CN107142321A - A kind of specific microsatellite locus of mongolian amygdalus seed and its application - Google Patents
A kind of specific microsatellite locus of mongolian amygdalus seed and its application Download PDFInfo
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Abstract
Present invention firstly discloses the specific microsatellite sequence in the 23 of mongolian amygdalus seed and 23 pairs of specific micro-satellite primers.These sites play an important role in the analysis of genetic diversity of mongolian amygdalus seed, 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 locus of mongolian amygdalus seed and its application.
Background technology
Mongolian amygdalus seed (Amygdalus mongolica) is under the jurisdiction of rose family peach category, it is that the protection in imminent danger of national three-level is planted
Thing, belongs to the ancient deleted species of Mongolian plat.The main low mountains and hills for being grown in desert and Desert Grassland, rocky slopes and
The habitat conditions such as dry wash more harsh area.Islanding with the habitat of mongolian amygdalus seed, distributed area area constantly reduces, and leads
It is mainly that mankind's mining, log, land development, industrialization and urbanization etc. are movable to its habitat to cause the reason for its is endangered
Severe jamming and destruction, the distribution and population quantity of mongolian amygdalus seed are all being reduced.
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 mongolian amygdalus seed, including
TR10339、TR10936、TR12298、TR12728、TR16484、TR17663、TR17745、TR18640、TR18807、
TR20374、TR21328、TR21918、TR23600、TR24122、TR3320、TR27527、TR29327、TR31156、
The one or more of TR31399, TR37605, TR37811, TR41171 and TR47552 nucleotide sequence, the nucleotide sequence
The sequence as shown in SEQ ID NO1~SEQ ID NO23.
Present invention also offers the specific primer pair of above-mentioned mongolian amygdalus seed microsatellite locus, the mongolian amygdalus seed specificity
The nucleotide sequence of the specific primer pair of microsatellite locus is respectively：
TR10339 primer pair sequence is as shown in SEQ ID NO.24 and SEQ ID NO.25；
TR10936 primer pair sequence is as shown in SEQ ID NO.26 and SEQ ID NO.27；
TR12298 primer pair sequence is as shown in SEQ ID NO.28 and SEQ ID NO.29；
TR12728 primer pair sequence is as shown in SEQ ID NO.30 and SEQ ID NO.31；
TR16484 primer pair sequence is as shown in SEQ ID NO.32 and SEQ ID NO.33；
TR17663 primer pair sequence is as shown in SEQ ID NO.34 and SEQ ID NO.35；
TR17745 primer pair sequence is as shown in SEQ ID NO.36 and SEQ ID NO.37；
TR18640 primer pair sequence is as shown in SEQ ID NO.38 and SEQ ID NO.39；
TR18807 primer pair sequence is as shown in SEQ ID NO.40 and SEQ ID NO.41；
TR20374 primer pair sequence is as shown in SEQ ID NO.42 and SEQ ID NO.43；
TR21328 primer pair sequence is as shown in SEQ ID NO.44 and SEQ ID NO.45；
TR21918 primer pair sequence is as shown in SEQ ID NO.46 and SEQ ID NO.47；
TR23600 primer pair sequence is as shown in SEQ ID NO.48 and SEQ ID NO.49；
TR24122 primer pair sequence is as shown in SEQ ID NO.50 and SEQ ID NO.51；
TR3320 primer pair sequence is as shown in SEQ ID NO.52 and SEQ ID NO.53；
TR27527 primer pair sequence is as shown in SEQ ID NO.54 and SEQ ID NO.55；
TR29327 primer pair sequence is as shown in SEQ ID NO.56 and SEQ ID NO.57；
TR31156 primer pair sequence is as shown in SEQ ID NO.58 and SEQ ID NO.59；
TR31399 primer pair sequence is as shown in SEQ ID NO.60 and SEQ ID NO.61；
TR37605 primer pair sequence is as shown in SEQ ID NO.62 and SEQ ID NO.63；
TR37811 primer pair sequence is as shown in SEQ ID NO.64 and SEQ ID NO.65；
TR41171 primer pair sequence is as shown in SEQ ID NO.66 and SEQ ID NO.67；
TR47552 primer pair sequence is as shown in SEQ ID NO.68 and SEQ ID NO.69.
Present invention also offers the kit for identifying mongolian amygdalus seed microsatellite locus, including archaeal dna polymerase, dNTPs,
Reaction buffer, primer pair and DNA profiling, wherein, the primer pair is the one or more of following primer pair：
TR10339 primer pair sequence is as shown in SEQ ID NO.24 and SEQ ID NO.25；
TR10936 primer pair sequence is as shown in SEQ ID NO.26 and SEQ ID NO.27；
TR12298 primer pair sequence is as shown in SEQ ID NO.28 and SEQ ID NO.29；
TR12728 primer pair sequence is as shown in SEQ ID NO.30 and SEQ ID NO.31；
TR16484 primer pair sequence is as shown in SEQ ID NO.32 and SEQ ID NO.33；
TR17663 primer pair sequence is as shown in SEQ ID NO.34 and SEQ ID NO.35；
TR17745 primer pair sequence is as shown in SEQ ID NO.36 and SEQ ID NO.37；
TR18640 primer pair sequence is as shown in SEQ ID NO.38 and SEQ ID NO.39；
TR18807 primer pair sequence is as shown in SEQ ID NO.40 and SEQ ID NO.41；
TR20374 primer pair sequence is as shown in SEQ ID NO.42 and SEQ ID NO.43；
TR21328 primer pair sequence is as shown in SEQ ID NO.44 and SEQ ID NO.45；
TR21918 primer pair sequence is as shown in SEQ ID NO.46 and SEQ ID NO.47；
TR23600 primer pair sequence is as shown in SEQ ID NO.48 and SEQ ID NO.49；
TR24122 primer pair sequence is as shown in SEQ ID NO.50 and SEQ ID NO.51；
TR3320 primer pair sequence is as shown in SEQ ID NO.52 and SEQ ID NO.53；
TR27527 primer pair sequence is as shown in SEQ ID NO.54 and SEQ ID NO.55；
TR29327 primer pair sequence is as shown in SEQ ID NO.56 and SEQ ID NO.57；
TR31156 primer pair sequence is as shown in SEQ ID NO.58 and SEQ ID NO.59；
TR31399 primer pair sequence is as shown in SEQ ID NO.60 and SEQ ID NO.61；
TR37605 primer pair sequence is as shown in SEQ ID NO.62 and SEQ ID NO.63；
TR37811 primer pair sequence is as shown in SEQ ID NO.64 and SEQ ID NO.65；
TR41171 primer pair sequence is as shown in SEQ ID NO.66 and SEQ ID NO.67；
TR47552 primer pair sequence is as shown in SEQ ID NO.68 and SEQ ID NO.69.
