CN108998561B - Method for developing elderberry SSR (simple sequence repeat) primers based on transcriptome sequencing - Google Patents
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Abstract
The invention provides a method for developing an elderberry SSR primer based on transcriptome sequencing, which comprises the following steps: s1, constructing an elderberry transcriptome library and sequencing, splicing and assembling sequencing results into a complete transcriptome, taking the longest transcript in each gene as a Unigene, and splicing to obtain a mixed Unigenes library; s2, carrying out SSR detection on the mixed Unigenes library in the step S1; and S3, designing an SSR primer, and identifying polymorphism of the SSR primer. 64148 pairs of SSR specific primers are designed in the invention, an optimal fluorescence SSR-PCR system of Sambucus nigra is obtained by a test optimization scheme, and 25 pairs of primers in 100 pairs of synthesized fluorescence labeled SSR primers have good polymorphism. The method for developing the elderberry SSR primer based on transcriptome sequencing is quick and accurate.
Description
Technical Field
The invention belongs to the technical field of molecular biology and bioinformatics, and particularly relates to a method for developing an elderberry SSR primer based on transcriptome sequencing.
Background
Simple Sequence Repeat (SSR) is a class of Simple tandem repeat sequences consisting of 1-6 bases and is widely distributed in eukaryotic and prokaryotic genomes. The SSR can be divided into EST-SSR and genome SSR according to SSR sources, the traditional development of SSR markers by utilizing genome data has the defects of high cost, complicated test steps, few sites and the like, in recent years, with the reduction of sequencing cost, transcriptome sequencing has become one of the short and fast ideas for researching related contents of molecular biology, transcriptome data of different plants are developed and utilized successively, and the research for developing SSR markers of different plants based on the transcriptome sequencing is more and more.
The elder (Sambucus Linn) is Caprifoliaceae (Caprifoliaceae) plant, is deciduous tree or shrub, and is about 20 kinds, and is distributed in temperate and subtropical regions. About 6 species in China, and the seeds are produced in both south and north. Sambucus williamsii is a precious medicinal plant, and a plurality of kinds of fruits have high oil content (35-44%), and is one of woody oil plants published by the Ministry of agriculture. China has abundant elderberry resources, strong adaptability, cold resistance, drought resistance and disease resistance, most of elderberry is still asleep in mountain forest and is not utilized, the development and utilization of elderberry plants are still in the primary stage, the resource utilization is insufficient, the medicine is more, other comprehensive utilization is less, and related reports such as marker development based on transcriptome sequencing and molecular marker assisted breeding are not seen, so that microsatellite markers are necessary to be further developed, and a foundation is laid for the genetic diversity, linkage map construction and functional gene excavation of elderberry germplasm resources by utilizing SSR markers.
Disclosure of Invention
Aiming at the defects of the prior art, the inventor provides a method for developing an elderberry SSR primer based on transcriptome sequencing through long-term technical and practical exploration.
One of the purposes of the invention is to provide an elderberry SSR primer pair.
The invention also aims to provide an elderberry SSR primer group.
The invention also aims to provide application of the elderberry SSR primer pair and/or primer group.
The fourth purpose of the invention is to provide a method for obtaining an SSR molecular marker of elderberry.
The fifth purpose of the invention is to provide a method for developing an elderberry SSR primer based on transcriptome sequencing.
Specifically, the invention adopts the following technical scheme:
in a first aspect of the invention, provided is an elderberry SSR primer pair, the sequence of which is shown in SEQ ID NO.1-2, SEQ ID NO.3-4, SEQ ID NO.5-6, SEQ ID NO.7-8, SEQ ID NO.9-10, SEQ ID NO.11-12, SEQ ID NO.13-14, SEQ ID NO.15-16, SEQ ID NO.17-18, SEQ ID NO.19-20, SEQ ID NO.21-22, SEQ ID NO.23-24, SEQ ID NO.25-26, SEQ ID NO.27-28, SEQ ID NO.29-30, SEQ ID NO.31-32, SEQ ID NO.33-34, SEQ ID NO.35-36, As shown in SEQ ID NO.37-38, as shown in SEQ ID NO.39-40, as shown in SEQ ID NO.41-42, as shown in SEQ ID NO.43-44, as shown in SEQ ID NO.45-46, as shown in SEQ ID NO.47-48, or as shown in SEQ ID NO. 49-50;
note that M is added to the 3' end of the forward primer sequence in each primer pair13A joint; m13The sequence of the joint is as follows: 5'-TGTAAAACGACGGCCAGT-3', respectively;
