An SNP molecular marker related to the pedicel hardness trait of Oncidium and its application

Abstract

The present invention discloses an SNP molecular marker related to the pedicel hardness trait of Oncidium and its application. The nucleotide sequence of the molecular marker is shown as SEQ ID NO.1, wherein the SNP locus is at the 501bp of the sequence shown as SEQ ID NO.1, with A / G polymorphism. Through the simplified genome sequencing data of 106 Oncidium germplasm resources and combined with the phenotypic data related to the pedicel hardness of Oncidium, the present invention conducts a genome-wide association analysis, locates and screens out an SNP molecular marker related to the pedicel hardness. This molecular marker plays a key role in the regulation of the pedicel hardness trait of Oncidium, can be used for map-based cloning and molecular marker-assisted selection, so as to effectively screen out Oncidium varieties with stronger pedicel hardness at the seedling stage or non-flowering stage, accelerate the breeding process of Oncidium cut flower varieties, and is of great significance to the protection of germplasm resources and the breeding of new varieties.

Description

Technical Field

[0001] The present invention belongs to the technical fields of genetic breeding and molecular biology, and particularly relates to an SNP molecular marker related to the pedicel hardness trait of Oncidium and its application. Background Art

[0002] Oncidium hybridum belongs to the Orchidaceae family and contains more than 750 native species. Its flower shape is elegant, just like a young girl with a light dancing posture, so it is often called the "dancing orchid" or "auspicious orchid", and it occupies an important position in the Orchidaceae family. The origin of Oncidium hybridum is in regions such as Brazil, Mexico, and Guyana in Central and South America, mainly distributed in tropical regions. As one of the most important cut flower varieties in the world, since its introduction and cultivation in China in the 1990s, the cut flower industry across the country has gradually expanded, and the selection of cultivated varieties is related to the yield and quality of cut flowers. The grade of Oncidium cut flowers depends on the quality of the pedicel. In the "Quality Grade Standard for Oncidium Cut Flower Products" in Hainan Province, it is clearly required that fresh cut flowers need to have a complete appearance, fresh fragrance, and tough pedicels. On the premise of a complete appearance, the better the quality of the pedicel, the higher the value of the cut flower.

[0003] Pedicel hardness refers to the hardness degree of the stem or pedicel of a flower, that is, its resistance to bending or compression under mechanical action. Pedicel hardness is closely related to fresh cut flowers and has an important impact on the quality and lifespan of fresh cut flowers. Pedicel hardness determines the supporting ability of fresh cut flowers after being inserted into a vase. A harder pedicel can effectively support the flower, keep the flower upright and stable, and enable the flower to show the best appearance effect. In contrast, a softer pedicel is prone to bending, drooping, or breaking, resulting in the flower being unable to show the ideal shape and posture. A harder pedicel usually has larger ducts and better water and nutrient conduction abilities, which can promote the absorption of water and nutrients and extend the lifespan of fresh cut flowers. On the contrary, a soft pedicel may cause the absorption of water and nutrients to be blocked, shortening the lifespan of fresh cut flowers. A pedicel with a higher hardness is more resistant to external pressure and extrusion, which helps to maintain the integrity and freshness of the flower. In addition, a harder pedicel has stronger wind resistance and can reduce the shaking and damage of the flower in the wind. Therefore, pedicel hardness is an important evaluation index when selecting and processing Oncidium fresh cut flowers.

[0004] Genome-wide association studies (GWAS) is a commonly used genetic research method for finding associations between genes and specific traits. Single Nucleotide Polymorphisms (SNPs) are common forms of variation in genetics, referring to positions in the genome where a single nucleotide has mutated. There is a close relationship between GWAS and SNPs. In GWAS, a large number of SNPs are detected to find associations with traits of interest. By comparing the differences between individuals carrying different SNP variations, the degree of association between certain SNPs and specific traits can be determined. At the same time, SNPs are also the most commonly used markers in genome-wide association studies. Summary of the Invention

[0005] In view of this, the present invention aims to develop SNP markers associated with the pedicel hardness trait of Oncidium through GWAS to promote the breeding of Oncidium cut flower varieties.

[0006] The first aspect of the present invention provides an SNP molecular marker related to the pedicel hardness trait of Oncidium. The nucleotide sequence of this molecular marker is shown in SEQ ID NO.1, where the SNP locus is at the 501bp position of the sequence shown in SEQ ID NO.1, with A / G polymorphism.