Preferably, the present invention is used for the kit for identifying mongolian amygdalus seed 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 mongolian amygdalus seed microsatellite locus
In kit, including 50-100ng DNA profilings, 2 × EasyTaqPCR of 7.5ul Supermix (TransGenBiotech),
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 are 95 DEG C of pre- changes
Property 5min；Anneal 30sec under 95 DEG C of denaturation 30sec, suitable annealing temperature, and 72 DEG C of extension 30sec react 32 circulations；72℃
Extend 10min；Wherein, the suitable annealing temperature is 50 DEG C~61 DEG C；Most preferably, the suitable annealing temperature is 50
℃、51℃、52℃、53℃、54℃、55℃、56℃、57℃、58℃、59℃、60℃、61℃。
It is preferred that, the identification of the mongolian amygdalus seed comprises the following steps：With genome of the foregoing kit to testing sample
DNA enters performing PCR amplification, and amplified production is contrasted with microsatellite sequence：If containing at least one described in above-mentioned technical proposal
Microsatellite molecular marker is planted, then judges the testing sample as mongolian amygdalus seed；If not containing any described in above-mentioned technical proposal
A kind of microsatellite molecular marker, then it is not mongolian amygdalus seed to judge the testing sample.
Another aspect of the invention is the screening of the microsatellite locus and its specific primer pair that provide above-mentioned mongolian amygdalus seed
Method, comprises the following steps：
1) collect mongolian amygdalus seed 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 mongolian amygdalus seed and its screening technique of specific primer pair of the present invention, institute
State step 1) in extract DNA steps be CTAB methods；
Preferably, in the microsatellite locus of mongolian amygdalus seed and its screening technique of specific primer pair of the present invention, institute
State step 2) in RNA-seq cDNA libraries high-flux sequence using the microarray datasets of lumina Hiseq 3000 carry out high flux
Sequencing；
Preferably, in the microsatellite locus of mongolian amygdalus seed and its screening technique of specific primer pair of the present invention, institute
State step 3) in using batchprimer3 on-line analyses instrument to the step 2) the Sequence Detection microsatellite position that is completed
Point sequence simultaneously carries out design of primers on the flanking sequence of microsatellite locus；It is highly preferred that the bar of the microsatellite locus detection
Part 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 needs big
In 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 locus
The principle of specific primer design be that amplification purpose fragment is 100~200bp, 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.
Preferably, in the microsatellite locus of mongolian amygdalus seed and its screening technique of specific primer pair of the present invention, institute
State step 4) in the method for the screening microsatellite specific primer be：Using mongolian amygdalus seed DNA to step 3) obtain it is special
Property primer pair enter performing PCR and expand and detect, screening, which can stablize, amplify target stripe and the primer pair with polymorphism；
Preferably, in the microsatellite locus of mongolian amygdalus seed and its screening technique of specific primer pair of the present invention, institute
State 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 3730DNA sequenators；The knot of Genotyping
Fruit is read out using GENMARKER softwares, and inputs Excel file；Use Excel Mircosatellite Toolkit
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, the invention provides the microsatellite locus of mongolian amygdalus seed and its specific primer in Chenopodiaceae ceratoides plant
Genetic marker, positioning and qtl analysis, cultivar identification, the application in population and Study on Evolution, molecular mark.
The present invention compared with prior art, present invention firstly discloses the specific microsatellite sequence in the 23 of mongolian amygdalus seed and
23 pairs of specific micro-satellite primers.These sites are to the analysis of genetic diversity of mongolian amygdalus seed, conservative genetics research, germplasm
Played an important role in 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
32 mongolian amygdalus seed samples are gathered, are gathered from Alxa Zuoqi, Nei Mongol.Adopt it is lower after immediately use preservation in silica, adopt
Mongolian amygdalus seed genome DNA extraction is carried out with 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 mongolian amygdalus seed standard rna-seq cDNA libraries；It is sequenced using illumina Hiseq 3000
The both-end sequencing pattern of platform carries out high-flux sequence to the RNA-seq cDNA libraries of structure.To after Quality Control Analysis
High-quality sequence carry out sample assembling.
103923 transcript isomers are obtained after sequence is assembled, and are defined as 73225 original series
unigene。
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, 19498 microsatellite sequences are detected altogether, are designed into 14586 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 14586 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 mongolian amygdalus seed samples is template, grads PCR amplification is carried out to this 50 primer pairs.Grads PCR
Reaction system is as follows：50-100ng DNA profilings, 7.5ul 2 × EasyTaq PCR Supermix (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 mongolian amygdalus seed sample is expanded.PCR reaction systems are：50-100ng DNA profilings, 2 × EasyTaq of 7.5ul PCR
Supermix (TransGenBiotech), 10 μM of each 0.5ul of upstream and downstream primer, uses ultra-pure water polishing to 15ul.It is circularly set
For：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
Individual circulation；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 23 stable high polymorphic micro-satellite sites, the microsatellite locus be named as TR10339,
TR10936、TR12298、TR12728、TR16484、TR17663、TR17745、TR18640、TR18807、TR20374、
TR21328、TR21918、TR23600、TR24122、TR3320、TR27527、TR29327、TR31156、TR31399、
TR37605, TR37811, TR41171 and TR47552；The TR10339, TR10936, TR12298, TR12728, TR16484,
TR17663、TR17745、TR18640、TR18807、TR20374、TR21328、TR21918、TR23600、TR24122、
TR3320, TR27527, TR29327, TR31156, TR31399, TR37605, TR37811, TR41171 and TR47552 nucleosides
The acid sequence sequence as shown in SEQ ID NO1~SEQ ID NO23 respectively.