in a second aspect of the present invention, an elderberry SSR primer set is provided, which is composed of two or more of the following primer pairs: primer pairs according to SEQ ID NO.1-2, primer pairs according to SEQ ID NO.3-4, primer pairs according to SEQ ID NO.5-6, primer pairs according to SEQ ID NO.7-8, primer pairs according to SEQ ID NO.9-10, primer pairs according to SEQ ID NO.11-12, primer pairs according to SEQ ID NO.13-14, primer pairs according to SEQ ID NO.15-16, primer pairs according to SEQ ID NO.17-18, primer pairs according to SEQ ID NO.19-20, primer pairs according to SEQ ID NO.21-22, primer pairs according to SEQ ID NO.23-24, primer pairs according to SEQ ID NO.25-26, primer pairs according to SEQ ID NO.27-28, primer pairs according to SEQ ID NO.29-30, primer pairs according to SEQ ID NO.31-32, primer pairs according to SEQ ID NO.33-34, The primer pair as shown in SEQ ID NO.35-36, the primer pair as shown in SEQ ID NO.37-38, the primer pair as shown in SEQ ID NO.39-40, the primer pair as shown in SEQ ID NO.41-42, the primer pair as shown in SEQ ID NO.43-44, the primer pair as shown in SEQ ID NO.45-46, the primer pair as shown in SEQ ID NO.47-48, and the primer pair as shown in SEQ ID NO. 49-50;
preferably, the elderberry SSR primer group consists of the following primer pairs: primer pairs according to SEQ ID NO.1-2, primer pairs according to SEQ ID NO.3-4, primer pairs according to SEQ ID NO.5-6, primer pairs according to SEQ ID NO.7-8, primer pairs according to SEQ ID NO.9-10, primer pairs according to SEQ ID NO.11-12, primer pairs according to SEQ ID NO.13-14, primer pairs according to SEQ ID NO.15-16, primer pairs according to SEQ ID NO.17-18, primer pairs according to SEQ ID NO.19-20, primer pairs according to SEQ ID NO.21-22, primer pairs according to SEQ ID NO.23-24, primer pairs according to SEQ ID NO.25-26, primer pairs according to SEQ ID NO.27-28, primer pairs according to SEQ ID NO.29-30, primer pairs according to SEQ ID NO.31-32, primer pairs according to SEQ ID NO.33-34, The primer pair shown in SEQ ID NO.35-36, the primer pair shown in SEQ ID NO.37-38, the primer pair shown in SEQ ID NO.39-40, the primer pair shown in SEQ ID NO.41-42, the primer pair shown in SEQ ID NO.43-44, the primer pair shown in SEQ ID NO.45-46, the primer pair shown in SEQ ID NO.47-48 and the primer pair shown in SEQ ID NO. 49-50.
In a third aspect of the invention, the application of the elderberry SSR primer pair and/or primer group is provided; the application comprises the following steps:
1) the application in the elderberry genetic diversity analysis;
2) the application in the construction of elderberry genetic maps;
3) the application in elderberry germplasm identification;
4) the application in the analysis of the genetic relationship of elderberry;
5) the application in the excavation of functional genes of elderberry;
6) the application in elderberry molecular marker assisted breeding.
In a fourth aspect of the present invention, a method for obtaining an SSR molecular marker of elderberry is provided, which comprises:
s1, taking elderberry genome DNA as a template, and carrying out PCR amplification by using the elderberry SSR primer pair or the elderberry SSR primer group to obtain a PCR amplification product;
s2, performing capillary electrophoresis detection on the PCR amplification product obtained in the step S1 to obtain the SSR molecular marker of the elderberry.
In a fifth aspect of the invention, there is provided a method for developing an elderberry SSR primer based on transcriptome sequencing, comprising:
s1, constructing an elderberry transcriptome library and sequencing, splicing and assembling sequencing results into a complete transcriptome, taking the longest transcript in each gene as a Unigene, and splicing to obtain a mixed Unigenes library;
s2, carrying out SSR detection on the mixed Unigenes library in the step S1;
and S3, designing an SSR primer, and identifying polymorphism of the SSR primer.
The invention has the beneficial technical effects that:
the invention designs 64148 specific primers for SSR. The fluorescence SSR-PCR optimal system of the elderberry is obtained by a test optimization scheme, and among 100 pairs of synthesized fluorescence labeled SSR primers, 80 pairs of primers are amplified to have strips, the effective amplification rate is 80%, and 25 pairs of primers (SEQ ID NO.1-50) among 80 pairs of primers with amplified strips have good polymorphism.
The method for developing the elderberry SSR primer based on transcriptome sequencing is quick and accurate, and provides a new idea for developing the elderberry SSR primer. The invention provides effective technical support for high-density elderberry genetic linkage map construction, elderberry germplasm identification, elderberry genetic diversity analysis, elderberry genetic relationship analysis, elderberry important economic trait QTL positioning, elderberry molecular marker-assisted breeding and the like.
