CN116042891A - Duck grass cpsSR labeled primer and application thereof - Google Patents

Duck grass cpsSR labeled primer and application thereof Download PDF

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CN116042891A
CN116042891A CN202211397714.1A CN202211397714A CN116042891A CN 116042891 A CN116042891 A CN 116042891A CN 202211397714 A CN202211397714 A CN 202211397714A CN 116042891 A CN116042891 A CN 116042891A
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冯光燕
张新全
焦永娟
边昊阳
李顺凤
郝飞翔
黄琳凯
聂刚
余国辉
许肖恒
王苗利
杨忠富
李丹丹
汪霞
黄婷
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Sichuan Agricultural University
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Abstract

The invention discloses a group of festuca arundinacea cpSSR marked primers and application thereof. The labeled primer comprises 4 pairs of polymorphic primers, and the nucleotide sequences of the polymorphic primers are respectively shown as SEQ ID NO.1 and 2, SEQ ID NO.3 and 4, SEQ ID NO.5 and 6 and SEQ ID NO.7 and 8. The marking primer fills the blank of marking information of the festuca arundinacea cpSSR, can be applied to the group structure and species identification of the festuca arundinacea Mao Yachong, provides convenience for distinguishing and identifying the ducks Mao Yachong, and lays a foundation for researches such as genetic diversity evaluation, germplasm identification and molecular marker assisted breeding of the festuca arundinacea.

Description

Duck grass cpsSR labeled primer and application thereof
Technical Field
The invention relates to the technical field of plant biology, in particular to a group of festuca arundinacea cpSSR marked primers and application thereof.
Background
The festuca arundinacea (Dactylis glomerata L.) is also called orchard grass (Orchardgrass), belongs to the genus festuca arundinacea of the subfamily poaceae, has the advantages of high yield of leaves, strong negative resistance, good palatability, high nutritive value and the like, and shows good feeding value and ecological value. As a highly variant grass, this genus contains many subspecies, and the major species of cogongrass can be classified into three major categories depending on ploidy levels: diploids (2n=2x=14), tetraploids (2n=2x=28) and hexaploids (2n=2x=42). The classification of this genus is made difficult by the wide variety of ducks Mao Yachong, lack of taxonomic diagnostic features and high morphological similarity between subspecies. While previous studies have attempted to classify cogongrass at a cytological and genetic level, there is still a lack of uniform criteria for classifying subspecies thereof, and classification identification and characterization of this genus has been a major challenge and problem faced by plant taxonomies. Thus, the accurate, rapid and efficient identification of duck Mao Yachong is not only an urgent need for plant taxonomies, but also has great significance for better development and utilization of duck Mao Ziyuan for further research of classification of this genus.
The chloroplast microsatellite technology is a novel and efficient molecular marking technology developed in recent years, and has the characteristics of small chloroplast genome molecular weight, multiple copies, simple structure, maternal inheritance and the like, and has the advantages of co-dominance, high polymorphism, simple operation, wide distribution and the like of microsatellites. Because of the advantages of SSR (microsatellite sequence) markers and the characteristics of chloroplast genome, cpSSR (chloroplast microsatellite sequence) can be used for identifying species and species with close relatedness, and describing genetic differences at regions and individual levels. However, at present, research on the molecular identification technology of cogongrass generally adopts nuclear genome information of cogongrass, but a method for identifying the cogongrass Mao Yachong by adopting chloroplast SSR markers is not reported yet, and further development of the molecular identification technology of cogongrass is severely limited. Therefore, based on chloroplast genome data of cogongrass, SSR markers are identified and developed, and screening of the chloroplast SSR markers for identification of cogongrass molecules is necessary for promoting classification and identification of ducks Mao Yachong; and constructing a duck Mao Zhiwen map based on the cpsSR molecular marker, and providing a reference for variety protection.
Disclosure of Invention
The invention aims to provide a group of duck cogongrass cpSSR marked primers for distinguishing ducks Mao Yachong, which can fill the blank of duck cogongrass cpSSR marked information and can also be used for identifying the structure and species of a duck Mao Yachong population.
