CN113151399A - SSR (simple sequence repeat) marker primer group of cyperus esculentus developed based on Super-GBS (Super-GBS) technology and application of SSR marker primer group - Google Patents
SSR (simple sequence repeat) marker primer group of cyperus esculentus developed based on Super-GBS (Super-GBS) technology and application of SSR marker primer group Download PDFInfo
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Abstract
The invention relates to the technical field of molecular biology, in particular to a cyperus esculentus SSR marker primer group developed based on a Super-GBS technology and application thereof. The invention comprises the following steps: (1) performing Super-GBS sequencing on the cyperus esculentus, clustering and assembling sequencing data by using Stack software, and constructing a reference-free genome sequence of the cyperus esculentus; (2) carrying out SSR marker development on the genome sequence obtained in the step (1) by using MISA software to obtain an SSR marker covering the cyperus esculentus genome; (3) designing a primer based on the SSR marker obtained in the step (2) to obtain an SSR marker primer group. The primer group designed by the invention can realize accurate identification and screening of the germplasm resources or varieties of the cyperus esculentus, and has the characteristics of rapidness, simplicity, convenience, low cost and the like.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to a cyperus esculentus SSR marker primer group developed based on a Super-GBS technology and application thereof.
Background
The cyperus esculentus is the only known crop which accumulates a large amount of grease in underground tubers, and due to wide introduction and breeding, the original genetic basis of the cyperus esculentus cannot be traced, and the germplasm resources are deficient and disordered; in addition, the cyperus esculentus is mainly bred through underground tubers, so that the tillering force is strong, and mechanical mixing is easily caused during harvesting. At present, the identification of the germplasm resources of the cyperus esculentus is mainly realized by distinguishing the phenotypes such as tuber shape, size, color and the like, and the accuracy and the reliability are poor; meanwhile, the appearance is susceptible to environmental factors, and the identification error can be caused. The similarity and the source tracing of the form characteristics of the cyperus esculentus are unclear, and great difficulty is caused for accurate identification and reasonable utilization of the cyperus esculentus resources, particularly for seed selection and variety right protection of the cyperus esculentus varieties.
The microsatellite marker SSR (simple Sequence repeats) is a DNA Sequence which takes 1-6 nucleotides as units and is in tandem repetition. Compared with other molecular markers, the SSR marker has the characteristics of codominance, high polymorphism, good repeatability, simple operation and the like, is widely applied to the research in the fields of biodiversity research, germplasm resource or variety classification, genetic map construction, molecular marker-assisted selective breeding, comparative genomics and the like, and is taken as one of the first-choice DNA fingerprint marking technologies by the International New breed conservation Association. However, the whole genome sequence of the cyperus esculentus is not published at present, the existing research mainly utilizes random-designed RAPD or SRAP primers for detection, the specificity and stability of amplified fragments are poor, and data integration among different molecular markers is difficult, so that the cyperus esculentus is not widely applied. On the basis, a set of SSR markers with abundant polymorphism and high coverage are urgently needed to be developed, and scientific basis is provided for identification of cyperus esculentus germplasm resources, variety breeding and protection, purity detection and the like.
Disclosure of Invention
The invention aims to provide a set of SSR markers with abundant polymorphism and high coverage, and particularly relates to a cyperus esculentus SSR marker primer group developed based on a Super-GBS technology and application thereof.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a method for developing an SSR labeled primer group of cyperus esculentus based on a Super-GBS technology, which comprises the following steps:
(1) performing Super-GBS sequencing on the cyperus esculentus, clustering and assembling sequencing data by using Stack software, and constructing a reference-free genome sequence of the cyperus esculentus;
(2) carrying out SSR marker development on the genome sequence obtained in the step (1) by using MISA software to obtain an SSR marker covering the cyperus esculentus genome;
(3) designing a primer based on the SSR marker obtained in the step (2) to obtain an SSR marker primer group.
Preferably, the parameters for designing the primer in step (3) are as follows: product size range: 100-600 bp; number to return: 3; max 3' stability: 2 kcal/mol; max mistrating: 8; PrimerTm is 50-65 bp; maximum Tm difference: 5 ℃ is adopted.
The invention also provides a cyperus esculentus SSR marker primer group constructed by the method, which is shown as SEQ ID No. 1-24.
The invention also provides application of the primer group in genetic diversity analysis of cyperus esculentus germplasm resources.