Drawings
FIG. 1 is a SSR locus profile of Sambucus nigra, wherein: the X coordinate is the SSR type, the Y coordinate value is the coordinate, the specific repeated times are corresponding to the legend according to the color, and the Z coordinate is the SSR number;
FIG. 2 shows the genotypes of 8 elderberry samples at site No. 5; wherein FIGS. 2(a) - (c) are three samples of Sambucus canadensis; FIG. 2(d) is a sample of Sambucus nigra; FIG. 2(e) is an elderberry sample; FIG. 2(f) is a sample of ramulus Sambuci Williamsii; fig. 2(g) - (h) are two samples of elderberry.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
The present invention is further illustrated by reference to specific examples, which are intended to be illustrative only and not limiting. If the specific conditions are not specified in the examples, the conditions are generally in accordance with the conventional conditions or in accordance with the conditions recommended by the sales company; the present invention is not particularly limited, and may be commercially available.
As mentioned above, China has abundant elderberry resources, but China is still in the primary stage for development and utilization of elderberry plants, resource utilization is insufficient, only a lot of medicines are used, other comprehensive utilization is less, and related reports such as development of markers based on transcriptome sequencing and molecular marker-assisted breeding are not seen.
In view of this, in one specific embodiment of the present invention, an elderberry SSR primer pair is provided, the sequence of which is shown in SEQ ID NO.1-2, SEQ ID NO.3-4, SEQ ID NO.5-6, SEQ ID NO.7-8, SEQ ID NO.9-10, SEQ ID NO.11-12, SEQ ID NO.13-14, SEQ ID NO.15-16, SEQ ID NO.17-18, SEQ ID NO.19-20, SEQ ID NO.21-22, SEQ ID NO.23-24, SEQ ID NO.25-26, SEQ ID NO.27-28, SEQ ID NO.29-30, SEQ ID NO.31-32, SEQ ID NO.33-34, As shown in SEQ ID NO.35-36, SEQ ID NO.37-38, SEQ ID NO.39-40, SEQ ID NO.41-42, SEQ ID NO.43-44, SEQ ID NO.45-46, SEQ ID NO.47-48, or SEQ ID NO. 49-50;
note that M is added to the 3' end of the forward primer sequence in each primer pair13A joint; m13The sequence of the joint is as follows: 5'-TGTAAAACGACGGCCAGT-3', respectively;
in another specific embodiment of the present invention, an elderberry SSR primer set is provided, which is composed of two or more of the following primer pairs: primer pairs according to SEQ ID NO.1-2, primer pairs according to SEQ ID NO.3-4, primer pairs according to SEQ ID NO.5-6, primer pairs according to SEQ ID NO.7-8, primer pairs according to SEQ ID NO.9-10, primer pairs according to SEQ ID NO.11-12, primer pairs according to SEQ ID NO.13-14, primer pairs according to SEQ ID NO.15-16, primer pairs according to SEQ ID NO.17-18, primer pairs according to SEQ ID NO.19-20, primer pairs according to SEQ ID NO.21-22, primer pairs according to SEQ ID NO.23-24, primer pairs according to SEQ ID NO.25-26, primer pairs according to SEQ ID NO.27-28, primer pairs according to SEQ ID NO.29-30, primer pairs according to SEQ ID NO.31-32, primer pairs according to SEQ ID NO.33-34, The primer pair as shown in SEQ ID NO.35-36, the primer pair as shown in SEQ ID NO.37-38, the primer pair as shown in SEQ ID NO.39-40, the primer pair as shown in SEQ ID NO.41-42, the primer pair as shown in SEQ ID NO.43-44, the primer pair as shown in SEQ ID NO.45-46, the primer pair as shown in SEQ ID NO.47-48, and the primer pair as shown in SEQ ID NO. 49-50;
in another specific embodiment of the present invention, the SSR primer set of elderberry consists of the following primer pairs: primer pairs according to SEQ ID NO.1-2, primer pairs according to SEQ ID NO.3-4, primer pairs according to SEQ ID NO.5-6, primer pairs according to SEQ ID NO.7-8, primer pairs according to SEQ ID NO.9-10, primer pairs according to SEQ ID NO.11-12, primer pairs according to SEQ ID NO.13-14, primer pairs according to SEQ ID NO.15-16, primer pairs according to SEQ ID NO.17-18, primer pairs according to SEQ ID NO.19-20, primer pairs according to SEQ ID NO.21-22, primer pairs according to SEQ ID NO.23-24, primer pairs according to SEQ ID NO.25-26, primer pairs according to SEQ ID NO.27-28, primer pairs according to SEQ ID NO.29-30, primer pairs according to SEQ ID NO.31-32, primer pairs according to SEQ ID NO.33-34, The primer pair shown in SEQ ID NO.35-36, the primer pair shown in SEQ ID NO.37-38, the primer pair shown in SEQ ID NO.39-40, the primer pair shown in SEQ ID NO.41-42, the primer pair shown in SEQ ID NO.43-44, the primer pair shown in SEQ ID NO.45-46, the primer pair shown in SEQ ID NO.47-48 and the primer pair shown in SEQ ID NO. 49-50.
