CN103937786B - Jute EST microsatellite DNA mark - Google Patents
Jute EST microsatellite DNA mark Download PDFInfo
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- CN103937786B CN103937786B CN201410088219.1A CN201410088219A CN103937786B CN 103937786 B CN103937786 B CN 103937786B CN 201410088219 A CN201410088219 A CN 201410088219A CN 103937786 B CN103937786 B CN 103937786B
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Abstract
The present invention relates to a kind of molecular marking technique, be specifically related to a kind of jute EST Microsatellite DNA molecular marker.Its step includes, develops new microsatellite locus, the primer sequence of amplification microsatellite locus and microsatellite locus polymorphism analysis.The invention is characterized in that, jute EST is downloaded from GenBank public database, separate and identify 66 new microsatellite markers of jute, screen 10 microsatellite markers CcSSR008, CcSSR013, CcSSR016, CcSSR024, CcSSR029, CcSSR040, CoSSR049, CoSSR050, CoSSR052 and CoSSR053 of wherein rich polymorphism.The invention provides the new microsatellite locus of 66 jutes and expand primer sequence and the amplification method of these 66 microsatellite locus, can be applicable to the researchs such as the analysis of genetic diversity of the most of the same race of Corchorus, genetic linkage maps structure and molecular mark.
Description
Technical field
The present invention relates to a kind of molecular marking technique, be specifically related to a kind of jute EST microsatellite DNA molecule
Mark.
Background technology
The short sequence tandem sequence repeats that microsatellite (being also called simple repeated sequence, SSR) is made up of 1-6 nucleotides is repeatedly
And the section of DNA formed；Wherein, it is most commonly that dinucleotides and Trinucleotide repeats unit, such as (CT) n, (AT) n, (ACC)
N, (CGA) n and (AGA) n.Owing to microsatellite flanking sequence is the most conservative, designing primer according to its flanking sequence, available PCR expands
Increase the sequence microsatellite locus.Owing in microsatellite, the number of repetition of repetitive is different, thus the microsatellite sequence amplified
The length of row presents polymorphism.Microsatellite sequence is widely present in eukaryotic gene group, and random distribution, have polymorphic
Property higher, repeatability and the feature such as good stability, be highly effective molecular labeling, be widely used in genetic map construction,
The fields such as the assignment of genes gene mapping, genetic affinity analysis, cultivar identification.As by the Origin and evoluation relation of Microsatellite marker analysis jute
(Kundu etc., 2013, Journal of Plant Biochemistry and Biotechnology, 22: 372-381),
The patent application pig microsatellite DNA mark of classification " be suitable to pig variety " carrying out pig variety classification with microsatellite DNA mark is (public
The number of opening CN1370834A).
Jute is Tiliaceae (Tiliaceae) Corchorus (Corchorus) annual herb plant, is important bast fiber
Crop.Jute yield and cultivated area are only second to cotton, mainly in Asia (India, Bangladesh, China and Thailand) in the world
Plant with Latin America.In production, have long fruit kind (Corchorus olitorius L.) and the circle fruit of cultivation plant (Corchorus
Capsularis L.), chromosome number is 2n=2X=14.Tossa is soft, strength is big, good hygroscopicity, aproll fast, can spin
Property is good, and purposes is the most wide.
So far nearly 1000 microsatellite markers found in jute genome, meanwhile, jute microsatellite DNA exists
Application in the assignment of genes gene mapping and analysis of genetic diversity spreads out the most rapidly, and the patent about jute microsatellite marker is fought for the most progressively
Launch.But, the research of China's jute microsatellite is the most extremely limited, and only only a few laboratory has started relevant work.In view of Huang
Fiber crops microsatellite number is many, the most substantial amounts of undiscovered, and they have important theory and using value, particularly
GenBank public database there is likely to be substantial amounts of microsatellite locus.Therefore, carry out in China jute microsatellite point
From, identify and application study, exploitation has the microsatellite marker of Intellectual Property Right in China and seems the most urgent and necessary.
