1. A cpPSSR marker primer group developed based on peony chloroplast genome is characterized in that: the primer group comprises the following primers:

cpPSSR-1 marker primer: the forward sequence is shown as SEQ ID NO: 1, and the reverse sequence is shown as SEQ ID NO: 2 is shown in the specification;

cpPSSR-4 labeled primer: the forward sequence is shown as SEQ ID NO: 3, the reverse sequence is shown as SEQ ID NO: 4 is shown in the specification;

cpPSSR-5 labeled primer: the forward sequence is shown as SEQ ID NO: 5, the reverse sequence is shown as SEQ ID NO: 6 is shown in the specification;

cpPSSR-6 labeled primer: the forward sequence is shown as SEQ ID NO: 7, and the reverse sequence is shown as SEQ ID NO: 8 is shown in the specification;

cpPSSR-7 labeled primer: the forward sequence is shown as SEQ ID NO: 9, the reverse sequence is shown as SEQ ID NO: 10 is shown in the figure;

cpPSSR-8 labeled primer: the forward sequence is shown as SEQ ID NO: 11, and the reverse sequence is shown as SEQ ID NO: 12 is shown in the specification;

cpPSSR-9 labeled primer: the forward sequence is shown as SEQ ID NO: 13, and the reverse sequence is shown as SEQ ID NO: 14 is shown in the figure;

cpPSSR-11 marker primer: the forward sequence is shown as SEQ ID NO: 15, and the reverse sequence is shown as SEQ ID NO: 16 is shown in the figure;

cpPSSR-12 labeled primer: the forward sequence is shown as SEQ ID NO: 17, and the reverse sequence is shown as SEQ ID NO: 18 is shown in the figure;

cpPSSR-15 labeled primer: the forward sequence is shown as SEQ ID NO: 19, and the reverse sequence is shown as SEQ ID NO: 20 is shown in the figure;

cpPSSR-17 labeled primer: the forward sequence is shown as SEQ ID NO: 21, and the reverse sequence is shown as SEQ ID NO: 22;

cpPSSR-19 labeled primer: the forward sequence is shown as SEQ ID NO: 23, and the reverse sequence is shown as SEQ ID NO: shown at 24;

cpPSSR-21 marker primer: the forward sequence is shown as SEQ ID NO: 25, and the reverse sequence is shown as SEQ ID NO: 26 is shown;

cpPSSR-23 labeled primer: the forward sequence is shown as SEQ ID NO: 27, the reverse sequence is shown in SEQ ID NO: 28 is shown;

cpPSSR-25 labeled primer: the forward sequence is shown as SEQ ID NO: 29, and the reverse sequence is shown as SEQ ID NO: 30 is shown in the figure;

cpPSSR-26 labeled primer: the forward sequence is shown as SEQ ID NO: 31, and the reverse sequence is shown as SEQ ID NO: shown at 32.

2. The cpSSR-tagged primer set of claim 1, wherein: obtained by screening according to the following method:

step one, extracting total DNA of a plant tissue sample;

step two, detecting the integrity, purity and concentration of the total DNA;

step three, fragmenting the plant total DNA obtained in the step two, and carrying out high-throughput sequencing on the library fragments by using a second generation sequencing platform Illumina Xten to obtain original sequencing data of a plant sample;

step four, carrying out sequence splicing and verification on the plant chloroplast genome data obtained in the step three;

step five, detecting potential microsatellite repetitive sequences in batch for the sequence data spliced in the step four, and obtaining a cpSSR primer sequence through a formulated screening standard;

step six, filtering the cpPSSR primer obtained by screening in the step five, and deleting a repeated primer sequence;

seventhly, synthesizing primers of the cpPSSR primer obtained by filtering in the sixth step, diluting and storing for later use;

step eight, collecting disease-free tender leaves of a plurality of plant samples with large differences of plant phenotypes and plant sources, wherein the number of the plant samples is generally not more than 80, extracting total DNA of the plant leaves, detecting the integrity, purity and concentration of the DNA, diluting and storing for later use;

