Primer pair for distinguishing between once-flowering and continuous-flowering cymbidium varieties, and application and kit thereof

Abstract

The present application relates to the fields of molecular biology and plant breeding technology, and particularly relates to a primer pair for distinguishing between one-flowering and continuous-flowering Cymbidium varieties, application and kit thereof. The primer pair comprises FRI-M-F and FRI-M-R. The molecular marker FRI-M-F / FRI-M-R provided by the present application can accurately and efficiently distinguish between one-flowering Cymbidium varieties and continuous-flowering Cymbidium varieties, and can be used for molecular marker assisted screening and identification of the continuous-flowering trait of Cymbidium, thereby significantly improving the breeding efficiency.

Description

Technical Field

[0001] This invention relates to the fields of molecular biology and plant breeding technology, and in particular to a primer pair for distinguishing between single-flowering and continuously flowering Cymbidium varieties, their applications, and a kit. Background Technology

[0002] Orchids are important commercial flowers globally. Among them, the small-flowered terrestrial species of the genus *Cymbidium*, native to my country, are collectively known as Chinese orchids and are one of my country's ten traditional famous flowers, boasting a long cultivation history. Most Chinese orchid varieties, including *Cymbidium goeringii* and *Cymbidium sinense*, bloom only once a year (single-flowering type), with their flowering period concentrated in spring. Furthermore, the development of flower buds from induction to bloom requires more than six months and a prolonged period of low-temperature accumulation before normal flowering; otherwise, the flower buds will fail to bloom. In stark contrast, *Cymbidium ensifolium* varieties, a type of Chinese orchid, do not require low-temperature accumulation and can rapidly develop flower buds within half a month, achieving the characteristic of continuous flowering multiple times a year (continuous flowering type). With the continuous expansion of the market and the ongoing development of the industry, the limitations of most Chinese orchid varieties—long flower development time and single-season flowering—are becoming increasingly prominent, leading to sluggish sales during peak bloom and scarcity during peak season. Therefore, there is an urgent need to cultivate new varieties that do not require dormancy and can bloom continuously to meet market demand.

[0003] With the rapid development and application of modern bio-breeding technology, it is of great significance to utilize key genes and loci controlling flowering to achieve molecular marker-assisted selection and the breeding application of functional genes, and to cultivate varieties that can flower continuously multiple times a year. Studies in the model plant Arabidopsis thaliana have found that allele variation at the FRIDIDA (FRI) locus is the main determinant of natural variation in flowering time. The FRI gene encodes a plant-specific scaffold protein that does not directly bind to the DNA sequence but regulates gene expression through interactions with other proteins. It can effectively activate the expression of the flowering repressor gene FLC, thereby inhibiting the transcription of the flowering initiator gene to achieve flowering repression. The diversity of flowering time in Arabidopsis thaliana is mainly due to allele mutations in the FRIDIDA (FRI) gene and its downstream target gene FLOWERING LOCUS C (FLC). Winter-flowering Arabidopsis thaliana typically possesses functional FRI and FLC genes, while most early-flowering ecotypes carry different deletions in the FRI allele. It is precisely these loss-of-function mutations in FRI that have provided the basis for the evolution of many early-flowering ecotypes.

[0004] To promote the application of the FRI gene in molecular marker-assisted breeding of Cymbidium goeringii, improve breeding efficiency, and quickly breed new Cymbidium goeringii varieties that can flower continuously, this invention utilizes the gene to develop a functional molecular marker that can quickly and accurately identify traits that do not require overwintering and can flower continuously. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a primer pair for distinguishing between single-flowering and continuously flowering Cymbidium varieties, along with its application and a kit. This invention effectively distinguishes between single-flowering and continuously flowering Cymbidium varieties by detecting a 27bp deletion in the coding region of the CeFRI gene (SEQ ID No. 1). This invention also provides specific primer pairs (FRI-MF and FRI-MR, SEQ ID No. 5 and 6) for detecting this molecular marker, and their application in Cymbidium breeding.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] This invention provides a primer pair for distinguishing between single-flowering and continuously flowering Cymbidium varieties, including FRI-MF and FRI-MR;

[0008] The nucleotide sequence of the FRI-MF is shown in SEQ ID No. 5;

[0009] The nucleotide sequence of the FRI-MR is shown in SEQ ID No. 6.

