CN117051155A - SNP molecular marker related to hardness traits of oncidium stems and application thereof - Google Patents
SNP molecular marker related to hardness traits of oncidium stems and application thereof Download PDFInfo
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Abstract
The invention discloses an SNP molecular marker related to the hardness property of a broccoli stem and application thereof, wherein the nucleotide sequence of the molecular marker is shown as SEQ ID NO.1, and the SNP locus is 501bp of the sequence shown as SEQ ID NO.1 and has an A/G polymorphism. According to the invention, through 106 parts of the simplified genome sequencing data of the oncidium germplasm resources, the full genome association analysis is carried out by combining with the phenotype data related to the oncidium flower stalk hardness, and a SNP molecular marker related to the flower stalk hardness is selected in a positioning way, the molecular marker plays a key role in regulating and controlling the oncidium flower stalk hardness character, and can be used as map cloning and molecular marker auxiliary selection, so that oncidium varieties with stronger flower stalk hardness can be effectively screened in a seedling stage or a florescence-free stage, the fresh cut flower variety breeding process of oncidium is accelerated, and the method has important significance for germplasm resource protection and new variety breeding.
Description
Technical Field
The invention belongs to the technical fields of genetic breeding and molecular biology, and particularly relates to an SNP molecular marker related to the hardness trait of a broccoli stem and application thereof.
Background
The oncidium (Oncidium hybridum) belongs to the orchid family, contains over 750 primordial species, and is graceful in flower morphology, just like a light dancing girl, and is therefore also often called "dancing orchid" or "Ji Xianglan", which takes on an important role in the orchid family. The original places of the oncidium are Brazil, mexico and Guiana in the south America, and are mainly distributed in tropical areas, and are used as one of the most important cut flower varieties in the world, after introduction and cultivation in China from the 90 th century, the cut flower industry in the whole country is gradually expanded, and the selection of the cultivated varieties is related to the yield and quality of cut flowers. The grade of the cut flowers of the oncidium is dependent on the quality of the pedicel, and in the product quality grade standard of the cut flowers of the oncidium of the Hainan province, the fresh cut flowers are clearly required to have complete appearance, fresh aroma and tough pedicel, and on the premise of complete appearance, the better the quality of the pedicel is, the higher the cut flower value is.
The stem stiffness refers to the degree of stiffness of the stem or stem of the flower, i.e. its resistance to bending or compression under mechanical action. The hardness of the pedicel has close relation with the fresh cut flowers, and has important influence on the quality and the service life of the fresh cut flowers. The stem hardness determines the support capacity of the cut flowers after flower arrangement. The harder pedicel can effectively support the flowers, keep the flowers upright and stable, and enable the flowers to show the best appearance effect. In contrast, weaker pedicel tends to bend, sag or break, resulting in flowers that do not exhibit the desired morphology and posture. The harder pedicel usually has larger guide pipe and better water and nutrient guiding capability, can promote the absorption of moisture and nutrient, and prolongs the service life of the fresh cut flowers. In contrast, weak pedicel may cause resistance to absorption of moisture and nutrients, shortening the life of the cut flowers. The flower stalks with higher hardness can resist external pressure and extrusion, and are favorable for keeping the integrity and vividness of flowers. In addition, the harder pedicel has more wind resistance, and can reduce the shaking and damage of flowers in wind. Thus, the stem hardness of the oncidium is an important evaluation index when selecting and processing fresh cut flowers.
Genome-wide association analysis (Genome-wide association studies, GWAS) is a commonly used genetic research method to find associations between genes and specific traits. Single nucleotide polymorphisms (Single Nucleotide Polymorphisms, SNPs) are a common form of variation in genetics, which refers to the location in the genome where a single nucleotide is mutated. There is a close relationship between GWAS and SNPs, where 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 of certain SNPs with specific traits can be determined, and SNPs are also the most commonly used markers in genome association studies.