step nine, after DNA of 8-10 plants with large differences in phenotype and source is randomly selected from the step eight and equivalently mixed, DNA screening is carried out on the cpSSR primer obtained in the step seven by adopting a PCR amplification program;

tentatively detecting the amplification efficiency of the product by using 1% agarose gel electrophoresis;

and step eleven, carrying out secondary screening on the cpsR primers selected in the step eleven by using the DNA of the plant sample in the step eight through a capillary electrophoresis technology, and reserving the primers with high amplification efficiency and polymorphism and good stability.

3. The cpSSR-tagged primer set of claim 2, wherein: fifthly, carrying out cpSSR site excavation on the sequence obtained by sequencing, wherein the screening condition is that the number of the repetitive units is 5 or more, the size range of the repetitive fragments is 2-6 bp, and further screening the SSR sites by using the following constraint conditions after primer information is obtained preliminarily: the size of the amplification product is 150-400 bp; annealing temperature is 60 ℃, and the annealing temperature of the forward and reverse primers is close to that of the forward and reverse primers; length of the flanking region is less than or equal to 2000 bp; and fourthly, deleting the repetitive primer sequence.

4. The use of the cpSSR-tagged primer set of claim 1 for peony fingerprint construction and variety genetic relationship analysis.

Background

Microsatellite markers (or SSRs) are methods for amplifying plant genomic DNA by designing specific primers for relatively conserved sequences flanking repeated sequences between different alleles. As the marker has the characteristics of codominance, stable amplification, large information amount, low price and high efficiency, the marker is widely applied as an ideal marker selection for plant genetic diversity analysis and research (Lishansan et al, 2014; Yanghai Pingyi et al, 2017; Ebrahimi et al, 2017). The chloroplast genome is independent of the nuclear genome, and is one of the organelle genomes, which exhibit a high degree of conservation in sequence and structure. Compared with nuclear genome, chloroplast genome has simple structure and smaller molecular weight and copy number, and the gene structure sequence and gene content of chloroplast genome have the characteristics of high conservation, low gene replacement rate and the like, thereby being more beneficial to the construction of physical maps, the separation and identification of specific genes, sequence determination, evolution research and the like.

The complete sequence of the chloroplast genome is obtained for the first time in the research of liverworts and tobaccos (1986), the chloroplast genome is one of organelle genomes, is independent of a nuclear genome, and presents high conservation in sequence and structure, thereby providing feasibility for developing chloroplast microsatellite markers. Plant chloroplast genomes are generally four-segment in structure: a large single copy region (LSC), a small single copy region (SSC) and two Inverted repeat regions (IRs). The chloroplast genome structure of plants is highly conserved, typically encoding about 110-130 genes. These genes are mainly classified into chloroplast self-replication related genes, photosynthesis related genes, and other genes according to their functions.

Chloroplast microsatellite marker (cpPSSR) sites are usually distributed in a non-coding region, show higher variability compared with a coding region, and have the advantages of low evolution rate and almost zero recombination rate. In addition, the regions flanking the cpsrs locus are highly conserved, with greater versatility in closely related species.

Disclosure of Invention

Aiming at the defects of nuclear gene markers, the invention aims to provide a chloroplast microsatellite marker based on a plant chloroplast whole genome sequence, which not only can make up the defects of the number of nuclear genome markers, but also can research the characteristics of plant maternal inheritance, better eliminate the interference of a paternal genome, and the detection and development of the cpsR marker can provide a simple and reliable method for group genetics research, particularly the identification and the identification of related species.

