CN101392253B - Rana spinosa microsatellite DNA marker and uses thereof - Google Patents
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Abstract
The invention discloses a Rana spinosa microsatellite DNA marker and a nucleotide sequence marked by a microsatellite DNA is any one of eight sequences respectively in SEQ ID NO: 1 to SEQ ID NO: 8. Simultaneously, the invention also provides applicaitons of the Rana spinosa microsatellite DNA marker: the marker is used for carrying out genetic analysis, germplasm resource survey and assisted breeding to the Rana spinosa.
Description
Technical field
The present invention relates to molecular marking technique, be specifically related to dna molecular genetic marker of a kind of Ranaspinosa David. and uses thereof.
Background technology
Little satellite (is called simple repeated sequence again, the section of DNA that the short sequence series connection of SSR) being made up of 1-6 Nucleotide repeats repeatedly to form.Because little satellite flanking sequence is conservative relatively, according to its flanking sequence design primer, available pcr amplification goes out the sequence of microsatellite locus.Because the multiplicity of repeating unit is different in little satellite, thereby the length of the microsatellite sequence that amplifies presents polymorphum.Microsatellite sequence extensively is present in the eukaryotic gene group, and stochastic distribution, has the height polymorphum; Neutral in the selection; Characteristics such as codominance are highly effective genetic markers, are widely used in fields such as genetic map construction, the assignment of genes gene mapping, genetic affinity analysis, cultivar identification.As analyze the sibship (Zhou etc. of ramee variety with microsatellite marker; 2005; Progress in Natural Science, 15:137-142), the patented claim of carrying out pig variety classification with microsatellite DNA mark " be suitable for the pig microsatellite DNA mark of pig variety classification " (publication number CN1370834A), carry out the patented claim " SSR mark and the application thereof of silkworm " (publication number CN 1970792A) that silkworm molecule linkage inheritance is analyzed with microsatellite DNA mark.
Ranaspinosa David. (Paa spinosa David) belongs to amphibia, Anura, Ranidae.Ranaspinosa David. is individual big, and fine and tender taste is pure white, and delicious flavour has very high nutritive value and pharmaceutical use, have the laudatory title of " kings of hundred frogs ".Because the people is the deterioration of catching and killing with physical environment, wild Ranaspinosa David. resource is seriously damaged, and carries out Ranaspinosa David. and propagates artificially and both help to protect wild resource, can meet the need of market again.Now; Ranaspinosa David. is one of important breed batrachia of China; But because its fundamental research is weak, realize that breeding scale also has many problem demanding prompt solutions, it carried out genetic affinity analysis, germ plasm resource investigation, marker-assisted breeding etc. comprising the effective molecule marker of needs one cover.
Summary of the invention
The technical problem that the present invention will solve provides a kind of Rana spinosa microsatellite DNA marker, utilizes these molecule markers to carry out genetic analysis, germ plasm resource investigation and assistant breeding to Ranaspinosa David..
In order to solve the problems of the technologies described above, the present invention provides a kind of Rana spinosa microsatellite DNA marker, and the nucleotide sequence of this microsatellite DNA mark is respectively any one in these 8 kinds of sequences of SEQ ID NO:1～SEQ ID NO:8; Little satellite that said SEQID NO:1 is corresponding is numbered Psp16; Little satellite that SEQ ID NO:2 is corresponding is numbered Psp17, and little satellite that SEQ IDNO:3 is corresponding is numbered Psp18, and little satellite that SEQ ID NO:4 is corresponding is numbered Psp19; Little satellite that SEQ ID NO:5 is corresponding is numbered Psp20; Little satellite that SEQ ID NO:6 is corresponding is numbered Psp21, and little satellite that SEQ ID NO:7 is corresponding is numbered Psp22, and little satellite that SEQ ID NO:8 is corresponding is numbered Psp23.