In order to achieve the above object, the present invention provides a group of festuca arundinacea cpSSR marker primers comprising 4 pairs of polymorphic marker primers, the nucleotide sequences of the primers are as follows:
the cpSSR1 forward primer is shown as SEQ ID NO. 1;
the reverse primer of the cpSSR1 is shown as SEQ ID NO. 2;
the cpSSR2 forward primer is shown as SEQ ID NO. 3;
the cpsSR2 reverse primer is shown as SEQ ID NO. 4;
the cpSSR3 forward primer is shown as SEQ ID NO. 5;
the cpSSR3 reverse primer is shown as SEQ ID NO. 6;
the cpSSR4 forward primer is shown as SEQ ID NO. 7;
the reverse primer of the cpSSR4 is shown as SEQ ID NO. 8.
The invention also provides a kit containing the festuca arundinacea cpSSR labeled primer.
The invention also provides a preparation method of the festuca arundinacea cpSSR marked primer, which comprises the following steps:
1) Extracting chloroplast DNA of the festuca arundinacea;
2) Carrying out high-throughput Illumina sequencing on the chloroplast DNA obtained in the step 1);
3) Filtering the raw data obtained by sequencing in the step 2) by using fastp software;
4) Assembling chloroplast genome of the data obtained by filtering in the step 3) by adopting SPades 2 software;
5) Searching SSR sites distributed in the duck Mao Yachong chloroplast genome obtained in the step 4) by using MISA perl script software;
6) And (3) designing a cpSSR Primer by using Primer Premier 5 software, and screening to obtain 4 pairs of festuca arundinacea cpSSR marked primers.
Wherein, parameters of MISA perl script software in step 5) are set as follows: a single nucleotide repeat sequence, the repeat unit is more than or equal to 8; a dinucleotide repeat sequence, the repeat unit is more than or equal to 5; trinucleotide repeat sequence, the repeat unit is more than or equal to 3; 4. five and six nucleotide repetitive sequences, and the repetitive units are more than or equal to 3.
The design principle of the cpSSR primer design in the step 6) is as follows: primer length ranges from 20 bases; the annealing temperature is 50-60 ℃; the product length is 200-300 bp.
The cogongrass cpsSR marker primer provided by the invention can be applied to the identification of the structure and species of the duck Mao Yachong population.
The invention also provides a method for identifying the structure and species of the duck Mao Yachong population based on the duck cogongrass cpSSR labeled primer, which comprises the following steps:
1) Extracting genome DNA from young leaves of the duck Mao Yachong to be detected;
2) Performing PCR amplification by using the genome DNA obtained in the step 1) as a template and using the 4 pairs of cpsSR labeled primers;
3) Carrying out polymorphism detection on the product obtained by amplification in the step 2) through electrophoresis, and carrying out statistics on clear bands;
4) And 3) constructing a germplasm population structure diagram according to the statistical data obtained in the step 3), namely obtaining the result of the duck Mao Yachong population structure and species identification.
Preferably, the PCR amplification in step 2) of the aforementioned duck Mao Yachong population structure and species identification method has a PCR amplification system of 10. Mu.L: comprises 1. Mu.L of template DNA, 0.5. Mu.L of 5 pmol. Mu.L -1 5. Mu.L of 2X Taq PCR MasterMix II, 3. Mu.L of ddH2O; the PCR amplification procedure is as follows: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, annealing at 57℃for 30s, extension at 72℃for 35s, for a total of 35 cycles; extending at 72deg.C for 5min, and preserving at 4deg.C.
Preferably, the electrophoresis buffer in step 3) of the duck Mao Yachong colony structure and species identification method is 1 XTBE, and the electrophoresis apparatus parameters are 200V stabilized for 20min and 280V stabilized for 4h.
The invention provides a fingerprint constructed based on the festuca arundinacea cpSSR labeled primer, which can be applied to identification of different subspecies of festuca arundinacea.