Preferably, the method for analyzing genetic diversity of the cyperus esculentus germplasm resources comprises the following steps:
(1) amplifying cyperus esculentus genomic DNA by using the primer set according to claim 3 to obtain an amplification product;
(2) carrying out fluorescence electrophoresis detection on the amplification product to obtain an SSR locus polymorphism amplification band;
(3) and (3) carrying out genetic diversity analysis on the cyperus esculentus according to the SSR locus polymorphism amplification band.
Preferably, the primer group for amplifying the Cyperus esculentus genomic DNA further comprises a modification step;
the modification is that M13 adaptor sequence with fluorescent group is added at the 5' end of the forward primer in the primer group;
the fluorescent group is HEX and FAM;
the M13 linker sequence is shown in SEQ ID No. 25.
The invention also provides application of the primer group in constructing the DNA fingerprint of the cyperus esculentus.
Preferably, the method for constructing the DNA fingerprint of the cyperus esculentus comprises the following steps:
(1) amplifying cyperus esculentus genomic DNA by using the primer set according to claim 3 to obtain an amplification product;
(2) carrying out fluorescence electrophoresis detection on the amplification product to obtain an SSR locus polymorphism amplification band;
(3) and constructing a DNA fingerprint of the cyperus esculentus according to the polymorphism amplification band of the SSR locus.
Preferably, the primer group for amplifying the Cyperus esculentus genomic DNA further comprises a modification step;
the modification is that M13 adaptor sequence with fluorescent group is added at the 5' end of the forward primer in the primer group;
the fluorescent group is HEX and FAM;
the M13 linker sequence is shown in SEQ ID No. 25.
The invention provides a cyperus esculentus SSR marker primer group developed based on a Super-GBS technology and application thereof. The primer design method of the invention can obtain 12 pairs of SSR primer groups, which have the advantages of high coverage rate, abundant polymorphism, stable amplification and easy identification of amplified bands. The method realizes identification and screening of the cyperus esculentus germplasm or variety by combining joint fluorescent labeling, capillary electrophoresis separation and fluorescent detection technologies, realizes simple, reliable and high-throughput detection, can be applied to researches such as cyperus esculentus genetic diversity research, germplasm resource classification, DNA (deoxyribonucleic acid) map construction, variety purity identification and the like, and provides a basis for cyperus esculentus germplasm resource identification, cyperus esculentus variety right confirmation and the like on a molecular level.
Drawings
FIG. 1 is a diagram of a detection peak of a cyperus esculentus resource (wherein the upper diagram is JYD-35, the middle diagram is JYD-36, the lower diagram is JYD-42, and amplification primers are F1).
FIG. 2 is a phylogenetic tree plotting genetic distance of Cyperus esculentus resource Nei's.
Detailed Description
The resource information of 42 parts of cyperus esculentus described in the examples of the present invention is shown in table 1.
TABLE 1 Cyperus esculentus germplasm resource information
The method for extracting the genomic DNA of the cyperus esculentus, which is described in the example, comprises the following steps:
1) sample treatment: weighing 50mg of cyperus esculentus leaf tissue, fully grinding the cyperus esculentus leaf tissue by using liquid nitrogen, putting the ground cyperus esculentus leaf tissue into a centrifuge tube filled with 800 mu l of preheated 2 xCTAB extraction buffer solution, adding 60 mu l of mercaptoethanol, uniformly mixing, treating at 60 ℃ for 30min, and uniformly mixing for 3 times;
2) the tube was removed and cooled in ice for 5min, then 800. mu.l chloroform was added: isoamyl alcohol (24:1), shaking and mixing evenly, and centrifuging at 12000rpm for 15 min;
3) the supernatant was aspirated into a 2ml centrifuge tube, and an equal volume of chloroform was added: isoamyl alcohol (24:1), shaking and mixing evenly, and centrifuging at 12000rpm for 15 min;
4) sucking the supernatant into a new 2ml centrifuge tube, adding 1 × CTAB precipitation solution with the volume of 1.5 times, standing at room temperature for 20-30 min, and centrifuging at 12000rpm for 15 min;
5) discarding the supernatant, standing the precipitate at room temperature for 10min, and adding 200. mu.l TE buffer solution for dissolution;
6) adding 400 μ l 95% ethanol, 20 μ l 3M NaAC, precipitating at-20 deg.C for 1 hr, centrifuging at 4 deg.C, and centrifuging at 12000rpm for 15 min;
7) discarding the supernatant, adding 500 μ l of 75% ethanol to wash the precipitate, and centrifuging at 12000rpm for 10 min;
8) and removing the supernatant, placing the precipitate at room temperature for 10-5 min, and adding 30-50 mu l of TE buffer solution for dissolving to obtain the cyperus esculentus DNA.
