CN110951911B - Tilia EST-SSR primer based on transcriptome as well as screening method and application thereof - Google Patents
Tilia EST-SSR primer based on transcriptome as well as screening method and application thereof Download PDFInfo
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Abstract
The invention discloses a set of EST-SSR molecular markers developed based on a Tilia miqueliana full-length transcription group sequence, and relates to 27 pairs of primers, wherein the nucleic acid sequences of the EST-SSR molecular markers are shown in a sequence table SEQ ID NO. 1-54. The 27-pair EST-SSR marker is an SSR amplification primer which is designed by screening candidate SSR sites based on a transcriptome sequence, and is a clear SSR marker by taking different species of the genus as materials and selecting genome DNA for amplification verification, and has the advantages of rich polymorphism, stable amplification, good repeatability, codominance, easiness in detection and the like. The group of markers fills the blank of the common marker of the tilia, and can greatly promote the research, development and application of the tilia resources in the fields of species identification, genetic pedigree analysis, germplasm resource protection, auxiliary breeding and the like of the tilia.
Description
Technical Field
The invention relates to a transcriptome-based Tilia EST-SSR primer as well as a screening method and application thereof, belonging to the technical field of development and application of molecular marker technology.
Background
Tilia (Tilia L.) and deciduous tree belong to a special and unique northern hemisphere distribution genus of Malvaceae (Malvaceae), are one of characteristic and dominant plants in northern temperate deciduous to evergreen broadleaf forests, and are centered on species diversity in China. The plant has a long cultivation history in the world, has the functions of materials, fibers, medicines, landscapes, honey sources and the like, and has a very wide development and application prospect.
Tilia plants have obvious characteristics, and inflorescences and infructescence have large bracts with unique leaf shapes, so that the Tilia plants are easily distinguished from other groups. Recent studies have divided worldwide tilia plants into 23 species, 2 species in north america, 4 species from europe to western asia, 17 species in east asia, including 15 species peculiar to china. However, the germ isolation is lacked among species, the phenomenon of germ plasm infiltration is relatively common, the morphological character variation and crossing phenomenon among the related species are not well explained, and the relationship and evolution history among the tilia species are still unclear. The problem of phylogeny of chloroplast is difficult to completely solve only by means of chloroplast phylogeny genomics, so that starting from nuclear genes, more information sites are obtained, and a basis is provided for comprehensively explaining the evolution of tilia species. For a long time, efficient, reliable, stable and co-dominant nuclear gene SSR markers are lacked in the tilia plants. The SSR marker is not only beneficial to disclosing the problem of the systemic development of the linden, but also beneficial to the diversity analysis of the plant resources of the linden and the promotion of molecular marker-assisted breeding work, and has important significance for the efficient utilization and development of the linden plant resources in future.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a Tilia EST-SSR primer based on a transcriptome as well as a screening method and application thereof. The provided tilia specific SSR molecular marker can be applied to the work of species classification and evolution research, resource genetic diversity analysis, core germplasm construction, auxiliary breeding and the like in the genus, and fills the blank of the tilia co-dominant SSR marker.
In order to solve the technical problems, the invention provides a transcriptome-based Tilia EST-SSR primer, which comprises 27 pairs of primers, wherein the sequence of the primers is shown as SEQ ID NO. 1-54.
The linden plant can be any one or more of the following species in the linden, but is not limited to the following species, and can be popularized and applied to 23 species in the linden: tilia miqueliana Maxim, Tilia miqueliana (yellow leaf), Tilia miqueliana (Tilia henyana Szyszyl.), Tilia tomentosa (Tilia tomentosa Moench), Tilia amurensis Rupr (Tilia amurensis Rupr), Tilia manicularia Maxim (Tilia mangshurica Maxim.), Tilia tomentosa (Tilia cordata Mill.).
