CN105512513B - A kind of method for differentiating Amygdalus plant germplasm based on SSR molecular marker - Google Patents

A kind of method for differentiating Amygdalus plant germplasm based on SSR molecular marker Download PDF

Info

Publication number
CN105512513B
CN105512513B CN201510870315.6A CN201510870315A CN105512513B CN 105512513 B CN105512513 B CN 105512513B CN 201510870315 A CN201510870315 A CN 201510870315A CN 105512513 B CN105512513 B CN 105512513B
Authority
CN
China
Prior art keywords
sense primer
ssr
temperature
mark
amygdalus
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Fee Related
Application number
CN201510870315.6A
Other languages
Chinese (zh)
Other versions
CN105512513A (en
Inventor
曾斌
李疆
田嘉
夏江宏
刘梦雯
李伟阳
刘楠楠
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Xinjiang Agricultural University
Original Assignee
Xinjiang Agricultural University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Xinjiang Agricultural University filed Critical Xinjiang Agricultural University
Priority to CN201510870315.6A priority Critical patent/CN105512513B/en
Publication of CN105512513A publication Critical patent/CN105512513A/en
Application granted granted Critical
Publication of CN105512513B publication Critical patent/CN105512513B/en
Expired - Fee Related legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B40/00—ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding

Abstract

The present invention relates to a kind of method for differentiating Amygdalus plant germplasm based on SSR molecular marker, this method carries out SSR-PCR amplified reactions, electrophoresis detection, statistical analysis by selecting the high SSR primers of polymorphism, to 55 parts of Amygdalus plant germplasm materials.The result shows that：Use the strong primer of 10 pairs of polymorphisms in 100 pairs of SSR primers screened from kernel approaches, SSR PCR amplifications are carried out to the genomic DNA of Amygdalus plant germplasm material, coamplification goes out 63 amplified bands, wherein 60, polymorphic band, polymorphic rate is 95.2%, obtains preferable amplification.By carrying out similarity factor and cluster analysis, genetic similarity 0.4133-0.9600, average similarity coefficient 0.6867；Hereditary difference between 55 parts of amygdalae plant germplasms shows：Genetic distance between not of the same race and different cultivars is different, and when similarity factor is less than 0.68, most of cultivars gather can cluster together for one kind, most of cultivars of same Provenance Region.Important channel is provided to further discriminate between amygdalae plant germplasm resource affiliation distance.