In the PCR amplification of Cyperus esculentus described in the examples, the PCR system (25. mu.l): 10 XPCR Buffer 2.5. mu.l, dNTPs (10mM) 0.5. mu.l, upstream and downstream primers (10. mu.M) 0.5. mu.l each, DNATaq enzyme (5U/. mu.l) 0.5. mu.l, template DNA 2.0. mu.l, and ddH2O to 25. mu.L.
PCR reaction procedure: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 45s, extension at 72 ℃ for 30s, 35 cycles, and extension at 72 ℃ for 10 min.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1 Cyperus esculentus genome sequence SSR marker development
And performing Super-GBS sequencing by using 42 portions of cyperus esculentus resources collected at home and abroad. Sample DNA extraction, library construction and sequencing are finished by Shanghai Europe and Yi biomedicine science and technology Limited, and the sequencing platform is Illumina Hiseq XtenPE 150. And (3) filtering and quality controlling original sequencing data, and clustering and assembling by utilizing an ustacks module of Stack software to construct a reference-free genome sequence of the cyperus esculentus.
SSR marker development was performed on Cyperus esculentus genomic sequences using MISA (v2.0, https:// weblast. ipk-gatersleen. de/MISA /) software. A total of 540618 SSR markers were obtained distributed over 79 scaffolds, with an average of 6843 SSR markers per Scaffold. Wherein, the number of the labels of the mononucleotide which is repeated more than 10 times accounts for 73.4 percent, the number of the labels of the dinucleotide which is repeated more than 6 times accounts for 16.4 percent, the number of the labels of the trinucleotide which is repeated more than 5 times accounts for 6.7 percent, the number of the labels of the tetranucleotide which is repeated more than 5 times accounts for 2.2 percent, the number of the labels of the pentanucleotide which is repeated more than 5 times accounts for 0.9 percent, and the number of the labels of the hexanucleotide which is repeated more than 5 times accounts for 0.4 percent.
In order to further improve the SSR precision, a sequence with intersection of an SSR marker in a genome sequence and an InDel sequence is extracted. The InDel sequence parameters are: the number of the variant bases is more than or equal to 5, and the variant bases are positioned in the sequence within 5bp of the upstream and downstream of the SSR region. After repeated marker sequences are removed, 58 Scaffold and 512 SSR markers covering the Cyperus esculentus genome are finally obtained, wherein the number of markers of mononucleotide repeated for more than 10 times accounts for 59.2%, the number of markers of dinucleotide repeated for more than 6 times accounts for 20.3%, the number of markers of trinucleotide repeated for more than 5 times accounts for 8.8%, the number of markers of tetranucleotide repeated for more than 5 times accounts for 6.4%, the number of markers of pentanucleotide repeated for more than 5 times accounts for 3.1%, and the number of markers of hexanucleotide repeated for more than 5 times accounts for 2.1%.
Example 2 Cyperus esculentus genome SSR marker primer design
According to the 512 SSR marker sites obtained in example 1, 71 maximum SSR sequences were selected from 58 scaffold. Primers were designed using primer (v3.2.5.0) software. The parameters are set as follows: product size range: 100-600 bp; number to return: 3; max 3' stability: 2 kcal/mol; max mistrating: 8; the Primer Tm is 50-65 bp; maximum Tm difference: 5 ℃ is adopted. The sequence of the 71 pairs of primers obtained by design is synthesized by Beijing ancient China Changsheng biotechnology Limited liability company.
And performing PCR amplification and capillary electrophoresis fluorescence detection on the randomly selected 15 cyperus esculentus resources by using the designed 71-pair SSR marker primer group. The result shows that the designed 71 pairs of SSR marker primers can be effectively amplified in the DNA of the cyperus esculentus genome, wherein more than 80 percent of primer amplification bands show abundant polymorphism, and the SSR marker developed according to the cyperus esculentus genome is effective. On the basis, 12 pairs of core SSR marker primers with rich polymorphism and good stability are selected. The sequences of the 12 pairs of SSR marker primers are shown in SEQ ID No. 1-24 and Table 2.
