CN109280694A - Curcuma alismatifolia polymorphism SSR primer and kit based on the sequencing exploitation of overall length transcript profile - Google Patents
Curcuma alismatifolia polymorphism SSR primer and kit based on the sequencing exploitation of overall length transcript profile Download PDFInfo
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Abstract
The invention discloses a kind of curcuma alismatifolia polymorphism SSR primers and kit based on the sequencing exploitation of overall length transcript profile.The primer includes 20 pairs of primers, and as described in SEQ ID NO.1-SEQ ID NO.40, a kind of kit that identifying curcuma alismatifolia affiliation includes the primer.20 pairs of primers provided by the invention can be applied between more curcuma alismatifolia kinds and kind in-group, carries out the analysis of cultivar identification, Genetic Diversity of Germplasm and affiliation, applies also for the hair molecular mark of curcuma alismatifolia.
Description
Technical field
The present invention relates to SSR primers, and the curcuma alismatifolia polymorphism SSR specifically based on the sequencing exploitation of overall length transcript profile draws
Object and kit, belong to field of biotechnology.
Background technique
Curcuma alismatifolia originates in Thailand, has the characteristics that flower pattern is unique, pattern is gorgeous, the florescence is long, Vase time is long, due to not
It educates bract likeness in form lotus and for Zingiber, therefore referred to as curcuma alismatifolia, has lotus and the dual beauty of Radix Curcumae, also known as tropical tulip
(summer tulip).In recent years, China had also introduced curcuma alismatifolia, and gradually applied on cut-flower, potted flower and gardens presbyopic glasses.By
In curcuma alismatifolia be novel flower and ornamental value height is favored by people, and more and more foreign countries' kinds are introduced into, many germplasm moneys
It is tamed increasingly to adapt to domestic climatic environment at home in source.Numerous variety sources there are synonym, homonym and
The unclear problem of product Interspecific relationship is unfavorable for subsequent curcuma alismatifolia new varieties to variety collection, preservation and using affecting
Cultivate the expansion of work.Therefore exploitation polymorphic molecular marker understands product interspecies relation in depth, for subsequent development curcuma alismatifolia kind
Matter resource innovation work is necessary.
With the development of molecular biology, molecular marking technique is widely used in Genetic Diversity of Germplasm and auxiliary is educated
In kind work.ISSR, RAPD, AFLP codominance label are in spite of without knowing genomic information, advantage at low cost, but with
Machine is strong, and stability is poor.Compared with other molecular labelings, SSR marker has the advantages that codominance, reproducible, be at present into
The first choice of row analysis of genetic diversity and genetic map construction etc..
The SSR application of curcuma alismatifolia is seldom at present, there are no the SSR marker developed specifically for the species, only one
The hereditary difference between curcuma alismatifolia original kind and gamma ray mutating variety is detected using SSR marker, but the primer is to use for reference
Belong to medicinal plant turmeric (Curcuma longa).Since the species specificity of SSR marker is strong, for the exploitation of this species
Label has better stability and higher polymorphism certainly.
Summary of the invention
For overcome the deficiencies in the prior art, it is more to provide a kind of curcuma alismatifolia based on the sequencing exploitation of overall length transcript profile by the present invention
State property SSR primer and kit.