The microsatellite locus TR10339, TR10936, TR12298, TR12728, TR16484, TR17663,
TR17745、TR18640、TR18807、TR20374、TR21328、TR21918、TR23600、TR24122、TR3320、
TR27527, TR29327, TR31156, TR31399, TR37605, TR37811, TR41171 and TR47552 specific primer
To being followed successively by：
TR10339 primer pair sequence is as shown in SEQ ID NO.24 and SEQ ID NO.25；
TR10936 primer pair sequence is as shown in SEQ ID NO.26 and SEQ ID NO.27；
TR12298 primer pair sequence is as shown in SEQ ID NO.28 and SEQ ID NO.29；
TR12728 primer pair sequence is as shown in SEQ ID NO.30 and SEQ ID NO.31；
TR16484 primer pair sequence is as shown in SEQ ID NO.32 and SEQ ID NO.33；
TR17663 primer pair sequence is as shown in SEQ ID NO.34 and SEQ ID NO.35；
TR17745 primer pair sequence is as shown in SEQ ID NO.36 and SEQ ID NO.37；
TR18640 primer pair sequence is as shown in SEQ ID NO.38 and SEQ ID NO.39；
TR18807 primer pair sequence is as shown in SEQ ID NO.40 and SEQ ID NO.41；
TR20374 primer pair sequence is as shown in SEQ ID NO.42 and SEQ ID NO.43；
TR21328 primer pair sequence is as shown in SEQ ID NO.44 and SEQ ID NO.45；
TR21918 primer pair sequence is as shown in SEQ ID NO.46 and SEQ ID NO.47；
TR23600 primer pair sequence is as shown in SEQ ID NO.48 and SEQ ID NO.49；
TR24122 primer pair sequence is as shown in SEQ ID NO.50 and SEQ ID NO.51；
TR3320 primer pair sequence is as shown in SEQ ID NO.52 and SEQ ID NO.53；
TR27527 primer pair sequence is as shown in SEQ ID NO.54 and SEQ ID NO.55；
TR29327 primer pair sequence is as shown in SEQ ID NO.56 and SEQ ID NO.57；
TR31156 primer pair sequence is as shown in SEQ ID NO.58 and SEQ ID NO.59；
TR31399 primer pair sequence is as shown in SEQ ID NO.60 and SEQ ID NO.61；
TR37605 primer pair sequence is as shown in SEQ ID NO.62 and SEQ ID NO.63；
TR37811 primer pair sequence is as shown in SEQ ID NO.64 and SEQ ID NO.65；
TR41171 primer pair sequence is as shown in SEQ ID NO.66 and SEQ ID NO.67；
TR47552 primer pair sequence is as shown in SEQ ID NO.68 and SEQ ID NO.69.
The microsatellite locus TR10339, TR10936, TR12298, TR12728, TR16484, TR17663,
TR17745、TR18640、TR18807、TR20374、TR21328、TR21918、TR23600、TR24122、TR3320、
TR27527, TR29327, TR31156, TR31399, TR37605, TR37811, TR41171 and TR47552 microsatellite locus
Genetic diversity data 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 table
<110>Inner Mongolia Autonomous Region Academy of Agricultural and Livestock Husbandry
<120>A kind of specific microsatellite locus of mongolian amygdalus seed and its application
<160> 69
<170> PatentIn version 3.5
<210> 1
<211> 314
<212> DNA
<213>Mongolian amygdalus seed
<400> 1
tcaatttctt cttgttacag gtaggtaggg gctgctcacc ttctttttgt caggattctc 60
ttgccagtct ctaaagtata acaaaaggtg ttgcaagcaa acaataaaaa cacccccata 120
taaccactag aatagaagca actcaatcag atctttctcc atctttttaa ctcttctctc 180
cattgagtcc catagtcata ccaaccaatt aattaacctc tgtagacttc cactcctttg 240
ctctctctct ctctctctct ctctctctct ctctctctct tcctaaattt cccagatgag 300
gacaattatg gccg 314
<210> 2
<211> 321
<212> DNA
<213>Mongolian amygdalus seed
<400> 2
aaagtagaaa cgaaggaaaa gaaagaaaat gcaaatagaa gagagaagca aactgcgaag 60
gcaaacctct gaaaaaccct aaaaccggcg aatgaaaaca tcattcacga agaagaagaa 120
aaatggtaaa gaagatagag ggatgggagt gttggacctc acgcttcgtt agcttggtga 180
cgtcagcaga gaccgccgtc attgtcttgt cacaagaacc taagtgtgtg tgacagagag 240
agagagagag agagagagag agagagagag ctgttggctg tttggttttg gttgttgcag 300
agagggaggg gggggggggg g 321
<210> 3
<211> 399
<212> DNA
<213>Mongolian amygdalus seed
<400> 3
tttggtcgtc ctatgatgtt tgagttattg ctggtgtatc tcattggctc attggctcac 60
cttagaattt gcgaacgttt aaatgcattt ctgtccattt tcttgttcaa catattaatc 120
tatatcagct tagcatatat atatctatgc ccaatagcca aaaccctctc cacagccact 180
ccgtcttctt cacccaaacc ccttctctct ctctctctct ctctctctct ctctctctca 240
gagacagtta agctctgaga cggtgctgtg agaaactcgg ggagacatag agaaatggaa 300
ggcaaatcgt accagaggct accgcgggtc aaaatccgtg aaatgcgcga cgactacatg 360
aagttcgagc tccgcgacac cgacgccagc gtcgccaac 399
<210> 4
<211> 385
<212> DNA
<213>Mongolian amygdalus seed
<400> 4
ggatgctatt gaaatctaag cccacgtaca tcatgagaga gagagagaga gagagagaga 60
gagagagaga attaagaatg gaacgtttaa tataaattgc ttggcctgat tcaggaattg 120
gtccttgtat tagtgaatat acaagaaaaa tctgattatg agttaccagg gtgttacagc 180
actgtacaac cagttgatta gcaacaaggc aattagagac cagcatcatc ctcctcctca 240
ctggtttttg ccactggagt ttatcctgat gcctgaaaat gttggactgc aaattaagcc 300
