CN102424826B - Preparation method and application for Miscanthus Genic-SSR mark - Google Patents

Preparation method and application for Miscanthus Genic-SSR mark Download PDF

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CN102424826B
CN102424826B CN 201110443963 CN201110443963A CN102424826B CN 102424826 B CN102424826 B CN 102424826B CN 201110443963 CN201110443963 CN 201110443963 CN 201110443963 A CN201110443963 A CN 201110443963A CN 102424826 B CN102424826 B CN 102424826B
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ssr
awns
genic
sequence
primer
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CN102424826A (en
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刁英
郑兴飞
胡小虎
胡中立
游永宁
余作平
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HUBEI GUANGMANG ENERGY PLANT CO Ltd
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HUBEI GUANGMANG ENERGY PLANT CO Ltd
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Abstract

The invention discloses a preparation method and application for a Miscanthus Genic-SSR mark. The method comprises the following steps: A, acquiring Miscanthus transcriptomic sequence, that is, selecting young and tender Miscanthus leaves, extracting total RNA respectively so as to obtain transcriptome Reads, removing the pollution of carrier RNA sequence and carrying out splicing so as to obtain the Miscanthus transcriptomic sequence; B, identifying SSR sites in Miscanthus ESR sequence, that is, searching SSR sites in obtained non-redundant Unigene-EST sequence by using SSR retrieval software, with repeats being 2 to 6 bases, so as to obtain a series of Unigene sequences containing information about the SSR sites; C, designing Miscanthus Genic-SSR primers, that is, using primer design software to design primers according to the obtained Unigene sequences containing the SSR sites so as to obtain a series of the Miscanthus Genic-SSR primers. According to the invention, the method is simple, has high efficiency and is simple to operate; a huge amount of marks are obtained; EST sequence obtained through transcriptomic sequencing lays a foundation for genetic research, molecular improvement and the like of the critical energy plant Chinese silvergrass.

Description

A kind of preparation method and application of awns platymiscium Genic-SSR mark
Technical field
The invention belongs to technical field of molecular biology, more specifically relate to a kind of preparation method of awns platymiscium Genic-SSR mark, also relate to simultaneously a kind of purposes of awns platymiscium Genic-SSR mark.
Technical background
The awns platymiscium belongs to Gramineae C4 per nnial herb, mainly comprises awns, Caulis Miscanthis floriduli, reed, Nan Di, Qi Gang, concentrates to be distributed in east Asia and the southeast.The awns platymiscium has the characteristics such as strong, the photosynthetic utilising efficiency height of vitality, strong stress resistance, can be used for pharmacy, papermaking, soil conservation and restoration of the ecosystem etc.Along with the mankind to the improving constantly of energy demand, the continuous minimizing of global energy reserves, the awns platymiscium more and more is subject to showing great attention to of people as a kind of potential energy-source plant.At present, realize the extensively strange hilllock of plantation the America and Europe, annual output can reach annual 10～40 tons/hectare, is used to thermal power generation and fuel ethanol production.Along with deepening continuously of the work such as the genetic diversity Journal of Sex Research of awns platymiscium, hereditary and selection improvement and genetic map drafting, also increasing to the demand of awns platymiscium molecule marker.But the output, resistance, the eurytropy that belong to energy-source plant by the excavation Effective Raise awns of genetic improvement or important gene.Therefore, excavate the molecular genetic marker of awns platymiscium, great to awns platymiscium Research Significance.
Simple sequence repeats (simple sequence repeat, SSR) be the tumor-necrosis factor glycoproteins that a class 1-6bp Nucleotide motif consists of, be distributed widely in coding region, the non-coding region of eukaryotic gene group, the flank conserved sequence design primer that the simple sequence of utilizing the SSR mark repeats, through pcr amplification, reflect the polymorphism of dna sequence dna according to the size of band.Compare with other molecule marker such as RFLP, AFLP, ISSR etc., the SSR mark has the characteristics such as polymorphism height, codominant inheritance, good reproducibility, high specificity, becomes in recent years the fields such as genetic diversity Journal of Sex Research, genetic mapping, the critical function assignment of genes gene mapping, marker assisted selection and uses maximum a kind of marks.The exploitation of tradition SSR mark, normal operation screening libraries, magnesphere and utilize among the NCBI existing est database information excavating etc.Wherein, front two kinds of methods waste time and energy, cost of development high, and the third method depends on again EST data among the existing NCBI, and are very limited for its application of species that the EST order-checking is few.
By the end of on November 21st, 2011, the awns platymiscium est sequence that can retrieve in ncbi database only had 5, was difficult to satisfy the needs of exploitation EST-SSR mark; And in the existing research, awns platymiscium molecular markers development only is confined to utilize paramagnetic particle method or sets up the library and excavate, can't satisfy now awns platymiscium Research Requirements, therefore, obtain a large amount of est sequence information by transcribing the group order-checking, utilize information biology to excavate SSR and be marked as for a kind of efficient technique means.
Summary of the invention
The objective of the invention is to be to provide a kind of preparation method of awns platymiscium Genic-SSR mark, easy to implement the method, easy and simple to handle, obtain est sequence by transcribing the group order-checking, excavate SSR site information, design primer, make up awns platymiscium specificity Genic-SSR mark system, for the genetics research of important energy-source plant Chinese silvergrass and molecular improvement etc. lay the foundation.
Another object of the present invention is to be to provide a kind of awns platymiscium Genic-SSR to be marked at application in the awns platymiscium genetic diversity Journal of Sex Research, because the quantity of Genic-SSR mark is many and good stability, analytical results accurately and reliably.
The present invention is achieved through the following technical solutions:
A kind of preparation method of awns platymiscium Genic-SSR mark the steps include:
1), the awns platymiscium is transcribed the acquisition of group sequence:
Choose the young leaflet tablet of five kinds of awns platymiscium (awns, Caulis Miscanthis floriduli, reed, Nan Di, Qi Gang), extract respectively total RNA, group sequence measurement (Illumina Solexa HiSeq2000) is transcribed in employing, obtain transcribing group Reads, remove the RNA sequence pollutions such as carrier, utilize splicing software (such as SOAP, iAssembler etc.) to finish sequence assembly, finally obtain the nonredundancy Unigene-EST sequence of five kinds of awns platymiscium.Described awns platymiscium is wherein any one of awns, Caulis Miscanthis floriduli, reed, Nan Di, strange hilllock.
