CN101225443B - Molecule detecting method for rapidly identifying salt-tolerant iris species - Google Patents
Molecule detecting method for rapidly identifying salt-tolerant iris species Download PDFInfo
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- CN101225443B CN101225443B CN2007101922389A CN200710192238A CN101225443B CN 101225443 B CN101225443 B CN 101225443B CN 2007101922389 A CN2007101922389 A CN 2007101922389A CN 200710192238 A CN200710192238 A CN 200710192238A CN 101225443 B CN101225443 B CN 101225443B
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- 238000000034 method Methods 0.000 title claims abstract description 12
- 108091092878 Microsatellite Proteins 0.000 claims abstract description 13
- 108020004414 DNA Proteins 0.000 claims abstract description 8
- 239000003147 molecular marker Substances 0.000 claims description 4
- 238000007400 DNA extraction Methods 0.000 claims description 2
- 239000003795 chemical substances by application Substances 0.000 claims description 2
- 238000010367 cloning Methods 0.000 claims description 2
- 150000003839 salts Chemical class 0.000 abstract description 24
- 239000003550 marker Substances 0.000 abstract description 8
- 102000053602 DNA Human genes 0.000 abstract description 2
- 238000001514 detection method Methods 0.000 description 13
- 230000003321 amplification Effects 0.000 description 5
- 238000003199 nucleic acid amplification method Methods 0.000 description 5
- 238000009938 salting Methods 0.000 description 4
- 241000894007 species Species 0.000 description 4
- 238000000137 annealing Methods 0.000 description 3
- 230000004069 differentiation Effects 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 238000005070 sampling Methods 0.000 description 3
- 241000768494 Polymorphum Species 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000007619 statistical method Methods 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 238000010413 gardening Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 230000015784 hyperosmotic salinity response Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 235000019600 saltiness Nutrition 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
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Abstract
The invention relates to a detecting and distinguishing method of molecule biology in the biology field, which is characterized in that: DNA is extracted from the aimed plant iris, the nine special primer sequences are processed with DNA expansion to acquire microsatellite molecule marker data, the molecule maker date is analyzed by using the software Arlequin 3.11 to acquire the plant population and classification. The detecting and distinguishing method of molecule biology has the advantages that: the molecule detecting and distinguishing can be carried out fast to judge the iris belongs to the salt tolerant or non-salt tolerant population, the detecting and distinguishing method provides microsatellite DNA molecule of the iris salt tolerant population to mark the nine primer sequence and expansive conditions and parameters.
Description
One, technical field
The invention belongs to molecular biological detection and method of discrimination in the field of biology.Relate in particular to Molecular Detection and the differentiation of Jris salt-tolerant plant population, also relate to Diversity Detection, evolution, genetic map construction and the QTL (quantitative character gene locus therefor) that are applied to iris simultaneously and detect etc.
Two, background technology
Iris is one of flowers that have long enjoyed a good reputation the earliest in the gardening, and its pattern is abundant, the flower type is various, and different sizes, and strong stress resistance have very high ornamental value.The iris of most of kinds can not be grown on the saltings, or receives behind the salt stress growth restriction even occur dead.Wherein one type of kind that is called salt-tolerant iris not only has above-mentioned advantage but also has very strong salt resistance ability, can surpass more than 0.3% in saltiness, normal growth and breeding on the saltings of PH8-10.This veriety is to the greening in coastal cities and beautify, be positioned at the greening of the field area in saltings, the soil improvement in saltings has vital role.
But; Generally be difficult to distinguish salt tolerant and non-salt-tolerant plants from phenotypic characteristic; Especially filter out the salt tolerant individuality from non-salt-tolerant iris and salt-tolerant iris filial generation; Usually to carry out the test of physiological measurement and salt tolerant, so not only time-consuming, effort but also possibly destroy plant, the physical signs judged result usually also exists uncertain in addition.
