] Currently, there is no effective method for early sex determination of *Ilex crenata* in horticultural production. Traditional methods rely on the characteristics of mature flowers followed by transplanting and planting, which are costly and inefficient. Therefore, finding an effective method for early sex determination of *Ilex crenata* is a pressing technical problem that needs to be solved in this field. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing technologies and provide primer combinations and their applications for early (seedling stage) identification of the sex of the bird-attracting plant *Ilex chinensis*. This invention utilizes population-level simplified genome deep sequencing of male and female plants from different sources to identify sex-associated DNA tags (RAD-tags), and designs corresponding polymerase chain reaction (PCR) primers to achieve rapid early (seedling stage) sex identification of *Ilex chinensis*.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] In a first aspect, the present invention provides a primer combination for early identification of the sex of Ilex chinensis using molecular markers, the primer combination comprising an upstream primer and a downstream primer; the nucleotide sequence of the upstream primer is shown in SEQ ID No: 7; the nucleotide sequence of the downstream primer is shown in SEQ ID No: 8.

[0008] This invention discovers a specific nucleotide fragment in the genome of male *Ilex cornuta* individuals, which is absent in the genome of female individuals. The inventors found that this difference can be utilized by extracting the leaf genome to identify the sex of *Ilex cornuta*, eliminating the need to wait until the plant matures and flowers. Therefore, this invention allows for sex identification of *Ilex cornuta* before flowering by amplifying this sequence fragment from individual leaves, shortening the time required and saving significant economic costs. In this invention, this molecular marker is named MSL26. Primer combinations designed with nucleotide sequences for MSL26, as shown in SEQ ID No: 7-8, can amplify MSL26. Therefore, the primer combinations of this invention can be used to identify the sex of *Ilex cornuta* at an early stage.

[0009] Secondly, the present invention provides the application of the primer combination of the molecular markers described in the first aspect in the early identification of the sex of Ilex chinensis.

[0010] As a preferred embodiment of the second aspect, the application includes the following steps:

[0011] A. Primer synthesis: Synthesize the primer combination as described in claim 4;

[0012] B. DNA extraction: Genomic DNA was extracted from the leaves of Ilex chinensis.

[0013] C. PCR amplification: Based on genomic DNA, PCR amplification is performed using the primer combination synthesized in step A;

[0014] D. Individuals that produce amplified products are classified as male, and individuals that do not produce amplified products are classified as female.

[0015] In a preferred embodiment of the fourth aspect, the DNA extraction in step B includes the following steps:

[0016] (1) Grind the leaves into powder and add DNA extraction solution into a water bath. After water bath, centrifuge and collect the supernatant.

[0017] (2) Add an equal volume of a mixture of chloroform and isoamyl alcohol, centrifuge, and collect the supernatant;

[0018] (3) Add sodium acetate and anhydrous ethanol to precipitate overnight, centrifuge, collect the precipitate, and obtain the genomic DNA of the leaves of Ilex chinensis.

[0019] As a preferred embodiment of the second aspect, in step (2), the volume ratio of chloroform to isoamyl alcohol in the mixed solution of chloroform and isoamyl alcohol is: chloroform: isoamyl alcohol = 24:1.

[0020] As a preferred embodiment of the second aspect, the PCR amplification program in step C is as follows: pre-denaturation 95°C, 5 min; denaturation 95°C, 30 s; annealing 54°C, 30 s; extension 72°C, 30 s (36 cycles); 72°C for 5 min; 12°C for 2 min.

[0021] As a preferred embodiment of the second aspect, the PCR amplification reaction system in step C is as follows:

[0022] The DNA template concentration was 20 ng / μL, and the volume was 3 μL; the upstream primer concentration was 10 μM, and the volume was 1 μL; the downstream primer concentration was 10 μM, and the volume was 1 μL; the 2×PrimeSTAR Max Premix volume was 15 μL; and the ddH2O volume was 10 μL.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0024] This invention enables rapid sex identification of Ilex chinensis at an early stage (seedling stage) by performing simplified genome deep sequencing at the population level on male and female plants from different sources, identifying sex-associated DNA tags (RAD-tags), and designing corresponding polymerase chain reaction (PCR) primers. Attached Figure Description

[0025] Figure 1 Images showing the characteristics of female and male *Ilex chinensis* plants;

[0026] Figure 2 A schematic diagram showing the detection results of amplification products at 11 male-specific sites (M: male plant; F: female plant);

[0027] Figure 3 A schematic diagram showing the amplification and validation results of the MSL26 molecular marker in other male and female plants (M: male plant; F: female plant). Detailed Implementation

[0028] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0029] Example 1

[0030] 1. Identify the source and sex information of Ilex chinensis in the sample.

