A molecular marker for early identification of petal color in cattleya and its application

Abstract

The application belongs to the field of plant molecular biology, and discloses a molecular marker for early identification of petal color of Asian lotus and application thereof. Through detection of 36 Asian lotus with known flower color by molecular markers PC1 and PC2, single plants with red and white petals can be accurately distinguished, and the accuracy can reach 90.9%. The molecular marker and the identification method provided by the application can also be used for early identification of petal color in the process of molecular assisted breeding of new germplasm of Asian lotus, and can accurately distinguish single plants with red and white petals. The application has high reliability and good stability, can significantly shorten the breeding period, improve the breeding efficiency, save the cost, and has high social and economic value and very broad application prospect.

Description

Technical Field

[0001] This invention belongs to the field of plant molecular biology, specifically relating to an early molecular marker for identifying the color of Asian lotus petals and its application. Background Technology

[0002] Lotus is an ancient relict plant belonging to the genus Nelumbo in the family Nelumbocephalae. There are only two species worldwide: the Asian lotus (Nelumbo nucifera) and the American lotus (N. lutea). The Asian lotus has a wider distribution, ranging from southern Russia in the north to northern Australia in the south, exhibiting extremely rich genetic diversity in plant shape, flower shape, rhizome, and seeds. The American yellow lotus is mainly distributed in the United States and exists in the wild. It has a smaller plant size, single-petaled flowers, and yellow petals. Through long-term artificial domestication and selection, the Asian lotus has developed into three cultivated types for different agricultural purposes: ornamental lotus (mainly used for ornamental purposes), rhizome lotus (mainly used for rhizome production), and seed lotus (mainly used for seed production). Among them, the ornamental lotus, with its varied flower shapes and vibrant colors, is deeply loved by the public and is an important component of garden water feature design.

[0003] Flower color is an important indicator for evaluating the ornamental value of Hualien. Although wild Asian lotus resources only have pink and white varieties, Hualien varieties bred through intraspecific hybridization of Asian lotus and interspecific hybridization with American yellow lotus have five flower colors: red, pink, white, yellow, and multi-colored. Currently, over 1000 Hualien varieties have been bred, with red accounting for 35%, pink for 20%, white for 20%, yellow for 6%, and multi-colored for 13%. In the process of breeding Hualien varieties with different flower colors, flower color identification is always done by observing flower morphology during the flowering period. Developing molecular markers or methods for early flower color identification in the seedling stage would save significant breeding costs in terms of manpower, resources, and time, improve breeding efficiency, and accelerate the targeted breeding process of Hualien. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides a molecular marker and method for early identification of petal color in Asian lotus or for molecular-assisted breeding of petal color in new Asian lotus germplasm.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] Application of molecular markers in early identification of petal color in Asian lotus or in molecular-assisted breeding of petal color in new Asian lotus germplasm: This includes molecular markers PC1 and PC2. The nucleotide sequence of molecular marker PC1 is shown in SEQ ID NO.1, and the nucleotide sequence of molecular marker PC2 is shown in SEQ ID NO.2. Pure recessive plants with white petals contain only molecular marker PC1, pure dominant plants with red petals contain only molecular marker PC2, and heterozygous plants with red petals or heterozygous plants with both red and white petals contain both molecular markers PC1 and PC2.

[0007] An early method for identifying the petal color of Asian lotus: white-petaled Asian lotuses contain only the molecular marker PC1 with the sequence shown in SEQ ID NO.1, while red or red-and-white bicolor Asian lotuses contain the molecular marker PC2 shown in SEQ ID NO.2. Specifically, the method includes the following steps: using the genomic DNA of the Asian lotus to be tested as a template, the molecular marker PC1 is amplified using primers 1 shown in SEQ ID NO.3 and 4, and the molecular marker PC2 is amplified using primers 2 shown in SEQ ID NO.5 and 6. The petal color is determined based on the amplification products. If primer 1 amplifies a band of 2079 bp and primer 2 does not amplify a band, the lotus to be tested has white petals; if primer 2 amplifies a band of 3630 bp, the lotus to be tested has red petals or red-and-white bicolor petals.

[0008] Compared with traditional methods of identifying patterns based on appearance, this invention has the following advantages:

[0009] 1. High Efficiency: This invention provides molecular markers and their identification methods for rapid and accurate identification and screening of red and white lotus germplasm resources, with clear selection targets and high efficiency. In the embodiments of this invention, the accuracy rate of identifying lotus germplasm of different flower colors using molecular markers can reach 90.9%.

[0010] 2. Saves breeding time: The molecular markers and their identification methods provided by this invention can select petal colors at the DNA level and complete early identification of flower colors in the seedling stage. They can be used for molecular marker-assisted breeding of new red and white lotus varieties, significantly shortening the breeding time. Attached Figure Description

[0011] Figure 1 Electrophoresis diagrams showing the detection of molecular markers PC1 and PC2 in 24 lotus germplasm resources; where A is the electrophoresis diagram of molecular marker PC1 and B is the electrophoresis diagram of molecular marker PC2. The lane numbers in the diagrams are in the same order as the sample numbers in Table 2, and M is D5000 Ladder. Detailed Implementation

[0012] The following examples are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the examples were conducted under standard experimental conditions, such as those described in *Molecular Cloning: A Laboratory Manual* (2012) by Michael R. Greenhe and Joseph Sambrook, or as recommended in the manufacturer's instructions.

