CN101974617B - Lagerstroemia caudate microsatellite DNA molecular markers and application thereof - Google Patents
Lagerstroemia caudate microsatellite DNA molecular markers and application thereof Download PDFInfo
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Abstract
The invention provides lagerstroemia caudate microsatellite DNA molecular markers which comprise 15 lagerstroemia caudate microsatellite loci. The invention further provides a method for screening the lagerstroemia caudate microsatellite DNA molecular markers, and an application of the method in researching lagerstroemia caudate genetic diversity. In addition, the invention further provides primer sequences for amplifying 15 lagerstroemia caudate microsatellite loci and an amplification method thereof and establishes a lagerstroemia caudate microsatellite DNA molecular marker technical system for lagerstroemia caudate genetic diversity analysis, fingerprint establishment and molecular-marker-assisted breeding, thus the DNA molecular markers are reliable and effective and have good repeatability.
Description
Technical field
The present invention relates to the dna molecular marker technology, specifically, relate to a kind of caudal lobe crape myrtle microsatellite DNA molecule marker and application thereof.
Background technology
Caudal lobe crape myrtle (Lagerstroemia caudata) belongs to Lythraceae (Lythraceae) Lagerstroemia fallen leaves megaphanerophyte, and is high 18～30 meters, about 40 centimetres of the diameter of a cross-section of a tree trunk 1.3 meters above the ground; Bark is smooth, brown, and slabbing peels off; Complete stool does not have hair; The florescence 4-5 month, the fruit phase 7-10 month.The caudal lobe crape myrtle blooms dense, and aromatic flavour is the good parent who cultivates fragrant crape myrtle kind.It is well-grown on the hillside that the ls tor covers slightly, and sprout tiller strength is stronger, is one of good green tree species of ls tor.Simultaneously, caudal lobe crape myrtle timber is hard, texture is careful, and is faint yellow, is suitable for doing first-class furniture, interior decoration, fine workmanship or engraving etc. and uses material.The caudal lobe crape myrtle originates in China, has been classified as threatened plant (" the red register of Chinese species " first roll, Higher Education Publishing House, 2004).
At present only relevant for the research (Huang Chenhui etc. of the single population structure of caudal lobe crape myrtle; 2008; Caudal lobe crape myrtle population structure and dynamic preliminary study, Tropical Forest, 36 (4); 24～26), and do not appear in the newspapers as yet about the research of aspects such as the resource distribution of caudal lobe crape myrtle, population genetic diversity and plasm resource protection.Simple repeated sequence (simple sequence repeat SSR), also claims microsatellite DNA (microsatellite), be a kind of be that the unit multiple of repeatedly connecting reaches tens even the sequence of a hundreds of Nucleotide by 2～5 Nucleotide.These sequences extensively are present on the different seats of eukaryotic gene group, and the quantity of each seat repeating unit maybe be incomplete same, thereby form polymorphum.Because it has rich polymorphism, advantages such as repeatability height, codominant inheritance, be widely used in the fields such as colony and evolutionary genetics research, germ plasm resource analysis of genetic diversity, fingerprint map construction of plant.Therefore, exploitation caudal lobe crape myrtle microsatellite marker utilizes its research caudal lobe crape myrtle resource genetic diversity and population structure, to the protection of caudal lobe crape myrtle germ plasm resource with utilize significant.
Summary of the invention
The purpose of this invention is to provide a kind of caudal lobe crape myrtle microsatellite DNA molecule marker.
Another object of the present invention provides the screening method of above-mentioned dna molecular marker.
Further object of the present invention provides the application of above-mentioned dna molecular marker in the research of caudal lobe crape myrtle genetic diversity.
In order to realize the object of the invention, a kind of caudal lobe crape myrtle microsatellite DNA molecule marker of the present invention, it is right that the primer of the said molecule marker that wherein increases is selected from following primer:
1)I1GF：5′-GTCACAGGTTACCGAATC-3′
I1GR：5′-ATGTAAATGGTGAGGAGG-3′
2)I2BF：5′-TTCTTGTCTTGGGTATCGC-3′
I2BR：5′-GAGCCAGTATTGTCTTCACG-3′
3)I2GF：5′-TTCTTCCACTTCCTCCTT-3′
I2GR：5′-CAGCCCACATTAACTTTT-3′
4)I2HF：5′-AAAGACGCAGAAGGATGG-3′
I2HR：5′-CGATTAGTTTCAGCTCGT-3′
5)I3GF：5′-ATGTGGTGAATGGGAACT-3′
I3GR：5′-TTGGGCTAAAGATATGGA-3′
6)I9HF：5′-GGACCAGATTGTAAATGC-3′
I9HR：5′-CTGCTCCTAATATCAGTGTC-3′
7)I10HF：5′-ATTCGATCTGCCCTCTTG-3′
I10HR：5′-ACTCGGGTTTACGTGGTG-3′
8)I12HF：5′-TCCCTTTAACGAGGAATG-3′
I12HR：5′-AAGTTTCTCGACGGCTTT-3′
9)II1CF：5′-GTGCTGGAGGAACTGCTA-3′
II1CR：5′-CACGCTATTCTAAGACAAGG-3′
10)II2CF：5′-TTTGGTGGTAGTGGGAGT-3′
II2CR：5′-GTGTCTGCATGGCTGTAA-3′
11)II3AF：5′-CCTAACAAGAAAGGAACAG-3′
II3AR：5′-TTTCAGGACATCAGCACC-3′
12)II3HF：5′-CCTCCTCCTGCCACTCCTCT-3′
II3HR：5′-CCCGTCGTCTCCTCAGTTCTC-3′
13)II6AF：5′-AGTTACTTATCTCCGCATTC-3′
II6AR：5′-GACCATTTTACCCTTTCA-3′
14)II8AF：5′-AAGATATGCTGTTGAGTTT-3′
II8AR：5′-TTCACAAAAGAAAGGAAG-3′
15)II12AF：5′-CAACAGTAAAATTGGAGC-3′
II12AR：5′-AGTAGTGATTCGGGTGGA-3′
Aforesaid dna molecular marker; It is selected from the nucleotide sequence that numbering is respectively I1G, I2B, I2G, I2H, I3G, I9H, I10H, I12H, II1C, II2C, II3A, II3H, II6A, II8A, II12A, and said nucleotide sequence is shown in SEQ ID No.1 to SEQ ID No.15.
