Gene McAP23a related to formation of double petals of ornamental malus spectabilis as well as molecular marker and application of gene McAP23a

Abstract

The invention discloses a gene McAP23a related to formation of double petals of ornamental malus spectabilis as well as a molecular marker and application of the gene McAP23a. The nucleotide sequence of the gene McAP23a disclosed by the invention is as shown in SEQ ID NO: 1. When the petals of the ornamental crabapple are double-petal varieties, in the double-petal varieties, the gene McAP23a is a double-petal allele McAP23a-D, and the nucleotide sequence of the gene McAP23a is as shown in SEQ ID NO: 2; or when the ornamental Chinese flowering crabapple petals are single-petal varieties, in the single-petal varieties, the gene McAP23a is a single-petal allele McAP23a-S, and the nucleotide sequence of the gene McAP23a is shown as SEQ ID NO: 3. The invention finds that the miR172l negatively regulates the target gene McAP23a to mediate the formation of double petals of the ornamental malus spectabilis.

Description

Technical Field

[0001] The present invention relates to a field, and in particular to a gene McAP2_3a related to the formation of double-petaled flowers of ornamental crabapple, and a molecular marker and application thereof. Background Art

[0002] Begonia belongs to the genus Malus (Rosaceae). Malus ) is a general term for plants with relatively small fruits (≤5 cm). There are many varieties of ornamental crabapples, with diverse flower colors, rich leaf colors, a long viewing period, outstanding ornamental characteristics, and high ornamental value. They are important foliage, flower, and fruit ornamental plants, and are widely used in landscaping and various landscape creation. The cultivation and application history of ornamental crabapples in my country exceeds 2,000 years, and the variety resources are rich, covering three types of flower types: single petals, semi-double petals, and double petals. However, double-petal varieties account for only 14%. Double flowers are the core ornamental traits and main breeding targets of ornamental plants. They are directly related to the ornamental quality and application value. Improving the double petals of ornamental crabapples is urgent.

[0003] MicroRNAs (miRNAs) are a class of endogenous small RNAs, approximately 20 to 24 nucleotides in length, that play a variety of important regulatory roles within cells. Each miRNA can have multiple target genes, and several miRNAs can regulate the same gene. This complex regulatory network allows a single miRNA to regulate the expression of multiple genes, while a combination of miRNAs can finely regulate the expression of a single gene. miRNAs also play a crucial role in plant nutrient homeostasis, such as miR399, which regulates phosphorus uptake in Arabidopsis thaliana. Among the many plant miRNAs, miR172 was discovered relatively early, and its mode of action and biological function are among the most thoroughly studied.

[0004] However, there are few studies on the target genes of miR172 family members in ornamental crabapple, and there is an urgent need to conduct in-depth research on ornamental crabapple through the target genes of miR172 family members. Summary of the Invention

[0005] The present invention aims to overcome the shortcomings of the prior art and provides a gene McAP2_3a related to double flower formation in ornamental crabapple, its molecular marker and application. The present invention identifies 16 miR172 family members in the ornamental crabapple genome and predicts their target genes for the miR172 family members. McAP2_3a The present invention found that miR1721 negatively regulates the target gene McAP2_3a to mediate the formation of double-petaled flowers in ornamental crabapple.

[0006] To achieve the above purpose, the technical solution designed by the present invention is as follows: The present invention provides a gene related to the formation of double petals of ornamental crabapple McAP2_3a , the gene McAP2_3a The nucleotide sequence is shown in SEQ ID NO: 1.

[0007] Furthermore, when the ornamental crabapple petals are double petal varieties, in the double petal varieties, the gene McAP2_3a Double allele McAP2_3a-D, The nucleotide sequence is shown in SEQ ID NO: 2; Alternatively, when the ornamental crabapple petals are single-petal varieties, in the single-petal variety, the gene McAP2_3a Single petal allele McAP2_3a-S , the nucleotide sequence of which is shown in SEQ ID NO: 3; The above-mentioned related genes McAP2_3a Negatively correlated with miR172l expression, miR172l negatively regulates target genes McAP2_3a Mediate the formation of double flowers in ornamental crabapple.