In order to achieve the purpose, the invention adopts the specific scheme that:

a cpSSR-tagged primer set developed based on the peony chloroplast genome, the primer set comprising the following primers:

cpPSSR-1 marker primer: the forward sequence is shown as SEQ ID NO: 1, and the reverse sequence is shown as SEQ ID NO: 2 is shown in the specification;

cpPSSR-4 labeled primer: the forward sequence is shown as SEQ ID NO: 3, the reverse sequence is shown as SEQ ID NO: 4 is shown in the specification;

cpPSSR-5 labeled primer: the forward sequence is shown as SEQ ID NO: 5, the reverse sequence is shown as SEQ ID NO: 6 is shown in the specification;

cpPSSR-6 labeled primer: the forward sequence is shown as SEQ ID NO: 7, and the reverse sequence is shown as SEQ ID NO: 8 is shown in the specification;

cpPSSR-7 labeled primer: the forward sequence is shown as SEQ ID NO: 9, the reverse sequence is shown as SEQ ID NO: 10 is shown in the figure;

cpPSSR-8 labeled primer: the forward sequence is shown as SEQ ID NO: 11, and the reverse sequence is shown as SEQ ID NO: 12 is shown in the specification;

cpPSSR-9 labeled primer: the forward sequence is shown as SEQ ID NO: 13, and the reverse sequence is shown as SEQ ID NO: 14 is shown in the figure;

cpPSSR-11 marker primer: the forward sequence is shown as SEQ ID NO: 15, and the reverse sequence is shown as SEQ ID NO: 16 is shown in the figure;

cpPSSR-12 labeled primer: the forward sequence is shown as SEQ ID NO: 17, and the reverse sequence is shown as SEQ ID NO: 18 is shown in the figure;

cpPSSR-15 labeled primer: the forward sequence is shown as SEQ ID NO: 19, and the reverse sequence is shown as SEQ ID NO: 20 is shown in the figure;

cpPSSR-17 labeled primer: the forward sequence is shown as SEQ ID NO: 21, and the reverse sequence is shown as SEQ ID NO: 22;

cpPSSR-19 labeled primer: the forward sequence is shown as SEQ ID NO: 23, and the reverse sequence is shown as SEQ ID NO: shown at 24;

cpPSSR-21 marker primer: the forward sequence is shown as SEQ ID NO: 25, and the reverse sequence is shown as SEQ ID NO: 26 is shown;

cpPSSR-23 labeled primer: the forward sequence is shown as SEQ ID NO: 27, the reverse sequence is shown in SEQ ID NO: 28 is shown;

cpPSSR-25 labeled primer: the forward sequence is shown as SEQ ID NO: 29, and the reverse sequence is shown as SEQ ID NO: 30 is shown in the figure;

cpPSSR-26 labeled primer: the forward sequence is shown as SEQ ID NO: 31, and the reverse sequence is shown as SEQ ID NO: shown at 32.

Further, the cpSSR labeled primer set is obtained by screening according to the following method:

step one, extracting total DNA of a plant tissue sample;

step two, detecting the integrity, purity and concentration of the total DNA;

step three, fragmenting the plant total DNA obtained in the step two, and carrying out high-throughput sequencing on the library fragments by using a second generation sequencing platform Illumina Xten to obtain original sequencing data of a plant sample;

step four, carrying out sequence splicing and verification on the plant chloroplast genome data obtained in the step three;

step five, detecting potential microsatellite repetitive sequences in batches for the sequence data spliced in the step four, obtaining a cpSSR primer sequence through a set screening standard, wherein the specific screening condition is that the repetitive units are more than 5, the size range of repetitive fragments is 2-6 bp, and further screening SSR sites by using the following constraint conditions after primer information is obtained preliminarily: the size of the amplification product is 150-400 bp; annealing temperature is 60 ℃, and the annealing temperature of the forward and reverse primers is close to that of the forward and reverse primers; length of the flanking region is less than or equal to 2000 bp; and fourthly, deleting the repetitive primer sequence.