Description

A kind of method for differentiating Amygdalus plant germplasm based on SSR molecular marker
Technical field
The invention belongs to the field of molecular marker of molecular biology, and in particular to one kind is differentiated flat based on SSR molecular marker The method of peach family plant seed germplasm.
Background technology
Almond, is commonly called as almond, is rose family Lee's subfamily peach category almond platymiscium, the almond platymiscium of the existing distribution in China Germ plasm resource includes wild and totally 6 kinds of cultigen, the Kaxgar Prefecture being mainly distributed on the south Xinjiang Tianshan；The quasi- Ge Er basins in Xinjiang The mountainous regions such as western Ba Er Lukes mountain, Part of Tarbagatai Mountain and Altai Mountains；Inner Mongol Wula Shan Mountain, Daqunshan Mountains；Helan Mountain in Ningxia； The ground such as Qinghai-Tibet the east.It is common almond Amygdalus.communis L., Xinjiang Wild Almonds respectively Amygdalus.ledebouriana Schlecht., mongolian amygdalus seed Amygdalus.mongolica Moxim., Amygdalus pedunculata Amygdalus.pedunculata Pall., Xikang almond Amygdalus.tangutica Batal., flowering plum Amygdalus.triloba(Lindl)Ricker.。
Almond is very long as the history of common almond cultigen, and about before 4000, almond is in its original producton location west Subregion is just by introducing and planting.Due to the limitation of the conditions such as climatic environment, worldwide there was only Xinjiang, China at present, it is beautiful State adds the ground such as Li Fuliya states, the Iran of West Asia, the Italian Xi Banya in southern Europe to have cultivation.Cultivate common almond and have in China The history of more than 1300 years, is mainly distributed on the ground such as the Yingjisha of Kaxgar Prefecture and Shache county on the south Xinjiang Tianshan.Due to long-term reality Raw breeding and natural evolvement, form abundant resource, cultivar, strain and fine individual plant have more or less a hundred, these resources Come in every shape, relation is complicated, and most kind pedigrees are unclear, and the phenomenon of homonym or synonym is universal, this divides to almond Class, cultivation and breed breeding bring very big difficulty.
Emphasis of the present invention based on Xinjiang almond resource, including be distributed in 5 different groups in Xinjiang Xinjiang open country it is flat The type of peach, while to introducing U.S.'s almond variety source (i.e. so-called U.S.'s jordan almond) of cultivation and being distributed in the flat of China Peach family plant seed kind carries out SSR molecular marker and cluster analysis.SSR is also referred to as microsatellite marker, and it by length is 1-6 base to be Primitive, what tandem sequence repeats formed, it is distributed widely in genome.SSR is the sequence the most rapid that makes a variation in genome, it is connected The mutation rate of repeat number is very high, so as to generate many allele, forms the high polymorphism of SSR.SSR marker is to be based on The molecular labeling of PCR amplification, in codominance, primer is developed by the genome sequence of species to be checked, and species specificity is strong, no Easily influenced by Nei Sheng and inoculating microbe genome, as a result accurately and reliably, be based on these advantages, International Plant kind power SSR was determined as building the mark of DNA fingerprint database by protective tissue (UPOV) in 2007 together with the SNP marker of latest development Note method.
It is contemplated that research China's amygdalae plant germplasm includes the affiliation between each kind of introduction, kind. The identification that a set of easy, quick, reliable Amygdalus plant germplasm identification technology system is applied to the category germ plasm resource is established, into Row germplasm and kind rights protection, the interests for safeguarding breeder and the producer, ensure that the industry develops in a healthy way, have important And the meaning of reality.
The content of the invention
It is an object of the invention to：A kind of method for being differentiated Amygdalus plant germplasm based on SSR molecular marker technology is provided, This method carries out SSR-PCR expansions by selecting the high SSR primers of polymorphism, to domestic and international 55 parts of Amygdalus plant germplasms material Increase reaction, electrophoresis detection, statistical analysis.The result shows that：It is polymorphic using 10 couple in 100 pairs of SSR primers screened from kernel approaches The strong primer pair Amygdalus plant germplasm material of property, including the gene of domestic and international cultivar and not of the same race 55 individual specimens Group DNA carries out SSR-PCR amplifications, obtains preferable amplification.10 pairs of primer coamplifications go out 63 amplified bands, wherein more 60, state band, polymorphic rate 95.2%.By carrying out similarity factor and cluster analysis, genetic similarity 0.4133- 0.9600, average similarity coefficient 0.6867；A little class germplasm materials are divided into 6 major classes by cluster analysis, and 55 parts of amygdalaes are planted Hereditary difference between species matter shows：Genetic distance between not of the same race and different cultivars is different, is less than 0.68 in similarity factor When, most of cultivars gather for one kind, and in cultivar, most of cultivars of same Provenance Region can cluster together. Affiliation between common cultivation almond and Xinjiang Wild Almonds is than between Amygdalus pedunculata, mongolian amygdalus seed, Xikang almond and flowering plum Affiliation closer to providing important channel to further discriminate between amygdalae plant germplasm resource affiliation distance.The party Method is more easy, directly perceived, true than conventional methods such as conventional morphology, cytology, biochemical marker identifications, it is convenient to should Distinguish, identify, check, exchange, manage, utilize for Amygdalus plant germplasm.Its opening be more conducive to in the ranks exchange and It is shared, provide identification technology system for further good breeding purpose.
The present invention provides a kind of method for differentiating Amygdalus plant germplasm based on SSR molecular marker technology, follows these steps Carry out：
Prepare Amygdalus plant germplasm material：
A, early spring, chooses the fresh tender leaf of healthy and strong, no disease and pests harm 55 parts of samples of almond platymiscium, every part of sample is with liquid It is put into -70 DEG C of ultra low temperature freezers of temperature and saves backup after chilled nitrogen；
The extraction of Amygdalus plant germplasm DNA：
B, it is rapidly added 1000 μ L, 65 DEG C of preheatings of temperature contain 2% cetyl trimethylammonium bromide, 1.4M sodium chloride, The buffering of 20mM tetraacethyl diamino-vinyls, 100mM trishydroxymethylaminomethanes, the buffer solution of pH8.0 and 8% mercaptoethanol Liquid, gently shakes up；Warm bath 60min in 65 DEG C of water-baths of temperature is placed in, gently teetertotter every 10min shakes up 1 time therebetween, makes Solution fully cracks；Centrifuged using desk centrifuge, 21 DEG C, rotating speed 10000r/min, time 15min of temperature, Aspirate supernatant, Supernatant mean allocation is transferred in two other 1500 new μ L centrifuge tube, wherein containing on 480 μ L in each centrifuge tube Clear liquid；Isometric chloroform will be added in each centrifuge tube containing supernatant:Isoamyl alcohol=24:1, gently teetertotter and shake Even, centrifugation, 21 DEG C, rotating speed 10000r/min, time 15min of temperature, Aspirate supernatant, the supernatant in each centrifuge tube is closed And be transferred in another 1500 new μ L centrifuge tube, 600 μ L of supernatant liquid are contained in centrifuge tube；By equipped with 600 μ L of supernatant liquid from Isometric chloroform is added in heart pipe:Isoamyl alcohol=24:1, gently teetertotter and shake up, centrifuge, 21 DEG C of temperature, rotating speed 10000r/min, time 15min, then Aspirate supernatant, supernatant are transferred in another 1500 new μ L centrifuge tube, centrifuge tube In contain 400 μ L of supernatant liquid；The freezing absolute ethyl alcohol of 2 times of volumes will be added in centrifuge tube equipped with 400 μ L of supernatant liquid, be put into temperature - 20 DEG C of deep freezers are spent, time 30min, centrifuges, and has after 21 DEG C, rotating speed 10000r/min, time 15min of temperature clear milky white The DNA white cotton fibers precipitation of color, removes upper liquid；Milky DNA white cotton fibers precipitation is washed into 2-3 with the ethanol that concentration is 70% again It is secondary, after natural air drying, it is dissolved in 50 μ L and contains 10mM trishydroxymethylaminomethanes, 1mM tetraacethyl diamino-vinyls, pH8.0's is slow Rush in solution, -20 DEG C of refrigerators of temperature preserve；
C, DNA purity is detected with 1.5% agarose gel electrophoresis；The obtained DNA concentrations of step b are diluted to 50ng/ μ After l, it is placed in spare in -20 DEG C of refrigerators of temperature；
D, SSR primer screenings：
Searched for according on ncbi database, from from 100 pairs of SSR primers of tone fruit trees crop by primary dcreening operation and Secondary screening, finally selects that 10 pairs of product master tapes are obvious and the preferable primer of polymorphism carries out SSR microsatellite molecular marker analyses, its Middle SSR 10 to mark for：
Mark 1BPPCT002, its sense primer 5 ' TCGACAGCTTGATCTTGACC, anti-sense primer 5 ' CAATGCCTACGGAGATAAAAGAC；
Mark 2BPPCT004, its sense primer 5 ' CTGAGTGATCCATTTGCAGG, anti-sense primer 5 ' AGGGCATCTAGACCTCATTGTT；