TABLE 2.12 pairs of SSR core primer sets for Cyperus esculentus
| Primer numbering | ScaffoldID | Forward primer (5'-3') | Reverse primer (5'-3') |
| F12 | Scaffold_05 | GTTGATGATGCCTTTTAGAAAGCC | ACCAAAAGAAAAGAAAGGGACCAG |
| F41 | Scaffold_28 | ATGATCTTAGGGTCGACCAACAAA | ATGTTGTTTGATCTTGGGTCAGGT |
| F3 | Scaffold_01 | GCAGAGAGGGAAAACAAATTCAGA | GGACAGTGGAAACAAATTAATCCG |
| F69 | Scaffold_56 | CCAGCAATCCAAGATGTTAAAAGG | GTTGAAACTTGAAACAGCTGCAGA |
| F62 | Scaffold_49 | GTAAAAACGACCGGTAAAGTGTGC | TCCCCGGGTATCCTCTAACAGTAT |
| F38 | Scaffold_25 | TCACAAAGTAGTTCCGCCACTGTA | AATCCCTTCTTTATGGGTCCACAT |
| F36 | Scaffold_23 | TACAAGTGACCTGAGGTGGGAAAT | ATTCGTCAGGTTGTGTCAAACGTA |
| F31 | Scaffold_19 | TAGGTGCATTGTAATGACCGAATG | GGCTCATTACCCAAAACATGAAAG |
| F1 | Scaffold_01 | TGTGAAGTTGATACATTGCCGTCT | GAACTCCATCTGTTGATGTGTTGC |
| F67 | Scaffold_54 | TATTGGGGGCAGTACAGTTCAAGT | GTTAAGTGGTTCTCATGCCAGGTT |
| F42 | Scaffold_29 | CGGTCCTCTCCAAATAAAGCTTTT | ACACCAAGTCCATGAATTAGCACA |
| F71 | Scaffold_71 | ACTCAGAAAAAGGCAAAACTTCCC | TTGAGTCAATGCTTCATGCTTCTC |
Example 3 application of SSR-labeled primer group in genetic diversity analysis of cyperus esculentus resources
42 parts of cyperus esculentus genome DNA is extracted, and the concentration of the extracted cyperus esculentus genome DNA is diluted to 50 ng/. mu.L to be used as a PCR amplification template.
M13 linker sequence was added to the 5' -end of the forward primers in the 12 primer sets obtained in example 2, and M13 linker sequence with two fluorophores (HEX and FAM) was synthesized. And carrying out PCR amplification on 42 parts of cyperus esculentus resource genome DNA by using primers with different fluorescent group joint sequences to obtain an amplification product.
Mu.l of the amplified product, 9.5. mu.l of formamide and 0.5. mu.l of ROX500 molecular weight internal standard were added to a 96-well loading plate. Automated fluorescence electrophoresis detection was performed using an ABI3730X genetic Analyzer, the raw data was band-typed using the software GeneMarkerV2.2.0, and the fragment size at each site was recorded. A partial Cyperus esculentus resource sequencing peak diagram is shown in FIG. 1.
Data were analyzed by automated statistics using GeneScan 500LIZ Size Standard analysis software and valid peaks were converted to "0, 1" original matrices. The polymorphism Information Content index (PIC) of each SSR locus is calculated by using Cervus software, and the polymorphism degree of each allele is counted. Genetic distances of Cyperus esculentus resources Nei's are calculated by using GeneAIEx software, and a phylogenetic tree is drawn by using a UPGMA method of Phy1ip software, and the results of the phylogenetic tree are shown in FIG. 2.
The results show that: the PIC value of the detected 42 cyperus esculentus resources is between 0.19 and 0.712 (the average value is 0.44), which indicates that different cyperus esculentus resources have higher genetic diversity, and different alleles have different contributions to the cyperus esculentus polymorphism. 42 portions of cyperus esculentus resources can be divided into 3 groups, wherein JYD-35 and JYD-36 are divided into one group, JYD-14 and JYD-16 are divided into one group, and 38 portions of resources are divided into 1 group. The genetic distance between 3 populations is 0.39-0.91, showing large genetic diversity. In addition, the cyperus esculentus resource JYD-22 from Jiangsu province is the same as the band type JYD-23 from Jilin province in size, is consistent with the phenotype and possibly is the same germplasm resource.