The invention also provides a screening method of the Tilia EST-SSR primer based on the transcriptome, which comprises the following steps:
construction of transcriptome libraries: respectively extracting RNAs of 5 tissues of root, stem, leaf, flower and bract of Tilia miqueliana Maxim, mixing the RNAs in equal amount, performing reverse transcription to synthesize full-length cDNA, constructing cDNA libraries with different sizes, performing quality control, and sequencing by using a PacBio RSII platform to obtain a full-length non-chimeric sequence;
performing cluster analysis on the full-length non-chimeric sequence, correcting to obtain a high-quality transcript sequence, and performing redundancy removal analysis to obtain a high-quality redundancy-removed transcript sequence;
carrying out single-base repeated SSR, double-base repeated SSR, three-base repeated SSR, four-base repeated SSR, five-base repeated SSR, six-base repeated SSR and mixed SSR locus search on the high-quality redundancy-removed transcript sequence in Tilia miqueliana transcriptome data by adopting MISA software;
adopting Primer3 software to design SSR Primer, wherein the length of the Primer sequence is 18-27bp, the expected length of the amplification product is 100-280bp, the GC content is 40-60%, the annealing temperature is 57-63 ℃, and the difference between the annealing temperature values of the upstream Primer and the downstream Primer is not more than 2 ℃;
extracting the DNA of the leaves of different species of the linden, carrying out PCR identification of the effectiveness of a designed primer, and screening an SSR primer, wherein the primer is an effective primer if an amplification product with the length similar to that of a predicted amplification product exists.
Further, the quality control comprises performing end repair and joint connection on the full-length cDNA, performing secondary library screening by using Bluepippin, and performing qualitative and quantitative detection by using Qubit2.0 and Agilent 2100.
Further, the reaction system for PCR identification of the effectiveness of the designed primer is as follows: 2 x 8 muL of PCR mix, 0.5 muL of upstream primer and downstream primer with the concentration of 10 mumol/L, 2-5 ng of genome DNA and 10 muL of total volume.
Further, the reaction procedure for performing PCR identification of the effectiveness of the designed primers was: 2min at 98 ℃; 10s at 98 ℃, 30s at Tm +2 ℃ and 10s at 72 ℃ for 30 cycles; 10min at 72 ℃; and separating the amplification product by 8 percent polyacrylamide denatured gel, and carrying out silver staining and color development after electrophoresis.
The invention also provides application of the Tilia EST-SSR primer based on the transcriptome in the fields of species identification of the Tilia plants, genetic pedigree analysis, germplasm resource protection and auxiliary breeding.
The invention achieves the following beneficial effects: the 27-pair EST-SSR marker is an SSR amplification primer which is designed by screening candidate SSR sites based on a transcriptome sequence, and is a clear SSR marker by taking different species of the genus as materials and selecting genome DNA for amplification verification, and has the advantages of rich polymorphism, stable amplification, good repeatability, codominance, easiness in detection and the like. The group of markers fills the blank of the common marker of the tilia, and can greatly promote the research, development and application of the tilia resources in the fields of species identification, genetic pedigree analysis, germplasm resource protection, auxiliary breeding and the like of the tilia.
Drawings
FIG. 1 shows the result of total DNA extraction of a sample;
FIG. 2 shows the results of partial polymorphism primer screening;
fig. 3 is a graph of cluster analysis among samples tested.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The invention provides a method for searching SSR sequence and designing and verifying SSR marker primer based on full-length transcriptome sequencing by combining a bioinformatics method, which comprises the following specific implementation modes:
1. assembly and screening of transcriptome data
1.1RNA extraction and detection
5 tissue RNA materials of roots, stems, leaves, flowers and bracts of Tilia miqueliana Maxim are extracted, and the determination of the RNA quality and concentration is completed by adopting the following method: a. detecting the purity (OD260/280), concentration and nucleic acid absorption peak of the RNA by using Nanodrop; accurately detecting the integrity of RNA by Agilent 2100, wherein the detection indexes comprise an RIN value, 28S/18S, the presence or absence of an uplifted spectrum base line and a 5S peak; c. electrophoresis is used for detecting whether the RNA sample is polluted by the genome DNA.