Improvement as Rana spinosa microsatellite DNA marker of the present invention: it is right that the Rana spinosa microsatellite DNA marker primer is selected from following primer, and nucleotides sequence wherein classifies 5 ' → 3 as ',
Psp16 forward: GTATCATTTTGTGTTTTTT
Oppositely: TTTGAATTGTCTTTTGTC
Psp17 forward: AGTTATGTGGCAGAGAAAC
Oppositely: AAGAGCACAGGAGAGTCA
Psp18 forward: GCTTATTGTTGGGCTTCA
Oppositely: TCACTGGCATCAGGTCTG
Psp19 forward: TCACAATCGACGTAATGG
Oppositely: ACACACAGAGGGTCACACT
Psp20 forward: TTGGCTCATCAACTACCC
Oppositely: AGTCACATAACATACCCCCT
Psp21 forward: ACTCTCGCCTCAATCC
Oppositely: ATGTAGCTCAGCGTACTG
Psp22 forward: ATCTTAGGTGCAGCAACTT
Oppositely: TCCCTTGTGTAGTGTTCAGT
Psp23 forward: TCGACGATTGCCAGAGACC
Oppositely: GCTGGTGGGGAAACTACATTAT.
The present invention also provides the purposes of such Rana spinosa microsatellite DNA marker simultaneously: be used for Ranaspinosa David. is carried out genetic analysis, germ plasm resource investigation or assistant breeding.
The technical scheme that realizes foregoing invention comprise Ranaspinosa David. (CA) n enriched microsatellite library structure, contain the screening and the order-checking of the positive colony of microsatellite sequence, confirmed microsatellite marker Psp16, Psp17, Psp18, Psp19, Psp20, Psp21, Psp22, the Psp23 of 8 rich polymorphism.
The present invention is intended to the separating clone microsatellite DNA mark, set up Ranaspinosa David. microsatellite DNA technical system and utilize these molecule markers to carry out investigation of genetic analysis, germ plasm resource and assistant breeding.Therefore; The invention provides the dna sequence dna of 8 Ranaspinosa David. microsatellite locus and the primer sequence and the amplification method of above-mentioned 8 the Ranaspinosa David. microsatellite locus that increase, 8 Ranaspinosa David. microsatellite locus are used for the research of Ranaspinosa David. germ plasm resource, genetic affinity analysis; Auxiliary seed selection of mark and marker-assisted breeding; Good reproducibility, polymorphum is high, is a kind of reliable and effective molecule marker.
Description of drawings
Do further explain below in conjunction with the accompanying drawing specific embodiments of the invention.
Fig. 1 is the structure dendrogram (K=3) of 13 Ranaspinosa David.s nature populations that the present invention relates to.
Embodiment
Embodiment 1, Ranaspinosa David. (CA)
nThe structure of enriched microsatellite library
Carry out following steps successively:
1), extracts Ranaspinosa David. (LONGSHENG IN GUANGXI) genomic dna with classical phenol-chloroform extraction process.Cut the Ranaspinosa David. genomic dna with restriction enzyme Sau3AI enzyme, enzyme is cut product electrophoresis on 1.5% sepharose, and UV-light downcuts the dna fragmentation of 300-1000bp size down and reclaims test kit purifying and recovering enzyme with gel and cut product.
2), (it is synthetic that the worker is given birth in Shanghai with the oligonucleotide Sau3AI Oligo A:5 '-GGCCAGAGACCCCAAGCTTCG-3 ' of base complementrity and Oligo B:5 '-pGATCCGAAGCTTGGGGTCTCTGGCC-3 '; HPLC level purifying; 200pmol/ μ l) respectively gets 10 μ l spiral mixing gently; 80 ℃ of sex change are 10 minutes on the PCR appearance; At room temperature make the general sour chain of few nuclear about 1 hour of the renaturation of slowly annealing then, adding 60 μ l high purity waters, to be prepared into concentration be that 25pmol/ μ l has the joint that the Sau3AI enzyme is cut recognition site.
3), with above-mentioned steps 1) enzyme of gained cuts product and step 2) joint of gained uses T
4Dna ligase connects, and the connection liquid of gained is used the cleaning agents box purifying of tieing up special clean company, removes salt ion, albumen and unnecessary linker fragment, is dissolved in the 50 μ l TE damping fluids; Must connect product.
