CN107201406A - A kind of specific microsatellite molecular marker of arborescent ceratoidses and its application - Google Patents
A kind of specific microsatellite molecular marker of arborescent ceratoidses and its application Download PDFInfo
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Abstract
Present invention firstly discloses the specific microsatellite sequence in the 22 of arborescent ceratoidses and 22 pairs of specific micro-satellite primers.These sites play an important role in the analysis of genetic diversity of arborescent ceratoidses, conservative genetics research, Germplasm Resource Investigation and assistant breeding, and the also research to study plant drought mechanism provides basic material.
Description
Technical field
The invention belongs to field of molecular marker, and in particular to a kind of specific microsatellite molecular marker of arborescent ceratoidses and its
Using.
Background technology
Arborescent ceratoidses (Ceratoides arborescens) are subordinate to Chenopodiaceae ceratoides, are Chinese endemic plant, its
Nutritious, palatability is good, and is arid, the excellent forage grass in semiarid zone with characteristics such as drought resisting, cold-resistant, impoverishment tolerants,
Be also water and soil conservation, check winds and fix drifting sand, the important herbage of land management.Therefore, arborescent ceratoidses in Pasture improvement, prevent grassland
Have great importance in " three change " and the upgrading synergy application of desert steppe.It is to the basic scientific researches of arborescent ceratoidses pair
It, which carries out resource management, population reconstruction and utilization of resources etc., great importance, but the direction that it is studied is focused mostly at present
In terms of Physiology and biochemistry, still lack and the germ plasm resource such as genetic diversity and Genetic Constitution of Population genetic background tune is carried out to it
Look into, germ plasm resource genetic background investigation result further instructs formulation and assistant breeding of protection scheme etc..And understand North China
The germ plasm resource genetic background of ceratoides latens, primary work is for the efficient specific molecular labeling of its exploitation.
Microsatellite (Microsatellite) is also referred to as SSR (Simple Sequence Repeats), is by 1~6 core
Thuja acid for repeat unit group into the tandem repetitive sequence up to tens nucleotides, be distributed widely in eukaryotic gene group
In.Because it has, many, the special PCR of quantity are expanded, stability is good, be evenly distributed in genome, polymorphism information is abundant, easily
Turn into one of widely used molecular labeling in recent years many advantages, such as the characteristics such as detection.
The content of the invention
In view of this, it is an object of the invention to provide a kind of specific microsatellite locus of arborescent ceratoidses, including T1,
T6、T11、T13、T15、T16、T17、T20、T22、T23、T25、T26、T28、T29、T30、T31、T34、T35、T36、T37、
The one or more of T38 and T39 nucleotide sequences, nucleotide sequence sequence as shown in SEQIDNO1~SEQIDNO22.
It is described special present invention also offers the specific primer pair for identifying above-mentioned arborescent ceratoidses microsatellite locus
The nucleotide sequence of property primer pair is respectively：
T1 primer pairs sequence is as shown in SEQ ID NO.23 and SEQ ID NO.24；
T6 primer pairs sequence is as shown in SEQ ID NO.25 and SEQ ID NO.26；
T11 primer pairs sequence is as shown in SEQ ID NO.27 and SEQ ID NO.28；
T13 primer pairs sequence is as shown in SEQ ID NO.29 and SEQ ID NO.30；
T15 primer pairs sequence is as shown in SEQ ID NO.31 and SEQ ID NO.32；
T16 primer pairs sequence is as shown in SEQ ID NO.33 and SEQ ID NO.34；
T17 primer pairs sequence is as shown in SEQ ID NO.35 and SEQ ID NO.36；
T20 primer pairs sequence is as shown in SEQ ID NO.37 and SEQ ID NO.38；
T22 primer pairs sequence is as shown in SEQ ID NO.39 and SEQ ID NO.40；
T23 primer pairs sequence is as shown in SEQ ID NO.41 and SEQ ID NO.42；
T25 primer pairs sequence is as shown in SEQ ID NO.43 and SEQ ID NO.44；
T26 primer pairs sequence is as shown in SEQ ID NO.45 and SEQ ID NO.46；
T28 primer pairs sequence is as shown in SEQ ID NO.47 and SEQ ID NO.48；
T29 primer pairs sequence is as shown in SEQ ID NO.49 and SEQ ID NO.50；
T30 primer pairs sequence is as shown in SEQ ID NO.51 and SEQ ID NO.52；
T31 primer pairs sequence is as shown in SEQ ID NO.53 and SEQ ID NO.54；
T34 primer pairs sequence is as shown in SEQ ID NO.55 and SEQ ID NO.56；
T35 primer pairs sequence is as shown in SEQ ID NO.57 and SEQ ID NO.58；
T36 primer pairs sequence is as shown in SEQ ID NO.59 and SEQ ID NO.60；
T37 primer pairs sequence is as shown in SEQ ID NO.61 and SEQ ID NO.62；
T38 primer pairs sequence is as shown in SEQ ID NO.63 and SEQ ID NO.64；
T39 primer pairs sequence is as shown in SEQ ID NO.65 and SEQ ID NO.66.
Another object of the present invention is to provide the kit for identifying arborescent ceratoidses microsatellite locus, including DNA
Polymerase, dNTPs, reaction buffer, primer pair and DNA profiling, wherein the primer pair is one kind or several of following primer pair
Kind：
T1 primer pairs sequence is as shown in SEQ ID NO.23 and SEQ ID NO.24；
T6 primer pairs sequence is as shown in SEQ ID NO.25 and SEQ ID NO.26；
T11 primer pairs sequence is as shown in SEQ ID NO.27 and SEQ ID NO.28；
T13 primer pairs sequence is as shown in SEQ ID NO.29 and SEQ ID NO.30；
T15 primer pairs sequence is as shown in SEQ ID NO.31 and SEQ ID NO.32；
T16 primer pairs sequence is as shown in SEQ ID NO.33 and SEQ ID NO.34；
T17 primer pairs sequence is as shown in SEQ ID NO.35 and SEQ ID NO.36；
T20 primer pairs sequence is as shown in SEQ ID NO.37 and SEQ ID NO.38；
T22 primer pairs sequence is as shown in SEQ ID NO.39 and SEQ ID NO.40；
T23 primer pairs sequence is as shown in SEQ ID NO.41 and SEQ ID NO.42；
T25 primer pairs sequence is as shown in SEQ ID NO.43 and SEQ ID NO.44；
T26 primer pairs sequence is as shown in SEQ ID NO.45 and SEQ ID NO.46；
T28 primer pairs sequence is as shown in SEQ ID NO.47 and SEQ ID NO.48；
T29 primer pairs sequence is as shown in SEQ ID NO.49 and SEQ ID NO.50；
T30 primer pairs sequence is as shown in SEQ ID NO.51 and SEQ ID NO.52；
T31 primer pairs sequence is as shown in SEQ ID NO.53 and SEQ ID NO.54；
T34 primer pairs sequence is as shown in SEQ ID NO.55 and SEQ ID NO.56；
T35 primer pairs sequence is as shown in SEQ ID NO.57 and SEQ ID NO.58；
T36 primer pairs sequence is as shown in SEQ ID NO.59 and SEQ ID NO.60；
T37 primer pairs sequence is as shown in SEQ ID NO.61 and SEQ ID NO.62；
T38 primer pairs sequence is as shown in SEQ ID NO.63 and SEQ ID NO.64；
T39 primer pairs sequence is as shown in SEQ ID NO.65 and SEQ ID NO.66.
