CN108034696B - Method for developing SSR primers based on transcriptome sequencing - Google Patents
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Abstract
The invention discloses a method for developing SSR primers based on transcriptome sequencing. The method screens candidate SSR loci and designs SSR amplification primers aiming at the genomic exon sequences based on the existing whole genome sequence data and the annotations thereof. And then carrying out ePCR verification based on the sequencing data of a plurality of individual transcriptomes, finally screening SSR sites with good polymorphism and obtaining amplification primers thereof. Compared with the laboratory PCR and polyacrylamide gel electrophoresis adopted in the past, the method provided by the invention greatly reduces the development period and manpower, and compared with the screening method adopting the laboratory PCR and sequencing typing, the method greatly reduces the development period and development cost. The method provided by the invention is not only suitable for the development of the SSR primers, but also suitable for the development of all SSR primers with reference genome species, and has wide application prospect.
Description
Technical Field
The invention belongs to the technical field of SSR primer development, and particularly relates to a novel method for developing SSR primers.
Background
Microsatellite markers (STRs), also known as Short Tandem Repeats (STRs) or simple sequence repeats (simple sequence repeats), are simple repeats that are uniformly distributed in eukaryotic genomes, and consist of tandem repeats of 2-6 nucleotides, and are highly variable and abundant in number among individuals due to the number of repeats of the repeat unit.
The most difficult to separate microsatellite markers is the acquisition of primers, unlike genetic markers such as RAPD, AF L P, and the like, microsatellite research firstly needs to know the sequence information of sites so as to design primers from conserved sequences flanking both ends of repeated sequences, so the development of the microsatellite primers is the key to the application of the technology.
Microsatellite loci are currently amplified by PCR, and the amplified products are detected by electrophoretic analysis or sequencing analysis and allele separation by size. In fact, SSR sites developed from large genomic data tend to be large in number and poorly polymorphic. This poses a problem in that the recognition of SSR loci is still very labor intensive. Researchers often need to design hundreds of pairs of primers, amplify thousands of samples, and perform electrophoresis or sequencing verification on them one by one, and not only the cost of manpower, material resources and financial resources is not a little great, but also the finally obtained effective SSR sites are very limited (dozens to dozens of pairs). Therefore, developing a set of efficient and rapid SSR primer development method has great practical significance for developing the research of SSR loci.
In addition, existing SSR marker development methods are divided into genome-based SSR development and SSR development based on expressed sequence tags. The SSR marker developed based on the expression sequence has better conservation and higher efficiency in the related species. Moreover, the SSR marker based on the expression data is based on a gene expression sequence in a certain period, is directly related to gene functions and trait phenotypes, and has important research value in the aspects of precious or endangered animal and plant germplasm resources, genetic diversity evaluation and the like. With the big outbreak of sequencing data, mining and developing new SSR markers from transcriptome data becomes a simple and efficient way. However, the current methods for developing SSR markers based on transcriptome sequencing still have the situation that a large number of verification tests are required.
Disclosure of Invention
The invention aims to provide a novel method for developing SSR marker primers based on transcriptome sequencing, which is characterized in that SSR sites are searched from exon sequences of a genome, corresponding PCR amplification primers are designed, ePCR verification is performed through exons, and finally ePCR verification is performed through transcripts obtained by individual non-reference assembly, so that SSR markers with good polymorphism and primers thereof are obtained finally. The method provides a new efficient and rapid way for researching the genetic diversity of species and identifying the germplasm resources.
In order to achieve the technical purpose, the method for developing SSR primers based on transcriptome sequencing comprises the following steps:
(1) collecting samples of at least 2 different individuals, respectively extracting total RNA and constructing a transcriptome sequencing library;
(2) transcriptome sequencing and data filtering;
(3) independently carrying out transcript non-parameter assembly on each sample to obtain all transcripts of each individual;
(4) SSR screening and primer design based on exon sequences: downloading a reference genome sequence from a database, extracting all exon sequences, identifying SSR loci by using GMATA2.0 software, and designing a primer; carrying out ePCR on the exon sequence of the obtained primer by using the ePCR function of GMATA2.0, and excluding primer pairs with poor specificity;
(5) ePCR validation of SSR primers: and (3) carrying out ePCR on the SSR primer screened in the step (4) by using the ePCR function of GMATA2.0 on the transcript of each individual obtained in the step (3), and screening the primer from the result.