Mark 3BPPCT032, its sense primer 5 ' TTAAGCCACAACATCCATGAT, anti-sense primer 5 ' AATGGTCTAAGGAGCACACG；
Mark 4BPPCT036, its sense primer 5 ' AAGCAAAGTCCATAAAAACGC, anti-sense primer 5 ' GGACGAAGACGCTCCATT；
Mark 5BPPCT040, its sense primer 5 ' ATGAGGACGTGTCTGAATGG, anti-sense primer 5 ' AGCCAAACCCCTCTTATACG；
Mark 6Pchgms1, its sense primer 5 ' GGGTAAATATGCCCATTGTGCAATC, anti-sense primer 5 ' GGATCATTGAACTACGTCAATCCTC；
Mark 7Pchgms3, its sense primer 5 ' ACGGTATGTCCGTACACTCTCCATG, anti-sense primer 5 ' CAACCTGTGATTGCTCCTATTAAAC；
Mark 8Pchgms10, its sense primer 5 ' CGTCACGCATCCTTTCATTT, anti-sense primer 5 ' GACACCTCCATTTGTATCAAAGC；
Mark 9Pchgms11, its sense primer 5 ' TTGAGGCCCACTTATTAGCC, anti-sense primer 5 ' CCCCCATTATTCAAACTTCTG；
Mark 10Pchgms21, its sense primer 5 ' ACCACCATTTTGGCTCTCTG, anti-sense primer 5 ' CATGCAACCCAAAACCATCT；
E, mono- pcr amplification reactions of SSR, electrophoresis detection：20 μ l reaction systems of SSR-PCR reaction systems include：10 × Taq enzyme Supporting buffer solution, 1.5mmol/L MgCl2, 0.25mmol/L dNTPS, SSR upstream and downstream primer are respectively 0.2mol/ μ l, 0.5UTaq archaeal dna polymerases, DNA profiling 30ng, corresponding amplification program are：94 DEG C of pre-degenerations of temperature, 4min, followed by temperature 94 DEG C of denaturation 60s, 55 DEG C of renaturation 40s of temperature, 72 DEG C of extension 60s of temperature, after totally 34 circulations, 72 DEG C of extension 8min of temperature are mended Together, 6 times of Loadingbuffer2.0 μ l are added in amplified production, 2.5 μ l loadings is taken, is coagulated with 8% non denatured polypropylene phthalein amine Gel electrophoresis detect, silver staining colour developing；
F, statistical analysis：Carried out based on the cluster analysis for not weighting matched group algorithmic approach with NTSYS-pc 2.10e softwares Data analysis, selects clear and legible electrophoresis band, and sample has band to be then denoted as 1, and no band is then denoted as 0, builds two condition data matrix；It is right Original matrix seeks S with SimQual programsSMSimilarity factor and genetic distance matrix, and carried out with the UPGMA methods in SAHN programs Cluster analysis, the cluster analysis based on Wagner parsimony principles SEQBOOT, MIX, CONSENSE etc. in PHYLIP3.65 software kits Program makes uniformity tree graph, and with the brief figure of TreeView1.6 Program Generatings, builds molecular evolution genealogical tree.
A kind of method for differentiating Amygdalus plant germplasm based on SSR molecular marker of the present invention, involved in this method Preparation Amygdalus plant germplasm material：The Xinjiang Wild for picking up from Xinjiang, China Tacheng and Altay Prefecture's different distributions population is flat 5 parts of peach (in Burqin, Habahe County, Tacheng, Tuoli, randomly selects, respectively respectively in the 5 different distributions population centers that make people rich Numbering is Xinjiang Wild Almonds 1, No. 2, No. 3, No. 4, No. 5 single plants)；Pick up from Xinjiang, China South Sinkiang Kaxgar Prefecture Yingjisha and Shache county 30 parts of the Xinjiang place cultivation almond kind in county；Xinjiang, China South Sinkiang Kaxgar Prefecture Shache County is picked up from from California, USA Excellent 16 parts of the cultivation almond kind introduced；Pick up from the mongolian amygdalus seed, Xikang almond, length in Xinjiang Academy of Forestry's forest fruit resource garden 4 parts of the germ plasm resource of the almond platymiscium such as handle almond and flowering plum；55 parts of sample materials are amounted to, cover Amygdalus plant substantially The germ plasm resource of thing.
A kind of method for differentiating Amygdalus plant germplasm based on SSR molecular marker of the present invention, passes through this method institute Obtain result and analysis：
Using the granted patent of the applicant's early period：" one kind is suitable for arid biogeographic zone fruit tree crop extracting genome DNA technology Method " (the patent No.：201310236983.4) DNA of various Amygdalus plant germplasm materials is extracted, band clearly becomes clear, nothing Signs of degradation, DNA mass integrality is good, and spectrophotometer detection finds OD260/OD280Between 1.8-2.0, concentration exists ratio 350-700ng/ μ l, can meet the requirement of SSR-PCR.
Use the strong primer pair Amygdalus plant germplasm of 10 pairs of polymorphisms in 100 pairs of SSR primers screened from kernel approaches Material, including the genomic DNA of domestic and international cultivar and 55 individual specimens not of the same race carry out SSR-PCR amplifications, obtain Preferable amplification.10 pairs of primer coamplifications go out 63 amplified bands, wherein 60, polymorphic band, polymorphic rate 95.2%.
Hereditary difference and Genetic relationship：The result of calculation of genetic similarty and genetic distance shows：Almond platymiscium Germplasm is not of the same race and different cultivars between genotypic difference it is different：In all almond cultivars for trying, each genotype Between genetic similarty it is universal larger compared with inter-species, and it is also larger to make a variation, wherein the heredity of late rich kind and short rich kind Similarity factor maximum (0.9600), affiliation is nearest between illustrating both, and the yellow bar denier kind of sharp mouth and Thompson kinds Genetic similarity it is minimum (0.4500), reflection between both affiliation it is farthest.
To including 47 almond cultivars (home and abroad)；Xinjiang Wild Almonds No. 1, No. 2, No. 3, No. 4 and No. 5；Long handle Amygdalus plant germplasm inter-species including almond, mongolian amygdalus seed, Xikang almond, flowering plum etc. carries out analysis of genetic diversity, hair Existing genetic similarity index between almond cultivar and Xinjiang Wild Almonds No. 1, No. 2, No. 3, No. 4 and No. 5 0.5067~ Between 0.7067, average value 0.6067, more than between almond cultivar and Amygdalus pedunculata, mongolian amygdalus seed and Xikang almond Similitude (0.4400~0.6133, average 0.5266).That is the parent between almond cultivar and Xinjiang Wild Almonds Edge relation than the affiliation between Amygdalus pedunculata, mongolian amygdalus seed, Xikang almond closer to.
Between 0.4133-0.5433, average is genetic similarity between 5 kinds of plants of flowering plum kind and Amygdalus 0.4783, affiliation is farther, and therefore, when cluster is gathered in outer most edge.
Cluster analysis：Likeness coefficient between 55 samples is formed matrix and carries out cluster analysis structure dendrogram, as a result Show：Almond cultivar gathers when similarity factor is less than 0.68 for a major class inside and outside tested host country, shows that almond is planted Training kind has similar genetic background.
At the same time with almond cultivar similar in amygdalae plant species be divided into almond under the similarity factor (0.68) Cultivar group (domestic and international cultivar), Xinjiang Wild Almonds group (Xinjiang Wild Almonds No. 1, No. 2, No. 3, No. 4 and No. 5), almond Kind group (Amygdalus pedunculata, mongolian amygdalus seed, Xikang almond) and flowering plum.The result basic explanation each almond kind or kind The common point and variation diversity of genotype genetic background.
In cultivar group, the kind from California, USA Provenance Region can largely be classified as one kind, lead in addition Together with easily sorting out for the variety protection from areal (Xinjiang Yingjisha and Shache County) of Asia Provenance Region, but Also there are the crossing instances of part, which indicates the affiliation and hereditary variation situation between each kind of almond substantially.
The beneficial effects of the present invention are：It is contemplated that China's amygdalae plant germplasm is studied by SSR molecular marker Each kind including introduction, the affiliation between kind.Establish a set of easy, quick, reliable Amygdalus plant germplasm Identification technology system is applied to the identification of the category germ plasm resource, carries out germplasm and kind rights protection, is educated to be further excellent Kind purpose provides identification technology system.Interests for safeguarding breeder and the producer, ensure that the industry develops in a healthy way, have weight Want and the meaning of reality.
Brief description of the drawings
Fig. 1 builds the SSR dendrograms of 55 parts of almond platymisciums, numbering corresponding table 1 wherein in figure for UPGMA methods of the present invention；
Fig. 2 is the SSR dendrograms that Wagner parsimony principles of the present invention build 55 parts of almond platymisciums, numbers and corresponds to wherein in figure Table 1.
Embodiment
The invention will be further described below in conjunction with the accompanying drawings.
Embodiment
Prepare Amygdalus plant germplasm material：Pick up from Xinjiang, China Tacheng and the Xinjiang of Altay Prefecture's different distributions population Wild 5 parts of almond is (random respectively in the 5 different distributions population centers that make people rich to select in Burqin, Habahe County, Tacheng, Tuoli Take, numbering is Xinjiang Wild Almonds 1, No. 2, No. 3, No. 4, No. 5 single plants respectively)；Pick up from Xinjiang, China South Sinkiang Kaxgar Prefecture Ying Ji Husky and Shache County 30 parts of Xinjiang place cultivation almond kind；Pick up from Xinjiang, China South Sinkiang Kaxgar Prefecture Shache County and add profit from the U.S. Excellent 16 parts of the cultivation almond kind that Fu Niya states are introduced；Pick up from mongolian amygdalus seed, the west in Xinjiang Academy of Forestry's forest fruit resource garden 4 parts of the germ plasm resource of the almond platymiscium such as health almond, Amygdalus pedunculata and flowering plum；55 parts of sample materials are amounted to, are covered substantially The germ plasm resource of almond platymiscium, in early spring, choose the new fresh and tender of healthy and strong, no disease and pests harm almond platymiscium 55 parts of samples Leaf, every part of sample are saved backup (table 1) with being put into after liquid nitrogen frozen in -70 DEG C of ultra low temperature freezers of temperature；