[0008] The present invention also provides a molecular marker related to the formation of double flowers of ornamental crabapple. The sequence of the molecular marker contains a SNP site, which is located at the 1234th base in the nucleotide sequence shown in SEQ ID NO: 1; the allele of this site is T / C.

[0009] Furthermore, the sequence of the molecular marker is shown in SEQ ID NO: 4; and the SNP site is located at the 112th base in the sequence; The double-petal variety in the ornamental crabapple has C as the dominant allele, and the single-petal variety in the ornamental crabapple has T as the dominant allele.

[0010] The present invention also provides an application of the molecular marker in auxiliary selection of double-petal traits of ornamental crabapples.

[0011] The present invention also provides a primer pair for amplifying the molecular marker, wherein the primer pair is: 3a-F: 5'-ACGGAGAAGAGTATGGATGTTAACT-3'; 3a-R: 5'-TCAGCTTCTGCAATAGAAATGG-3'.

[0012] The present invention also provides a method for detecting SNPs related to the formation of double-petaled flowers of ornamental crabapples, wherein the method uses the primer pair for amplification and sequencing comparison to complete the detection.

[0013] The present invention also provides an application of the primer pair in identifying the double-petal trait of ornamental crabapple.

[0014] The present invention also provides a kit for identifying double-petal varieties / single-petal varieties of ornamental crabapples, and the kit comprises the primer pair.

[0015] The present invention also provides a method for identifying double-petal varieties / single-petal varieties of ornamental crabapples, comprising the following steps: 1) Extract genomic DNA from the sample to be tested and set aside; 2) using the genomic DNA as a template, performing PCR amplification using the primer pair or the kit; 3) Sequencing for identification, the identification criteria are as follows: When the 112th base is T, the tested variety is a single-petal ornamental crabapple; Alternatively, when the 112th base is C, the variety to be tested is a double-petaled ornamental crabapple.

[0016] Beneficial effects of the present invention: (1) The present invention provides McAP2_3a The gene has sequence differences between single-petal and double-petal ornamental crabapple varieties. McAP2_3a The gene and its corresponding amplification primers, the SNP molecular marker corresponding to the gene and its detection primers can be used to identify the double-petal trait of a hybrid population, thereby realizing rapid and accurate identification of single-petal and double-petal traits during the seedling stage of ornamental crabapple, shortening the breeding cycle, greatly reducing the breeding workload, and accelerating the breeding process of double-petal ornamental crabapple.

[0017] (2) The present invention provides the double-petal allele McAP2_3a-D and the single-petal allele McAP2_3a-S. Transgenic plants were obtained by constructing an overexpression vector and introducing it into tobacco. Overexpression of the double-petal allele McAP2_3a-D in tobacco resulted in an increase in petals and petalization of stamens. However, floral organs such as stamens and pistils in tobacco transformed with the single-petal gene McAP2_3a-S showed no significant differences compared to wild-type tobacco. The double-petal allele McAP2_3a-D provides a molecular biological basis for enhancing plant double petal characteristics through plant gene function. Application of this gene will contribute to the double petal characteristics of plants, enhance their ornamental value, and cultivate varieties with superior traits.

