CN113142048A - Effective method for screening double-petal mutants of cone stone flower - Google Patents

Abstract

The invention discloses an effective method for screening double-petal mutants of cone stone flower, which comprises the following steps: grouping the seeds of the cone stone flower, and performing EMS mutagenesis on each group of seeds corresponding to different time to obtain EMS-mutagenized cone stone flower seeds; sowing the EMS-mutagenized conical stone flower seeds on a matrix of a mixture of peat and perlite, and culturing until the seeds germinate and grow into conical stone flower seedlings; cultivating the conical stone flower seedlings to mutant plants; and performing data statistical analysis on the mutant plant, and separating to obtain the double petal mutant of the cone stone flower. The effective method for screening the double-petal mutants of the cone stone flower identifies three double-petal mutants with different variations on flower types and flower colors and a plurality of other phenotypes by screening forward genetic mutants. The result of EMS mutagenesis of the invention is consistent with the expectation of a plurality of mutants discovered, and shows that the method of the invention successfully mutates the cone stone flower seeds.

Description

Effective method for screening double-petal mutants of cone stone flower

Technical Field

The invention relates to the technical field of biological breeding, in particular to an effective method for screening double-petal mutants of cone stone flower.

Background

Conium paniculatum (Gypsophila paniculata) is a flowering plant of the genus Geotrichum, the only species used as cut flowers in this genus of plants (Li et al, 2019). Cone stonecrop is one of the most important cut flowers in commercial flower culture worldwide because of its ornamental value (Zvi et al, 2008). However, breeding genetic variation of a specific floral trait using traditional breeding methods is a major obstacle in the breeding of Erythrochloe paniculata (Wang et al, 2013). As a result, there is a lack of variation between commercial varieties in the world's floral market (Zvi et al, 2008). Therefore, in the last decade, there has been a great demand for new varieties with new characteristics of ornamental traits.

The generation of multiple mutants with altered phenotypes provides a key tool for identifying and characterizing the biological functions of the relevant genes. Various mutagenesis approaches and methods have been developed, such as T-DNA insertion, irradiation, and chemical methods (Alonso et al, 2003; Belfield et al, 2012; Jansen et al, 1997). Each method has the advantages and disadvantages of generating mutations, but chemical mutagenesis is readily available and produces more subtle changes than other methods. For example, Ethyl Methane Sulfonate (EMS) induces chemical modifications of nucleotides, resulting in mismatches and base pair changes. EMS-induced alkylation of guanine (G) residues results in thymine (T) pairing, but not cytosine (C) pairing. Subsequently, the original G/C pair was replaced by an A/T pair by DNA repair (Greene et al, 2003). EMS mutagenesis in Arabidopsis thaliana (Arabidopsis) produces mutations evenly distributed throughout the genome, mainly inducing C to T changes, resulting in C/G to T/a substitutions, less G/C to C/G or G/C to T/a transversions or a/T to G/C conversions (Kim et al, 2006). Loss-of-function or gain-of-function mutants can be isolated due to the predominant presence of single nucleotide polymorphisms or substitutions. In combination with forward genetics methods, changes in a large number of randomly mutagenized individuals can be monitored and detected (Alonso and Ecker, 2006).

The flower type is one of the key ornamental characters and is the first thing in the breeding of the cone stone flower. Breeders have issued several commercial varieties of Echinacea having a heavy petal pattern, such as "Million Stars", "Bristol Fairy", "Snowball" and "Huixing 1" (Li et al, 2020; Shibuya et al, 2017). Given the challenges associated with genetic engineering of specific ornamental traits, EMS mutagenesis is an ideal alternative for efficient induction of mutants that may lead to improved traits. In addition, several specific mutant phenotypes have been identified by EMS mutagenesis, such as petaloid mutants of Portulaca grandiflora (Portulaca grandiflora) (Lokeshwar and Bhalla, 1982), stunted and variegated plants of ornamental ginger (Sakhanokho et al, 2012), and valuable ornamental mutants of four muscadine (Silene) species (Jiang and Dunn, 2017). Therefore, the induction of gene mutation by EMS provides a simple method for developing ornamental plant breeding by creating beneficial mutation.

Disclosure of Invention

Therefore, the invention provides an effective method for screening the double-petal mutants of the cone stone flower.