atatgaagaa acagaaaatt gatttgagca atgggactat agatctctcc tcatttgcac 360
tggcgctaca atagactcaa caatt 385
<210> 5
<211> 564
<212> DNA
<213>Mongolian amygdalus seed
<400> 5
gacggcaagt cttgaggctc aagcatatgc aaaccgggaa tactagtaac atcaccacca 60
tgatacttca tatagtaaat ataattgacc gtgcaggttt gagtaaaaaa ggcagcggac 120
gctagaccaa actgtttggc tacatccaga gcccagggca aaaatggatc atatattatg 180
caattaatag ggtggggtga gttgctgtac tttgtaatga gctcgattag ggttctggag 240
ccctcggctt ccatccggtc tagataagca tcgatgcttt cagctcgggc aaagccttcc 300
tcatcgtagc catcggatat ggtgtcgaat tggacggaac cgcttgattt gagcggcaat 360
gacttgctga tgaagatggt gttggctaga gtgactttga ggcccttaga ggctaagcgc 420
ttggagaact ggagcaaggg gtttatgtga ccttgagcag gataaggaat tgccaagacg 480
tggcttctat ccatttgaga tctctctctc tctctctctc tctctctctc tctcaaaaac 540
ttaggtgtgg cggtgctgtg caga 564
<210> 6
<211> 455
<212> DNA
<213>Mongolian amygdalus seed
<400> 6
atcacttcca atatttcaaa atcatctcct ctaccttttt ccttcatccc tctctctctc 60
tctctctctc tctctctctc tctctctgtg tctctggggt atgcatattt acaataggaa 120
taacatttac atataaaagc gtcaaacgca ccgtatcaag cagagatcat gaccatggac 180
cgaccgcaac cgagctcaga ctcccccacc cgagagccga aatcccctct ggtgctgtcc 240
atcgaatgcc tcaaaggcag ctcgaaagcc gacgagtgga ctggagacat gctccagact 300
gcagacatcg tcgaggagct ccgcatcggc agctccgtca ccgttaagtc gccgtttaag 360
aacggaaaga gcggcgtcca gaagatcctc cacgcctcct ttaaagccaa ggacacttct 420
atcctcgtca aagtccgacg cggccgagac gagtt 455
<210> 7
<211> 302
<212> DNA
<213>Mongolian amygdalus seed
<400> 7
tgcagatgtc cctatgggct gcctctctga gaatttgttg gctcccagcc tcacatcttc 60
ttctttccct tccatttctc tgttctctct ctctttctct ctctctctct ctctctctct 120
ctctctctct ctctctcaag aactctggtt ttggccttcc ctctctacaa agttgaaagc 180
tttggtttct ggagtgagag ctatgatttg gttgagtgag caatggagtg aggtttataa 240
agggaaacaa atattatgag tgtgagctgt taaagtatgt taaagaaatt atgaaaagca 300
ga 302
<210> 8
<211> 326
<212> DNA
<213>Mongolian amygdalus seed
<400> 8
ttgctcatca aatcagcccc ctgatattcg tggatgttga ggcggcggag ctgttgctgc 60
tgccattttc cggcgataga gagagaacga gaggtaagct gcttcagtaa tcctctcacc 120
attgtttaaa gctctcaaac cctaaagcaa caactagaga gagagacgca cacagcgaga 180
gagagagaga gagagagaga gagagagaga gagagagaga gagagagaga gagagactgc 240
gtttgccgga cgcagaggcg attttttttt tttttggacg cagaggcgat tgggtctgag 300
aatgcgaaag aacccctacc ctagtg 326
<210> 9
<211> 464
<212> DNA
<213>Mongolian amygdalus seed
<400> 9
gtccttaagc agcccagtag agcagccctc tctctctctc tctctctctc tctctctctc 60
tctctctgat acaaccacaa attcaccgtg cccggaggga catggcgtat aaaggcctgg 120
accgtatcac cgtctctgac atcgaagctc tgggaatcga atcagaggct gccaagagac 180
tccatgcaac tctcactaac atcatccaaa attacggtcc tgccactccc cacacgtggc 240
gcaatatcac tgcccatgtc ctcagcccgg agcttccttt ctccttccac cagatgttgt 300
actacggttg ctataaggac ttcggacccg acccgccggc ctggctaccc gactcggaga 360
ctgcaaattt aacaaatgtt ggccagctac tggaaaggcg gggaaaagag ttcttgggat 420
ctagatataa ggatcccatg tccagctttt ccgattttca agaa 464
<210> 10
<211> 876
<212> DNA
<213>Mongolian amygdalus seed
<400> 10
cttccatcca cacttacatg agagagagag agagagagag agagagagag agagagaaag 60
agagagatac cattaggttc ccagctccgt tggtcagcac aaggtttcca agaagaagca 120
ggaacaaatg gctccagcca caaatctttc ggctcaggtt tattcagtcc ctctgccaac 180
gcatgcgcag ctaaatccaa catccttccg taattacctt tctgcgcctt tttttcttta 240
ccaagttttc tccatttttc ccgaacggta accgataccg aagccacaga cgaattcccg 300
gaaaacccag attcagaatg gagcccatga tcttgtagcc gtaaaagcat aacccaattc 360
atcaagaaaa acaaaaccaa taatccaatc acacccgcaa caaactttac tcgctttctg 420
ctataccaag ccctccctac tttcctcttg gccctcctct gcatcttcag ctccttttcg 480
atccccaaaa acaacggcgt atattgtgaa tcacacgtgt ccgagttggc gaatcgagtg 540
gggccgagtc gcggcgagtt accaggtgag gattggcggc tcctcggagt ggtggagcca 600
gagccggagc ggtaatgcaa caggctggga aaggaggtgt tggggtcggg ctgagctgaa 660
ggtccatctt cttcttcttc tgagactggt ttaacttgaa tggcgtttaa ggacattgat 720
ttgtgctcgg ctcctctgga tttggcccca agagctcaga gagagagcct gaggaagaaa 780
cgctaagaag ccggaagcga aacgagtgcg ataagtctgc tcttcgagtc ctttggctaa 840
aatgaatgtc cgcaatttgc taaaaatgga agaacg 876
<210> 11
<211> 351
<212> DNA
<213>Mongolian amygdalus seed
<400> 11
acgccggacc tcctgagtgg tttgggtgaa aaggccattg cccattgcac atttcacttt 60
gagggaggac tttaagctgc tctggttgac ttttttgggg agaaatgagt gaagaaagta 120
ttgctttctg gagagcgaag aaagctggtc tgcttttgtg tggggtaagg tgaagggtga 180
agatactaaa gaagccattg ttaatcagcc tctgtatctc accaccagaa cctcctctgt 240
ttcaaaattg aggggtggaa aaatgagaga gagagagaga gagagagaga gagagaagca 300