1.2 full-Length cDNA library construction and sequencing
After the detection is qualified, 5 tissues of RNA with the same quantity are mixed, the SMARTer TM PCR cDNA Synthesis Kit is used for synthesizing the full-length cDNA of mRNA, Blue Pippin is used for screening the full-length cDNA library to construct cDNA libraries with different sizes, then the screened full-length cDNA is amplified through PCR amplification, the tail end of the full-length cDNA is repaired, an SMRT joint is connected for exonuclease digestion, finally Blue Pippin is used for performing secondary library screening, and qualitative and quantitative detection is performed through the qubit2.0 and Agilent 2100. After the assay was qualified, transcriptome sequencing was performed using PacBio RSII.
1.3 sequencing data and quality control
Co-acquisition of off-line data via PacBio rsi sequencing platform co-acquisition of 511,632 Polymerase (Polymerase Read) sequences.
1.3.1 filtering sequences with the length of the Polymerase Read fragment being less than 50bp and the sequence accuracy being less than 0.75, breaking the residual sequences from the joints and filtering out the joint sequences to obtain subreads, wherein the filtering length is less than 50bp, the residual subreads are clean data, the data volume is 10.84Gb, and the number of the subreads is 4,388,533. 307,278 ROI sequences were extracted from the original sequence according to the condition fullpases > 0 and sequence accuracy greater than 0.75. By detecting whether the ROI sequence contains a 5 'primer, a 3' primer and a polyA tail, the sequence can be divided into a full-length sequence (containing the 5 'primer, the 3' primer and the polyA tail) and a non-full-length sequence.
1.3.2 removing cDNA primer sequence and polyA sequence in ROI sequence by using classify process to obtain insertion sequence in library construction, determining strand synthesis direction according to difference of primers at two ends in library construction, and dividing sequence into full-length sequence and non-full-length sequence, chimeric sequence and non-chimeric sequence. Thereby obtaining 165,812 full-length non-chimeric sequences.
1.3.3 use the IsoSeq module of SMRT Analysis software to carry out cluster Analysis to the full-length sequence, obtain 92,732 consistent transcripts, use the high quality transcript sequence after the non-full-length sequence correction 69,475.
SSR locus identification and SSR primer design
2.1 screening transcripts with the length of more than 500bp, carrying out single-base repeat SSR, double-base repeat SSR, three-base repeat SSR, four-base repeat SSR, five-base repeat SSR, six-base repeat SSR and mixed SSR locus search on 62189 high-quality transcript sequences in Tilia miqueliana transcriptome data by adopting MISA software, and obtaining SSR analysis results, which are shown in Table 1.
TABLE 1 SSR analysis results statistics
2.2 SSR Primer design was performed using Primer3 software. Design parameters of the primers are as follows: designing primers based on sequences before and after the SSR repeat unit, wherein each SSR generates 5 primers, the length of the sequence of the primers is 18-27bp, the length of the expected amplification product is 100-280bp, the GC content is 40-60%, the annealing temperature is 57-63 ℃, and the difference between the annealing temperature values of the upstream primer and the downstream primer is not more than 2 ℃. The PCR primers for obtaining 29894 SSR sites are designed.
764 pairs of primer sequences were randomly selected from the above primers, and the synthesis of the primers was carried out by Nanjing Optimalaceae Biotechnology Ltd.
SSR primer screening and diversity analysis
3.1 plant Material selection
The test plant DNA template material in this example was obtained from 7 parts of Tilia (see Table 2), including 6 species and 1 Tilia miqueliana yellow bud transformed plant. In the plant collection process, leaf materials are respectively placed in paper tea bags and then placed in self-sealing bags, and allochroic silica gel is added according to the mass ratio of 1:5 to prepare dry samples for later use.