1. amygdalae plant germplasm material information of table
Note：★ Chinese varieties, ◆ external kind, ● kind；
The extraction of Amygdalus plant germplasm DNA：1000 μ L are rapidly added, 65 DEG C of preheatings of temperature contain 2% cetyl three Methyl bromide ammonium, 1.4M sodium chloride, 20mM tetraacethyl diamino-vinyls, 100mM trishydroxymethylaminomethanes, the buffering of pH8.0 The buffer solution of liquid and 8% mercaptoethanol, gently shakes up；Warm bath 60min in 65 DEG C of water-baths of temperature is placed in, therebetween every 10min Gently teetertotter and shake up 1 time, solution is fully cracked；Centrifuged using desk centrifuge, 21 DEG C of temperature, rotating speed 10000r/ Min, time 15min, Aspirate supernatant, supernatant mean allocation is transferred in two other 1500 new μ L centrifuge tube, wherein Contain 480 μ L of supernatant liquid in each centrifuge tube；Isometric chloroform will be added in each centrifuge tube containing supernatant:Isoamyl Alcohol=24:1, gently teetertotter and shake up, centrifuge, 21 DEG C, rotating speed 10000r/min, time 15min of temperature, Aspirate supernatant, Supernatant in each centrifuge tube is merged, is transferred in another 1500 new μ L centrifuge tube, 600 μ L of supernatant is contained in centrifuge tube Liquid；Isometric chloroform will be added in centrifuge tube equipped with 600 μ L of supernatant liquid:Isoamyl alcohol=24:1, gently teetertotter and shake up, Centrifugation, 21 DEG C, rotating speed 10000r/min, time 15min of temperature, then Aspirate supernatant, it is new to be transferred to another by supernatant In 1500 μ L centrifuge tubes, 400 μ L of supernatant liquid are contained in centrifuge tube；2 times of volumes will be added in centrifuge tube equipped with 400 μ L of supernatant liquid Freezing absolute ethyl alcohol, be put into -20 DEG C of deep freezers of temperature, time 30min, centrifugation, 21 DEG C, rotating speed 10000r/min of temperature, There is clear milky DNA white cotton fibers precipitation after time 15min, remove upper liquid；Milky DNA white cotton fibers are precipitated with dense again Spend and washed 2-3 times for 70% ethanol, after natural air drying, be dissolved in 50 μ L and contain 10mM trishydroxymethylaminomethanes, 1mM tetraacethyls Diamino-vinyl, in the buffer solution of pH8.0, -20 DEG C of refrigerators of temperature preserve；
DNA purity is detected with 1.5% agarose gel electrophoresis；The obtained DNA concentrations of step b are diluted to 50ng/ μ l Afterwards, it is placed in spare in -20 DEG C of refrigerators of temperature；
SSR primer screenings：
Searched for according on ncbi database, from from 100 pairs of SSR primers of tone fruit trees crop by primary dcreening operation and Secondary screening, finally selects that 10 pairs of product master tapes are obvious and the preferable primer of polymorphism carries out SSR microsatellite molecular marker analyses, its Middle SSR 10 to mark for：
Mark 1BPPCT002, its sense primer 5 ' TCGACAGCTTGATCTTGACC, anti-sense primer 5 ' CAATGCCTACGGAGATAAAAGAC；
Mark 2BPPCT004, its sense primer 5 ' CTGAGTGATCCATTTGCAGG, anti-sense primer 5 ' AGGGCATCTAGACCTCATTGTT；
Mark 3BPPCT032, its sense primer 5 ' TTAAGCCACAACATCCATGAT, anti-sense primer 5 ' AATGGTCTAAGGAGCACACG；
Mark 4BPPCT036, its sense primer 5 ' AAGCAAAGTCCATAAAAACGC, anti-sense primer 5 ' GGACGAAGACGCTCCATT；
Mark 5BPPCT040, its sense primer 5 ' ATGAGGACGTGTCTGAATGG, anti-sense primer 5 ' AGCCAAACCCCTCTTATACG；
Mark 6Pchgms1, its sense primer 5 ' GGGTAAATATGCCCATTGTGCAATC, anti-sense primer 5 ' GGATCATTGAACTACGTCAATCCTC；
Mark 7Pchgms3, its sense primer 5 ' ACGGTATGTCCGTACACTCTCCATG, anti-sense primer 5 ' CAACCTGTGATTGCTCCTATTAAAC；
Mark 8Pchgms10, its sense primer 5 ' CGTCACGCATCCTTTCATTT, anti-sense primer 5 ' GACACCTCCATTTGTATCAAAGC；
Mark 9Pchgms11, its sense primer 5 ' TTGAGGCCCACTTATTAGCC, anti-sense primer 5 ' CCCCCATTATTCAAACTTCTG；
Mark 10Pchgms21, its sense primer 5 ' ACCACCATTTTGGCTCTCTG, anti-sense primer 5 ' CATGCAACCCAAAACCATCT；
Table 2.SSR primer sequences and title
Mono- pcr amplification reactions of SSR, electrophoresis detection：20 μ l reaction systems of SSR-PCR reaction systems include：10 × Taq enzyme is matched somebody with somebody Cover buffer solution, 1.5mmol/L MgCl2, 0.25mmol/L dNTPS, SSR upstream and downstream primer are respectively 0.2mol/ μ l, 0.5UTaq Archaeal dna polymerase, DNA profiling 30ng, corresponding amplification program are：94 DEG C of pre-degenerations of temperature, 4min, followed by 94 DEG C of denaturation of temperature 60s, 55 DEG C of renaturation 40s of temperature, 72 DEG C of extension 60s of temperature, after totally 34 circulations, 72 DEG C of extension 8min polishings of temperature, amplification production 6 times of Loadingbuffer2.0 μ l are added in thing, take 2.5 μ l loadings, are examined with 8% non denatured polyacrylamine gel electrophoresis Survey, silver staining colour developing；
Statistical analysis：In not weighting matched group algorithmic approach (unweighted pair group method with Arithmetic mean, UPGMA) algorithm cluster analysis NTSYS-pc 2.10e (numerical taxonomy and Multivariate analysis system, NTSYS) software progress data analysis.Select clear and legible electrophoresis band, sample There is band to be then denoted as 1, no band is then denoted as 0, builds two condition data matrix；In order to further compare and study Amygdalus plant germplasm money The hereditary difference of source difference inter-species and different cultivars, S is sought to original matrix with SimQual programsSMSimilarity factor and genetic distance Matrix, and carry out cluster analysis with the UPGMA methods in SAHN programs.Based on Wagner parsimony principles (Wagner Parsimony) Cluster analysis SEQBOOT, MIX in PHYLIP3.65 (Phylogeny Inference Package) software kit, The programs such as CONSENSE make uniformity tree graph, and with the brief figure of TreeView1.6 Program Generatings, build molecular evolution system Tree；
As a result with analysis：
By extracting the DNA of various Amygdalus plant germplasm materials in the method for the invention, band clearly becomes clear, no drop Phenomenon is solved, DNA mass integrality is good, and spectrophotometer detection finds OD260/OD280Between 1.8-2.0, concentration exists ratio 350-700ng/ μ l, can meet the requirement of SSR-PCR；
Use the strong primer pair Amygdalus plant germplasm of 10 pairs of polymorphisms in 100 pairs of SSR primers screened from kernel approaches Material, including the genomic DNA of domestic and international cultivar and not of the same race 55 individual specimens carry out SSR-PCR amplifications, obtain Preferable amplification.10 pairs of primer coamplifications go out 63 amplified bands, wherein 60, polymorphic band, polymorphic rate 95.2%；
Hereditary difference and Genetic relationship：The result of calculation of genetic similarty and genetic distance shows：Almond platymiscium Germplasm is not of the same race and different cultivars between genotypic difference it is different:In all almond cultivars for trying, each genotype Between genetic similarty it is universal larger compared with inter-species, and it is also larger to make a variation, wherein the heredity of late rich kind and short rich kind Similarity factor maximum (0.9600), affiliation is nearest between illustrating both, and the yellow bar denier kind of sharp mouth and Thompson kinds Genetic similarity it is minimum (0.4500), reflection between both affiliation it is farthest；
To including 47 almond cultivars (home and abroad)；Xinjiang Wild Almonds No. 1, No. 2, No. 3, No. 4 and No. 5；Long handle Amygdalus plant germplasm inter-species including almond, mongolian amygdalus seed, Xikang almond, flowering plum etc. carries out analysis of genetic diversity, hair Existing genetic similarity index between almond cultivar and Xinjiang Wild Almonds No. 1, No. 2, No. 3, No. 4 and No. 5 0.5067~ Between 0.7067, average value 0.6067, more than between almond cultivar and Amygdalus pedunculata, mongolian amygdalus seed and Xikang almond Similitude (0.4400~0.6133, average 0.5266).That is the parent between almond cultivar and Xinjiang Wild Almonds Edge relation than the affiliation between Amygdalus pedunculata, mongolian amygdalus seed, Xikang almond closer to；
Between 0.4133-0.5433, average is genetic similarity between 5 kinds of plants of flowering plum kind and Amygdalus 0.4783, affiliation is farther, and therefore, when cluster is gathered in outer most edge；
Cluster analysis：Likeness coefficient between 55 samples is formed matrix and carries out cluster analysis structure dendrogram, is based on UPGMA methods (Fig. 1) are very similar (Fig. 2) with Wagner parsimony principles cluster result.The result shows that：Almond inside and outside tested host country Cultivar gathers when similarity factor is less than 0.68 for a major class, shows that almond cultivar has similar genetic background；
At the same time with almond cultivar similar in amygdalae plant species be divided into almond under the similarity factor (0.68) Cultivar group (domestic and international cultivar), Xinjiang Wild Almonds group (Xinjiang Wild Almonds No. 1, No. 2, No. 3, No. 4 and No. 5), almond Kind group (Amygdalus pedunculata, mongolian amygdalus seed, Xikang almond) and flowering plum.The result basic explanation each almond kind or kind The common point and variation diversity of genotype genetic background.