In another embodiment of the present invention, there is provided the use of the SSR primer pair and/or primer set described above for elderberry; the application comprises the following steps:
1) the application in the elderberry genetic diversity analysis;
2) the application in the construction of elderberry genetic maps;
3) the application in elderberry germplasm identification;
4) the application in the analysis of the genetic relationship of elderberry;
5) the application in the excavation of functional genes of elderberry;
6) the application in elderberry molecular marker assisted breeding.
In another embodiment of the present invention, a method for obtaining SSR molecular markers of elderberry is provided, which comprises:
s1, taking elderberry genome DNA as a template, and carrying out PCR amplification by using the elderberry SSR primer pair or the elderberry SSR primer group to obtain a PCR amplification product;
s2, performing capillary electrophoresis detection on the PCR amplification product obtained in the step S1 to obtain the SSR molecular marker of the elderberry.
In another embodiment of the present invention, in step S1,
the specific conditions for PCR amplification are as follows: amplifying by adopting a two-step fluorescent primer PCR amplification method;
(1) adding M13Linker PCR amplification system:
10 μ L of PCR system contained 1 μ L of DNA stock solution, 2 Xmix 5 μ L of DNA stock solution, 0.1 μ L of 10 μ M each of primers, and H2O3.8 mu L; the PCR amplification procedure is shown in table 1:
TABLE 1PCR amplification procedure
(2) Fluorescent primer PCR amplification system: 20 μ L of PCR system contains 2 μ L of DNA stock solution of the first-step amplification product, 2 XMix 10 μ L, 10 μ M M13 adaptor (with fluorescent label) and reverse primer 0.15 μ L each, H2O7.7 mu L; the fluorescent primer PCR amplification procedure is shown in table 2:
TABLE 2 fluorescent primer PCR amplification procedure
In another embodiment of the present invention, the elder in the above method may be any one or more of Sambucus Canadensis (Sambucus Canadensis), Sambucus Canadensis (Sambucus Canadensis 'Aurea'), Sambucus floridus (Sambucus nigra 'Variegata'), Sambucus pinnata (Sambucus racemosa 'pluronic Aurea'), and Sambucus williamsii Hance;
in another embodiment of the present invention, there is provided a method for developing an elderberry SSR primer based on transcriptome sequencing, comprising:
s1, constructing an elderberry transcriptome library and sequencing, splicing and assembling sequencing results into a complete transcriptome, taking the longest transcript in each gene as a Unigene, and splicing to obtain a mixed Unigenes library;
s2, carrying out SSR detection on the mixed Unigenes library in the step S1;
and S3, designing an SSR primer, and identifying polymorphism of the SSR primer.
In another embodiment of the present invention, in step S1,
the elderberry is Sambucus nigra (Sambucus nigra);
splicing by adopting Trinity software, wherein main splicing parameters are SS _ lib _ type, min _ kmer _ cov and min _ glue;
in another embodiment of the present invention, in step S2,
adopting MISA software to search SSR locus, wherein the search criteria are as follows: the minimum number of repeats of dinucleotide, trinucleotide, tetranucleotide, pentanucleotide and hexanucleotide is 6, 5 and 5 times respectively;
in another embodiment of the present invention, in step S3,
the SSR Primer design is carried out by adopting Primer 3.0 software, and the length of the SSR locus flanking sequence is more than or equal to 50 bp. The main parameters of primer design:
(1) the annealing temperature (Tm) is between 55 and 75 ℃, and the difference of Tm values of the upstream primer and the downstream primer is less than or equal to 5 ℃;
(2) the size of the PCR amplification product is between 50 and 500bp, and the length of the primer is between 15 and 30 bp;
(3) the GC content is between 40 and 60 percent, and no primer dimer and secondary structure appear between the upstream primer and the downstream primer;
performing Blast verification on the designed SSR primer in a Unigenes library.
Examples
1 materials and methods
1.1 sources of transcriptome data
Sambucus nigra transcriptome data was derived from Illumina HiSeq 2000 high-throughput sequencing of an adult sambucus nigra fruit in 2016 of this group. In the sequencing, fruits in 3 developmental stages (olive, medium-maturing and late-maturing) were collected, RNA was extracted, RNA-Seq transcriptome sequencing was performed by Beijing Nozao genetic Gene Co, and 257975 Unigenes were obtained by assembling by the De Novo method as analysis background data.