The screening method of a kind of caudal lobe crape myrtle microsatellite DNA molecule marker of the present invention, it may further comprise the steps:
(1) extracts caudal lobe crape myrtle genomic dna, carry out enzyme with restriction endonuclease Rsa I and cut, obtain the dna fragmentation that size is 300～1000bp;
(2) moles such as the SuperSNX24R of oligonucleotide SuperSNX24F (5 '-GTTTAAGGCCTAGCTAGCAGAATC-3 ') and terminal phosphateization (5 '-pGATTCTGCTAGCTAGGCCTTAAACAAAA-3 ') are mixed, form joint;
(3) joint of step (2) being cut product with the enzyme of step (1) is connected;
(4) with SuperSNX24F be primer, the connection product of step (3) is a template, carries out the PCR reaction, and the enrichment size is the dna fragmentation of 300～1000bp;
(5) 20 kinds of the probes of synthetic 3 ' Biotin mark are with its [Group2=(AG) as requested
12, (TG)
12, (AAC)
6, (AAG)
8, (AAT)
12, (ACT)
12, (ATC)
8Group3=(AAAC)
6, (AAAG)
6, (AATC)
6, (AATG)
6, (ACAG)
6, (ACCT)
6, (ACTC)
6, (ACTG)
6Group4=(AAAT)
8, (AACT)
8, (AAGT)
8, (ACAT)
8, (AGAT)
8] equal-volume mixing (all concentration and probe concentration are 100 μ M), 100 times of the probe dilution that mixes is for use;
(6) dna fragmentation of step (4) is hybridized with (5) three groups of probes of step respectively;
(7) add magnetic bead in the solution after hybridization, the washing magnetic bead;
(8) add TE in the magnetic bead after washing, obtain containing the segmental supernatant of microsatellite DNA;
(9) with SuperSNX24F be primer, the supernatant of step (7) is a template, carries out the PCR reaction, obtains the purpose fragment;
(10) amplified production with step (8) is converted in the intestinal bacteria, and the screening transformant carries out pcr amplification, is that the bacterium liquid of 500～1200bp checks order to the product size, obtains 50 microsatellite sequences, wherein comprises 60 of microsatellite locus;
(11) be that template is carried out the SSR-PCR amplification according to the flanking sequence design primer at microsatellite locus two ends and with 8 strain caudal lobe crape myrtle genes of individuals group DNA; Obtain 15 of caudal lobe crape myrtle microsatellite markers; Its numbering is respectively I1G, I2B, I2G, I2H, I3G, I9H, I10H, I12H, II1C, II2C, II3A, II3H, II6A, II8A, II12A, and its nucleotide sequence is shown in SEQ ID No.1 to SEQ ID No.15.
Utilize the genetic differences between caudal lobe crape myrtle microsatellite DNA molecular marker analysis caudal lobe crape myrtle individuality of the present invention, specific analytical method is:
(1) respectively 5 ' end of the forward primer of the Auele Specific Primer in I1G, I2B, I2G, I2H, I3G, I9H, I10H, I12H, II1C, II2C, II3A, II3H, II6A, II8A, II12A site is carried out 6-FAM or JOE mark;
(2) genomic dna of extraction caudal lobe crape myrtle Different Individual;
(3) respectively with the Auele Specific Primer in the I1G behind the mark, I2B, I2G, I2H, I3G, I9H, I10H, I12H, II1C, II2C, II3A, II3H, II6A, II8A, II12A site, the genomic dna of the 20 strain caudal lobe crape myrtles of increasing.
The sequence of said Auele Specific Primer is respectively:
I1GF：5′-
JOEGTCACAGGTTACCGAATC-3′
I1GR：5′-ATGTAAATGGTGAGGAGG-3′
I2BF：5′-
6-FAMTTCTTGTCTTGGGTATCGC-3′
I2BR：5′-GAGCCAGTATTGTCTTCACG-3′
I2GF：5′-
JOETTCTTCCACTTCCTCCTT-3′
I2GR：5′-CAGCCCACATTAACTTTT-3′
I2HF：5′-
6-FAMAAAGACGCAGAAGGATGG-3′
I2HR：5′-CGATTAGTTTCAGCTCGT-3′
I3GF：5′-
6-FAMATGTGGTGAATGGGAACT-3′
I3GR：5′-TTGGGCTAAAGATATGGA-3′
I9HF：5′-
6-FAMGGACCAGATTGTAAATGC-3′
I9HR：5′-CTGCTCCTAATATCAGTGTC-3′
I10HF：5′-
JOEATTCGATCTGCCCTCTTG-3′
I10HR：5′-ACTCGGGTTTACGTGGTG-3′
I12HF：5′-
6-FAMTCCCTTTAACGAGGAATG-3′
I12HR：5′-AAGTTTCTCGACGGCTTT-3′
II1CF：5′-
JOEGTGCTGGAGGAACTGCTA-3′
II1CR：5′-CACGCTATTCTAAGACAAGG-3′
II2CF：5′-
6-FAMTTTGGTGGTAGTGGGAGT-3′
II2CR：5′-GTGTCTGCATGGCTGTAA-3′
II3AF：5′-
6-FAMCCTAACAAGAAAGGAACAG-3′
II3AR：5′-TTTCAGGACATCAGCACC-3′
II3HF：5′-
JOECCTCCTCCTGCCACTCCTCT-3′
II3HR：5′-CCCGTCGTCTCCTCAGTTCTC-3′
II6AF：5′-
6-FAMAGTTACTTATCTCCGCATTC-3′
II6AR：5′-GACCATTTTACCCTTTCA-3′
II8AF：5′-
JOEAAGATATGCTGTTGAGTTT-3′
II8AR：5′-TTCACAAAAGAAAGGAAG-3′
II12AF：5′-
6-FAMCAACAGTAAAATTGGAGC-3′
II12AR：5′-AGTAGTGATTCGGGTGGA-3′
(4) characteristic of analysis PCR product and caudal lobe crape myrtle DNA polymorphic site.