[0007] The present invention performs genome-wide association analysis on the reduced genome sequencing data of 106 Oncidium germplasm resources, combined with the phenotypic data of Oncidium pedicel hardness, locates and screens out molecular markers related to pedicel hardness, and finds that the SNP molecular marker Orc.scaffolds:p122266884 at the 122266884bp position of the scaffolds has a high contribution rate to Oncidium pedicel hardness and plays a key role in the regulation of Oncidium pedicel hardness.

[0008] The second aspect of the present invention provides a method for developing and obtaining the above SNP molecular marker, including the following steps:

[0009] a) Using Oncidium materials with genetic differences from different regions to form an association population;

[0010] b) Extracting the total DNA of the leaves of each material in the association population, and then performing reduced genome sequencing on the DNA of the population to identify the SNP genotype information of the population;

[0011] c) Filtering and screening high-quality population SNP data sets through SNP data quality filtering;

[0012] d) Investigating the phenotypic data of the pedicel hardness of all Oncidium materials in the association population;

[0013] e) Combine the genotype and pedicel hardness phenotype data to conduct a genome-wide association study, identify the QTL loci significantly associated with pedicel hardness, and obtain SNP molecular markers related to the pedicel hardness trait of Oncidium.

[0014] In the above method, in step d), the pedicel hardness phenotype data for at least two consecutive years should be investigated.

[0015] The third aspect of the present invention provides the application of the SNP molecular markers of the present invention, which is A) or B):

[0016] A) Detect or identify the strength of the pedicel hardness of Oncidium.

[0017] B) Breeding of Oncidium with strong pedicel hardness.

[0018] The fourth aspect of the present invention provides a method for detecting or identifying the strength of the pedicel hardness of Oncidium at the seedling stage or non-flowering stage, including the following steps:

[0019] S1. Extract the genomic DNA of the Oncidium material to be tested.

[0020] S2. Using the genomic DNA described in S1 as a template, amplify the target fragment containing the sequence shown in SEQ ID NO.1 and then perform sequencing, or directly perform reduced-representation genome sequencing on the genomic DNA to determine the base type at the 501bp position of the sequence shown in SEQ ID NO.1 of the Oncidium material to be tested.

[0021] In the above method, when the base type of the Oncidium material to be tested is AA, the pedicel of this material is soft, easy to bend but not easy to break; when the base type of the Oncidium material to be tested is AG, this material has a medium-hard pedicel with a certain degree of elasticity and softness; when the base type of the Oncidium material to be tested is GG, the pedicel of this material is hard, difficult to bend or deform, and has strong supporting force.

[0022] Preferably, in the above method, in step S2, primers shown in SEQ ID NO.2 - 3 are used for amplification. More preferably, the PCR amplification system in step S2 is: 1 μL of 50 ng / μL template DNA, 5 μL of 2×PCR Master Mix, 0.5 μL each of 10 μmol / L forward and reverse primers, and 3 μL of ddH2O, for a total of 10 μL.

[0023] The fifth aspect of the present invention provides a molecular marker-assisted breeding method for Oncidium with strong pedicel hardness, specifically: by detecting the base type at the 501bp position of the sequence shown in SEQ ID NO.1 of the Oncidium material, select the Oncidium material with the base type of GG for assisted breeding.

[0024] The research of the present invention shows that when the base type of the Oncidium material to be tested is AA, it is the variety with the strongest pedicel hardness; when the base type of the Oncidium material to be tested is AG, the pedicel hardness of this Oncidium is weaker; when the base type of the Oncidium material to be tested is GG, the pedicel hardness of this Oncidium is the weakest. Therefore, by using molecular marker-assisted selection to select Oncidium varieties with stronger pedicel hardness, the breeding process of Oncidium cut flower varieties can be accelerated.

[0025] The sixth aspect of the present invention provides a detection kit for detecting or identifying the pedicel hardness of Oncidium during the seedling stage or the non-flowering period. The kit includes substances for detecting the SNP locus of the Oncidium material, and the SNP locus is at the 501bp of the sequence shown in SEQ ID NO.1.

[0026] Preferably, in the above detection kit, primers with nucleotide sequences as shown in SEQ ID NOs. 2-3 are included.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0028] Through the reduced-representation genome sequencing data of 106 Oncidium germplasms and combined with the phenotypic data of the pedicel hardness of Oncidium, the present invention conducts a genome-wide association analysis, and for the first time locates the QTL locus affecting the pedicel hardness in Oncidium, and develops a SNP molecular marker Orc.scaffolds:p122266884. The contribution rate of this SNP molecular marker to the pedicel hardness of Oncidium is 52.03%, that is, the SNP molecular marker plays a key regulatory role in the trait of the pedicel hardness of Oncidium. Based on this SNP molecular marker, primers are designed, and the analysis results are objective and accurate, not affected by subjective factors, the detection is convenient and fast, the breeding efficiency is high, and the breeding process of Oncidium varieties with strong pedicel hardness is accelerated, which is of great significance for screening Oncidium cut flowers. Description of the Drawings