gagctctctc tgtgattgtg ttttgtgcct gagcttgagt ttcaggtctt a 351
<210> 12
<211> 423
<212> DNA
<213>Mongolian amygdalus seed
<400> 12
tcttttcttc ttccttccct ccttctgtct ctctcttcac gccactacag attcaaccat 60
caactcaccc tctctctctc tctctctctc tctctctctc tctctctagt tccactaatt 120
aatggttgtg gtggttcgat tgggtagtgc tggtggttta actgggtatg tggtggctgg 180
tgatgatggt ttgattgact aattgggtga tggtgatggt tcgattgggt atgtggtggc 240
tggttatgtg tttgatggtg gtggtctgat ggttgtggca tcgtggtgat ggtggtatgt 300
ttgatgtttc gggctcgtgt gtttgatggt agaggtggta ttcagccgga ctgtgtgttc 360
aaaatagcat cctcgctatt ttgcgaaccc agttttgggc tcgctatatt aaacaagcga 420
gtc 423
<210> 13
<211> 1118
<212> DNA
<213>Mongolian amygdalus seed
<400> 13
gcttctcttt tagggaagag aaaaggctag agaacaaccc ctttttcttc tttttctctc 60
tctctctctc cctctctctc cttctctctc cttttagcat ctctgttatc agcagcaacc 120
ttttgtttga aaccctaatt gatcttaacc caaaaatagt aaaaaagaag aggggcagaa 180
gagctagcta gctttcatat gtatttttga aacagtctct ttctgcaacc atcaatttcc 240
gtaggctgtt tattctcttc ttcacgcata cccttttgca gctcccagat aaagcacaag 300
aaccaaacca aaccaaagca ctgtactcct ttgctttgca gatctcacag agctagcttt 360
catatatggc ttctcatagt tggtactttc tcatcagctt acctcacagt ttcagctctc 420
tctttctctc tcctcttctt aattgagctt tgtattttaa gtaaccaaat atctctcacc 480
tcctccatat ctttgtcccc cttcacgtct ttccatttct ctctctctct ctctctctct 540
ctctctctct ctctctcatg gattctggta gtggaagtat gcaatcctcc agcggcggcg 600
atgacgagta cgactcgcgg gccgagtcaa tctccgctct actgagtaac ccaccgagtc 660
aactcggcca catgtctagc cacgcaccgc cacatcacca ccaccaccac caccaacaaa 720
cccatcacca cctagaccct ctatcaaaca tgttcgatcc attatcctcc aggctcacga 780
acccgaaccc gcttctcaat ttcgacatgg cgtggtccaa aaccctaaga tccgacccga 840
atcccaccga tctcggtggc ctaagccaac cctttttgac caaccccaat attaatcaat 900
taggacaaag cagaggcggc ggcggcggcg gaggcggctc gtctactttt gcggctcttc 960
agatcccgca tgatcaccaa aacgtttcgg cttcttcttc ggctccaaac aaccaaaccc 1020
acaacatcaa cagtaacagc aacaacaaca acagcaacag caacggagtc gttcggaacc 1080
cgaagaagag gtcgagagcg tcgaggcgcg caccgacg 1118
<210> 14
<211> 534
<212> DNA
<213>Mongolian amygdalus seed
<400> 14
ctaacgctgc attgtactcg actagccaat ttgaatctgc agtgaagttg aaatactgga 60
ataagattcc caagaccaag aaaacaattc caacaacaat acccaatgac tcaggcactt 120
tgacagtgcc ctgcggggta ccctttttgt taatgtcata accaaagagg ttgcgcttga 180
gaacgtatct ggaagccacg gggatcatct taacggtgac aaagaacccg gcaaggctga 240
gagccgcatt gatgagaatc gattttcgaa gctcatgttc gatcttgtag tgatagaaga 300
tgagataaaa gtatgggact gagaagaaga gcgtgagctt gagaattaca cctaactttg 360
gcggtgcaat tggcgggtct gttaatggag gttcttgggg tttgggtcgt gtggtggtat 420
cgtttggttt ggtttgtgcg gacgagtctt ttgaagctcg ctttcgagct gccatggctc 480
tctctctctc tctctctctc tctctctctc tctgtctctc tctcaatatt ggtg 534
<210> 15
<211> 436
<212> DNA
<213>Mongolian amygdalus seed
<400> 15
ctcgcctcct ctctctctct atctctctct ctctctctct ctctctctct ctctctctcc 60
tttttgccct atctgatagg gttttcatgt ccttgtaagc tcctatatct catgcataac 120
tgcatccaag cactatacgc cgttgttgta gccttaatct tttctgggtc tctctttttt 180
gctctctttc gagggttttt cggtggaaag ctttgcgggt tcgacctcct ggggtttttt 240
gagctgtacc catgtcaaag ctccttggtt tcgctggtaa tgatgatttt tgccctgggg 300
ggtcaattta tacaaacccc aaagaaccta gcctcttttt gtcccgtggt aatcatgcgg 360
atgtcttttt tactcctcgc aagaggtctc gcatcagtgg tccgtttgtt ttcagtgaag 420
aaggttttga gcagaa 436
<210> 16
<211> 556
<212> DNA
<213>Mongolian amygdalus seed
<400> 16
ccctaccttc tattcccaat ctctctctct cttaatattt ccctcaccat ccaccaaacc 60
tttctctctc ttctctctct gctctctctc tctctctctc tctctctctc tctctctctc 120
ttttaacagc tatggctaac aatggcgtac cggcgaccgg aaaaggcctg atcgtgagtt 180
tcggcgagat gctgatcgac tttgtgccca ctgaatcggg cgtttcgttg gcggaggctc 240
cgggcttcct caaggccccc ggtggtgccc ctgctaacgt ggcgattgct gtggcccgcc 300
tcggcgggaa ggcggcgttc gtcggaaaac tcggcgacga cgagttcggc cacatgctcg 360
ccgacatcct caagcaatac ggcgtcaccg gagagggcat cctcttcgat cagggcgccc 420
gcaccgccct ggccttcgtt accctacgcg ccgacggaga gcgcgagttc atgttctacc 480
gcaaccctag cgcagatatg ctgctcaagc ccgacgagct caacttcgag ctcatcaaat 540
ccgccaaggt gttcca 556
<210> 17
<211> 316
<212> DNA
<213>Mongolian amygdalus seed
<400> 17
caggtatctc tctctttctt ctctataatt gtttctctca tctctatatc tcatggtctc 60
tcaatctctt tcccaggctt cgtttcaaaa ccaaacccat accaaaaatc tcagccccaa 120
tctaaacccc cattttgctc gatccctgcc gtctatctct ctctctcaat cgtttcagcc 180
ttcactctcc gtctctcact cgattctatt tctctatagc tctgttttgc tctctctctc 240
tctctctctc tctctctctc tctctctctc tctcttagat acgagcacca aacttgggtg 300
ggcacaaatc tgtgag 316
