An SNP molecular marker related to the diameter of Oncidium flower stalk and its application

Abstract

The present invention discloses an SNP molecular marker related to the diameter of Oncidium flower stalk and its application. The nucleotide sequence of the molecular marker is shown as SEQ ID NO.1, wherein the SNP locus is at the 401bp of the sequence shown as SEQ ID NO.1, with G / A polymorphism. The present invention performs a genome-wide association analysis on the reduced-representation genome sequencing data of 106 Oncidium germplasm resources and the phenotypic data related to the diameter of Oncidium flower stalk, and locates and screens out the SNP molecular marker Orc.chr05:p56360532 related to the diameter of flower stalk. This molecular marker plays a key role in the regulation of the diameter trait of Oncidium flower stalk, and can be used for map-based cloning and molecular marker-assisted selection, so as to effectively screen out Oncidium varieties with thicker flower stalks 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 diameter of Oncidium flower stalks and its application. Background Art

[0002] Oncidium hybridum belongs to the Orchidaceae family, which contains more than 750 native species. Its flower shape is elegant, just like a young girl with graceful dancing postures, so it is often called the "dancing orchid" or "auspicious orchid" by people. 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 it was introduced and cultivated 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 flower stalks. 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 flower stalks. On the premise of a complete appearance, the better the quality of the flower stalks, the higher the value of the cut flowers.

[0003] The diameter of the flower stalk refers to the cross-sectional diameter of the flower stalk, that is, the widest distance from one side to the other side. The diameter of the flower stalk can vary according to specific circumstances, and the diameter ranges of flower stalks of different plants also vary. The requirements for the diameter of fresh cut flower stalks usually depend on the size and weight of the flowers. Thicker flower stalks can usually provide better support, enabling the flowers to remain upright and not easily fall or break. At the same time, generally, thicker flower stalks are needed to ensure the stability of the flowers; the flower stalk is the channel for transporting water and nutrients to the flowers, and thicker flower stalks can usually provide more water and nutrients, which is very important for maintaining the freshness and lifespan of the flowers; thicker flower stalks help slow down water evaporation and nutrient loss, thereby prolonging the lifespan of the flowers. An appropriate size of the flower stalk diameter can ensure that the flowers can remain fresh and have a long-lasting flowering period after being cut. Therefore, screening for Oncidium varieties with thick flower stalks is beneficial to the development of the Oncidium fresh cut flower market and has important breeding value.

[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 diameter 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 diameter of Oncidium. The nucleotide sequence of this molecular marker is shown in SEQ ID NO.1, where the SNP locus is at the 401bp position of the sequence shown in SEQ ID NO.1, with G / A polymorphism.

[0007] The present invention performs genome-wide association analysis on the reduced-representation genome sequencing data of 106 Oncidium germplasm resources, combined with the phenotypic data of the pedicel diameter of Oncidium, to locate and screen molecular markers related to the pedicel diameter. It is found that the SNP molecular marker Orc.chr05:p56360532 at the position of 56360532bp on chromosome 05 has a high contribution rate to the pedicel diameter of Oncidium and plays a key role in the regulation of the pedicel diameter of Oncidium.

[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-representation genome sequencing on the DNA of the population to identify the SNP genotype information of the population;

[0011] c) Filtering the SNP data quality to screen a high-quality population SNP data set;

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

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

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

[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 thickness of the pedicel diameter of Oncidium.

[0017] B) Breeding of the pedicel diameter of Oncidium.

[0018] The fourth aspect of the present invention provides a method for detecting or identifying the thickness of the pedicel diameter 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 to obtain a target fragment containing the sequence shown in SEQ ID NO.1 and then conduct sequencing, or directly perform reduced-representation genome sequencing on the genomic DNA to determine the base type at the 401bp position of the sequence shown in SEQ ID NO.1 of the Oncidium material to be tested.