4), with biotin labeled oligonucleotide probe (CA)
15Hybridize with the product D NA fragment that is connected of step 3) gained.Concrete operations are: (6 * SSC, 0.1%SDS) the middle 500ng of adding connects product and the biotin labeled oligonucleotide probe of 100pmol, and at 95 ℃ of following sex change 10min, hybridizes 1 hour for 60 ℃ at TV 100 μ l hybridization solutions.
5), in the step 4) gains, add 100 μ l (10 μ g/ μ l) magnetic beads (Dynabeads M-280 is available from Dynal Biotech company), and placed 30 minutes in room temperature.
6), on the magnetic force frame, adsorb magnetic bead, abandon supernatant.(6 * SSC 0.1%SDS) washed 15 minutes down at 60 ℃ magnetic bead, at room temperature used 2 * SSC and 1 * SSC solution washing twice then respectively, and abandoned supernatant with 400 μ l lavation buffer solutions.
7), in the step 6) gains, add 50 μ l sterilized waters, 90 ℃ of insulations 10 minutes down, carefully draw supernatant, supernatant is the single stranded DNA fragment that contains (CA) n Tumor-necrosis factor glycoproteins.With Oligo A is primer, is that template is carried out pcr amplification and obtained double-stranded purpose fragment with the single stranded DNA fragment.
25 μ lPCR reaction systems comprise: about 100ng single stranded DNA fragment, 20mm Tris-HCI (pH8.3), 100mmKCI, 3mm MgCl
2, each 1000 μ m of dNTP, 3pmol Oligo A, 0.8units Taq polymerase (Shanghaipromega).
The PCR cycling program is set to: 95 ℃ of preparatory sex change 3 minutes, and then 5 circulations comprise 95 ℃ of sex change 30s, 60 ℃ of annealing 30s; 72 ℃ are extended 45s, carry out 92 ℃ of sex change 30s of other 30 round-robin then, 60 ℃ of annealing 30s; 72 ℃ are extended 55s, extend below 10 minutes in 72 ℃ at last.Detect the PCR product and use the purification kit purifying of tieing up special clean company, product is dissolved in the 30 μ l TE damping fluids.
8), be connected on the PMD18-T carrier behind the amplified production purifying with the step 7) gained, will connect product then and be converted into the intestinal bacteria recipient cell, transformed bacteria liquid is coated on and carries out blue hickie screening on the LB solid medium flat board that contains IPTG and X-gal.
Embodiment 2, the screening that contains the positive colony of microsatellite sequence, order-checking and micro-satellite primers design
Carry out following steps successively:
1), the white colony of picking embodiment 1 gained and being inoculated in the 3ml LB nutrient solution, 37 ℃ of overnight cultures are extracted plasmid then.
2), be template with the DNA, take the three-primer method (Gardner etc., 1999, Journal of Heredity, 90,301-304) screening contains the positive colony of (CA) n microsatellite sequence.
3), with positive colony order-checking, analyze whether contain (CA) n Tumor-necrosis factor glycoproteins, and whether have enough flanking sequences to be used for design of primers.
4), according to the conserved sequence of little satellite Tumor-necrosis factor glycoproteins both sides, with primer premier5.0 software design primer, generally between 16-22 bases, the target fragment of selecting amplification is between 150-350bp for primer length.
Embodiment 3, Rana spinosa microsatellite DNA marker stability and polymorphum detect
With 32 Ranaspinosa David.s individual (Zhejiang) of microsatellite marker amplification of the foregoing description 2 gained from same natural population, confirmed 8 rich polymorphism, be suitable for the microsatellite marker (table 1) of Ranaspinosa David. Diversity Detection.Specific operation process is following:
1), use classical 32 Ranaspinosa David. DNA of individual of phenol-chloroform method extracting as template.
