A method for identifying a female-determining factor S gene of a self-incompatible character of pitaya and a detection kit thereof

Abstract

The present application belongs to the field of modern agricultural technology, and particularly relates to a method for identifying a female-determining factor S gene of a self-incompatible character of pitaya and a detection kit. According to the female-determining factor S gene of the self-incompatible character of pitaya, a specific primer is designed, and on this basis, a PCR amplification system is established. The DNA of different pitaya varieties to be tested is used as a template, a set of primers is used to analyze and amplify the results, and the self-incompatible female-determining factor S gene of each pitaya variety to be tested is determined. A pair of primers, 2 × Phanta Max Master Mix and ddH2O are collectively used to form a complete detection kit for the self-incompatible female-determining factor S gene of pitaya. The kit is simple to operate, can quickly identify the self-incompatible female-determining factor S gene of unknown pitaya varieties, and can select appropriate pitaya varieties for pollination according to the results, thereby providing a basis for the selection of new varieties.

Description

Technical Field

[0001] This invention belongs to the field of modern agricultural technology, specifically relating to a method and detection kit for identifying the S gene, a female determinant of self-incompatibility traits in dragon fruit. Background Technology

[0002] Dragon fruit belongs to the cactus family (Cactaceae). Cactaceae ) *Hylocereus* genus ( Hylocereus ) or genus *Cypripedium* ( Selenieureus Dragon fruit is an important tropical fruit, and its global consumption continues to grow. By the end of 2020, the cultivation area in my country had exceeded one million mu, making it an important economic crop in the production area. In terms of production, existing varieties can be divided into two categories: (1) self-compatible types, such as 'Jindu No. 1', 'Dahong', and 'Fuguihong', which can naturally self-pollinate and bear fruit, have low management costs, and are suitable for large-scale planting; (2) self-incompatible types, such as 'Cusha', 'Wucihuanglong', and 'Mibao', which have advantages in flavor, appearance, or resistance, but must rely on artificial pollination. The characteristic of dragon fruit flowering once at night and flowering period of only a few hours means that artificial pollination must be completed under short-term, rainless, and suitable temperature conditions at night, which is labor-intensive and costly, and seriously restricts the industrial use of incompatible varieties. Therefore, elucidating the molecular mechanism of self-incompatibility and developing molecular marker-assisted breeding technology are of great significance for reducing costs and accelerating the promotion of new varieties.

[0003] Previous studies have classified dragon fruit as a gametophyte self-incompatible (GSI) plant (Lichtenzveig et al., 2000), and its pistil-specific expression product, S-RNase, is considered a female-determining factor. After being absorbed into the cell via the pollen tube, S-RNase degrades pollen rRNA, inhibiting pollen tube elongation. The pollen-side SLF / SFB proteins mediate S-RNase degradation via the ubiquitin-26S proteasome pathway, thus achieving "self--non-self" recognition and affinity regulation. This mechanism was first discovered in *Nicotiana solanaceae* and has since been validated in plants from multiple families, including Rosaceae, Plantaginaceae, and Scrophulariaceae, with its evolution dating back approximately 90 million years. However, the functional sites, expression patterns, and regulatory networks of S-RNase and SLF / SFB in dragon fruit remain unresolved, hindering high-throughput identification of affinity / incompatibility and the progress of molecular breeding. Summary of the Invention

[0004] To address the aforementioned technical issues, this invention has for the first time isolated and identified the full-length cDNA and promoter sequence of the S-RNase gene in the pistil of dragon fruit, providing a molecular marker that can be used for genotyping of varieties and for assisting in the breeding of new self-compatible varieties, thus providing technical support for reducing pollination costs and accelerating the promotion of superior varieties.

[0005] The first objective of this invention is to provide a female-determining gene for the self-incompatibility trait in dragon fruit.

[0006] The second aspect of the present invention aims to provide the application of the female determinant S gene of the self-incompatibility trait in dragon fruit in the identification of dragon fruit varieties with the self-incompatibility trait, as described in the first aspect of the present invention.