In cultivar group, the kind from California, USA Provenance Region can largely be classified as one kind, lead in addition Together with easily sorting out for the variety protection from areal (Xinjiang Yingjisha and Shache County) of Asia Provenance Region, but Also there are the crossing instances (table 5) of part：
Kind and interracial genetic similarty and genetic distance table between 5 almond platymiscium of table
Colony 1 2 3 4 5 6 7 8 9 10 11
1 **** 0.8267 0.7733 0.7600 0.7467 0.6533 0.7467 0.7467 0.6600 0.8533 0.7200
2 0.1904 **** 0.7067 0.7200 0.7067 0.6667 0.7600 0.7867 0.6933 0.7333 0.6533
3 0.2570 0.3472 **** 0.8000 0.7867 0.7200 0.7067 0.6800 0.8000 0.7600 0.7333
4 0.2744 0.3285 0.2231 **** 0.7200 0.7867 0.6933 0.6400 0.7333 0.6933 0.7467
5 0.2921 0.3472 0.2400 0.3285 **** 0.8000 0.7867 0.7067 0.6933 0.7333 0.7867
6 0.4257 0.4055 0.3285 0.2400 0.2231 **** 0.8267 0.6667 0.6267 0.6667 0.6933
7 0.2921 0.2744 0.3472 0.3662 0.2400 0.1904 **** 0.7867 0.6933 0.7067 0.6533
8 0.2921 0.2400 0.3857 0.4463 0.3472 0.4055 0.2400 **** 0.7467 0.7067 0.6800
9 0.2744 0.3662 0.2231 0.3102 0.3662 0.4673 0.3662 0.2921 **** 0.8000 0.7200
10 0.1586 0.3102 0.2744 0.3662 0.3102 0.4055 0.3472 0.3472 0.2231 **** 0.7867
11 0.3285 0.4257 0.3102 0.2921 0.2400 0.3662 0.4257 0.3857 0.3285 0.2400 ****
12 0.3102 0.4463 0.2570 0.3857 0.2231 0.3102 0.3662 0.4055 0.2066 0.1904 0.1904
13 0.3285 0.3857 0.2744 0.3662 0.2400 0.2921 0.3472 0.4257 0.2570 0.2400 0.2066
14 0.2921 0.3857 0.2066 0.1904 0.3857 0.3285 0.3857 0.4673 0.2231 0.3102 0.3102
15 0.2400 0.2570 0.3662 0.3857 0.4055 0.4257 0.4463 0.3662 0.3102 0.2921 0.3662
16 0.3102 0.2921 0.3285 0.4673 0.4055 0.4673 0.3285 0.2921 0.3857 0.4055 0.4888
17 0.3472 0.4463 0.4463 0.4257 0.5798 0.4257 0.5798 0.4463 0.5108 0.4055 0.5333
18 0.3662 0.5108 0.2744 0.3285 0.2744 0.5333 0.4673 0.3857 0.2921 0.4257 0.2400
19 0.3472 0.4055 0.3662 0.3472 0.3285 0.3102 0.4888 0.2921 0.2744 0.3285 0.2921
20 0.3472 0.4888 0.3285 0.2400 0.4463 0.3857 0.4463 0.3662 0.2066 0.3285 0.3285
21 0.4055 0.5563 0.3857 0.4888 0.4257 0.4055 0.4257 0.4257 0.4463 0.3857 0.3472
22 0.4888 0.4673 0.3472 0.4463 0.3102 0.3285 0.4257 0.4257 0.4055 0.3857 0.3857
23 0.2400 0.2921 0.3285 0.3102 0.4055 0.5563 0.3662 0.3285 0.2744 0.3285 0.3662
24 0.3472 0.3662 0.3285 0.3857 0.3662 0.3857 0.4888 0.3285 0.3472 0.3285 0.3285
25 0.4888 0.5563 0.3102 0.2921 0.3857 0.3662 0.4673 0.6039 0.3285 0.3857 0.3857
26 0.5563 0.6286 0.5333 0.5563 0.4888 0.3857 0.4055 0.5333 0.5563 0.6286 0.5798
27 0.4055 0.5563 0.3102 0.2921 0.3857 0.4463 0.4257 0.5108 0.2921 0.3857 0.3472
28 0.4888 0.4673 0.4673 0.3662 0.3857 0.4463 0.5108 0.4673 0.3662 0.5108 0.3857
29 0.6039 0.4888 0.4888 0.5563 0.6286 0.5563 0.4888 0.4463 0.4257 0.7340 0.7340
30 0.3102 0.3285 0.4055 0.3857 0.4888 0.5563 0.3285 0.4463 0.3857 0.3662 0.4055
31 0.3662 0.5108 0.2744 0.2921 0.3857 0.4888 0.4673 0.4673 0.2570 0.3102 0.3102
32 0.4257 0.4888 0.4463 0.3857 0.5798 0.6539 0.5798 0.4463 0.3857 0.5333 0.4463
33 0.5563 0.5798 0.5333 0.5563 0.4888 0.4257 0.4888 0.2570 0.4673 0.5333 0.4463
34 0.4257 0.5798 0.4463 0.3857 0.6286 0.4673 0.4888 0.4463 0.4673 0.5798 0.4888
35 0.3857 0.4888 0.5333 0.5108 0.4463 0.4257 0.3285 0.3662 0.4257 0.4888 0.4055
36 0.4257 0.6799 0.4888 0.5108 0.5798 0.6039 0.5798 0.5333 0.3472 0.4055 0.3662
37 0.7066 0.7340 0.5798 0.4257 0.3472 0.3102 0.6286 0.4673 0.5108 0.5798 0.5798
38 0.4673 0.5333 0.4463 0.4673 0.6799 0.4257 0.5333 0.3662 0.5563 0.6286 0.5798
39 0.4257 0.5333 0.4055 0.4673 0.5798 0.4673 0.5798 0.4055 0.4673 0.4888 0.4888
40 0.4055 0.5108 0.4673 0.4888 0.5563 0.4888 0.6039 0.5563 0.5798 0.4673 0.4257
41 0.4257 0.4463 0.5333 0.5563 0.6286 0.5563 0.4888 0.3285 0.6039 0.6799 0.6286
42 0.2400 0.3285 0.3662 0.4673 0.3662 0.5108 0.4055 0.3662 0.4673 0.4055 0.4055
43 0.4888 0.6039 0.4257 0.4463 0.6539 0.4888 0.6039 0.4673 0.5798 0.6539 0.5108
44 0.3857 0.6286 0.4888 0.4673 0.6799 0.6539 0.7340 0.5798 0.4673 0.4055 0.4055
45 0.3662 0.4257 0.3857 0.4888 0.4673 0.6286 0.5108 0.3857 0.4888 0.5563 0.4673
46 0.3285 0.4673 0.4257 0.4055 0.4257 0.5333 0.5563 0.4257 0.4463 0.5563 0.3472
47 0.7340 0.8835 0.7066 0.7911 0.9502 0.8518 0.7621 0.6539 0.5798 0.7621 0.8210
48 0.5333 0.6039 0.6039 0.6799 0.6539 0.7911 0.6539 0.5563 0.5333 0.6039 0.5108
49 0.5563 0.5798 0.6286 0.6039 0.6286 0.5108 0.4888 0.5333 0.7621 0.5798 0.4888
50 0.5798 0.6539 0.6039 0.4888 0.6539 0.6286 0.6039 0.6539 0.6286 0.6039 0.5563
51 0.5798 0.4673 0.4673 0.4463 0.5108 0.4888 0.5563 0.6039 0.4055 0.3857 0.4673
52 0.6039 0.5333 0.4463 0.5108 0.5798 0.5108 0.5333 0.7340 0.4673 0.4888 0.4888
53 0.4888 0.4673 0.4673 0.4055 0.5563 0.4888 0.4673 0.6039 0.4463 0.3472 0.4257
54 0.6286 0.5563 0.4673 0.4888 0.5108 0.4463 0.4673 0.6039 0.4055 0.4673 0.4257
55 0.3857 0.4055 0.4888 0.6039 0.5333 0.6539 0.4888 0.4888 0.3857 0.3662 0.4888
Continuous upper table
Colony 12 13 14 15 16 17 18 19 20 21 22
1 0.7333 0.7200 0.7467 0.6867 0.7333 0.7067 0.6933 0.7067 0.7067 0.6667 0.6133
2 0.6400 0.6800 0.6800 0.7733 0.7467 0.6400 0.6000 0.6667 0.6133 0.5733 0.6267
3 0.7733 0.7600 0.8133 0.6933 0.7200 0.6400 0.7600 0.6933 0.7200 0.6800 0.7067
4 0.6800 0.6933 0.8267 0.6800 0.6267 0.6533 0.7200 0.7067 0.7867 0.6133 0.6400
5 0.8000 0.7867 0.6800 0.6667 0.6667 0.5600 0.7600 0.7200 0.6400 0.6533 0.7333
6 0.7333 0.7467 0.7200 0.6533 0.6267 0.6533 0.5867 0.7333 0.6800 0.6667 0.7200
7 0.6933 0.7067 0.6800 0.6400 0.7200 0.5600 0.6267 0.6133 0.6400 0.6533 0.6533
8 0.6667 0.6533 0.6267 0.6933 0.7467 0.6400 0.6800 0.7467 0.6933 0.6533 0.6533
9 0.8133 0.7733 0.8000 0.6333 0.6800 0.6000 0.7467 0.7600 0.8133 0.6400 0.6667
10 0.8267 0.7867 0.7333 0.7467 0.6667 0.6667 0.6533 0.7200 0.7200 0.6800 0.6800
11 0.8267 0.8133 0.7333 0.6933 0.6133 0.5867 0.7867 0.7467 0.7200 0.7067 0.6800
12 **** 0.9600 0.8533 0.7867 0.6533 0.6533 0.7733 0.8133 0.7867 0.8000 0.8000
13 0.0408 **** 0.8667 0.7733 0.6667 0.6400 0.7333 0.8000 0.8000 0.7867 0.8133
14 0.1586 0.1431 **** 0.7200 0.6400 0.6667 0.7600 0.7467 0.8533 0.7333 0.7067
15 0.2400 0.2570 0.3285 **** 0.6800 0.7600 0.6400 0.8133 0.7067 0.6667 0.7467
16 0.4257 0.4055 0.4463 0.3857 **** 0.7333 0.6400 0.6267 0.6800 0.6933 0.6933
17 0.4257 0.4463 0.4055 0.2744 0.3102 **** 0.5867 0.7600 0.6800 0.7200 0.7733
18 0.2570 0.3102 0.2744 0.4463 0.4463 0.5333 **** 0.7200 0.6933 0.7333 0.6533
19 0.2066 0.2231 0.2921 0.2066 0.4673 0.2744 0.3285 **** 0.8133 0.6933 0.8267
20 0.2400 0.2231 0.1586 0.3472 0.3857 0.3857 0.3662 0.2066 **** 0.7200 0.7467
21 0.2231 0.2400 0.3102 0.4055 0.3662 0.3285 0.3102 0.3662 0.3285 **** 0.7333
22 0.2231 0.2066 0.3472 0.2921 0.3662 0.2570 0.4257 0.1904 0.2921 0.3102 ****
23 0.3472 0.3285 0.3285 0.3102 0.2400 0.3857 0.3285 0.3472 0.2400 0.3662 0.2921
24 0.2400 0.2231 0.2921 0.1744 0.3472 0.2066 0.4055 0.1128 0.2066 0.3285 0.1278
25 0.2231 0.2066 0.2066 0.4463 0.4463 0.4463 0.3472 0.3662 0.2231 0.2744 0.2066
26 0.3857 0.4463 0.5333 0.5108 0.4257 0.3857 0.4055 0.4673 0.4673 0.3285 0.3285
27 0.2921 0.3472 0.2400 0.4055 0.4888 0.5333 0.2400 0.3662 0.2231 0.3857 0.3472
28 0.4055 0.3857 0.4257 0.4463 0.4055 0.5333 0.4673 0.4055 0.3285 0.5563 0.3857
29 0.5563 0.5798 0.4463 0.5108 0.3102 0.4673 0.4463 0.5563 0.4257 0.4888 0.4888
30 0.3857 0.3285 0.3662 0.3472 0.2744 0.5563 0.4055 0.5108 0.3857 0.4463 0.4463
31 0.2231 0.2744 0.2400 0.4055 0.4888 0.4463 0.1431 0.3662 0.3285 0.2400 0.3472
32 0.4257 0.4463 0.4055 0.2744 0.4673 0.4257 0.4055 0.3102 0.3857 0.4055 0.4055
33 0.5563 0.6286 0.5798 0.6039 0.4257 0.4673 0.5333 0.3857 0.4257 0.4463 0.4888
34 0.4673 0.4463 0.3285 0.3857 0.4257 0.3472 0.4888 0.4257 0.2400 0.2921 0.5333
35 0.4257 0.3662 0.4888 0.4257 0.4257 0.4673 0.4888 0.4257 0.3857 0.4055 0.5798
36 0.2744 0.3285 0.3285 0.3472 0.5108 0.3472 0.4055 0.3857 0.3102 0.2921 0.4463
37 0.4673 0.4673 0.7340 0.4673 0.5333 0.6286 0.3472 0.5108 0.6286 0.4257 0.3102
38 0.5108 0.5333 0.4055 0.3472 0.4257 0.2744 0.5333 0.3472 0.4257 0.4055 0.5333
39 0.4673 0.4888 0.4055 0.3857 0.5108 0.3472 0.4888 0.3472 0.4257 0.5333 0.5333
40 0.3662 0.3102 0.3102 0.3662 0.4463 0.2570 0.4673 0.3285 0.3662 0.3472 0.4257
41 0.7066 0.6799 0.5798 0.4257 0.3102 0.3102 0.5798 0.4673 0.5108 0.5798 0.4888
42 0.4673 0.4463 0.4888 0.3472 0.3472 0.3472 0.4055 0.3857 0.5108 0.5798 0.3662
43 0.5333 0.5108 0.4257 0.4055 0.3662 0.2921 0.4673 0.4055 0.4055 0.4257 0.5108
44 0.3472 0.3662 0.3662 0.3857 0.4257 0.2400 0.4463 0.3857 0.3102 0.3285 0.4055
45 0.5333 0.5108 0.4673 0.4463 0.3285 0.3662 0.4257 0.4888 0.4463 0.6039 0.4673
46 0.3662 0.3857 0.3857 0.3285 0.4055 0.3662 0.2744 0.3662 0.4463 0.3857 0.5108
47 0.7340 0.7066 0.7066 0.8518 0.5333 0.7340 0.7066 0.8518 0.6286 0.6039 0.8210
48 0.5333 0.5108 0.6039 0.5798 0.4888 0.6286 0.5108 0.6286 0.5333 0.5563 0.6039
49 0.7066 0.6286 0.6799 0.7621 0.6039 0.6039 0.6286 0.7066 0.6539 0.5333 0.6799
50 0.6286 0.6539 0.5563 0.6799 0.5798 0.4888 0.4673 0.7340 0.5798 0.4257 0.6539
51 0.3662 0.3472 0.4257 0.4888 0.5333 0.5333 0.5563 0.4888 0.3662 0.4673 0.5108
52 0.3857 0.3662 0.3662 0.5108 0.5563 0.6039 0.4888 0.6539 0.4673 0.4463 0.5798
53 0.4463 0.4257 0.4257 0.5333 0.5798 0.6286 0.5563 0.5798 0.4055 0.4673 0.5563
54 0.3662 0.3472 0.3857 0.5798 0.6286 0.6286 0.4257 0.5333 0.4055 0.4257 0.5563
55 0.4257 0.4463 0.4888 0.4673 0.3472 0.5563 0.5333 0.6539 0.4673 0.5798 0.6799
Continuous upper table
Colony 23 24 25 26 27 28 29 30 31 32 33
1 0.7867 0.7067 0.6133 0.5733 0.6667 0.6133 0.5467 0.7333 0.6933 0.6533 0.5733
2 0.7467 0.6933 0.5733 0.5333 0.5733 0.6267 0.6133 0.7200 0.6000 0.6133 0.5600
3 0.7200 0.7200 0.7333 0.5867 0.7333 0.6267 0.6133 0.6667 0.7600 0.6400 0.5867
4 0.7333 0.6800 0.7467 0.5733 0.7467 0.6933 0.5733 0.6800 0.7467 0.6800 0.5733
5 0.6667 0.6933 0.6800 0.6133 0.6800 0.6800 0.5333 0.6133 0.6800 0.5600 0.6133