Preferably, the present invention is used for the kit for identifying arborescent ceratoidses microsatellite locus, including 2 × EasyTaq PCR
Supermix, primer pair, DNA profiling and ultra-pure water；It is highly preferred that the present invention is used to identify arborescent ceratoidses microsatellite locus
Kit in, including 50-100ngDNA templates, 7.5ul2 × EasyTaq PCR Supermix (TransGen
Biotech), 10 μM of each 0.5ul of primer pair, use ultra-pure water polishing to 15ul；It is highly preferred that the PCR conditions of the kit
For 95 DEG C of pre-degeneration 5min；Anneal 30sec under 95 DEG C of denaturation 30sec, suitable annealing temperature, and 72 DEG C of extension 30sec react 32
Circulation；72 DEG C of extension 10min；Wherein, the suitable annealing temperature is 50 DEG C~61 DEG C；Most preferably, it is described suitable to move back
Fiery temperature is 50 DEG C, 51 DEG C, 52 DEG C, 53 DEG C, 54 DEG C, 55 DEG C, 56 DEG C, 57 DEG C, 58 DEG C, 59 DEG C, 60 DEG C, 61 DEG C.
It is preferred that, the identification of the arborescent ceratoidses comprises the following steps：With gene of the foregoing kit to testing sample
Group DNA enters performing PCR amplification, and amplified production is contrasted with microsatellite sequence：If containing described in above-mentioned technical proposal at least
A kind of microsatellite molecular marker, then judge the testing sample as arborescent ceratoidses；If not containing described in above-mentioned technical proposal
Any one microsatellite molecular marker, then it is not arborescent ceratoidses to judge the testing sample.
Another aspect of the invention is the microsatellite locus of the above-mentioned arborescent ceratoidses of offer and its sieve of specific primer pair
Choosing method, comprises the following steps：
1) collect arborescent ceratoidses sample and extract DNA；
2) to step 1) obtain DNA, build RNA-seq cDNA libraries, and to the RNA-seq cDNA libraries carry out
High-flux sequence；
3) detect microsatellite locus and design the primer pair of microsatellite locus；
4) screen and detect microsatellite locus and its specific primer pair.
Preferably, in the microsatellite locus of arborescent ceratoidses and its screening technique of specific primer pair of the present invention,
The step 1) in extract DNA steps be CTAB methods；
Preferably, in the microsatellite locus of arborescent ceratoidses and its screening technique of specific primer pair of the present invention,
The step 2) in RNA-seq cDNA libraries high-flux sequence using lumina Hiseq3000 microarray datasets carry out high pass
Measure sequence；
Preferably, in the microsatellite locus of arborescent ceratoidses and its screening technique of specific primer pair of the present invention,
The step 3) in using batchprimer3 on-line analyses instrument to the step 2) the Sequence Detection microsatellite that is completed
Site sequence simultaneously carries out design of primers on the flanking sequence of microsatellite locus；It is highly preferred that the microsatellite locus detection
Condition is repeat unit when being 2 bases, and number of repetition need to be more than or equal to 6；When repeat unit is 3 bases, number of repetition is needed
More than or equal to 5；When repeat unit is 4,5 and 6 bases, number of repetition need to be more than or equal to 4；It is highly preferred that the microsatellite position
The principle of the specific primer design of point is that amplification purpose fragment is 100~200bp, and primer G/C content is 40~60%, up and down
Primer annealing temperature (Tm) is swum between 50~60 DEG C and difference is less than 5 DEG C.
Preferably, in the microsatellite locus of arborescent ceratoidses and its screening technique of specific primer pair of the present invention,
The step 4) in the method for the screening microsatellite specific primer be：Using arborescent ceratoidses DNA to step 3) obtain
Specific primer is expanded and detected to entering performing PCR, and screening, which can stablize, amplify target stripe and the primer pair with polymorphism；
Preferably, in the microsatellite locus of arborescent ceratoidses and its screening technique of specific primer pair of the present invention,
The step 4) in the detection microsatellite specific primer method be：Using the fluorescence molecule amount standards of Tamara 350 in ABI
Genotyping is carried out to the primer pair screened using polyacrylamide gel in 3730 DNA sequencers；The knot of Genotyping
Fruit is read out using GENMARKER softwares, and inputs Excel file；Use Excel Mircosatellite Tool kit
Program counts expectation heterozygosity (the Expected Heterozygosity, H of each microsatellite locusE), observation heterozygosity
(Observed Heterozygosity, HO) and polymorphism information content (Polymorphic information content,
PIC), to detect the polymorphism situation of the microsatellite locus, filter out above three polymorphism numerical value and be all higher than 0.5 as micro-
The special primer pair in satellite site.
Therefore, planted the invention provides the microsatellite locus of arborescent ceratoidses and its specific primer in Chenopodiaceae ceratoides
Application in genetic marker, positioning and the qtl analysis of strain, cultivar identification, population and Study on Evolution, molecular mark.
Compared with prior art, present invention firstly discloses the specific microsatellite sequence in the 22 of arborescent ceratoidses by the present invention
And 22 pairs of specific micro-satellite primers.These sites studying the analysis of genetic diversity of arborescent ceratoidses, conservative genetics,
Played an important role in Germplasm Resource Investigation and assistant breeding, the also research to study plant drought mechanism provides basic material.
Embodiment
Below in conjunction with the embodiment in the present invention, the technical scheme in the present invention is clearly and completely described.It is aobvious
So, described embodiment is only a part of embodiment of the invention, rather than whole embodiments.Based on the reality in the present invention
Example is applied, the every other embodiment that those of ordinary skill in the art are obtained under the premise of creative work is not made all belongs to
In the scope of protection of the invention.
Embodiment 1
1st, DNA is extracted
Preservation in silica is used after sample collection, uses to 32 parts of arborescent ceratoidses samples, is gathered from Alashan Area, inner Mongolia Autonomous Region altogether
Zuo Qi.Sample carries out DNA extractings using CTAB methods.
2nd, Jian Ku, high-flux sequence and assembling
Use illumina TruSeq Stranded Total RNA Sample Prep Kit (brilliant energy biotechnologys
(Shanghai) Co., Ltd.) build arborescent ceratoidses standard rna-seqcDNA libraries；Surveyed using illumina Hiseq 3000
The both-end sequencing pattern of sequence platform carries out high-flux sequence to the RNA-seq cDNA libraries of structure.To passing through Quality Control Analysis
High-quality sequence afterwards carries out sample assembling.92629 original series (unigene) are obtained after sequence is assembled.
3rd, microsatellite sequence detection and design of primers
The Sequence Detection microsatellite locus sequence being completed using batchprimer3 on-line analyses instrument to the step 2
Arrange and design of primers is carried out on the flanking sequence of microsatellite locus；The condition of the microsatellite locus detection is that repeat unit is
During 2 bases, number of repetition need to be more than or equal to 6；When repeat unit is 3 bases, number of repetition need to be more than or equal to 5；Repeat single
Member is 4,5 and 6 bases when, number of repetition need to be more than or equal to 4；The principle of the design of primers of the microsatellite locus is amplification mesh
Fragment be 100~200bp, primer G/C content be 40~60%, upstream and downstream primer annealing temperature (Tm) is between 50~60 DEG C
And difference is less than 5 DEG C.