2), the evaluation in SSR site in the awns platymiscium ESR sequence:
The nonredundancy Unigene-EST sequence that adopts SSR retrieval software (as: can select SSR hunter, BatchPrimer3.0, MISA etc.) that above-mentioned steps (1) is obtained is carried out the SSR site and is searched.The qualifications of retrieval is: repeating motif is 2-6 base, and number of repeat unit is successively greater than 6 times, 5 times, and 4 times, 4 times, 3 times; The compound SSR that interrupts for the interval base of middle quilt≤50bp counts a SSR site, thereby obtains the Unigene sequence (asking for an interview table 1) of a series of SSR of containing site informations.
The Unigene-EST Statistical information that table 1 order-checking produces
Plant name (writing a Chinese character in simplified form) Unigene number/bar Design primer sum/bar The SSR sum/
Nan Di (LT) 64663 2347 3381
Reed (TS) 103114 2916 4463
Awns (MS) 97043 2576 3925
Caulis Miscanthis floriduli (MF) 67323 2351 3425
Strange hilllock (MG) 70021 2153 3211
3), the design of awns platymiscium Genic-SSR primer:
The Unigene sequence that contains the SSR site that obtains according to above-mentioned steps (2), adopt primer-design software (Primer3.0, Primer Premier 5.0 etc.) to carry out design of primers, obtain a series of Genic-SSR primers (asking for an interview table 1, table 2).The design of primers parameter mainly comprises: primer length is 18-25bp, and annealing temperature is 50-65 ℃, and GC content is 45-65%, and pcr amplification product length is 90-300bp.
4), amplification and the screening of awns platymiscium Genic-SSR primer:
Choose be used to five materials of transcribing the group order-checking, for detection of amplification situation and the transferability of awns platymiscium Genic-SSR mark.The spire 50-100mg that chooses five materials places the 2.0ml centrifuge tube, after the cooled with liquid nitrogen, rapidly sample is milled into Powdered with grinding rod.Genome extraction and application TIANGEN plant genome DNA extracts test kit (DP305-03).Utilize the quality of the agarose gel electrophoresis detection genomic dna of 1% (mass volume ratio).
Adopt the synthetic primer of above-mentioned steps (3), increase with the genomic dna of five materials, to detect reliability, amplification pattern and the versatility thereof of design of primers.According to amplification, the awns that screening can effectively be increased belongs to the Genic-SSR primer.According to amplification, filter out 60 pairs of Genic SSR primers can stablizing amplification, specifically see Table 2.
The synthetic awns platymiscium Genic-SSR primer sequence of design among table 2 the present invention
The primer title Repeat motif Upstream primer (5 '-3 ') Downstream primer (5 '-3 ')
ms-It-1 (CGATGA)3 CACCAGTGTTGGATGCTGAC TGTTGTCTAGCCTCCCGTCT
ms-It-2 (GA)9 ACACTGTCCCAAATCCTTCC AAGAAATCTCACTCCTCTCCCC
ms-It-3 (CGTGCT)3 CTCACAGGACAAGAGAGGGG GCATTCCAAAAGCATCCAGT
ms-It-4 (GCT)5 TGGAGCTGTGATCCTCTTCC CGGCTCAAAAACCACAAAAT
ms-It-5 (GCA)5 TCCAATCTAACCTCGTTGCC TGAGGTCTCGAACTGCAAAA
ms-It-6 (AGG)5 CACCGTCGAGTAGGGTGG CTGACGAGGGCACCAGAC
ms-It-8 (GCTGGT)3 ACCGGGGTCAGGAAGTAGAA TGTTCGTCTACAACGCCACT
ms-It-9 (CTCCGC)3 CCAGCTTCTCCCACACCC CACCGAAGAGCCCCTCTC
ms-It-10 (GTACGA)3 CTTGCAGTGCCTCCGTATG CCTCATCGTCGACTTCAACA
ms-It-12 (AGAGAC)3 AGTGATGGAACTGCTGGAGG CTTTGAGCGTCGATCCATTT
ms-It-13 (CAG)6 TGATGGTGTTGGTGATTTGG ATTTGCTGTTGCTGCTGTTG
ms-It-18 (TCACTA)3 TGGCTCCCACTATCACTTCC CTTGCTGCAACAGATGGAAA
ms-It-19 (CCT)5 CATGGACTCGGATCAGGACT CGAGGAGGAACTTGGTGGAT
ms-It-20 (CT)15 GCAGGTTGCGATCCTAAGAC GATCACCTGAAAAGGCAAGC
ms-It-21 (CGCCA)4 CCAAGCTCTAAACAGGCTCG GATGGGAGTTTACGTGGGG
ms-It-23 (GCACCG)3 CAACCTCCTCTCCCTGATGA AGACGGAGATGAATCGTTGG
ms-It-24 (CGC)5 CATGGTTGAGTTCTACGCCC AGAAGAGGATGGTGGGGAAG
ms-It-26 (GAG)5 GGTCCATGACGAAGTCGAAG CCCGGAGTTCTACTGCTACG
ms-It-27 (GAGC)4 AAACATGCGTGCTTTGACTG TTGCCTCTTTCGTGCTACCT
ms-It-28 (CTG)5 ACCACCAAAACCAAACGAAG TAGAGGGGAAGAAGGGGAAG
ms-It-29 (CCTT)4 GGTCTAATCGCCCCGTAGAT CCCTTTACTCCCGAGGAGAC
mf-ts-1 (AG)7 CAGATGAGACCAGCAGTGGA ACGCCAGTCAACCATCTCTT
mf-ts-2 (AC)6 ACAGGGAGAGGGGAGAAGAA TCCATCGTTTTCTGGTGTGA
mf-ts-4 (CCGCCT)3 GTGCTCTTCCGCCTGAACTA TCCTCCTCCTCACCATCATC
mf-ts-5 (TCCGCG)4 CAAATGGTCGTCCCAAAGAC AGAGGAAGAATGGGTGGGAG
mf-ts-6 (GCGACG)3 ATGGCTACCACTCAGTTCGC GTCGTCGTCCTCTTCAAGG
mf-ts-9 (TGCATC)3 ACCGTCCATCCATCTGTAGC GCTTCCTGGACGACTCTGTT
