CN106636325A - Method for detecting phalaenopsis amabilis mutants - Google Patents

Method for detecting phalaenopsis amabilis mutants Download PDF

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CN106636325A
CN106636325A CN201610739795.7A CN201610739795A CN106636325A CN 106636325 A CN106636325 A CN 106636325A CN 201610739795 A CN201610739795 A CN 201610739795A CN 106636325 A CN106636325 A CN 106636325A
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seq
primer
iris
mutant
primer sequences
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CN106636325B (en
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肖文芳
李佐
吕复兵
陈和明
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Environmental Horticulture Institute of Guangdong Academy of Agricultural Sciences
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    • C12Q1/686—Polymerase chain reaction [PCR]
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Abstract

The invention belongs to the technical field of monitoring of plant varieties and in particular discloses a method for detecting phalaenopsis amabilis mutants. Four varieties including 'HongMantianHong', 'Guangmangsishe', V31 and 'Hongzhenzhu' and mutants thereof can be completely distinguished through utilizing combination of ten pairs of primers including SSR1, SSR2, SSR3, SSR4, SSR5, SSR6, SSR7, SSR8, SSR9 and SSR10; sequences of the ten pairs of primers including the SSR1, the R2, the SSR3, the SSR4, the SSR5, the SSR6, the SSR7, the SSR8, the SSR9 and the SSR10 are shown as SEQ: 1 to 20. The detection method disclosed by the invention can be used for screening the mutants, which do not meet growth requirements, at a bottle seedling period, so that the production of large-scale production is saved.