A kind of curcuma alismatifolia polymorphism SSR primer based on the sequencing exploitation of overall length transcript profile, the primer include 20 pairs of primers,
It is respectively as follows:
P2 F:AGCAATGTTGACGAAATGGAAA (SEQ ID NO.1),
P2 R: AAGCAATCTCTTAGGAAATCAG (SEQ ID NO.2)；
P5F:CTGGTCACCCACCTAAATCCTT (SEQ ID NO.3),
P5R: ACGCCGTGGAGAAGAGGCTATT (SEQ ID NO.4)；
P6F:GGAGGGACTACGGGAAAGGGAA (SEQ ID NO.5),
P6R: GCAGGACAGTTACGATTGATGA (SEQ ID NO.6)；
P7F:GAAAGCGACAAGATGCTGAGGG (SEQ ID NO.7),
P7R: ACCTTCCTCTTCTCCACCTGAT (SEQ ID NO.8)；
P9F:GAGCCAGAAGTCACAATCAGCG (SEQ ID NO.9),
P9R: TTGTCCTGGCTTCCTCTCTGGG (SEQ ID NO.10)；
P11F:CTGAGTGTAGGACTTGTCGGTG (SEQ ID NO.11),
P11R: TAGCCATTGACGAACAAAAAGC (SEQ ID NO.12)；
P12F:GAAGAGCAAAAGACAGGCAGAT (SEQ ID NO.13),
P12R: CACAGACAAAGAATGAAAACTA (SEQ ID NO.14)；
P13F:AGGGCGTCTTCAAGGTCTGGAT (SEQ ID NO.15),
P13R: ACAACAACGGCAGCAGCAGTAT (SEQ ID NO.16)；
P14F:GCCTTTGGTATCTCTGTTCTAA (SEQ ID NO.17),
P14R: TAATGCGGTAGGAGGGGAACAA (SEQ ID NO.18)；
P16F:TCTATGTTCTTCGCTACCTTCG (SEQ ID NO.19),
P16R: ACCTTTATCCTCTTGGTCTCGC (SEQ ID NO.20)；
P17F:CAAAACGAGGAAAGAAACCAAA (SEQ ID NO.21),
P17R: TATGCTTGTTGGAGGCTATCAC (SEQ ID NO.22)；
P18F:AGGGCAAAACTCAAAAGGGTAG (SEQ ID NO.23),
P18R: CTCCTTGTAGTCCTTGTCCTGC (SEQ ID NO.24)；
P19F:AAACACAGACTGCCCTCCTTGA (SEQ ID NO.25),
P19R: TTCCCATCAGAAGATAACAACG (SEQ ID NO.26)；
P21F:CGTTTCTCCAGACAATGACCGC (SEQ ID NO.27),
P21R: CGTCAACTAACTCTACGCTAAC (SEQ ID NO.28)；
P23F:CCCTGGCGTAACCTCCATTTCC (SEQ ID NO.29),
P23R: GATGGCGTGGAGTATGGCGTCT (SEQ ID NO.30)；
P25F:CCTGGCGTAACCTCCATTTCCG (SEQ ID NO.31),
P25R: GATGGCGTGGAGTATGGCGTCT (SEQ ID NO.32)；
P26F:ACTCGCCAATCTCTTACACAGC (SEQ ID NO.33),
P26R: TCGGGGTAATGGTGACGGTAAG (SEQ ID NO.34)；
P27F:TTTCACCAGGAGTAAGAGGGAT (SEQ ID NO.35),
P27R: AAACCTCAGCCTTTTCTCTTCA (SEQ ID NO.36)；
P31F:CAGCCGTTTGTTATGCTACTTA (SEQ ID NO.37),
P31R: TTGAAACTTTACCAAGAACAGG (SEQ ID NO.38)；
P33F:GTCAAACTTCTCAAACCACACG (SEQ ID NO.39),
P33R: ATCATACAATACCCTCAACAAT (SEQ ID NO.40)。
A kind of kit for identifying curcuma alismatifolia affiliation, it includes the primer.
Beneficial effects of the present invention:
20 pairs of SSR primers of the invention are obtained by 10 curcuma alismatifolia screening varieties, are all had in 10 curcuma alismatifolia kinds
Polymorphism, therefore the cultivar identification of curcuma alismatifolia can be used as, SSR marker is the new label being stabilized, and can directly be sent out this
20 primer pairs provided by bright are applied in more curcuma alismatifolia kinds, carry out cultivar identification, Relationships among Germplasm Resources and something lost
Diversity analysis is passed, is had laid a good foundation for curcuma alismatifolia molecular mark.
Detailed description of the invention
Fig. 1 is part primer electrophoretogram；
Fig. 2 is the dendrogram for 10 kinds done based on amplified band polymorphism.
Specific embodiment
One, curcuma alismatifolia ' Chiang Mai powder ' overall length transcript profile is sequenced
Sequencing material is leaf, the flower of growth conditions good one plant curcuma alismatifolia of the plantation in academy of agricultural sciences of Zhejiang Province flowers research center
Stem, green bract, pink colour bract, small petal extract the sample mixing after RNA respectively.Sequencing experiment is limited by Tianjin biochip technology
Responsible company completes, and the genetic fragment of 64 471 de-redundancy is obtained after sequencing, and sequence total length is 132 833 kb.