<210> 18
<211> 1132
<212> DNA
<213>Mongolian amygdalus seed
<400> 18
tgcttgtctc atttccgata aggagatacc tattcttttt ataaatcaca atttgataca 60
tatgtaaaaa taattcatca aaaacacact aaaaaaacac atatgtcaca atgctaaggt 120
aagagctcct acatagtaga gagatgtatt ttctaaagag atatttatat taaataatcg 180
aaaaaaacaa agttacaaac tccccaaatc cactgccgag gagaaagaca aaataatccc 240
tcagtagtta actaaatatg aacccaccct ctcctgatac tcacatcaca taagtgagga 300
attctcagac aaaccatata tatgttcata atattaagct acaaacactt ttacataaac 360
cgacgacttt actttttttt tctctctcat atattcacta acaagtctga tgtaaacttg 420
ttcattgctt cacgcaacac atgttggcag caagcagctt gaaaagaagc caaaccctct 480
cttctttctg gttttagcat agagagtctc tggtgatatt ttgtacaagt cttcatccac 540
aggcttgggg gtaggcctgt gcctgggagg agggcttggt ggctccttca catcactcac 600
cacccaactt tgctcttttg cttctttcac atttgactga cgacttttgt tccttctgcg 660
aggaacaaca atcatggcag gtgtgacgac gtcgttctca tacaaatcac cagcaacata 720
taaatcacga tcctcatcag agtaactggt gtagcgcaga agcccagctt gcgtagctga 780
ctcaaagcac tgagtaaatg ggaggtcttg gttccaatcc cagctcccaa aggctggcac 840
ttggcttctc atcatcctaa agtcttgatc catttgagct taaagttcgc aacgttgaag 900
aaacagaaga ttttttacct cagagcctca aagggttttc aaaatgcgtt gtctttctct 960
gcctctttct cttctcttcc ttttcttggc tgtgaatgta agcgtgggtt tatgataacc 1020
tatttaaaag gaaagatggg aagagagaga gagagagaga gagagagaga gagagagaga 1080
gagggggggc aagaaagtag ggggatggga gagcgggaga gcacacgcgt ga 1132
<210> 19
<211> 430
<212> DNA
<213>Mongolian amygdalus seed
<400> 19
aagaagaaca agaagggcaa gcccaagtgc ccttgaactc aacatcatca actatttcac 60
acagagagag agagagagag agagagagag agagagagag agatttggtt tgagtgtgag 120
tgtgagtgtt aatctggtgg tgttagtgtt gtagaaagtc agtttaaaaa atctatggtg 180
agtgttttag tgagagagag ttgtgggctc tgcttagcaa gagttggttg ttgatcatcc 240
tttgtgtctt gatgggtctg gtaatagttt atgtgtgatc gtgatgatta atgtccaatg 300
gccgttggat ttgcataaaa tgaattatat tagtagttat gcctactact ttagtatatg 360
ccttgaataa gtttgattat tcatataaat cttgaataag tgtgtaatta tattgtaaat 420
aatacatgat 430
<210> 20
<211> 425
<212> DNA
<213>Mongolian amygdalus seed
<400> 20
gacacttcct cctcctccct ttctaacccc tctgtgtctc ttctctctct ctctctctct 60
ctctctctct ctctctctct ctcgtttcag agagcagaaa caagaccaag acacaaaaca 120
cacacagaga gccagccatg gactcagacg aaggtaagct gttcatagga gggctggcct 180
gggacaccac agaggacaag ctcaccgact acttcaacca gtacggtgac gtcacccaga 240
ccgtcatcat gcgcgacaag accactggcc ggcccagggg cttcggcttc gttgtcttct 300
ctgatccctc tgtgctcgat agggtcctca acgacaagca caccattgat ggccgagtgg 360
tggaagcaaa aaaggcctta tcaagagaag aacagcagac atccaataga agtggaaatt 420
ttaat 425
<210> 21
<211> 530
<212> DNA
<213>Mongolian amygdalus seed
<400> 21
agaagaggtt ggttacaata tacagttctt gagaacccga ctccttggat tgggcgagac 60
gcctctgtct cactggcttt tggcttttgg cagaatgtgt aaagctggct ctgccggagc 120
tttgccctta tctttcctcc tgccatttct atcaatccca gccaaatcca attataattg 180
gcacatactc attaaagaaa ggactcaaat ctttgtcctc gccgtaggat taatggtacc 240
cttgtctcca agttctcctt aaaaatgcct gccatcaggg gccttggtcc atcttcaaga 300
caaattaaga agaacccatt tagcagaaag ggaacgagcg aactttgagc tgcagatcaa 360
ggaggaagac aaagtctaga gagagagaga gagagagaga gagagagaga gagagagaga 420
gagagagagg agggaacaga aagaaagaaa agaaagaaaa gaaagaatga gcttcctctt 480
tctctctctc tgcccgtcgt gaagtccaaa ccaatggcag gaatttccgg 530
<210> 22
<211> 263
<212> DNA
<213>Mongolian amygdalus seed
<400> 22
cggcgggatc ctcttgacaa tccggagctc cggctcgcct ttagaaacta tgtgcgtgtg 60
tgtacgtgag agagagagag agagagagag agagagagag agagagagag agagagcgat 120
atacagagtg gagagacgaa gatgagagag agaattcgaa acgaagccga atttatagcc 180
tccctgaaga tatgatcgtt tccagagcct tcgaaaacga aagcgtcagg tccaccggtt 240
tctttctgtt gcttctcaat tga 263
<210> 23
<211> 573
<212> DNA
<213>Mongolian amygdalus seed
<400> 23
atctgcacaa tctcttcgtc tctggttggc tgctgtctct caaagttttc aactttgctg 60
aaatagaaaa acgaaaaaca gagagagaga gagagagaga gagagagaga gagagaggag 120
atgttgaagc ttacaacgta cactggagcg gaccaaattc atggcgtggc ccaatgccac 180
tccttgggcc catcgaaacc caaccccact ttccctcgcc ggaaagacgg catttctctt 240
gtgccatgca cttctgcgcc ttcgaaaacg ccggttctcg ccgtcgtttc ggaccggggg 300
gccgggagtg tgcgctcgga gactggaccg ggtcgggtcg agcattcggt tagcctggcg 360
gaccggttga ggcaggggag cttggcggag gacgggaagt catataagga gtgctttata 420
gtgaggtgct atgaggttgg gatgaacaag actgccactg ttgagaccat tgccaatctc 480
ttgcaggagg ttggatgcaa ccatgctcaa gttgttggct tttcaactga tggttttgcg 540
accaccacca ccatgaggaa aatgcatctc ata 573
<210> 24
<211> 21
<212> DNA
<213>Artificial sequence
<400> 24
