Primer combination of molecular marker for early identification of sex of Ilex rotunda L. as bird inducing plant and application of primer combination

Abstract

The invention relates to a primer combination of a molecular marker for early identification of sex of a bird inducing plant ilex rotunda and application of the primer combination, and belongs to the technical field of molecular biology. A primer combination of the molecular marker for early identification of the sex of Ilex rotunda L. as a bird inducing plant comprises an upstream primer and a downstream primer; the nucleotide sequence of the upstream primer is as shown in SEQ ID No: 7; the nucleotide sequence of the downstream primer is as shown in SEQ ID No: 8. According to the present invention, the rapid identification of the sex of Ilex rotunda in the early stage (seedling stage) is achieved by carrying out the simplified genome deep sequencing at the population level on the male and female plants from different sources, identifying the restricted associated DNA tag fragment (RAD-tag) associated with the sex, and designing the corresponding polymerase chain reaction (PCR) primer, and the rapid identification of the sex of Ilex rotunda in the early stage (seedling stage) is achieved.

Description

Technical Field

[0001] The invention relates to the technical field of molecular biology, and in particular to a primer combination for early identification of the sex of a bird-attracting plant, Ilex ferox, and an application thereof. Background Art

[0002] Ilex rotunda Thunb is an evergreen shrub or tree of the genus Ilex in the Aquifoliaceae family. It is a common native tree species in southern my country. It is evergreen all year round and has a beautiful tree shape. In particular, it has a lot of red fruits in autumn, which are as red as red pearls and have great ornamental value. At the same time, its fruits can attract a variety of wild birds, create micro-habitats in urban green spaces, and effectively improve the ecological benefits of green spaces. It is widely used in gardening and landscape construction.

[0003] Ilex truncatum is dioecious. When it is propagated by seedlings, it is impossible to determine their gender and randomly select them, which will lead to insufficient female plants and insufficient fruiting, affecting the landscape creation effect and the realization of ecological benefits. In production, it is generally necessary to wait until the plants are adults and bloom, and then transplant and configure them again according to gender, which increases the cost of garden production and reduces production efficiency. Therefore, the development of gender-specific molecular markers for Ilex truncatum can realize the early identification of the gender of seedlings, so as to accurately configure male and female plants and improve the landscape creation effect and ecological benefits.

[0004] There is no effective early sex identification method for Ilex oleracea in garden production. Traditionally, it is transplanted and arranged according to the characteristics of adult flowers, which is costly and inefficient. Therefore, seeking an effective method for early sex identification of Ilex oleracea is a technical problem to be solved in this field. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a primer combination and application of molecular markers for early (seedling) identification of the sex of the bird-attracting plant Ilex oleraceus. The present invention performs population-level simplified genome deep sequencing on male and female plants from different sources, identifies restriction-associated DNA tags (RAD-tags) associated with sex, and designs corresponding polymerase chain reaction (PCR) primers to achieve rapid identification of the sex of Ilex oleraceus at an early stage (seedling stage).

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

[0007] In a first aspect, the present invention provides a primer combination for early identification of the sex of Ilex oleifera by 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] It was found in the present invention that a specific nucleotide fragment exists in the genome of male individuals of Ilex rotunda, while this nucleotide fragment does not exist in the genome of female individuals. The inventor found that this difference can be used to identify the gender of Ilex rotunda by extracting the genomic DNA from leaves, without having to wait until the plant reaches adulthood and flowers to know its gender. Therefore, the present invention can identify the gender of Ilex rotunda before flowering by means of whether the sequence fragment can be amplified from the individual leaves, shortening the time and saving more economic costs. In the present invention, this molecular marker is named: MSL26. By designing primer combinations with nucleotide sequences as shown in SEQ ID No: 7-8 for the molecular marker MSL26, the molecular marker MSL26 can be amplified. Therefore, the gender of Ilex rotunda can be identified at an early stage by the primer combination of the present invention.

[0009] In a second aspect, the present invention provides the use of the primer combination of the molecular marker described in the first aspect in the early identification of the gender of Ilex rotunda.

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

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

[0012] B. DNA extraction: Extract the genomic DNA of Ilex rotunda leaves;

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

[0014] D. Determine the individuals with amplified products as male, and the individuals without amplified products as female.

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

[0016] (1) Grind the leaves into powder, add DNA extraction buffer, perform water bath, and then centrifuge after the water bath to collect the supernatant;

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

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

[0019] As a preferred embodiment of the second aspect, in the mixed solution of chloroform and isoamyl alcohol in step (2), the volume ratio of chloroform to 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: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 54°C for 30 s, extension at 72°C for 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 is 20 ng / μL and the volume is 3 μL; the upstream primer concentration is 10 μM and the volume is 1 μL; the downstream primer concentration is 10 μM and the volume is 1 μL; the volume of 2×PrimeSTAR Max Premix is 15 μL; ddH 2 O volume is 10 μL.