Disclosure of Invention
In view of this, the present invention aims to develop SNP markers associated with the hardness trait of the flower stems of the oncidium by GWAS to promote the breeding of fresh cut flower varieties of oncidium.
The invention provides a SNP molecular marker related to the hardness property of the aragonite orchid stems, the nucleotide sequence of the molecular marker is shown as SEQ ID NO.1, wherein the SNP locus is the 501bp of the sequence shown as SEQ ID NO.1, and the SNP locus has an A/G polymorphism.
According to the invention, through 106 parts of simplified genome sequencing data of the oncidium germplasm resources, full genome association analysis is performed by combining phenotype data of oncidium flower stalk hardness, molecular markers related to the flower stalk hardness are selected in a positioning mode, and researches find that SNP molecular markers Orc.scanfords at 122266884bp positions of scanfords have high contribution rate of p122266884 to the oncidium flower stalk hardness, and play a key role in regulating and controlling the oncidium flower stalk hardness.
The second aspect of the present invention provides a method for developing and obtaining the above SNP molecular marker, comprising the steps of:
a) Constructing a related population using the oncidium material having genetic differences from different regions;
b) Extracting total DNA of leaves of each material of the related population, and then using the DNA of the population to carry out simplified genome sequencing to identify SNP genotype information of the population;
c) Screening high-quality population SNP data sets by filtering SNP data quality;
d) Investigating the pedicel hardness phenotype data of all the oncidium materials in the associated population;
e) Combining genotype and pedicel hardness phenotype data, carrying out whole genome association analysis, and identifying QTL sites with obvious correlation of pedicel hardness to obtain SNP molecular markers with correlation of the pedicel hardness characters of the oncidium;
in the above method, step d) is performed by examining the stem hardness phenotype data for at least two consecutive years.
In a third aspect, the invention provides the use of the SNP molecular markers of the invention, which are A) or B):
a) Detecting or identifying the hardness of the stems of the oncidium;
b) And (5) breeding the strong pedicel hardness of the oncidium.
The fourth aspect of the invention provides a method for detecting or identifying the hardness of the broccoli stems in the seedling stage or the florescence-free stage, which comprises the following steps:
s1, extracting genome DNA of a to-be-detected oncidium material;
s2, amplifying the genome DNA of the S1 to obtain a target fragment containing a sequence shown in SEQ ID NO.1, and then sequencing, or directly performing simplified genome sequencing by using the genome DNA to determine the base type of the to-be-detected oncidium material at the 501bp position of the sequence shown in SEQ ID NO. 1.
In the method, if the base type of the oncidium material to be detected is AA, the pedicel of the material is soft, flexible and not easy to break; if the base type of the to-be-detected oncidium material is AG, the material is a medium-hardness pedicel and has certain elasticity and softness; if the base type of the to-be-detected oncidium material is GG, the pedicel of the material is hard and difficult to bend or deform, and has strong supporting force.
Preferably, in the above method, step S2 is performed using primers shown as SEQ ID NOS.2 to 3. More preferably, the PCR amplification system in step S2 is: 50 ng/. Mu.L template DNA 1. Mu.L, 2 XPCR Master Mix 5. Mu.L, 10. Mu. Mol/L forward and reverse primers 0.5. Mu.L each, ddH 2 O3. Mu.L, 10. Mu.L in total.
The fifth aspect of the invention provides a molecular marker assisted breeding method for strong pedicel hardness of oncidium, which specifically comprises the following steps: by detecting the base type of the oncidium material at the 501bp position of the sequence shown in SEQ ID NO.1, the oncidium material with the base type GG is selected for auxiliary breeding.
The research of the invention shows that when the base type of the to-be-detected oncidium material is AA, the to-be-detected oncidium material is the variety with the strongest pedicel hardness; when the base type of the to-be-detected oncidium material is AG, the hardness of the oncidium stems is weaker; when the base type of the to-be-detected oncidium material is GG, the hardness of the oncidium stem is the weakest. Therefore, the molecular marker is used for assisting in selecting the oncidium varieties with stronger pedicel hardness, so that the breeding process of the fresh cut flower varieties of the oncidium can be accelerated.