mf-ts-10 (GAC)5 CTCGAGCGAGCTGTTCATTT TGAAGATACCGTGGAGGAGG
mf-ts-11 (CGA)5 CTTGTCCCTCTTCACCAAGC CCTTTCTTCCAGTGGCTCAA
mf-ts-13 (GAA)5 CCAGAGGGATAGGAGGAGGA CACGTGTAGCAGGTCCTGAA
mf-ts-14 (GCC)5 GCTCTCCGAGGTCGTGTC CCTGTCTGAGGATGGTCCC
mf-ts-15 (CTC)9 ACGTCCTTCTTTCTGCTGGA CGTCTCTGCTCCTGGCTTTA
mf-ts-16 (CGT)6 GGAACGGGGAAACCTCTG TCTCTTCTCTTCTCCGTCGC
mf-ts-17 (CGG)5 AAAGGGTACGGCACTGAGGT GCAACGTGAACTCTGTCTGC
mf-ts-18 (GC)6 GAGGGGATCTAGATGAGGGC CCAGCTCGTTTCTTCCTCAG
mf-ts-19 (GCA)5 CGTCTGCGACACAAGAACAT ATGATGTTGCTGGCCTTGAT
mf-ts-21 (CAC)5 GGACACCTACACGGTCACCT CTGCTGGCTGGTGGTGTT
mf-ts-22 (CGC)5 CATGGTTGAGTTCTACGCCC AGAAGAGGATGGTGGGGAAG
mf-ts-23 (TCTTCA)3 CGCACAAAAGAATGCATCAC GGAACGCCACTTTCTAGCAC
mf-ts-24 (GGA)5 ACCAGCACCGTCATCATCAG GAACAGGTTGCCGGTGTATC
mf-ts-25 (CTC)5 CTACGACCGCTGCTGCTAGT TCTCCATACTGCTCTGGGCT
mf-ts-28 (CGG)5 TTCCGATCTCAAGGACCAAC AACCCTCCTGTCCATGATGA
mf-ts-29 (CTCTTC)3 CTGCAAGTTTCTTTGCCCTC TCTGAAAAGTGGTGGTGTGC
mg-1 (AG)8 ATCAGAGAGAGGCACCTGGA GAGTGAGGAGATGACCCGAC
mg-2 (AC)10 AGAAACGTGAAACCTGCTGAA AAGCTGACAGCACAACAACG
mg-5 (CTCATC)4 GGCGGATAATCAGCAAGTGT GAAGAGTGGCTGGGTTGAAG
mg-6 (GGC)5 GGAGAGTGAGAGCAGGGTTG GTTGCCACAAATCTGGCTG
mg-7 (CCTGAA)3 ACCAAGACCAAATCTGCCAC ACACAGCTACATCGGGTTCC
mg-8 (CA)6 AACTCTTTTACAACGTCAATTTGC CAGCCTTTGGTTAGAGCACC
mg-17 (AG)6 AATAGGAGGCTGGCTGGTTT GCAATTTGCTCCTGCACATA
mg-18 (AG)13 GCAGCAGAGAGGAGAGGAGA CCCGTCGTGTTGATGTACTG
mg-19 (GTGGCA)3 TCTCCTCCAGGTTCACCATC AGGTGCAAATCATCTACCGC
mg-21 (CTG)6 ACAGGAACGGAGGAACTTGA GATACGCCGTCGCTGAAG
mg-22 (TG)7 GATCGCTCCTTCCATTGTGT AATCTCAAGTCCTCCTCCCC
mg-23 (TCA)6 TGAGATTCCTCCCTCCATTG GCGCTCTACTCCAGGAAAAA
mg-25 (ATA)6 GCCCCTCAGATGGTTGAATA TGTGGTACATCTCCCTGCAA
mg-26 (CCT)5 GTCACTCTCCCCTCCCATTC TGCACCCTTTACGAACTCCT
mg-27 (TA)6 CCATGGCTAAGCCTCACTTC GCGCACACACACACACTACT
mg-29 (GGCGCG)3 AGCGTAAGAGGGAGGATGGT GGAGCTCCTCCTCGGTGT
mg-30 (GGC)7 TTGACGGTGACGATGAGGTA TCAAATCTCATCAGTTCCCAAA
A kind of awns platymiscium Genic-SSR is marked at the application in the awns platymiscium analysis of genetic diversity, the steps include:
1), experiment material:
Choose that awns belongs to and river eight kings belong to totally 17 parts of materials, be marked at validity in the awns platymiscium analysis of genetic diversity for detection of awns platymiscium Genic-SSR, concrete material information sees table 3 for details.
Be used for carrying out the material information of primer screening and analysis of genetic diversity among table 3 the present invention
Material number Belong to-kind/the kind name Material source Kind of information
1 Awns genus-Mang Chang Ling town, Ezhou, Hubei Province Wild species
2 Awns genus-Mang Chang Ling town, Ezhou, Hubei Province Wild species
3 Awns genus-spire awns Wuhan University's Germplasm Resources Cultivar
4 Awns genus-zebra grass Wuhan University's Germplasm Resources Cultivar
5 Awns genus-morning twilight awns Wuhan University's Germplasm Resources Cultivar
6 Awns genus-floral leaf awns Wuhan University's Germplasm Resources Cultivar
7 Awns genus-Caulis Miscanthis floriduli Liang Zi island, Ezhou, Hubei Province Wild species
8 Awns genus-Caulis Miscanthis floriduli Liang Zi island, Ezhou, Hubei Province Wild species
9 Awns genus-Mang Liang Zi island, Ezhou, Hubei Province Wild species
10 Awns genus-Mang Liang Zi island, Ezhou, Hubei Province Wild species
11 Awns genus-reed Chang Ling town, Ezhou, Hubei Province Wild species
12 Awns genus-reed Liang Zi island, Ezhou, Hubei Province Wild species
13 Awns genus-Nan Di The Hunan Yueyang Dongting lake Wild species
14 Awns genus-Nan Di The Yun County, Hubei Province Wild species
15 Awns genus-Qi hilllock Source, Wuhan University Germplasm Resources Beijing Cultivar
16 Awns genus-Qi hilllock Source, Wuhan University Germplasm Resources Nanjing Cultivar
17 Eight king genus-rivers, river, eight kings Chang Ling town, Ezhou, Hubei Province Wild species
2), genome extracts:
Every part of material selection young tender leaf agreement that contracts a film or TV play to an actor or actress 50-100mg places the 2.0ml centrifuge tube, after the cooled with liquid nitrogen, mills rapidly with grinding rod, and cooling is milled until powdered repeatedly.Utilize the TIANGEN plant genome DNA to extract test kit and extract genome.Utilize the quality of the agarose gel electrophoresis detection genomic dna of 1% (mass volume ratio).
