Gerbera petal-specific SSR marker primers and their applications

Abstract

This invention discloses SSR marker primers specific to the filamentous petals of gerberas and their applications. This invention utilizes SSR molecular markers to effectively identify different filamentous petal traits in gerberas, resulting in an SSR marker primer that can rapidly and accurately identify the petal traits of gerbera plants with different filamentous petals and the F1 generation of interspecific hybrids. The SSR marker primers provided by this invention can effectively distinguish between filamentous and non-filamentous petals in gerberas and exhibit significant polymorphism, with an accuracy greater than 65%. Furthermore, compared with traditional morphological trait observation methods, the identification method provided by this invention has better repeatability, saves time, and has high specificity. It can replace traditional morphological identification methods, effectively improving the molecular-level breeding process of gerberas and reducing the identification cycle of traditional morphological trait observation. It also allows for early screening to obtain gerbera offspring with superior filamentous varieties, which is of great significance for gerbera breeding.

Description

Technical Field

[0001] This invention belongs to the field of biomolecular science. More specifically, it relates to SSR marker primers specific to the petals of the gerbera daisy and their applications. Background Technology

[0002] Gerbera (Gerbera jamesonii Bolus) is a herbaceous plant belonging to the genus Gerbera in the family Asteraceae. Native to southern Africa, it is now widely cultivated in my country. Gerbera boasts numerous varieties, vibrant colors, a long flowering period, and tolerance for long-distance transport, making it primarily used for cut flowers and potted ornamental purposes. It is one of the world's five major cut flowers. Developing unique flower shapes has always been a primary goal of gerbera genetic improvement. The filamentous flower type is currently a popular gerbera variety, and breeding for this trait to obtain superior filamentous offspring is an important direction in gerbera breeding. Early screening of gerbera hybrids with filamentous traits using molecular markers would be of great significance in gerbera breeding.

[0003] Currently, traditional gerbera breeding mainly relies on hybridization between varieties and superior lines, phenotypic selection of new or improved offspring, and tissue culture of variant varieties. This identification process suffers from problems such as long cycle, low accuracy, and great influence from personal subjectivity. However, the method of using molecular marker-assisted identification to identify hybrid offspring is not affected by the environment and can accurately and quickly identify the genetic traits of hybrid offspring. Its diversity analysis is superior to conventional methods based on phenotypic data. However, compared with other ornamental plants, the number of SSR markers in gerbera is very limited (Lin Fazhuang, Li Jinye, An Huizhen, et al. SSR locus information analysis based on gerbera transcriptome sequencing [J]. Fujian Agricultural Science and Technology 2020, No.363(11):1-6.DOI:10.13651 / j.cnki.fjnykj.2020.11.001.). Moreover, the existing technology has only disclosed the use of SSR molecular markers to identify intergeneric hybrids of the genus Chrysanthemum and the genus Aster. So far, there have been no reports on using SSR molecular marker technology to identify the specificity of gerbera petals. Therefore, there is an urgent need to provide an SSR molecular marker for early screening of hybrid offspring with stringy traits, so as to improve breeding efficiency and shorten the breeding cycle, which is of great significance to gerbera breeding. Summary of the Invention

[0004] The technical problem to be solved by this invention is to overcome the defects and shortcomings of traditional morphological identification methods for identifying filamentous gerberas. This invention provides a specific SSR marker primer for filamentous gerberas and a method for identifying the petal specificity of the offspring of the F1 population of interspecific hybrids between filamentous white and revolutionary purple gerberas using this SSR marker. It has the characteristics of good repeatability, time saving and strong specificity, and can replace traditional morphological identification methods to effectively screen the offspring of gerbera hybrids at an early stage.

[0005] The purpose of this invention is to provide an SSR marker primer for identifying the specificity of gerbera flower petals.

[0006] Another object of the present invention is to provide the application of the primers in the identification of gerbera plants with stringy petals.

[0007] Another objective of this invention is to provide a method for identifying gerbera plants with stringy petals using SSR markers.

[0008] Another object of the present invention is to provide a product for identifying gerbera plants with stringy petals.