[0021] In the above method, if the base type of the Oncidium material to be tested is GG, the material has a thin pedicel, which is thin and weak; if the base type of the Oncidium material to be tested is AA, the pedicel diameter of the material is thick and has strong supporting force.

[0022] Preferably, in the above method, step S2 uses primers shown in SEQ ID NO.2 - 3 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 of 10 μmol / L forward and reverse primers each, 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 the pedicel diameter of Oncidium, specifically: by detecting the base type at the 401bp position of the sequence shown in SEQ ID NO.1 of the Oncidium material, select the Oncidium material with the base type of AA for assisted breeding.

[0024] The sixth aspect of the present invention provides a detection kit for detecting or identifying the thickness of the pedicel diameter of Oncidium during the seedling stage or the non-flowering stage. The kit includes substances for detecting SNP loci of Oncidium materials, and the SNP locus is at the 401bp of the sequence shown in SEQ ID NO.1.

[0025] Preferably, in the above detection kit, primers with nucleotide sequences as shown in SEQ ID NO.2-3 are included.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0027] Through the reduced-representation genome sequencing data of 106 Oncidium germplasms and combined with the phenotypic data of the pedicel diameter of Oncidium, a genome-wide association analysis was carried out. For the first time, the QTL locus affecting the pedicel diameter in Oncidium was located, and the SNP molecular marker Orc.chr05:p56360532 was developed. The contribution rate of this SNP molecular marker to the pedicel diameter trait of Oncidium is 58.65%, that is, it plays a key role in regulating the pedicel diameter of Oncidium. Based on this SNP molecular marker, primers were designed. The analysis results are objective and accurate, not affected by subjective factors, the detection is convenient and fast, the breeding efficiency is high, accelerating the breeding process of thick-pedicel Oncidium varieties, and it has important significance for screening Oncidium cut flowers. Description of the Drawings

[0028] Figure 1 It is a schematic diagram of the distribution results of the pedicel diameter trait of Oncidium in Example 1 of the present invention for two years of data in 2021 and 2022;

[0029] Figure 2 It is a corresponding relationship diagram between different genotypes of the SNP molecular marker Orc.chr05:p56360532 and the pedicel diameter phenotype in Example 2 of the present invention. Detailed Embodiments

[0030] 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.

[0031] In the following embodiments, unless otherwise specified, they are all conventional methods; the reagents and materials, unless otherwise specified, can all be obtained from commercial channels.

[0032] Example 1 Mapping of the major QTL locus for the pedicel diameter of Oncidium

[0033] (1) Phenotypic determination of the pedicel diameter trait of Oncidium.

[0034] In this example, a total of 106 Oncidium germplasm resources were used, which were from Hainan (20), Guangzhou (26), Fujian (29), Taiwan (27), Guangdong (1), Yunnan (1), Guizhou (1), and Shanghai (1) respectively.

[0035] Screen 106 germplasm resources from the Oncidium germplasm resource nursery. Randomly select 5 healthy adult plants from each germplasm, for a total of 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 with a 2000-fold solution of balanced fertilizer once a month. The average annual temperature in the resource nursery is 25℃ - 35℃, the annual relative humidity is 70 - 80%, and the light intensity is 20000 - 30000 LX.

[0036] In 2021 and 2022 for two consecutive years, select Oncidium plants at the full-bloom stage (the number of flowers reaches more than 50%) as sampling materials. Refer to the "Descriptive Specification and Data Standard for Oncidium Germplasm Resources" for trait observation, and use a vernier caliper to measure the pedicel diameter.

[0037] Table 1 Average pedicel diameter of 106 materials in 2021 and 2022

[0038]

[0039]

[0040] Note: PD represents the average pedicel diameter (Pedicel diameter, PD) of Oncidium in 2021 and 2022.