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

[0024] By performing reduced-representation genome deep sequencing at the population level on male and female plants from different sources, the present invention identifies restriction-associated DNA marker fragments (RAD-tags) associated with gender, designs corresponding polymerase chain reaction (PCR) primers, and realizes the rapid identification of the gender of Ilex rotunda Thunb. at an early stage (seedling stage). Description of the Drawings

[0025] Figure 1 are characteristic diagrams of female and male plants of Ilex rotunda Thunb.;

[0026] Figure 2 are schematic diagrams of the detection results of the amplification products of 11 male-specific loci (M: male plants; F: female plants);

[0027] Figure 3 are schematic diagrams of the verification results of the amplification of the MSL26 molecular marker in other male and female plant individuals (M: male plants; F: female plants). Detailed Embodiments

[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 gender information of Ilex rotunda Thunb. in the identification samples

[0031] In this example, 11 female plants and 12 male plants of Ilex rotunda Thunb. were collected from the South China Botanical Garden and the Dongguan Botanical Garden. According to the fruiting records of the current year and previous years, the plants identified as fruiting were female plants, and those not fruiting were male plants ( Figure 1 ).

[0032] 2. Extract the genomic DNA of Ilex rotunda

[0033] Use the CTAB method to extract the total DNA of dry leaves. The steps are as follows:

[0034] For different individuals, take 1 - 2 young and healthy leaves, mark them clearly, and use discoloring silica gel to fully dry them for 2 - 3 days. Add liquid nitrogen and grind them into powder. Add 800 μL of 1×CTAB extraction buffer, mix well by shaking, and then incubate in a water bath at 65°C for 30 minutes, mixing constantly during this period. Subsequently, centrifuge at 12000g at room temperature for 6 minutes using a common centrifuge (Sigma, 1 - 14, Germany), and aspirate the supernatant. Add an equal volume of a mixed solution of chloroform and isoamyl alcohol (volume ratio of chloroform:isoamyl alcohol = 24:1), mix well, centrifuge at 12000g at room temperature for 6 minutes using a centrifuge (Sigma, 1 - 14, Germany), take the supernatant, and repeat 1 - 2 times. Finally, take the supernatant, add 1M sodium acetate and an equal volume of absolute ethanol, and precipitate overnight. After centrifugation, wash the obtained precipitate twice with 70% ethanol, dry it, and add 50 - 100 μL of TE buffer for storage and standby.

[0035] 3. Construct a reduced - representation genomic ddRAD library

[0036] 3a. In each sample DNA with adjusted concentration and volume, add 10 μL of a premixed double - digestion (EcoR I product number R3101L + Mse I product number R0525L from NEB) mixture, and mix well by pipetting thoroughly with a 200 μL pipette tip. Then place it in a PCR instrument for digestion, set the temperature to 37°C for 8 hours, 65°C for 20 minutes, and keep it at 12°C; the reaction system is as follows:

[0037]

[0038] 3b. Aspirate 5 μL of the digested product for agarose gel electrophoresis detection. If there is no obvious main band of total DNA and the digested product shows a complete smear band in the lane, it can be judged that the digestion is complete;

[0039] 3c. After adding the EcoR I - end adapter with a specific tag (barcode) (Nextera XT IndexKit V2 set A, Illunmia Inc) to the digested product of each sample, then add a ligation mixture containing a universal Mse I adapter (the ligation uses T4 DNA ligase from NEB), and mix well by pipetting thoroughly with a 200 μL pipette tip. Then place it in a PCR instrument for digestion, set the temperature to 16°C for 8 hours, 65°C for 20 minutes, and keep it at 12°C; the reaction system is as follows:

[0040]

[0041] 3d. 1 times volume of magnetic beads to remove small fragment linkers.

[0042] 3e. After the ligation is completed, the sample digestion / ligation products with different barcodes are mixed in equal volumes, and then subjected to gel excision and recovery under agarose gel electrophoresis (Omega gel recovery kit, catalog number D2500), and the screening fragment range is 300-550bp.

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

[0044] 3g. Further purify the library 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, the sequencing mode was PE150, and the average sequencing volume of a single sample was 1.0 GB of raw data.

[0046] 4. Ilex simplifies genomic data analysis

[0047] 4a. Use the process_radtags module in stacks (version 1.48) to perform data quality control on the offline data. The specific operating 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 file in the split data, merge the single individual double-end data, and use the ustacks program to cluster the single individual data, where m is set to 3 and M is set to 4.