[0009] The above-mentioned objective of this invention is achieved through the following technical solution:

[0010] Based on previous analysis of SSR locus information obtained from the transcriptome of 'Jinxiang' gerbera, our team identified 48 pairs of SSR primers with good polymorphism. From these, we developed the SSR molecular marker primer GEM201, which effectively distinguishes between the filamentous and non-filamentous traits of gerbera. Its upstream primer is 5'-GGTTACTTCAA TCTCCCAATGC-3', and its downstream primer is 5'-AGTGGCAGCTCGATAAAAGTG-3'. The GEM201 marker primer exhibits significant polymorphism, enabling rapid and accurate identification of petal traits in the F1 generation of interspecific hybrids between filamentous white and revolutionary purple gerberas. Furthermore, it was able to identify over 65% of the filamentous gerberas in 140 hybrid progeny.

[0011] Therefore, this invention protects the application of GEM201 primers in the identification of gerbera plants with stringy petals, in the preparation of products for identifying gerbera plants with stringy petals, and in the breeding of gerbera plants with stringy petals.

[0012] This invention also provides a method for identifying gerbera plants with stringy petals using SSR markers, comprising the following steps:

[0013] S1. Extract genomic DNA from the sample to be tested;

[0014] S2. Using the DNA of the sample to be tested as a template, SSR-PCR amplification is performed using the primers described in claim 1, and the amplification products are detected by agarose gel electrophoresis.

[0015] S3. Statistical analysis of the band patterns and sizes of the detection results in step S2 is performed. Based on the band pattern analysis results, the gerbera plant with stringy petals is identified. When the amplification product shows two bands at the 100-120bp position, it is a gerbera plant with stringy petals. When the amplification product shows a single band at the 100-120bp position, it is a gerbera plant with non-stringy petals.

[0016] Compared with traditional morphological observation methods, the method improved by this invention has the advantages of being convenient, fast, quick, and reliable, and can effectively improve the efficiency of gerbera petal identification and reduce the identification cycle of traditional morphological observation.

[0017] Preferably, in step S1, fresh, tender leaves of the plant to be tested are extracted.

[0018] Preferably, the amplification reaction system in step S2 is as follows: 1.0 μL of 30-50 ng·μL-1 template DNA, 10.0 μL of 2×Taq PCRStarMix, 1.0 μL each of 10 mM upstream and downstream primers, and ultrapure water to a final volume of 20 μL.

[0019] Preferably, the amplification program in step S2 is as follows: 94℃ pre-denaturation for 2 min; 94℃ denaturation for 45 s, 58℃ annealing for 30 s, 72℃ extension for 30 s, 35 cycles; 72℃ repair extension for 7 min.

[0020] The present invention also provides a product for identifying plants with stringy petals of gerberas, containing the above-mentioned SSR marker primers for identifying the specificity of stringy petals of gerberas.

[0021] Preferably, the product further contains reagents for extracting genomic DNA from samples and reagents for SSR-PCR amplification.

[0022] The present invention has the following beneficial effects:

[0023] This invention utilizes multiple pairs of SSR molecular markers to effectively identify different filamentous petal traits in gerberas, resulting in an SSR marker primer specifically for identifying filamentous petal types in gerberas. This primer can rapidly and accurately identify gerbera plants with different filamentous petal types, as well as the petal traits of F1 generation from interspecific hybrids. The SSR marker primer provided by this invention can effectively distinguish between filamentous and non-filamentous traits in gerberas and exhibits significant polymorphism, identifying over 65% of filamentous offspring. Compared to traditional morphological identification methods, this invention's method, using SSR markers to identify gerbera plants with different filamentous petal types and the petal traits of F1 generation from interspecific hybrids, offers advantages such as good repeatability, time-saving, and high specificity. It can replace traditional morphological identification methods, enabling earlier identification of gerbera plants with filamentous petal traits, effectively accelerating the molecular-level breeding process of gerberas, and reducing the identification cycle of traditional morphological observation. Attached Figure Description

[0024] Figure 1 This refers to some F1 offspring of a cross between Silky White and Revolutionary Purple;

[0025] Figure 2The results of 11 primer pairs at the amplification sites of the parental white and revolutionary purple lines are shown (Note: a is white, b is revolutionary purple).

[0026] Figure 3 The results of amplification sites of 11 primer pairs in some progeny are shown (Note: a is white with stringiness; b is purple; LG03, LG09, LG23, LG27, and LG79 are gerberas with stringiness; LG02, LG74, LG138, LG148, and LG169 are gerberas without stringiness).