3), Genic-SSR primer amplification:
Utilize the Genic-SSR primer of screening among the embodiment 1, the genomic dna that amplification above-mentioned steps (2) obtains.The employing amplification system is as follows:
Contain in the 15ul amplification system: 1x PCR buffer, MgCl 21.6mmol/L, dNTPs (ancient cooking vessel state) 100pmol/L, forward and reverse primer 0.5umol/L, genomic templates DNA50ng and Taq archaeal dna polymerase (ancient cooking vessel state) 0.5U.
(America) increases on the bio-RAD thermal cycler, and the pcr amplification program is:
A, 94 ℃ of denaturation 4min;
B, 94 ℃ of sex change 30s, 56 ℃ of annealing 30s, 72 ℃ of extension 45s;
C, 72 ℃ of extension 6min;
d、16min，5min。
The PCR product that amplification obtains directly adds 6X loading buffer 10ul, place 95 ℃ of sex change 5min of thermal cycler, ice bath 5min, in 6% (mass volume ratio) polyacrylamide denaturing gel electrophoresis, the electrophoresis parameter is 3000v, 60mA, the permanent power electrophoresis of 68W 40min, the colour developing of employing silver staining method, the Taking Pictures recording result.Fig. 1 is primer mf-ts-9, mf-ts-10 amplification.
17 parts for the examination materials in, 60 pairs of Genic-SSR coamplifications 245 sites, average 4.1 sites of every pair of primer.And most of SSR primers all can effectively increase between different genera, transferable rate 71%-100%, and average transferable rate is 97.5%, illustrates that Genic-SSR has very high versatility in awns genus and relative genus thereof, can be used for further diversity analysis.
4), Genic-SSR primer amplification result statistics and analysis of genetic diversity:
According to the step among the embodiment 1 (3) amplification, there is band to count 1 according to amplified band, count 0 without band, obtain one 0,1 binary matrix, its input NTSYS-pc software processes is analyzed, Jaccard similarity factor with between SIMQUAL sub-routine calculating sample then based on the similarity factor matrix, utilizes SHAN sub-routine UPGMA algorithm to carry out cluster analysis.Cluster analysis result is seen Fig. 2.
To carrying out cluster analysis for the examination material, the result shows the genetic distance maximum (cofficient=0.71) of river eight kings and other awns platymisciums with the UPGMA algorithm, illustrates that Genic-SSR can clearly distinguish the awns genus and river eight kings belong to; In addition, it is a subgroup that each kind that awns belongs to do not have obvious cluster, as: material 11-reed and awns are poly-to be a branch, and material 12-reed and material 13-south reed are poly-to be a branch, illustrates within awns belongs to have abundant diversity and the similarity in the heredity between each kind.
The present invention compared with prior art has the following advantages and effect:
Method is simple, and efficient is high, and easy and simple to handle, the marker number of acquisition is huge, and the Genic-SSR mark that the present invention obtains is more reliable and more stable than marks such as AFLP, SRAP, ISSR.Obtain est sequence by transcribing group order-checking, excavate SSR site information, design primer, make up awns platymiscium specificity Genic-SSR mark system, for the genetics research of important energy-source plant Chinese silvergrass and molecular improvement etc. lay the foundation.
Description of drawings
Fig. 1 be a kind of primer mf-ts-9 (left side) and mf-ts-10 (right side) to the amplification situation of 17 parts of materials, M is pBR322maker.
Primer mf-ts-9 among the present invention and mf-ts-10 are to the amplification situation of 17 parts of materials.
Fig. 2 is the similarity factor dendrogram that a kind of primer analysis awns genus and river eight kings belong to 17 parts of materials.
Primer analysis awns genus of the present invention and river eight kings belong to the similarity factor dendrogram of 17 parts of materials.
Embodiment
The present invention is described in further detail below in conjunction with embodiment.
Embodiment 1:
Present embodiment is preparation and the screening method of awns platymiscium Genic-SSR mark, mainly comprises following key step:
1, the acquisition of awns platymiscium est sequence:
Choose the young leaflet tablet of five kinds of awns platymiscium (awns, Caulis Miscanthis floriduli, reed, Nan Di, Qi Gang), extract total RNA, the Solexa sequence measurement of employing standard, receive and transcribe group Reads, adopt Repeat Masker to remove the RNA sequence pollutions such as carrier, utilize SOAP splicing software to finish sequence assembly, finally obtain the nonredundancy Unigene-EST sequence of five cover awns platymisciums, see table 1 for details.
The Unigene-EST Statistical information that table 1 order-checking produces
Plant name (writing a Chinese character in simplified form) Unigene number/bar Design primer sum/bar The SSR sum/
Nan Di (LT) 64663 2347 3381
Reed (TS) 103114 2916 4463
Awns (MS) 97043 2576 3925
Caulis Miscanthis floriduli (MF) 67323 2351 3425
Strange hilllock (MG) 70021 2153 3211
2, the screening in SSR site in the awns platymiscium est sequence:
Adopt little satellite acquisition software MISA, the five cover nonredundancy Unigene-EST sequences that above-mentioned steps (1) obtains are carried out the retrieval of SSR site, the retrieval restricted condition comprises: primer length is 18-25bp, annealing temperature is 50-65 ℃, GC content is 45-65%, and pcr amplification product length is 90-300bp; Retrieval obtains a series of Genic-SSR site, sees Table 1.
3, the design of awns platymiscium Genic-SSR primer is with synthetic:
The Genic-SSR that obtains according to above-mentioned steps (2), utilize EMBOSS software, the primer that prediction awns and southern reed, Caulis Miscanthis floriduli and reed are total, in conjunction with Unigene-EST flank region sequence, use Primer3.0 software and carry out design of primers, the significant parameter of design of primers comprises: GC content 45-65%, 45-65 ℃ of Tm value, primer length 18-25bp, expection amplified production length 90-300bp.Last choose at random that awns is total with southern reed, Caulis Miscanthis floriduli is total with reed, very the hilllock respectively 30 synthesizes (90 pairs) primer totally.