On this condition, 7534 microsatellite sequences are detected altogether, are designed into 5978 pairs of primer pairs.
4th, the preliminary screening of micro-satellite primers
50 primer pairs of selection are entered performing PCR amplification condition and determined in 5978 pairs of successful primer pairs of design.The original of selection
It is then：It is preferred that it is 2 or 3 (this kind of microsatellite polymorphism is higher) to repeat base units；It is preferred that repetitive sequence is without nucleotide variation, nothing
Insert or lack；Preferred repeat units number big (but no more than 50).
The use of the DNA of 8 arborescent ceratoidses samples is template, grads PCR amplification is carried out to this 50 primer pairs.Gradient
PCR reaction systems are as follows：50-100ngDNA templates, 7.5ul2 × EasyTaq PCRSupermix (TransGen
Biotech), 10 μM of each 0.5ul of upstream and downstream primer, use ultra-pure water polishing to 15ul.Grads PCR reaction cycle sets as follows：
95 DEG C of pre-degeneration 5min；95 DEG C are denatured in the range of 30sec, 45~65 DEG C the 30sec that annealed under 12 gradient temperatures, 72 DEG C of extensions
15sec, reacts 32 circulations；Last 72 DEG C of extensions 10min.12 gradients of the present invention are preferably 50 DEG C, 51 DEG C, 52 DEG C,
53℃、54℃、55℃、56℃、57℃、58℃、59℃、60℃、61℃。
The amplified production that grads PCR is obtained uses 2% agarose-EB detected through gel electrophoresis, and selection meets following condition
Primer pair be used as preliminary screening primer pair：(1) amplified production is single band；(2) clip size meets purpose fragment size；
(3) amplification obtains consistent results in 8 individuals.Record meets PCR annealing temperatures during above-mentioned condition, is designated as the primer pair
Most suitable annealing temperature.
On this condition, 25 primary dcreening operation primer pairs are obtained.
4th, the polymorphic detection of microsatellite locus
Fluorescence labeling (FAM/HEX/TAMRA) is added to the end of sense primer 5 ' of above-mentioned primary dcreening operation primer pair, and uses 32
The DNA of arborescent ceratoidses sample is expanded.PCR reaction systems are：50-100ngDNA templates, 7.5ul2 × EasyTaq PCR
Supermix (TransGen Biotech), 10 μM of each 0.5ul of upstream and downstream primer, uses ultra-pure water polishing to 15ul.Circulation is set
It is set to：95 DEG C of pre-degeneration 5min；Anneal 30sec under 95 DEG C of denaturation 30sec, most suitable annealing temperature, 72 DEG C of extension 15sec, reaction
35 circulations；72 DEG C of extension 10min.
Amplified production uses polypropylene using the fluorescence molecule amount standards of Tamara 350 in the DNA sequencers of ABI 3730
Acrylamide gel carries out Genotyping.The result of genetic analysis is read out using GENMARKE softwares, and inputs Excel file.
Using Excel Mircosatellite Tool kit programs calculate expect heterozygosity (Expected Heterozygosity,
HE), observation heterozygosity (Observed Heterozygosity, HO) and polymorphism information content (Polymorphic
Information content, PIC), to detect the polymorphism situation of the microsatellite locus, select above three polymorphism
Numerical value is all higher than 0.5 site.
Finally obtain 22 stable high polymorphic micro-satellite sites, the microsatellite locus be named as T1, T6, T11,
T13, T15, T16, T17, T20, T22, T23, T25, T26, T28, T29, T30, T31, T34, T35, T36, T37, T38 and T39；
The T1, T6, T11, T13, T15, T16, T17, T20, T22, T23, T25, T26, T28, T29, T30, T31, T34, T35,
T36, T37, T38 and T39 the nucleotide sequence sequence as shown in SEQIDNO1~SEQIDNO22 respectively；The T1, T6, T11,
T13, T15, T16, T17, T20, T22, T23, T25, T26, T28, T29, T30, T31, T34, T35, T36, T37, T38 and T39；
The T1, T6, T11, T13, T15, T16, T17, T20, T22, T23, T25, T26, T28, T29, T30, T31, T34, T35,
T36, T37, T38 and T39 primer pair are followed successively by：
T1 primer pairs sequence is as shown in SEQ ID NO.23 and SEQ ID NO.24；
T6 primer pairs sequence is as shown in SEQ ID NO.25 and SEQ ID NO.26；
T11 primer pairs sequence is as shown in SEQ ID NO.27 and SEQ ID NO.28；
T13 primer pairs sequence is as shown in SEQ ID NO.29 and SEQ ID NO.30；
T15 primer pairs sequence is as shown in SEQ ID NO.31 and SEQ ID NO.32；
T16 primer pairs sequence is as shown in SEQ ID NO.33 and SEQ ID NO.34；
T17 primer pairs sequence is as shown in SEQ ID NO.35 and SEQ ID NO.36；
T20 primer pairs sequence is as shown in SEQ ID NO.37 and SEQ ID NO.38；
T22 primer pairs sequence is as shown in SEQ ID NO.39 and SEQ ID NO.40；
T23 primer pairs sequence is as shown in SEQ ID NO.41 and SEQ ID NO.42；
T25 primer pairs sequence is as shown in SEQ ID NO.43 and SEQ ID NO.44；
T26 primer pairs sequence is as shown in SEQ ID NO.45 and SEQ ID NO.46；
T28 primer pairs sequence is as shown in SEQ ID NO.47 and SEQ ID NO.48；
T29 primer pairs sequence is as shown in SEQ ID NO.49 and SEQ ID NO.50；
T30 primer pairs sequence is as shown in SEQ ID NO.51 and SEQ ID NO.52；
T31 primer pairs sequence is as shown in SEQ ID NO.53 and SEQ ID NO.54；
T34 primer pairs sequence is as shown in SEQ ID NO.55 and SEQ ID NO.56；
T35 primer pairs sequence is as shown in SEQ ID NO.57 and SEQ ID NO.58；
T36 primer pairs sequence is as shown in SEQ ID NO.59 and SEQ ID NO.60；
T37 primer pairs sequence is as shown in SEQ ID NO.61 and SEQ ID NO.62；
T38 primer pairs sequence is as shown in SEQ ID NO.63 and SEQ ID NO.64；
T39 primer pairs sequence is as shown in SEQ ID NO.65 and SEQ ID NO.66.
The T1, T6, T11, T13, T15, T16, T17, T20, T22, T23, T25, T26, T28, T29, T30, T31,
T34, T35, T36, T37, T38 and T39；The T1, T6, T11, T13, T15, T16, T17, T20, T22, T23, T25, T26,
The genetic diversity data of T28, T29, T30, T31, T34, T35, T36, T37, T38 and T39 microsatellite locus are shown in Table 1.
Table 1：Microsatellite locus Diversity Detection result
It can be seen from the data of table 1 provided by the present invention for identification microsatellite locus molecular labeling polymorphism number
According to being all higher than 0.5, genetic diversity sex investigation, Genetic Constitution of Population analysis, individual identification and affiliation mirror may be incorporated for
Surely genetic analysis are waited.
Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
SEQUENCE LISTING
<110>Inner Mongolia Autonomous Region Academy of Agricultural and Livestock Husbandry
<120>A kind of specific microsatellite molecular marker of arborescent ceratoidses and its application
<130>A Sida
<160> 66
<170> PatentIn version 3.5
<210> 1
<211> 499
<212> DNA
<213>Arborescent ceratoidses
<400> 1
cgttcaaaga aatgacatcg gaaaaacctt tgaaaggtgt ctggtgtggc tatatgtcat 60
ctaccgcgac catgattatt attattatta ttattattat tattattatt attattattg 120
tgtactacat cgttttgtaa gctgttggcg aatggataag ccctggaaga agcagtggtg 180
gtagaaaatg atgagaatcc tgatgatgct gcagttgaca acaattggac ttgggttgca 240
gtaccaccaa ttccacctcc acttcctcct tgaccttggt tttgcatgta ccattgccag 300
tttggtagac ttgctgagcc tgccattgct tctagctttt catgatcatt aactttgtcc 360
tcataagctg cagaccgacc tggaggtaag ccagaccaca taggaggttg tcgcgatgcc 420
aatggctgac catagagtgg tggtgcacca ggagtaaagt atcccccatt cacaggccat 480
ttcccattag caacatgaa 499
<210> 2
<211> 375
<212> DNA
<213>Arborescent ceratoidses
<400> 2
tctcggcggt ttttcttctc ctcccccggc cgctccaact ccatcgttga ctccctctcc 60
ctctcctcct ccaacctact attatcttca ccaccgcggg aggttgaggc ggaggcggag 120
gcagagttat catcctcctc ctcctcctcc tccacggtgg aggtggccgg gggagtaccc 180
gtgcagacgt actcccccga cccctacgtg gacttcagac aatccatgca agagatggtg 240
gaagcacgtg agttaggaaa tctaggggtg aaacaagcat gggaatattt gcaagagtta 300
ctgttgagtt acctagcact gaatccaaag aacactcaca agtacattgt tggagctttt 360
actgaccttg ttctt 375
<210> 3
<211> 499
<212> DNA
<213>Arborescent ceratoidses
<400> 3
ccgtggtggg aaatggaaga ggagaaaagg cgcggagatg agcggagagg acacgtggca 60
ggagatgatg aggaggagga ggaggaggag aagggtggtg gtgtggcagt tgcaaagggt 120
aaacgaaggt tttcaagtaa ggcgcacgat agcggctacc atacgctaac aaaggagatg 180
ctgatgctga tattattgat gaatgtagta gaaatggcat ctcttttttt tcttgaatta 240
tttccttttt ttaaaaaaaa aaaaaatcaa gggtggatga agataatgga taaagagaag 300
gcagccattg ttgaaggctt gaagcaagga agagaggaca tttgtaatat ctgaatgaag 360
taaatactta tctagacctg gcaaaacaga ccggcaccaa ttacccgacc tgacataccc 420
attctaccca aaaatgccaa aatatgctga cccccgaacg atacctgaac cgaactgacc 480
cgacaataaa tgtgtgtaa 499
<210> 4
<211> 481
<212> DNA
<213>Arborescent ceratoidses
<400> 4
agtaggcctg tagcagtttc attggagttc catccgagca agtcgaacaa catcaagtgc 60
agcatagttg gtagttgtat ttttcatttt ttggttaatg ctgttttatt tgtgtttcct 120
accgtctgct tctagagtct gcatttcaaa atttccagtt tcctgtggcc atactatgat 180
gatgatgatg atgatgaaaa gctctcctgc atggggtatc gattgcaatg gggctacagc 240
gattataaaa ttctttttct tgcaaattta ggaccaggag aatgatttag agtactttgg 300
tactctcaat catgtgtgtt tttcttgtaa ataggattgt taatttggtc ggtcaagaat 360
aaaaatgttc taaatttatg tactttttta aaggggtcaa tgaacatttc taccttcagc 420
tgtgtacatt tgccatatag gagtaattct tgagctcgat cttgttttat tttaccttgg 480
t 481
<210> 5
<211> 451
<212> DNA
<213>Arborescent ceratoidses
<400> 5
catgagagat agagctacaa cctaatacct ctcatgcata tataacaaac tactaaaaca 60
tcaaaccaat aaaattctaa acccataata ataataataa taataataat aataatatgg 120
acctccaact acatacgatt aatacgaact cctattttat aacataaagg aaccgagggt 180
acgtgcaagt atttaataat ttgtaaaaac gtaataatat aagaccaact cctatattac 240
gtacgcacca tatagtacgt actcaataat ctttatttat ggaatagctg gaagaccacc 300
attcccagga ctagatccaa ctccaggacc accaaaacct cccaaaccac caccaggact 360
accgcccaag ccacctagtc ctcccaaacc accaggacta ccaccaccta aagacccaaa 420
accaccaagt ccaccaggat taccaccacc a 451
<210> 6
<211> 274
<212> DNA
<213>Arborescent ceratoidses
<400> 6
cgggcggagc tgcggcggca aacacggcgg atgtgggtaa tccggtggtg gcggaattgt 60
tgagggagga gtttaaagga gctgagggat tgagatttgg gagaagaaga gtgagaaagc 120
atgctttgag tggagccatg gaagattgct tgagaacagg ctgccattga agagttgaag 180
agagattgaa tctatgaatg ttgttgttgt tgttgttgtt gttgttgttg taatgtggaa 240
gacagtgtta gatatggggg tcgtttgttc ttcg 274
<210> 7
<211> 499
<212> DNA
<213>Arborescent ceratoidses
<400> 7
gccgccggca ccaccgtcta ccagctccta ccccctaccc tcccccccct ctccaccacc 60
actgccatct tttgtcccac caccaccacc accaccacca cctctccacc tccacccgag 120
caaccatcag ataaaatcct cgacaacggg gtcataaaac gatccttcta ccccattggg 180
accgccgcat ataacttcgt attcatgtca gcttatcgcg gcggtcccaa cactttcgct 240
gtcataggtc tatcatccaa aacccttcaa gacttcggtc atcctacata cacatgtgaa 300
taccaatcaa acaataatac ctcttcttca tatatagtac caggttataa gcttcctatg 360
gatgactttg gttacgcacg aatgtacatt atagcagtcg taaactgtac atttcctaca 420
caaaccgacc cttccaccgg cggccggctg ctcctccacg ccgccaccac caacggcggt 480
gtaaactcaa ccgacacca 499
<210> 8
<211> 542
<212> DNA
<213>Arborescent ceratoidses
<400> 8
tgacatctcg tcactgaacc ttaatgaaat cccgataaat aaataaataa ataaataaac 60
ttggtacagt gacattgagg aatgaggtta ctatcaaaaa tgttgcagtg aacaggtttg 120
caaatttcca atgaatattc atctacgttt ctgtgagggc tgtggcatgt tattgctgga 180
ggattacagt tggtacggaa cagatatagc cataaaattt acccagatca ttcaatagtc 240
aagtgtctac gatacaaggc gtattttaga agttcatagg acgaaactac aatcccaact 300
gaaggcccag cacgggcaac tcgaggactg attcctgtaa atagcccttt ccaacctcca 360
tccctccata tctccatcaa tgtatgtcgt gttgtcatcc tcatctgtct agctggatct 420