6 0.5733 0.6800 0.6933 0.6800 0.6400 0.6400 0.5733 0.5733 0.6133 0.5200 0.6533
7 0.6933 0.6133 0.6267 0.6667 0.6533 0.6000 0.6133 0.7200 0.6267 0.5600 0.6133
8 0.7200 0.7200 0.5467 0.5867 0.6000 0.6267 0.6400 0.6400 0.6267 0.6400 0.7733
9 0.7600 0.7067 0.7200 0.5733 0.7467 0.6933 0.6533 0.6800 0.7733 0.6800 0.6267
10 0.7200 0.7200 0.6800 0.5333 0.6800 0.6000 0.4800 0.6933 0.7333 0.5867 0.5867
11 0.6933 0.7200 0.6800 0.5600 0.7067 0.6800 0.4800 0.6667 0.7333 0.6400 0.6400
12 0.7067 0.7867 0.8000 0.6800 0.7467 0.6667 0.5733 0.6800 0.8000 0.6533 0.5733
13 0.7200 0.8000 0.8133 0.6400 0.7067 0.6800 0.5600 0.7200 0.7600 0.6400 0.5333
14 0.7200 0.7467 0.8133 0.5867 0.7867 0.6533 0.6400 0.6933 0.7867 0.6667 0.5600
15 0.7333 0.8400 0.6400 0.6000 0.6667 0.6400 0.6000 0.7067 0.6667 0.7600 0.5467
16 0.7867 0.7067 0.6400 0.6533 0.6133 0.6667 0.7333 0.7600 0.6133 0.6267 0.6533
17 0.6800 0.8133 0.6400 0.6800 0.5867 0.5867 0.6267 0.5733 0.6400 0.6533 0.6267
18 0.7200 0.6667 0.7067 0.6667 0.7867 0.6267 0.6400 0.6667 0.8667 0.6667 0.5867
19 0.7067 0.8933 0.6933 0.6267 0.6933 0.6667 0.5733 0.6000 0.6933 0.7333 0.6800
20 0.7867 0.8133 0.8000 0.6267 0.8000 0.7200 0.6533 0.6800 0.7200 0.6800 0.6533
21 0.6933 0.7200 0.7600 0.7200 0.6800 0.5733 0.6133 0.6400 0.7867 0.6667 0.6400
22 0.7467 0.8800 0.8133 0.7200 0.7067 0.6800 0.6133 0.6400 0.7067 0.6667 0.6133
23 **** 0.7867 0.7467 0.6533 0.6933 0.7467 0.6800 0.8133 0.7200 0.7333 0.6000
24 0.2400 **** 0.7200 0.6533 0.6667 0.7200 0.6267 0.6800 0.6667 0.7067 0.6533
25 0.2921 0.3285 **** 0.6933 0.8133 0.7067 0.6133 0.6933 0.7867 0.6400 0.5867
26 0.4257 0.4257 0.3662 **** 0.6667 0.6400 0.7600 0.5733 0.6667 0.6000 0.6000
27 0.3662 0.4055 0.2066 0.4055 **** 0.6533 0.6133 0.6933 0.7867 0.6667 0.5867
28 0.2921 0.3285 0.3472 0.4463 0.4257 **** 0.7200 0.6933 0.5733 0.6133 0.6667
29 0.3857 0.4673 0.4888 0.2744 0.4888 0.3285 **** 0.6267 0.6400 0.6000 0.6267
30 0.2066 0.3857 0.3662 0.5563 0.3662 0.3662 0.4673 **** 0.6933 0.6800 0.5467
31 0.3285 0.4055 0.2400 0.4055 0.2400 0.5563 0.4463 0.3662 **** 0.6667 0.4800
32 0.3102 0.3472 0.4463 0.5108 0.4055 0.4888 0.5108 0.3857 0.4055 **** 0.6000
33 0.5108 0.4257 0.5333 0.5108 0.5333 0.4055 0.4673 0.6039 0.7340 0.5108 ****
34 0.4257 0.3857 0.4055 0.4673 0.3662 0.4888 0.3857 0.4673 0.4888 0.3472 0.3472
35 0.4257 0.4673 0.5798 0.5108 0.5333 0.5333 0.5563 0.3857 0.4888 0.4257 0.4673
36 0.4257 0.3857 0.3662 0.4257 0.4463 0.4888 0.5108 0.5108 0.3285 0.3472 0.4673
37 0.5563 0.3857 0.6286 0.5563 0.5333 0.6286 0.5108 0.5563 0.5798 0.3472 0.3472
38 0.6039 0.4257 0.6799 0.6039 0.4888 0.5798 0.5563 0.5563 0.4888 0.3857 0.3857
39 0.6039 0.4257 0.6799 0.6039 0.4888 0.5798 0.5563 0.5563 0.4888 0.3857 0.3857
40 0.4888 0.2570 0.4257 0.5333 0.5563 0.4673 0.5798 0.4463 0.4673 0.4888 0.4888
41 0.4257 0.4257 0.6799 0.4257 0.5333 0.5798 0.3472 0.5108 0.6286 0.4257 0.3857
42 0.3472 0.3472 0.6286 0.5563 0.5333 0.5333 0.6039 0.3472 0.4055 0.3857 0.6039
43 0.5333 0.4055 0.5563 0.5333 0.5108 0.5563 0.4463 0.4888 0.5108 0.4055 0.4055
44 0.4257 0.3472 0.4055 0.4673 0.4463 0.4463 0.5563 0.4673 0.4055 0.3857 0.5108
45 0.4055 0.4055 0.6039 0.6286 0.4673 0.5108 0.4888 0.4055 0.5108 0.4055 0.4888
46 0.4055 0.4055 0.5108 0.4888 0.4673 0.4257 0.4888 0.4463 0.3857 0.3285 0.4888
47 0.6286 0.7911 0.7066 0.7340 0.8835 0.6539 0.7340 0.6286 0.7066 0.7340 0.5798
48 0.4888 0.5798 0.6039 0.6799 0.7066 0.7066 0.5798 0.5333 0.4673 0.5798 0.6799
49 0.6539 0.6539 0.7340 0.6539 0.7340 0.7911 0.6539 0.6539 0.5333 0.8210 0.5563
50 0.6286 0.6286 0.5563 0.5798 0.5108 0.7066 0.6286 0.6286 0.4257 0.6799 0.5798
51 0.4888 0.4888 0.3857 0.6286 0.6039 0.5563 0.6799 0.4888 0.4257 0.5798 0.6799
52 0.7066 0.6039 0.4055 0.5563 0.4055 0.6799 0.6539 0.5108 0.3662 0.6539 0.8835
53 0.4888 0.5333 0.3857 0.6286 0.4673 0.6539 0.7911 0.4055 0.3472 0.5333 0.7340
54 0.6286 0.5798 0.4257 0.5798 0.4257 0.6539 0.6799 0.5333 0.3857 0.5798 0.6799
55 0.3857 0.5563 0.5333 0.6039 0.6286 0.5798 0.6039 0.4257 0.5333 0.6539 0.6539
Continuous upper table
Colony 34 35 36 37 38 39 40 41 42 43 44
1 0.6533 0.6800 0.6533 0.6533 0.6267 0.6533 0.6667 0.6533 0.7867 0.6133 0.6800
2 0.5600 0.6133 0.5067 0.6400 0.5867 0.5867 0.6000 0.6400 0.7200 0.5467 0.5333
3 0.6400 0.5867 0.6133 0.5867 0.6400 0.6667 0.6267 0.5867 0.6933 0.6533 0.6133
4 0.6800 0.6000 0.6000 0.5467 0.6267 0.6267 0.6133 0.5733 0.6267 0.6400 0.6267
5 0.5333 0.6400 0.5600 0.4800 0.5067 0.5600 0.5733 0.5333 0.6933 0.5200 0.5067
6 0.6267 0.6533 0.5467 0.5733 0.6533 0.6267 0.6133 0.5733 0.6000 0.6133 0.5200
7 0.6133 0.7200 0.5600 0.6133 0.5867 0.5600 0.5467 0.6133 0.6667 0.5467 0.4500
8 0.6400 0.6933 0.5867 0.7467 0.6933 0.6667 0.5733 0.7200 0.6933 0.6267 0.5600
9 0.6267 0.6533 0.7067 0.6000 0.5733 0.6267 0.5600 0.5467 0.6267 0.5600 0.6267
10 0.5600 0.6133 0.6667 0.5333 0.5333 0.6133 0.6267 0.5067 0.6667 0.5200 0.6667
11 0.6133 0.6667 0.6933 0.4800 0.5600 0.6133 0.6533 0.5333 0.6667 0.6000 0.6667
12 0.6267 0.6533 0.7600 0.4933 0.6000 0.6267 0.6933 0.4933 0.6267 0.5867 0.7067
13 0.6400 0.6933 0.7200 0.4800 0.5867 0.6133 0.7333 0.5067 0.6400 0.6000 0.6933
14 0.7200 0.6133 0.7200 0.5600 0.6667 0.6667 0.7333 0.5600 0.6133 0.6533 0.6933
15 0.6800 0.6533 0.7067 0.6533 0.7067 0.6800 0.6933 0.6533 0.7067 0.6667 0.6800
16 0.6533 0.6533 0.6000 0.7067 0.6533 0.6000 0.6400 0.7333 0.7067 0.6933 0.6533
17 0.7067 0.6267 0.7067 0.7333 0.7600 0.7067 0.7733 0.7333 0.7067 0.7467 0.7867
18 0.6133 0.6133 0.6667 0.5333 0.5867 0.6133 0.6267 0.5600 0.6667 0.6267 0.6400
19 0.6533 0.6533 0.6800 0.6267 0.7067 0.7067 0.7200 0.6267 0.6800 0.6667 0.6800
20 0.7867 0.6800 0.7333 0.6000 0.6533 0.6533 0.6933 0.6000 0.6000 0.6667 0.7333
21 0.7467 0.6667 0.7467 0.5600 0.6667 0.5867 0.7067 0.5600 0.5600 0.6533 0.7200
22 0.5867 0.5600 0.6400 0.5600 0.5867 0.5867 0.6533 0.6133 0.6933 0.6000 0.6667
23 0.6533 0.6533 0.6533 0.6267 0.5733 0.5467 0.6133 0.6533 0.7067 0.5867 0.6533
24 0.6800 0.6267 0.6800 0.6267 0.6800 0.6533 0.7733 0.6533 0.7067 0.6667 0.7067
25 0.6667 0.5600 0.6933 0.4800 0.5333 0.5067 0.6533 0.5067 0.5333 0.5733 0.6667
26 0.6267 0.6000 0.6533 0.6267 0.5733 0.5467 0.5867 0.6533 0.5733 0.5867 0.6267
27 0.6933 0.5867 0.6400 0.5867 0.5867 0.6133 0.5733 0.5867 0.5867 0.6000 0.6400
28 0.6133 0.5867 0.6133 0.5333 0.5333 0.5600 0.6267 0.5600 0.5867 0.5733 0.6400
29 0.6800 0.5733 0.6000 0.7067 0.6000 0.5733 0.5600 0.7067 0.5467 0.6400 0.5733
30 0.6267 0.6800 0.6000 0.6000 0.5733 0.5733 0.6400 0.6000 0.7067 0.6133 0.6267
31 0.6133 0.6133 0.7200 0.5333 0.5600 0.6133 0.6267 0.5333 0.6667 0.6000 0.6667
32 0.7067 0.6533 0.7067 0.6533 0.7067 0.6800 0.6133 0.6533 0.6800 0.6667 0.6800
33 0.7067 0.6267 0.6267 0.7333 0.7067 0.6800 0.6133 0.6800 0.5467 0.6667 0.6000
34 **** 0.7867 0.7867 0.7333 0.8400 0.7600 0.7733 0.7067 0.5733 0.8267 0.7600
35 0.2400 **** 0.7333 0.7067 0.7333 0.7067 0.6933 0.7067 0.7067 0.7467 0.6533
36 0.2400 0.3102 **** 0.6000 0.7067 0.7067 0.8000 0.5733 0.6000 0.6933 0.8400
37 0.3102 0.3472 0.5108 **** 0.8133 0.7867 0.6400 0.9200 0.7067 0.7467 0.5733
38 0.1744 0.3102 0.3472 0.2066 **** 0.8667 0.7733 0.7600 0.6800 0.9333 0.7333
39 0.2744 0.3472 0.3472 0.2400 0.1431 **** 0.7733 0.7333 0.7333 0.8267 0.7067
40 0.2570 0.3662 0.2231 0.4463 0.2570 0.2570 **** 0.6400 0.6667 0.7867 0.8267
41 0.3472 0.3472 0.5563 0.0834 0.2744 0.3102 0.4463 **** 0.7600 0.7733 0.5733
42 0.5563 0.3472 0.5108 0.3472 0.3857 0.3102 0.4055 0.2744 **** 0.7200 0.6533
43 0.1904 0.2921 0.3662 0.2921 0.0690 0.1904 0.2400 0.2570 0.3285 **** 0.7733
44 0.2744 0.4257 0.1744 0.5563 0.3102 0.3472 0.1904 0.5563 0.4257 0.2570 ****
45 0.3285 0.3285 0.4463 0.2570 0.2921 0.2231 0.3472 0.2231 0.1586 0.2744 0.3285
46 0.3285 0.3285 0.3285 0.3662 0.2231 0.2921 0.3102 0.3285 0.2570 0.2066 0.2921
47 0.5798 0.6286 0.5798 0.5798 0.5798 0.5798 0.6539 0.6799 0.6799 0.6039 0.5798
48 0.5333 0.5333 0.4463 0.6799 0.6286 0.5333 0.5108 0.6286 0.4463 0.5108 0.4463
49 0.5563 0.5108 0.6039 0.6039 0.7066 0.5563 0.4888 0.6039 0.5108 0.6286 0.6539
50 0.4888 0.6286 0.5333 0.6799 0.6286 0.5798 0.5563 0.7340 0.6286 0.5563 0.4888
51 0.4888 0.3662 0.4463 0.8518 0.5798 0.5798 0.4673 0.9163 0.6286 0.5563 0.4055
52 0.4673 0.4257 0.4673 0.7621 0.5563 0.5563 0.4463 0.8210 0.6539 0.5333 0.4257
53 0.5333 0.4055 0.4463 0.7911 0.6286 0.5798 0.4673 0.7911 0.5798 0.6039 0.4888
54 0.4463 0.3285 0.4463 0.7911 0.5333 0.4888 0.4673 0.8518 0.6286 0.5108 0.4055
55 0.5108 0.3472 0.4257 0.6039 0.5563 0.6039 0.4888 0.6539 0.5108 0.5333 0.4257
Continuous upper table
Explanation:* upper is genetic similarty (I)；* lower is genetic distance (D)；VIII numbering 1-55 of annex corresponds to table 1 in text
Result above indicates the affiliation and hereditary variation situation between each kind of almond.
Title：A kind of method for differentiating Amygdalus plant germplasm based on SSR molecular marker
Applicant：Xinjiang Agricultural Univ
SSR 10 to mark for：
1 BPPCT002 of mark, its sense primer 5’TCGACAGCTTGATCTTGACC, anti-sense primer 5’ CAATGCCTACGGAGATAAAAGAC；
2 BPPCT004 of mark, its sense primer 5’CTGAGTGATCCATTTGCAGG, anti-sense primer 5’ AGGGCATCTAGACCTCATTGTT；
3 BPPCT032 of mark, its sense primer 5’TTAAGCCACAACATCCATGAT, anti-sense primer 5’ AATGGTCTAAGGAGCACACG；
4 BPPCT036 of mark, its sense primer 5’AAGCAAAGTCCATAAAAACGC, anti-sense primer 5’ GGACGAAGACGCTCCATT；
5 BPPCT040 of mark, its sense primer 5’ATGAGGACGTGTCTGAATGG, anti-sense primer 5’AGCCAA ACCCCTCTTATACG；
6 Pchgms1 of mark, its sense primer 5’GGGTAAATATGCCCATTGTGCAATC, anti-sense primer 5’ GGATCATTGAACTACGTCAATCCTC；
7 Pchgms3 of mark, its sense primer 5’ACGGTATGTCCGTACACTCTCCATG, anti-sense primer 5’ CAACCTGTGATTGCTCCTATTAAAC；
8 Pchgms10 of mark, its sense primer 5’CGTCACGCATCCTTTCATTT, anti-sense primer 5’ GACACCTCCATTTGTATCAAAGC；
9 Pchgms11 of mark, its sense primer 5’TTGAGGCCCACTTATTAGCC, anti-sense primer 5’ CCCCCATTATTCAAACTTCTG；
Mark 10Pchgms21, its sense primer 5’ACCACCATTTTGGCTCTCTG, anti-sense primer 5’ CATGCAACCCAAAACCATCT。