Two, the screening of SSR primer
The site SSR in gene order is scanned for using MISA software, parameter setting is as follows: 1 ~ 6 nucleotide at least repeats
Number is respectively 10,6,5,5,5,5.The maximum interval base number of composite S SR is 100 bp.50 pairs are designed with Primer 5.0
Primer, design of primers is according to following principle: 1, primer is located at microsatellite sequence both ends, primer length 18 bp ~ 25 bp；2, expected
Pcr amplified fragment length 100 bp ~ 250 bp；3, annealing temperature is 52 DEG C ~ 62 DEG C；4, G/C content is 40% ~ 68%；5, it avoids drawing
Occur dimer, hairpin structure and mispairing in object.
Three, the extraction of kind DNA
10 curcuma alismatifolia kind (table 1) genomic DNAs are extracted with CTAB method.
Table 1. is used for 10 kinds of SSR primer polymorphism screening
Four, agarose gel primary dcreening operation and polyacrylamide gel further screen
PCR amplification is carried out with 50 pairs of primer pairs, 10 curcuma alismatifolia kinds, amplified production is carried out just with agarose gel electrophoresis first
Step screening, there is further being detected with polyacrylamide gel electrophoresis for purpose product band.Polyacrylamide gel electrophoresis
Experimental procedure is as follows: (1) glue
By glass plate wash clean, be vertically arranged dry it is spare.Spacer bar, lamina affixad and backboard are assembled into gel interlayer device, it is horizontal
It places, clips fixation in two sides with clip.50 mL, 6 % denaturing polyacrylamide gel solution is measured, 100 mL, 20 % is added
APS and 50 mL TEMED, is poured into interlayer after mixing gently using encapsulating bottle, and the concordant end of shark tooth comb is inserted into two
5 ~ 6 mm of glue between plate, placing 1 ~ 1.5 h at room temperature makes gel sets.
(2) prerunning
After gel polymerisation, clip is unloaded, device of gel is installed on electrophoresis tank, 1 × TBE that 1 000 mL or so are added is slow
Fliud flushing, liquid level need to not cross glue surface certain altitude.Meanwhile 1 × the tbe buffer liquid for 400 mL being added in electrophoresis tank or so.It pulls out
Shark tooth comb out, opens electrophoresis apparatus, and invariable power is 85 W, 20 ~ 30 min of prerunning.
(3) it is denaturalized
The centrifugation of 5~10 mL sample-loading buffers is added in pcr amplification product to mix.After 95 DEG C of 5 min of denaturation, takes out and place rapidly
In on ice.Cooling 10 min are stand-by.
(4) electrophoresis
After prerunning, suspend electrophoresis apparatus, blow and beat glue surface with suction pipe, remove bubble and impurity, avoids blocking loading wells, take suitable
Amount denatured sample (4.5 ~ 6.5 mL) clicks and enters well, and 85 W of invariable power starts electrophoresis.After 1.5 h, electrophoresis is terminated.
(5) silver staining
The glass plate glue surface for adhering to glue is put into upward in 2 000 mL fixers, is placed on the shaking table of 50 ~ 60 rpm of revolving speed light
20 min or so are shaken, gel glass plate is taken out from fixer, rinse 3 min with deionized water.It takes out gel glass plate and is placed in 2
000 mL silver nitrate solution is placed in 30 ~ 40 min of jog on the shaking table of 50 ~ 60 rpm of revolving speed, keeps dyeing abundant.Take out gel glass
Glass plate is put into 2 000 mL deionized waters, rinses 30 ~ 60 s.Gel glass plate is taken out to be put into 2 000 mL developing solutions, in
Develop the color 4 ~ 7 min on the shaking table of 60 rpm of revolving speed, until DNA band is clear.It takes out gel glass plate and is placed in 2 000 mL distilled water
3 min of middle rinsing, are placed in dry place and dry after taking-up.Offset plate is scanned on the scanner.Part primer electrophoretogram is shown in figure
1。
One, primer polymorphism analysis
Polyacrylamide gel the result shows that, have that 20 pairs of primer polymorphisms are good, amplified band is clear.This 20 primers are counted
Amplification polymorphism result (table 2) has done the dendrogram (Fig. 2) of 10 kinds based on amplified band polymorphism.The topology of clustering tree
The affiliation that structure can embody 10 kinds is far and near, and the green angel of C7 is the kind of only one green petal, and dendrogram is aobvious
Show that the kind and other kind relationships are farthest.The nearest c2 kind sunset of relationship and c3 kind Dutch Red pattern are closest, all
It is deep rose, the kind purpurine spirit (c10) that white snow (c8) and the natural variation of the field Bai Xue screen also is demonstrated by with each other most
Close affiliation, cluster result shows that 20 pairs of primers of exploitation not only have polymorphism, but also can react relationship between kind
Relationship.