In a sixth aspect, the present invention provides a detection kit for detecting or identifying the hardness of a stem of oncidium in a seedling stage or a flowering stage, wherein the kit comprises a substance for detecting an SNP locus of oncidium material, namely, the 501bp position of a sequence shown as SEQ ID NO. 1.
Preferably, the detection kit comprises primers with nucleotide sequences shown as SEQ ID NO. 2-3.
Compared with the prior art, the invention has the beneficial effects that:
according to the invention, through 106 parts of simplified genome sequencing data of the oncidium germplasm, the whole genome association analysis is carried out by combining with phenotype data of the oncidium flower stalk hardness, a QTL site influencing the flower stalk hardness in the oncidium is obtained by first positioning, and an SNP molecular marker Orc.scaffoldes: p122266884 is developed, wherein the contribution rate of the SNP molecular marker to the oncidium flower stalk hardness is 52.03%, namely the SNP molecular marker plays a key role in regulating and controlling the character China of the oncidium flower stalk hardness. The primer is designed based on the SNP molecular marker, the analysis result is objective and accurate, the influence of subjective factors is avoided, the detection is convenient and rapid, the breeding efficiency is high, the breeding process of the strong-pedicel hardness variety of the oncidium is accelerated, and the method has important significance for screening fresh cut flowers of the oncidium.
Drawings
FIG. 1 is a graph showing the results of two years of data distribution of the hardness of the heart blue stems in 2021 and 2022 in example 1 of the present invention;
FIG. 2 is a diagram showing the correspondence between the stem hardness phenotype data of the two years 2021 and 2022 of the stem hardness trait of Chinese heart orchid and the different genotypes of SNP molecular markers Orc. Scaffoldes: p122266884 in example 2 of the present invention.
Detailed Description
The technical scheme of the present invention will be clearly and completely described in the following examples. It should be understood that the detailed description and specific examples, while indicating and illustrating the invention, are not intended to limit the invention.
In the following examples, unless otherwise specified, the methods are conventional; the reagents and materials described, unless otherwise specified, are commercially available.
Example 1 major QTL site positioning of the floral peduncles of the Wenzhong
(1) Phenotype determination of the hardness property of the aragonite orchid stems.
The germplasm resources of the oncidium used in this example were 106 parts oncidium from southwest (20 parts), guangzhou (26 parts), fujian (29 parts), taiwan (27 parts), guangxi (1 part), yunnan (1 part), guizhou (1 part), shanghai (1 part).
106 parts of germplasm resources are selected from a oncidium planting resource garden, 5 adult plants which grow well are randomly selected from each part of germplasm, 530 plants are counted, and the plants are planted and managed under the same environmental conditions. Watering is carried out once every 2 days in summer and once every 3 days in winter. The high nitrogen fertilizer is sprayed 1 time per week in the vegetative growth period, and the balance fertilizer is used after 5-6 times of nitrogen fertilizer, so that the overgrowth is prevented, and the balance fertilizer with 2000 times of liquid is used for root irrigation once per month in the reproductive growth period. The average annual temperature in the resource nursery is 25-35 ℃, the annual relative humidity is 70-80%, and the illumination intensity is 20000-30000 LX.
The culture of oncidium plants with positive full bloom (with flower number of more than 50%) was selected as sampling material for two years in 2021 and 2022. The characteristic observation refers to 'the description specification and data standard of the germplasm resources of the oncidium', the measurement of the hardness of the pedicel refers to a method for testing the quality of the stalk in the corn lodging resistance evaluation literature, namely, the stalk is inclined by pulling force from different angles and forms a certain included angle with the ground, and the lodging resistance of the stalk is evaluated by using the pulling force value measured at the moment. The specific method comprises the following steps: the digital Wei Du tension meter tester is used for hooking the stem of the oncidium, the stem of oncidium is inclined by 45 degrees with force from the vertical direction, and the value is read and averaged.