[0013] Example 1: Development and Method of a Molecular Marker for Early Identification of the Color of Asian Lotus Petals

[0014] (1) Development of molecular markers

[0015] The inventors analyzed the genome sequences of 86 red, 36 white, and 5 red-and-white bicolor lotus germplasm resources and discovered a 90 kb insertion / deletion variant at 32 Mb on chromosome 4. This 90 kb fragment was present in the genome sequences of 97.7% of the red lotus germplasm resources and 80.0% of the bicolor lotus germplasm resources, while it was absent in the genome sequences of 83.3% of the white lotus germplasm resources (Table 1). Molecular markers PC1 (SEQ ID NO.1) and PC2 (SEQ ID NO.2) were developed based on the flanking and internal sequences of this 90 kb fragment, respectively.

[0016] The primer sequence for the molecular marker PC1 is as follows:

[0017] Forward primer PC1F: 5'-GGAAGGATTATGCAATAGATCCGAGC-3' (SEQ ID NO.3);

[0018] Reverse primer PC1R: 5'-CGAACTGGTTCAGACCCAAAATTAATTATT-3 (SEQ ID NO.4)'.

[0019] The primer sequences for the molecular marker PC2 are as follows:

[0020] Forward primer PC2F: 5'-GAAGTTTTTGGAGTTGATTACGTGTGAC-3' (SEQ ID NO.5);

[0021] Reverse primer PC2R: 5'-TCAATAACTCCACCACCTATGATTCAT-3' (SEQ ID NO.6).

[0022] Table 1. Insertion and deletion of 90 kb sequence fragments in the genomes of 127 lotus germplasm resources.

[0023]

[0024] (2) Extraction of genomic DNA from lotus germplasm resources with known petal colors

[0025] Tender, fresh leaves from 36 lotus germplasm resources (14 white, 19 red, and 3 red-white bicolor) were selected, and genomic DNA was extracted using a modified CTAB method. The specific steps are as follows:

[0026] I. Take about 0.5 grams of fresh sample and put it into a mortar in an ice bath. Add 1 mL of CTAB extraction solution and grind the leaves into a paste. Then transfer the paste into a 2.0 mL centrifuge tube.

[0027] II. Place the centrifuge tubes in a 65°C water bath for 60 minutes, mixing 2-3 times during the process. Centrifuge at 12000 rpm for 10 minutes, and transfer the supernatant to a new centrifuge tube.

[0028] III. Add an equal volume of 24:1 (v:v) chloroform:isoamyl alcohol, gently invert to mix thoroughly; then centrifuge at 12000 rpm for 10 minutes to separate the layers; aspirate the supernatant and repeat the process of adding an equal volume of 24:1 (v:v) chloroform:isoamyl alcohol once more.

[0029] IV. Gently aspirate the supernatant into a clean centrifuge tube, add 800 µL of pre-cooled anhydrous ethanol at -20°C, and freeze at -20°C for 30 minutes to precipitate the DNA.

[0030] V. Remove the white, flocculent DNA and soak it in 200 μL of 75% ethanol for 5 hours. Discard the ethanol, repeat the process of adding 75% ethanol once, soaking for 1 hour each time, and then place the centrifuge tube in a fume hood to dry.

[0031] Add 100 µL of TE solution to dissolve the DNA;

[0032] VII. DNA quality and concentration were determined using 0.8% agarose gel electrophoresis and a OneDrop spectrophotometer;

[0033] VIII. Dilute the DNA concentration to 50 ng / μL and store at -20°C for later use.

[0034] (3) PCR amplification

[0035] DNA from 36 lotus germplasm resources was amplified by PCR using primers with molecular markers PC1 and PC2. The PCR reaction volume (per 10 μL) consisted of: 50 ng DNA template, 2 μL 10×PCR buffer, 1 U Taq DNA polymerase, 0.2 mmol / L dNTPs, and 4 μmol / L each of forward and reverse primers. The PCR amplification program was as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 45 s, 58–60℃ annealing for 30 s, 72℃ extension for 45 s, for a total of 35 cycles; final extension at 72℃ for 10 min, and storage at 10℃.

[0036] (4) Detection of PCR products

[0037] PCR products were added to 2 μL of 6× loading buffer and detected by 1.5% agarose gel electrophoresis, with a D5000 Ladder used as a standard molecular weight control. Electrophoresis was performed at 120 V and 48 mA, and images were taken using a gel imaging system.

[0038] (5) Analysis of electrophoresis results

[0039] PCR amplification was performed on 36 tested lotus germplasm resources using primers for molecular markers PC1 and PC2, and the results are shown in Table 2. Primers for molecular marker PC1 amplified a 2079 bp band in 13 white-flowered, 2 red-flowered, and 2 multi-colored germplasm resources, but no band was amplified in 17 red-flowered and 1 multi-colored germplasm resource. Although primers for molecular marker PC1 amplified a band in the white germplasm resource 'Bai Long Xi Zhu', the fragment size differed from other white lotus germplasm resources. Primers for molecular marker PC2 amplified a 3630 bp band in all red-flowered and multi-colored germplasm resources, but no band was amplified in any white-flowered germplasm resource.