[0031] In this embodiment, 11 female *Ilex chinensis* plants and 12 male plants were collected from the South China Botanical Garden and the Dongguan Botanical Garden. Based on the fruiting records of the current year and several years, the plants that bore fruit were identified as female, and those that did not bear fruit were identified as male. Figure 1 ).

[0032] 2. Extraction of genomic DNA from Ilex chinensis

[0033] Total DNA was extracted from dried leaves using the CATB method, as follows:

[0034] One to two young, healthy leaves from different individuals were clearly marked, thoroughly dried in silica gel for 2 to 3 days, and ground into powder using liquid nitrogen. 800 μL of 1×TAB extraction buffer was added, and the mixture was shaken to mix thoroughly. The mixture was then incubated in a 65°C water bath for 30 minutes, mixing continuously. Subsequently, the mixture was centrifuged at 12000g for 6 minutes at room temperature using a standard centrifuge (Sigma, 1-14, Germany), and the supernatant was collected. An equal volume of a mixture of chloroform and isoamyl alcohol (chloroform:isoamyl alcohol = 24:1) was added, and the mixture was centrifuged again at 12000g for 6 minutes at room temperature using a centrifuge (Sigma, 1-14, Germany). This process was repeated 1 to 2 times. Finally, the supernatant was collected, and 1M sodium acetate and an equal volume of anhydrous ethanol were added. The mixture was allowed to precipitate overnight. After centrifugation, the precipitate was washed twice with 70% ethanol, dried, and then stored in 50–100 μL of TE buffer for later use.

[0035] 3. Constructing a simplified genome ddRAD library

[0036] 3a. To each adjusted sample DNA volume, add 10 μL of premixed double enzyme digestion buffer (NEB EcoRI, catalog number R3101L + MseI, catalog number R0525L), and thoroughly mix using a 200 μL pipette tip. Then, place the sample in a PCR instrument for enzyme digestion treatment, setting the reaction temperature to 37℃ for 8 hours, 65℃ for 20 minutes, and incubation at 12℃; the system is as follows:

[0037]

[0038] 3b. Take 5 μL of the enzyme digestion product and perform agarose gel electrophoresis. If there is no obvious total DNA main band and the enzyme digestion product shows a complete diffuse band in the lane, the enzyme digestion is considered complete.

[0039] 3c. Add the EcoR I end adapter with a specific tag (barcode) (Nextera XT IndexKit V2 set A, Illinois Inc.) to the enzyme digestion product of each sample, then add the ligation mixture containing the universal Mse I adapter (ligation was performed using NEB-produced T4 DNA ligase), and thoroughly mix with a 200 μL pipette tip. Then, place the sample in a PCR instrument for enzyme digestion, setting the reaction temperature to 16℃ for 8 hours, 65℃ for 20 minutes, and incubation at 12℃; the system is as follows:

[0040]

[0041] 3d.1 times volume magnetic beads for removing small fragment joints.

[0042] 3e. After ligation, mix equal volumes of the enzyme digestion / ligation products of samples with different barcodes, and then perform gel extraction and recovery under agarose gel electrophoresis (Omega Gel Extraction Kit, catalog number D2500). The selection fragment range is 300-550bp.

[0043] 3f. Amplify the recovered library to achieve the required concentration for sequencing.

[0044] 3g. Further purification of the library was performed using 0.8 times the volume of magnetic beads to remove the adapter dimer.

[0045] 3h. The library was sequenced using the Illumina Novaseq platform in PE150 mode, with an average sequencing volume of 1.0 GB of raw data per sample.