Description

A kind of method of detection iris mutant
Technical field
The present invention relates to plant variety monitoring technical field, in particular it relates to a kind of method of detection iris mutant.
Background technology
Iris is the class orchid for originating in Tropical Asian area, and flower-shape is peculiar, and pattern enriches, with high Ornamental values and the economic values.At present mainly by the approach breeding seedling of tissue culture, this makes for the large-scale production of iris Its production cost is higher, growth cycle is longer, and going out bottle seedling from tissue culture and selling to blooming probably needs 18 months.Then, due to Many mutagens during tissue culture, iris can produce a certain amount of mutant during tissue culture, and mutant is not simultaneously inconsistent The requirement of commodity production is closed, production cost can be increased again, it is very unfavorable to large-scale commercial production.So efficient iris Mutant detection technique is very necessary.
There is RAPD to detect currently used for the detection method of iris mutant, such as 2013 Xiao Wenfang disclose several frequently seen The RAPD detection methods of iris tissue culture mutant, type can be incised using 7 random primers by ' red red ' iris and its flower Mutant is distinguished, and can be distinguished ' all over the sky red ' iris and its pattern mosaic type mutant using 6 primers, but not Relevant primer can be found from 50 random primers by ' red red ' iris and its petal lip mutant.Although RAPD is detected Method is equally applicable to mutant detection, but, the detection method has unsurmountable defect：（1）Experimental repeatability and steady Qualitative extreme difference；（2）Gel electrophoresiss can only separate the larger DNA fragmentation of difference in length, and can not separate those molecular weight it is identical or The sample of difference very little.
The content of the invention
The invention aims to overcome the deficiencies in the prior art, there is provided a kind of method of detection iris mutant, The mutant for not meeting Production requirement is just screened out in the bottle seedling phase, the production cost of large-scale production is saved.
To achieve these goals, the present invention is achieved by the following technical programs.
Primer is made up of SSR1, SSR2, SSR3, SSR4, SSR5, SSR6, SSR7, SSR8, SSR9 and SSR10 ten, SSR1 primer sequences such as SEQ ID：Shown in 1～2, SSR2 primer sequences such as SEQ ID：Shown in 3～4, SSR3 primer sequences such as SEQ ID：Shown in 5～6；SSR4 primer sequences such as SEQ ID：Shown in 7～8；SSR5 primer sequences such as SEQ ID：Shown in 9～10；SSR6 Primer sequence such as SEQ ID：Shown in 11～12；SSR7 primer sequences such as SEQ ID：Shown in 13～14；SSR8 primer sequences such as SEQ ID：Shown in 15～16；SSR9 primer sequences such as SEQ ID：Shown in 17～18；SSR10 primer sequences such as SEQ ID：19～20 institutes Show；The kind of the iris is red, shining, V31 all over the sky and red Margarita.
The present invention just can completely will ' red over the sky ', ' shining ', V31 and ' red Margarita ' using the combination of the 10 pairs of primers 4 kinds and its mutant are all distinguished.
It is prominent in SSR fluorescent labeling capillary electrophoresis detections iris that the present invention is also claimed primer combination as above Application in variant.
A kind of method of detection iris mutant, comprises the steps：Extract the STb gene of iris to be measured, with SSR1, SSR2, SSR3, SSR4, SSR5, SSR6, SSR7, SSR8, SSR9 and SSR10 ten enters respectively performing PCR, PCR primer point to primer SSR fluorescent labeling capillary electrophoresis detections are not carried out, the result of the testing result of iris to be measured and normal iris is carried out Contrast, if both results are differed, judges iris to be measured as mutant.
Preferably, the reaction system of the PCR be the L of 2.5 × Buffer V 6.0, upstream and downstream primer（5 µM） 1.0 L, Taq enzyme（5 U·µL-1）0.1 L, DNA 1.0 L, ddH2O polishings are to 15 L.
Preferably, the response procedures of the PCR be 95 DEG C of 5 min, 94 DEG C of 30 sec, 58 DEG C of 35 sec 35 Circulation, 72 DEG C of 35 sec, 60 DEG C of 30 min.
Compared with prior art, the present invention has the advantages that：
The present invention is using SSR1, SSR2, SSR3, SSR4, SSR5, SSR6, SSR7, SSR8, SSR9 and SSR10 ten to primer Combination just can completely will ' over the sky red ', ' shining ', V31 and ' red Margarita ' 4 kind and its mutant are all distinguished, Sequence such as SEQs of SSR1, SSR2, SSR3, SSR4, SSR5, SSR6, SSR7, SSR8, SSR9 and the SSR10 ten to primer：1～20 It is shown.The detection method of the present invention can just screen out the mutant that do not meet Production requirement in the bottle seedling phase, save extensive raw The production cost of product.
Description of the drawings
Fig. 1 is using the detection figure of primer SSR8.
Specific embodiment
The present invention is made with reference to Figure of description and specific embodiment further being elaborated, the embodiment It is served only for explaining the present invention, is not intended to limit the scope of the present invention.Test method used in following embodiments is such as without spy Different explanation, is conventional method；Material, reagent for being used etc., are the reagent for commercially obtaining if no special instructions And material.
' all over the sky red ', ' shining ', V31 and ' red Margarita ' 4 cultivar origin is in market purchase.
Embodiment 1
（1）The extraction of STb gene：Take 0.2 g ' respectively all over the sky red ', ' shining ', V31 and ' red Margarita ' the four kind iris tip of a root Tissue, according to the general extraction methods or conventional kit of this area STb gene extract ' all over the sky red ', ' shining ', V31 and ' the STb gene of red Margarita ' four kind iris.
（2）PCR reacts：With SSR1, SSR2, SSR3, SSR4, SSR5, SSR6, SSR7, SSR8, SSR9 and SSR10 ten couples Primer enters respectively performing PCR.
PCR reaction systems be the L of 2.5 × Buffer V 6.0, upstream and downstream primer（5 µM）1.0 L, Taq enzyme（5 U·µL-1）0.1 L, DNA 1.0 L, ddH2O polishings are to 15 L；
Response procedures are 95 DEG C of 5 min, 94 DEG C of 30 sec, and 58 DEG C of 35 sec 35 is circulated, 72 DEG C of 35 sec, 60 ℃ 30 min。
SSR1, SSR2, SSR3, SSR4, SSR5, SSR6, SSR7, SSR8, SSR9 and SSR10 ten is shown in the sequence of primer Table 1.
The primer sequence information table of table 1
（3）3730XL sequenators are detected and analyzed：Every hole adds molecular weight internal standard and Methanamide mixed liquor (0.5 in 96 orifice plates: 8.5) 9 μ L, the μ L of PCR primer 1.0, after 95 DEG C of min of degeneration 3, upper machine testing.The raw data file that detection is obtained is led Enter and be analyzed in analysis software.
Using iris of the molecular marker of present invention offer to 4 different cultivars（' all over the sky red ', ' shining ', V31 ' red Margarita '）And its mutant（' all over the sky red ', ' shining ' and V31 are Flower color mutant, and ' red Margarita ' is florescence mutation Body）Detected, as a result as shown in table 2.
Table 2 experiment 4 butterfly orchid varieties and its detection data of mutant
As seen from the results in Table 2：10 SSR fluorescent labelinies do not have to there was only SSR4 in the detection of 4 butterfly orchid varieties and its mutant There is polymorphism, remaining primer will can be distinguished to a certain extent between each kind or normal strain and mutant, utilize The wherein combination of 3 couples of primer SSR3+SSR8+SSR9 just completely can all distinguish 4 kinds and its mutant.This 10 SSR fluorescent dye primers are produced during tissue culture using more than 400 butterfly orchid variety and part kind collected from market The DNA of mutant screen the SSR fluorescent dye primers for being commonly used for iris, although not to further types of mutation Body is detected, but from the point of view of 4 mutant detection efficiencies of example, the detection to iris mutant is more suitable for, energy Enough distinguish the upper relatively conventional iris mutant of some productions.
（4）Extract the STb gene of iris to be measured, with SSR1, SSR2, SSR3, SSR4, SSR5, SSR6, SSR7, SSR8, SSR9 and SSR10 ten enters respectively performing PCR to primer, and PCR primer carries out respectively SSR fluorescent labeling capillary electrophoresis detections, will treat The result of the testing result and normal iris of surveying iris is contrasted, if both results are differed, judges butterfly to be measured Phalaenopsis is mutant.