The specific process for extracting total RNA and constructing the transcriptome sequencing library in the step (1) comprises the steps of extracting the total RNA by using QIAGEN RNeasy protective Animal Blood Kit (73224) and checking the quality of the RNA by agarose gel electrophoresis, and constructing the transcriptome sequencing library by using VAHTSTM mRNA-seq v 2L infectious Prep Kit for
(NR602-01) kit, wherein 1. mu.g of total RNA is taken from each sample, mRNA is obtained by separating and purifying with poly (A) magnetic beads, and the mRNA is fragmented by treating divalent cations in VAHTS Frag/Prime Buffer at 98 ℃ for 8 minutes; purification followed by first and second strand cDNA synthesis, followed by end filling, dATP endcapping and linker ligation; agarose gel electrophoresis was performed to select the 150-and 200-bp fragment for magnetic bead purification, followed by amplification of the RCR library: denaturation at 98 ℃ for 10s, binding at 60 ℃ for 30s, extension at 72 ℃ for 30s, and cyclic amplification for 12 times; finally, the library quality is checked and confirmed before sequencing.
The concrete process of transcriptome sequencing and data filtering in the step (2) is as follows: sequencing the mRNA library by adopting IlluminaHiseq 2500; and filtering out low-quality sequences and adaptor-polluted sequences in sequencing data, wherein the low-quality sequences are sequences with N content exceeding 30% or low-quality base content exceeding 10%, and then performing quality detection by using fastqc software default parameters to ensure that the data are effective.
The specific process of assembling the transcript in the step (3) without reference is as follows: and (3) adopting a Trinity reference-free genome assembly process, independently assembling the transcript to each sample by default parameters, and obtaining the own Trinity.
The search criteria of the SSR identified in the step (4) are as follows: the minimum repeat motif is a dinucleotide, the maximum repeat motif is a decade of nucleotides; a minimum of 5 repeats in the exon sequence.
The conditions for designing the primers in the step (4) are as follows: the length of the amplified product is between 120bp and 400 bp; the length of the product flanking sequence is 400 bp; the optimal annealing temperature Tm is 60 ℃; the maximum template length is 2000 bp.
The ePCR process parameters in the step (4) and the step (5) are as follows: the word length is 12, the continuous word length is 1, the maximum deletion is 1, the maximum mismatch is 0, and the length of the amplification product is 100-1000 bp.
The standard of the screening primer in the step (5) is as follows: the difference of the amplified product fragments in at least two different samples is integral multiple of the length of the SSR repeat unit motif.
Compared with the prior art, the technical scheme of the invention has the following beneficial technical effects: based on transcriptome sequencing, double ePCR is utilized to carry out polymorphism detection and verification on SSR primers, compared with the existing method, the method can greatly save manpower, material resources and financial resources, and is a novel method for efficiently developing SSR markers.
Drawings
FIG. 1 shows the results of electrophoresis of two pairs of primers obtained by screening according to the present invention.
Detailed Description
The present invention is further illustrated by the following examples, which should not be construed as limiting the scope of the invention as claimed. All kit operations are carried out according to instructions, and the non-instruction part of the transcriptome library building process is carried out according to a standard process.
Example 1
First, extraction of total RNA and construction of transcriptome library
The 4 golden monkey samples of this example were collected from Hubei Shen agricultural frame national park, after the golden monkeys were anesthetized by the method of anesthetic blow tube, hind limb venous Blood was taken and stored at-80 ℃ for subsequent total RNA extraction, total RNA was extracted using QIAGENNeasy protective Animal Blood Kit (73224), and RNA quality was checked by agarose gel electrophoresis
(NR602-01) kit. Briefly, 1. mu.g of total RNA was taken from each sample, and then mRNA was isolated and purified using poly (A) beads, and fragmented by treatment with divalent cations at high temperature in VAHTS Frag/Prime Buffer (98 ℃ for 8 minutes). Purification is followed by first and second strand cDNA synthesis, followed by end filling, dATP endcapping and linker ligation. Agarose gel electrophoresis was performed to select the 150-and 200-bp fragment for magnetic bead purification, followed by amplification of the RCR library: denaturation at 98 ℃ for 10s, binding at 60 ℃ for 30s, extension at 72 ℃ for 30s, and cyclic amplification for 12 times. Finally, library quality was measured on-machine and finally quantified using Agilent2100Bioanalyzer (Agilent Technologies) and qPCR.