Example 4 application of SSR marker primer group in construction of cyperus esculentus resource fingerprint
The 12 pairs of primer sets were fluorescently labeled according to the method of example 3, 42 cyperus esculentus resources were subjected to PCR amplification, and fluorescence detection by capillary electrophoresis. Each pair of SSR primers can amplify different polymorphic bands. According to the fragment size of each locus, the allele of each locus of each cyperus esculentus resource is encoded into a fingerprint in a 01 matrix form, and the fingerprint of the cyperus esculentus resource is obtained by statistics and is shown in table 3.
TABLE 3 Cyperus esculentus resource fingerprint
The embodiments show that the invention provides a cyperus esculentus SSR marker primer group developed based on a Super-GBS technology and application thereof. The 12-pair SSR primer group of the cyperus esculentus provided by the invention has the advantages of rich polymorphism, high coverage, stable amplification and easiness in identification. The method combines joint fluorescent labeling, capillary electrophoresis separation and fluorescent detection technologies, realizes simple, reliable and high-throughput detection, can be applied to researches on genetic diversity of the cyperus esculentus, germplasm resource classification, DNA (deoxyribonucleic acid) map construction, variety purity identification and the like, and provides a basis for cyperus esculentus germplasm resource identification, cyperus esculentus variety authentication and the like on a molecular level.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
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Claims (9)
1. A method for developing SSR labeled primer groups of cyperus esculentus based on a Super-GBS technology is characterized by comprising the following steps:
(1) performing Super-GBS sequencing on the cyperus esculentus, clustering and assembling sequencing data by using Stack software, and constructing a reference-free genome sequence of the cyperus esculentus;
(2) carrying out SSR marker development on the genome sequence obtained in the step (1) by using MISA software to obtain an SSR marker covering the cyperus esculentus genome;
(3) designing a primer based on the SSR marker obtained in the step (2) to obtain an SSR marker primer group.
2. The method of claim 1, wherein the parameters for designing the primer in step (3) are: product size range: 100-600 bp; number to return: 3; max 3' stability: 2 kcal/mol; max mistrating: 8; the Primer Tm is 50-65 bp; maximum Tm difference: 5 ℃ is adopted.
3. The SSR labeled primer group of the cyperus esculentus constructed by the method according to claim 1 or 2 is characterized by being shown in SEQ ID No. 1-24.
4. The use of the primer set of claim 3 in genetic diversity analysis of Cyperus esculentus germplasm resources.
5. The use according to claim 4, wherein the method for genetic diversity analysis of Cyperus esculentus germplasm resources comprises the following steps:
(1) amplifying cyperus esculentus genomic DNA by using the primer set according to claim 3 to obtain an amplification product;
(2) carrying out fluorescence electrophoresis detection on the amplification product to obtain an SSR locus polymorphism amplification band;
(3) and (3) carrying out genetic diversity analysis on the cyperus esculentus according to the SSR locus polymorphism amplification band.
6. The use according to claim 5, wherein the primer set for amplifying Cyperus esculentus genomic DNA further comprises a modification step;
the modification is that M13 adaptor sequence with fluorescent group is added at the 5' end of the forward primer in the primer group;
the fluorescent group is HEX and FAM;
the M13 linker sequence is shown in SEQ ID No. 25.
7. Use of the primer set of claim 3 for constructing a DNA fingerprint of Cyperus esculentus.
8. The use according to claim 7, wherein the cyperus esculentus DNA fingerprint is constructed by a method comprising the following steps:
(1) amplifying cyperus esculentus genomic DNA by using the primer set according to claim 3 to obtain an amplification product;
(2) carrying out fluorescence electrophoresis detection on the amplification product to obtain an SSR locus polymorphism amplification band;
(3) and constructing a DNA fingerprint of the cyperus esculentus according to the polymorphism amplification band of the SSR locus.
9. The use according to claim 8, wherein the primer set for amplifying the genomic DNA of Cyperus esculentus further comprises a modification step;
the modification is that M13 adaptor sequence with fluorescent group is added at the 5' end of the forward primer in the primer group;
the fluorescent group is HEX and FAM;
the M13 linker sequence is shown in SEQ ID No. 25.
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