[0040] The results show that 92.9% of the white lotus germplasm resources could only be amplified with primers using molecular marker PC1, yielding a 2079 bp band; 89.5% of the red lotus germplasm resources could only be amplified with primers using molecular marker PC2, yielding a 3630 bp band; and the two multi-colored lotus germplasm resources could be amplified with primers using molecular markers PC1 and PC2, yielding 2079 bp and 3630 bp bands respectively. Therefore, molecular markers PC1 and PC2 can accurately identify the white and red colors of lotus germplasm petals, with an accuracy rate of 90.9%.

[0041] Table 2. Amplification results of molecular markers in 36 tested lotus germplasm resources.

[0042]

[0043] Example 2: Early identification of petal color in lotus hybrid offspring

[0044] Early identification of flower color was performed on 50 F2 generation individual plants obtained from crosses of red and white lotus germplasm resources using molecular markers PC1 and PC2, including the following steps:

[0045] (1) Red and white lotus germplasm resources were used for hybridization and self-pollination to obtain F2 offspring seeds. The seeds were germinated and when they grew to 3 leaves, a tender lotus leaf was taken and genomic DNA was extracted. Fifty F2 generation plants were planted in a container with a diameter of 80 cm.

[0046] (2) Using the extracted genomic DNA as a template, PCR amplification was performed using the molecular markers PC1 and PC2 primer pairs, and the results were detected by 1.5% agarose gel electrophoresis, as in Example 1.

[0047] (3) Investigate the petal color of 50 F2 generation individual plants during the lotus period.

[0048] Table 3 shows the PCR amplification results of 50 F2 generation plants using primers with molecular markers PC1 and PC2, and the petal colors of the plants. Thirteen F2 generation plants amplified a 2079 bp band using primers with only PC1, and all had white petals; twelve F2 generation plants amplified a 3630 bp band using primers with only PC2, and all had red petals; 25 F2 generation plants amplified bands with both molecular markers PC1 and PC2, and all had red petals.

[0049] The results show that the phenotypic segregation ratio of white to red was 1:3, and the genotypic segregation ratio was 1:2:1. Therefore, red is the dominant trait, and the genotypic segregation ratio is consistent with the F2 generation. This demonstrates the accuracy of using molecular markers PC1 and PC2 to identify the petal color of lotus hybrid offspring.

[0050] Therefore, molecular markers PC1 and PC2 can be used for early identification and molecular-assisted selection of the petal color of Asian lotus. If the primers for molecular marker PC1 amplify a 2079 bp band while the primers for molecular marker PC2 do not amplify a band, it indicates that the tested plant is purely recessive and has white petals. If the primers for molecular marker PC2 amplify a 3630 bp band while the primers for molecular marker PC1 do not amplify a band, it indicates that the tested plant is purely dominant and has red petals. If both primers for molecular markers PC1 and PC2 amplify bands, it indicates that the tested plant is heterozygous and has red petals.

[0051] Table 3. Petal colors of 50 individual lotus F2 plants

[0052]

[0053] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. The application of reagents for detecting molecular markers in the early identification or molecular-assisted breeding of red, white, or red-white bicolor petals of Asian lotus, characterized in that, The sample includes molecular markers PC1 and PC2. The nucleotide sequence of molecular marker PC1 is shown in SEQ ID NO.1, and the nucleotide sequence of molecular marker PC2 is shown in SEQ ID NO.

2. Asian lotus with white petals contains only molecular marker PC1, while Asian lotus with red or red-white petals contains molecular marker PC2.

2. Primers for early identification of the color of red, white, or red-and-white bicolor petals of Asian lotus, characterized in that, It includes two pairs of primers, the nucleotide sequences of primer pair 1 are shown in SEQ ID NO.3 and 4, and the nucleotide sequences of primer pair 2 are shown in SEQ ID NO.5 and 6.

3. An early identification method for the color of Asian lotus petals, characterized in that, The petals are white, red, or red and white. Asian lotus with white petals contains only the molecular marker PC1 with the sequence shown in SEQ ID NO.1, while Asian lotus with red or red and white petals contains the molecular marker PC2 shown in SEQ ID NO.

2.

4. The method according to claim 3, characterized in that... Includes the following steps: Using the genomic DNA of the Asian lotus to be tested as a template, the molecular marker PC1 was amplified using primer pair 1 shown in SEQ ID NO.3 and 4, and the molecular marker PC2 was amplified using primer pair 2 shown in SEQ ID NO.5 and 6. The color of the petals was determined based on the amplification products: if primer pair 1 amplified a band of 2079 bp and primer pair 2 did not amplify a band, the lotus to be tested had white petals; if primer pair 2 amplified a band of 3630 bp, the lotus to be tested had red petals or red and white bicolor petals.

Images