[0046] 4. Simplified genome data analysis of Ilex chinensis

[0047] 4a. Use the process_radtags module in stacks (version 1.48) to perform data quality control on the offline data. The specific running parameters are as follows:

[0048] process_radtags-1xxx.R1.fq.gz-2xxx.R2.fq.gz-b barcode.txt--renz-1ecoRI--renz-2mseI-cqro pro_out--len_limit 140-t 135

[0049] 4b. Remove the rem files from the split data and merge the end-to-end data of each individual. Use the ustacks program to cluster the individual data, setting m to 3 and M to 4.

[0050] 4c. Use the cstacks program to further cluster the results generated by ustacks to obtain the catalog reference sequence for all individuals.

[0051] 4d. Then use sstacks to paste the results of ustacks back to the catalog reference.

[0052] 4e. Using R scripts, we analyzed the sequence depth information of each individual posted to the catalog and filtered out RAD-tags that appeared only in one sex and were missing in the other. We found a total of 110 RAD-tags (MSL_specific.fa) that appeared only in all male individuals; no RAD-tags specific to female individuals were found.

[0053] 4f. Using the bwa software (Version: 0.7.18-r1243-dirty), the 110 tags from 4e were used as target reference fragments, and all female individuals' sequencing data were aligned to them. We found that only 12 RAD-tags (F_missloci.fa) were not present in more than 10 female individuals (a total of 11 female individuals and 12 male individuals were used for sequencing). Further investigation of the opposite end of these RAD-tags in the male data revealed that RAD-tags 2 and 27 belonged to the paired ends of the same read, therefore these two tags represent one locus. Thus, a total of 11 male-specific loci (MSLs) remain for further validation.

[0054] 4g. Using the 11 male-specific loci mentioned above, the data from the other end were found and merged into a single read before primer design. Primer design was performed using the online website https: / / primer3.ut.ee / .

[0055] The 11 MSL loci selected above are named as follows: MSL2-27, MSL14, MSL20, MSL26, MSL30, MSL35, MSL41, MSL77, MSL98, MSL100, and MSL103.

[0056] Table 1: MSL sites and amplification primers

[0057]

[0058] 4h. Primers were synthesized and validated for the 11 MSL loci mentioned above. Four female and four male individuals were selected for the assay. The polymerase chain reaction (PCR) program was as follows: pre-denaturation 95℃, 5 min; denaturation 95℃, 30 s; annealing 54℃, 30 s; extension 72℃, 30 s (36 cycles); 72℃ for 5 min; 12℃ for 2 min. The PCR reaction system is as follows:

[0059]

[0060]

[0061] The verification results are as follows Figure 2As shown, the target product was amplified at all 11 male-specific loci (MSLs). Figure 2 The results showed that the amplification product of about 380 base pairs was clear and stable in male individuals but absent in female individuals. Therefore, the MSL26 site was selected as the marker fragment for this invention.

[0062] Application example: Universality of male-specific site fragments in Ilex chinensis

[0063] Universality verification was performed on male and female *Ilex cornuta* individuals from 12 plants at the South China Botanical Garden (SCBG) and 11 plants at the Dongguan Botanical Garden (DGBG), Chinese Academy of Sciences. The procedure is as follows:

[0064] (1) Extract genomic DNA from Ilex chinensis leaves;

[0065] (2) Amplify genomic DNA using primer combinations from MSL26;

[0066] The reaction program was as follows: pre-denaturation at 95℃ for 5 min; denaturation at 95℃ for 30 s; annealing at 54℃ for 30 s; extension at 72℃ for 30 s (36 cycles); 72℃ for 5 min; 12℃ for 2 min.

[0067] The PCR reaction system is as follows:

[0068]

[0069] Amplification results as follows Figure 3 The results showed that at the 380bp position, the MSL26 fragment was amplified in 7 male individuals out of 12 SCBG plants, but not in 5 female individuals; similarly, the MSL26 fragment was amplified in 5 male individuals out of 11 DGBG plants, but not in 6 female individuals. Therefore, the MSL26 marker fragment and its amplification primers of this invention can be used for early (seedling stage, before flowering) sex identification of *Ilex chinensis*.

[0070] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A primer set for early identification of the sex of Ilex chinensis, characterized in that, The primer combination includes an upstream primer and a downstream primer; the nucleotide sequence of the upstream primer is shown in SEQ ID No: 7; the nucleotide sequence of the downstream primer is shown in SEQ ID No: 8.

Images