2. 20 pairs of curcuma alismatifolia SSR primer information of table
Polymorphism primer provided by the invention can be used for analysis of genetic diversity between curcuma alismatifolia kind, genetic map construction, point
Sub- marker-assisted breeding etc..
Sequence table
<110>Zhejiang Province Xiaoshan Cotton and Flax Research Institute
<120>curcuma alismatifolia polymorphism SSR primer and kit based on the sequencing exploitation of overall length transcript profile
<141> 2018-10-23
<160> 40
<170> SIPOSequenceListing 1.0
<210> 1
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
agcaatgttg acgaaatgga aa 22
<210> 2
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
aagcaatctc ttaggaaatc ag 22
<210> 3
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
ctggtcaccc acctaaatcc tt 22
<210> 4
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
acgccgtgga gaagaggcta tt 22
<210> 5
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
ggagggacta cgggaaaggg aa 22
<210> 6
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
gcaggacagt tacgattgat ga 22
<210> 7
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
gaaagcgaca agatgctgag gg 22
<210> 8
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
accttcctct tctccacctg at 22
<210> 9
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
gagccagaag tcacaatcag cg 22
<210> 10
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 10
ttgtcctggc ttcctctctg gg 22
<210> 11
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 11
ctgagtgtag gacttgtcgg tg 22
<210> 12
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 12
tagccattga cgaacaaaaa gc 22
<210> 13
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 13
gaagagcaaa agacaggcag at 22
<210> 14
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 14
cacagacaaa gaatgaaaac ta 22
<210> 15
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 15
agggcgtctt caaggtctgg at 22
<210> 16
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 16
acaacaacgg cagcagcagt at 22
<210> 17
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 17
gcctttggta tctctgttct aa 22
<210> 18
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 18
taatgcggta ggaggggaac aa 22
<210> 19
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 19
tctatgttct tcgctacctt cg 22
<210> 20
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 20
acctttatcc tcttggtctc gc 22
<210> 21
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 21
caaaacgagg aaagaaacca aa 22
<210> 22
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 22
tatgcttgtt ggaggctatc ac 22
<210> 23
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 23
agggcaaaac tcaaaagggt ag 22
<210> 24
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 24
ctccttgtag tccttgtcct gc 22
<210> 25
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 25
aaacacagac tgccctcctt ga 22
<210> 26
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 26
ttcccatcag aagataacaa cg 22
<210> 27
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 27
cgtttctcca gacaatgacc gc 22
<210> 28
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 28
cgtcaactaa ctctacgcta ac 22
<210> 29
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 29
ccctggcgta acctccattt cc 22
<210> 30
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 30
gatggcgtgg agtatggcgt ct 22
<210> 31
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 31
cctggcgtaa cctccatttc cg 22
<210> 32
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 32
gatggcgtgg agtatggcgt ct 22
<210> 33
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 33
actcgccaat ctcttacaca gc 22