Claims (5)

1. it is a kind of detection iris mutant primer combination, it is characterised in that by SSR1, SSR2, SSR3, SSR4, SSR5, SSR6, SSR7, SSR8, SSR9 and SSR10 ten is constituted to primer, SSR1 primer sequences such as SEQ ID：Shown in 1～2, SSR2 draws Thing sequence such as SEQ ID：Shown in 3～4, SSR3 primer sequences such as SEQ ID：Shown in 5～6；SSR4 primer sequences such as SEQ ID：7 Shown in～8；SSR5 primer sequences such as SEQ ID：Shown in 9～10；SSR6 primer sequences such as SEQ ID：Shown in 11～12；SSR7 draws Thing sequence such as SEQ ID：Shown in 13～14；SSR8 primer sequences such as SEQ ID：Shown in 15～16；SSR9 primer sequences such as SEQ ID：Shown in 17～18；SSR10 primer sequences such as SEQ ID：Shown in 19～20；The kind of the iris is red, radiance all over the sky Four penetrate, V31 and red Margarita.
2. the primer described in claim 1 combines the application in SSR fluorescent labeling capillary electrophoresis detection iris mutants.
3. a kind of method of SSR fluorescent labelinies capillary electrophoresis detection iris mutant, it is characterised in that including following step Suddenly：Extract the STb gene of iris to be measured, with SSR1, SSR2, SSR3, SSR4, SSR5, SSR6, SSR7, SSR8, SSR9 and SSR10 ten enters respectively performing PCR to primer, and PCR primer carries out respectively SSR fluorescent labeling capillary electrophoresis detections, by butterfly to be measured Blue testing result and the result of normal iris is contrasted, if both results are differed, judge iris to be measured as Mutant.
4. method according to claim 3, it is characterised in that the reaction system of the PCR is 2.5 × Buffer V 6.0 L, upstream and downstream primer（5 µM）1.0 L, Taq enzyme（5 U·µL-1）0.1 L, DNA 1.0 L, ddH2O polishings are to 15 µL。
5. the method according to claim 3 or 4, it is characterised in that the response procedures of the PCR are 95 DEG C, 5 min, 94 DEG C, 30 sec, 58 DEG C, 35 sec 35 circulations, 72 DEG C, 35 sec, 60 DEG C, 30 min.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109156352A (en) * 2018-10-09 2019-01-08 青岛农业大学 One cultivate peanut mutant rapid screening method and application
CN114085921A (en) * 2021-10-27 2022-02-25 海南省农业科学院热带园艺研究所 Genetic marker of phalaenopsis based on RTE (terminal-to-terminal insertion deletion) polymorphism and application

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109156352A (en) * 2018-10-09 2019-01-08 青岛农业大学 One cultivate peanut mutant rapid screening method and application
CN114085921A (en) * 2021-10-27 2022-02-25 海南省农业科学院热带园艺研究所 Genetic marker of phalaenopsis based on RTE (terminal-to-terminal insertion deletion) polymorphism and application
CN114085921B (en) * 2021-10-27 2023-10-24 海南省农业科学院热带园艺研究所 Genetic marker of phalaenopsis based on RTE indel polymorphism and application

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