4, amplification and the screening of awns platymiscium Genic-SSR primer:
Choose be used to five materials of transcribing the group order-checking, for detection of amplification situation and the transferability of awns platymiscium Genic-SSR mark.The spire 50-100mg that chooses five materials places the 2.0ml centrifuge tube, after the cooled with liquid nitrogen, rapidly sample is milled into Powdered with grinding rod.Genome extraction and application TIANGEN plant genome DNA extracts test kit (DP305-03).Utilize the quality of the agarose gel electrophoresis detection genomic dna of 1% (mass volume ratio).
Adopt the synthetic primer of above-mentioned steps (3), increase with the genomic dna of five materials, to detect reliability, amplification pattern and the versatility thereof of design of primers.According to amplification, the awns that screening can effectively be increased belongs to the Genic-SSR primer.
In this example, when each material genomic dna is carried out pcr amplification, adopt following amplification system:
Contain in the 15ul amplification system: 1x PCR buffer, MgCl 21.6mmol/L, dNTPs (ancient cooking vessel state) 100pmol/L, forward and reverse primer 0.5umol/L, genomic templates DNA50ng and Taq archaeal dna polymerase (ancient cooking vessel state) 0.5U.
(America) increases on the bio-RAD thermal cycler, and the pcr amplification program is:
A, 94 ℃ of denaturation 4min;
B, 94 ℃ of sex change 30s, 56 ℃ of annealing 30s, 72 ℃ of extension 45s;
C, 72 ℃ of extension 6min;
d、16min，5min。
After pcr amplification reaction is finished, directly in the PCR product, add 6 * loading buffer 10ul mixing, place 95 ℃ of sex change 5min of thermal cycler, ice bath 5min, detect in 8% (mass volume ratio) denaturing polyacrylamide gel, the electrophoresis parameter is 3000v, 60mA, the permanent power electrophoresis of 68W 40min, adopts the silver staining method colour developing, the Taking Pictures recording result.According to amplification, filter out 60 pairs of Genic-SSR primers can stablizing amplification, specifically see Table 2.
The synthetic awns platymiscium Genic-SSR primer sequence of design among table 2 the present invention
The primer title Repeat motif Upstream primer (5 '-3 ') Downstream primer (5 '-3 ')
ms-It-1 (CGATGA)3 CACCAGTGTTGGATGCTGAC TGTTGTCTAGCCTCCCGTCT
ms-It-2 (GA)9 ACACTGTCCCAAATCCTTCC AAGAAATCTCACTCCTCTCCCC
ms-It-3 (CGTGCT)3 CTCACAGGACAAGAGAGGGG GCATTCCAAAAGCATCCAGT
ms-It-4 (GCT)5 TGGAGCTGTGATCCTCTTCC CGGCTCAAAAACCACAAAAT
ms-It-5 (GCA)5 TCCAATCTAACCTCGTTGCC TGAGGTCTCGAACTGCAAAA
ms-It-6 (AGG)5 CACCGTCGAGTAGGGTGG CTGACGAGGGCACCAGAC
ms-It-8 (GCTGGT)3 ACCGGGGTCAGGAAGTAGAA TGTTCGTCTACAACGCCACT
ms-It-9 (CTCCGC)3 CCAGCTTCTCCCACACCC CACCGAAGAGCCCCTCTC
ms-It-10 (GTACGA)3 CTTGCAGTGCCTCCGTATG CCTCATCGTCGACTTCAACA
ms-It-12 (AGAGAC)3 AGTGATGGAACTGCTGGAGG CTTTGAGCGTCGATCCATTT
ms-It-13 (CAG)6 TGATGGTGTTGGTGATTTGG ATTTGCTGTTGCTGCTGTTG
ms-It-18 (TCACTA)3 TGGCTCCCACTATCACTTCC CTTGCTGCAACAGATGGAAA
ms-It-19 (CCT)5 CATGGACTCGGATCAGGACT CGAGGAGGAACTTGGTGGAT
ms-It-20 (CT)15 GCAGGTTGCGATCCTAAGAC GATCACCTGAAAAGGCAAGC
ms-It-21 (CGCCA)4 CCAAGCTCTAAACAGGCTCG GATGGGAGTTTACGTGGGG
ms-It-23 (GCACCG)3 CAACCTCCTCTCCCTGATGA AGACGGAGATGAATCGTTGG
ms-It-24 (CGC)5 CATGGTTGAGTTCTACGCCC AGAAGAGGATGGTGGGGAAG
ms-It-26 (GAG)5 GGTCCATGACGAAGTCGAAG CCCGGAGTTCTACTGCTACG
ms-It-27 (GAGC)4 AAACATGCGTGCTTTGACTG TTGCCTCTTTCGTGCTACCT
ms-It-28 (CTG)5 ACCACCAAAACCAAACGAAG TAGAGGGGAAGAAGGGGAAG
ms-It-29 (CCTT)4 GGTCTAATCGCCCCGTAGAT CCCTTTACTCCCGAGGAGAC
mf-ts-1 (AG)7 CAGATGAGACCAGCAGTGGA ACGCCAGTCAACCATCTCTT
mf-ts-2 (AC)6 ACAGGGAGAGGGGAGAAGAA TCCATCGTTTTCTGGTGTGA
mf-ts-4 (CCGCCT)3 GTGCTCTTCCGCCTGAACTA TCCTCCTCCTCACCATCATC
mf-ts-5 (TCCGCG)4 CAAATGGTCGTCCCAAAGAC AGAGGAAGAATGGGTGGGAG
mf-ts-6 (GCGACG)3 ATGGCTACCACTCAGTTCGC GTCGTCGTCCTCTTCAAGG
mf-ts-9 (TGCATC)3 ACCGTCCATCCATCTGTAGC GCTTCCTGGACGACTCTGTT
mf-ts-10 (GAC)5 CTCGAGCGAGCTGTTCATTT TGAAGATACCGTGGAGGAGG
mf-ts-11 (CGA)5 CTTGTCCCTCTTCACCAAGC CCTTTCTTCCAGTGGCTCAA
mf-ts-13 (GAA)5 CCAGAGGGATAGGAGGAGGA CACGTGTAGCAGGTCCTGAA
mf-ts-14 (GCC)5 GCTCTCCGAGGTCGTGTC CCTGTCTGAGGATGGTCCC