Claims (1)

  1. A kind of 1. method for differentiating Amygdalus plant germplasm based on SSR molecular marker, it is characterised in that follow these steps to carry out：
    Prepare Amygdalus plant germplasm material：
    A, early spring, the fresh tender leaf of healthy and strong, no disease and pests harm 55 parts of samples of almond platymiscium is chosen, every part of sample is cold with liquid nitrogen It is put into -70 DEG C of ultra low temperature freezers of temperature and saves backup after jelly；
    The extraction of Amygdalus plant germplasm DNA：
    B, 1000 μ L are rapidly added, 65 DEG C of preheatings of temperature contain 2% cetyl trimethylammonium bromide, 1.4M sodium chloride, 20mM tetra- The buffer solution of acetic acid diamino-vinyl, 100 mM trishydroxymethylaminomethanes, the buffer solution of pH8.0 and 8% mercaptoethanol, gently Shake up；60 min of warm bath in 65 DEG C of water-baths of temperature is placed in, gently teetertotter every 10 min shakes up 1 time therebetween, makes solution Fully cracking；Centrifuged using desk centrifuge, 21 DEG C of temperature, 10000 r/min of rotating speed, 15 min of time, Aspirate supernatant, will Supernatant mean allocation is transferred in two other 1500 new μ L centrifuge tube, wherein containing 480 μ L of supernatant in each centrifuge tube Liquid；Isometric chloroform will be added in each centrifuge tube containing supernatant:Isoamyl alcohol=24:1, gently teetertotter and shake up, from The heart, 21 DEG C of temperature, 10000 r/min of rotating speed, 15 min of time, Aspirate supernatant, the supernatant in each centrifuge tube is merged, It is transferred in another 1500 new μ L centrifuge tube, 600 μ L of supernatant liquid is contained in centrifuge tube；By the centrifugation equipped with 600 μ L of supernatant liquid Isometric chloroform is added in pipe:Isoamyl alcohol=24:1, gently teetertotter and shake up, centrifuge, 21 DEG C of temperature, 10000 r/ of rotating speed Min, 15 min of time, then Aspirate supernatant, supernatant is transferred in another 1500 new μ L centrifuge tube, is contained in centrifuge tube 400 μ L of supernatant liquid；The freezing absolute ethyl alcohol of 2 times of volumes will be added in centrifuge tube equipped with 400 μ L of supernatant liquid, be put into temperature -20 DEG C deep freezer, time 30min, centrifugation, 21 DEG C of temperature, has after rotating speed 10000 r/min, time 15min clear milky DNA white cotton fibers precipitate, and remove upper liquid；Milky DNA white cotton fibers precipitation is washed 2-3 times with the ethanol that concentration is 70% again, After natural air drying, it is dissolved in 50 μ L and contains 10 mM trishydroxymethylaminomethanes, 1mM tetraacethyl diamino-vinyls, the buffering of pH8.0 In solution, -20 DEG C of refrigerators of temperature preserve；
    C, DNA purity is detected with 1.5% agarose gel electrophoresis；After the obtained DNA concentrations of step b are diluted to 50ng/ μ l, It is placed in spare in -20 DEG C of refrigerators of temperature；
    D, SSR primer screenings：
    Searched for according on ncbi database, by primary dcreening operation and again from from 100 pairs of SSR primers of tone fruit trees crop Sieve, finally selects that 10 pairs of product master tapes are obvious and the preferable primer of polymorphism carries out SSR microsatellite molecular marker analyses, wherein SSR 10 to mark for：
    1 BPPCT002 of mark, its sense primer 5’TCGACAGCTTGATCTTGACC, anti-sense primer 5’ CAATGCCTACGGAGATAAAAGAC；
    2 BPPCT004 of mark, its sense primer 5’CTG AGT GATCCATTTGCAGG, anti-sense primer 5’ AGGGCATCTAGACCTCATTGTT；
    3 BPPCT032 of mark, its sense primer 5’TTAAGCCACAACATCCATGAT, anti-sense primer 5’ AATGGTCTAAGGAGCACACG；
    4 BPPCT036 of mark, its sense primer 5’AAGCAAAGTCCATAAAAACGC, anti-sense primer 5’ GGACGAAGACGCTCCATT；
    5 BPPCT040 of mark, its sense primer 5’ATGAGGACGTGTCTGAATGG, anti-sense primer 5’AGCCAA ACCCCTCTTATACG；
    6 Pchgms1 of mark, its sense primer 5’GGGTAAATATGCCCATTGTGCAATC, anti-sense primer 5’ GGATCATTGAACTACGTCAATCCTC；
    7 Pchgms3 of mark, its sense primer 5’ACGGTATGTCCGTACACTCTCCATG, anti-sense primer 5’ CAACCTGTGATTGCTCCTATTAAAC；
    8 Pchgms10 of mark, its sense primer 5’CGTCACGCATCCTTTCATTT, anti-sense primer 5’ GACACCTCCATTTGTATCAAAGC；
    9 Pchgms11 of mark, its sense primer 5’TTGAGGCCCACTTATTAGCC, anti-sense primer 5’ CCCCCATTATTCAAACTTCTG；
    Mark 10Pchgms21, its sense primer 5’ACCACCATTTTGGCTCTCTG, anti-sense primer 5’ CATGCAACCCAAAACCATCT；
    E, mono- pcr amplification reactions of SSR, electrophoresis detection：20 μ l reaction systems of SSR-PCR reaction systems include：10 × Taq enzymes are matched somebody with somebody Cover buffer solution, 1.5 mmol/L MgCl2, 0.25mmol/L dNTPS, SSR upstream and downstream primer are respectively 0.2mol/ μ l, 0.5U Taq DNA polymerases, DNA templates 30ng, corresponding amplification program are：94 DEG C of pre-degenerations of temperature, 4 min, followed by temperature 94 DEG C of denaturation 60s, 55 DEG C of 40 s of renaturation of temperature, 72 DEG C of extension 60s of temperature, after totally 34 circulations, 72 DEG C of extensions 8 of temperature Min polishings, 6 times of Loadingbuffer2.0 μ l are added in amplified production, 2.5 μ l loadings are taken, with 8% non denatured polypropylene phthalein Amine detected through gel electrophoresis, silver staining colour developing；
    F, statistical analysis：Data are carried out with NTSYS-pc 2.10e softwares based on the cluster analysis for not weighting matched group algorithmic approach Analysis, selects clear and legible electrophoresis band, and sample has band to be then denoted as 1, and no band is then denoted as 0, builds two condition data matrix；To original Matrix seeks S with SimQual programsSMSimilarity factor and genetic distance matrix, and clustered with the UPGMA methods in SAHN programs Analysis, SEQBOOT, MIX or CONSENSE program in PHYLIP3.65 software kits of the cluster analysis based on Wagner parsimony principles Make uniformity tree graph, and with the brief figure of TreeView1.6 Program Generatings, build molecular evolution genealogical tree.
CN201510870315.6A 2015-12-02 2015-12-02 A kind of method for differentiating Amygdalus plant germplasm based on SSR molecular marker Expired - Fee Related CN105512513B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201510870315.6A CN105512513B (en) 2015-12-02 2015-12-02 A kind of method for differentiating Amygdalus plant germplasm based on SSR molecular marker