1.2 transcriptome SSR site identification
Splicing the transcription group data of the Sambucus nigra by using Trinity software, storing the sequence information of the spliced transcripts in a FASTA format, wherein the main splicing parameters are SS _ lib _ type, min _ kmer _ cov and min _ glue. Obtaining a Unigenes library through splicing, and carrying out SSR locus search on the mixed Unigenes library by using an MISA program, wherein the search criteria are as follows: the minimum number of repeats of dinucleotide, trinucleotide, tetranucleotide, pentanucleotide and hexanucleotide is 6, 5 and 5 times, respectively.
1.3 SSR primer design
Primer design is carried out on the sequence containing the SSR locus by adopting a Primer 3.0 Primer design program (version 2.3.5, default parameters), and the length of the flanking sequence of the SSR locus is more than or equal to 50 bp; the main parameters of primer design: (1) the annealing temperature (Tm) is between 55 and 75 ℃, and the difference of Tm values of the upstream primer and the downstream primer is less than or equal to 5 ℃; (2) the size of the PCR amplification product is between 50 and 500bp, and the length of the primer is between 15 and 30 bp; (3) the GC content is between 40% and 60%, and no primer dimer and secondary structure are generated between the upstream primer and the downstream primer. Performing blast verification on the designed SSR primers in a Unigenes library.
1.4 plant Material and DNA extraction
Selecting mature leaves of 8 elderberries (3 Canadian elderberries, 1 Kigelelderberries, 1 Kadsura japonica, 1 Kigelelderberries, and 2 Sambucus nigra), and storing DNA by using a Ranunculus sieboldii biochemical technology (Shanghai) Limited plant tissue genome DNA extraction kit (paramagnetic particle method) at-70 ℃ for later use.
1.5 fluorescent PCR amplification System and program optimization
And (3) carrying out primer screening on the 8 mature elderberry leaves. The fluorescent primer PCR amplification system adopts semi-nested PCR, and obtains the optimal fluorescent PCR amplification system and program through optimization processes of reducing annealing temperature, performing gradient PCR amplification and the like.
And (3) detecting the amplification product by using capillary electrophoresis: mixing 0.3. mu.L of PCR product, 0.5. mu.L of molecular weight internal standard and 9.5. mu.L of deionized formamide, adding into a PCR plate, denaturing at 95 ℃ for 5min, cooling at 4 ℃, centrifuging, and detecting on a machine with 1 Xbuffer Buffer.
1.6 fluorescent SSR primer screening
100 pairs of fluorescent primers were derived from Primer 3.0 software design, synthesized by Rui Boxing, Bio-technologies, Inc. of Beijing, and purified by PAGE. The principle of primer screening is that the Tm value range is as follows: 55-62 ℃; number of repeated bases of primer: 2bp, such As (AG), (AT), etc.; number of repeated bases: 8-11 times; the PCR amplification system and procedure are selected from the optimal results obtained experimentally.
2 results and analysis
2.1 distribution and structural features of SSR in transcriptome
By searching 257975 Unigene sequences of the elderberry transcriptome (the total length of the sequences is 254175kb), it is found that 51336 Unigene sequences contain 64148 SSR sites, the occurrence frequency of the SSR sites is 24.87% (the ratio of the number of the SSR sites to the total number of the Unigene sites), 1 SSR appears in every 3.96kb on average, and the types of the SSR are rich, wherein single nucleotide repeats are main types and account for 51.13% of the total SSR. The second is a di-and trinucleotide repeat, accounting for 38.63% and 9.11% of the total SSR, respectively. A/T and AG/CT are the dominant repeat motifs of both single and dinucleotide. The tetranucleotide, pentanucleotide and hexanucleotide repeat types were fewer in number, accounting for 0.86%, 0.1% and 0.17% of the total (table 3, fig. 1), respectively.
TABLE 3 distribution of different SSR repeat unit types in Sambucus nigra transcriptome
2.2 fluorescent PCR amplification System and program optimization
The DNA of the leaves of Sambucus nigra is taken as a template, and a PCR system is optimized according to the basic conditions and the amplification program of SSR-PCR.
The fluorescent primer PCR amplification system and the program are as follows:
adding M13Linker PCR amplification system: 20 μ L of PCR system contained 1 μ L of DNA stock solution, 2 XMix 10 μ L, and 10 μ M of M13Linker and upstream and downstream primers 0.2. mu.L, 0.1. mu.L, 0.3. mu.L, H, respectively2O 8.4μL；
Fluorescent primer PCR amplification procedure: pre-denaturation at 95 ℃ for 5 min; then 35 cycles were performed, each cycle comprising denaturation at 95 ℃ for 30s, annealing at 57 ℃ for 30s (annealing temperature varied for different primers), and extension at 72 ℃ for 30 s; finally, extension is carried out for 10min at 72 ℃.