The festuca arundinacea cpSSR marked primer fills the blank of the festuca arundinacea cpSSR marked information and has the following advantages:
the 4 pairs of the festuca arundinacea cpSSR marker primers provided by the invention can be applied to the identification of the population structure and species of the ducks Mao Yachong, the population structure among the ducks Mao Yachong is analyzed by using the developed cpSSR marker primers, convenience is provided for the distinguishing and identification of the ducks Mao Yachong, meanwhile, the 4 pairs of the festuca arundinacea cpSSR marker primers provided by the invention have the characteristics of co-dominance, high polymorphism and the like of nuclear genome SSR markers, have the single-parent genetic mode of chloroplast genome DNA, are not easy to recombine, overcome the characteristics of low development efficiency and long period of the traditional molecular markers, and simultaneously fill the blank of the development of the festuca arundinacea cpSSR, and lay a foundation for the research of genetic diversity evaluation, germplasm identification, molecular marker assisted breeding and the like of the festuca arying.
Drawings
FIG. 1 is an electrophoretically detected map of 31 ducks Mao Yachong subjected to PCR amplification by the primer pair cpSSR 1.
FIG. 2 is an electrophoretically detected map of 31 ducks Mao Yachong subjected to PCR amplification by the primer set cpsSR 2.
FIG. 3 is an electrophoretically detected map of 31 ducks Mao Yachong subjected to PCR amplification by the primer pair cpSSR 3.
FIG. 4 is an electrophoretically detected pattern of primer set cpSSR4 after PCR amplification of 31 ducks Mao Yachong.
FIG. 5 is a block diagram of a duck Mao Yachong population constructed using 4 pairs of cogongrass cpSSR labeled primers.
FIG. 6 is a graph of duck Mao Zhiwen constructed using 4 pairs of cogongrass cpSSR labeled primers.
Detailed Description
The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Experimental example 1 development of Duck grass cpsSR marker primer
1. Extraction of chloroplast DNA of Duck grass
Extracting chloroplast DNA of festuca arundinacea, detecting the purity, concentration and integrity of the extracted chloroplast DNA, and removing DNA fragments with unqualified purity, concentration and integrity to obtain qualified DNA fragments; the purity, concentration, and integrity of the DNA fragment were judged as being acceptable from the following points: observing whether the appearance of the sample contains foreign matters; agarose electrophoresis detects whether the sample has degradation and DNA fragment size; detecting the purity of DNA by Nanodrop/Onedrop; qubit carries out accurate quantification on DNA; the appearance of the sample is free from foreign matters, the agarose electrophoresis detection is free from degradation, the DNA fragment is larger, the DNA purity reaches the standard, and the like, and the sample is used as a qualified basis.
The specific process for extracting the chloroplast DNA of the festuca arundinacea is as follows:
(1) Chloroplast crude extraction operation flow
(1) Selecting duck Mao Huanghua seedlings, preparing a buffer solution A according to the proportion of 20mL of buffer solution per gram of material (adding 0.2% BSA, 0.2% cysteine and 0.5% beta-mercaptoethanol before using, and then gently reversing, uniformly mixing or stirring, dissolving, and then placing on ice for precooling for later use). (2) 2g of green plant tissue (tender leaves and callus after removing veins) is transferred into a mortar by using a liquid nitrogen grinding method, added with liquid nitrogen to be ground into a powder state, timely supplemented with the liquid nitrogen to prevent deliquescence of the powder, then quickly transferred into a pre-cooled 50mL centrifuge tube filled with 10-20 mL of buffer solution A, and evenly mixed upside down. (3) The buffer was filtered through a layer of 300 mesh nylon mesh and the permeate was collected into a pre-chilled 50mL centrifuge tube via a pre-chilled funnel. (4) Centrifuging at 1000rpm for 5min at 4 ℃ on a refrigerated centrifuge, carefully transferring the supernatant into a new precooled 50mL centrifuge tube, temporarily storing on ice, adding 5mL of precooled buffer solution A into the precipitate, blowing and mixing uniformly or mixing