In order to achieve the above purpose, the invention provides the following technical scheme:

the embodiment of the invention provides an effective method for screening double petal mutants of cone stone flower, which comprises the following steps:

grouping the seeds of the cone stone flower, and performing EMS mutagenesis on each group of seeds corresponding to different time to obtain EMS mutagenesis seeds of the cone stone flower;

sowing the EMS mutagenesis conical stone flower seeds on a matrix of a mixture of peat and perlite, and culturing until the seeds germinate and grow into conical stone flower seedlings;

culturing the conical stone flower seedlings until the conical stone flower seedlings grow into mutant plants;

and analyzing the mutant plants, and separating to obtain the double petal mutants of the cone stone flower.

Preferably, the EMS concentration is 150mM EMS.

Preferably, the number of the conical stone flower seeds in each group is 1000.

Preferably, the weight of each group of the seeds of the cone stone flower is 0.641 +/-0.02 g.

Preferably, the EMS mutagenesis process is:

putting the dried cone stone flower seeds into a centrifuge tube, and pre-absorbing with 100mM potassium phosphate buffer solution at 4 ℃ overnight;

removing the potassium phosphate buffer solution, adding 10mL of 100mM potassium phosphate buffer solution into the centrifuge tube, and adding EMS to the potassium phosphate buffer solution to reach a final concentration of 150 mM/L;

and (3) incubating the cone stone flower seeds in a potassium phosphate buffer solution for 6h, then washing the cone stone flower seeds for 10 times within 30min by using 100mM/L sodium thiosulfate and distilled water in sequence, and drying the cone stone flower seeds.

Preferably, the different times are 0 hours, 4 hours, 6 hours, 8 hours and 12 hours.

Preferably, the EMS mutagenesis time is 6 h.

Preferably, the EMS mutagenic cone stone flower seeds are sown and cultivated within one week.

The double-petal flower mutant of the cone stone flower prepared by the method also belongs to the protection scope of the invention.

The invention has the following advantages:

the invention provides an effective method for screening the double-petal mutants of the cone stone flower by a forward genetics method, and identifies three double-petal mutants with different variations on flower type and flower color, and a plurality of other phenotypes. The result of EMS mutagenesis of the invention is consistent with the expectation of finding several mutants, which shows that the method of the invention successfully mutates the wild type seeds of the cone stone flower. In the absence of cone stone flower genome information and effective genetic modification, the invention provides a simple method for forming various cone stone flower mutants and provides a basic material for the genetic modification and molecular breeding of cone stone flower.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It should be apparent that the drawings in the following description are merely exemplary, and that other embodiments can be derived from the drawings provided by those of ordinary skill in the art without inventive effort.

FIG. 1 provides the phenotype of wild type plants and flowers of Conium, at a scale bar of 10cm, for an embodiment of the present invention;

fig. 2 is a graph of germination rates of M1 seeds at different treatment times in the cone stone flower using 150mM EMS, the average germination rate from three replicates, a one-way anova for statistical significance evaluation, and p-value calculated by Tukey's HSD test (α ═ 0.05);

fig. 3 is a table showing the phenotype of EMS mutants with different types of plants and leaves provided by the examples of the present invention, wherein a: a plurality of leaf types in the M1 population, each leaf collected from a different mutant; b: typical plant phenotype of mutants in the M1 population, scale bar 1 cm;

FIG. 4 is a phenotype of petaloid mutants with different flower types and flower colors according to the present invention, wherein the same longitudinal flower line is derived from a mutant plant with a scale bar of 1 cm;

FIG. 5 is an alignment of the amino acid sequences of AG in petaloid mutants with different flower types according to the present invention, wherein the same amino acids are shown in black background and the sequence differences are highlighted in white.

Detailed Description

The present invention is described in terms of particular embodiments, other advantages and features of the invention will become apparent to those skilled in the art from the following disclosure, and it is to be understood that the described embodiments are merely exemplary of the invention and that it is not intended to limit the invention to the particular embodiments disclosed. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Materials and methods: plant material, a wild type of cone stone flower with a single pink petal flower was selected for this study, as shown in figure 1. Seed of cone stone flower is obtained from Yuxi Yuxingxing Biotechnology GmbH (Yuxi, China). The thousand seed weight (TKW) of the stone bouquet seed is about 0.641g (n 10).

Example 1

This example provides an efficient method for screening for mutants of double petals of cone stone flower, comprising the steps of:

1. EMS mutagenesis of conical stone flower seeds

To determine the optimal treatment time for EMS mutagenesis, a large number of seeds were pre-treated with 150mM EMS at different times (0, 4, 6, 8 and 12 hours, 3 replicates each with 1000 seeds) in a gradient procedure before mutagenizing the seeds. The best treatment was selected based on criteria for visible traits (e.g. 50% seed germination control, albinism and trichome phenotype of seedlings) (Li, 2018). The germination data for each treatment is shown in table 1, using 150mM EMS for seeds at different mutagenesis times in the cone stone flower. One-way anova was used to assess statistical significance and p-values were calculated using Tukey's HSD test (α ═ 0.05). Significance.