[0041] Based on the above data, draw a schematic diagram of the pedicel diameter distribution results of the association population, as Figure 1 shown. The results show that the pedicel diameter performance distributions in 2021 and 2022 are continuously distributed and tend to a main peak, indicating that the pedicel diameter belongs to a quantitative trait and there are major gene loci. The heritability of the pedicel diameter calculated from the two-year data is 0.76, and the heritability is relatively high, indicating that the pedicel diameter is mainly controlled by genetics.

[0042] (2) Simplified genome sequencing of the Oncidium population.

[0043] Sampling was carried out in the germplasm resource nursery. For each germplasm, about 2 g of young leaves were taken, quickly frozen with liquid nitrogen and sent to the company for sequencing. The sequencing strategy was Illumina HiSeq2500 PE150. Quality control was performed on the sequencing data (raw data), and 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 Oncidium reference genome, the clean data were aligned to the reference genome using the BWA software; the alignment results were sorted using the SAMtools software; the duplicate markers generated during the library preparation process were processed using the Picard Tools software; and 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]".

[0044] (3) Genome-wide association analysis.

[0045] The plink software was used to convert the format of the vcf file; the EMMAX software was used to generate the Balding-Nichols kinship matrix, and the command was "emmax-kin-intel64 -v -d 10 -o [out_prefix] [tped_prefix]"; the command for performing association analysis was "emmax-intel64 -v -d 10 -t [tped_prefix] -p [trait_file] -k [kin_file] -o [out_prefix]". The P value of each locus in Oncidium was obtained from the association analysis results. When the P value was less than 0.5 / 66731 = 7.49277E-06, the SNP was a significant SNP. The materials were grouped according to the different allele types of the SNP in the population, and an analysis of variance was performed. The percentage of the ratio of the between-group variance to the total variance was the contribution rate of the SNP.

[0046] Through analysis, the SNP was chr05_56360532 (G / A), and the contribution rate of this QTL to the Oncidium pedicel diameter trait was 58.65% (the materials were grouped according to the different allele types of the SNP, and a one-way analysis of variance was performed. The percentage of the between-group variance divided by the total variance was the contribution rate).

[0047] Example 2 Development of SNP Molecular Markers for the Pedicel Diameter of Oncidium

[0048] According to the identified QTL and SNP for the pedicel diameter of Oncidium, SNP molecular markers were developed. The 400 bp sequences before and after each SNP were extracted as the characteristic sequences of the SNP molecular marker Orc.chr05:p56360532 (as shown in Sequence Listing SEQ ID NO.1, where the 401st bp is the SNP locus with a G / A mutation). Primers for the SNP molecular marker Orc.chr05:p56360532 were designed as follows:

[0049] Forward primer: 5’-TGAACTAGAAACCCTTGAACGT-3’ (SEQ ID NO.2);

[0050] Reverse primer: 5’-GATTTTACATTTCGCTGA-3’ (SEQ ID NO.3).

[0051] Through conventional PCR amplification and sequencing detection, the specific PCR amplification system was as follows: 50 ng / μL template DNA / 1 μL, 2×PCR Master Mix 5 μL, 0.5 μL each of 10 μmol / L forward and reverse primers, and 3 μL of ddH2O, for a total of 10 μL.

[0052] The detection results showed that the bases of the SNP molecular marker were divided into two types. When the base type of Orc.chr05:p56360532 was AA, the average pedicel diameters of the materials in 2021 and 2022 were 4.79 and 4.57 respectively, which were thick-pedicel Oncidium; when the base type was GG, the average pedicel diameters of the materials in 2021 and 2022 were 2.70 and 2.68 respectively, which were thin-pedicel Oncidium, and the pedicels were soft and easy to bend or deform.

[0053] The relationship between the pedicel diameter phenotypes corresponding to different genotypes was as Figure 2 shown.

[0054] The results showed that there were significant differences in the pedicel diameter phenotypes corresponding to different genotype types of the SNP molecular marker Orc.chr05:p56360532. Therefore, the thickness of the pedicel diameter of the Oncidium to be detected can be quickly and simply evaluated by identifying the type of this SNP molecular marker, so as to quickly identify the thickness of the pedicel diameter of the material.