ttctctatct gcctccgagt tcttgcaaca tctagagggc atgtgacagc tgcagcaagg 480
gtgcctgcaa tgaatccacc agcaaaattt gcacctaaaa cacttgtcgc attcatttca 540
tc 542
<210> 9
<211> 397
<212> DNA
<213>Arborescent ceratoidses
<400> 9
ttttagataa aattgcattt catttttttg ataaatttgg atttttttag gtgaatttta 60
tttttatttt ttattttttt aattttatac aatgcagttg caaattattg ggttttcaat 120
tttttgttaa atcttacgga ttactgattt atttttatga atttggtttt ttttattagt 180
ttagaatttt agagaaaaaa tatcgttcaa cgatttgtag caattttgct cgctctcata 240
gaatctgatg gagctctcct tgaaagtgtc ttctttttaa ttaaaagaga tagattataa 300
ataggaataa aaataaataa ataaataaat aaataaaagg gatgggcggt tataaatagg 360
agttgggacg cagacgtagt tcaaattaaa attgaag 397
<210> 10
<211> 376
<212> DNA
<213>Arborescent ceratoidses
<400> 10
ttaaacggat agtcaggagg gaaatggatc gtcactagaa atactcctcc agcataaggg 60
ctatcagcag gacccattat tgttgcctgc caatgaaaca tgtcttcagc aacaggacca 120
gcgctgcaag aagtaggagg atccttctgg agatccttga gctccttcaa gatccgcttg 180
gaagccatcg ctggagaacg gcaccctaat tttcagtccg acggcggaaa tcgtttggaa 240
ggagggttga atttgaatga aggttgaatg aatgaatgaa tgaatgaaag aaaacccaaa 300
tccaaattat aagggagatt gattgtggtg tggtgtgggg taggctgctg cttcttcacg 360
cggacctgat ttttgg 376
<210> 11
<211> 448
<212> DNA
<213>Arborescent ceratoidses
<400> 11
tggaagccat ttgttgttgc cagtttggtt gttgaggatt ctgcctcatc tgttgttgct 60
gttgttgttg ttgttgctgc tgctgctgct gctgctgtaa caaaacacgc tgcatcggtg 120
gcatattctg caacatttga tgctgctgtc gtagcctctg ctgctgaatg atagcatgca 180
actgcgggtt atggctttgc aactgcattg gatcctgtct ctgcagcaat tgatgtaaga 240
cttgctgctg caaaatatct tcttgcttaa tatccagccg cggttttttt ctgcagatgg 300
gatagattag caggatcttg catgaatgaa ttgggattcc ttgaccccat agaatgtgtg 360
agttggcctg tttgagatgc tggcaataat gaagcaccag aagcaccttg cggaaactga 420
tgttgactct gaatagcttg ttgagagt 448
<210> 12
<211> 435
<212> DNA
<213>Arborescent ceratoidses
<400> 12
tggatatacc tagtcgggcc aactgctgga gctatatctg gtgcaatggt gtacaatctt 60
ataagattta cggaaaaacc attgcgcgag atcaccaaaa ccggatcttt ccttaggact 120
aagggttcgt ctgtaagtcg taatggttct tcccagtact aggatttaga ggagaaacaa 180
cttaatttta gagggtttta tttgatcata aaaagtgtaa aatcctttta actcaaaatg 240
tatgtcaact aagtcttcca tctttaaata ataagttaga tggttgatat atataatgta 300
gttgcattat gtacgtacgt atgtatgtat gtatgtatgt atgttatgtg ccgtgcctgc 360
tgtattagtt tagtgatgtt ttataaggtt tttaattgtg aggaagatgg ttgagctatt 420
gttatttgag tagat 435
<210> 13
<211> 524
<212> DNA
<213>Arborescent ceratoidses
<400> 13
tatagcaaga agtaaacatc aaataaatag aaaatcacaa aaataaataa ataaataaat 60
aaattcatta aataagagcc taatcaaaca tttcccaatt tcaagttctt aaattccagt 120
gaatcaactc acgcaacatt agtccaagtg tattcgcacc atttccatca ttcatacaaa 180
cattcaaata cgaaagcaga aattaaacag aaatcagaac caaaacctca aatccccaaa 240
aatgaattaa taacccaaga ataaattaaa aaaagaaaaa aaaaggtaca gcggagattg 300
aagaagggtt ttcggcgatg atagctatgg cggaaacgga gtacttgaat cctacccgaa 360
cctctttgaa ttcgtcgttt cactgcgaag acggtgtgac ggctgggctt ccggcgagaa 420
ggagagagga caccgtgacg atgggcggac gacgtgacag tgggcggaga aagcaagctg 480
cagctgtggt gtatttctta gtaggaggat gatatcctct tgtg 524
<210> 14
<211> 559
<212> DNA
<213>Arborescent ceratoidses
<400> 14
aataaagtca atgccttgta aaataatcaa caaaaacatt tagtccaact actcattttt 60
ttgttgtgaa gaacaaagat taagctcgct cgctcgctcg ctcgctccca ctcgtactgc 120
gcatttcatc ctctctcctc cttcctcatc aatggcgagc agtaatctcc ctcgcagaat 180
catcaaggaa actcagcgtc tcctcagtga accagcaccg ggaataagtg cttcaccatc 240
agaagacaat atgcggtatt ttaatgtcat gattcttggc ccttcacagt ctccatatga 300
aggaggagtg tttaagttgg aattattttt gcctgaggaa tatccaatgg cggctcctaa 360
ggttcgattt ctcactaaaa tatatcatcc caacattgat aagctaggga ggatatgcct 420
tgatattctt aaagacaagt ggagtcctgc tctccagata cgtactgtac tcctgagtat 480
tcaagcattg cttagtgctc caaaccctga tgaccctctg tctgagaaca ttgctaagca 540
ttggaaaacg gatgaagca 559
<210> 15
<211> 460
<212> DNA
<213>Arborescent ceratoidses
<400> 15
tgctccatgt ttattgagga acaacttata tgatgaaggc agagattctt gcttcaatat 60
gtaggcagac aatatttggg aggaagaaag acacatgagg aagatgtctc catgagccca 120
agttaacggt ttgcaaaagc ggccaaaccg cttgcttttg attccacagc gagatgccaa 180
tacagctgct cgcatgagga tatatatagc caagctatta tgttgtgtgt ttaacccagt 240
taatagcata gaaggcccag ctattccacc tgccagtaat gccctccatt ttgcagtcct 300
gaattacagt gtatccgact caaaacacta acacaagtgc tagaatatac aagtagaata 360
tttcattatg ctatccaaat ttatttattt atttatttat ttttaaaaaa acaccaacag 420
tgagggaagt taagtttgta tggccattaa aactcagaac 460
<210> 16
<211> 481
<212> DNA
<213>Arborescent ceratoidses
<400> 16
tcacgtcagc atcaggcagc aaatacaaat tcacacttgg atattggttc aactggatac 60
agattactat ttatggagat gattgaagga ttgtaaacca gtaaagaatc ttacaggaca 120
gtctacctag aacaagctta tcaattaagg cttcaggttc aactggatac agagtactgt 180
ttatggaagg tatttgaagg attagaaact gggaaagcct atccagaaca actttgatca 240
ttaaggcttc aaggaaaaaa aaaaaacaca cacacacaca cacacacaca taggcatgtg 300
cttatacctg tgagcaatga cacgtcttat gttactcaca tcaagctatc atgtctgcca 360
agaacgatta aatcatatag gaaagaccct ccagcagcac aatatatgca atggcagcta 420