Based on the amplification system and the procedure, the optimal fluorescence PCR amplification system and the procedure are obtained by reducing the annealing temperature and carrying out optimization processes such as gradient PCR amplification and the like as follows:
the two-step fluorescent primer PCR amplification method comprises the following steps:
(1) adding M13Linker PCR amplification system: 10 μ L of PCR system contained 1 μ L of DNA stock solution, 2 Xmix 5 μ L of DNA stock solution, 0.1 μ L of 10 μ M each of primers, and H2O3.8 mu L; the PCR amplification procedure is shown in Table 1.
(2) Fluorescent primer PCR amplification system: 20 μ L of PCR system contains 2 μ L of DNA stock solution of the first-step amplification product, 2 XMix 10 μ L, 10 μ M M13 adaptor (with fluorescent label) and reverse primer 0.15 μ L each, H2O7.7 mu L; the fluorescent primer PCR amplification procedure is shown in Table 2.
2.3 fluorescent SSR marker screening
51336 Unigene sequences of the SSR locus are subjected to primer design, and 64148 primers specific to SSR are designed in total. In order to verify the effectiveness of the primers, 100 pairs of fluorescent SSR primers with dinucleotide repeat motifs are randomly selected and synthesized, 8 different elderberry individuals are subjected to fluorescent SSR amplification by using the 100 pairs of screened primers (shown in Table 4), and the result shows that 80 pairs of primers have bands in amplification, namely the amplification efficiency is 80%. Among the 80 pairs of primers successfully amplified, 25 pairs of primers (SEQ ID NO.1-50) are better in polymorphism, namely the primer polymorphism efficiency is 31.25%. FIG. 2 shows the amplification of primer No.5 in 8 different elder individuals.
TABLE 4
In the invention, 257975 Unigenes in a cDNA library obtained by transcriptome sequencing of a mature sambucus nigra fruit are subjected to SSR search to obtain 64148 SSR sites, the occurrence frequency is 24.87%, dinucleotides and trinucleotides are the most common SSR repeated types in most species, and from the structure of the SSR sites of sambucus nigra, the occurrence frequency of mononucleotides (51.13%) is the highest and is obviously higher than that of dinucleotides (38.63%) and trinucleotides (9.11%); the dinucleotide dominant repeat motif in the Sambucus nigra transcriptome is AG/CT.
And (3) carrying out primer design according to 257975 Unigene sequences, and totally designing 64148 pair of SSR specific primers. The optimized fluorescent SSR-PCR system of Sambucus nigra is obtained through an experimental optimization scheme, and among 100 pairs of synthesized fluorescent labeled SSR primers, 80 pairs of primers are amplified to form a band, the effective amplification rate is 80%, and partial primer amplification failures can be related to the quality of the primers and templates (20 pairs of primers with amplification failures are 13 th, 14 th, 18 th, 20 th, 23 th, 29 th, 34 th, 36 th, 50 th, 52 th, 57 th, 58 th, 59 th, 83 th, 88 th, 89 th, 93 th, 94 th, 98 th and 100 pairs of primers in Table 4 respectively). Among 80 pairs of primers with amplified bands, 25 pairs of primers have better polymorphism, the polymorphism ratio is 31.25%, and the size of the polymorphism ratio can be related to the type of the marker, the material to be tested and the quantity.
In consideration of SSR locus polymorphism potential, 64148 designed by the research has higher availability to SSR primers, and is feasible for elderberry genetic variation research, and the research result also shows that an SSR marker developed by using western elderberry transcriptome data lays a foundation for elderberry germplasm resource genetic diversity analysis, molecular marker assisted breeding, genetic map construction, functional gene excavation and the like. The next step is to enlarge the primer screening range, and a large number of SSR primers obtained by transcriptome sequencing can provide more and more effective molecular markers for researches on resource classification, gene localization, comparative genomics and the like of elderberry and other Caprifoliaceae plants.