[0007] The third objective of this invention is to provide a method for identifying dragon fruit varieties with self-incompatible traits.

[0008] The fourth aspect of this invention aims to provide primer pairs for identifying the S gene, a female-determining factor for the self-incompatibility trait in dragon fruit.

[0009] The fifth aspect of this invention is to provide a detection kit.

[0010] The sixth aspect of this invention aims to provide applications of the reagent kits of the fifth and sixth aspects of this invention.

[0011] The seventh aspect of this invention aims to provide a more specific method for identifying dragon fruit varieties with self-incompatible traits.

[0012] To achieve the above-mentioned objectives of this invention, the technical solution adopted by this invention is as follows:

[0013] The first aspect of the present invention provides a female-determining factor S gene for the self-incompatibility trait in dragon fruit. The gene is 981 bp in length, contains two introns, and contains three conserved domains of the RNase-T2 super family. The CDs are 690 bp in length and contain one conserved domain of the RNase-T2 super family.

[0014] In some embodiments of the present invention, the sequence of the gene is shown in SEQ ID NO: 1.

[0015] In some embodiments of the present invention, the CDS sequence of the gene is shown in SEQ ID NO: 2.

[0016] In some embodiments of the present invention, the protein sequence of the gene is shown in SEQ ID NO: 3.

[0017] In a second aspect, the invention provides the application of the female determinant S gene of the self-incompatibility trait in dragon fruit, as described in the first aspect of the invention, in the identification of dragon fruit varieties with the self-incompatibility trait.

[0018] A third aspect of the present invention provides a method for identifying dragon fruit varieties with self-incompatibility traits, comprising the following steps:

[0019] The presence of the female-determining factor S gene for the self-incompatibility trait of dragon fruit as described in the first aspect of this invention is detected in the dragon fruit sample. If it is present, the self-incompatibility trait is determined to exist.

[0020] In some embodiments of the present invention, it is possible to detect the entire gene field, the CDS sequence, or the protein it encodes.

[0021] A fourth aspect of the invention provides primer pairs for identifying the S gene, a female-determining factor for the self-incompatibility trait in dragon fruit, the sequences of which are as follows:

[0022] Hu S-RNase-F (5'-3'): ATGGCAAAGATAATTATTGTTGCT (SEQ ID NO: 4);

[0023] Hu S-RNase-R (5'-3'): TTAATTAGGAAACATGACAACTGG (SEQ ID NO: 5).

[0024] A fifth aspect of the present invention provides a detection kit comprising the primer pair of the fourth aspect of the present invention.

[0025] In some embodiments of the present invention, the kit may further include reagents for extracting genomic DNA from dragon fruit materials, universal reagents for PCR reactions (e.g., DNA polymerase, dNTPs, reaction buffer, etc.), related consumables (e.g., centrifuge tubes), and a kit instruction manual describing the female-determining factors for analyzing and identifying self-incompatible traits in dragon fruit. Hu Methods for screening S-RNase-1 genes (genomic DNA extraction methods, PCR systems and procedures).

[0026] In some embodiments of the present invention, the form of the detection reagent includes, but is not limited to, powder, suspension, galactoemulsion, solution, etc.

[0027] The sixth aspect of the present invention provides the application of the reagent kits of the fifth and sixth aspects of the present invention in identifying dragon fruit varieties with self-incompatible traits, and in the preliminary identification of dragon fruit varieties with self-incompatible traits in batch production.

[0028] A seventh aspect of the present invention provides a method for identifying dragon fruit varieties with self-incompatibility traits, comprising the following steps:

[0029] DNA was extracted from the dragon fruit sample, and the sample was amplified using the primer pair described in the fourth aspect of the present invention or the kit described in the fifth aspect of the present invention; if the result was positive, it was determined that the sample had self-incompatibility.

[0030] In some embodiments of the present invention, the methods for determining positive samples include, but are not limited to, electrophoresis detection or sequencing.

[0031] In some embodiments of the present invention, the dragon fruit sample includes any tissue part such as dragon fruit stem, flower or fruit.