2), the synthetic back of designed primer is to the checking that experimentizes of primer exactness and feasibility, with a plurality of templates of thermograde pcr amplification.Contain about 50ng genomic dna in the PCR reaction system of 15 μ l, 10mm Tris-HCI (pH8.3), 50mm KCI, 1.5mm MgCl
2, each 500 μ m of dNTP, 1pmol micro-satellite primers, 0.5units Taq polymerase (Shanghai promega).Confirm that according to experimental result which primer can amplify stable purpose band, and definite optimum annealing temperature (seeing table one).Finishing screen is selected 8 sites, respectively called after: Psp16, Psp17, Psp18, Psp19, Psp20, Psp21, Psp22, Psp23.
3), use 8 pairs of primers (primer sequence is seen table 1) in above-mentioned 8 sites this several Ranaspinosa David. DNA of individual that increase respectively.5 ' end of wherein any SSR primer is coupled with one section does not have the fluorescently-labeled M13 sequence of far infrared, and the M13 primer of another IRDye mark is included in this reaction system simultaneously.By above-mentioned steps 2) reaction conditions carry out pcr amplification.The PCR product carries out gene type on LI-COR4300s heredity automatic analyser, be used built-in STR molecular weight standard (STR Marker, LI-COR Bioseienee) and soft SA GA
GTThe automatic interpretation band is aided with manual synchronizing.
4), use software CERVUS 2.0 statistics allelotrope numbers, polymorphism information content (PIC), the observation heterozygosity (Observed heterozygosities, Ho) with the expectation heterozygosity (Expected heterozygosities, He).Hardy-Weinberg equilibrium (Hardy-Weinberg equilibrium; HWE) linkage disequilibrium that departs between test and site is checked (Linkagedisequilibrium; LD) use GENEPOP software version3.4 to accomplish, be aided with successive Bonefermni afterwards and proofread and correct.Use the amorphs situation in each site of MICRO-CHEKER software estimation.
Finally confirmed following 8 Rana spinosa microsatellite DNA markers, be respectively:
Be numbered the microsatellite DNA mark of Psp16, its nucleotide sequence is shown in SEQ ID NO:1;
Be numbered the microsatellite DNA mark of Psp17, its nucleotide sequence is shown in SEQ ID NO:2;
Be numbered the microsatellite DNA mark of Psp18, its nucleotide sequence is shown in SEQ ID NO:3;
Be numbered the microsatellite DNA mark of Psp19, its nucleotide sequence is shown in SEQ ID NO:4;
Be numbered the microsatellite DNA mark of Psp20, its nucleotide sequence is shown in SEQ ID NO:5;
Be numbered the microsatellite DNA mark of Psp21, its nucleotide sequence is shown in SEQ ID NO:6;
Be numbered the microsatellite DNA mark of Psp22, its nucleotide sequence is shown in SEQ ID NO:7;
Be numbered the microsatellite DNA mark of Psp23, its nucleotide sequence is shown in SEQ ID NO:8.
Table 1, primer property list: primer sequence, annealing temperature, clip size
Embodiment 4, Ranaspinosa David. population genetic diversity and genetic construction analysis
From 13 sampling points, 210 individuals altogether, detect its genetic diversity and Genetic Constitution of Population with the 8 pairs of micro-satellite primers amplification of the foregoing description 3 gained.Specific operation process is following:
1) 210 in the Ranaspinosa David. sample that, is used for the population genetic analysis.Sampling point and code name thereof see table 2 for details.