mf-ts-15 (CTC)9 ACGTCCTTCTTTCTGCTGGA CGTCTCTGCTCCTGGCTTTA
mf-ts-16 (CGT)6 GGAACGGGGAAACCTCTG TCTCTTCTCTTCTCCGTCGC
mf-ts-17 (CGG)5 AAAGGGTACGGCACTGAGGT GCAACGTGAACTCTGTCTGC
mf-ts-18 (GC)6 GAGGGGATCTAGATGAGGGC CCAGCTCGTTTCTTCCTCAG
mf-ts-19 (GCA)5 CGTCTGCGACACAAGAACAT ATGATGTTGCTGGCCTTGAT
mf-ts-21 (CAC)5 GGACACCTACACGGTCACCT CTGCTGGCTGGTGGTGTT
mf-ts-22 (CGC)5 CATGGTTGAGTTCTACGCCC AGAAGAGGATGGTGGGGAAG
mf-ts-23 (TCTTCA)3 CGCACAAAAGAATGCATCAC GGAACGCCACTTTCTAGCAC
mf-ts-24 (GGA)5 ACCAGCACCGTCATCATCAG GAACAGGTTGCCGGTGTATC
mf-ts-25 (CTC)5 CTACGACCGCTGCTGCTAGT TCTCCATACTGCTCTGGGCT
mf-ts-28 (CGG)5 TTCCGATCTCAAGGACCAAC AACCCTCCTGTCCATGATGA
mf-ts-29 (CTCTTC)3 CTGCAAGTTTCTTTGCCCTC TCTGAAAAGTGGTGGTGTGC
mg-1 (AG)8 ATCAGAGAGAGGCACCTGGA GAGTGAGGAGATGACCCGAC
mg-2 (AC)10 AGAAACGTGAAACCTGCTGAA AAGCTGACAGCACAACAACG
mg-5 (CTCATC)4 GGCGGATAATCAGCAAGTGT GAAGAGTGGCTGGGTTGAAG
mg-6 (GGC)5 GGAGAGTGAGAGCAGGGTTG GTTGCCACAAATCTGGCTG
mg-7 (CCTGAA)3 ACCAAGACCAAATCTGCCAC ACACAGCTACATCGGGTTCC
mg-8 (CA)6 AACTCTTTTACAACGTCAATTTGC CAGCCTTTGGTTAGAGCACC
mg-17 (AG)6 AATAGGAGGCTGGCTGGTTT GCAATTTGCTCCTGCACATA
mg-18 (AG)13 GCAGCAGAGAGGAGAGGAGA CCCGTCGTGTTGATGTACTG
mg-19 (GTGGCA)3 TCTCCTCCAGGTTCACCATC AGGTGCAAATCATCTACCGC
mg-21 (CTG)6 ACAGGAACGGAGGAACTTGA GATACGCCGTCGCTGAAG
mg-22 (TG)7 GATCGCTCCTTCCATTGTGT AATCTCAAGTCCTCCTCCCC
mg-23 (TCA)6 TGAGATTCCTCCCTCCATTG GCGCTCTACTCCAGGAAAAA
mg-25 (ATA)6 GCCCCTCAGATGGTTGAATA TGTGGTACATCTCCCTGCAA
mg-26 (CCT)5 GTCACTCTCCCCTCCCATTC TGCACCCTTTACGAACTCCT
mg-27 (TA)6 CCATGGCTAAGCCTCACTTC GCGCACACACACACACTACT
mg-29 (GGCGCG)3 AGCGTAAGAGGGAGGATGGT GGAGCTCCTCCTCGGTGT
mg-30 (GGC)7 TTGACGGTGACGATGAGGTA TCAAATCTCATCAGTTCCCAAA
Embodiment 2:
A kind of awns platymiscium Genic-SSR is marked at the application in the awns platymiscium analysis of genetic diversity, the steps include:
1, experiment material:
Choose that awns belongs to and river eight kings belong to totally 17 parts of materials, be marked at validity in the awns platymiscium analysis of genetic diversity for detection of awns platymiscium Genic-SSR, concrete material information sees table 3 for details.
Be used for carrying out the material information of primer screening and analysis of genetic diversity among table 3 the present invention
Material number Belong to-kind/the kind name Material source Kind of information
1 Awns genus-Mang Chang Ling town, Ezhou, Hubei Province Wild species
2 Awns genus-Mang Chang Ling town, Ezhou, Hubei Province Wild species
3 Awns genus-spire awns Wuhan University's Germplasm Resources Cultivar
4 Awns genus-zebra grass Wuhan University's Germplasm Resources Cultivar
5 Awns genus-morning twilight awns Wuhan University's Germplasm Resources Cultivar
6 Awns genus-floral leaf awns Wuhan University's Germplasm Resources Cultivar
7 Awns genus-Caulis Miscanthis floriduli Liang Zi island, Ezhou, Hubei Province Wild species
8 Awns genus-Caulis Miscanthis floriduli Liang Zi island, Ezhou, Hubei Province Wild species
9 Awns genus-Mang Liang Zi island, Ezhou, Hubei Province Wild species
10 Awns genus-Mang Liang Zi island, Ezhou, Hubei Province Wild species
11 Awns genus-reed Chang Ling town, Ezhou, Hubei Province Wild species
12 Awns genus-reed Liang Zi island, Ezhou, Hubei Province Wild species
13 Awns genus-Nan Di The Hunan Yueyang Dongting lake Wild species
14 Awns genus-Nan Di The Yun County, Hubei Province Wild species
15 Awns genus-Qi hilllock Source, Wuhan University Germplasm Resources Beijing Cultivar
16 Awns genus-Qi hilllock Source, Wuhan University Germplasm Resources Nanjing Cultivar
17 Eight king genus-rivers, river, eight kings Chang Ling town, Ezhou, Hubei Province Wild species
2, genome extracts:
Every part of material selection young tender leaf agreement that contracts a film or TV play to an actor or actress 50-100mg places the 2.0ml centrifuge tube, after the cooled with liquid nitrogen, mills rapidly with grinding rod, and cooling is milled until powdered repeatedly.Utilize the TIANGEN plant genome DNA to extract test kit and extract genome.Utilize the quality of the agarose gel electrophoresis detection genomic dna of 1% (mass volume ratio).