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201510870315.6A CN105512513B (en) 2015-12-02 2015-12-02 A kind of method for differentiating Amygdalus plant germplasm based on SSR molecular marker

Publications (2)

Publication Number Publication Date
CN105512513A CN105512513A (en) 2016-04-20
CN105512513B true CN105512513B (en) 2018-04-13

Family

ID=55720490

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201510870315.6A Expired - Fee Related CN105512513B (en) 2015-12-02 2015-12-02 A kind of method for differentiating Amygdalus plant germplasm based on SSR molecular marker

Country Status (1)

Country Link
CN (1) CN105512513B (en)

Families Citing this family (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105969902B (en) * 2016-07-27 2019-10-11 新疆农业大学 Xinjiang Wild Almonds TP-M13-SSR core primers and its application
CN107190053B (en) * 2017-03-13 2021-01-08 北京林业大学 Cypress microsatellite molecular marker combination, primer screening method and application thereof
CN107142321B (en) * 2017-06-22 2019-11-26 内蒙古自治区农牧业科学院 A kind of mongolian amygdalus seed specificity microsatellite locus and its application
CN108220402B (en) * 2017-12-25 2020-07-07 山东省农业科学院蔬菜花卉研究所 Method for identifying pedigree relationship between Chinese cabbage germplasm and variety
CN108998567B (en) * 2018-05-24 2022-05-13 内蒙古农业大学 Molecular marker primer and method for identifying amygdalus pedunculata pall, amygdalus mongolicus and prunus ulmaria
CN109666725A (en) * 2018-12-26 2019-04-23 中国环境科学研究院 A method of identification mongolian amygdalus seed
CN110643736B (en) * 2019-11-15 2022-12-30 中国农业科学院蔬菜花卉研究所 Garlic germplasm resource classification method based on SSRseq molecular marker
CN111078732A (en) * 2019-12-31 2020-04-28 新疆农业科学院园艺作物研究所 Method for identifying almond variety
CN111363844B (en) * 2020-04-15 2023-05-26 广西壮族自治区农业科学院 Water chestnut SSR primer group and application thereof
CN111444984B (en) * 2020-04-24 2021-05-28 江苏省农业科学院宿迁农科所 Intelligent insect defining algorithm in ecological breeding system

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102031251A (en) * 2010-11-04 2011-04-27 浙江农林大学 Amygdalus communis EST (expressed sequence tag) microsatellite marker screening method
CN103266107A (en) * 2013-06-14 2013-08-28 新疆农业大学 Extraction method for genomic DNAs (deoxyribonucleic acids) of fruit tree crops in arid regions
CN103614374A (en) * 2013-12-04 2014-03-05 广东省昆虫研究所 Platysternon megacephalum microsatellite marked specific primer and detection method thereof
EP2813141A1 (en) * 2013-06-14 2014-12-17 Keygene N.V. Directed strategies for improving phenotypic traits
CN104263835A (en) * 2014-10-09 2015-01-07 凯里学院 Method for identifying varieties of lilium brownii by adopting SSR (simple sequence repeat) molecular marking technology

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102031251A (en) * 2010-11-04 2011-04-27 浙江农林大学 Amygdalus communis EST (expressed sequence tag) microsatellite marker screening method
CN103266107A (en) * 2013-06-14 2013-08-28 新疆农业大学 Extraction method for genomic DNAs (deoxyribonucleic acids) of fruit tree crops in arid regions
EP2813141A1 (en) * 2013-06-14 2014-12-17 Keygene N.V. Directed strategies for improving phenotypic traits
CN103614374A (en) * 2013-12-04 2014-03-05 广东省昆虫研究所 Platysternon megacephalum microsatellite marked specific primer and detection method thereof
CN104263835A (en) * 2014-10-09 2015-01-07 凯里学院 Method for identifying varieties of lilium brownii by adopting SSR (simple sequence repeat) molecular marking technology

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
中国荻属植物种质资源遗传多样性的SSR分析;刘晶;《中国优秀硕士学位论文全文数据库基础科学辑》;20111215(第S1期);第A006-280页 *
扁桃属植物种质资源鉴定的SSR分析研究;曾斌 等;《新疆农业科学》;20090210;第46卷(第1期);第18-22页摘要,第1-2小节,表1 *
新疆核桃8个天然群体遗传多样性的SSR评价;曾斌 等;《新疆农业科学》;20130110;第49卷(第12期);第2180-2188页 *
新疆核桃亲缘关系的ISSR分析;李超 等;《新疆农业科学》;20100930;第47卷(第9期);第1722-1727页 *
用SSR标记分析柿属种质资源的遗传多样性;耿攀;《中国优秀硕士学位论文全文数据库农业科技辑》;20101115(第11期);第D048-131页 *
硅胶干燥野扁桃叶片制备DNA样品及其SSR反应体系的建立;曾斌 等;《经济林研究》;20090315;第27卷(第1期);第1-6页 *

Also Published As

Publication number Publication date
CN105512513A (en) 2016-04-20

Similar Documents

Publication Publication Date Title
CN105512513B (en) A kind of method for differentiating Amygdalus plant germplasm based on SSR molecular marker
Que et al. Genetic analysis of diversity within a Chinese local sugarcane germplasm based on start codon targeted polymorphism
CN106676102A (en) Single nucleotide polymorphism (SNP) molecular marker related to content of arachidonic acid in grease of camellia oleifera seeds, and application of SNP molecular marker
CN105925721B (en) For identifying single nucleotide polymorphism site, primer, kit and the application of Peach fruits epidermis coloring character
Abid et al. Genetic relationship and diversity analysis of faba bean (Vicia faba L. var. Minor) genetic resources using morphological and microsatellite molecular markers
CN106755480A (en) A kind of SSR molecular marker I for identifying Gala apple Progeny plants and its application
CN105087768B (en) A kind of method of the anti-bean weevil kind of molecular marking supplementary breeding mung bean
CN102140506B (en) Molecular marker linked with gummy stem blight resistance gene Gsb-2 and application thereof
Cañas-Gutiérrez et al. Microsatellites revealed genetic diversity and population structure in Colombian avocado (Persea americana Mill.) germplasm collection and its natural populations
Wang et al. Establishment and application of an SNP molecular identification system for grape cultivars
Ebadi et al. Genetic diversity of Iranian and some European grapes as revealed by nuclear and chloroplast microsatellite and SNP molecular markers
Tesfamichael et al. Genetic diversity analysis of Eritrean sorghum (Sorghum bicolor (L.) Moench) germplasm using SSR markers
Lazović et al. Intra-varietal variability and genetic relationships among the homonymic East Adriatic olive (Olea europaea L.) varieties
Noda et al. Development of Indel markers for the selection of Satsuma mandarin (Citrus unshiu Marc.) hybrids that can be used for low-cost genotyping with agarose gels
CN109280709A (en) One kind molecular labeling relevant to pig growth and reproductive trait and application
Abdelhamid et al. Molecular characterization and genetic relationships of cultivated Tunisian olive varieties (Olea europaea L.) using SSR markers
Drikvand et al. Investigation of genetic diversity among bread wheat cultivars (Triticum aestivum L.) using SSR markers
CN106676180B (en) Molecular marker for regulating and controlling maize tassel branch number major QTL and application thereof
CN104805081A (en) Wheat grain heavy molecular marker and application thereof
CN106801105A (en) The SSR marker being associated with ramee fineness and application
CN104293774A (en) Functional SSR labels obviously related with wood quality characters in populus CesAs gene, and applications and kit thereof
CN112725521B (en) Dendrobium chrysotoxum SSR molecular marker primer composition and application thereof
Mathavaraj et al. Genetic diversity and structure revealed by genomic microsatellite markers in Centella asiatica (L.) Urb., a plant with medicinal potential
Liu et al. ISSR and SRAP markers in the genetic relationship analysis among Pinellia in China
CN111100946B (en) Molecular marker primer of rape grain weight character major gene locus and application

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant
CF01 Termination of patent right due to non-payment of annual fee

Granted publication date: 20180413

Termination date: 20181202

CF01 Termination of patent right due to non-payment of annual fee