The salt-tolerance character of plant receives controlled by multiple genes, and there are certain otherness in salt-tolerant plants and non-salt-tolerant plants on genome.Different with physical signs, in the relatively short time, genome has good stability, and is not affected by environment basically, and draw materials convenience, trace.Therefore, through being fully feasible in the detection of genome difference property and the difference of following the trail of on its proterties to plant.
Dna molecular marker is one of important method of rapid detection biological gene group difference; Wherein microsatellite marker is to have plurality of advantages such as polymorphum height, good stability, simple operation; Through the target plant population is carried out the detection of microsatellite marker, can distinguish the individuality that on proterties, has notable difference.In case after confirming specific markers, we just can differentiate the target plant.
The relevant report of at present domestic and international still unmatchful iris salt tolerant population molecule rapid detection.
Three, summary of the invention
The present invention seeks to: provide a cover to be used in nondestructive sampling iris salt tolerant population molecule Fast Detection Technique and method down, accurate, save time, laborsaving.
The present invention seeks to realize like this: obtain iris salt tolerant population molecule Fast Detection Technique and method; According to 9 primer sequences of iris microsatellite DNA molecule marker and amplification condition and the parameter that provide; The target plant of participating in the experiment is carried out the amplification of DNA extraction and microsatellite marker, adopt the statistical software analysis to draw detection and the classification of plant salt tolerant and non-salt tolerant population to molecular data then.
Scheme more specifically of the present invention is:
*Wherein the sequence of 9 micro-satellite primers and mark amplification condition are:
(1) primer sequence
01
Forward?Primer(5′to?3′) GAA?GTT?TTG?TAG?AGG?TCT?GGT?G
Reverse?Primer(5′to?3′) GCT?ACA?ACT?AAA?GAG?TCC?AGT?C
02
Forward?Primer(5′to?3′) GAG?AAA?GAA?GGA?GGA?GGA?AGG
Reverse?Primer(5′to?3′) CAG?CAA?CTG?TGA?GGA?GAA?AG
03
Forward?Primer(5′to?3′) GCA?CAT?AAC?CTC?TCT?TGT?CC
Reverse?Primer(5′to?3′) ACC?AGA?CTA?CGA?ATC?ATT?ACC?C
04
Forward?Primer(5′to?3′) TTC?AAG?CCT?CTA?CTA?GAG?AGA?C
Reverse?Primer(5′to?3′) GCA?ACC?TTG?TAA?CCA?TCC?C
05
Forward?Primer(5′to?3′) TTT?CCT?CCC?TCT?ACT?CAT?TCC
Reverse?Primer(5′to?3′) CTA?GCT?AAG?CGA?GCT?AAC?AC
06
Forward?Primer(5′to?3′) AGC?GGA?AAG?AAT?GAA?CAC?AAG
Reverse?Primer(5′to?3′) GCG?AAG?AAG?GAG?CAA?ATA?GTA?G
07
Forward?Primer(5′to?3′) CCT?CCT?TCT?TCA?GAC?CCG?TAG
Reverse?Primer(5′to?3′) CGG?CGG?CGA?GAA?AAG?TAG
08
Forward?Primer(5′to?3′) TGG?TCC?TAA?GTG?ACT?GAT?AGT?G
Reverse?Primer(5′to?3′) GGA?GTA?GAG?AGT?GAC?ATG?GAG
09
Forward?Primer(5′to?3′) GTG?CGA?GAT?ATA?AGG?GAA?AGA?C
Reverse?Primer(5′to?3′) GAT?ATT?ACC?CAT?GCT?AAG?GCA?G
(2) amplification condition
The annealing temperature of 1-5 primer is 50 degree; No. 6 is 60 degree; 7-9 number is 65 degree;
(3) PCR reaction system:
10X Buffer?w/15mM?MgCl
2(1.0ul)
10mM?dNTP?mix(0.2ul)
10uM?Forward?primer(0.25ul)
10uM Reverse?primer(0.25ul)
Taq?polymeras(5U/ul)(0.05ul)
dH
2O(7.25ul)
DNA(～15ng)(1.0ul)
Characteristics of the present invention are: according to good stability, 9 microsatellite marker sequence informations that polymorphum is strong; The plant that target is participated in the experiment carries out DNA cloning, behind the acquisition molecular marker data, utilizes statistical analysis software to obtain the differentiation of population; Compare with traditional physiology method, direct cultivation etc.; Obviously reduced workload and efficient improves greatly, under the prerequisite of nondestructive sampling, obtained genomic information fast, differentiated accuracy rate simultaneously up to 97.5%.The Jris microsatellite DNA molecular marking technique that provides through this invention can be realized the rapid detection and the differentiation of the salt-enduring species mass-planting strain under the sampling of Jris population nondestructive; Accurately, save time and laborsaving, greatly reduce workload that adopts other method of discrimination and the error that possibly bring.