The invention has the advantages that; The primer sequence and the amplification method of the microsatellite locus of 15 caudal lobe crape myrtles and these 15 microsatellite locus that increase are provided; Set up the microsatellite DNA molecular marking technique system of caudal lobe crape myrtle; And utilizing these marks to carry out caudal lobe crape myrtle analysis of genetic diversity, fingerprint map construction and molecular mark, good reproducibility is a kind of reliable and effective dna molecular marker.
Description of drawings
Fig. 1 is the STR somatotype figure of 20 caudal lobe crape myrtles of primer amplification genomic dna in I1G of the present invention site;
Fig. 2 is the STR somatotype figure of 20 caudal lobe crape myrtles of primer amplification genomic dna in I2B of the present invention site;
Fig. 3 is the STR somatotype figure of 20 caudal lobe crape myrtles of primer amplification genomic dna in I2G of the present invention site;
Fig. 4 is the STR somatotype figure of 20 caudal lobe crape myrtles of primer amplification genomic dna in I2H of the present invention site;
Fig. 5 is the STR somatotype figure of 20 caudal lobe crape myrtles of primer amplification genomic dna in I3G of the present invention site;
Fig. 6 is the STR somatotype figure of 20 caudal lobe crape myrtles of primer amplification genomic dna in I9H of the present invention site;
Fig. 7 is the STR somatotype figure of 20 caudal lobe crape myrtles of primer amplification genomic dna in I10H of the present invention site;
Fig. 8 is the STR somatotype figure of 20 caudal lobe crape myrtles of primer amplification genomic dna in I12H of the present invention site;
Fig. 9 is the STR somatotype figure of 20 caudal lobe crape myrtles of primer amplification genomic dna in II1C of the present invention site;
Figure 10 is the STR somatotype figure of 20 caudal lobe crape myrtles of primer amplification genomic dna in II2C of the present invention site;
Figure 11 is the STR somatotype figure of 20 caudal lobe crape myrtles of primer amplification genomic dna in II3A of the present invention site;
Figure 12 is the STR somatotype figure of 20 caudal lobe crape myrtles of primer amplification genomic dna in II3H of the present invention site;
Figure 13 is the STR somatotype figure of 20 caudal lobe crape myrtles of primer amplification genomic dna in II6A of the present invention site;
Figure 14 is the STR somatotype figure of 20 caudal lobe crape myrtles of primer amplification genomic dna in II8A of the present invention site;
Figure 15 is the STR somatotype figure of 20 caudal lobe crape myrtles of primer amplification genomic dna in II12A of the present invention site;
Wherein, among Fig. 1～Figure 15: 1～20 representes 20 different caudal lobe crape myrtle individual plants respectively, and X-coordinate is represented the amplified production size, ordinate zou representative amplification intensity.
Embodiment
Following examples are used to explain the present invention, but are not used for limiting scope of the present invention.
The structure in the little satellite of embodiment 1 caudal lobe crape myrtle library
Comprise the steps:
(1) extracts caudal lobe crape myrtle genomic dna (DNA secure Plant Kit, biological), carry out enzyme with restriction endonuclease Rsa I and Xmn I and cut available from the sky root; The enzyme system of cutting is: 2.5 μ L connect damping fluid (NEB), 0.25 μ L, 100 * BSA (NEB), 0.25 μ L NaCl (5M); 1 μ LRsa I (NEB), 1 μ L Xmn I (NEB), 20 μ L DNA (100ng/L); In 37 ℃, enzyme was cut 40 hours, obtained the dna fragmentation that size is 300～1000bp;
(2) SuperSNX24R of 10 μ M oligonucleotide SuperSNX24F (5 '-GTTTAAGGCCTAGCTAGCAGAATC-3 ') and terminal phosphateization (5 '-pGATTCTGCTAGCTAGGCCTTAAACAAAA-3 ') equal-volume is mixed; 95 ℃ of sex change 10 minutes; Slowly cool to room temperature, form joint;
(3) the joint 7 μ L of step (2), T4DNA ligase enzyme (NEB) 2 μ L and 10 * be connected damping fluid 1 μ L are mixed after, the enzyme that joins (1) is cut in the product, 22 ℃ connect 2 hours after, place 4 ℃ to connect more than 40 hours;
(4) with SuperSNX24F be primer, the connection product of step (3) is a template, carries out the PCR reaction, and system is 2.5 μ L, 10 * PCR damping fluid, 2.5 μ L BSA, 1.3 μ LSuperSNX24F (10 μ M), 1.5 μ L dNTPs (each 2.5mM), 2 μ L MgCl
2(25mM), 13 μ L dH