uniformly upside down by a pipetting gun, centrifuging at 1000rpm for 5min, collecting the supernatant obtained by the two centrifugation, centrifuging at 4000rpm for 10min, and transferring the supernatant into the new precooled 50mL centrifuge tube. (5) Centrifuging at 4deg.C for 20min in a refrigerated centrifuge at 12000g, and discarding supernatant to obtain precipitate as chloroplast crude extract. (6) 10mL of pre-chilled buffer B (50 mM Tris-Cl, 25mM EDTA, 0.1% BSA, 400mM sucrose, pH adjusted to 8.0, 0.2% BSA was added before use, and then gently inverted to mix or stirred to dissolve and then placed on ice for pre-chilled use), the chloroplast pellet was gently resuspended by gentle beating with a gun head or soft pen, and no vigorous shaking was possible, otherwise the chloroplast was prone to rupture. (7) Centrifugation was performed for 20min at 12000g at 4℃on a refrigerated centrifuge, and the precipitate was formed into chloroplasts, and the supernatant was carefully discarded. (8) To the pellet was added 2mL of pre-chilled buffer B (0.2% BSA, 0.2% cysteine added) and the chloroplast pellet was gently resuspended by gentle pipetting with a gun head without vigorous shaking, otherwise the chloroplasts were prone to rupture. (9) The integrity of chloroplasts in the resuspension obtained in the previous step was examined under a microscope. The specific method is that about 50 mu L of chloroplast heavy suspension is firstly dripped on a glass slide, and then 50 mu L of janus dye solution green B dye solution is dripped for 20min, and the blue-green particles are chloroplasts after observation under an optical microscope. Adding 80 mu LDNase I working solution into a centrifuge tube: each reaction was terminated by adding 10. Mu.L of DNase I (15U/. Mu.L) to 70. Mu.L of DNase Buffer, gently mixing, standing on ice for 1h, then adding 80. Mu.L of 0.5M EDTA, mixing, and standing for 10min. The above solution was carefully spread on buffer C (4 mL of buffer C pre-chilled per tube), centrifuged at 12000g for 20min at 4℃and precipitated as chloroplasts with the nuclear DNA removed. If long-term preservation is needed, the mixture is directly preserved at-80 ℃.
(2) Chloroplast DNA extraction procedure (Tiangen DP305 kit, operation reference instruction manual)
(1) 700 mu L of a preheated lysate at 65 ℃ is added into chloroplast sediment, the mixture is immediately and fully blown and evenly mixed by a pipetting gun, the centrifuge tube is placed in a water bath at 65 ℃ for 30min, and the centrifuge tube is inverted and evenly mixed for a plurality of times. (2) mu.L of RNase A (25 mg/mL) solution was added thereto, and the mixture was allowed to stand at room temperature for 10 minutes after mixing. (3) 700. Mu.L of chloroform was added thereto, and the mixture was thoroughly mixed and centrifuged at 12000rpm for 5 minutes. (4) The upper aqueous phase (about 650 μl) was carefully transferred to a new centrifuge tube to avoid touching the white film in the middle layer, and then 700 μl of binding solution or 0.5 volumes of absolute ethanol/isopropanol was added and thoroughly mixed. (5) Transferring the uniformly mixed liquid into an adsorption column, centrifuging at 12000rpm for 30s, and discarding the waste liquid. (6) To the column, 500. Mu.L of the washing solution was added, and the mixture was centrifuged at 12000rpm for 30s, and the waste solution was discarded. (7) 600. Mu.L of the rinse solution was added to the column, centrifuged at 12000rpm for 30s, and the waste solution was discarded. (8) Repeating the above steps. (9) Centrifuging at 12000rpm for 2min, transferring the adsorption column to a new centrifuge tube, and standing at room temperature for several minutes after opening the cover to thoroughly dry the residual rinse liquid in the adsorption material. Then, about 50. Mu.L of eluent TE is suspended and dripped into the middle part of the adsorption film, the solution is placed at room temperature for 2 to 5min and centrifuged at 12000rpm for 2min, and the solution is collected into a centrifuge tube. In order to increase the DNA yield, the solution obtained by centrifugation may be added to an adsorption column again, left to stand at room temperature for 2min, and centrifuged at 12000rpm for 2min. And (5) concentration measurement and agarose gel electrophoresis detection.