TABLE 1 average of the two year pedicel hardness of 106 parts of materials 2021 and 2022
Note that: PH represents the average of two year Pedicel Hardness (PH) values of the Wendelia miq 2021 and 2022.
According to the data, a schematic diagram of the distribution result of the stem hardness of the related population is drawn, as shown in fig. 1, and the result shows that the distribution of the stem hardness of 2021 and 2022 is continuously distributed, and the distribution deviates to a main peak, which indicates that the stem hardness belongs to quantitative characters and has main effective gene sites. The genetic force of the pedicel hardness is 0.70 through two years of data calculation, and the genetic force is high, which indicates that the pedicel hardness is mainly controlled by genetic control.
(2) The oncidium population simplifies genomic sequencing.
Samples were taken in germplasm resource nursery, and about 2g of tender leaves were taken from each germplasm, quick frozen with liquid nitrogen and sent to the company for sequencing with a sequencing strategy of Illumina HiSeq2500 PE150. Quality control is carried out on sequencing machine-off data (raw data), and the filtering standard is as follows: (1) filtering the sequence containing the linker; (2) filtering a pair of sequences having an N content exceeding 10% in the single-ended sequence; (3) a pair of sequences having a low quality (Q.ltoreq.5) base number exceeding 50% are filtered. And filtering the joint sequence, the inaccurate sequence and the low-quality sequence to obtain clean data, and performing next sequence comparison. After constructing an index file for a 'bee' reference genome, comparing clean data to the reference genome by using BWA software; ranking the alignment results using SAMtools software; processing the repeat tag generated during library preparation using Picard Tools software; the SNPs were further screened and filtered using GATK software (analysis of population variation information. VCFtools software, command "vccftools- -vcf [ vcf_file ] - -max-missing 0.5- -maf 0.05- -mac 3- -minQ 30- -minDP 3- -min-kernels 2- -max-kernels 2- -recode- -recode-INFO-all- -out_prefix ]".
(3) Whole genome association analysis.
Converting the format of the vcf file by using plink software; generating a sizing-Nichols affinity matrix using EMMAX software, commanded to "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 [ track_file ] -k [ kin_file ] -o [ out_prefix ]". And (3) obtaining a P value of each position of the oncidium by the association analysis result, wherein when the P value is smaller than 0.5/66731 = 7.49277E-06 SNP is a significant SNP, grouping materials according to different allele types of the SNP in the population, performing variance analysis, and obtaining the contribution rate of the SNP position as the percentage of the ratio of the inter-group variance to the total variance.
Through analysis, the SNP is scaffoldes_ 122266884 (A/G), the contribution rate of the QTL to the hardness trait of the aragonite orchid stems is 52.03% (the materials are grouped according to different allele types of the SNP, single-factor variance analysis is performed, and the percentage of the group variance divided by the total variance is the contribution rate).
Example 2 development of SNP molecular markers for the hardness trait of the flower stems of Wenzhong orchid
The SNP molecular markers are developed according to the stem hardness QTL and SNP identified in example 1, 500bp sequences before and after the SNP are extracted as the characteristic sequences of the SNP molecular markers Orc.scaffoldes: p122266884 (shown as a sequence table SEQ ID NO.1, wherein the 501 th bp is the SNP site, and A/G mutation exists), and primers are designed for the SNP molecular markers as follows:
forward primer: 5'-CTGCAAATGGATTTGTAGGATA-3' (SEQ ID NO. 2);
reverse primer: 5'-TACCCTCAATGACCTCCCTAT-3' (SEQ ID NO. 3).