[0032] Specifically, using the extracted dragon fruit DNA as a template, PCR amplification was performed using the primer pair described above. After agarose gel electrophoresis of the amplification products, the presence and size of the products were used to determine the female-determining gene S for the self-incompatibility trait in the dragon fruit to be identified. Hu S-RNase.

[0033] If adopted Hu If PCR amplification using the S-RNase primer set yields bands of the correct size, then the dragon fruit to be identified contains the S gene, a female-determining factor for the self-incompatible trait. Hu S-RNase (981bp). Furthermore, if the dragon fruit to be identified contains the S-gene, a female-determining factor... Hu S-RNase can be used to determine if it has a self-incompatible trait.

[0034] The beneficial effects of this invention are:

[0035] This invention utilizes the S gene, a female-determining factor for self-incompatibility in dragon fruit, to design a specific gene. Based on this, a PCR amplification system was established. Using DNA from different dragon fruit varieties as templates, a set of primers was used to analyze the amplification results, identifying the S gene for self-incompatibility in each variety. A matching kit for detecting the S gene for self-incompatibility in dragon fruit was developed, consisting of a primer pair, 2× Phanta Max Master Mix, and ddH2O. This kit is simple to use and can quickly identify the S gene for self-incompatibility in unknown dragon fruit varieties. Based on the results, suitable dragon fruit varieties can be selected for pollination, providing a basis for the selection of new varieties. Attached Figure Description

[0036] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0037] Figure 1 for Hu Schematic diagram of the conserved domains of the S-RNase protein.

[0038] Figure 2 for Hu Agarose gel electrophoresis image of the S-RNase gene in some dragon fruit varieties. Detailed Implementation

[0039] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0040] Example 1 Hu Isolation verification of S-RNase

[0041] Utilizing high-resolution time-series transcriptome data (covering pre-pollination stigma and pollen, and key stages at 0.5, 2, 4, and 12 hours post-pollination) of the self-incompatible dragon fruit variety 'Cu sha' and the self-compatible variety 'Jin duNo.1' obtained by our team, and integrating complete genomic information from the dragon fruit genome database, the inventors designed and implemented a systematic screening process. Using "specific presence or high expression in self-incompatible materials, and absence or low expression in self-compatible materials" as the core criterion, in-depth analysis was conducted on the dragon fruit S-RNase gene family (14 members in total). This preliminary finding was further validated on a large scale using resequencing data from 51 dragon fruit germplasm resources.

[0042] Using this strategy, the inventor successfully identified HuS-RNase (S-RNase13) gene. Further population validation confirmed that this gene was fully present and highly expressed in self-incompatible varieties; however, it was found to be absent in the corresponding genomic region of all self-compatible varieties. This finding clarifies that... HuS-RNase (S-RNase13) is a core factor determining the self-incompatibility phenotype of dragon fruit.

[0043] Should HuS-RNase The gene sequence is as follows:

[0044] (SEQ ID NO:1).

[0045] The cds sequence is as follows:

[0046] ATGGCAAAGATAATTATTGTTGCTATTTTGATAGCTTTTCTCTTTCCGGCTTCCCATTGCGTATATGACTACCTGATCTTTGTGGAAACTTGGCCGCCGTCCTATTGCTTGATAAATCCTTGTTCAGGGCCTAACATTAATAACAGATTCACAATTCACGGTGTCTGGGATGCGTACAATAATGGACAGTATGGTGTTGGTCCTTGCAGCTCAACTAATTTCAGCCGAGCTCTGGTAGCCAATGTAGAGAGGCAATTGAATATGAGTTGGCCCAACCTAAAAGATCAAAATGACAACGAATGGATATGGAACCATGAATGGGATAAACATGGAACATGCTTAGTGCCACCCTTCAATGTGCCTACTTATTTCCAGCTGGGACTTGATTTGAATCGCCGATATCCCATACTCAGCTACCTGACAGCAAGGGGTTACATTCCTGGCCGGGGAAGGATGTACGACACAAACCAGATGAGAAATGCAATCAGAATGATGGTTGGCACTGAGCCTCTGCTTATATGTAGAAATAGCAATAGGAATAGGAATAGGAATAGGAACAGGAATGCTCGCAATCAAACATATCCTTTCCTAATAGAAGTTCGCATTTGTTTTGATAGTCAGGGGTCACAACCTGTACCTTGTGCTGCCGGCGGACACTGTCAATCTCCAGTTGTCATGTTTCCTAATTAA (SEQ ID NO: 2).