Summary of the invention
It is an object of the invention to: separate and identify jute EST microsatellite DNA mark, set up that jute is micro-to be defended
The technical system of star DNA also utilizes these molecular labelings to carry out Genetic Diversity of Jute analysis.
The technical scheme that the present invention realizes:
Described jute microsatellite DNA mark numbering CcSSR001-CcSSR045, CoSSR046-CoSSR066, corresponding
Jute microsatellite DNA mark primer sequence is shown in SEQ ID NO.1-132.
Described jute microsatellite DNA mark numbering CcSSR001-CcSSR045 is corchorus capsularis EST-SSR,
CoSSR046-CoSSR066 is corchorus olitorius EST-SSR.
Described jute microsatellite DNA mark numbering CcSSR001-CcSSR045, CoSSR046-CoSSR066, it is corresponding
EST GenBank ID be 112136062,112136096,112136110,112136120,112136121,
112136129,194395676,194395679,194395680,194395697,194395700,194395710,
194395722,194395729,194395733,194395738,194395740,194395754,194395756,
194395782,194395791,194395793,194395795,194395797,194395807,194395858,
194395868,194395881,222876067,222876073,222876092,222876097,222876103,
222876143,260098990,260099036,260099099,365732691,365732703,365732731,
375150553,375150558,375150561,375150562,375150586,311146658,311157368,
311157371,150174518,1112000549,1112000555,1112000578,1112000581,1112000583,
1112000590,1112000595,1112000596,1112000618,1112008416,1112008423,1112008466,
1112008468,1112008500,1112008512,1112000586,1112000637.
From GenBank public database, download jute EST, utilize SSRPrimer software that it is carried out
SSR site is searched, and utilizes Primer 3.0 Software for Design SSR primer, utilizes these SSR primers to Corchorus 6 differences of 2 kinds
Types of material, i.e. long fruit Wild related germplasm jute, long fruit wild species jute, long fruit cultigen jute, circle fruit cultigen jute and
Circle fruit wild species jute, expands.The PCR being studied these SSR primers by 1.5% agarose gel electrophoresis expands feature.?
To 66 microsatellite markers, it is determined that the microsatellite marker CcSSR008 of 10 rich polymorphism, CcSSR013, CcSSR016,
CcSSR024, CcSSR029, CcSSR040, CoSSR049, CoSSR050, CoSSR052 and CoSSR053.
The present invention comprises the concrete steps that:
(1) jute EST SSR primer development: from GenBank public database (http: //
Misuse.ncbi.nih.gov/) jute EST EST (expressed sequence tags) 838 is downloaded in
Bar.Utilize online SSRPrimer instrument (http://hornbill.cspp.latrobe.edu.au/
Ssrdiscovery.html) SSR that these sequences are comprised is searched for.The standard of its screening SSR is as follows: dinucleotides repeats secondary
Number > 6 times, Trinucleotide repeats number of times > 4 times, tetranucleotide repeat number of times > 3 times, pentanucleotide number of repetition > 2 times.Utilize
Primer3 software carries out the lookup of SSR site to it, and designs 66 to primer.The most numbered CcSSR001-CcSSR045 is
Corchorus capsularis EST-SSR, CoSSR046-CoSSR066 are corchorus olitorius EST-SSR, as shown in table 1.
The primer numbers of table 1 jute EST-SSR and source
(2) extracting genome DNA of different jute kinds: use CTAB method to extract 2 kinds of Corchorus 6 dissimilar
Material genomic DNA, these 6 materials include long fruit Wild related germplasm jute, long fruit wild species jute, long fruit cultigen jute,
Circle fruit cultigen jute and circle fruit wild species jute (table 2).