Step six, filtering the cpPSSR primer obtained by screening in the step five, and deleting a repeated primer sequence;

seventhly, synthesizing primers of the cpPSSR primer obtained by filtering in the sixth step, diluting and storing for later use;

step eight, collecting disease-free tender leaves of a plurality of plant samples with large differences of plant phenotypes and plant sources, wherein the number of the plant samples is generally not more than 80, extracting total DNA of the plant leaves, detecting the integrity, purity and concentration of the DNA, diluting and storing for later use;

step nine, after DNA of 8-10 plants with large differences in phenotype and source is randomly selected from the step eight and equivalently mixed, DNA screening is carried out on the cpSSR primer obtained in the step seven by adopting a PCR amplification program;

tentatively detecting the amplification efficiency of the product by using 1% agarose gel electrophoresis;

and step eleven, carrying out secondary screening on the cpsR primers selected in the step eleven by using the DNA of the plant sample in the step eight through a capillary electrophoresis technology, and reserving the primers with high amplification efficiency and polymorphism and good stability.

The invention also provides application of the cpsSR marker primer group in peony fingerprint map construction and variety genetic relationship analysis.

Has the advantages that:

according to the invention, a group of cpsR marker loci with high polymorphism and good universality are obtained by screening a large number of SSR loci in a chloroplast genome sequence, the developed cpsR markers can not only make up for the defects of nuclear genome markers, but also can research the characteristics of plant maternal inheritance, better exclude the interference of male parent genomes, and the detection and development of the cpsR markers can provide a simple and reliable method for population genetics research, especially the identification and identification of closely related species.

Drawings

FIG. 1 is a capillary electrophoresis of the cpPSSR-6 primer in a portion of a peony variety sample.

Detailed Description

A method for rapidly and effectively developing chloroplast microsatellite markers based on plant chloroplast whole genome technology comprises the following steps:

firstly, extracting total DNA of a plant tissue sample by adopting an improved CTAB method, and specifically operating the following steps:

1. putting a proper amount of peony tender leaves into a precooled mortar filled with a proper amount of liquid nitrogen, quickly grinding the peony tender leaves into powder (in the process, the liquid nitrogen in the mortar is always kept sufficient to ensure the quality of extracted DNA), and quickly transferring the proper amount of powder into a 2ml centrifugal tube precooled by the liquid nitrogen;

2. adding 1500 mul of CTAB preheated at 65 ℃ and 100 mul of beta-mercaptoethanol into a centrifuge tube, tightly covering the centrifuge tube, violently shaking and uniformly mixing, ensuring that the powder at the bottom of the centrifuge tube is uniformly dispersed in CTAB buffer solution, and placing the centrifuge tube in a 65 ℃ water bath kettle for 50 minutes. During the period, taking out the centrifuge tube every 5min and violently shaking for 30 s;

3. after the water bath is finished, taking out the centrifuge tube, cooling the centrifuge tube to room temperature, putting the centrifuge tube into a high-speed refrigerated centrifuge, and centrifuging the centrifuge tube for 12min at the temperature of 4 ℃ and the rpm of 12000;

4. sucking the supernatant into another 2ml centrifuge tube, adding the chloroform/isoamylol with the same volume as 24:1, and violently shaking for 10min by turning upside down;

5. after the high-speed refrigerated centrifuge centrifuges for 15min at 4 ℃ and 12000rpm, repeating the step 4;

6. repeating steps 4 and 5 twice to sufficiently remove proteins and polyphenols;

7. taking the supernatant into another 2ml centrifuge tube, adding equal volume of anhydrous ethanol precooled at-20 ℃, slightly reversing the volume from top to bottom, mixing the mixture evenly, and then placing the mixture in a refrigerator at-20 ℃ for 30min to ensure that the DNA is fully precipitated;

8. picking out flocculent DNA precipitate in a centrifuge tube by 200 mul of snatching into another centrifuge tube of 1.5ml, adding 1000 mul of 70% absolute ethyl alcohol to rinse the precipitate, flicking with fingers to make the precipitate leave the bottom of the centrifuge tube, slightly reversing the precipitate for a plurality of times, and repeatedly cleaning for 3 times;

9. and (3) placing the DNA centrifuge tube with the rinsed DNA into an ultraclean workbench, airing to be semitransparent, adding 200-300 mu l of sterile double distilled water for dissolving, adding 2 mu l of RNase to remove RNA in the total DNA, carrying out water bath at 37 ℃ for 1h, and storing at-20 ℃ for later use.