II, transcriptome sequencing and data filtering:
mRNA libraries were sequenced using Illumina Hiseq 2500. Low-quality sequences with N contents of more than 30% or low-quality base contents of more than 10% in the raw data were first filtered out. Linker contamination was removed using the cutadapt version 1.9. And after low-quality data and joint pollution are filtered out from sequencing data, performing quality detection by using fastqc software to ensure that the data are effective. High-quality data are assembled by adopting Trinity 2.2.0 default parameters without reference genome transcripts, and each sample is independently assembled to obtain respective Trinity. Statistics on transcript assembly are shown in table 1.
Third, SSR site search and amplification primer design
The golden monkey's genomic reference sequence (Rox v1) and the corresponding annotation files were downloaded from a RefSeq database (http:// www.ncbi.nlm.nih.gov/RefSeq). Firstly, writing perl script to extract all exon sequences from the genome sequence file according to the intron information in the annotation file. Then, the software of Windows GMATA2.0 is adopted to carry out SSR search, 2-10 nucleotide repeating units are searched, and the number of times of repetition is more than or equal to 5 SSR sites. The results collectively searched for 5011 dinucleotide repeats, 2733 trinucleotide repeats, 217 tetranucleotide repeats, 60 pentanucleotide repeats, 42 hexanucleotide repeats and 2 octanucleotide repeats in the exon region. Primer design was performed using MGATA embedded Primer 3 with the following parameters: the length of the amplified product is between 120bp and 400 bp; the length of the product flanking sequence is 400 bp; the optimal annealing temperature Tm is 60 ℃; the maximum template length is 2000 bp. Results a total of successful primer 5279 pairs were designed, randomly selected 5 pairs are shown in table 2.
Fourth, screening of SSR markers and validation of polymorphisms
ePCR was performed using GMATA embedded ePCR program and genomic exon sequences extracted in the previous step as template. The ePCR process parameters were: the word size is 12, the continuous word size is 1, the maximum deletion maxindels is 1, the maximum mismatch max mismatch is 0, and the length of the amplification product is 100-1000 bp. Then, PCR primers with multiple amplified bands were filtered out to obtain 5253 pairs of SSR primers. The process aims to eliminate unqualified primers amplified to multiple bands in the candidate SSR primers and SSR sites sharing the same pair of primers so as to avoid interfering with subsequent SSR polymorphism verification. Then, ePCR amplification is carried out on all SSR primers by taking a transcript sequence assembled from each sample as a template. The numbers of SSR markers successfully amplified by the four samples are respectively as follows: 1520 samples S1 in total; 1502 samples S2; 888 samples S3; 755 samples S4; there were 1305 SSR sites that were successfully amplified in at least two samples. SSR sites with polymorphic products in all samples in the statistical results. Criteria for screening for well polymorphic primers were: the amplified product fragments differ in at least two different samples. As a result, a total of 17 pairs of SSR primers and amplification primers having polymorphisms were obtained (Table 3, Table 4). Product fragment length the more polymorphic fragments in all samples the better the SSR primer.
Fifth, verification of polymorphism of SSR primer in 9 golden monkeys
And in addition, 9 golden monkey sample transcriptome data are respectively assembled into transcripts, 17 pairs of polymorphic EST-SSR primers screened in the fourth step are subjected to ePCR amplification, and the amplification results are shown in a table 5. The results showed that 17 EST-SSR sites were phenotypically polymorphic to varying degrees in these golden monkey individuals. Two pairs of primers were randomly selected for PCR experiments on 9 samples, and the results of polyacrylamide gel electrophoresis are shown in FIG. 1. The method for screening SSR markers based on transcriptome data is accurate and effective, and the primers can be applied to genetic diversity detection and genetic relationship identification of golden monkeys.