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
SEQUENCE LISTING
<110> scientific research institute of forestry in Shandong province
<120> method for developing elderberry SSR primer based on transcriptome sequencing
<130>
<160> 50
<170> PatentIn version 3.3
<210> 1
<211> 38
<212> DNA
<213> Artificial sequence
<400> 1
tgtaaaacga cggccagtaa aagggtttgg cttgggga 38
<210> 2
<211> 20
<212> DNA
<213> Artificial sequence
<400> 2
gtcaccccca caaaccctag 20
<210> 3
<211> 38
<212> DNA
<213> Artificial sequence
<400> 3
tgtaaaacga cggccagtaa aatcgtctcg gaggctcc 38
<210> 4
<211> 20
<212> DNA
<213> Artificial sequence
<400> 4
agcgcccttt atcagaaccc 20
<210> 5
<211> 38
<212> DNA
<213> Artificial sequence
<400> 5
tgtaaaacga cggccagtaa acgatgggtt gctgttgc 38
<210> 6
<211> 21
<212> DNA
<213> Artificial sequence
<400> 6
gatctgcttg taaggcactg c 21
<210> 7
<211> 38
<212> DNA
<213> Artificial sequence
<400> 7
tgtaaaacga cggccagtaa agggcttgac cgacttgt 38
<210> 8
<211> 20
<212> DNA
<213> Artificial sequence
<400> 8
tcatctttgc tccctcggaa 20
<210> 9
<211> 38
<212> DNA
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<400> 9
tgtaaaacga cggccagtaa caaccccact cagagtcg 38
<210> 10
<211> 20
<212> DNA
<213> Artificial sequence
<400> 10
taggcggtta gggtccacat 20
<210> 11
<211> 38
<212> DNA
<213> Artificial sequence
<400> 11
tgtaaaacga cggccagtaa cctggtgtcc acgaagtc 38
<210> 12
<211> 20
<212> DNA
<213> Artificial sequence
<400> 12
ttgttctagg ccccactcca 20
<210> 13
<211> 38
<212> DNA
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<400> 13
tgtaaaacga cggccagtaa cgtgagcatc aaccccaa 38
<210> 14
<211> 21
<212> DNA
<213> Artificial sequence
<400> 14
tcaattgtcg gccttcactg t 21
<210> 15
<211> 38
<212> DNA
<213> Artificial sequence
<400> 15
tgtaaaacga cggccagtaa ctcactgcca ccactagc 38
<210> 16
<211> 20
<212> DNA
<213> Artificial sequence
<400> 16
attcagccgc ctccaatctc 20
<210> 17
<211> 38
<212> DNA
<213> Artificial sequence
<400> 17
tgtaaaacga cggccagtaa ctgtagcgtg aggccaaa 38
<210> 18
<211> 20
<212> DNA
<213> Artificial sequence
<400> 18
tatacccgtg ctctggggaa 20
<210> 19
<211> 38
<212> DNA
<213> Artificial sequence
<400> 19
tgtaaaacga cggccagtaa cttcaaacag catcgccg 38
<210> 20
<211> 20
<212> DNA
<213> Artificial sequence
<400> 20
agcgtgattg cagggaaaga 20
<210> 21
<211> 38
<212> DNA
<213> Artificial sequence
<400> 21
tgtaaaacga cggccagtaa gaactgtagc gtgaggcc 38
<210> 22
<211> 20
<212> DNA
<213> Artificial sequence
<400> 22
tatacccgtg ctctggggaa 20
<210> 23
<211> 38
<212> DNA
<213> Artificial sequence
<400> 23
tgtaaaacga cggccagtaa gaccctgttt ggttgggg 38
<210> 24
<211> 20
<212> DNA
<213> Artificial sequence
<400> 24
cctgttctcg tggagccttt 20
<210> 25
<211> 38
<212> DNA
<213> Artificial sequence
<400> 25
tgtaaaacga cggccagtaa gatcccatcg ccggaaag 38
<210> 26
<211> 20
<212> DNA
<213> Artificial sequence
<400> 26
tgggcaagga tgactttggg 20
<210> 27
<211> 38
<212> DNA
<213> Artificial sequence
<400> 27
tgtaaaacga cggccagtaa gcgcgtcact ttcccata 38
<210> 28
<211> 20
<212> DNA
<213> Artificial sequence
<400> 28
agacactggg cacaagttgt 20
<210> 29
<211> 38
<212> DNA
<213> Artificial sequence
<400> 29
tgtaaaacga cggccagtaa gcgcttgggc atttgttt 38
<210> 30
<211> 20
<212> DNA
<213> Artificial sequence
<400> 30
acgggagagt tgcatgacag 20
<210> 31
<211> 38
<212> DNA
<213> Artificial sequence
<400> 31
tgtaaaacga cggccagtaa ggcggcaata ccccaaat 38
<210> 32
<211> 22
<212> DNA
<213> Artificial sequence
<400> 32
accatgtatg caaggtacaa ca 22
<210> 33
<211> 38
<212> DNA
<213> Artificial sequence
<400> 33
tgtaaaacga cggccagtaa ggctatccaa gtgagcgg 38
<210> 34
<211> 20
<212> DNA
<213> Artificial sequence
<400> 34
ttccgagacg cacgtattcc 20
<210> 35
<211> 38
<212> DNA
<213> Artificial sequence
<400> 35
tgtaaaacga cggccagtaa gggttcgggt tcctaagg 38
<210> 36
<211> 22
<212> DNA
<213> Artificial sequence
<400> 36
gaggttggag gtctttcaca ga 22
<210> 37
<211> 38
<212> DNA