Detection by conventional PCR amplification and sequencing, wherein the PCR amplification system is 10. Mu.L, specifically comprises: 50 ng/. Mu.L of leaf DNA 1. Mu.L, 2 XPCR Master Mix 5. Mu.L, 10. Mu. Mol/L forward and reverse primers 0.5. Mu.L each, ddH 2 O 3μL。
The base of SNP molecular marker is divided into three types, when Orc. Scaffoldes: p122266884 base type is GG, the average hardness of pedicel of 2021 year and 2022 year materials is 0.56 and 0.55 respectively, namely the pedicel is strong in hardness, the pedicel is difficult to bend or deform, and the pedicel has strong supporting force; when the base type is AG, the average hardness of the pedicel of the materials in 2021 and 2022 is 0.48, namely the pedicel with medium hardness, and the pedicel has certain elasticity and softness; when the base type is GG, the average hardness of the pedicel of the materials in 2021 and 2022 is 0.33, namely the pedicel is soft and flexible, but not easy to break. A schematic representation of the stem hardness phenotypes corresponding to the different genotypes is shown in FIG. 2.
The result shows that the flower stalk hardness character phenotype corresponding to the different genotype types of the SNP molecular marker Orc.scanfolds: p122266884 has obvious difference. Therefore, the type of the SNP molecular marker can be identified to rapidly and simply evaluate the intensity of the stem hardness character of the oncidium to be detected, so as to rapidly identify the stem hardness capability of the material.
In addition to using the primer amplification sequence of this example, the material was subjected to simplified genomic sequencing, and SNP genotyping was performed on the SNP molecular marker Orc. Scaffoldes: p122266884 provided by the invention according to the method in example 1, and the genotype of SNP was determined, thereby rapidly evaluating the stem hardness of the material.
Example 3 application of SNP molecular marker Orc. Scaffoldes: p122266884 in identification of hardness trait of aragonite orchid stems
The early known strong peduncle materials, wenyan (305), lemon yellow (leaf art) and HOM-6, were extracted and young leaf DNA was sent to sequencing company for simplified genomic sequencing, SNP genotypes of the three materials were identified, respectively, and peduncle hardness measurements were performed on the three materials according to the method of example 1.
Meanwhile, simplified genome sequencing was performed on three materials of medium-hardness pedicel material ONZ 9004, golden No.2 and lemon yellow (SH) previously known, tender leaf DNA was extracted and sent to a sequencing company, SNP genotypes of the three materials were respectively identified, and pedicel hardness traits of the three materials were identified according to the method of example 1.
Meanwhile, the DNA of young leaves extracted from three materials of Luo Manxiang, red peach and crape myrtle, which were previously known soft pedicel, was sent to a sequencing company for simplified genome sequencing, SNP genotypes of the three materials were identified, respectively, and pedicel hardness of the three materials was measured according to the method of example 1.
The average value of the SNP genotypes at 122266884bp positions of the scaffoldes and the pedicel hardness traits of the nine materials are shown in Table 2.
TABLE 2 Orc.scaffoldes: p122266884 locus genotype in materials with different pedicel hardness traits and pedicel hardness thereof
Note that: PH represents the average value of the stem hardness.
The results show that, for the known materials of strong pedicel hardness, wenyan blue (305), lemon yellow (leaf art) and HOM-64, the SNP genotypes at the 122266884bp position of the scaffoldes (i.e., at the 501bp position of the sequence shown in SEQ ID NO. 1) are GG, and the pedicel hardness phenotype values are 0.68, 0.61 and 0.60, respectively, which are consistent with the strong pedicel hardness trait corresponding to the GG genotypes of the SNP molecular markers in example 2. Similarly, for the known medium hardness pedicel materials ONZ 9004, golden No.2 and lemon yellow (SH), the SNP genotypes at the 122266884bp position of the scaffoldes (i.e., at the 501bp position of the sequence shown in SEQ ID NO. 1) were AG, and the pedicel hardness traits had phenotypes of 0.54, 0.47 and 0.46, respectively, consistent with the weaker pedicel hardness trait corresponding to the AG genotypes of the SNP molecular markers in example 2. Similarly, for the known pedicel softness Luo Manxiang, red peach and crape myrtle, the SNP genotypes at the 122266884bp positions of the scaffoldes (i.e., at the 501bp position of the sequence shown in SEQ ID NO. 1) are all AA, and the pedicel hardness traits have phenotypes of 0.25, 0.34 and 0.37, respectively, consistent with the weak pedicel hardness traits corresponding to the AA genotypes of the SNP molecular markers in example 2.