2O, 0.2 μ L Taq enzyme (5U/ μ L), 2 μ L connect product, and response procedures is 95 ℃ of 2min; 95 ℃ of 20sec, 60 ℃ of 20sec, 72 ℃ of 1.5min, 35 circulating reactions; 15 ℃ of insulations.With 2% agarose gel electrophoresis detection reaction product, the enrichment size is the dna fragmentation of 300～1000bp;
(5) 20 kinds of the probes of synthetic 3 ' Biotin mark are divided into three groups, respectively according to [Group2=(AG)
12, (TG)
12, (AAC)
6, (AAG)
8, (AAT)
12, (ACT)
12, (ATC)
8Group3=(AAAC)
6, (AAAG)
6, (AATC)
6, (AATG)
6, (ACAG)
6, (ACCT)
6, (ACTC)
6, (ACTG)
6Group4=(AAAT)
8, (AACT)
8, (AAGT)
8, (ACAT)
8, (AGAT)
8] equal-volume mixing (all concentration and probe concentration are 100 μ M), 100 times of the probe dilution that mixes is for use;
(6) dna fragmentation of step (4) is hybridized with three groups of probes of step (5) respectively; Crossover process is: with 25 μ L, 2 * hybridization solution (12 * SSC, 0.2%SDS), the biotin labeled one group of probe of 10 μ L, the dna fragmentation of 101 μ L steps (4), 5 μ L dH
2O mixes, and in the PCR appearance, hybridizes; The hybridization program is: 95 ℃ of 5min, cool to 70 ℃ then rapidly, and the speed with 0.04 ℃/sec cools to 50 ℃ again, and at 50 ℃ of insulation 10min, the speed with 0.1 ℃/sec cools to 40 ℃ again, cools to 15 ℃ of insulations at last rapidly then;
(7) (Invitrogen), room temperature jog 30 minutes separates magnet stand to the magnetic bead after the adding 50 μ L balances, removes solution for 10mg/mL, Dynabeads M-280;
(8) with 400 μ L W1 washing lotions (2 * SSC, cleaning magnetic bead 0.1%SDS) 2 times is abandoned solution; (1 * SSC 0.1%SDS) cleans magnetic bead 2 times, abandons solution with 400 μ L W2 washing lotions; Add 400 μ L W2 washing lotions, 40 ℃ of jogs 2 minutes are abandoned solution; Add 400 μ LW2 washing lotions, 50 ℃ of jogs 2 minutes are abandoned solution; Add 400 μ L W2 washing lotions, 45 ℃ of jogs 2 minutes are abandoned solution;
(9) repeating step (8) once;
(10) add 200 μ L TE, 95 ℃ are incubated 5 minutes, and magnet stand obtains containing the segmental supernatant of microsatellite DNA after separating;
(11) with SuperSNX24F be primer, the supernatant of step (10) is a template, carries out the PCR reaction according to the reaction system of step (4) and response procedures, obtains the purpose fragment;
(12) amplified production with step (11) is connected on pCR2.1-TOPO (the InvitrogenTOPO Cloning Kit) carrier; To connect product transformed into escherichia coli TOP10 competent cell then, and transformed bacteria liquid will be coated on carry out blue hickie screening on the LB solid medium flat board that contains 50 μ g/mL penbritins and 40mg/mLX-gal.
Comprise the steps:
(1) with 72 white colonies of toothpick picking and be inoculated in the LB substratum that contains 50 μ g/mL penbritins, 37 ℃, 150rmp cultivated 40 hours.With bacterium liquid is template, carries out the PCR reaction;
The PCR reaction system is: 2.5 μ L, 250 μ g/mL BSA; 2.5 μ L 10 * PCR damping fluid; 1 μ L 10mM primer M13F:5 '-GTAAAACGACGGCCAG-3 ', 1 μ L 10mM primer M13R:5 '-CAGGAAACAGCTATGAC-3 ', 2 μ L 25mM MgCl
2, 1.5 μ L 2.5mM dNTP, 0.2 μ L TaqDNA polysaccharase, 1.5 μ L bacterium liquid, 12.8 μ LdH
2O; Response procedures: 95 ℃ of 3min; 95 ℃ of 20sec, 50 ℃ of 20sec, 72 ℃ of 1.5min, 35 circulating reactions; 15 ℃ of insulations;
(2) with 1.5% agarose gel electrophoresis detection reaction product, it is single to choose the PCR band, and the product size is that the bacterium liquid of 500～1200bp checks order, and obtains 50 microsatellite sequences, wherein comprises 60 of microsatellite locus;
(3) be that template is carried out the SSR-PCR amplification according to the flanking sequence design primer at microsatellite locus two ends and with 8 strain caudal lobe crape myrtle genes of individuals group DNA, 10 μ L reaction systems are for comprising the 10ng genomic dna, 1 * PCR damping fluid (10mM Tris-HCl; 50mM KCl, pH 8.3), dNTPs (each 250 μ M); MgCl2 (1.5mM); Forward and reverse primer (each 0.5 μ M), Taq enzyme (0.5U), response procedures are 95 ℃ of 3min; 95 ℃ of 30sec, optimum annealing temperature (48～60 ℃) 30sec, 72 ℃ of 1min, 30 circulating reactions; 72 ℃ of 5min, 15 ℃ of insulations.Each PCR product is detected each primer polymorphum on 6% denaturing polyacrylamide gel; Filter out the amplification of non-specific band less, 15 of the microsatellite markers of stable, the rich polymorphism of result; Be respectively I1G, I2B, I2G, I2H, I3G, I9H, I10H, I12H, II1C, II2C, II3A, II3H, II6A, II8A, II12A, its nucleotide sequence is shown in SEQ ID No.1 to SEQ ID No.15.