TABLE 2 statistics table of experimental materials for Tilia SSR primer screening
| Numbering | Name (R) | Origin of origin | Time of acquisition |
| A | Tilia miqueliana Maxim | Nanjing Zhongshan botanical garden classification garden | 2018.04.14 |
| B | Tilia miqueliana (yellow leaf variation individual) | Nanjing Zhongshan plant garden nursery | 2018.04.30 |
| C | Tilia miqueliana Maxim | Nanjing Zhongshan botanical garden | 2018.04.30 |
| D | Tilia tomentosa (Miquel.) Maxim | Nanjing Zhongshan plant garden nursery | 2018.04.30 |
| Z | Tilia amurensis | Chuzhou city copper town nursery garden of Anhui province | 2018.05.08 |
| K | Tilia amurensis | One-east nursery garden in Hongze area of Huaian city, Jiangsu province | 2018.05.09 |
| H | Tilia heartwood (Gray) | One-east nursery garden in Hongze area of Huaian city, Jiangsu province | 2018.05.09 |
3.2 DNA extraction
The DNA of the sample is extracted by a novel rapid plant genome DNA extraction kit of Beijing Baitacg Biotechnology GmbH. The DNA sample is placed in a refrigerator at the temperature of 20 ℃ below zero for standby after being detected to be qualified by 2 percent agarose gel electrophoresis, and the electrophoresis result is shown in figure 1.
3.3 PCR amplification
The randomly selected primers were amplified with 7 DNA fragments in Table 2, in sequence, using 764 pairs of random selection primers. The total PCR amplification system was 10. mu.L: wherein the PCR mix comprises 8 muL of 2 XPrimataceae gold PCR mix, 0.5 muL of upstream primer and downstream primer (concentration is 10 ng/muL), and 2-5 ng of 1 muL of template DNA; the amplification reaction is completed on an ABI Veriti96 PCR instrument, and the specific steps are as follows: : pre-denaturation at 98 ℃ for 2 min; denaturation at 98 ℃ for 10s, annealing at Tm +2 ℃ for 30s, extension at 72 ℃ for 10s, and 30 cycles; extension at 72 ℃ for 1min and storage at 4 ℃. The detection of the product adopts 8 percent polyacrylamide denatured gel for separation, and silver staining is carried out for color development after electrophoresis. The primer annealing temperatures are shown in Table 3, and the partial amplification results are shown in FIG. 2.
3.4 PCR primer screening
The primers with clear bands and polymorphic products are obtained by selecting 7 templates from 100bp to 400bp, and the sequence information is shown in Table 3.
TABLE 3 Tilia EST-SSR primer characteristics (SEQ ID NO. 1-54).
And (3) counting and respectively judging the amplification results of 27 pairs of primers, counting the mobility of the polymorphic bands, judging according to the sizes and the sizes of the polymorphic bands, counting the number of the bands as 1 and the number of the bands as 0, and establishing an original matrix.
The secondary matrix was analyzed by PowerMarker V3.25 software, and the number of allelic loci (Na, number of allolines), gene diversity index (H, expected heterologous) and Polymorphism Information Content (PIC) of each pair of primers were calculated.