TABLE 1

To avoid exposure to toxic and carcinogenic EMS, the mutagenesis procedure was performed in a Plexiglas glove box in a fume hood. Approximately 30,000 well-dried seeds (19.23g) were placed in 10ml centrifuge tubes and treated with 100mM potassium phosphate buffer overnight at 4 ℃. After removing the potassium phosphate buffer, 10mL of fresh 100mM potassium phosphate buffer was added. Then 154.4. mu.L EMS was added to a final concentration of 150 mM. The seeds were incubated in solution for 6 hours at room temperature with gentle shaking. After incubation, the seeds were washed thoroughly 10 times with 100mM sodium thiosulfate and distilled water sequentially over 30 minutes. The seeds were then carefully transferred to filter paper and allowed to dry completely overnight. The seeds treated by mutagenesis need to be sown as soon as possible, since the M1 seeds treated by EMS mutagenesis (first generation seeds treated with mutagen) are very weak and cannot be stored for a long time (even after 1 week, the seeds all die).

2. Screening of conical stone flower seed mutants

After EMS mutagenesis of the bougainvillea cone seeds, all seeds were sown on a matrix of peat and perlite mixture for germination on a breeding bed. The mutagenized seedlings were then grown in a solar greenhouse under natural photoperiod in an experimental farm of the academy of agricultural sciences of Yunnan province. This example investigates dominant mutants that show any variation compared to the wild type. Randomly planted plants, including total plants and the number of dominant mutants, were observed and studied in five plots (one square meter each).

3. Results

3.1EMS mutagenesis method, the germination rate of control seeds (0 hours) was 71.13% (n-3, as shown in fig. 2) in pretreatment for EMS mutagenesis, while the germination rate of seeds decreased significantly with increasing mutagenesis time (α -0.05). After 6 hours of treatment, the seed germination was reduced to 38.47% (n-3), which is about half of the seed germination control. At the same time, some visible traits such as albino plants and trichome phenotype were also determined in the mutagenized seedlings that had been treated for 6 hours. This indicates that 6 hour treatment with 150mM EMS is an ideal method for EMS mutagenesis of Ericatum paniculatum.

It is estimated that a population of 5,000 EMS mutagenized M1 would be sufficient to generate a mutation in any given gene in arabidopsis, since EMS would generate multiple point mutations in each individual (Qu and Qin, 2014). There are more than 56000 genes in the genome of Echinacea paniculata, twice as many as those of Arabidopsis thaliana (27655 genes) (Initiative, 2000). Therefore, at least 10000M 1 plants are needed for EMS mutagenesis of Erysiphe conotica to generate at least one mutation in any gene. Thus, this example was subjected to 6 hours of mutagenic treatment of 30,000 seeds with 150mM EMS to achieve the desired minimum M1 population. These seeds were sown on a greenhouse breeding bed for further mutation screening. As shown in fig. 2, seed germination rates of the M1 population at different treatment times in the cone stone flower were determined using 150mM EMS. The average germination rate was from three replicates. One-way anova was used to assess statistical significance and p-values were calculated by Tukey's HSD test (α ═ 0.05).

3.2EMS mutant screening

After germination of the seeds, plants with different mutant phenotypes were observed and studied. To determine the effectiveness of this EMS processing, the present embodiment performs a spot survey at five sites. Dominant mutants were counted and the dominant mutation rate was about 54.63% (n-5, table 2, dominant mutation rate in EMS mutagenized population). Thus, the EMS mutagenesis of this example generated enough mutants to produce multiple phenotypes in the cone of echeveria.

TABLE 2

For example, several mutants with different types of plants and leaves were isolated, as shown in FIG. 3, e.g., dwarf plants, plants with altered shape and color of the leaves. The result shows that EMS mutagenesis can cause the texture, shape and color variation of the cone stone flower leaves, and the pale yellow leaf variegated mutant is a new ornamental character of cone stone flower. Furthermore, the identification of albino and yellowish seedlings indicates that Phytoene Desaturase (PDS), a key enzyme in carotenoid synthesis, is likely to be targeted and lose its function. In addition, a number of abortive mutants were identified, confirming the adequacy of this EMS mutagenesis.

In summary, the protocol of this example provides a simple and efficient method to generate useful mutations in Erinaceus paniculatus, since there is no efficient method for molecular genetic engineering of Erinaceus paniculatus, such as a library of T-DNA insertion mutants in Arabidopsis thaliana or a gene editing system.