Specific analytical method is:
(1) respectively 5 ' end of the forward primer of the Auele Specific Primer in I1G, I2B, I2G, I2H, I3G, I9H, I10H, I12H, II1C, II2C, II3A, II3H, II6A, II8A, II12A site is carried out 6-FAM or JOE mark;
(2) genomic dna of the different individual plants of extraction caudal lobe crape myrtle (DNA secure PlantKit, biological) available from the sky root;
(3) use the Auele Specific Primer in I1G, I2B, I2G, I2H, I3G, I9H, I10H, I12H, II1C, II2C, II3A, II3H, II6A, II8A, II12A site respectively, the individual genomic dna of 20 caudal lobe crape myrtles increases.10 μ L reaction systems are for comprising the 10ng genomic dna, 1 * PCR damping fluid (pH 8.3 for 10mM Tris-HCl, 50mM KCl), dNTPs (each 250 μ M), MgCl
2(1.5mM), forward and reverse primer (each 0.5 μ M), Taq enzyme (0.5U), response procedures are 95 ℃ of 3min; 95 ℃ of 30sec, optimum annealing temperature (48～60 ℃) 30sec, 72 ℃ of 1min, 30 circulating reactions; 72 ℃ of 5min, 15 ℃ of insulations;
The sequence of said Auele Specific Primer is respectively:
I1GF：5′-
JOEGTCACAGGTTACCGAATC-3′
I1GR：5′-ATGTAAATGGTGAGGAGG-3′
I2BF：5′-
6-FAMTTCTTGTCTTGGGTATCGC-3′
I2BR：5′-GAGCCAGTATTGTCTTCACG-3′
I2GF：5′-
JOETTCTTCCACTTCCTCCTT-3′
I2GR：5′-CAGCCCACATTAACTTTT-3′
I2HF：5′-
6-FAMAAAGACGCAGAAGGATGG-3′
I2HR：5′-CGATTAGTTTCAGCTCGT-3′
I3GF：5′-
6-FAMATGTGGTGAATGGGAACT-3′
I3GR：5′-TTGGGCTAAAGATATGGA-3′
I9HF：5′-
6-FAMGGACCAGATTGTAAATGC-3′
I9HR：5′-CTGCTCCTAATATCAGTGTC-3′
I10HF：5′-
JOEATTCGATCTGCCCTCTTG-3′
I10HR：5′-ACTCGGGTTTACGTGGTG-3′
I12HF：5′-
6-FAMTCCCTTTAACGAGGAATG-3′
I12HR：5′-AAGTTTCTCGACGGCTTT-3′
II1CF：5′-
JOEGTGCTGGAGGAACTGCTA-3′
II1CR：5′-CACGCTATTCTAAGACAAGG-3′
II2CF：5′-
6-FAMTTTGGTGGTAGTGGGAGT-3′
II2CR：5′-GTGTCTGCATGGCTGTAA-3′
II3AF：5′-
6-FAMCCTAACAAGAAAGGAACAG-3′
II3AR：5′-TTTCAGGACATCAGCACC-3′
II3HF：5′-
JOECCTCCTCCTGCCACTCCTCT-3′
II3HR：5′-CCCGTCGTCTCCTCAGTTCTC-3′
II6AF：5′-
6-FAMAGTTACTTATCTCCGCATTC-3′
II6AR：5′-GACCATTTTACCCTTTCA-3′
II8AF：5′-
JOEAAGATATGCTGTTGAGTTT-3′
II8AR：5′-TTCACAAAAGAAAGGAAG-3′
II12AF：5′-
6-FAMCAACAGTAAAATTGGAGC-3′
II12AR：5′-AGTAGTGATTCGGGTGGA-3′
The annealing temperature of said Auele Specific Primer is respectively: 50 ℃, 50 ℃, 50 ℃, 50 ℃, 50 ℃, 50 ℃, 50 ℃, 50 ℃, 52 ℃, 54 ℃, 52 ℃, 60 ℃, 48 ℃, 52 ℃, 54 ℃.
(4) the PCR product is carried out the str locus somatotype with ABI377 genetic analyzer (Applied Biosystem company), use the allelic concrete numerical value of GeneMapper 4.0 softwares (Applied Biosystem company) interpretation;
(5) use the value of Popgen 1.32 calculation expectation heterozygosities, observation heterozygosity, polymorphic quantity of information to describe the characteristic of caudal lobe crape myrtle DNA polymorphic site, the result is as shown in table 1:
The characteristic in table 115 a caudal lobe crape myrtle microsatellite polymorphism site
Can find out that by Fig. 1～15 15 microsatellite sequence length of the present invention show variety in 20 caudal lobe crape myrtle kinds that supply examination, this shows that microsatellite marker of the present invention can be used for the fingerprint map construction and the genetic affinity analysis of caudal lobe crape myrtle kind.
Though, the present invention has been done detailed description in the preceding text with general explanation and specific embodiments, on basis of the present invention, can to some modifications of do or improvement, this will be apparent to those skilled in the art.Therefore, these modifications or the improvement on the basis of not departing from spirit of the present invention, made all belong to the scope that requirement of the present invention is protected.