Secondly, detecting the integrity, purity and concentration of the total DNA: the integrity of the DNA was checked by electrophoresis on a 1% agarose gel and the purity and concentration were determined using a Nanodrop 2000 microspectrophotometer.

Thirdly, fragmenting the total plant DNA obtained in the second step by adopting a VAHTSTM Universal DNA Library Prep Kit for Illumina V2 Kit, and carrying out high-throughput sequencing on the Library fragments by using a second generation sequencing platform Illumina Xten so as to obtain the original sequencing data of the plant sample.

And fourthly, performing sequence splicing and verification on the plant chloroplast genome data obtained in the third step.

And fifthly, detecting potential microsatellite repetitive sequences in batches from the sequence data spliced in the fourth step, and establishing strict screening standards to obtain the sequence of the cpSSR primer.

Sixthly, filtering the cpPSSR primer obtained by screening in the step five, and deleting a repeated primer sequence.

Seventhly, synthesizing primers of the cpPSSR primer obtained by filtering in the sixth step in Biotechnology Limited of Beijing Ruiboxke, diluting the primers according to the standard on a primer centrifuge tube, and immediately storing the diluted primers at-20 ℃ for later use.

Eighthly, collecting young leaves of a plurality of plant samples with large differences of plant phenotypes and sources (the number of the plant samples is generally not more than 80), wherein the collected young leaves are required to be complete and free of diseases, and extracting the total DNA of the plant leaves by using an improved CTAB method.

And ninthly, detecting the integrity, purity and concentration of the DNA by using the method described in the step two so as to meet the experimental requirements.

Ten, the total DNA was diluted in aliquots (20 ng/. mu.L) with the elution buffer TB (Tiangen) and immediately stored at-20 ℃ until use.

Eleven, randomly selecting DNA of 8-10 plants with large differences in phenotype and source from the step eight, equivalently mixing, and screening the cpSSR primer obtained from the step seven through the DNA of the sample to be tested by adopting a PCR program.

Twelfth, the amplification efficiency of the product in the eleventh step is preliminarily detected by using 1% agarose gel electrophoresis.

Thirteen, using the cPSS primer selected in the step twelve, and using the DNA of the plant sample in the step eight to perform secondary screening on the cPSR primer by a capillary electrophoresis technology, and reserving the primer with high polymorphism and good stability.

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention.

Aiming at mass SSR marker sequences obtained by high-throughput sequencing data, a plurality of effective markers are rapidly obtained from the sequences, and the sequences are very necessary to be used for basic researches such as plant population genetics, identification and identification of closely related species and the like. The method provided by the invention is time-saving and labor-saving, has high amplification efficiency of the marker, and is not only suitable for the research of the diversity and the population structure of the plant population, but also suitable for the relevant research of the core germplasm construction of the plant population and the like.

② it is noted that the method is applicable to the development of plant microsatellite markers. The plant material used in the invention is illustrated by taking peony as an example. Other plants may also be used in the practice of the present application without conflict.

③ using liquid nitrogen to store the DNA sample of the plant tissue for constructing the chloroplast whole genome library, and carrying back the experiment when collecting the material, and immediately storing the DNA sample at-80 ℃ for later use.

And fourthly, when the leaves of 60 peony plant samples are sampled in an experimental place, putting the leaves into a self-sealing bag, wrapping the leaves with tinfoil, quickly freezing the leaves in liquid nitrogen, and taking the leaves back to the laboratory for storage at-80 ℃ for later use.