TABLE 1 results of separate assembly of 4 Simian sample transcriptomes in example 1
| Sample ID | S1 | S2 | S3 | S4 |
| Total number of transcripts | 182210 | 173808 | 81807 | 71073 |
| Length (bp) of N50 | 1504 | 1346 | 869 | 699 |
TABLE 2 part of candidate SSR markers identified based on exon sequences of golden monkey genome and corresponding amplification primers in example 1
TABLE 3 partial results of screening validation of candidate SSR markers in example 1 based on Simian Sichuan-Reptilia-transcriptome sequencing
TABLE 4 17 pairs of SSR with polymorphisms and amplification primer sequences screened in example 1
TABLE 5
Sequence listing
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<213> Unknown (Unknown)
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Claims (7)
1. A method for developing SSR primers based on transcriptome sequencing is characterized in that: the method comprises the following steps:
(1) collecting samples of at least 2 different individuals, respectively extracting total RNA and constructing a transcriptome sequencing library;
(2) transcriptome sequencing and data filtering; sequencing the mRNA library by adopting Illumina Hiseq 2500; filtering low-quality sequences and adaptor-contaminated sequences from sequencing data, wherein the low-quality sequences are sequences with N content exceeding 30% or low-quality base content exceeding 10%, and then performing quality detection by using fastqc software default parameters to ensure that the data are effective;
(3) independently carrying out transcript non-parameter assembly on each sample to obtain all transcripts of each individual;
(4) SSR screening and primer design based on exon sequences: downloading a reference genome sequence from a database, extracting all exon sequences, identifying SSR loci by using GMATA2.0 software, and designing a primer; carrying out ePCR on the exon sequence of the obtained primer by using the ePCR function of GMATA2.0, and excluding primer pairs with poor specificity;
(5) ePCR validation of SSR primers: and (3) carrying out ePCR on the SSR primer screened in the step (4) by using the ePCR function of GMATA2.0 on the transcript of each individual obtained in the step (3), and screening the primer from the result.
2. A method for developing SSR primers based on transcriptome sequencing according to claim 1, characterized in that:
the specific process of extracting total RNA and constructing a transcriptome sequencing library comprises the following steps of extracting the total RNA by using a QIAGENNeasy protective Animal Blood Kit 73224, checking the quality of the RNA by agarose gel electrophoresis, constructing the transcriptome sequencing library by using a VAHTSTM mRNA-seq v 2L ibrary Prep Kit for Illumina NR602-01 Kit, taking 1 mu g of total RNA from each sample, separating and purifying by using poly-A magnetic beads to obtain mRNA, fragmenting by using divalent cations in VAFrag/Prime Buffer at 98 ℃ for 8 minutes, synthesizing cDNA first strands and second strands after purification, carrying out terminal flattening, connecting a dATP end and a joint, selecting a 150 bp fragment by agarose gel electrophoresis, purifying by using magnetic beads, carrying out RCR library amplification at 98 ℃ for 10s denaturation, combining at 60 ℃ for 30s, extending at 72 ℃ for 30s, circularly amplifying for 12 times, and finally, testing the quality of the library and confirming.
3. A method for developing SSR primers based on transcriptome sequencing according to claim 1, characterized in that:
the specific process of assembling the transcript in the step (3) without reference is as follows: and (3) adopting a Trinity reference-free genome assembly process, independently assembling the transcript to each sample by default parameters, and obtaining the own Trinity.
4. A method for developing SSR primers based on transcriptome sequencing according to claim 1, characterized in that:
the search criteria of the SSR identified in the step (4) are as follows: the minimum repeat motif is a dinucleotide, the maximum repeat motif is a decade of nucleotides; a minimum of 5 repeats in the exon sequence.
5. A method for developing SSR primers based on transcriptome sequencing according to claim 1 wherein the conditions for primer design in step (4) are: the length of the amplification product is between 120bp and 400 bp; the length of the product flanking sequence is 400 bp; an annealing temperature Tm of 60oC; the maximum template length is 2000 bp.
6. A method for developing SSR primers based on transcriptome sequencing according to claim 1, characterized in that: the ePCR process parameters in the step (4) and the step (5) are as follows: the word length is 12, the continuous word length is 1, the maximum deletion is 1, the maximum mismatch is 0, and the length of the amplification product is 100-1000 bp.
7. A method for developing SSR primers based on transcriptome sequencing according to claim 1, characterized in that: the standard of the screening primer in the step (5) is as follows: the difference of the amplified product fragments in at least two different samples is integral multiple of the length of the SSR repeat unit motif.
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| CN110791573B (en) * | 2019-12-05 | 2022-06-03 | 云南大学 | Microsatellite locus and primer suitable for identifying golden monkey individual |
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