[0047] The encoded amino acid sequence is as follows:

[0048] MAKIIIVAILIAFLFPASHCVYDYLIFVETWPPSYCLINPCSGPNINNRFTIHGVWDAYNNGQYGVGPCSSTNFSRALVANVERQLNMSWPNLKDQNDNEWIWNHEWDKHGTCLVP PFNVPTYFQLGLDLNRRYPILSYLTARGYIPGRGRMYDTNQMRNAIRMMVGTEPLLICRNSNRNRNRNRNRNARNQTYPFLIEVRICFDSQGSQPVPCAAGGHCQSPVVMFPN (SEQ ID NO: 3).

[0049] Conserved sequence analysis of this protein was performed using NCBI's CDD module, and the results are as follows: Figure 1 As shown.

[0050] This encoded protein belongs to the standard RNase T2 superfamily. Its conserved domain contains the core catalytic module essential for the function of this family of hydrolases. Notably, the key amino acid residues responsible for catalytic activity are fully preserved within this domain, which is highly consistent with typical S-RNase functional characteristics. This analysis confirms at the protein primary structure level that the S-RNase13 gene we identified encodes a functional protein with the potential for full RNase activity, and its function as a self-incompatible "toxic effector" relies on this intact conserved domain.

[0051] Example 2: Method for identifying the S gene, a female-determining factor for the self-incompatibility trait in dragon fruit.

[0052] In this embodiment, specific primers were designed based on the S gene, the female determinant of the self-incompatibility trait in dragon fruit, and a PCR amplification system was established based on these primers.

[0053] The method for identifying the S gene, the female determinant of the self-incompatibility trait in dragon fruit, includes the following specific steps:

[0054] (1) Extracting dragon fruit genomic DNA.

[0055] (2) Using DNA as a template, the gene was amplified by mixing enzymes with 2 × Phanta Max Master Mix.

[0056] The PCR reaction system is shown in Table 1 (50 µL).

[0057] Table 1

[0058]

[0059] Primers designed for the S gene are as follows:

[0060] Primer forward sequence: Hu S-RNase-F (5'-3'): ATGGCAAAGATAATTATTGTTGCT (SEQ ID NO: 4);

[0061] Primer reverse sequence: Hu S-RNase-R (5'-3'): TTAATTAGGAAACATGACAACTGG (SEQ ID NO: 5).

[0062] The PCR reaction procedure is shown in Table 2.

[0063] Table 2

[0064]

[0065] (3) After PCR amplification, the product was detected by 1% agarose gel electrophoresis. The amplification product is a single band, and the target band is approximately 981 bp in size.

[0066] (4) Use a PCR product recovery kit to cut the gel and recover the target band.

[0067] (5) Use the pMD™19-T Vector Cloning Kit to ligate the gel-recovered target fragment to the pMD19-TVector vector. The specific procedure is as follows: add the reaction mixture to a 600 µL PCR tube, gently tap to mix, and incubate at 16 °C for 30 min. Use 10 µL of the reaction mixture for ligation.

[0068] (6) Add the ligation product to 100 µL of DH5α Escherichia coli competent cells, gently mix, and let stand on ice for 30 min.

[0069] (7) Heat shock in a 42 ℃ metal water bath for 45 s, then place on ice for 2 min.

[0070] (8) Add 1 mL of antibiotic-free LB liquid culture medium, and incubate at 37 °C for 1 h at 200 rpm.

[0071] (9) Centrifuge at 8000 rpm for 1 min at room temperature. Discard 950 µL of supernatant, resuspend, and spread onto a medium containing 100 µg / mL Ampicillin. + Incubate the antibiotic on LB solid medium at a constant temperature of 37 °C for 16-20 h.