In conclusion, the SNP molecular marker Orc.scaffoldes: p122266884 plays a key role in regulating and controlling the hardness of the flower stalks of the oncidium, and the genotype of the SNP molecular marker Orc.scaffoldes: p122266884 can be used for effectively identifying or predicting the hardness of the flower stalks of the oncidium material, so that a solid foundation is laid for subsequent breeding work.
The foregoing description of the preferred embodiments of the present invention should not be taken as limiting the scope of the invention, and it should be noted that any modifications, equivalents, improvements and others within the spirit and principles of the present invention will become apparent to those of ordinary skill in the art.
Claims (10)
1. A SNP molecular marker related to the hardness property of the peduncles of the oncidium is characterized in that the nucleotide sequence of the molecular marker is shown as SEQ ID NO.1, wherein the SNP locus is 501bp of the sequence shown as SEQ ID NO.1 and has an A/G polymorphism.
2. The use of the SNP molecular marker according to claim 1 for detecting or identifying the hardness of the stems of the broccoli.
3. A method for detecting or identifying the hardness of a peduncle of a heart orchid during a seedling stage or a florless stage, comprising the steps of:
s1, extracting genome DNA of a to-be-detected oncidium material;
s2, amplifying the genome DNA of the S1 to obtain a target fragment containing a sequence shown in SEQ ID NO.1, and then sequencing, or directly performing simplified genome sequencing by using the genome DNA to determine the base type of the to-be-detected oncidium material at the 501bp position of the sequence shown in SEQ ID NO. 1.
4. A method according to claim 3, wherein if the base type of the material of the oncidium to be tested is AA, the pedicel of the material is soft, pliable but not breakable; if the base type of the to-be-detected oncidium material is AG, the material is a medium-hardness pedicel; if the base type of the to-be-detected oncidium material is GG, the pedicel of the material is hard, difficult to bend or deform and has strong supporting force.
5. The method according to claim 3, wherein the amplification in step S2 is performed using primers shown in SEQ ID NOS.2 to 3.
6. A test kit for detecting or identifying the hardness of a stem of a broccoli at the seedling stage or the florescence stage, comprising a substance for detecting the SNP site according to claim 1.
7. The detection kit according to claim 6, wherein the kit comprises primers with nucleotide sequences shown in SEQ ID NO. 2-3.
8. The use of the SNP molecular marker according to claim 1 in molecular marker-assisted breeding of the strong pedicel hardness of the oncidium.
9. A molecular marker assisted breeding method for the strong pedicel hardness of oncidium is characterized in that the basic group type of the oncidium material at the 501bp position of the sequence shown in SEQ ID NO.1 is detected, and the oncidium material with the basic group type GG is selected for assisted breeding.
10. A method for obtaining SNP molecular markers related to the hardness traits of the aragonite orchid stems, comprising the following steps:
a) Constructing a related population using the oncidium material having genetic differences from different regions;
b) Extracting total DNA of leaves of each material of the related population, and then using the DNA of the population to carry out simplified genome sequencing to identify SNP genotype information of the population;
c) Screening high-quality population SNP data sets by filtering SNP data quality;
d) Investigating the pedicel hardness phenotype data of all the oncidium materials in the associated population;
e) Combining genotype and pedicel hardness phenotype data, carrying out whole genome association analysis, and identifying QTL sites with obvious correlation of pedicel hardness to obtain SNP molecular markers with correlation of the pedicel hardness characters of the oncidium;
the SNP molecular marker is as set forth in claim 1.
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