[0027] Figure 4 Comparison of amplification sites marked with GEM201 in hybrid progeny (Note: a is white; b is purple; 1, 3, 6-9, 15-16, 18, 20-23, 26-27, 32, 43, 45, 49-50, 52, 54, 56, 58, 60-61, 67, 69, 71, 73, 77-80, 83, 86, 88-89, 91, 96, 98-99, 101-103, 109-110, 112, 114, 119, 121, 124, 130, 133-134, 142-144, 146, 150-151, 155, 157-159, 161, ...). 165 and 170 are filamentous gerberas; 2, 4-5, 10-14, 17, 19, 25, 28-31, 33-35, 37-40, 42, 44, 46-48, 51, 53, 55, 57, 59, 63-66, 70, 74-75, 81-82, 84-85, 93-95, 97, 100, 105-106, 111, 113, 118, 122, 125-125, 129, 131, 137-138, 140, 145, 147-148, 152-153, 156, 160, 162-163, 167, and 169 are non-filamentous gerberas. Detailed Implementation

[0028] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0029] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0030] The parent gerberas used in this invention are two varieties: Lasi White and Revolution Purple, both introduced from Yunnan Province and currently preserved as resources at the South China Agricultural University Flower Base. Revolution Purple is a small-flowered potted variety with vibrant colors; while potted, its flower shape is relatively ordinary. Lasi White is a lasi-petaled gerbera variety with unique white flowers and a single flower color.

[0031] Example 1: Identification based on traditional morphological observations

[0032] The parent plants used in this invention, namely, Lasibai and Revolution Purple, as well as the F1 offspring obtained through hybridization, were all planted at the Flower Research Center of South China Agricultural University.

[0033] The traditional morphological characteristics of gerberas were observed using the industry standard "Guidelines for Testing the Distinctiveness, Uniformity and Stability of New Plant Varieties - Gerbera" issued by the Ministry of Agriculture and Rural Affairs of my country. The flower shape characteristics of the parent plants bred in this invention and their F1 hybrid offspring during the full bloom period were observed. The main observation indicators included the length of the outer ligules, the number of outer ligule petals, and the number of outer ligule lobes.

[0034] During cultivation, the F1 generation of hybrids (female *Lazy White* × male *Revolutionary Purple*) was designated LG, and the main observation indicators of the parents and the F1 generation were statistically analyzed. When the plants reached full bloom, the main morphological traits of the silky and non-silky types in the F1 generation differed. Analysis was conducted using some F1 hybrid samples LG12, LG105, LG169, LG9, LG109, and LG159 as examples.

[0035] The main morphological characteristics of the flowers of the parent plants, Lass White and Revolution Purple, and some hybrid offspring are as follows: Figure 1 As shown, LG12, LG105, and LG169 are non-spinning gerberas, while LG9, LG109, and LG159 are pinning gerberas. The morphological traits of the parental pinning gerberas (e.g., Lapinning White, Revolutionary Purple) and some F1 hybrids were compared, and the results are shown in Table 1. Among them, the F1 hybrids obtained by crossing Lapinning White as the female parent and Revolutionary Purple as the male parent (based on...) Figure 1 Taking the offspring as an example, various types of gerberas with different flower colors, both filamentous and non-filamentous, appeared. The number of petals in the outer whorl of the ray florets was basically the same for both types. The outer whorl of the ray florets in the filamentous gerbera showed lobes, with most petals having 3 or 4 lobes. The outer whorl of the ray florets in the filamentous gerbera had slightly pointed edges after lobing, while the outer whorl of the ray florets in the non-filamentous gerbera was oblong and shorter than that of the filamentous gerbera.

[0036] Table 1. Comparison of morphological traits of some F1 offspring from the cross between Lasibai and Revolutionary Purple.