[0018] (3) The present invention identified 16 miR172 family members in the ornamental crabapple genome and predicted their target genes. The present invention discovered that miR172l negatively regulates the target gene FlameC04AgG010340 (McAP2_3a), mediating the formation of double flowers in ornamental crabapples. The present invention overexpressed miR172l and McAP2_3a genes in ornamental crabapples and found that McAP2_3a gene expression was significantly decreased, indicating that miR172l targeted cleavage of McAP2_3a in flower buds. Subsequently, dual-luciferase assays revealed that the fluorescence intensity of the double-petal allele, McAP2_3a-D, was significantly higher than that of the single-petal allele, McAP2_3a-S, indicating that miR172l cleaves McAP2_3a less efficiently in double-petal varieties than in single-petal varieties. The present invention discovered a molecular marker associated with double-petal flower formation in ornamental crabapples. The marker sequence contains a single nucleotide polymorphism (SNP) located at base 1234 in the nucleotide sequence of the related gene McAP2_3a. The allele at this site is T / C. In double-petal varieties of ornamental crabapples, C is the dominant allele, while in single-petal varieties, T is the dominant allele. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 To view the results of the mature sequence alignment of the crabapple miR172 family members; Figure 2 This is the result of phylogenetic analysis of mature sequences of miR172 family members in eight plant species; Figure 3 To view the precursor sequence alignment results of the Malus miR172 family members; Figure 4 This is the result of phylogenetic analysis of the plant euAP2 gene subfamily; Figure 5 The results of expression analysis of six McAP2s genes and miR172 family members in single and double petal flower buds of ornamental crabapple are shown in the figure. In the figure, HBL-S and JSYD-S represent the single-petal cultivars 'Red Barron' and 'Golden Raindrop' crabapple, respectively, and BLD-D and KL-D represent the double-petal cultivars 'Brandy' and 'Corey' crabapple, respectively; Figure 6 This is the result of alignment of CDS sequences of single-petal variety McAP2_3a-S and double-petal variety McAP2_3a-D genes; Figure 7 The results of AP2 evolution analysis of crabapple, apple and Arabidopsis are shown in the figure; Figure 8 Figure 2 is the result of the targeting relationship between miR1721 and McAP2_3a allele; In the figure, A: Prediction of miR172 target sites of McAP2_3a-D and McAP2_3a-S genes, B: Expression level of McAP2_3a gene in ornamental crabapple flower buds, C: Dual luciferase verification of the interaction between miR172l and McAP2_3a-D and McAP2_3a-S; Figure 9 The phenotype of tobacco plants overexpressing McAP2_3a-D and McAP2_3a-S alleles is shown; In the figure, A: From left to right, floral phenotypes of WT wild-type tobacco, transgenic tobacco 35S::McAP2_3a-S, and transgenic tobacco 35S::McAP2_3a-D; B: stamen petalization phenomenon of wild-type WT and transgenic tobacco 35S::McAP2_3a-D; Figure 10 The figure shows the sequence alignment results of McAP2_3a gene in four single-petal and two double-petal ornamental crabapple varieties; In the figure, HBL-S, JSYD-S, MGD-S and HZB-S represent the single-petal varieties 'Red Barron' crabapple, 'Golden Raindrop' crabapple, 'Rose Column' crabapple and 'Red Jewel' crabapple, respectively; BLD-D and KL-D represent the double-petal varieties 'Brandy' crabapple and 'Corey' crabapple, respectively. DETAILED DESCRIPTION

[0020] The present invention is further described in detail below with reference to specific embodiments so that those skilled in the art can understand.

[0021] Example 1 Identification of ornamental crabapple miR172 family members The precursor and mature sequences of apple miR172s (mdm-miR172a-o) were searched and downloaded from the miRBase database. Combined with the high-quality chromosome-level reference genome sequence of the ornamental crabapple "Flame", a total of 16 miR172 family members were identified in the ornamental crabapple genome, namely miR172a, miR172b, miR172c, miR172d, miR172e, miR172f, miR172g, miR172h, miR172i, miR172j, miR172k, miR172l, miR172m, miR172n, miR172o and miR172p.

[0022] The results of the mature sequence alignment of the ornamental crabapple miR172 family members are as follows Figure 1 As shown in Figure 2, the mature sequences of miR172 family members all contain GAAUCUUGAUGAUGCUGCA. Evolutionary analysis of miR172 family members was performed, and the results were as follows. Figure 2As shown in Figure 2, all miR172 members of the ornamental crabapple family were clustered into one branch. The precursor sequences of the ornamental crabapple miR172 family members were aligned, and the results were as follows: Figure 3 As shown in the data, the precursor sequence lengths of the 16 ornamental crabapple miR172 family members ranged from 104 to 181 nt, and the sequence variation was large, indicating that the functions of the ornamental crabapple miR172 family members were relatively conserved during evolution, but there was also functional differentiation.