[0055] In addition to amplifying the sequence using the primers provided in this example, the material can also be subjected to reduced-representation genome sequencing. According to the method in Example 1, SNP genotyping is performed on the SNP molecular marker Orc.chr05:p56360532 disclosed by the present invention to determine the genotype type of the SNP, and then the thickness of the pedicel diameter of the material can be rapidly evaluated.

[0056] Example 3 Application of SNP Molecular Marker Orc.chr05:p56360532 in the Identification of Oncidium Pedicel Diameter

[0057] Extract the DNA of young leaves from three previously known thick-pedicel materials, ONZ 9007, Volcano Queen (cut flower), and Nancy, 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 diameters 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 thin-pedicel materials, White Dream Fragrance, Red Ladybug, and Red Child, 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 diameters of the three materials according to the method in Example 1.

[0059] The SNP genotypes of the above six materials at the 56360532bp position on chromosome 05 and the average pedicel diameter are shown in Table 2.

[0060] Table 2 Genotypes at the Orc.chr05:p56360532 Locus and Their Pedicel Diameter Traits in Materials with Different Pedicel Diameter Traits

[0061]

[0062] Note: PD represents the average pedicel diameter (unit: mm).

[0063] The results show that for the previously known thick-pedicel diameter materials ONZ 9007, Volcano Queen (cut flower), and Nancy, their SNP genotypes at the 56360532bp position on chromosome 05 (i.e., the 401bp of the sequence shown in SEQ ID NO.1) are all AA, and the phenotypic values of the pedicel diameters are 4.80, 5.01, and 4.10 respectively, which are consistent with the thick-pedicel diameter traits corresponding to the AA genotype of the SNP molecular marker in Example 2. Similarly, for the previously known thin-pedicel White Dream Fragrance, Red Ladybug, and Red Child, their SNP genotypes at the 56360532bp position on chromosome 05 (i.e., the 401bp of the sequence shown in SEQ ID NO.1) are all GG, and the phenotypes of the pedicel diameter traits are 1.62, 1.82, and 2.43 respectively, which are consistent with the thin-pedicel diameter traits corresponding to the GG genotype of the SNP molecular marker in Example 2.

[0064] In summary, the SNP molecular marker Orc.chr05:p56360532 provided by the present invention plays a key role in regulating the pedicel diameter of Oncidium. Based on this SNP molecular marker, the pedicel diameters of different Oncidium materials can be effectively identified or predicted, so as to screen specific varieties and retain Oncidium materials with different pedicel thicknesses as needed, laying a solid foundation for subsequent breeding work.

[0065] The above is the preferred embodiment of the present invention, and the scope of the rights of the present invention cannot be limited thereby. 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 principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An SNP molecular marker related to the diameter of Oncidium flower stalk, characterized in that, The nucleotide sequence of the molecular marker is shown in SEQ ID NO.1, where the SNP locus is at the 401bp of the sequence shown in SEQ ID NO.1, with G / A polymorphism.

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

3. A method for detecting or identifying the pedicel diameter 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 genomic sequencing on the genomic DNA to determine the base type at the 401bp 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 GG, the material has a thin pedicel; if the base type of the Oncidium material to be tested is AA, the material has a thick pedicel.

5. A detection kit for detecting or identifying the diameter of the pedicel of Oncidium in the seedling stage or non-flowering stage, characterized in that, It includes substances for detecting the SNP locus according to claim 1.

6. The application of the SNP molecular marker according to claim 1 in molecular marker-assisted breeding of the thick pedicel diameter of Oncidium.

7. A molecular marker-assisted breeding method for the diameter of Oncidium thick flower stalks, characterized in that, By detecting the base type at the 401bp of the sequence shown in SEQ ID NO.1 of the Oncidium material, select the Oncidium material with the base type of AA for assisted breeding.

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