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

[0051] 4d. Use sstacks to post the results of ustacks back to the catalog reference.

[0052] 4e. Use R scripts to count the sequence depth information of each individual replied to the catalog, and screen out RAD-tags that only appear in one sex and are missing in the other sex. We found a total of 110 RAD-tags (MSL_specific.fa) that only appear in all male individuals, and no specific RAD-tags were found in female individuals.

[0053] 4f. Use the bwa software (Version: 0.7.18-r1243-dirty) to take the 110 tags in 4e as the target reference fragments, and align the downloaded data of all female individuals to them. We found that only 12 RAD-tags (F_missloci.fa) did not appear in more than 10 female individuals (a total of 11 female individuals and 12 male individuals were sequenced). By finding the other-end data of these RAD-tags in the male individual data, it was found that RAD-tags No. 2 and No. 27 belong to the paired-end data of the same read, so these two tags are at one locus. Therefore, there are still 11 male-specific loci (Male specific Loci, MSL) in total that can be used for subsequent verification.

[0054] 4g. Use the above 11 male-specific loci, find the other-end data and merge them into one read, and then design primers. The primer design is carried out using the online website https: / / primer3.ut.ee / .

[0055] Name the above 11 MSL loci as: MSL2-27, MSL14, MSL20, MSL26, MSL30, MSL35, MSL41, MSL77, MSL98, MSL100, MSL103.

[0056] Table 1: MSL loci and amplification primers

[0057]

[0058] 4h. Synthesize primers to verify the primers of the above 11 MSL loci. Select 4 females and 4 males for verification. The polymerase chain reaction (PCR) reaction program is as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 54°C for 30 s, extension at 72°C for 30 s (36 cycles); 72°C for 5 min, 12°C for 2 min; The PCR reaction system is as follows:

[0059]

[0060]

[0061] The verification results are as Figure 2As shown, target products were amplified from all 11 male specific loci (MSL). According to Figure 2 the results shown, the amplification products of approximately 380 base pairs were clear and stable in male individuals, and absent in female plants. Therefore, the MSL26 locus was selected as the marker fragment of the present invention.

[0062] Application Example: Universality of the Male Specific Locus Fragment of Ilex rotunda

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

[0064] (1) Extract the genomic DNA of Ilex rotunda leaves;

[0065] (2) Amplify the genomic DNA using the primer combination of MSL26;

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

[0067] The PCR reaction system was as follows:

[0068]

[0069] The amplification results were as Figure 3 shown. At the 380 bp position, the MSL26 fragment was amplified in 7 male individuals among 12 plants in SCBG, and not amplified in 5 female plants; the MSL26 fragment was amplified in 5 male individuals among 11 plants in DGBG, and not amplified in 6 female plants. Thus, it can be seen that the MSL26 marker fragment of the present invention and its amplification primers can be used for early (seedling stage, before flowering) identification of the gender of Ilex rotunda.

[0070] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A primer combination for early identification of the sex of Ilex ferox, 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.

2. Use of the molecular marker primer combination according to claim 1 in early identification of the sex of Ilex oleifera.

3. The use according to claim 2, characterized in that: The steps include: A. Primer synthesis: synthesizing the primer combination described in claim 1; B. DNA extraction: Extract genomic DNA from Ilex ferruginosa leaves; C. PCR amplification: Based on genomic DNA, PCR amplification is performed using the primer combination synthesized in step A; D. Individuals that produce amplification products are judged as males, and individuals that do not produce amplification products are judged as females.

4. The use according to claim 3, characterized in that The extraction of DNA in step B comprises the following steps: (1) Grind the leaves into powder and add them into a water bath with DNA extraction solution. Centrifuge after water bath and collect the supernatant. (2) Add equal volumes of a mixed solution of chloroform and isoamyl alcohol, centrifuge, and collect the supernatant; (3) Sodium acetate and anhydrous ethanol were added to precipitate overnight, and the precipitate was collected by centrifugation to obtain the genomic DNA of Ilex ferruginosa leaves.

5. The use according to claim 4, characterized in that The volume ratio of chloroform to isoamyl alcohol in the mixed solution of chloroform and isoamyl alcohol in step (2) is: chloroform:isoamyl alcohol=24:

1.

6. The use according to claim 3, characterized in that The PCR amplification program in step C is: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 54°C for 30 s, extension at 72°C for 30 s (36 cycles); 72°C for 5 min, 12°C for 2 min.

7. The use according to claim 3, characterized in that The PCR amplification reaction system in step C is as follows: 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.

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