TABLE 4.27 analysis of the polymorphism information content of the primers
| Primer name | Number of alleles (Na) | Gene diversity (H) | Polymorphism Information Content (PIC) |
| B2405 | 6.0000 | 0.8163 | 0.7913 |
| C80 | 4.0000 | 0.6939 | 0.6414 |
| B2975 | 3.0000 | 0.5714 | 0.5015 |
| B1340 | 6.0000 | 0.8163 | 0.7913 |
| B2550 | 3.0000 | 0.6122 | 0.5298 |
| B55 | 6.0000 | 0.8163 | 0.7913 |
| D150 | 5.0000 | 0.7755 | 0.7397 |
| B2410 | 4.0000 | 0.7347 | 0.6847 |
| C840 | 4.0000 | 0.6939 | 0.6414 |
| C3380 | 7.0000 | 0.8571 | 0.8397 |
| C3235 | 6.0000 | 0.8163 | 0.7913 |
| B485 | 6.0000 | 0.8163 | 0.7913 |
| B1045 | 2.0000 | 0.4898 | 0.3698 |
| C2155 | 3.0000 | 0.6531 | 0.5798 |
| B75 | 7.0000 | 0.8571 | 0.8397 |
| B595 | 7.0000 | 0.8571 | 0.8397 |
| B2145 | 5.0000 | 0.7755 | 0.7397 |
| B90 | 6.0000 | 0.8163 | 0.7913 |
| C280 | 6.0000 | 0.8163 | 0.7913 |
| B2915 | 7.0000 | 0.8571 | 0.8397 |
| B505 | 5.0000 | 0.7347 | 0.6997 |
| C2700 | 5.0000 | 0.7347 | 0.6997 |
| C1050 | 4.0000 | 0.7347 | 0.6847 |
| C110 | 4.0000 | 0.6122 | 0.5698 |
| E5 | 2.0000 | 0.4082 | 0.3249 |
| C3260 | 4.0000 | 0.6939 | 0.6414 |
| C3150 | 6.0000 | 0.8163 | 0.7913 |
| Mean | 4.9259 | 0.7362 | 0.6940 |
Genetic distances were calculated between 7 samples tested using the POPGene 1.32 software (see Table 5).
TABLE 5 genetic distance between samples
| A | B | C | D | Z | K | H | |
| A | 1.0000000 | 0.4383562 | 0.5777778 | 0.5810811 | 0.5625000 | 0.5542169 | 0.5844156 |
| B | 0.4383562 | 1.0000000 | 0.5301205 | 0.5000000 | 0.5657895 | 0.5925926 | 0.6081081 |
| C | 0.5777778 | 0.5301205 | 1.0000000 | 0.6219512 | 0.4625000 | 0.4941176 | 0.5903614 |
| D | 0.5810811 | 0.5000000 | 0.6219512 | 1.0000000 | 0.6111111 | 0.5616438 | 0.6376812 |
| Z | 0.5625000 | 0.5657895 | 0.4625000 | 0.6111111 | 1.0000000 | 0.3802817 | 0.5555556 |
| K | 0.5542169 | 0.5925926 | 0.4941176 | 0.5616438 | 0.3802817 | 1.0000000 | 0.5270270 |
| H | 0.5844156 | 0.6081081 | 0.5903614 | 0.6376812 | 0.5555556 | 0.5270270 | 1.0000000 |
Clustering was performed by unweighted pair arithmetic mean (UPGMA) using SHAN module (sequential, hierarchical, and hierarchical clustering) provided by NTSYS-pc 2.1 software, and a dendrogram was plotted (see FIG. 3).
The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.
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<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>46
atacccccgg cgttatactt 20
<210>47
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>47
agttcactcc gatcagcagc 20
<210>48
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>48
tggttctaac ggattgggag 20
<210>49
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>49
gctggtctaa ctggagacgc 20
<210>50
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>50
gcagccttgc taaggtcaag 20
<210>51
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>51
gcttgttttc aagagtcggc 20
<210>52
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>52
atggggaaaa ttgtatggca 20
<210>53
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>53
ttgctgtttc attctgctgg 20
<210>54
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>54
agtttcgaat agcgccttga 20
Claims (2)
1. The Tilia EST-SSR primers based on the transcriptome are characterized by comprising 27 pairs of primers, wherein the sequences of the primers are shown in a table 3 and SEQ ID NO. 1-54.
2. The use of the transcriptome-based EST-SSR primer of claim 1 in the fields of species identification of Tilia plants, genetic pedigree analysis, protection of germplasm resources or assisted breeding.
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