(3) Chloroplast DNA sequencing of Duck grass plants
Chloroplast DNA of festuca arundinacea plants was obtained by high-throughput Illumina combined nanopore sequencing (Nanopore sequencing), while raw data was filtered by using fastp (v0.20.0) software, with the following filtration criteria:
(1) the sequencing adaptors and primer sequences in the Reads are excised.
(2) Reads with average homogeneity value less than Q5 are filtered out.
(3) N reads greater than 5 are filtered out.
(4) And (5) recovering the obtained qualified DNA fragments by using a BluePIPP full-automatic nucleic acid recovery instrument.
2. Purification, treatment and DNA library construction of chloroplast DNA of festuca arundinacea
The purification, treatment and DNA library construction process of the chloroplast DNA of the festuca arundinacea comprises the following steps:
(1) Performing primary purification treatment on the recovered qualified DNA fragments by using magnetic beads;
(2) Performing damage and end repair on the DNA fragment subjected to primary purification treatment;
(3) Purifying the repaired DNA fragment by using magnetic beads again to obtain target DNA;
(4) Using a sequencing joint in a SQK-LSK109 kit to connect the target DNA fragments obtained through the treatment in the step 5) to obtain a DNA library;
(5) The DNA library obtained in step 6) was precisely quantified using Qubit.
3. Obtaining of Duck grass chloroplast complete genome sequence
Adding a DNA library with certain concentration and volume into the Flow cell, transferring the Flow cell to a Oxford Nanopore PromethION sequencer for real-time single-molecule sequencing to obtain the full length of a chloroplast genome, correcting the full length by a high-throughput Illumina sequencing result, splicing corrected three-generation data by using three-generation assembly software canu, setting the genome size to be 5M, correcting error=0.03 to obtain a contig sequence, comparing the contig sequence with a plant chloroplast gene database by using blast v2.6, using the contig of the aligned chloroplast gene as a seed sequence, extending and cyclizing the original data to obtain a circular dominant structure, correcting the assembly result by using NextPolish1.3.1 by using three-generation data, and correcting the assembly result by using pilot software by using second-generation data to obtain a circular festuca chloroplast genome; the high-flux Illumina sequencing combined nanopore sequencing (Nanopore sequencing) technology is utilized to obtain the festuca arundinacea chloroplast whole genome sequence, and original data is provided for development of festuca arundinacea cp SSR markers.
Wherein, SPADes 2 (v3.10.1) software is adopted to assemble chloroplast genome, and the detailed flow comprises the following 7 steps:
step1: assembling the cpDNA sequence through SPades software to obtain a SEED sequence of a chloroplast genome;
step2: kmer iterative extend seed if Step2 results in a contig, then the result is defined as a pseudo genome sequence, and Step6 is performed directly;
step3: using SSPACE (v 2.0)) software, connecting the contig sequences obtained by Step2 to obtain scaffoldes;
step4: GAP compensation was performed on the scaffolders sequence obtained in Step3 using Gapfiller (v2.1.1) software;
step5: if GAP still exists after the operation is finished, designing a primer, sequencing by PCR, and then assembling until a complete pseudogenome sequence is obtained;
step6: aligning the sequenced sequences to pseudogenome for genome correction;
step7: and (3) according to the structure of chloroplasts, carrying out coordinate rearrangement on the corrected pseudogenome to obtain a complete chloroplast circular genome sequence.
Meanwhile, because chloroplast genome has the characteristics of conservation, rearrangement and the like, the accuracy of an assembly result is ensured by carrying out quality control on 3 aspects of assembled chloroplast genome, and the method comprises the following steps of:
reads back ratio genome, statistics of genome coverage, insert size, etc.;
b. comparing the genome with a reference sequence, and checking the conservation, rearrangement and other colinear analysis of the genome;
c. genome alignment reference sequence structure information, and comparing the difference between the two.