Sequence table
< 110>Beijing Forestry University
< 120>a kind of caudal lobe crape myrtle microsatellite DNA molecule marker and application
<130>KHP09113884.4
<160>49
<170>PatentIn?version?3.5
<210>1
<211>627
<212>DNA
<213>Lagerstroemia?caudata
<400>1
tttaagctgg?cgtggagctg?atgtggcact?gacatggcac?taaattgtct?tagtgccacg 60
tcagcgaaaa?gactcgatgt?tataatgtgg?attacatata?ggacttgatt?taacactttg 120
aaacatttag?gacttacctg?caaggatcgt?caaacatata?ggactaccga?tgtcattctc 180
ccatatactt?ttttattttc?tcttcgttga?atcctctctt?aacttacgag?tcttttttta 240
acttaaggca?tacaaaaaaa?gatgtctaaa?aaaatgtgaa?tgcttgcttg?accaaatcat 300
catattatgc?ggccaatgga?tgtcacaggt?taccgaatcg?ggttaaacaa?actgtgctgt 360
gaaactaatg?cgtctatatt?taattgatca?atcaatcaat?caatcaatcg?aactgcttaa 420
caatacgaat?atttttttaa?aaaaaagaag?acttttgttg?acttatatac?aaatttaggg 480
tttacaatca?acgattgccc?atcagataca?taaccctacc?tcgaagaaaa?ctttgatgtc 540
atctttgtgt?tcgatccctc?ctcaccattt?acataagtcc?ctattttctt?ttccatgaca 600
acgagaaaca?ttttaccaat?ttccggt 627
<210>2
<211>530
<212>DNA
<213>Lagerstroemia?caudata
<400>2
ccatcttcgc?tcaccttttc?gacctcaata?gcttggcctc?tacactacag?ctaaaggcac 60
gggatcttgg?tttttggtgc?tccagccatg?tattcttgtc?ttgggtatcg?ctacactatg 120
caatgcaatg?caatagtaat?cagctttcag?tcacatgcct?gatctcaata?ctacgtgctg 180
gactgactga?cttgactgac?ttttagccaa?ttgtgcctgc?cttccttcct?tccttccttc 240
cttccttctt?aggacaataa?aaagaaatca?tgggcttgtt?actattgcct?ttgcctacgg 300
cgtgaagaca?atactggctc?ccgagggaca?attagatgct?gttgttcaca?gttactaaca 360
tttcacttga?caaattaaaa?gaacaaggtg?agtgaaatag?actttggctc?tactcacaac 420
agctcatatc?catttccatc?tccacgcaaa?tattattcaa?aagatcaacc?ccaagcaagg 480
caatgcagca?caaaatagtc?aacactgttt?tatttgagaa?tgtcccccgt 530
<210>3
<211>244
<212>DNA
<213>Lagerstraemia?caudata
<400>3
tcttcaaact?tgggagtctt?cttccacttc?ctccttaact?cctcgacgtt?tcagagggag 60
gattaattca?tcggttggtg?tagtattgag?gttcagaggc?gatacattaa?tgtatactac 120
tattatacaa?tacagtagtc?agagagagag?agagagagag?agagggagag?aattttcaca 180
atcacatttc?acagtagaga?gaaaaaaagt?taatgtgggc?tgacagagag?agagagagtt 240
gagt 244
<210>4
<211>658
<212>DNA
<213>Lagerstroemia?caudata
<400>4
tctggaggag?catgtgtgct?gaatggacat?acaatattca?caccttgtgc?aggaggttaa 60
gggagtctga?gcttcctctg?agtgcatctc?acgaaagacg?cagaaggatg?gagatctctt 120
ctgtggtttc?taacgttgga?gtctgtttct?ctatgctcag?aaaaaggaaa?aaaaaagcca 180
aaacagagag?agaaagagag?agagagagag?agagagagag?agagagagag?agaggtgttt 240
tcaaaggttc?agtgagggac?caaaaatggg?agacagttca?cacactgatg?gaaaggagag 300
acatacagta?gaaagggaag?atgggaaggg?aggggagaga?gtggaatggg?agaaaatatt 360
ggacgactgt?ttttgttttc?tgctttttag?aattttgtat?gcccagctga?tctttttttc 420
cccttcaatt?tgttgatttt?ttctttcttt?tttcatgatt?tacttctaat?aattatcgtg 480
agatattctt?agcatacgag?ctgaaactaa?tcgtatgaga?gtcggcttaa?cccgaaccac 540
gagatataag?actattttca?ttcactcaat?tttgcatatg?tttttttttt?tttgtagaaa 600
tatacgtaaa?gaatcaaagt?atatgattgg?tttcaagggc?aatgcatata?cagtgtgt 658
<210>5
<211>568
<212>DNA
<213>Lagerstroemia?caudata
<400>5
acgccattca?aaatgttttc?gccaggtatt?gcctattcgc?tgactcctcc?atttattaat 60
ttttcaattc?attctgcagt?ttagttttgt?tgttctctct?ctctctctct?ttcatatcta 120
tcttgttatg?tattattact?tatttatcaa?aaattgataa?ttataagtgg?aatgtaatta 180
tgcatttggg?aatgtggtga?atgggaactg?agaaggtgac?ccgatcagag?aggagtttgt 240
ttgaataata?aagatgggta?tgatgatgat?atataaccaa?ccttatccgg?tcttctgata 300
ggcagctgtg?gctgtagttg?tagctctgtt?tttccatatc?tttagcccaa?aatgaggaga 360
ccgatgagct?gagcgtctca?gagatgctaa?ttataacaga?ttaacttaga?aaacaagaag 420
gaaaagaaag?acaactcttc?atggttggtc?ggttggttcg?ttcggttcgg?gttcagtcac 480
gagtgtttgt?tgaccattta?tgttcgccca?atttatcttt?gtccagacaa?tgtgctgtgc 540
tgggctgggc?gttgggtagg?tggggaag 568
<210>6
<211>321
<212>DNA
<213>Lagerstroemia?caudata
<400>6
acagggacca?gattgtaaat?gcacaaggac?ttgtgtgaac?tataagaaaa?ataagggggg 60
attcgtgtaa?aatcctagat?ctgagccgtt?taagcagtta?acagaggccc?tatataatga 120
tgtcaagggg?ctctgtcaca?gtgtggaata?acagatctgg?acttccctct?cattctcgag 180