The present invention has important value and application prospect in the salt-tolerant iris plant is quick, technological thought is applicable to that too the rapid molecular of other species difference proterties detects.The present invention has been applied to obtain in Jiangsu Province education department " jiangsu coast is fitted and given birth to the seed selection of the salt-tolerant iris new lines " project (JHB05-01) effect of efficient sorting and accuracy.
Four, description of drawings
Fig. 1 is a flow sheet of the present invention
Five, embodiment
Referring to Fig. 1.Instance data is 120 iris populations (each 60 individuals of salt tolerant and non-salt tolerant) leaf samples.After this technical Analysis, plant is differentiated result and accuracy rate:
Table 120 an iris species plant is differentiated accuracy rate % (in the bracket is number of individuals)
| Population | The salt tolerant population | Non-salt tolerant population |
| The non-salt tolerant population of salt tolerant population (n=60) (n=60) | 0.98(59) 0.03(2) | 0.02(1) 0.97(58) |
Annotate: 60 salt tolerant individualities have 59 correct decisions to the salt tolerant population in the table, have only 2 individuals to declare non-salt tolerant population; 60 non-salt tolerant individualities have 58 correct decisions to non-salt tolerant population, have only 1 individuals to declare the salt tolerant population.
Said 9 primer sequences all are the peculiar primer that iris is distinguished salt-tolerant iris species; The software that statistical method embodies is non-commercialization software, is the software that use in the present technique field; The fundamental operation principle of the software that statistical method embodies: the principle that has similarity according to the genomic information (dna fragmentation for microsatellite marker in the present case is big or small) of similar properties; Just the similarity of plant is high more; The similarity of mark is just big; Software calculates the similarity on label information between plant and the plant according to the characteristic of the microsatellite marker of each plant, sorts out according to the size of similarity.