The invention adopts two methods to annotate chloroplast genome to improve annotation accuracy, comprising: 1) Chloroplast CDS was annotated with prodigal (v2.6.3), rRNA was predicted using hmmer (v3.1b2) software, and tRNA was predicted using argorn (v1.2.38). 2) Based on closely related species already published on NCBI, its gene sequences were extracted and then the assembled sequences were aligned using blast (v 2.6) to obtain a second annotation result. Then, the two annotation results were manually checked for differential genes, erroneous annotations and redundant annotations were removed, and the multi-exon boundaries were determined, thereby obtaining final annotations, and chloroplast genome annotation information statistics are shown in tables 1 and 2 below:
TABLE 1 chloroplast Gene functional taxonomy information Table
Figure BDA0003934297670000081
Figure BDA0003934297670000091
Note that: gene represents a Gene containing one intron; gene represents a Gene comprising two introns; gene (2) represents a Gene having a copy number of more than 1, the copy number being in brackets; genes a Indicating that ycf15 is characteristic of D.g subsp.
TABLE 2 statistical table of chloroplast gene annotation information
Figure BDA0003934297670000092
Note that: numberofgenes indicates the number of genes; tRNA means the number on tRNA annotation; rRNA represents the number on rRNA annotation; mRNA represents the number of mRNAs.
4. SSR locus distributed in duck grass chloroplast genome
Searching SSR sites distributed in the festuca arundinacea chloroplast genome by using MISSferl script software, wherein parameters are set as follows: a single nucleotide repeat sequence, the repeat unit is more than or equal to 8; a dinucleotide repeat sequence, the repeat unit is more than or equal to 5; trinucleotide repeat sequence, the repeat unit is more than or equal to 3; 4. five and six nucleotide repetitive sequences, the repetitive unit is more than or equal to 3; the statistics of the searched dactylogyrus cpSSR site information are shown in table 3 below.
TABLE 3 Duck grass cpsSR site information
Figure BDA0003934297670000101
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Figure BDA0003934297670000111
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Figure BDA0003934297670000121
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Figure BDA0003934297670000131
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Figure BDA0003934297670000141
5. cpSSR primer design
The cpSSR Primer design was performed using Primer Premier 5 software according to the following Primer design principles: the primer length ranges from 20 to 22 bases; the annealing temperature is 50-60 ℃; the product length is 200-300 bp, 280 pairs of cpSR primers are designed according to the cpSR locus, the annealing temperature is preferably selected, the primer length is similar, the comprehensive experiment efficiency is realized, 30 pairs of cpSR primers are selected, the primer sequences are synthesized by Kangshen biotechnology Co., ltd (Hangzhou), 4 pairs of cpSR labeled primers of clear and high-polymorphism electrophoresis bands can be amplified in the cogongrass subspecies by screening the 30 pairs of cpSR primers, and the specific primer information is shown in the following table 4.
TABLE 4 Duck grass cpsSR marker primer information
Figure BDA0003934297670000142
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Figure BDA0003934297670000151
Experimental example 2 application verification of labeled primer
In order to verify the applicability of the 4 pairs of the festuca arundinacea cpSSR marker primers provided by the invention to the distinction of ducks Mao Yachong, the experiment utilizes the developed polymorphic cpSSR primers to carry out group structure analysis on 31 ducks Mao Yachong, and the specific steps are as follows:
1) Extraction of genomic DNA: firstly, 31 parts of tender leaves of duck Mao Yachong are collected, plant total DNA is respectively extracted, DNA integrity is detected by 1% agarose gel electrophoresis, purity detection and concentration quantification are carried out on the extracted DNA by using Nanodrop 2000, and finally, the DNA concentration of each sample is diluted to 20 ng/. Mu.L.
The source information of the ducks Mao Yachong with 31 young leaves is shown in the following table 5:
table 5 31 ducks Mao Yachong sample information
Figure BDA0003934297670000152
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Figure BDA0003934297670000161
2) cpSSR-PCR reaction: performing PCR amplification using the 31 sample DNAs to be tested extracted in step 1) as templates and using 4 pairs of the labeled primers (cpSSR 1, cpSSR2, cpSSR3, cpSSR 4) shown in the above Table 3 to obtain PCR amplification products; a total of 10. Mu.L of the PCR amplification system contained 1. Mu.L of the template DNA, 0.5. Mu.L of the upstream and downstream primers (5 pmol. Mu.L) -1 )，5μL 2×TaqPCR MasterMix II，3μL ddH 2 O; the PCR amplification procedure was as follows: pre-heating at 94 °cDenaturation for 5min; then denaturation at 94 ℃ for 30s, annealing at 57 ℃ for 30s and extension at 72 ℃ for 35s, for 35 cycles; finally, the mixture is extended at 72 ℃ for 5min and stored at 4 ℃.