[0010] This invention also provides the application of the primer pairs described in the above technical solution in distinguishing between single-flowering and continuously flowering Cymbidium varieties.

[0011] Preferably, the application includes the following steps:

[0012] 1) Total RNA was extracted from Cymbidium orchid buds and reverse transcribed into cDNA;

[0013] 2) Using the cDNA obtained in step 1) as a template, PCR amplification is performed using the primer pair described in claim 1 to obtain the amplification product;

[0014] 3) Perform agarose gel electrophoresis on the amplification product described in step 2). When a 319bp band is amplified, the Cymbidium goeringii is a single-flowering variety.

[0015] When a 292bp band is amplified, Cymbidium goeringii is a continuously flowering variety.

[0016] Preferably, the PCR amplification system in step 2) is: 12.5 µL of 2 × Phanta Flash Master Mix (DyePlus), 1 µL each of 10 µM upstream and downstream primers, 2 µL of cDNA, and ddH2O to bring the total to 25 µL.

[0017] Preferably, the PCR amplification program in step 2) is as follows: 94℃ for 3 min; 94℃ for 30 sec, 55℃ for 30 sec, 72℃ for 1 min, 30 cycles; 72℃ for 3 min.

[0018] This invention also provides the application of the primer pairs described in the above technical solution in Cymbidium orchid breeding.

[0019] This invention also provides a kit for distinguishing between single-flowering and continuously flowering Cymbidium varieties, containing the primer pairs described in the above technical solution.

[0020] This invention also provides the application of the kit described in the above technical solution in distinguishing between single-flowering and continuously flowering Cymbidium varieties.

[0021] The beneficial effects of this invention are:

[0022] The molecular markers FRI-MF / FRI-MR provided by this invention can accurately and efficiently distinguish between Cymbidium goeringii and Cymbidium faberi varieties that flower only once and Cymbidium ensifolium varieties that flower continuously. They can be used for molecular marker-assisted screening and identification of continuous flowering traits in Cymbidium goeringii, significantly improving breeding efficiency. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0024] Figure 1 The RNA of Cymbidium ensifolium cultivar 'Qingshan Yuquan' (A) and the CeFRI gene of Cymbidium ensifolium (B);

[0025] Figure 2 Analysis of conserved domains of the CeFRI gene in Cymbidium ensifolium;

[0026] Figure 3 Multiple sequence alignment of amino acids from CeFRI of Cymbidium ensifolium;

[0027] Figure 4 Phylogenetic evolutionary tree of FRI proteins in Cymbidium ensifolium and other plants;

[0028] Figure 5 Multiple sequence alignment of the FRI gene in Cymbidium cultivars;

[0029] Figure 6 The images show the agarose gel electrophoresis results of PCR products from the FRI gene deletion sites of 12 Cymbidium orchid varieties. Lanes 2-7 show the electrophoresis results of Yipinmei, Cuiyu Mudan, Qingshan Yuquan, Samojin, Jinsi Mawei and Yuhuanfei, respectively. Lanes 9-14 show the electrophoresis results of Qihei, Zhizun, Huanqiu Heding, Huanghemei, Yushizi and Fucui, respectively.

[0030] Figure 7 The sequencing results validated the FRI gene deletion sites in 12 Cymbidium varieties. Detailed Implementation

[0031] This invention provides a primer pair for distinguishing between single-flowering and continuously flowering Cymbidium varieties, including FRI-MF and FRI-MR; the nucleotide sequence of FRI-MF is shown in SEQ ID No. 5; and the nucleotide sequence of FRI-MR is shown in SEQ ID No. 6.