Table 2 is for trying variety name and the source of jute germ plasm resource
(3) PCR of SSR reacts and electrophoresis: PCR reaction volume 10 μ L, reaction system consists of 50 ng μ L–1 DNA
2.0 μ L, 10 μm ol μ L–1Primer 0.5 μ L, 0.5 U Taq enzyme 0.1 μ L, 10 mmol L–1 DNTPs 0.2 μ L, 10 ×
PCR buffer 1 μ L, 50 mmol L–1 Mg2+ 0.8 μ L, dd H2O 5.4 μL.PCR response procedures is: 94 DEG C of denaturations
3 min；94 DEG C of sex change 30 sec, 60 DEG C of annealing 30 sec, 72 DEG C extend 45 sec, totally 10 circulations, each cycle annealing
Temperature reduces by 0.5 DEG C；94 DEG C of sex change 30 sec, 55 DEG C of annealing 30 sec, 72 DEG C extend 45 sec, totally 35 circulations；Last 72
DEG C extend 10 min, 10 DEG C preserve 10 min.The reagent such as Taq enzyme, dNTP is purchased from Shanghai Sheng Gong Bioisystech Co., Ltd, electrophoresis
Method is 1.5% agarose gel electrophoresis.
(5) polymorphism of SSR and amplification efficiency analysis: utilize above-mentioned 66 pairs of SSR primers to 6 inhomogeneities of above-mentioned jute
Shaped material expands.The polymorphism of SSR and amplification efficiency are as shown in table 3 and Fig. 1.64 pairs of primers (97.0%) obtain strong and clear
Clear PCR primer, at least there is hereditary difference at 2 storerooms, shows polymorphism in 42 pairs of primers (63.6%).Wherein, 10 is right
Between primer shows kind in these 6 materials and Interspecific polymorphism, such as: CcSSR008, CcSSR013, CcSSR016,
CcSSR024, CcSSR029, CcSSR040, CoSSR049, CoSSR050, CoSSR052 and CoSSR053 etc..These primer tables
Reveal abundant polymorphism.
Accompanying drawing explanation
Fig. 1 SSR marker (CoSSR050-CoSSR053) electrophoretogram to 6 different kind of material amplifications of jute,
Wherein 1: jute 179；2: like the wild jute in shop；3: sweet fiber crops；4: wide leaf length fruit；5: fine jade Guangdong is blue or green；6: the wild long fruit of Mali；M:
DL2000。
Fig. 2 Genetic Diversity of Jute is analyzed.
Detailed description of the invention
Below by embodiment, the present invention is further illustrated, and its purpose is only that and is best understood from present disclosure
Rather than limit the scope of the invention.
Embodiment 1: develop 66 and be applicable to the new microsatellite marker that Genetic Diversity of Jute is analyzed.These 66 microsatellites
Marker number is respectively as follows: CcSSR001-CcSSR045 (corchorus capsularis EST-SSR), CoSSR046-CoSSR066, and (long fruit is planted
Jute EST-SSR), as shown in table 1.
Embodiment 2: determine the microsatellite marker CcSSR008 of 10 rich polymorphism, CcSSR013, CcSSR016,
CcSSR024, CcSSR029, CcSSR040, CoSSR049, CoSSR050, CoSSR052 and CoSSR053.Such as table 3 and Fig. 1 institute
Show.
The polymorphism of table 3 jute EST-SSR and amplification efficiency
ABCD represents the clear grade of electrophoresis banding pattern.
Embodiment 3: utilize the analysis of genetic diversity of the Microsatellite marker analysis Corchorus of above-mentioned exploitation.Material to be tested is
From the jute length fruit Wild related germplasm jute of 11 countries and regions, long fruit wild species jute, long fruit cultigen jute, circle fruit
Cultigen jute and circle fruit wild species jute totally 48 parts (table 4).NTsys software is utilized to carry out heredity to 48 parts for examination jute material
Similarity factor calculates.Its Computational Methods is non-Weighted Cluster Analysis (UPGMA), and 0,1 mode is taked in tape reading, and 0 indicates without carrying, 1
Indicate band.Cluster analysis result shows, these 10 microsatellite markers can perform well in the analysis of genetic diversity of jute.Poly-
Alanysis result as in figure 2 it is shown, be divided into two big classes by 48 parts of jute materials, and a class is planted for circle fruit, and a class is planted for long fruit.