The specific condition of amplification:
(1) annealing temperature of primer
The annealing temperature of 1-5 primer is 50 degree; No. 6 is 60 degree; 7-9 number is 65 degree;
(2) PCR reaction system:
10X?Buffer?w/15mM?MgCl
2(1.0ul)
10mM?dNTP?mix(0.2ul)
10uM?Forward?primer(0.25ul)
10uM?Reverse?primer(0.25ul)
Taq?polymeras(5U/ul)(0.05ul)
dH
2O(7.25ul)
DNA(～15ng)(1.0ul)
Claims (1)
1. the molecular detecting method of Rapid identification salt-tolerant iris species; It is characterized in that according to the target plant that provides be that iris carries out DNA extraction; Distinctive 9 primer sequences are carried out DNA cloning; Obtain the microsatellite molecular marker data, adopt Arlequin 3.11 softwares to analyze molecular marker data, the population that obtains plant is differentiated and classification; The sequence of 9 micro-satellite primers wherein:
Primer sequence
01 forward primer 5 ' hold 3 ' end GAA GTT TTG TAG AGG TCT GGT G
Reverse primer 5 ' hold 3 ' end GCT ACA ACT AAA GAG TCC AGT C
02 forward primer 5 ' hold 3 ' end GAG AAA GAA GGA GGA GGA AGG
Reverse primer 5 ' hold 3 ' end CAG CAA CTG TGA GGA GAA AG
03 forward primer 5 ' hold 3 ' end GCA CAT AAC CTC TCT TGT CC
Reverse primer 5 ' hold 3 ' end ACC AGA CTA CGA ATC ATT ACC C
04 forward primer 5 ' hold 3 ' end TTC AAG CCT CTA CTA GAG AGA C
Reverse primer 5 ' hold 3 ' end GCA ACC TTG TAA CCA TCC C
05 forward primer 5 ' hold 3 ' end TTT CCT CCC TCT ACT CAT TCC
Reverse primer 5 ' hold 3 ' end CTA GCT AAG CGA GCT AAC AC
06 forward primer 5 ' hold 3 ' end AGC GGA AAG AAT GAA CAC AAG
Reverse primer 5 ' hold 3 ' end GCG AAG AAG GAG CAA ATA GTA G
07 forward primer 5 ' hold 3 ' end CCT CCT TCT TCA GAC CCG TAG
Reverse primer 5 ' hold 3 ' end CGG CGG CGA GAA AAG TAG
08 forward primer 5 ' hold 3 ' end TGG TCC TAA GTG ACT GAT AGT G
Reverse primer 5 ' hold 3 ' end GGA GTA GAG AGT GAC ATG GAG
09 forward primer 5 ' hold 3 ' end GTG CGA GAT ATA AGG GAA AGA C
Reverse primer 5 ' hold 3 ' end GAT ATT ACC CAT GCT AAG GCA G.
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Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101654707B (en) * | 2009-09-21 | 2012-02-29 | 安徽师范大学 | Polygonatum cyrtonema microsatellite markers |
| CN102634513B (en) * | 2009-12-31 | 2013-07-03 | 北京林业大学 | Development and application of microsatellite DNA molecular markers of lagerstroemia caudate |
| CN102634512B (en) * | 2009-12-31 | 2013-07-03 | 北京林业大学 | Microsatellite DNA molecular markers of lagerstroemia caudate |
| CN106967793B (en) * | 2017-03-10 | 2020-12-11 | 广东省林业科学研究院 | Molecular detection method for rapidly identifying cornus wisoniana population |
| CN110111845A (en) * | 2018-09-03 | 2019-08-09 | 广东美立康生物科技有限公司 | A kind of method of quick screening species identification DNA molecular marker |
| CN115242287A (en) * | 2022-01-04 | 2022-10-25 | 北京电子工程总体研究所 | Ground measurement, operation and control method and system for satellite constellation |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1670218A (en) * | 2004-03-17 | 2005-09-21 | 中国科学院植物研究所 | Method for screening salt-tolerant medicinal dandelion herb and its special primer |
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| CN1670218A (en) * | 2004-03-17 | 2005-09-21 | 中国科学院植物研究所 | Method for screening salt-tolerant medicinal dandelion herb and its special primer |
Non-Patent Citations (4)
| Title |
|---|
| 张敏，黄苏珍，仇硕，孙延东.鸢尾属植物遗传多样性的RAPD和ISSR分析.植物资源与环境学报16 2.2007,16(2),6-11. |
| 张敏,黄苏珍,仇硕,孙延东.鸢尾属植物遗传多样性的RAPD和ISSR分析.植物资源与环境学报16 2.2007,16(2),6-11. * |
| 王玲，卓丽环.基于ITS序列的鸢尾属植物部分种的系统分类.东北林业大学学报34 4.2006,34(4),54-56, 75. |
| 王玲,卓丽环.基于ITS序列的鸢尾属植物部分种的系统分类.东北林业大学学报34 4.2006,34(4),54-56, 75. * |
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