[0032] SEQ ID No. 5:

[0033] 5'-GAGGATAAGATGAGCGATATAG-3';

[0034] SEQ ID No. 6:

[0035] 5'-TAGCCTTCTCCAGGTTGT-3'.

[0036] This invention also provides the application of the primer pairs described in the above technical solution in distinguishing between single-flowering and continuously flowering Cymbidium varieties.

[0037] In this invention, the application preferably includes the following steps:

[0038] 1) Total RNA was extracted from Cymbidium orchid buds and reverse transcribed into cDNA;

[0039] 2) Using the cDNA obtained in step 1) as a template, PCR amplification is performed using the primer pair described in claim 1 to obtain the amplification product;

[0040] 3) Perform agarose gel electrophoresis on the amplification products described in step 2). When a 319bp band is amplified, the Cymbidium goeringii is a single-flowering variety; when a 292bp band is amplified, the Cymbidium goeringii is a continuously flowering variety.

[0041] This invention extracts total RNA from Cymbidium orchid buds and reverse transcribes it into cDNA. The method for extracting total RNA from Cymbidium orchid buds and for reverse transcription into cDNA is not particularly limited in this invention; conventional methods can be used by those skilled in the art.

[0042] This invention uses the cDNA as a template and performs PCR amplification using the primer pair described in claim 1 to obtain the amplified product. In this invention, the preferred PCR amplification system is: 12.5 µL of 2 × Phanta Flash Master Mix (Dye Plus), 1 µL each of 10 µM forward and reverse primers, 2 µL of cDNA, and ddH2O to a final volume of 25 µL. In this invention, the preferred PCR amplification program is: 94℃ for 3 min; 94℃ for 30 sec, 55℃ for 30 sec, 72℃ for 1 min, 30 cycles; 72℃ for 3 min.

[0043] In this invention, the amplification products are subjected to agarose gel electrophoresis. When a 319bp band is amplified, the Cymbidium goeringii is a single-flowering variety; when a 292bp band is amplified, the Cymbidium goeringii is a continuously flowering variety.

[0044] This invention also provides the application of the primer pairs described in the above technical solution in Cymbidium orchid breeding.

[0045] This invention also provides a kit for distinguishing between single-flowering and continuously flowering Cymbidium varieties, containing the primer pairs described in the above technical solution.

[0046] This invention also provides the application of the kit described in the above technical solution in distinguishing between single-flowering and continuously flowering Cymbidium varieties.

[0047] To further illustrate the present invention, the following detailed description is provided in conjunction with embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0048] Example 1

[0049] Cloning and sequence analysis of the FRI gene of Cymbidium ensifolium

[0050] 1. RNA extraction

[0051] Take 2g of flower buds from the Cymbidium ensifolium cultivar 'Qingshan Yuquan' and extract total RNA using the "Fastpure Universal Plant TotalRNA Isolation Kit". Figure 1 (A), and reverse transcribed into cDNA (HiScript III 1stStrand cDNA Synthesis Kit).

[0052] 2. Obtaining the target gene CeFRI

[0053] Using CeFRI-F (SEQ ID No. 3, ATGGCGTCGGCTGATTC) and CeFRI-R (SEQ ID No. 4, CTACCGATAATAAGCTTGCTGATAC) as primers, PCR was performed using the cDNA obtained in the previous step as a template. The PCR was performed with a high-fidelity enzyme (2x PhantaFlash Master Mix) under the following conditions: 95℃ for 30 seconds, followed by 35 cycles (95℃ for 10 seconds, 58℃ for 5 seconds, 72℃ for 5 seconds), and finally 72℃ for 10 minutes. The PCR products were detected by agarose gel electrophoresis, which showed a clear and single band, approximately 1560 bp in size. Figure 1 The target band was excised and recovered from the bacterium (B), and the recovered product was ligated into a cloning vector (5 min TA / Blunt-Zero Cloning Kit) and sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. Sequencing analysis revealed that the amplified fragment contained the complete CDs sequence of the target gene CeFRI, consisting of 1560 bases, as shown in SEQ ID No. 1. The CeFRI nucleotide sequence was translated into a protein sequence (as shown in SEQ ID No. 2) using SnapGene software. The encoded protein has an amino acid sequence of 520 residues and is named the orchid developmental regulatory protein FRI protein. The obtained *E. coli* containing the CeFRI gene is currently preserved at the Institute of Environmental Horticulture, Guangdong Academy of Agricultural Sciences.