<213> Artificial sequence
<400> 37
tgtaaaacga cggccagtaa gtgggcagaa gcaagtga 38
<210> 38
<211> 20
<212> DNA
<213> Artificial sequence
<400> 38
gagctgaatc tggtcgaggg 20
<210> 39
<211> 38
<212> DNA
<213> Artificial sequence
<400> 39
tgtaaaacga cggccagtaa tcgccgccat ctcttcat 38
<210> 40
<211> 20
<212> DNA
<213> Artificial sequence
<400> 40
tcctctaagc ctcctccgtt 20
<210> 41
<211> 38
<212> DNA
<213> Artificial sequence
<400> 41
tgtaaaacga cggccagtaa tgtcctaggc gaagtggg 38
<210> 42
<211> 20
<212> DNA
<213> Artificial sequence
<400> 42
cccactgaaa tgagggcagt 20
<210> 43
<211> 38
<212> DNA
<213> Artificial sequence
<400> 43
tgtaaaacga cggccagtaa ttgcgtggcc tcgaaaac 38
<210> 44
<211> 20
<212> DNA
<213> Artificial sequence
<400> 44
tgtttcaggg acgcaaagga 20
<210> 45
<211> 38
<212> DNA
<213> Artificial sequence
<400> 45
tgtaaaacga cggccagtac aaaaacctca gggctcgt 38
<210> 46
<211> 20
<212> DNA
<213> Artificial sequence
<400> 46
ccagtgatgc ttatgtgcgc 20
<210> 47
<211> 42
<212> DNA
<213> Artificial sequence
<400> 47
tgtaaaacga cggccagtac aaagggttac atgcactttt ct 42
<210> 48
<211> 21
<212> DNA
<213> Artificial sequence
<400> 48
tgcaacacct caaactgtcc t 21
<210> 49
<211> 38
<212> DNA
<213> Artificial sequence
<400> 49
tgtaaaacga cggccagtac aactaggttg gggagtgg 38
<210> 50
<211> 21
<212> DNA
<213> Artificial sequence
<400> 50
acaaccacaa atgccgttag c 21
Claims (4)
1. An elderberry SSR primer group, which is characterized by consisting of the following primer pairs: primer pairs according to SEQ ID NO.1-2, primer pairs according to SEQ ID NO.3-4, primer pairs according to SEQ ID NO.5-6, primer pairs according to SEQ ID NO.7-8, primer pairs according to SEQ ID NO.9-10, primer pairs according to SEQ ID NO.11-12, primer pairs according to SEQ ID NO.13-14, primer pairs according to SEQ ID NO.15-16, primer pairs according to SEQ ID NO.17-18, primer pairs according to SEQ ID NO.19-20, primer pairs according to SEQ ID NO.21-22, primer pairs according to SEQ ID NO.23-24, primer pairs according to SEQ ID NO.25-26, primer pairs according to SEQ ID NO.27-28, primer pairs according to SEQ ID NO.29-30, primer pairs according to SEQ ID NO.31-32, primer pairs according to SEQ ID NO.33-34, The primer pair as shown in SEQ ID NO.35-36, the primer pair as shown in SEQ ID NO.37-38, the primer pair as shown in SEQ ID NO.39-40, the primer pair as shown in SEQ ID NO.41-42, the primer pair as shown in SEQ ID NO.43-44, the primer pair as shown in SEQ ID NO.45-46, the primer pair as shown in SEQ ID NO.47-48 and the primer pair as shown in SEQ ID NO. 49-50.
2. An elderberry SSR primer set according to claim 1 for use in the analysis of the genetic diversity of elderberry.
3. Use of an elderberry SSR primer set according to claim 1 in the construction of an elderberry genetic map.
4. An application of the elderberry SSR primer group of claim 1 in elderberry molecular marker-assisted breeding.
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| CN103642912A (en) * | 2013-11-29 | 2014-03-19 | 中国农业科学院作物科学研究所 | Method for developing mung bean simple sequence repeat (SSR) primer based on transcriptome sequencing |
| CN106834510A (en) * | 2017-03-20 | 2017-06-13 | 西南林业大学 | A kind of method that pseudo-ginseng SSR primers are developed based on transcript profile sequence |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103642912A (en) * | 2013-11-29 | 2014-03-19 | 中国农业科学院作物科学研究所 | Method for developing mung bean simple sequence repeat (SSR) primer based on transcriptome sequencing |
| CN106834510A (en) * | 2017-03-20 | 2017-06-13 | 西南林业大学 | A kind of method that pseudo-ginseng SSR primers are developed based on transcript profile sequence |
Non-Patent Citations (2)
| Title |
|---|
| 接骨木属植物研究进展;徐亮等;《中国野生植物资源》;20101031;第29卷(第5期);第1-5页 * |
| 珍贵观赏树种接骨木的研究现状与展望;孙华等;《湖北农业科学》;20100331;第49卷(第3期);摘要 * |
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