Table 4 is for trying variety name and the source of jute germ plasm resource
The positive effect of the present invention is as follows:
(1) separate and identify the microsatellite marker of 66 jutes, through retrieving in international gene database, it was demonstrated that for newly
Microsatellite DNA mark.
(2) through above-mentioned microsatellite marker being used for jute polymorphism analysis, find that primer (97.0%) is obtained strong and clear by 64
Clear PCR primer, at least there is hereditary difference at 2 storerooms, shows polymorphism in 42 pairs of primers (63.6%).Wherein, 10 is right
Between primer shows kind in these 6 materials and Interspecific polymorphism, such as: CcSSR008, CcSSR013, CcSSR016,
CcSSR024, CcSSR029, CcSSR040, CoSSR049, CoSSR050, CoSSR052 and CoSSR053 etc..These primer tables
Reveal abundant polymorphism.Polymorphism analysis and amplification efficiency result are as shown in table 3 and Fig. 1.
(3) through above-mentioned microsatellite marker being used for the analysis of genetic diversity of Corchorus, it was demonstrated that these 66 microsatellite markers
Being applicable to Genetic Diversity of Jute research, cluster analysis result is as shown in Figure 2.
SEQUENCE LISTING
<110>University Of Agriculture and Forestry In Fujian
<120>jute EST microsatellite DNA mark
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<170> PatentIn version 3.3
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<210> 96
<211> 27
<212> DNA
<213>artificial sequence
<400> 96
agggacattc caaagtcctc atcatca 27
<210> 97
<211> 21
<212> DNA
<213>artificial sequence
<400> 97
ggcacgaggg aacatcaacc a 21
<210> 98
<211> 23
<212> DNA
<213>artificial sequence
<400> 98
aaaggagccg ccatagatct cca 23
<210> 99
<211> 21
<212> DNA
<213>artificial sequence
<400> 99
tggtgcccca tagccaccag a 21
<210> 100
<211> 20
<212> DNA
<213>artificial sequence
<400> 100
atgtgaacct aggcggcggg 20
<210> 101
<211> 23
<212> DNA
<213>artificial sequence
<400> 101
tcgttctcag cccttccatt cca 23
<210> 102
<211> 20
<212> DNA
<213>artificial sequence
<400> 102
cggccggggg attcgacaac 20
<210> 103
<211> 24
<212> DNA
<213>artificial sequence
<400> 103
actggctgct agactaccaa cgta 24
<210> 104
<211> 20
<212> DNA
<213>artificial sequence
<400> 104
tacgcccctg cagcatccct 20
<210> 105
<211> 20
<212> DNA
<213>artificial sequence
<400> 105
ttgccaggcc agcatggagc 20
<210> 106
<211> 19
<212> DNA
<213>artificial sequence
<400> 106
gtggccatta cggccgggg 19
<210> 107
<211> 20
<212> DNA
<213>artificial sequence
<400> 107
caccaccagc agcgacggaa 20
<210> 108
<211> 20
<212> DNA
<213>artificial sequence
<400> 108
ggcgctggtg cttcctctgg 20
<210> 109
<211> 20
<212> DNA
<213>artificial sequence
<400> 109
ttgcccgacc ccgatgacga 20
<210> 110
<211> 20
<212> DNA
<213>artificial sequence
<400> 110
cattacggcc gggggcgaag 20
<210> 111
<211> 20
<212> DNA
<213>artificial sequence
<400> 111
ccattacggc cggggcgaag 20
<210> 112
<211> 20
<212> DNA
<213>artificial sequence
<400> 112
atgggagtgc cgttttcgca 20
<210> 113