[0054] SEQ ID No. 1:

[0055]

[0056] SEQ ID No. 2:

[0057] MASADSTMPNSSAVKAGFAELERQRELISSCTALWKELSDHFSSVERNLELRSESLRSKRRSLDFHTQRKLASLYRREESIDGSVDLAIARVEELRAASLSRLSSADAADIPARVRGFCVKMDSDAFFDL IVSNRKESDLLRKEIPLALKECIDPAKFVMDAIGGVFPVDRRAVKSSSGDLGWVCVLLLEGLVSALADPELGAERSLVTRIVKERAEEMADAWKEEMEQRGGVENAKAQDVHAFLQHLVTFEIGVKEERE FYRKLVVSISWRKQMPKLAIALGLEDKMSDIVEELILKGQQLDAINFAYEAGLQDKYPPIPLLKSYLKDAKKAATLASENNTSRKEPSAIRAVMRCIEERKLEAEFPLENLQKRLDNLEKAKAEKRKTSG GPANKRTRANNGGPMPPAKAGRLTNNAYVSSFPAAAPTFVRSPSHTTYSSAPFSPYPYERTPGHGFYGSHSPTAMRDPYAYPTEEIVPAPITASYPSPPISYPTYAGYNSGMGAYSNGIPPVYQQAYYR*.

[0058] 3. CeFRI gene sequence analysis

[0059] Analysis of conserved domains of CeFRI protein using the Conserved Domain Search function in the NCBI database ( Figure 2 The study found that the CeFRI protein contains the highly conserved Frigida domain of the Frigida-like (FRL) protein family.

[0060] The CeFRI gene of Cymbidium ensifolium was searched for homologous sequences in NCBI, and the encoded amino acids of the homologous sequences were compared using MEGA software. Figure 3 ), and construct an evolutionary tree ( Figure 4The results showed that Cymbidium ensifolium CeFRI shared 85.77% and 75.43% homology with Phalaenopsis PeFRI (XP_020589242.1) and Cymbidium sinense AsFRI (PKA59748.1), respectively; 65.98% homology with Typha latifolia TlFRI (XP_073005253.1); and 56% homology with Iris tectorum IpFRI (KAJ6797808.1). It showed 0.23% homology with OgFRI (XP_052162269.1) of glutinous rice and 54.48% and 54.91% homology with ObFRI (XP_006658319.2) of short-flowered rice, respectively; 52.84% homology with PopFRI (KAJ6911057.1) of poplar; and 49.22% homology with ZmFRI (PWZ12688.1) of maize.

[0061] The phylogenetic results show that CeFRI and Phalaenopsis PeFRI cluster together, and orchids cluster together due to close kinship.

[0062] Example 2

[0063] Comparison of FRI gene sequences and identification of differential sites in Cymbidium varieties with different flowering habits.

[0064] The FRI gene sequence of several Cymbidium varieties was obtained by PCR amplification, and the obtained gene sequences were performed by multiple sequence alignment using ClustalW sequence alignment software. As shown in Figure 5, it was found that the CDS region of the "continuously flowering" Cymbidium ensifolium variety was missing compared with the "single-flowering" Cymbidium sinense and Cymbidium goeringii varieties. The gene had a 27 bp deletion at 1017 bp.