actcttctct ccattgagtc c 21
<210> 25
<211> 21
<212> DNA
<213>Artificial sequence
<400> 25
gtcctcatct gggaaattta g 21
<210> 26
<211> 21
<212> DNA
<213>Artificial sequence
<400> 26
agaagataga gggatgggag t 21
<210> 27
<211> 20
<212> DNA
<213>Artificial sequence
<400> 27
ctctctgcaa caaccaaaac 20
<210> 28
<211> 19
<212> DNA
<213>Artificial sequence
<400> 28
caatagccaa aaccctctc 19
<210> 29
<211> 21
<212> DNA
<213>Artificial sequence
<400> 29
gccttccatt tctctatgtc t 21
<210> 30
<211> 21
<212> DNA
<213>Artificial sequence
<400> 30
aaatctaagc ccacgtacat c 21
<210> 31
<211> 21
<212> DNA
<213>Artificial sequence
<400> 31
caccctggta actcataatc a 21
<210> 32
<211> 21
<212> DNA
<213>Artificial sequence
<400> 32
gtgttggcta gagtgacttt g 21
<210> 33
<211> 18
<212> DNA
<213>Artificial sequence
<400> 33
cgccacacct aagttttt 18
<210> 34
<211> 21
<212> DNA
<213>Artificial sequence
<400> 34
tcatctcctc tacctttttc c 21
<210> 35
<211> 21
<212> DNA
<213>Artificial sequence
<400> 35
ggtcatgatc tctgcttgat a 21
<210> 36
<211> 21
<212> DNA
<213>Artificial sequence
<400> 36
ccttccattt ctctgttctc t 21
<210> 37
<211> 21
<212> DNA
<213>Artificial sequence
<400> 37
cactcaacca aatcatagct c 21
<210> 38
<211> 21
<212> DNA
<213>Artificial sequence
<400> 38
aactagagag agagacgcac a 21
<210> 39
<211> 20
<212> DNA
<213>Artificial sequence
<400> 39
gttctttcgc attctcagac 20
<210> 40
<211> 19
<212> DNA
<213>Artificial sequence
<400> 40
gtccttaagc agcccagta 19
<210> 41
<211> 20
<212> DNA
<213>Artificial sequence
<400> 41
agagcttcga tgtcagagac 20
<210> 42
<211> 19
<212> DNA
<213>Artificial sequence
<400> 42
cttccatcca cacttacat 19
<210> 43
<211> 21
<212> DNA
<213>Artificial sequence
<400> 43
gagggactga ataaacctga g 21
<210> 44
<211> 21
<212> DNA
<213>Artificial sequence
<400> 44
aaggtgaagg gtgaagatac t 21
<210> 45
<211> 21
<212> DNA
<213>Artificial sequence
<400> 45
aaacacaatc acagagagag c 21
<210> 46
<211> 21
<212> DNA
<213>Artificial sequence
<400> 46
cctccttctg tctctctctt c 21
<210> 47
<211> 21
<212> DNA
<213>Artificial sequence
<400> 47
cagttaaacc accagcacta c 21
<210> 48
<211> 21
<212> DNA
<213>Artificial sequence
<400> 48
cagctctctc tttctctctc c 21
<210> 49
<211> 21
<212> DNA
<213>Artificial sequence
<400> 49
acttccacta ccagaatcca t 21
<210> 50
<211> 21
<212> DNA
<213>Artificial sequence
<400> 50
ggtctgttaa tggaggttct t 21
<210> 51
<211> 21
<212> DNA
<213>Artificial sequence
<400> 51
caccaatatt gagagagaga c 21
<210> 52
<211> 19
<212> DNA
<213>Artificial sequence
<400> 52
cgcctcctct ctctctcta 19
<210> 53
<211> 21
<212> DNA
<213>Artificial sequence
<400> 53
cccagaaaag attaaggcta c 21
<210> 54
<211> 21
<212> DNA
<213>Artificial sequence
<400> 54
caccaaacct ttctctctct t 21
<210> 55
<211> 20
<212> DNA
<213>Artificial sequence
<400> 55
acaaagtcga tcagcatctc 20
<210> 56
<211> 21
<212> DNA
<213>Artificial sequence
<400> 56
ctgccgtcta tctctctctc t 21
<210> 57
<211> 21
<212> DNA
<213>Artificial sequence
<400> 57
caagtttggt gctcgtatct a 21
<210> 58
<211> 21
<212> DNA
<213>Artificial sequence
<400> 58
ctctgcctct ttctcttctc t 21
<210> 59
<211> 18
<212> DNA
<213>Artificial sequence
<400> 59
atccccctac tttcttgc 18
<210> 60
<211> 19
<212> DNA
<213>Artificial sequence
<400> 60
gaagaacaag aagggcaag 19
<210> 61
<211> 21
<212> DNA
<213>Artificial sequence
<400> 61
caacactaac accaccagat t 21
<210> 62
<211> 21
<212> DNA
<213>Artificial sequence
<400> 62
ctttctaacc cctctgtgtc t 21
<210> 63
<211> 21
<212> DNA
<213>Artificial sequence
<400> 63
tatgaacagc ttaccttcgt c 21
<210> 64
<211> 21
<212> DNA
<213>Artificial sequence
<400> 64
gatcaaggag gaagacaaag t 21
<210> 65
<211> 21
<212> DNA
<213>Artificial sequence
<400> 65
gcagagagag agaaagagga a 21
<210> 66
<211> 21
<212> DNA
<213>Artificial sequence
<400> 66
tagaaactat gtgcgtgtgt g 21
<210> 67
<211> 21
<212> DNA
<213>Artificial sequence
<400> 67
cttcagggag gctataaatt c 21
<210> 68
<211> 21
<212> DNA
<213>Artificial sequence
<400> 68
acaatctctt cgtctctggt t 21
<210> 69
<211> 20
<212> DNA
<213>Artificial sequence
<400> 69
gctccagtgt acgttgtaag 20
Claims (10)
1. a kind of specific microsatellite locus of mongolian amygdalus seed, including TR10339, TR10936, TR12298, TR12728,
TR16484、TR17663、TR17745、TR18640、TR18807、TR20374、TR21328、TR21918、TR23600、
TR24122, TR3320, TR27527, TR29327, TR31156, TR31399, TR37605, TR37811, TR41171 and
The one or more of TR47552 nucleotide sequences, nucleotide sequence sequence as shown in SEQ ID NO1~SEQ ID NO23
Row.