[0037]

[0038] Example 2: Filtering SSR tags

[0039] The SSR markers used in this invention were developed based on the transcriptome data of 'Jinxiang' gerbera. 30,515 pairs of SSR primers were obtained based on the SSR locus information of the gerbera transcriptome. From these, 48 pairs of SSR primers with good polymorphism were screened and identified. Further, 11 pairs of SSR primers with good amplification effects were selected (preliminary screening of the 48 primer pairs revealed clear amplification bands and good polymorphism in the parent lines, selecting 11 pairs: GEM261, GEM130, GEM14, GEM65, GEM148, GEM203, P3-3, P3-19, GEM201, GEM57, and P3-4). The specific SSR marker primers used are detailed in Table 2. All SSR marker primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0040] Table 2 11 pairs of SSR marker primers

[0041]

[0042] The F1 hybrid samples LG03, LG09, LG23, LG27, LG79 (striped gerbera) and LG02, LG74, LG138, LG148, LG169 (non-striped gerbera) from Example 1 were analyzed. Genomic DNA was extracted from each parent in Example 1 and from some of the aforementioned interspecific hybrid F1 hybrids (using the Rapid Plant Genomic DNA Extraction Kit from Tiangen Biotech (Beijing) Co., Ltd.). The extracted DNA was amplified by PCR and analyzed in Veriti. TM The detection and analysis were performed on a 96well PCR instrument. The reaction system (20 μL) contained 30-50 ng / μL. -1 Template DNA 1.0 μL, 2×Taq PCRStarMix 10.0 μL, 1.0 μL each of 10 mM upstream and downstream primers, and ultrapure water 7.0 μL. The reaction program was: 94℃ pre-denaturation for 2 min; 94℃ denaturation for 45 s, 58℃ annealing for 30 s, 72℃ extension for 30 s, 35 cycles; 72℃ retrieval extension for 7 min. The SSR-PCR products were analyzed by 8% polyacrylamide gel electrophoresis.

[0043] After screening and analysis, the amplification sites of 11 primer pairs in the parental strains of *Streptomyces cerevisiae* and *Revolutionary Purple* were as follows: Figure 2 As shown, the 11 primer pairs produced clear and distinct amplification bands in both the parental plants (white and purple varieties), indicating that the parents possessed highly specific amplification sites for these 11 primer pairs. Five plants with stretchy filaments and five plants without stretchy filaments were then randomly selected for further screening and identification of these 11 primer pairs (using the same method as above). The results are shown below. Figure 3As shown, only the marker GEM201 can effectively distinguish between the spiny and non-spiny traits in these progeny, and can identify the petal specificity between spiny and non-spiny gerberas. Specifically, when the amplified product shows a double band at the 100-120bp position, it indicates a spiny-petaled gerbera plant; when the amplified product shows a single band at the 100-120bp position, it indicates a non-spiny-petaled gerbera plant. This indicates that the amplified sites of this marker are significantly different between spiny and non-spiny gerberas, exhibiting good polymorphism. Therefore, this invention selects the marker GEM201 as the primer for identifying the petal specificity of the F1 generation of interspecific hybrids between spiny white and revolutionary purple gerberas.

[0044] Example 3: Identification of genetic diversity in F1 hybrids of interspecific crosses between *Phyllostachys edulis* and *Purpledago virginiana* based on SSR markers

[0045] 1. Extraction of genomic DNA

[0046] Fresh, tender leaves were taken from the parent plants and F1 hybrid plants used in Example 1, as well as fresh, tender leaves from 140 plants of known petal traits. Genomic DNA was extracted using the rapid plant genomic DNA extraction kit from Tiangen Biotech (Beijing) Co., Ltd., with the specific steps strictly following the extraction instructions. The extracted genomic DNA was then detected by 1.0% agarose gel electrophoresis.

[0047] The results showed that the agarose gel electrophoresis images of the genomic DNA of the white, purple and some hybrid offspring were clear, with no diffusion or tailing, indicating that high-quality DNA was obtained and could be used for subsequent SSR-PCR amplification reactions.

[0048] 2. Polyacrylamide gel electrophoresis analysis

[0049] The primers for the SSR marker GEM201 obtained in Example 2 were used to perform SSR-PCR amplification with the DNA of the sample to be tested as a template. The amplification conditions and procedures were the same as in Example 2. Subsequently, the PCR amplification products were detected by agarose gel electrophoresis. After the electrophoresis detection was completed, the gel was placed on a transparent plate and the data was recorded by taking pictures.

[0050] 3. Comparison of amplification sites

[0051] The amplified bands of the F1 stretchable progeny and non-stretchable progeny from the above steps were analyzed to determine the accuracy of the marker primer in identifying petal specificity.