[0029] Figure 1 It is a schematic diagram of the distribution results of the pedicel hardness of Oncidium in Example 1 of the present invention for two years of data in 2021 and 2022;

[0030] Figure 2 It is a corresponding schematic diagram of the pedicel hardness trait of Oncidium in Example 2 of the present invention for the phenotypic data of the pedicel hardness in 2021 and 2022 and different genotypes of the SNP molecular marker Orc.scaffolds:p122266884. Detailed Embodiments

[0031] Next, the technical solutions of the present invention will be clearly and completely described in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0032] In the following examples, unless otherwise specified, all are conventional methods; unless otherwise specified, the reagents and materials can be obtained from commercial sources.

[0033] Example 1 Mapping of Major QTL Loci for the Hardness of Oncidium Pedicels

[0034] (1) Phenotypic determination of the hardness trait of Oncidium pedicels.

[0035] A total of 106 Oncidium germplasm resources used in this example are from Hainan (20), Guangzhou (26), Fujian (29), Taiwan (27), Guangxi (1), Yunnan (1), Guizhou (1), and Shanghai (1) respectively.

[0036] Select 106 germplasm resources from the Oncidium germplasm resource nursery. Randomly select 5 healthy adult plants from each germplasm, totaling 530 plants, and plant and manage them under the same environmental conditions. Water once every 2 days in summer and once every 3 days in winter. Spray high-nitrogen fertilizer once a week during the vegetative growth period, and use balanced fertilizer after 5 - 6 applications of nitrogen fertilizer to prevent excessive growth. During the reproductive growth period, irrigate the roots once a month with a 2000-fold solution of balanced fertilizer. The annual average temperature in the resource nursery is 25℃ - 35℃, the annual relative humidity is 70 - 80%, and the light intensity is 20000 - 30000 LX.

[0037] In 2021 and 2022, for two consecutive years, select Oncidium plants at the full-bloom stage (with the number of flowers reaching more than 50%) as sampling materials. For the trait observation, refer to the "Descriptive Specification and Data Standard for Oncidium Germplasm Resources". The measurement of pedicel hardness refers to the method of testing the stem quality in the literature on the evaluation of maize lodging resistance, that is, tilting the stem at a certain angle with the ground by applying tensile force from different angles, and using the tensile force value measured at this time to evaluate the lodging resistance of the stem. The specific method is as follows: Use a digital Weidu tensile force tester to hook at the center of the length of the Oncidium pedicel, and force it to tilt 45 degrees from the vertical direction, and read the value at this time and take the average.

[0038] Table 1 Average values of pedicel hardness of 106 materials in 2021 and 2022

[0039]

[0040]

[0041] Note: PH represents the average value of the pedicel hardness (Pedicel hardness, PH) of Oncidium in 2021 and 2022.

[0042] Based on the above data, draw a schematic diagram of the pedicel hardness distribution results of the association population, as Figure 1As shown, the results indicate that the distribution of pedicel hardness performance in 2021 and 2022 shows a continuous distribution, skewed towards a main peak, indicating that pedicel hardness is a quantitative trait and there are major gene loci. The heritability of pedicel hardness calculated from the two-year data is 0.70, with a relatively high heritability, indicating that pedicel hardness is mainly controlled by genetics.

[0043] (2) Simplified genome sequencing of Oncidium populations.

[0044] Samples were taken in the germplasm resource nursery. For each germplasm, approximately 2 g of young leaves were collected, quickly frozen in liquid nitrogen, and then sent to the company for sequencing. The sequencing strategy was Illumina HiSeq2500 PE150. Quality control was performed on the sequencing raw data. The filtering criteria were as follows: ① Filter sequences containing adapters; ② Filter a pair of sequences with an N content exceeding 10% in single-end sequences; ③ Filter a pair of sequences with a low-quality (Q≤5) base number exceeding 50%. After filtering adapter sequences, inaccurate sequences, and low-quality sequences, clean data were obtained for the next sequence alignment. After constructing an index file for the "Little Bee" reference genome, the clean data were aligned to the reference genome using the BWA software; the alignment results were sorted using the SAMtools software; duplicate markers generated during the library preparation process were processed using the Picard Tools software; the GATK software was used to analyze population variation information. The VCFtools software was used to further screen and filter SNPs, and the command was "vcftools --vcf [vcf_file] --max-missing 0.5 --maf 0.05 --mac 3 --minQ 30 --minDP 3 --min-alleles 2 --max-alleles 2 --recode --recode-INFO-all --out [out_prefix]".