2. a kind of specific primer pair for being used to identify mongolian amygdalus seed microsatellite locus as claimed in claim 1, its feature exists
In the nucleotide sequence of the specific primer pair is respectively：
TR10339 primer pair sequence is as shown in SEQ ID NO.24 and SEQ ID NO.25；
TR10936 primer pair sequence is as shown in SEQ ID NO.26 and SEQ ID NO.27；
TR12298 primer pair sequence is as shown in SEQ ID NO.28 and SEQ ID NO.29；
TR12728 primer pair sequence is as shown in SEQ ID NO.30 and SEQ ID NO.31；
TR16484 primer pair sequence is as shown in SEQ ID NO.32 and SEQ ID NO.33；
TR17663 primer pair sequence is as shown in SEQ ID NO.34 and SEQ ID NO.35；
TR17745 primer pair sequence is as shown in SEQ ID NO.36 and SEQ ID NO.37；
TR18640 primer pair sequence is as shown in SEQ ID NO.38 and SEQ ID NO.39；
TR18807 primer pair sequence is as shown in SEQ ID NO.40 and SEQ ID NO.41；
TR20374 primer pair sequence is as shown in SEQ ID NO.42 and SEQ ID NO.43；
TR21328 primer pair sequence is as shown in SEQ ID NO.44 and SEQ ID NO.45；
TR21918 primer pair sequence is as shown in SEQ ID NO.46 and SEQ ID NO.47；
TR23600 primer pair sequence is as shown in SEQ ID NO.48 and SEQ ID NO.49；
TR24122 primer pair sequence is as shown in SEQ ID NO.50 and SEQ ID NO.51；
TR3320 primer pair sequence is as shown in SEQ ID NO.52 and SEQ ID NO.53；
TR27527 primer pair sequence is as shown in SEQ ID NO.54 and SEQ ID NO.55；
TR29327 primer pair sequence is as shown in SEQ ID NO.56 and SEQ ID NO.57；
TR31156 primer pair sequence is as shown in SEQ ID NO.58 and SEQ ID NO.59；
TR31399 primer pair sequence is as shown in SEQ ID NO.60 and SEQ ID NO.61；
TR37605 primer pair sequence is as shown in SEQ ID NO.62 and SEQ ID NO.63；
TR37811 primer pair sequence is as shown in SEQ ID NO.64 and SEQ ID NO.65；
TR41171 primer pair sequence is as shown in SEQ ID NO.66 and SEQ ID NO.67；
TR47552 primer pair sequence is as shown in SEQ ID NO.68 and SEQ ID NO.69.
Be used to identifying the kit of mongolian amygdalus seed microsatellite locus 3. a kind of, including archaeal dna polymerase, dNTPs, reaction buffer,
Primer pair and DNA profiling, it is characterised in that the primer pair is the one or more of following primer pair：
TR10339 primer pair sequence is as shown in SEQ ID NO.24 and SEQ ID NO.25；
TR10936 primer pair sequence is as shown in SEQ ID NO.26 and SEQ ID NO.27；
TR12298 primer pair sequence is as shown in SEQ ID NO.28 and SEQ ID NO.29；
TR12728 primer pair sequence is as shown in SEQ ID NO.30 and SEQ ID NO.31；
TR16484 primer pair sequence is as shown in SEQ ID NO.32 and SEQ ID NO.33；
TR17663 primer pair sequence is as shown in SEQ ID NO.34 and SEQ ID NO.35；
TR17745 primer pair sequence is as shown in SEQ ID NO.36 and SEQ ID NO.37；
TR18640 primer pair sequence is as shown in SEQ ID NO.38 and SEQ ID NO.39；
TR18807 primer pair sequence is as shown in SEQ ID NO.40 and SEQ ID NO.41；
TR20374 primer pair sequence is as shown in SEQ ID NO.42 and SEQ ID NO.43；
TR21328 primer pair sequence is as shown in SEQ ID NO.44 and SEQ ID NO.45；
TR21918 primer pair sequence is as shown in SEQ ID NO.46 and SEQ ID NO.47；
TR23600 primer pair sequence is as shown in SEQ ID NO.48 and SEQ ID NO.49；
TR24122 primer pair sequence is as shown in SEQ ID NO.50 and SEQ ID NO.51；
TR3320 primer pair sequence is as shown in SEQ ID NO.52 and SEQ ID NO.53；
TR27527 primer pair sequence is as shown in SEQ ID NO.54 and SEQ ID NO.55；
TR29327 primer pair sequence is as shown in SEQ ID NO.56 and SEQ ID NO.57；
TR31156 primer pair sequence is as shown in SEQ ID NO.58 and SEQ ID NO.59；
TR31399 primer pair sequence is as shown in SEQ ID NO.60 and SEQ ID NO.61；
TR37605 primer pair sequence is as shown in SEQ ID NO.62 and SEQ ID NO.63；
TR37811 primer pair sequence is as shown in SEQ ID NO.64 and SEQ ID NO.65；
TR41171 primer pair sequence is as shown in SEQ ID NO.66 and SEQ ID NO.67；
TR47552 primer pair sequence is as shown in SEQ ID NO.68 and SEQ ID NO.69.
4. kit according to claim 3, it is characterised in that the kit includes 2 × EasyTaq PCR
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,
7.5ul 2 × EasyTaq PCR Supermix (TransGenBiotech), 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 mongolian amygdalus seed as claimed in claim 1 or 2 and its screening technique of specific primer pair, bag
Include following steps：
1) collect mongolian amygdalus seed 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 mongolian amygdalus seed DNA to step 3) obtain
Specific primer expand and detect to entering performing PCR, screening, which can stablize, amplify target stripe and the primer with polymorphism
It is right；
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 mongolian amygdalus seed as claimed in claim 1 or 2 belong to Mongolia in rose family peach
In genetic marker, positioning and the qtl analysis of almond plant, cultivar identification, population and Study on Evolution, molecular mark
Using.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109666725A (en) * | 2018-12-26 | 2019-04-23 | 中国环境科学研究院 | A method of identification mongolian amygdalus seed |
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