[0023] Example 2 Identification of target genes of miR1721 family members To understand the biological functions of the ornamental crabapple miR172 family members, the psRNATarget software was used to predict the target genes of the ornamental crabapple miR172 family members, and a total of 8 target genes were predicted. McAP2s , all of which are members of the AP2 gene family, namely FlameC02AgG013060, FlameC15AgG015790, FlameC0sAgG008980, FlameC11AgG010380, FlameC12AgG012650, FlameC04AgG010340, FlameC01AgG020840 and FlameC07AgG011140.

[0024] The above eight target genes were analyzed with AP2 protein sequences from other plants through phylogenetic tree analysis. Figure 4 As shown in the figure, phylogenetic analysis showed that FlameC02AgG013060 and FlameC15AgG015790 belonged to the AP2 branch, FlameC0sAgG008980 and FlameC11AgG010380 belonged to the TOE branch, and FlameC12AgG012650 and FlameC04AgG010340 belonged to the PETALOSA TOE type. These McAP2s genes are speculated to be involved in the formation of double flowers in ornamental crabapples.

[0025] To understand the expression characteristics of miR172 family members and their target genes in single and double flower buds of ornamental crabapples, this example screened six McAP2s genes and miR172 family members that may be involved in double flower formation based on phylogenetic trees and performed expression analysis in four varieties of ornamental crabapples. Figure 5As shown, FlameC04AgG010340 is significantly more expressed in the flower buds of double-petal varieties than in single-petal varieties and is negatively correlated with the expression of miR1721 family members. It is speculated that miR1721 family members may negatively regulate the target gene FlameC04AgG010340 to mediate double-petal flower formation in ornamental crabapple. The nucleotide sequence of the FlameC04AgG010340 gene is shown in SEQ ID NO: 1, where M is T or C.

[0026] Example 3 Genes Related to Double Flower Formation in Ornamental Begonia McAP2_3a To investigate whether the FlameC04AgG010340 gene has allelic variation, the present invention cloned and sequenced the gene in the double-petaled cultivars 'Brandy' and 'Corey', as well as the single-petaled cultivars 'Red Barron' and 'Golden Raindrop'. Figure 6 As shown, a single nucleotide polymorphism (SNP) allelic variation was found in the miR172 binding site of the gene in the double-petaled cultivars 'Brandy' and 'Corey', resulting in sequence complementary differences in the miR172 binding site of the gene, while no allelic variation was found in the single-petaled cultivars 'Red Barron' and 'Golden Raindrop'.

[0027] According to the evolutionary analysis of AP2 proteins in Arabidopsis, apple and ornamental crabapple, the results are as follows Figure 7 As shown in Figure 2, it was also found that FlameC04AgG010340 was distantly related to the Arabidopsis AP2-like proteins, but clustered in a small branch with apple MdAP2_3a. Therefore, the ornamental crabapple FlameC04AgG010340 gene was named McAP2_3a, wherein the FlameC04AgG010340 gene present in double-petal varieties is McAP2_3a-D, D refers to double flower, McAP2_3a-D is the double allele, and the nucleotide sequence of the double allele McAP2_3a-D is shown in SEQ ID NO: 2; the FlameC04AgG010340 gene present in single-petal varieties is McAP2_3a-S, S refers to single flower, McAP2_3a-S is the single allele, and the nucleotide sequence of the single allele McAP2_3a-S is shown in SEQ ID NO: NO:3 shown.

[0028] Example 4 Molecular markers associated with double flower formation in ornamental crabapple To further determine whether there are differences in miR172l target sites between McAP2_3a-D and McAP2_3a-S, the psRNATarget online tool was used to predict miRNA target sites. Figure 8As shown in A, a SNP allelic variation was found in the miR172l binding site of the McAP2_3a gene in the double-petal variety, resulting in sequence complementary differences in the miR172l binding site of the double-petal allele McAP2_3a-D.