[0052] The results of the identification are as follows Figure 4As shown, GEM201 can effectively distinguish between the spiny and non-spiny traits of the parents, and the spiny offspring have obvious amplified bands. Compared with the identification results of the spiny and non-spiny offspring of the parents and the hybrid F1 population used in this invention, as well as 140 offspring with known petal traits, in the GEM201 marker, 45 (66.18%) spiny offspring were identified as accurately spiny offspring, and 30 (41.67%) non-spiny offspring were identified as accurately non-spiny offspring.

[0053] In summary, the SSR molecular marker primers provided by this invention can effectively distinguish between the filamentous and non-filamentous traits of gerberas, enabling rapid and accurate identification of the specificity of filamentous petals in gerberas. Using the marker primer GEM201, over 65% of filamentous progeny can be identified. Compared to traditional morphological identification methods, the method of identifying filamentous petals in gerberas using SSR molecular markers in this invention has the following advantages:

[0054] Traditional methods for identifying the morphological traits of hybrid offspring from superior gerbera varieties suffer from drawbacks such as long cycles, significant environmental influences on offspring growth, and low accuracy. This invention utilizes SSR molecular markers to assist in identifying the petal specificity of hybrid offspring, saving substantial time and costs while maintaining high accuracy. Petal traits can be identified even before the hybrid offspring bloom, replacing traditional morphological identification methods and effectively advancing the molecular-level breeding process of gerberas. This invention can replace traditional morphological identification methods, effectively improving the molecular-level breeding process of gerberas and reducing the identification cycle of traditional morphological trait observation.

[0055] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The application of an SSR marker primer in identifying gerbera plants with stringy petals, characterized in that, The primers include an upstream primer 5'-GGTTACTTCAATCTCCCAATGC-3' and a downstream primer 5'-AGTGGCAGCTCGATAAAAGTG-3'.

2. The application of an SSR marker primer in the preparation and identification of gerbera plants with stringy petals, characterized in that, The primers include an upstream primer 5'-GGTTACTTCAATCTCCCAATGC-3' and a downstream primer 5'-AGTGGCAGCTCGATAAAAGTG-3'.

3. The application of an SSR marker primer in the breeding of gerbera plants with stringy petals, characterized in that, The primers include an upstream primer 5'-GGTTACTTCAATCTCCCAATGC-3' and a downstream primer 5'-AGTGGCAGCTCGATAAAAGTG-3'.

4. A method for identifying gerbera plants with stringy petals using SSR markers, characterized in that, Includes the following steps: S1. Extract genomic DNA from the sample to be tested; S2. Using the DNA of the sample to be tested as a template, SSR-PCR amplification was performed using SSR-labeled primers, and the amplification products were detected by agarose gel electrophoresis; the primers included the upstream primer 5'-GGTTACTTCAATCTCCCAATGC-3' and the downstream primer 5'-AGTGGCAGCTCGATAAAAGTG-3'. S3. Statistical analysis of the band patterns and sizes of the detection results in step S2 is performed. Based on the band pattern analysis results, the gerbera plant with stringy petals is identified. When the amplified product shows two bands at the 100-120bp position, it is a gerbera plant with stringy petals. When the amplified product shows a single band at the 100-120bp position, it is a gerbera plant with non-stringy petals.

5. The method according to claim 4, characterized in that, In step S1, fresh, tender leaves of the plant to be tested are extracted.

6. The method according to claim 4, characterized in that, The amplification reaction system in step S2 is: 30-50 ng μL -1 Template DNA 1.0 μL, 2x Taq PCR StarMix 10.0 μL, 10 mM upstream primer and downstream primer 1.0 μL each, and ultrapure water to 20 μL.

7. The method according to claim 4, characterized in that, The amplification program in step S2 is as follows: 94 °C pre-denaturation for 2 min; 94 °C denaturation for 45 s, 58 °C annealing for 30 s, 72 °C extension for 30 s, 35 cycles; 72 °C repair extension for 7 min.

8. The application of a product containing SSR marker primers in the identification of gerbera plants with stringy petals, characterized in that, The primers include an upstream primer 5'-GGTTACTTCAATCTCCCAATGC-3' and a downstream primer 5'-AGTGGCAGCTCGATAAAAGTG-3'.

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