Fifthly, extracting the total DNA of each plant sample by using a modified CTAB method, and carrying out the operation steps according to the step one in the implementation steps.

Sixthly, carrying out cpPSSR site excavation on the sequenced sequence of the fa. format file by using a GMATA v2.2 program (https:// github. com/Xuewen WangUGA/GMATA). The screening conditions are that the number of the repetitive units is 5 or more, the size range of the repetitive fragments is 2-6 bp, and after primer information is obtained preliminarily, SSR loci are further screened by using the following constraint conditions: the size of the amplification product is 150-400 bp; annealing temperature is 60 ℃, and the annealing temperature of the forward and reverse primers is close to that of the forward and reverse primers; length of the flanking region is less than or equal to 2000 bp; and fourthly, deleting the repetitive primer sequence.

The system for performing primary screening amplification on the cpsSR primer by using 1% agarose gel electrophoresis in the invention comprises the following steps: 10 μ L, including 2 μ L genomic DNA (20 ng/. mu.L), 5 μ L2 XTaq Plus Master Mix II (Dye Plus), 0.2 μ L (10 μ M) forward primer and 0.2 μ L (10 μ M) reverse primer, with dd H₂And (4) supplementing and finishing.

[ mu.L of the capillary electrophoresis PCR amplification System of the present invention, including 2. mu.L of genomic DNA (20 ng/. mu.L), 5. mu.L of 2 XTaq Plus Master Mix II (Dye Plus), 0.6. mu.L of M13 primer (10. mu.M; 5'-TGTAAAACGACGGCCAGT-3') of fluorescent Dye (FAM, HEX, ROX, TAMER), 0.2. mu.L (10. mu.M) of forward primer and 0.6. mu.L (10. mu.M) of reverse primer, using dd H₂And (4) supplementing and finishing.

Ninthly, amplifying the DNA of the sample to be detected by adopting a PCR program primer, wherein the amplification program is shown in the following table 1.

Table 1: and (3) performing a PCR amplification procedure.

Capillary electrophoresis detection of cpSSR markers obtained in (r) and (c) in 60 peony samples is shown in fig. 1.

Two screenings were carried out by (c) and (b) and the genetic parameters of each marker were calculated by means of the software GenAlEx v6.502 and PowerMarker v3.25 (see Table 2).

Table 2 analysis of genetic diversity of the cpPSSR markers in 60 peony varieties.

Note:N: the number of variation sites;Na: the number of equipotential points;Ne: the number of significant equipotential points;I: fragrance intensity information index;Ho: observing the heterozygosity;He: a desired heterozygosity;PICpolymorphism information content; H: a genetic polymorphism.

After comprehensively analyzing the table 2, primers with low amplification efficiency and monomorphism are deleted, and finally, a cpSSR primer marker with high polymorphism and good universality can be obtained and can be used for researches such as population genetics, identification and identification of closely related species and the like, and the cpSSR primer information developed by peony chloroplast genome is shown in the following table 3 and SEQ ID NO: 1-SEQ ID NO: shown at 32.

Table 3 cpSSR primer information.

According to the calculation result of the genetic parameters, the amplification efficiencies of the 16 pairs of markers are further ranked, wherein the amplification efficiency of the cpsSR-19 is particularly outstanding, and therefore, the markers can be used as the first-choice markers for fingerprint construction and genetic relationship analysis.

It should be noted that the above-mentioned embodiments illustrate rather than limit the scope of the invention, which is defined by the appended claims. It will be apparent to those skilled in the art that certain insubstantial modifications and adaptations of the present invention can be made without departing from the spirit and scope of the invention.

SEQUENCE LISTING

<110> university of Henan science and technology

<120> cpPSSR marker primer developed based on peony chloroplast genome sequence and application

<130> 1

<160> 32

<170> PatentIn version 3.3

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