3) And (3) electrophoresis detection: carrying out polymorphism detection on the amplification product obtained in the step 2) by 8% polyacrylamide gel electrophoresis, wherein an electrophoresis buffer solution is 1 XTBE, 200V is stabilized for 20min, and 280V is stabilized for 4h; the electrophoresis results of the amplified products of the four pairs of primers for 31 templates to be detected are respectively shown in the figures 1-4, wherein the figure 1 is the amplification electrophoresis result of the primer pair cpSSR1, the figure 2 is the amplification electrophoresis result of the primer pair cpSSR2, the figure 3 is the amplification electrophoresis result of the primer pair cpSSR3, the figure 4 is the amplification electrophoresis result of the primer pair cpSSR4, and the lane M is a 50bp DNA Ladder (Tiangen, MD 108); lanes 1-31 correspond to the 31 cogongrass germplasm described above, respectively. Counting clear bands in an electrophoresis result, and adopting a principle that the band is marked as '1', and the non-band is marked as '0'; the number of amplified electrophoresis bands of each pair of the festuca arundinacea cpSSR marker primers is 3 (cpSSR 3, cpSSR 4) to 9 (cpSSR 2), and the statistical result of the genetic diversity of the 4 pairs of the festuca arundinacea cpSSR marker primers is shown in table 6.
TABLE 6 statistics of genetic diversity of Duck grass cpSSR markers
Figure BDA0003934297670000171
As can be seen, the average of the amplified bands of each pair of primers is 5.25, the length of the actual amplified product is between 194 and 310bp, and the total amplified bands of 4 pairs of cpSSR primers are 21, wherein the number of polymorphic sites is 9, and the proportion of the polymorphic sites is 42.8%. The average number of primer polymorphic sites in each pair is 2.25, and the polymorphism ratio of each pair of cpSSR is 33.33-50%.
4) The result is shown in figure 5 by constructing a germplasm population STRUCTURE diagram by using STRUCTURE software, and the result shows that the 4 pairs of Duck grass cpSSR marker primers provided by the invention can cluster and distinguish ducks Mao Yachong.
5) Development of duck Mao Zhiwen map: based on the developed 4 pairs of cpSSR marker primers, a fingerprint for identifying different subspecies of festuca arundinacea was developed for 14 different ducks Mao Yachong, and the results are shown in fig. 6. It can be known that the 4 pairs of cpSSR marking primers provided by the invention have high-degree genetic variation in the cogongrass subspecies or among the species, have simple structure and easy development, have the characteristics of co-dominance and high variation of the nuclear genome SSR molecular markers, and have the characteristic of single parent genetic mode of cytoplasm at the same time, and are not easy to recombine.
In conclusion, the festuca arundinacea cpSSR polymorphism marker primer provided by the invention can be used for germplasm genetic diversity analysis of the festuca arundinacea Mao Yachong and related species thereof, can rapidly identify the duck Mao Yachong based on a developed cpSSR molecular marker fingerprint, provides convenience for distinguishing and identifying the duck Mao Yachong, and lays a foundation for researches such as genetic diversity evaluation, germplasm identification and molecular marker assisted breeding of the festuca arundinacea.
While the present invention has been described in detail through the foregoing description of the preferred embodiment, it should be understood that the foregoing description is not to be considered as limiting the invention. Many modifications and substitutions of the present invention will become apparent to those of ordinary skill in the art upon reading the foregoing. Accordingly, the scope of the invention should be limited only by the attached claims.