ctttcttctt?ctccaaatct?ctctctctct?ctctctctct?ctctctctcg?ttctttcaat 240
cttctacgta?acatccaacg?atggtagggg?aacgacactg?atattaggag?cagccgacga 300
taggggcggc?ggtggttgca?g 321
<210>7
<211>249
<212>DNA
<213>Lagerstroemia?caudata
<400>7
cacgcacgag?atcggtcatt?cgatctgccc?tcttgagcca?tgactgactg?actgactgac 60
cgacctcgac?cgaggcgtaa?tgtctttggg?acccattttt?tttaataggt?ttttctaaac 120
ttcaaatgcc?cacaaaattc?aagggtcaca?gaaactggtc?ttttttggtc?caatagggtt 180
gatttttttc?gggttctcga?tctttagagt?tttttacgcc?actaacatca?acaccacgta 240
aacccgagt 249
<210>8
<211>315
<212>DNA
<213>Lagerstroemia?caudata
<400>8
acgtgttaaa?gaaaatcagt?cagtttgaat?agaaattgag?tcttacaata?gtgaattctt 60
ctgtttatac?atattccctt?taacgaggaa?tgacgaaaga?attgaattca?agccatgaat 120
aaaattaaca?ccttcttctt?cttcttcttc?ttcttcctcc?tccgcctctt?cttcttcttc 180
ttcctcctgg?gcaaagccgt?cgagaaactt?aacccatatt?ttccaagaaa?cggcacccaa 240
atttccattg?aaaagctgaa?agcataaaac?gacagcattg?tcgaatcaag?actgttgttc 300
tcttcgggtg?gtgaa 315
<210>9
<211>384
<212>DNA
<213>Lagerstroemia?caudata
<400>9
acagctggag?tgagggaggt?gatattcttc?attttagggt?tctgtaattt?tctacattt?t 60
ctagccctaa?ttatcgagcg?ggttgattgg?tttgaaaaag?tttcaaatct?ggtgctggag 120
gaactgctaa?tggttgttac?agatagatag?atagatagat?agataggtag?gccatagatc 180
gtgggactgt?aatgtcctcc?gagatctcaa?actgattcca?acttgtttcc?atccttgtct 240
tagaatagcg?tgcgttttta?atgtctggaa?agatgcaagc?gttgtttttc?tcagaacaaa 300
acttcacgac?catagctata?atcacgtgtt?atagatatcc?tggttcgtga?ttgatggatg 360
gtatcgtttt?gtggtggttg?aatg 384
<210>10
<211>438
<212>DNA
<213>Lagerstroemia?caudata
<400>10
actcaaagga?gttattatcc?caataggaac?ttgggcaata?aatttgtctc?gtggtttcac 60
tgtttggtgg?tagtgggagt?tgtttctttt?ctttcctatg?aagttccttt?gaaggtatgc 120
tgtattgtct?gtctgtctgt?ctgtctgtct?gttaggtggt?gtactgaatt?cttgattttg 180
aggttgcttt?ctcactttat?atatgctgca?atttcaatct?cttgcattta?tgtttttttt 240
tttttttccc?tctgcaggat?ttctccgagc?agttgtagat?cgtgtgggtc?gggagagctt 300
caaggcagag?gcatcagtct?gctcaatttg?gagttctatc?taataggatt?tacagccatg 360
cagacaccaa?aagctcggtg?agatttcttc?tttgatcacg?ttctttctca?gtatcttcag 420
aaactttatg?cttggaat 438
<210>11
<211>556
<212>DNA
<213>Lagerstroemia?caudata
<400>11
tgctgaccta?acaagaaagg?aacagaatga?aatcaagaaa?gagaaactaa?cccagataga 60
gagagagaga?gagagagagg?ttggagggaa?cagagaaaga?gacgggaaga?agatgagaag 120
aacctttaaa?tcggtgctga?tgtcctgaaa?gcagaatgaa?gcaatcaagt?aggaggagga 180
ggaggaggga?tgaacaatga?gggggagaag?agaacgaaag?tgaagggggc?caggtacaga 240
gtgattctgc?tagctaggcc?ttttcaaggg?cgaatttcga?cccattgccg?gacgtttccg 300
gcggatccga?gctcgctacg?aggttgtgct?tgtgttcctc?attgttggtg?cctgtgggga 360
atgggtatac?tctcacactt?ccattttagt?tacggatcgc?cttcttgtgt?ggatagccgt 420
tcagacttat?gcgcgagctc?catactttaa?atgttctccg?ctcctgtcag?ctttggagcc 480
tttatctatg?agtttccctt?ttattgatcc?tgggcctcgt?gcgcgtcttt?ctaaccgctt 540
gttttcatgc?ccgtcg 556
<210>12
<211>347
<212>DNA
<213>Lagerstroemia?caudata
<400>12
ttttcttcat?cttcttgagc?atgttcgtcc?ctcctcctgc?cactcctctt?ctcactgtta 60
tccttagtag?gagacctgaa?ggccctcttg?acaagggtca?accaagaaga?agaagatgaa 120
gaagaggtgg?aggaagaggt?agaggatgaa?gaggtagacc?caatcttctt?tcccataatg 180
aaaggaagtc?acaccagaag?ctgagaactg?aggagacgac?gggctccttt?caccatttga 240
ggaatatatc?agtgagtgag?ggagttgagg?gtccccccga?agagcagtgg?aagaagaaga 300
acaagaaaag?agaagtgagt?tatcatggtg?aggagtggga?acagagt 347
<210>13
<211>283
<212>DNA
<213>Lagerstroemia?caudata
<400>13
acttttcatc?tgaagaatga?ctagtactat?atgagtttgg?tgttgaacat?tagttgtgca 60
gcaccataaa?taactattgc?tcagttactt?atctccgcat?tccttaaatc?aatctctctc 120
tctctctctc?tctctctctc?tctcttttac?gaaaacatgc?tttctttttc?ctcctttgct 180
ttttttgttt?cctggattca?agttgaggtg?gcttttgacc?tttgaattaa?aatgacattt 240
gaaagggtaa?aatggtctct?ggagattgat?gtgattgagt?gcc 283
<210>14
<211>394
<212>DNA
<213>Lagerstroemia?caudata
<400>14
tgttcatggc?tcttcacacc?taagactgcc?tctgatgatt?acctcttttc?atccgtgctc 60
ccccgatcct?ctagttctta?ctttcaagat?ctctgccagt?attctcgtga?ggatcatcct 120
tccttgccca?gtataaaaag?aacgagagag?agaagggaaa?tattgccgga?gacatccctt 180