<211> 23
<212> DNA
<213>artificial sequence
<400> 113
cctctcctga gtcctgtctg ggc 23
<210> 114
<211> 25
<212> DNA
<213>artificial sequence
<400> 114
tgtcagcaga tcctgttgca gttgt 25
<210> 115
<211> 21
<212> DNA
<213>artificial sequence
<400> 115
atggcggtga cattggtcca c 21
<210> 116
<211> 27
<212> DNA
<213>artificial sequence
<400> 116
aaggatgggg aagagagaaa gtagggc 27
<210> 117
<211> 25
<212> DNA
<213>artificial sequence
<400> 117
cgtacagtca tctctgcttg cttgt 25
<210> 118
<211> 20
<212> DNA
<213>artificial sequence
<400> 118
caccccagac cgttggcaca 20
<210> 119
<211> 20
<212> DNA
<213>artificial sequence
<400> 119
gagtggccat tacggccggg 20
<210> 120
<211> 20
<212> DNA
<213>artificial sequence
<400> 120
agtttgagcc ggcgctgtca 20
<210> 121
<211> 20
<212> DNA
<213>artificial sequence
<400> 121
acgaggaagg gccaagtgcg 20
<210> 122
<211> 20
<212> DNA
<213>artificial sequence
<400> 122
cggccggggg atctctcctt 20
<210> 123
<211> 23
<212> DNA
<213>artificial sequence
<400> 123
agcagagcca tctcagtcat gga 23
<210> 124
<211> 20
<212> DNA
<213>artificial sequence
<400> 124
acggccgggc acaacatagt 20
<210> 125
<211> 20
<212> DNA
<213>artificial sequence
<400> 125
ggcggcaagg acacgtggaa 20
<210> 126
<211> 20
<212> DNA
<213>artificial sequence
<400> 126
gagtggccat tacggccggg 20
<210> 127
<211> 25
<212> DNA
<213>artificial sequence
<400> 127
tgttggtggt caggacaagg tatgc 25
<210> 128
<211> 20
<212> DNA
<213>artificial sequence
<400> 128
ttgtgctggc tatgccccct 20
<210> 129
<211> 20
<212> DNA
<213>artificial sequence
<400> 129
tcctctcagc caccacggcg 20
<210> 130
<211> 21
<212> DNA
<213>artificial sequence
<400> 130
acgggtcagc cattccaacc a 21
<210> 131
<211> 22
<212> DNA
<213>artificial sequence
<400> 131
gcgaaaagtc caccaaagcc gc 22
<210> 132
<211> 20
<212> DNA
<213>artificial sequence
<400> 132
gagtggccat tacggccggg 20
Claims (1)
1. jute EST microsatellite DNA mark, it is characterised in that: described jute microsatellite DNA mark is numbered
CcSSR008、CcSSR013、CcSSR016、CcSSR024、CcSSR029、CcSSR040、CoSSR049、CoSSR050、
CoSSR052 and CoSSR053, the primer sequence of the jute microsatellite DNA mark corresponding to each numbering be SEQ ID NO.15,
16, shown in 25,26,31,32,47,48,57,58,79,80,97,98,99,100,103,104,105,106.
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| Application Number | Priority Date | Filing Date | Title |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410088219.1A CN103937786B (en) | 2014-03-12 | 2014-03-12 | Jute EST microsatellite DNA mark |
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| CN103937786A CN103937786A (en) | 2014-07-23 |
| CN103937786B true CN103937786B (en) | 2016-09-07 |
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| CN104328197B (en) * | 2014-11-14 | 2016-06-08 | 福建农林大学 | Bluish dogbane expressed sequence tag microsatellite DNA mark |
| CN104372096B (en) * | 2014-11-20 | 2016-06-08 | 福建农林大学 | A kind of Corchorus olitorius L. microsatellite DNA mark finger printing and application thereof |
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