[0045] (3) Genome-wide association analysis.

[0046] Use the plink software to convert the format of the vcf file; use the EMMAX software to generate the Balding-Nichols kinship matrix, and the command is "emmax-kin-intel64-v-d 10-o[out_prefix][tped_prefix]"; the command for association analysis is "emmax-intel64-v-d 10-t[tped_prefix]-p[trait_file]-k[kin_file]-o[out_prefix]". The P value of each locus of Oncidium was obtained from the association analysis results. When the P value is less than 0.5 / 66731 = 7.49277E-06, the SNP is a significant SNP. Group the materials according to the different allelic types of the SNP in the population, and perform an analysis of variance. The percentage of the ratio of the between-group variance to the total variance is the contribution rate of this SNP locus.

[0047] Through analysis, the SNP is scaffolds_122266884(A / G), and the contribution rate of this QTL to the pedicel hardness trait of Oncidium is 52.03% (group the materials according to the different allelic types of the SNP and perform a one-way analysis of variance. The percentage of the between-group variance divided by the total variance is the contribution rate).

[0048] Example 2 Development of SNP molecular markers for the pedicel hardness trait of Oncidium

[0049] Develop SNP molecular markers based on the pedicel hardness QTL and SNP identified in Example 1. Extract 500bp sequences before and after this SNP as the characteristic sequence of the SNP molecular marker Orc.scaffolds:p122266884 (as shown in Sequence Listing SEQ ID NO.1, where the 501bp is the SNP locus and there is an A / G mutation). Design primers for this SNP molecular marker as follows:

[0050] Forward primer: 5’-CTGCAAATGGATTTGTAGGATA-3’ (SEQ ID NO.2);

[0051] Reverse primer: 5’-TACCCTCAATGACCTCCCTAT-3’ (SEQ ID NO.3).

[0052] Detect by conventional PCR amplification and sequencing. The PCR amplification system is 10 μL, specifically including: 1 μL of 50 ng / μL leaf DNA, 5 μL of 2×PCR Master Mix, 0.5 μL of 10 μmol / L forward and reverse primers each, and 3 μL of ddH2O.

[0053] The bases of SNP molecular markers are divided into three types. When the base type of Orc.scaffolds:p122266884 is GG, the average pedicel hardness of the materials in 2021 and 2022 is 0.56 and 0.55 respectively, which is strong pedicel hardness. The pedicel is difficult to bend or deform and has strong supporting force. When the base type is AG, the average pedicel hardness of the materials in 2021 and 2022 is 0.48, which is medium-hard pedicel with certain elasticity and softness. When the base type is GG, the average pedicel hardness of the materials in 2021 and 2022 is 0.33, that is, the pedicel is soft and easy to bend but not easy to break. The schematic diagram of the pedicel hardness phenotypes corresponding to different genotypes is as Figure 2 shown.

[0054] The results show that there are significant differences in the pedicel hardness traits phenotypes corresponding to different genotype types of the SNP molecular marker Orc.scaffolds:p122266884. Therefore, the strength of the pedicel hardness trait of the to-be-detected Oncidium can be quickly and simply evaluated by identifying the type of this SNP molecular marker, so as to quickly identify the pedicel hardness strength ability of the material.

[0055] In addition to using the primer amplification sequence in this example, the material can also be subjected to reduced-representation genome sequencing, and the SNP genotype typing of the SNP molecular marker Orc.scaffolds:p122266884 provided by the present invention can be carried out according to the method in Example 1 to determine the genotype type of the SNP, and then quickly evaluate the strength of the pedicel hardness of the material.

[0056] Example 3 Application of SNP molecular marker Orc.scaffolds:p122266884 in the identification of Oncidium pedicel hardness trait

[0057] Extract the DNA of young leaves from three previously known materials with strong-hardness pedicels, namely Oncidium (305), Lemon Yellow (leaf art) and HOM-6, and send them to a sequencing company for reduced-representation genome sequencing. Identify the SNP genotypes of the three materials respectively, and measure the pedicel hardness of the three materials according to the method in Example 1.

[0058] At the same time, extract the DNA of young leaves from three previously known materials with medium-hardness pedicels, namely ONZ 9004, Golden No. 2 and Lemon Yellow (SH), and send them to a sequencing company for reduced-representation genome sequencing. Identify the SNP genotypes of the three materials respectively, and identify the pedicel hardness traits of the three materials according to the method in Example 1.