<210> 34
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 34
tcggggtaat ggtgacggta ag 22
<210> 35
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 35
tttcaccagg agtaagaggg at 22
<210> 36
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 36
aaacctcagc cttttctctt ca 22
<210> 37
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 37
cagccgtttg ttatgctact ta 22
<210> 38
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 38
ttgaaacttt accaagaaca gg 22
<210> 39
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 39
gtcaaacttc tcaaaccaca cg 22
<210> 40
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 40
atcatacaat accctcaaca at 22
Claims (2)
1. a kind of curcuma alismatifolia polymorphism SSR primer based on the sequencing exploitation of overall length transcript profile, which is characterized in that the primer includes
20 pairs of primers, are respectively as follows:
P2 F:AGCAATGTTGACGAAATGGAAA (SEQ ID NO.1),
P2 R: AAGCAATCTCTTAGGAAATCAG (SEQ ID NO.2)；
P5F:CTGGTCACCCACCTAAATCCTT (SEQ ID NO.3),
P5R: ACGCCGTGGAGAAGAGGCTATT (SEQ ID NO.4)；
P6F:GGAGGGACTACGGGAAAGGGAA (SEQ ID NO.5),
P6R: GCAGGACAGTTACGATTGATGA (SEQ ID NO.6)；
P7F:GAAAGCGACAAGATGCTGAGGG (SEQ ID NO.7),
P7R: ACCTTCCTCTTCTCCACCTGAT (SEQ ID NO.8)；
P9F:GAGCCAGAAGTCACAATCAGCG (SEQ ID NO.9),
P9R: TTGTCCTGGCTTCCTCTCTGGG (SEQ ID NO.10)；
P11F:CTGAGTGTAGGACTTGTCGGTG (SEQ ID NO.11),
P11R: TAGCCATTGACGAACAAAAAGC (SEQ ID NO.12)；
P12F:GAAGAGCAAAAGACAGGCAGAT (SEQ ID NO.13),
P12R: CACAGACAAAGAATGAAAACTA (SEQ ID NO.14)；
P13F:AGGGCGTCTTCAAGGTCTGGAT (SEQ ID NO.15),
P13R: ACAACAACGGCAGCAGCAGTAT (SEQ ID NO.16)；
P14F:GCCTTTGGTATCTCTGTTCTAA (SEQ ID NO.17),
P14R: TAATGCGGTAGGAGGGGAACAA (SEQ ID NO.18)；
P16F:TCTATGTTCTTCGCTACCTTCG (SEQ ID NO.19),
P16R: ACCTTTATCCTCTTGGTCTCGC (SEQ ID NO.20)；
P17F:CAAAACGAGGAAAGAAACCAAA (SEQ ID NO.21),
P17R: TATGCTTGTTGGAGGCTATCAC (SEQ ID NO.22)；
P18F:AGGGCAAAACTCAAAAGGGTAG (SEQ ID NO.23),
P18R: CTCCTTGTAGTCCTTGTCCTGC (SEQ ID NO.24)；
P19F:AAACACAGACTGCCCTCCTTGA (SEQ ID NO.25),
P19R: TTCCCATCAGAAGATAACAACG (SEQ ID NO.26)；
P21F:CGTTTCTCCAGACAATGACCGC (SEQ ID NO.27),
P21R: CGTCAACTAACTCTACGCTAAC (SEQ ID NO.28)；
P23F:CCCTGGCGTAACCTCCATTTCC (SEQ ID NO.29),
P23R: GATGGCGTGGAGTATGGCGTCT (SEQ ID NO.30)；
P25F:CCTGGCGTAACCTCCATTTCCG (SEQ ID NO.31),
P25R: GATGGCGTGGAGTATGGCGTCT (SEQ ID NO.32)；
P26F:ACTCGCCAATCTCTTACACAGC (SEQ ID NO.33),
P26R: TCGGGGTAATGGTGACGGTAAG (SEQ ID NO.34)；
P27F:TTTCACCAGGAGTAAGAGGGAT (SEQ ID NO.35),
P27R: AAACCTCAGCCTTTTCTCTTCA (SEQ ID NO.36)；
P31F:CAGCCGTTTGTTATGCTACTTA (SEQ ID NO.37),
P31R: TTGAAACTTTACCAAGAACAGG (SEQ ID NO.38)；
P33F:GTCAAACTTCTCAAACCACACG (SEQ ID NO.39),
P33R: ATCATACAATACCCTCAACAAT (SEQ ID NO.40)。
2. a kind of kit for identifying curcuma alismatifolia affiliation, which is characterized in that it includes primer as described in claim 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811233951.8A CN109280694B (en) | 2018-10-23 | 2018-10-23 | SSR (simple sequence repeat) primers and kit for curcuma alismatifolia polymorphisms developed based on full-length transcriptome sequencing |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811233951.8A CN109280694B (en) | 2018-10-23 | 2018-10-23 | SSR (simple sequence repeat) primers and kit for curcuma alismatifolia polymorphisms developed based on full-length transcriptome sequencing |
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| Publication Number | Publication Date |
|---|---|
| CN109280694A true CN109280694A (en) | 2019-01-29 |
| CN109280694B CN109280694B (en) | 2021-09-10 |
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