[0029] Therefore, this example discovered a molecular marker associated with double-petaled ornamental crabapple flowers. The sequence of the molecular marker contained a SNP site, which was located at base 1234 in the nucleotide sequence of the related gene McAP2_3a; the allele of this site was T / C.

[0030] The sequence of the molecular marker is shown in SEQ ID NO: 4, wherein M is T or C; and the SNP site is located at the 112th base in the sequence.

[0031] The dominant allele in double-petal varieties of ornamental crabapples is C, while the dominant allele in single-petal varieties of ornamental crabapples is T.

[0032] Example 5 Preliminary verification of the effect of allelic variation of McAP2_3a gene on its binding to miR1721 The miR172l and McAP2_3a genes were overexpressed in ornamental crabapple (McMIR172l-OE), and only the McAP2_3a gene was overexpressed in ornamental crabapple (McAP2_3a-OE). The expression level of McAP2_3a gene in the flower buds of ornamental crabapple was measured. The results are as follows: Figure 8 As shown in Figure 2, overexpression of miR1721 in the flower buds of ornamental crabapple also significantly decreased the expression of the McAP2_3a gene, indicating that miR1721 family members targeted the McAP2_3a gene in the flower buds.

[0033] Subsequently, dual luciferase assays were performed on the double-petal allele McAP2_3a-D and the single-petal allele McAP2_3a-S. Figure 8 As shown in Figure 3C, the fluorescence intensity of the double-petal allele McAP2_3a-D was significantly higher than that of the single-petal allele McAP2_3a-S, indicating that the efficiency of miR172l family members in cleaving McAP2_3a in double-petal varieties was lower than that in single-petal varieties, that is, the variation of the miR172l binding site in McAP2_3a reduced its binding to miR172l.

[0034] The above experimental results preliminarily verified the targeting relationship between miR172l family members and McAP2_3a gene, and at the same time, SNP allele variation reduced the interaction strength between miR172l and McAP2_3a.

[0035] Example 6 Identification of functional differences in McAP2_3a alleles in double flower formation The double-petal allele McAP2_3a-D and the simple-petal allele McAP2_3a-S were overexpressed in tobacco to obtain transgenic tobacco 35S::McAP2_3a-D and transgenic tobacco 35S::McAP2_3a-S, respectively.

[0036] The phenotype of transgenic tobacco during flowering was observed. Figure 9 As shown in the results, there was no significant difference in the floral organs such as stamens and pistils of all tobacco plants that overexpressed the single-petal gene McAP2_3a-S compared with wild-type tobacco. However, when the double-petal allele McAP2_3a-D was overexpressed in tobacco, it was observed that the floral organs became smaller, the flower color became lighter, the stamens increased, and the stamens became petalized.

[0037] The above results preliminarily revealed that the allelic variation of the miR172l binding site in McAP2_3a leads to the formation of double flowers, suggesting that the allelic variation of McAP2_3a plays a key role in the formation of double flowers.

[0038] Example 7 Primer pairs for amplifying molecular markers associated with double flower formation in ornamental crabapple Based on the sequences of the double-petal allele McAP2_3a-D and the simple-petal allele McAP2_3a-S, a primer pair was designed to amplify a molecular marker related to the formation of double petals in ornamental crabapple. The nucleotide sequences of the primer pair are as follows: 3a-F: 5'-ACGGAGAAGAGTATGGATGTTAACT-3'; 3a-R: 5'-TCAGCTTCTGCAATAGAAATGG-3'.

[0039] Example 8 Detection Method for SNPs Associated with Double Flower Formation in Ornamental Begonia This embodiment provides a method for detecting SNPs associated with double flower formation in ornamental crabapples. The method uses the primer pair in Example 7 for amplification and sequencing comparison to complete the detection.

[0040] Example 9 Kit for Identifying Double-petal Varieties and Single-petal Varieties of Ornamental Begonias This embodiment provides a kit for identifying double-petal varieties / single-petal varieties of ornamental crabapples, which includes the primer pair in Example 7.