[0065] Example 3

[0066] Development and application of functional molecular markers based on differentially expressed sites

[0067] Based on the differentially identified sites in Example 2, a pair of specific primers was designed:

[0068] FRI-MF (SEQ ID No. 5): 5'-GAGGATAAGATGAGCGATATAG-3';

[0069] FRI-M-R (SEQ ID No. 6): 5'-TAGCCTTCTCCAGGTTGT-3';

[0070] The molecular marker PCR amplification system was as follows: 12.5 μL of 2 × Phanta Flash Master Mix (Dye Plus), 1 μL each of 10 μM forward and reverse primers, 2 μL of cDNA, and ddH2O to a final volume of 25 μL. The PCR amplification conditions for the FRI-MF + FRI-MR primer pair were: 94 ℃ for 3 min; 94 ℃ for 30 sec, 55 ℃ for 30 sec, 72 ℃ for 1 min, for 30 cycles; 72 ℃ for 3 min.

[0071] Electrophoresis detection: Add 5 μl of sample loading buffer to the PCR amplification product, mix well, and then spot the mixture onto the wells of a 1% agarose gel containing SYBR Green dye. Perform electrophoresis in 1X TAE electrophoresis buffer at 10 V / cm, and then place the gel in a gel imaging system for photographic analysis.

[0072] Functional molecular markers were applied to 12 Cymbidium varieties using this marker. The electrophoresis and sequencing results are shown in Figures 6 and 7. The results showed that using FRI-MF and FRI-MR as primers, a single 319 bp specific band was stably amplified in the "single-flowering" Cymbidium sinense and Cymbidium goeringii varieties Qihei, Zhizun, Huanqiu Heding, Huanghemei, Yushizi, and Fucui. In contrast, in the "continuously flowering" Cymbidium ensifolium varieties Yipinmei, Cuiyu Mudan, Qingshan Yuquan, Satsuma Jin, "Jinsi Mawei", and Yuhuanfei, a characteristic band of 292 bp (corresponding to a 27 bp deletion homozygous type) was amplified. The above deletion was not observed in the single-flowering varieties.

[0073] The above results indicate that the molecular markers FRI-MF / FRI-MR provided by this invention can accurately and efficiently distinguish between Cymbidium goeringii and Cymbidium faberi varieties that flower only once and Cymbidium ensifolium varieties that flower continuously. They can be used for molecular marker-assisted screening and identification of continuous flowering traits in Cymbidium goeringii, significantly improving breeding efficiency.

[0074] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A primer pair for distinguishing between single-flowering and continuously flowering Cymbidium varieties, characterized in that, Including FRI-MF and FRI-MR; The nucleotide sequence of the FRI-MF is shown in SEQ ID No. 5; The nucleotide sequence of the FRI-MR is shown in SEQ ID No.

6.

2. The application of the primer pair described in claim 1 in distinguishing between single-flowering and continuously flowering Cymbidium varieties.

3. The application according to claim 2, characterized in that, The application includes the following steps: 1) Total RNA was extracted from Cymbidium orchid buds and reverse transcribed into cDNA; 2) Using the cDNA obtained in step 1) as a template, PCR amplification is performed using the primer pair described in claim 1 to obtain the amplification product; 3) Perform agarose gel electrophoresis on the amplification product described in step 2). When a 319bp band is amplified, the Cymbidium goeringii is a single-flowering variety. When a 292bp band is amplified, Cymbidium goeringii is a continuously flowering variety.

4. The application according to claim 3, characterized in that, Step 2) The PCR amplification system is as follows: 12.5 µL of 2 × Phanta Flash Master Mix (Dye Plus), 1 µL each of 10 µM upstream and downstream primers, 2 µL of cDNA, and ddH2O to bring the total to 25 µL.

5. The application according to claim 3, characterized in that, The PCR amplification program in step 2) is as follows: 94℃ for 3 min; 94℃ for 30 sec, 55℃ for 30 sec, 72℃ for 1 min, 30 cycles; 72℃ for 3 min.

6. The application of the primer pair according to claim 1 in Cymbidium breeding.

7. A kit for distinguishing between single-flowering and continuously flowering Cymbidium varieties, characterized in that, Contains the primer pair as described in claim 1.

8. The application of the kit according to claim 7 in distinguishing between single-flowering and continuously flowering Cymbidium varieties.

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