3, Genic-SSR primer amplification:
Utilize the Genic-SSR primer of screening among the embodiment 1, the genomic dna that amplification above-mentioned steps (2) obtains.The employing amplification system is as follows:
Contain in the 15ul amplification system: 1x PCR buffer, MgCl 21.6mmol/L, dNTPs (ancient cooking vessel state) 100pmol/L, forward and reverse primer 0.5umol/L, genomic templates DNA50ng and Taq archaeal dna polymerase (ancient cooking vessel state) 0.5U.
(America) increases on the bio-RAD thermal cycler, and the pcr amplification program is:
A, 94 ℃ of denaturation 4min;
B, 94 ℃ of sex change 30s, 56 ℃ of annealing 30s, 72 ℃ of extension 45s;
C, 72 ℃ of extension 6min;
d、16min，5min。
The PCR product that amplification obtains directly adds 6Xloading buffer 10ul, place 95 ℃ of sex change 5min of thermal cycler, ice bath 5min, in 6% (mass volume ratio) polyacrylamide denaturing gel electrophoresis, the electrophoresis parameter is 3000v, 60mA, the permanent power electrophoresis of 68W 40min, the colour developing of employing silver staining method, the Taking Pictures recording result.Fig. 1 is primer mf-ts-9, mf-ts-10 amplification.
17 parts for the examination materials in, 60 pairs of Genic-SSR coamplifications 245 sites, average 4.1 sites of every pair of primer.And most of SSR primers all can effectively increase between different genera, transferable rate 71%-100%, and average transferable rate is 97.5%, illustrates that Genic-SSR has very high versatility in awns genus and relative genus thereof, can be used for further diversity analysis.
4, Genic-SSR primer amplification result statistics and analysis of genetic diversity:
According to the step among the embodiment 1 (3) amplification, there is band to count 1 according to amplified band, count 0 without band, obtain one 0,1 binary matrix, its input NTSYS-pc software processes is analyzed, Jaccard similarity factor with between SIMQUAL sub-routine calculating sample then based on the similarity factor matrix, utilizes SHAN sub-routine UPGMA algorithm to carry out cluster analysis.Cluster analysis result is seen Fig. 2.
To carrying out cluster analysis for the examination material, the result shows the genetic distance maximum (cofficient=0.71) of river eight kings and other awns platymisciums with the UPGMA algorithm, illustrates that Genic-SSR can clearly distinguish the awns genus and river eight kings belong to; In addition, it is a subgroup that each kind that awns belongs to do not have obvious cluster, as: material 11-reed and awns are poly-to be a branch, and material 12-reed and material 13-south reed are poly-to be a branch, illustrates within awns belongs to have abundant diversity and the similarity in the heredity between each kind.

Claims (2)

1. the preparation method of an awns platymiscium Genic-SSR mark the steps include:
1), the awns platymiscium is transcribed the acquisition of group sequence:
Choose the young leaflet tablet of awns platymiscium, extract respectively total RNA, adopt and transcribe the group sequence measurement, obtain transcribing group Reads, remove the vector rna sequence and pollute, utilize splicing software to finish sequence assembly, finally obtain the nonredundancy Unigene-EST sequence of five kinds of awns platymiscium;
2), the evaluation in SSR site in the awns platymiscium est sequence:
The nonredundancy Unigene-EST sequence that adopts the SSR retrieval software that above-mentioned steps (1) is obtained is carried out the SSR site and is searched, and the qualifications of retrieval is: repeating motif is 2-6 base, and number of repeat unit is successively greater than 6 times, and 5 times, 4 times, 4 times, 3 times; The compound SSR that interrupts for the interval base of middle quilt≤50bp counts a SSR site, obtains a series of Unigene sequences that contain the SSR site information;
3), the design of awns platymiscium Genic-SSR primer:
The Unigene sequence that contains the SSR site that obtains according to above-mentioned steps (2), adopt primer-design software to carry out design of primers, obtain a series of Genic-SSR primers, the design of primers parameter mainly comprises: primer length is 18-25bp, annealing temperature is 50-65 ℃, GC content is 45-65%, and pcr amplification product length is 90-300bp;
4), amplification and the screening of awns platymiscium Genic-SSR primer:
Choose be used to five materials of transcribing the group order-checking, for detection of amplification situation and the transferability of awns platymiscium Genic-SSR mark; The spire 50-100mg that chooses five materials places the 2.0ml centrifuge tube, after the cooled with liquid nitrogen, rapidly sample is milled into Powdered with grinding rod, genome extraction and application TIANGEN plant genome DNA extracts test kit, utilizes the quality of the agarose gel electrophoresis detection genomic dna of 1% mass volume ratio;
Adopt the synthetic primer of above-mentioned steps (3), increase with the genomic dna of five materials, to detect reliability, amplification pattern and the versatility thereof of design of primers; According to amplification, the awns that screening can effectively be increased belongs to the Genic-SSR primer; According to amplification, filter out 60 pairs of Genic-SSR primers can stablizing amplification, specific as follows:
ms-lt-1
F:CACCAGTGTTGGATGCTGAC
R:TGTTGTCTAGCCTCCCGTCT
ms-lt-2
F:ACACTGTCCCAAATCCTTCC
R:AAGAAATCTCACTCCTCTCCCC
ms-lt-3
F:CTCACAGGACAAGAGAGGGG
R:GCATTCCAAAAGCATCCAGT
ms-lt-4
F:TGGAGCTGTGATCCTCTTCC
R:CGGCTCAAAAACCACAAAAT
ms-lt-5
F:TCCAATCTAACCTCGTTGCC
R:TGAGGTCTCGAACTGCAAAA
ms-lt-6
F:CACCGTCGAGTAGGGTGG
R:CTGACGAGGGCACCAGAC
ms-lt-8
F:ACCGGGGTCAGGAAGTAGAA
R:TGTTCGTCTACAACGCCACT
ms-lt-9
F:CCAGCTTCTCCCACACCC
R:CACCGAAGAGCCCCTCTC
ms-lt-10
F:CTTGCAGTGCCTCCGTATG
R:CCTCATCGTCGACTTCAACA
ms-lt-12
F:AGTGATGGAACTGCTGGAGG
R:CTTTGAGCGTCGATCCATTT
ms-lt-13
F:TGATGGTGTTGGTGATTTGG
R:ATTTGCTGTTGCTGCTGTTG
ms-lt-18
F:TGGCTCCCACTATCACTTCC
R:CTTGCTGCAACAGATGGAAA
ms-lt-19
F:CATGGACTCGGATCAGGACT
R:CGAGGAGGAACTTGGTGGAT
ms-lt-20
F:GCAGGTTGCGATCCTAAGAC
R:GATCACCTGAAAAGGCAAGC
ms-lt-21
F:CCAAGCTCTAAACAGGCTCG
R:GATGGGAGTTTACGTGGGG
ms-lt-23
F:CAACCTCCTCTCCCTGATGA
R:AGACGGAGATGAATCGTTGG
ms-lt-24
F:CATGGTTGAGTTCTACGCCC
R:AGAAGAGGATGGTGGGGAAG
ms-lt-26
F:GGTCCATGACGAAGTCGAAG
R:CCCGGAGTTCTACTGCTACG
ms-lt-27
F:AAACATGCGTGCTTTGACTG
R:TTGCCTCTTTCGTGCTACCT
ms-lt-28
F:ACCACCAAAACCAAACGAAG
R:TAGAGGGGAAGAAGGGGAAG
ms-lt-29
F:GGTCTAATCGCCCCGTAGAT
R:CCCTTTACTCCCGAGGAGAC
mf-ts-1
F:CAGATGAGACCAGCAGTGGA
R:ACGCCAGTCAACCATCTCTT
mf-ts-2
F:ACAGGGAGAGGGGAGAAGAA
R:TCCATCGTTTTCTGGTGTGA
mf-ts-4
F:GTGCTCTTCCGCCTGAACTA
R:TCCTCCTCCTCACCATCATC
mf-ts-5
F:CAAATGGTCGTCCCAAAGAC
R:AGAGGAAGAATGGGTGGGAG
mf-ts-6
F:ATGGCTACCACTCAGTTCGC
R:GTCGTCGTCCTCTTCAAGG
mf-ts-9
F:ACCGTCCATCCATCTGTAGC
R:GCTTCCTGGACGACTCTGTT
mf-ts-10
F:CTCGAGCGAGCTGTTCATTT
R:TGAAGATACCGTGGAGGAGG
mf-ts-11
F:CTTGTCCCTCTTCACCAAGC
R:CCTTTCTTCCAGTGGCTCAA
mf-ts-13
F:CCAGAGGGATAGGAGGAGGA
R:CACGTGTAGCAGGTCCTGAA
mf-ts-14
F:GCTCTCCGAGGTCGTGTC
R:CCTGTCTGAGGATGGTCCC
mf-ts-15
F:ACGTCCTTCTTTCTGCTGGA
R:CGTCTCTGCTCCTGGCTTTA
mf-ts-16
F:GGAACGGGGAAACCTCTG
R:TCTCTTCTCTTCTCCGTCGC
mf-ts-17
F:AAAGGGTACGGCACTGAGGT
R:GCAACGTGAACTCTGTCTGC
mf-ts-18
F:GAGGGGATCTAGATGAGGGC
R:CCAGCTCGTTTCTTCCTCAG
mf-ts-19
F:CGTCTGCGACACAAGAACAT
R:ATGATGTTGCTGGCCTTGAT
mf-ts-21
F:GGACACCTACACGGTCACCT
R:CTGCTGGCTGGTGGTGTT
mf-ts-22
F:CATGGTTGAGTTCTACGCCC
R:AGAAGAGGATGGTGGGGAAG
mf-ts-23
F:CGCACAAAAGAATGCATCAC
R:GGAACGCCACTTTCTAGCAC
mf-ts-24
F:ACCAGCACCGTCATCATCAG
R:GAACAGGTTGCCGGTGTATC
mf-ts-25
F:CTACGACCGCTGCTGCTAGT
R:TCTCCATACTGCTCTGGGCT
mf-ts-28
F:TTCCGATCTCAAGGACCAAC
R:AACCCTCCTGTCCATGATGA
mf-ts-29
F:CTGCAAGTTTCTTTGCCCTC
R:TCTGAAAAGTGGTGGTGTGC
mg-1
F:ATCAGAGAGAGGCACCTGGA
R:GAGTGAGGAGATGACCCGAC
mg-2
F:AGAAACGTGAAACCTGCTGAA
R:AAGCTGACAGCACAACAACG
mg-5
F:GGCGGATAATCAGCAAGTGT
R:GAAGAGTGGCTGGGTTGAAG
mg-6
F:GGAGAGTGAGAGCAGGGTTG
R:GTTGCCACAAATCTGGCTG
mg-7
F:ACCAAGACCAAATCTGCCAC
R:ACACAGCTACATCGGGTTCC
mg-8
F:AACTCTTTTACAACGTCAATTTGC
R:CAGCCTTTGGTTAGAGCACC
mg-17
F:AATAGGAGGCTGGCTGGTTT
R:GCAATTTGCTCCTGCACATA
mg-18
F:GCAGCAGAGAGGAGAGGAGA
R:CCCGTCGTGTTGATGTACTG
mg-19
F:TCTCCTCCAGGTTCACCATC
R:AGGTGCAAATCATCTACCGC
mg-21
F:ACAGGAACGGAGGAACTTGA
R:GATACGCCGTCGCTGAAG
mg-22
F:GATCGCTCCTTCCATTGTGT
R:AATCTCAAGTCCTCCTCCCC
mg-23
F:TGAGATTCCTCCCTCCATTG
R:GCGCTCTACTCCAGGAAAAA
mg-25
F:GCCCCTCAGATGGTTGAATA
R:TGTGGTACATCTCCCTGCAA
mg-26
F:GTCACTCTCCCCTCCCATTC
R:TGCACCCTTTACGAACTCCT
mg-27
F:CCATGGCTAAGCCTCACTTC
R:GCGCACACACACACACTACT
mg-29
F:AGCGTAAGAGGGAGGATGGT
R:GGAGCTCCTCCTCGGTGT
mg-30
F:TTGACGGTGACGATGAGGTA
R:TCAAATCTCATCAGTTCCCAAA
Described awns platymiscium is awns, Caulis Miscanthis floriduli, reed, Nan Di and Qi Gang.
2. the application of 60 pairs of awns platymiscium Genic-SSR primers claimed in claim 1 in awns platymiscium analysis of genetic diversity; Described awns platymiscium is awns, Caulis Miscanthis floriduli, reed, Nan Di, Qi Gang.
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