[0041] Example 10 Method for Identifying Double-petal Varieties and Single-petal Varieties of Ornamental Begonias This embodiment provides a method for identifying double-petal varieties / single-petal varieties of ornamental crabapples using the kit of Example 9, comprising the following steps: 1) Extract genomic DNA from the sample to be tested and set aside; 2) Using the above genomic DNA as a template, PCR amplification was performed using the primer pair of Example 7 or the kit of Example 9; 3) Sequencing for identification, the identification criteria are as follows: In the sequencing results, when the 112th base is T, there is no SNP variation in the sequence, and the variety to be tested is a single-petal ornamental crabapple; Alternatively, when the 112th base is C, a SNP mutation occurs in the sequence, and the variety to be tested is a double-petaled ornamental crabapple.

[0042] The above method was used to test the 'Red Barron' crabapple, 'Golden Raindrop' crabapple, 'Rose Column' crabapple, 'Red Jewel' crabapple, 'Brandy' crabapple and 'Corey' crabapple. The results are as follows: Figure 10 As shown, 'Red Barron', 'Golden Raindrop', 'Rose Column', and 'Red Jewel' crabapples are single-petal varieties, while 'Brandy' and 'Corey' crabapples are double-petal varieties. This result is consistent with known information, indicating that the SNP molecular markers of the present invention can be used to detect the variety of crabapple petals.

[0043] Although the above embodiments have been described in detail, they are only a part of the embodiments of the present invention, not all of them. People can also obtain other embodiments based on this embodiment without inventiveness, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A gene associated with double flower formation in ornamental crabapple McAP2_3a , characterized in that: The gene McAP2_3a The nucleotide sequence is shown in SEQ ID NO:

1.

2. The related gene according to claim 1 McAP2_3a , characterized in that: When the ornamental crabapple petals are double-petal varieties, in the double-petal varieties, the gene McAP2_3a Double allele McAP2_3a-D, The nucleotide sequence is shown in SEQ ID NO: 2; Alternatively, when the ornamental crabapple petals are single-petal varieties, in the single-petal variety, the gene McAP2_3a Single petal allele McAP2_3a-S , whose nucleotide sequence is shown in SEQ ID NO:

3.

3. A molecular marker associated with double flower formation in ornamental crabapple, characterized by: The sequence of the molecular marker contains a SNP site, which is located at the 1234th base in the nucleotide sequence shown in SEQ ID NO: 1; the allele of this site is T / C.

4. The molecular marker according to claim 3, characterized in that: The sequence of the molecular marker is shown in SEQ ID NO: 4; and the SNP site is located at the 112th base in the sequence; The double-petal variety in the ornamental crabapple has C as the dominant allele, and the single-petal variety in the ornamental crabapple has T as the dominant allele.

5. Use of the molecular marker according to claim 3 in assisted selection of double-petal traits in ornamental crabapples.

6. A primer pair for amplifying the molecular marker according to claim 3, characterized in that: The primer pairs are: 3a-F: 5'-ACGGAGAAGAGTATGGATGTTAACT-3'; 3a-R: 5'-TCAGCTTCTGCAATAGAAATGG-3'.

7. A method for detecting SNPs associated with double flower formation in ornamental crabapple, characterized by: The method uses the primer pair described in claim 6 for amplification and sequencing comparison to complete the detection.

8. Use of the primer pair according to claim 6 in identifying the double-petal trait of ornamental crabapple.

9. A kit for identifying double-petal varieties and single-petal varieties of ornamental crabapples, characterized by: The kit comprises the primer pair according to claim 6.

10. A method for identifying double-petal varieties and single-petal varieties of ornamental crabapples, characterized by: The following steps are involved: 1) Extract genomic DNA from the sample to be tested and set aside; 2) using the genomic DNA as a template, performing PCR amplification using the primer pair of claim 6 or the kit of claim 9; 3) Sequencing for identification, the identification criteria are as follows: When the 112th base is T, the tested variety is a single-petal ornamental crabapple; Alternatively, when the 112th base is C, the variety to be tested is a double-petaled ornamental crabapple.

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