[0072] (10) Pick a single white colony and add 1 mL of solution containing Amp +The antibiotic was cultured in LB liquid medium at 37 °C for 6-7 h at 200 rpm, and then colony PCR was performed for verification.

[0073] (11) Select positive clones, perform bidirectional sequencing and splicing to obtain the target gene sequence information.

[0074] The results showed that the primer pair could successfully amplify... Hu S-RNase gene.

[0075] Example 3 Hu Verification of S-RNase for the identification of self-incompatibility in dragon fruit

[0076] This embodiment was validated in different dragon fruit varieties.

[0077] 1. Collection of experimental materials

[0078] The test material was dragon fruit stem strips from Hainan. The sample information is shown in Table 3.

[0079] Table 3

[0080]

[0081] 2. Hu S-RNase gene detection

[0082] 2.1 DNA was extracted from the samples using a modified CTAB method.

[0083] 2.2 Using DNA as a template and HuS-RNase-F / R as primers, PCR amplification was performed. The reaction volume was 50 µL, as shown in Table 4.

[0084] Table 4

[0085]

[0086] The PCR reaction procedure is shown in Table 5.

[0087] Table 5

[0088]

[0089] 2.3 The amplified products were subjected to 1% 1×TAE agarose gel electrophoresis with a 2000 bp marker at 120 V for 25 min. The results were photographed and recorded using a gel imaging system.

[0090] The results are as follows Figure 2As shown, the band size of the S gene is approximately 981 bp, and it only appears in self-incompatible dragon fruit varieties. Therefore, the S gene of this invention and the primers used to identify the S gene can be used for the identification of self-incompatible dragon fruit varieties.

[0091] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. Application of the S gene, a female determinant of self-incompatibility in dragon fruit, in the identification of dragon fruit varieties with self-incompatibility traits; The sequence of the female-determining factor S gene for the self-incompatibility trait in dragon fruit is shown in SEQ ID NO: 1; The CDS sequence of the female-determining factor S gene for the self-incompatibility trait in dragon fruit is shown in SEQ ID NO: 2; When a dragon fruit variety possesses the S gene, the female-determining factor for the self-incompatibility trait in dragon fruit, it is identified as having the self-incompatibility trait.

2. A method for identifying dragon fruit varieties with self-incompatibility traits, comprising the following steps: The presence of the female-determining gene S for the self-incompatibility trait in dragon fruit samples was detected. If the gene was present, the self-incompatibility trait was determined to exist. The sequence of the female-determining factor S gene for the self-incompatibility trait in dragon fruit is shown in SEQ ID NO: 1; The CDS sequence of the female-determining factor S gene for the self-incompatibility trait in dragon fruit is shown in SEQ ID NO:

2.

3. Application of primer pairs and kits in identifying dragon fruit varieties with self-incompatible traits; The sequences of the primer pairs are shown in SEQ ID NO: 4 and 5; The kit includes primer pairs with sequences as shown in SEQ ID NO: 4 and 5; the DNA of the dragon fruit variety is amplified by PCR using the primer pairs or the kit, and if the product contains the sequence of the female-determining gene for the self-incompatible trait of dragon fruit, it is identified as having the self-incompatible trait. The sequence of the female-determining factor S gene for the self-incompatibility trait in dragon fruit is shown in SEQ ID NO:

1.

4. A method for identifying dragon fruit varieties with self-incompatibility traits, comprising the following steps: Extract DNA from dragon fruit samples and amplify the dragon fruit samples using the primer pairs or kits described in claim 3; if the dragon fruit sample contains the sequence of the female-determining gene for the self-incompatibility trait of dragon fruit, it is determined that it has the self-incompatibility trait. The sequence of the female-determining factor S gene for the self-incompatibility trait in dragon fruit is shown in SEQ ID NO:

1.

5. The method according to claim 4, characterized in that: The dragon fruit samples include dragon fruit stems, flowers, or fruits.

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