gaaacgaaag?atatgctgtt?gagtttgcat?ttttggcgaa?atcgaagttg?ctataagtta 240
ctatgtatat?ttgttgcgac?ctgtcattac?tgcaaggagc?ctttacttgc?tccatgttga 300
aaagtttgtt?ctactcccag?cattctttaa?gagaacttct?tgcatcttac?cgaactttct 360
ttctttcttt?ctttcttcct?ttcttttgtg?aaat 394
<210>15
<211>212
<212>DNA
<213>Lagerstroemia?caudata
<400>15
ccttcaacag?taaaattgga?gctcctttct?ttctttcttt?ctttctttct?ttccatggtt 60
tccacggaca?tggaagatga?gacggaactt?agtttggata?gaggggccta?ctccatagcg 120
tgacactgga?tccacccgaa?tcactactct?tcaattcaat?caagtcaggt?gactgataaa 180
attttggatt?tgcacattcg?atgtgaacat?gg 212
<210>16
<211>24
<212>DNA
< 213>artificial sequence
<400>16
gtttaaggcc?tagctagcag?aatc 24
<210>17
<211>28
<212>DNA
< 213>artificial sequence
<400>17
gattctgcta?gctaggcctt?aaacaaaa 28
<210>18
<211>16
<212>DNA
< 213>artificial sequence
<400>18
gtaaaacgac?ggccag 16
<210>19
<211>17
<212>DNA
< 213>artificial sequence
<400>19
caggaaacag?ctatgac 17
<210>20
<211>18
<212>DNA
< 213>artificial sequence
<400>20
gtcacaggtt?accgaatc 18
<210>21
<211>18
<212>DNA
< 213>artificial sequence
<400>21
atgtaaatgg?tgaggagg 18
<210>22
<211>19
<212>DNA
< 213>artificial sequence
<400>22
ttcttgtctt?gggtatcgc 19
<210>23
<211>20
<212>DNA
< 213>artificial sequence
<400>23
gagccagtat?tgtcttcacg 20
<210>24
<211>18
<212>DNA
< 213>artificial sequence
<400>24
ttcttccact?tcctcctt 18
<210>25
<211>18
<212>DNA
< 213>artificial sequence
<400>25
cagcccacat?taactttt 18
<210>26
<211>18
<212>DNA
< 213>artificial sequence
<400>26
aaagacgcag?aaggatgg 18
<210>27
<211>18
<212>DNA
< 213>artificial sequence
<400>27
cgattagttt?cagctcgt 18
<210>28
<211>18
<212>DNA
< 213>artificial sequence
<400>28
atgtggtgaa?tgggaact 18
<210>29
<211>18
<212>DNA
< 213>artificial sequence
<400>29
ttgggctaaa?gatatgga 18
<210>30
<211>18
<212>DNA
< 213>artificial sequence
<400>30
ggaccagatt?gtaaatgc 18
<210>31
<211>20
<212>DNA
< 213>artificial sequence
<400>31
ctgctcctaa?tatcagtgtc 20
<210>32
<211>18
<212>DNA
< 213>artificial sequence
<400>32
attcgatctg?ccctcttg 18
<210>33
<211>18
<212>DNA
< 213>artificial sequence
<400>33
actcgggttt?acgtggtg 18
<210>34
<211>18
<212>DNA
< 213>artificial sequence
<400>34
tccctttaac?gaggaatg 18
<210>35
<211>18
<212>DNA
< 213>artificial sequence
<400>35
aagtttctcg?acggcttt 18
<210>36
<211>18
<212>DNA
< 213>artificial sequence
<400>36
gtgctggagg?aactgcta 18
<210>37
<211>20
<212>DNA
< 213>artificial sequence
<400>37
cacgctattc?taagacaagg 20
<210>38
<211>18
<212>DNA
< 213>artificial sequence
<400>38
tttggtggta?gtgggagt 18
<210>39
<211>18
<212>DNA
< 213>artificial sequence
<400>39
gtgtctgcat?ggctgtaa 18
<210>40
<211>19
<212>DNA
< 213>artificial sequence
<400>40
cctaacaaga?aaggaacag 19
<210>41
<211>18
<212>DNA
< 213>artificial sequence
<400>41
tttcaggaca?tcagcacc 18
<210>42
<211>20
<212>DNA
< 213>artificial sequence
<400>42
cctcctcctg?ccactcctct 20
<210>43
<211>21
<212>DNA
< 213>artificial sequence
<400>43
cccgtcgtct?cctcagttct?c 21
<210>44
<211>20
<212>DNA
< 213>artificial sequence
<400>44
agttacttat?ctccgcattc 20
<210>45
<211>18
<212>DNA
< 213>artificial sequence
<400>45
gaccatttta?ccctttca 18
<210>46
<211>19
<212>DNA
< 213>artificial sequence
<400>46
aagatatgct?gttgagttt 19
<210>47
<211>18
<212>DNA
< 213>artificial sequence
<400>47
ttcacaaaag?aaaggaag 18
<210>48
<211>18
<212>DNA
< 213>artificial sequence
<400>48
caacagtaaa?attggagc 18
<210>49
<211>18
<212>DNA
< 213>artificial sequence
<400>49
agtagtgatt?cgggtgga 18
Claims (3)
1. a caudal lobe crape myrtle microsatellite DNA molecule marker is characterized in that it is the nucleotide sequence shown in SEQID No.3.
2. the primer of the described dna molecular marker of amplification claim 1 is right:
I2GF：5′-TTCTTCCACTTCCTCCTT-3′
I2GR：5′-CAGCCCACATTAACTTTT-3′。
3. the application of the described dna molecular marker of claim 1 in screening of caudal lobe crape myrtle or assistant breeding.
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