gcagattctt aacaccctcc taaaaaatga aagctacaca gcgctcaaaa ctattttaag 480
a 481
<210> 17
<211> 338
<212> DNA
<213>Arborescent ceratoidses
<400> 17
ctgacaaaag attggagctt gaatagtaaa gaaagaccta aagttgagga cattagtgaa 60
gtgttaagag agtgcttatt gtttgtggaa aaaatgaaca aaattcagat ctacggcttc 120
tcgtgatgtt tcttggtcgt ggccagaaga gcattcacgt gcaattttct ggggttgtgt 180
tgctgggatg ttccaagttg gttggttggt tggttggttg ttgtgatttg ttatggttgg 240
tttacgagcg tggaaaggat atatatcgat attactattc gagcagtttg ataacggatt 300
gccttttttt ttctccttgc ggtttgtctc tgtagttg 338
<210> 18
<211> 463
<212> DNA
<213>Arborescent ceratoidses
<400> 18
aagaaaatta aatactaaat aaaatcccca actcttccag actgagtgag tgagtgagtg 60
agtgagtaag aacaaccaca tttcttaaaa atcctaatat ttttttaggg tttttgttct 120
tcatattata tttatgagct tcaactatgg atacgcctga gcgtaatcag atcggaaccc 180
ccgtttccaa attcgaggag tctccatttt caaacttcat caacagtctg tctccgatca 240
acccaacgaa gacgatacac gtttcacaga cgtttggctc gcttagtttt acttcacctc 300
catctgtttt tacttcaccc catgtgagcc tccagaagga ttataagctc ttacgaaggc 360
atctagtttc agatcttgcg aaaactcagc ttgtgtctga tgaaggaaac aaagattctg 420
ctgcgaaaca ggtttcagaa gagccgcagt ctgacagtct cgc 463
<210> 19
<211> 563
<212> DNA
<213>Arborescent ceratoidses
<400> 19
gtgagctact catgttatca gtgaagagag cattgaccat tccacgttat ttcaatgcta 60
tcacattttt cattctcatt cattcattca ttcattcatt ctcttgtctc tctctatttc 120
tcactctcaa ttactcctac cgcattgatt gcattaacct aaacacgcat cttgttcaac 180
tccagatggc ggttgacgat gatatgtgtg gcgacggaac ccactgtgct gaccatccct 240
accgcaccac ctcccccggc ggcggcggta tttgcgcttt ctgcctccaa gacaaactcg 300
gtaaactcgt ctcctcctct tcctcctcct tcttcattgc ctcctcttcc tcttcctccc 360
cttccttcag atctgacctc ccttccgcca cttctacctc caatgtcgtc ggtgtcgggc 420
cacctcccac caccaccaac aatatcagta gtagtcggca tcaccatcaa aatagtagta 480
gtaatagtaa tagtaatagt aatagtagta ctaatcagca acatcaacat catgaagttg 540
gaagtgggca tcatcatcat cat 563
<210> 20
<211> 253
<212> DNA
<213>Arborescent ceratoidses
<400> 20
gttaaattcg tctcgataat atttttcaaa atatcatttt tttataatta tttacataag 60
tagattttgg gatgttaatg gttgaaaaag tgtatttgta aacatgataa aagaaacatg 120
tcaactaaag cggaagtatg attaacattg ccattttatg acttttaact agttggaatt 180
tttttccttt atttatttat ttatttattt atgtatgtaa tgtttagcat agtcagttca 240
aggaatattg gag 253
<210> 21
<211> 561
<212> DNA
<213>Arborescent ceratoidses
<400> 21
ataaccctaa acccatactt ttctctcacc agccgcttta ctctctcttc tctcatcatt 60
tcccctcttt tgatgatctt ctcatctctt tgatctaaca tttatagaac ttgaagcaaa 120
gagattaaaa aaaatgttaa gttccggcaa ttcctcggtg gttctgtcaa tttgtagcaa 180
ggaaatccaa gaatattgct tgcttgcttg cttgcttgct tgttgttcaa agagagcagc 240
cacgaagtat taaaacacac actcaccgac attacttact cacagtcaca aatgtaattg 300
taaaattcag atttaaaaaa tattagatct taaaaaaaaa agatagtaaa aagaatgata 360
tttagatcca aaataaaatg agtagttgtt ggtggagagt tgatgctata tccatggtac 420
cattaagtta actgaagatc ttggtgatga aattttgctg ctgatgactt cggtcaaatt 480
cctcggttgg tgttttgttg tttacatttt tttttgggta ggaaacaatt tgctttactt 540
aattttagtt gtagatctag t 561
<210> 22
<211> 612
<212> DNA
<213>Arborescent ceratoidses
<400> 22
aaataatgca taaacaaaca aaaaatttaa aatgttacaa aaaaaaaaag accgatgtat 60
attaatcgtc tacctgaaga ttatataagc tttttattcc ctttagatat ttctaagtac 120
aatttagcca acatcaaggc agtcaataat atttcttcac tctttttttc tttgtatgtg 180
tctatgcata attcaacaaa taaatttttc actatcttcc aatctataat atgcaatcta 240
tttttaccat ggtaaaatat ctgctcgtca agaatcttac tgctgattgt aacaagagaa 300
acaaatctct cttaacattc ctaatcttca gttctccaat taaggactgg ccgcaaaatc 360
accgcatgaa aggccaataa tatgtaaatg cagttttcag gttcttccag gaatttgtga 420
gaagcaaagg gaaaacttaa ctatgagaaa agtagaataa gattaatcca gatacagaat 480
acctccctgt aaatgcaact cttgcgcctg ggatgagaaa ctcatctccc caccaaaaaa 540
taaataaata aataaataaa taaaaaatga agagaaactc atcctctttc tttgggcttc 600
accaaaatga aa 612
<210> 23
<211> 21
<212> DNA
<213>Artificial sequence
<400> 23
gtgtctggtg tggctatatg t 21
<210> 24
<211> 21
<212> DNA
<213>Artificial sequence
<400> 24
ctcagcaagt ctaccaaact g 21
<210> 25
<211> 21
<212> DNA
<213>Artificial sequence
<400> 25
ctctcctcct ccaacctact a 21
<210> 26
<211> 20
<212> DNA
<213>Artificial sequence
<400> 26
atctcttgca tggattgtct 20
<210> 27
<211> 20
<212> DNA
<213>Artificial sequence
<400> 27
aaatggaaga ggagaaaagg 20
<210> 28
<211> 21
<212> DNA
<213>Artificial sequence
<400> 28
tcctttgtta gcgtatggta g 21
<210> 29
<211> 21
<212> DNA
<213>Artificial sequence
<400> 29
gcttctagag tctgcatttc a 21
<210> 30
<211> 21
<212> DNA
<213>Artificial sequence
<400> 30
ctaaatcatt ctcctggtcc t 21
<210> 31
<211> 21
<212> DNA
<213>Artificial sequence
<400> 31
acaacctaat acctctcatg c 21
<210> 32
<211> 21
<212> DNA
<213>Artificial sequence
<400> 32
ccctcggttc ctttatgtta t 21
<210> 33
<211> 21
<212> DNA
<213>Artificial sequence
<400> 33
ggagaagaag agtgagaaag c 21
<210> 34
<211> 21
<212> DNA
<213>Artificial sequence
<400> 34
acccccatat ctaacactgt c 21
<210> 35
<211> 19
<212> DNA
<213>Artificial sequence
<400> 35