[0059] Meanwhile, DNA of young leaves was extracted from three materials, namely Luomanxiang, Xiaotaohong, and Lagerstroemia indica, which were known to have soft flower stalks in the early stage, and sent to a sequencing company for reduced-representation genome sequencing. The SNP genotypes of the three materials were identified respectively, and the flower stalk hardness of the three materials was measured according to the method of Example 1.

[0060] The SNP genotypes at the position of 122266884bp on scaffolds and the average values of the flower stalk hardness traits of the above nine materials are shown in Table 2.

[0061] Table 2 Genotypes at the Orc.scaffolds:p122266884 locus and their flower stalk hardness in materials with different flower stalk hardness traits

[0062]

[0063]

[0064] Note: PH represents the average value of flower stalk hardness.

[0065] The results showed that for the known materials with strong flower stalk hardness, namely Oncidium (305), Lemon Yellow (leaf art), and HOM-64, their SNP genotypes at the position of 122266884bp on scaffolds (i.e., the 501bp of the sequence shown in SEQ ID NO.1) were all GG, and the phenotypic values of flower stalk hardness were 0.68, 0.61, and 0.60 respectively, which were consistent with the strong flower stalk hardness traits corresponding to the GG genotype of the SNP molecular marker in Example 2. Similarly, for the known materials with medium-hard flower stalks, namely ONZ 9004, Golden No. 2, and Lemon Yellow (SH), their SNP genotypes at the position of 122266884bp on scaffolds (i.e., the 501bp of the sequence shown in SEQ ID NO.1) were all AG, and the phenotypes of the flower stalk hardness traits were 0.54, 0.47, and 0.46 respectively, which were consistent with the weaker flower stalk hardness traits corresponding to the AG genotype of the SNP molecular marker in Example 2. Similarly, for the known materials with soft flower stalks, namely Luomanxiang, Xiaotaohong, and Lagerstroemia indica, their SNP genotypes at the position of 122266884bp on scaffolds (i.e., the 501bp of the sequence shown in SEQ ID NO.1) were all AA, and the phenotypes of the flower stalk hardness traits were 0.25, 0.34, and 0.37 respectively, which were consistent with the weak flower stalk hardness traits corresponding to the AA genotype of the SNP molecular marker in Example 2.

[0066] In summary, the SNP molecular marker Orc.scaffolds:p122266884 of the present invention plays a key role in regulating the pedicel hardness of Oncidium. According to the genotype type of the SNP molecular marker Orc.scaffolds:p122266884, the strength of the pedicel hardness of Oncidium materials can be effectively identified or predicted, laying a solid foundation for subsequent breeding work.

[0067] The above is the preferred embodiment of the present invention and should not be used to limit the scope of the rights of the present invention. It should be pointed out that for those of ordinary skill in the art of this technology, any modifications, equivalent substitutions, and improvements made within the spirit and principle of the present invention should be included within the protection scope of the present invention.

Claims

1. An SNP molecular marker related to the pedicel hardness trait of Oncidium, characterized in that, The nucleotide sequence of the molecular marker is shown in SEQ ID NO.1, where the SNP site is at the 501bp of the sequence shown in SEQ ID NO.1, with A / G polymorphism.

2. The application of the SNP molecular marker according to claim 1 in detecting or identifying the pedicel hardness of Oncidium.

3. A method for detecting or identifying the pedicel hardness of Oncidium at the seedling stage or non-flowering stage, characterized by comprising the following steps: S1. Extract the genomic DNA of the Oncidium material to be tested; S2. Using the genomic DNA described in S1 as a template, amplify the target fragment containing the sequence shown in SEQ ID NO.1 and then perform sequencing, or directly perform reduced-representation genome sequencing on the genomic DNA to determine the base type at the 501bp of the sequence shown in SEQ ID NO.1 of the Oncidium material to be tested.

4. The method according to claim 3, wherein If the base type of the Oncidium material to be tested is AA, the pedicel of this material is soft, easy to bend but not easy to break; if the base type of the Oncidium material to be tested is AG, this material has a medium-hard pedicel; if the base type of the Oncidium material to be tested is GG, the pedicel of this material is hard, difficult to bend or deform, and has strong supporting force.

5. A detection kit for detecting or identifying the pedicel hardness of Oncidium flowers at the seedling stage or non-flowering stage, characterized in that, It includes substances for detecting the SNP site described in claim 1.

6. The application of the SNP molecular marker according to claim 1 in molecular marker-assisted breeding for strong pedicel hardness of Oncidium.

7. A molecular marker-assisted breeding method for improving the pedicel hardness of Oncidium, characterized in that, By detecting the base type at the 501bp of the sequence shown in SEQ ID NO.1 of the Oncidium material, select the Oncidium material with the base type of GG for assisted breeding.

Images