3.3 identification of the multiple petal mutants

To identify petaloid mutants, mutants were screened for flower types in flowering M1 plants. As a result, three different types of petaloid mutants were isolated, as shown in fig. 4. The phenotypes of the three petaloid mutants showed unequal variation in flower type and color compared to wild-type flowers, including one white and two pink petaloids. More specifically, mutagenesis results in a weakening or even disappearance of the stamens, but the pistils appear to be unchanged. Further, no seeds were produced in all mutants, and it was speculated that extra petals might be transformed from the stamen, thereby causing sterility of the mutants. The use of a pollen-poor or pollen-free mutant as a female in breeding would facilitate cross breeding of phimosa paniculata based on our observations that this species has a high self-pollination rate.

The discovery of petaloid mutants that differ in flower type and flower color suggests that multiple base pairs or genes are targeted during EMS mutagenesis. Since the M1 generation contained only a dominant mutant, it was shown that one or more genes of interest that regulate the petaloid flower were dominant. Furthermore, the results are consistent with theoretical expectations, indicating successful EMS mutagenesis of wild type seeds of erichsen paniculatum.

3.4 genes regulating Bipetala in Conium

In order to further research the regulatory genes of the double petal in the cone stone flower, the invention clones AGAMOUS (AG) genes of three double petal mutants. Several SNP transitions or deletions in the AG gene, as well as synonymous substitutions, were identified from these mutants by gene sequencing. Although the mutant patterns were similar, the mutation sites detected in the AGa and AGb genes were different from each other. For example, a single base pair deletion at 540bp of the AGa gene (T /) was detected in M1, resulting in an amino acid change from leucine to serine (L181S), and a four amino acid substitution and a premature STOP codon (I185> STOP). In addition, the present invention identified a 6bp base pair deletion in AGb from M1 and M3 that resulted in the deletion of two non-synonymous amino acids compared to the reference genomic sequence. This suggests that the loss of function of the AG gene may cause a half-bivalve or a bivalve pattern. Table 3 shows the information on the mutation of the AG gene detected from the double-petal mutants of Echinacea purpurea. As shown in FIG. 5, the amino acid sequences of AG in the petaloid mutants having different patterns were aligned. Identical amino acids are shown on a black background, while sequence differences are highlighted in white.

TABLE 3

Although the invention has been described in detail above with reference to a general description and specific examples, it will be apparent to one skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

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Claims (8)

1. An efficient method for screening for mutants of double petals of cone stone flower, comprising:

grouping the cone stone flower seeds, and performing EMS mutagenesis on each group of cone stone flower seeds corresponding to different time to obtain EMS-mutagenized cone stone flower seeds;

sowing the EMS mutagenesis conical stone flower seeds on a matrix of a mixture of peat and perlite, and culturing until the seeds germinate and grow into conical stone flower seedlings;

cultivating the conical stone flower seedlings to mutant plants;

and analyzing the mutant plants, and separating to obtain the double petal mutants of the cone stone flower.

2. The efficient method for screening for mutants of double petals of cone stone flower according to claim 1,

the EMS concentration is 150mM/L EMS.

3. The efficient method for screening for mutants of double petals of cone stone flower according to claim 1,

the number of the conical stone flower seeds in each group is 1000.

4. An efficient method for screening for mutants of double petals of cone stone flower according to claim 3,

the weight of each group of conical stone flower seeds is 0.641 +/-0.02 g.

5. The efficient method for screening for mutants of double petals of cone stone flower according to claim 1,

the EMS mutagenesis process comprises the following steps:

putting the dried cone stone flower seeds into a centrifuge tube, and pre-absorbing with 100mM potassium phosphate buffer solution at 4 ℃ overnight;

removing the potassium phosphate buffer solution, adding 10mL of 100mM potassium phosphate buffer solution into the centrifuge tube, and adding EMS to the potassium phosphate buffer solution to reach a final concentration of 150 mM/L;

and (3) incubating the cone stone flower seeds in a phosphate buffer solution for 6h, then washing the cone stone flower seeds for 10 times within 30min by using 100mM/L sodium thiosulfate and distilled water in sequence, and drying the cone stone flower seeds.

6. The efficient method for screening for mutants of double petals of cone stone flower according to claim 1,

the different times are 0 hours, 4 hours, 6 hours, 8 hours and 12 hours.

7. The efficient method for screening for mutants of double petals of cone stone flower according to claim 5,

the EMS mutagenesis time is 6 h.

8. The efficient method for screening for mutants of double petals of cone stone flower according to claim 1,

the EMS mutagenized conical stone flower seeds are sowed and cultured within one week.

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