accactgcca tcttttgtc 19
<210> 36
<211> 21
<212> DNA
<213>Artificial sequence
<400> 36
gcgataagct gacatgaata c 21
<210> 37
<211> 21
<212> DNA
<213>Artificial sequence
<400> 37
acatctcgtc actgaacctt a 21
<210> 38
<211> 21
<212> DNA
<213>Artificial sequence
<400> 38
cctcacagaa acgtagatga a 21
<210> 39
<211> 21
<212> DNA
<213>Artificial sequence
<400> 39
agctctcctt gaaagtgtct t 21
<210> 40
<211> 22
<212> DNA
<213>Artificial sequence
<400> 40
ttttaatttg aactacgtct gc 22
<210> 41
<211> 19
<212> DNA
<213>Artificial sequence
<400> 41
ggcaccctaa ttttcagtc 19
<210> 42
<211> 21
<212> DNA
<213>Artificial sequence
<400> 42
accacaatca atctccctta t 21
<210> 43
<211> 20
<212> DNA
<213>Artificial sequence
<400> 43
cagtttggtt gttgaggatt 20
<210> 44
<211> 20
<212> DNA
<213>Artificial sequence
<400> 44
cagtttggtt gttgaggatt 20
<210> 45
<211> 23
<212> DNA
<213>Artificial sequence
<400> 45
tgtcaactaa gtcttccatc ttt 23
<210> 46
<211> 23
<212> DNA
<213>Artificial sequence
<400> 46
tgtcaactaa gtcttccatc ttt 23
<210> 47
<211> 20
<212> DNA
<213>Artificial sequence
<400> 47
agcaagaagt aaacatcaaa 20
<210> 48
<211> 21
<212> DNA
<213>Artificial sequence
<400> 48
gcgaatacac ttggactaat g 21
<210> 49
<211> 22
<212> DNA
<213>Artificial sequence
<400> 49
caacaaaaac atttagtcca ac 22
<210> 50
<211> 20
<212> DNA
<213>Artificial sequence
<400> 50
attctgcgag ggagattact 20
<210> 51
<211> 21
<212> DNA
<213>Artificial sequence
<400> 51
cattttgcag tcctgaatta c 21
<210> 52
<211> 21
<212> DNA
<213>Artificial sequence
<400> 52
caaacttaac ttccctcact g 21
<210> 53
<211> 21
<212> DNA
<213>Artificial sequence
<400> 53
atggaaggta tttgaaggat t 21
<210> 54
<211> 21
<212> DNA
<213>Artificial sequence
<400> 54
acataagacg tgtcattgct c 21
<210> 55
<211> 21
<212> DNA
<213>Artificial sequence
<400> 55
aaattcagat ctacggcttc t 21
<210> 56
<211> 21
<212> DNA
<213>Artificial sequence
<400> 56
ctcgtaaacc aaccataaca a 21
<210> 57
<211> 21
<212> DNA
<213>Artificial sequence
<400> 57
ctcgtaaacc aaccataaca a 21
<210> 58
<211> 20
<212> DNA
<213>Artificial sequence
<400> 58
ctcaggcgta tccatagttg 20
<210> 59
<211> 21
<212> DNA
<213>Artificial sequence
<400> 59
tccacgttat ttcaatgcta t 21
<210> 60
<211> 21
<212> DNA
<213>Artificial sequence
<400> 60
acaagatgcg tgtttaggtt a 21
<210> 61
<211> 21
<212> DNA
<213>Artificial sequence
<400> 61
gcggaagtat gattaacatt g 21
<210> 62
<211> 21
<212> DNA
<213>Artificial sequence
<400> 62
ctccaatatt ccttgaactg a 21
<210> 63
<211> 21
<212> DNA
<213>Artificial sequence
<400> 63
gtggttctgt caatttgtag c 21
<210> 64
<211> 21
<212> DNA
<213>Artificial sequence
<400> 64
aagtaatgtc ggtgagtgtg t 21
<210> 65
<211> 21
<212> DNA
<213>Artificial sequence
<400> 65
agaatacctc cctgtaaatg c 21
<210> 66
<211> 21
<212> DNA
<213>Artificial sequence
<400> 66
ccaaagaaag aggatgagtt t 21
Claims (10)
1. a kind of specific microsatellite locus of arborescent ceratoidses, including T1, T6, T11, T13, T15, T16, T17, T20, T22,
The one or more of T23, T25, T26, T28, T29, T30, T31, T34, T35, T36, T37, T38 and T39 nucleotide sequence, institute
State nucleotide sequence sequence as shown in SEQ ID NO1~SEQ ID NO22.
2. a kind of specific primer pair for being used to identify arborescent ceratoidses microsatellite locus as claimed in claim 1, its feature
It is, the nucleotide sequence of the specific primer pair is respectively：
T1 primer pairs sequence is as shown in SEQ ID NO.23 and SEQ ID NO.24；
T6 primer pairs sequence is as shown in SEQ ID NO.25 and SEQ ID NO.26；
T11 primer pairs sequence is as shown in SEQ ID NO.27 and SEQ ID NO.28；
T13 primer pairs sequence is as shown in SEQ ID NO.29 and SEQ ID NO.30；
T15 primer pairs sequence is as shown in SEQ ID NO.31 and SEQ ID NO.32；
T16 primer pairs sequence is as shown in SEQ ID NO.33 and SEQ ID NO.34；
T17 primer pairs sequence is as shown in SEQ ID NO.35 and SEQ ID NO.36；
T20 primer pairs sequence is as shown in SEQ ID NO.37 and SEQ ID NO.38；
T22 primer pairs sequence is as shown in SEQ ID NO.39 and SEQ ID NO.40；
T23 primer pairs sequence is as shown in SEQ ID NO.41 and SEQ ID NO.42；
T25 primer pairs sequence is as shown in SEQ ID NO.43 and SEQ ID NO.44；
T26 primer pairs sequence is as shown in SEQ ID NO.45 and SEQ ID NO.46；
T28 primer pairs sequence is as shown in SEQ ID NO.47 and SEQ ID NO.48；
T29 primer pairs sequence is as shown in SEQ ID NO.49 and SEQ ID NO.50；
T30 primer pairs sequence is as shown in SEQ ID NO.51 and SEQ ID NO.52；
T31 primer pairs sequence is as shown in SEQ ID NO.53 and SEQ ID NO.54；
T34 primer pairs sequence is as shown in SEQ ID NO.55 and SEQ ID NO.56；
T35 primer pairs sequence is as shown in SEQ ID NO.57 and SEQ ID NO.58；
T36 primer pairs sequence is as shown in SEQ ID NO.59 and SEQ ID NO.60；
T37 primer pairs sequence is as shown in SEQ ID NO.61 and SEQ ID NO.62；
T38 primer pairs sequence is as shown in SEQ ID NO.63 and SEQ ID NO.64；
T39 primer pairs sequence is as shown in SEQ ID NO.65 and SEQ ID NO.66.