Claims (7)

1. A set of festuca arundinacea cpSSR marker primers comprising 4 pairs of polymorphic marker primers having the nucleotide sequences shown below:
the cpSSR1 forward primer is shown as SEQ ID NO. 1;
the reverse primer of the cpSSR1 is shown as SEQ ID NO. 2;
the cpSSR2 forward primer is shown as SEQ ID NO. 3;
the cpsSR2 reverse primer is shown as SEQ ID NO. 4;
the cpSSR3 forward primer is shown as SEQ ID NO. 5;
the cpSSR3 reverse primer is shown as SEQ ID NO. 6;
the cpSSR4 forward primer is shown as SEQ ID NO. 7;
the reverse primer of the cpSSR4 is shown as SEQ ID NO. 8.
2. A kit comprising the cogongrass cpSSR marker primer of claim 1.
3. A method for preparing the cogongrass cpSSR labeled primer according to claim 1, comprising the steps of:
1) Extracting chloroplast DNA of the festuca arundinacea;
2) Carrying out high-throughput Illumina sequencing on the chloroplast DNA obtained in the step 1);
3) Filtering the raw data obtained by sequencing in the step 2) by using fastp software;
4) Assembling chloroplast genome of the data obtained by filtering in the step 3) by adopting SPades 2 software;
5) Searching SSR sites distributed in the duck Mao Yachong chloroplast genome obtained in the step 4) by using MISAPERL script software;
6) Designing a cpSSR Primer by using Primer Premier 5 software, and screening to obtain 4 pairs of festuca arundinacea cpSSR labeled primers according to claim 1;
wherein, the parameters of the MISAAperl script software in the step 5) are set as follows: a single nucleotide repeat sequence, the repeat unit is more than or equal to 8; a dinucleotide repeat sequence, the repeat unit is more than or equal to 5; trinucleotide repeat sequence, the repeat unit is more than or equal to 3; 4. five and six nucleotide repetitive sequences, the repetitive unit is more than or equal to 3;
the design principle of the cpSSR primer design in the step 6) is as follows: primer length ranges from 20 bases; the annealing temperature is 50-60 ℃; the product length is 200-300 bp.
4. The use of a cogongrass cpSSR marker primer according to claim 1 for the structural and species identification of a duck Mao Yachong population.
5. A method for identifying the structure and species of a population of ducks Mao Yachong based on the cogongrass cpSSR marker primer of claim 1, comprising the steps of:
1) Extracting genome DNA from young leaves of the duck Mao Yachong to be detected;
2) Performing PCR amplification using the genomic DNA obtained in step 1) as a template and 4 pairs of the cpsSR labeled primers as set forth in claim 1;
3) Carrying out polymorphism detection on the product obtained by amplification in the step 2) through electrophoresis, and carrying out statistics on clear bands;
4) And 3) constructing a germplasm population structure diagram according to the statistical data obtained in the step 3), namely obtaining the result of the duck Mao Yachong population structure and species identification.
6. The method according to claim 5, wherein the PCR amplification in the step 2) is performed with a PCR amplification system of 10. Mu.L: comprises 1. Mu.L of template DNA, 0.5. Mu.L of 5 pmol. Mu.L -1 5. Mu.L of 2X TaqPCR MasterMix II, 3. Mu.L of ddH 2 O; the PCR amplification procedure is as follows: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, annealing at 57℃for 30s, extension at 72℃for 35s, for a total of 35 cycles; extending at 72deg.C for 5min, and preserving at 4deg.C.
7. The method according to claim 5, wherein the electrophoresis buffer in step 3) is 1 XTBE and the electrophoresis apparatus parameters are 200V 20min and 280V 4h.
CN202211397714.1A 2022-11-09 2022-11-09 Duck grass cpsSR labeled primer and application thereof Pending CN116042891A (en)

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Citations (1)

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Publication number Priority date Publication date Assignee Title
JP2011211948A (en) * 2010-03-31 2011-10-27 Japan Grassland Farming Forage Seed Association Ssr primer pair useful for breed identification of dactylis glomerata, and utilization thereof

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JP2011211948A (en) * 2010-03-31 2011-10-27 Japan Grassland Farming Forage Seed Association Ssr primer pair useful for breed identification of dactylis glomerata, and utilization thereof

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