Table 2, Ranaspinosa David. sample message
| The sampling position | The population code name | Sample size | Observations |
| Mt. Huang in Anhui | HS | 15 | Wild |
| Jinhua, Zhejiang | JH | 14 | Wild |
| Lishui of Zhejiang | LS | 18 | Wild |
| The Jianyang, Fujian | JY | 13 | Wild |
| The Pingjiang River, Hunan | PJ | 23 | Wild |
| The Jiangxi Shinkansen | XG | 10 | Wild |
| The Wuyi, Fujian | WY | 19 | Wild |
| Jinggang Mountain, Jiangxi | JG | 24 | Wild |
| LONGSHENG IN GUANGXI | LH | 27 | Wild |
| The Yongfu, Guangxi | YF | 16 | Wild |
| Yangshan, Guangdong | YS | 17 | Wild |
| Screen limit, Yunnan | PB | 9 | Wild |
| Vietnam | VT | 5 | Wild |
2), the extraction of Ranaspinosa David. genomic dna
Through the protease K digesting and the extractive method of phenol/chloroform (Sambrook and Fritsch, et.al, 1989) of standard, extract genomic dna from leg muscle.
3), the pcr amplification of microsatellite DNA
(1) PCR reaction system
About 50ng genomic dna, 10mm Tris-HCI (pH8.3), 50mm KCI, 1.5mm MgCl
2, each 500 μ m of dNTP, 1pmolSTR primer, 0.5units Taq polymerase (Shanghai promega), 0.5pmol fluorescent mark M13 primer, IRD700 or IRD800 (LICOR Bioseienee).
(2) PCR response procedures
The PCR cycling condition is following: 95 ℃ of preparatory sex change 3min, follow 95 ℃ of sex change 30s of 35 round-robin, and locus specificity annealing temperature (seeing table one) 30s, 72 ℃ are extended 30s, and last 72 ℃ are prolonged chain 8min.
(3) detection of PCR reaction product
The PCR product carries out gene type on LI-COR4300s heredity automatic analyser, be used built-in STR molecular weight standard (STR Marker, LI-COR Bioseienee) and soft SA GA
GTThe automatic interpretation band is aided with manual synchronizing.
4), data analysis
Use software CERVUS2.0 (Marshall et al.1998) statistics allelotrope number, polymorphism information content (PIC), (observed heterozygosities is Ho) with expectation heterozygosity (expeeted heterozygosities, H for the observation heterozygosity
E).Hardy-Weinberg equilibrium (Hardy-Weinberg equilibrium; HWE) linkage disequilibrium that departs between test and site is checked (linkage disequilibrium; LD) use GENEPOP software version3.4, (Raymond & Rousset 2004) accomplishes.Carry out the population cluster analysis with software STRUCTURE (Pritchart et al.2000), the dendrogram that is generated is as shown in Figure 1.Among Fig. 1: each bar vertical line is represented body one by one, and three kinds of colors are represented a branch after the cluster, the probability that ordinate zou expresses possibility respectively.The color of vertical line is formed and is shown that this individuality possibly belong to software defined some branches.Show among the figure that can Mount Huang population (HS), Jinhua population (JH), Lishui population (LS), Jianyang population (JY) be gathered basically is one type; It is one type that Pingjiang River population (PJ), Shinkansen population (XG), Wuyi Mountain population (WY), Jinggang Mountain population (JG), Yongfu population (YF) gather; Dragon wins population (LH), Yangshan population (YS), screen limit population (PB) gathers is one type; Vietnam population (VT) cluster and practical situation are not inconsistent, maybe be because the limited error that causes of sample size.