3. a kind of kit for being used to identify arborescent ceratoidses microsatellite locus, including archaeal dna polymerase, dNTPs, reaction buffering
Liquid, primer pair and DNA profiling, it is characterised in that the primer pair is the one or more of following primer pair：
T1 primer pairs sequence is as shown in SEQ ID NO.23 and SEQ ID NO.24；
T6 primer pairs sequence is as shown in SEQ ID NO.25 and SEQ ID NO.26；
T11 primer pairs sequence is as shown in SEQ ID NO.27 and SEQ ID NO.28；
T13 primer pairs sequence is as shown in SEQ ID NO.29 and SEQ ID NO.30；
T15 primer pairs sequence is as shown in SEQ ID NO.31 and SEQ ID NO.32；
T16 primer pairs sequence is as shown in SEQ ID NO.33 and SEQ ID NO.34；
T17 primer pairs sequence is as shown in SEQ ID NO.35 and SEQ ID NO.36；
T20 primer pairs sequence is as shown in SEQ ID NO.37 and SEQ ID NO.38；
T22 primer pairs sequence is as shown in SEQ ID NO.39 and SEQ ID NO.40；
T23 primer pairs sequence is as shown in SEQ ID NO.41 and SEQ ID NO.42；
T25 primer pairs sequence is as shown in SEQ ID NO.43 and SEQ ID NO.44；
T26 primer pairs sequence is as shown in SEQ ID NO.45 and SEQ ID NO.46；
T28 primer pairs sequence is as shown in SEQ ID NO.47 and SEQ ID NO.48；
T29 primer pairs sequence is as shown in SEQ ID NO.49 and SEQ ID NO.50；
T30 primer pairs sequence is as shown in SEQ ID NO.51 and SEQ ID NO.52；
T31 primer pairs sequence is as shown in SEQ ID NO.53 and SEQ ID NO.54；
T34 primer pairs sequence is as shown in SEQ ID NO.55 and SEQ ID NO.56；
T35 primer pairs sequence is as shown in SEQ ID NO.57 and SEQ ID NO.58；
T36 primer pairs sequence is as shown in SEQ ID NO.59 and SEQ ID NO.60；
T37 primer pairs sequence is as shown in SEQ ID NO.61 and SEQ ID NO.62；
T38 primer pairs sequence is as shown in SEQ ID NO.63 and SEQ ID NO.64；
T39 primer pairs sequence is as shown in SEQ ID NO.65 and SEQ ID NO.66.
4. kit according to claim 3, it is characterised in that the kit includes 2 × EasyTaqPCR
Supermix, primer pair, DNA profiling and ultra-pure water.
5. kit according to claim 4, it is characterised in that the kit includes 50-100ng DNA profilings,
2 × EasyTaqPCR of 7.5ul Supermix (TransGen Biotech), 10 μM of each 0.5ul of primer pair, are mended using ultra-pure water
Together to 15ul.
6. kit according to claim 4, it is characterised in that the PCR conditions of the kit are 95 DEG C of pre-degenerations
5min；Anneal 30sec under 95 DEG C of denaturation 30sec, suitable annealing temperature, and 72 DEG C of extension 30sec react 32 circulations；72 DEG C are prolonged
Stretch 10min.
7. a kind of microsatellite locus of arborescent ceratoidses as claimed in claim 1 or 2 and its screening technique of specific primer pair,
Comprise the following steps：
1) collect arborescent ceratoidses sample and extract DNA；
2) to step 1) obtain DNA, build RNA-seq cDNA libraries, and to the RNA-seq cDNA libraries carry out high pass
Measure sequence；
3) detect microsatellite locus and design the primer pair of microsatellite locus；
4) screen and detect microsatellite locus and its specific primer pair.
8. method according to claim 7, it is characterised in that
The step 1) in extract DNA steps be CTAB methods.
9. method according to claim 7, it is characterised in that
The step 2) in RNA-seq cDNA libraries high-flux sequence use the microarray datasets of lumina Hiseq 3000 carry out
High-flux sequence；
The step 3) in using batchprimer3 on-line analyses instrument to the step 2) Sequence Detection that is completed is micro-
Satellite site sequence simultaneously carries out design of primers on the flanking sequence of microsatellite locus, wherein, the microsatellite locus detection
Condition is repeat unit when being 2 bases, and number of repetition need to be more than or equal to 6；When repeat unit is 3 bases, number of repetition is needed
More than or equal to 5；When repeat unit is 4,5 and 6 bases, number of repetition need to be more than or equal to 4；The specificity of the microsatellite locus
The principle of design of primers is that amplification purpose fragment is 100~200bp, and primer G/C content is 40~60%, upstream and downstream primer annealing
Temperature (Tm) is between 50~60 DEG C and difference is less than 5 DEG C；
The step 4) in the method for the screening microsatellite specific primer be：Using arborescent ceratoidses DNA to step 3) obtain
Specific primer expand and detects to entering performing PCR, screening, which can stablize, amplify target stripe and have drawing for polymorphism
Thing pair；
The step 4) in the detection microsatellite specific primer method be：Existed using the fluorescence molecule amount standards of Tamara 350
Genotyping is carried out to the primer pair screened using polyacrylamide gel in ABI 3730DNA sequenators；Genotyping
Result be read out using GENMARKER softwares, and input Excel file；Use Excel Mircosatellite Tool
Kit programs count expectation heterozygosity (the Expected Heterozygosity, H of each microsatellite locusE), observation heterozygosity
(Observed Heterozygosity, HO) and polymorphism information content (Polymorphic information content,
PIC), to detect the polymorphism situation of the microsatellite locus, filter out above three polymorphism numerical value and be all higher than 0.5 micro- and defend
Star special primer.
10. the microsatellite locus and its specific primer of arborescent ceratoidses as claimed in claim 1 or 2 are in Chenopodiaceae ceratoides
In genetic marker, positioning and the qtl analysis of plant, cultivar identification, population and Study on Evolution, molecular mark should
With.
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| CN111808982A (en) * | 2020-07-21 | 2020-10-23 | 神华神东煤炭集团有限责任公司 | Microsatellite locus of desert plant cotton thorn as well as amplification primer and application thereof |
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| CN105969767A (en) * | 2016-07-18 | 2016-09-28 | 黄冈师范学院 | SSR molecular marker primer based on transcriptome data of azalea as well as screening method and application of SSR molecular marker primer |
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| CN105969767A (en) * | 2016-07-18 | 2016-09-28 | 黄冈师范学院 | SSR molecular marker primer based on transcriptome data of azalea as well as screening method and application of SSR molecular marker primer |
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| 王普昶: "内蒙古野生华北驼绒藜种群遗传多样性和遗传分化研究", 《草业学报》 * |
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| CN111808982A (en) * | 2020-07-21 | 2020-10-23 | 神华神东煤炭集团有限责任公司 | Microsatellite locus of desert plant cotton thorn as well as amplification primer and application thereof |
| CN111808982B (en) * | 2020-07-21 | 2023-03-31 | 神华神东煤炭集团有限责任公司 | Microsatellite locus of desert plant cotton thorn as well as amplification primer and application thereof |
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