5), the result is following:
The sample size of table 3, each population, average allelotrope number, average expectation heterozygosity, average polymorphism information content (PIC)
| The population code name | Sample size | Na | HE | PIC |
| HS | 15 | 6.08 | 0.676 | 0.617 |
| JH | 14 | 6.08 | 0.687 | 0.623 |
| LS | 18 | 9.33 | 0.749 | 0.702 |
| JY | 13 | 9.58 | 0.797 | 0.745 |
| PJ | 23 | 11.17 | 0.742 | 0.705 |
| XG | 10 | 9.42 | 0.830 | 0.774 |
| WY | 19 | 14.58 | 0.892 | 0.855 |
| JG | 24 | 13.58 | 0.795 | 0.765 |
| LH | 27 | 13.00 | 0.828 | 0.793 |
| YF | 16 | 8.67 | 0.827 | 0.776 |
| YS | 17 | 12.08 | 0.826 | 0.786 |
| PB | 9 | 6.75 | 0.785 | 0.710 |
| VT | 5 | 3.92 | 0.700 | 0.574 |
Observation heterozygosity (the H in table 4, eight sites
O), expectation heterozygosity (H
E), polymorphism information content (PIC), allelotrope number (Na) and MV
| Site Locus | Observation heterozygosity (H O) | Expectation heterozygosity (H E) | Polymorphism information content PIC | Allelotrope is counted Na |
| Psp16 | 0.688 | 0.888 | 0.878 | 13 |
| Psp17 | 0.428 | 0.953 | 0.949 | 8 |
| Psp18 | 0.679 | 0.915 | 0.906 | 11 |
| Psp19 | 0.572 | 0.920 | 0.913 | 24 |
| Psp20 | 0.419 | 0.759 | 0.734 | 18 |
| Psp21 | 0.628 | 0.966 | 0.963 | 12 |
| Psp22 | 0.609 | 0.953 | 0.948 | 19 |
| Psp23 | 0.488 | 0.945 | 0.940 | 5 |
| Average mean | 0.5638 | 0.9123 | 0.9038 | 13.75 |
Expect heterozygosity, each population polymorphism information content in allelotrope quantity in 13 populations of table 5, eight microsatellite locus, the population
Annotate: in the table
*Expression significantly departs from Hardy-Weinberg equilibrium.
Can find out that by above-mentioned Fig. 1 and table 1～3 embody population genetic diversity highly by 8 microsatellite markers of the present invention, cluster analysis result can well reflect the actual state of these 13 populations.This shows that eight microsatellite markers of the present invention clone can well be used for the detection of Ranaspinosa David. population genetic diversity analysis and genetic construction, and are used for the investigation and the assistant breeding of germ plasm resource.
At last, it is also to be noted that what more than enumerate only is several specific embodiments of the present invention.Obviously, the invention is not restricted to above embodiment, many distortion can also be arranged.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention all should be thought protection scope of the present invention.
Sequence table
SEQ?ID?NO：1
SEQ?ID?NO：2
SEQ?ID?NO：3
SEQ?ID?NO：4
SEQ?ID?NO：5
SEQ?ID?NO：6
SEQ?ID?NO：7
SEQ?ID?NO：8
Claims (3)
1. Rana spinosa microsatellite DNA marker, it is characterized in that: the nucleotides sequence of this microsatellite DNA mark is classified SEQ IDNO:1 as, and this little satellite is numbered Psp16.
2. Rana spinosa microsatellite DNA marker according to claim 1 is characterized in that: said Rana spinosa microsatellite DNA marker primer is that following primer is right, nucleotides sequence wherein classifies 5 ' → 3 as ',
Psp16 forward: GTATCATTTTGTGTTTTTT
Oppositely: TTTGAATTGTCTTTTGTC.
3. the purposes of Rana spinosa microsatellite DNA marker according to claim 1 and 2 is characterized in that: be used for Ranaspinosa David. is carried out genetic analysis, germ plasm resource investigation or assistant breeding.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2008101219523A CN101392253B (en) | 2008-10-23 | 2008-10-23 | Rana spinosa microsatellite DNA marker and uses thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2008101219523A CN101392253B (en) | 2008-10-23 | 2008-10-23 | Rana spinosa microsatellite DNA marker and uses thereof |
Related Child Applications (3)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2010101623482A Division CN101805734B (en) | 2008-10-23 | 2008-10-23 | Rana spinosa microsatellite DNA marker II and applications thereof |
| CN2010101620889A Division CN101805733B (en) | 2008-10-23 | 2008-10-23 | Rana spinosa microsatellite DNA marker III and applications thereof |
| CN2010101620484A Division CN101805732B (en) | 2008-10-23 | 2008-10-23 | Rana spinosa microsatellite DNA marker I and applications thereof |
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| Publication Number | Publication Date |
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| CN101392253A CN101392253A (en) | 2009-03-25 |
| CN101392253B true CN101392253B (en) | 2012-05-09 |
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