CN104782574B - Frankliniella occidentalis arrhenotoky inbreeding population establishing method - Google Patents
Frankliniella occidentalis arrhenotoky inbreeding population establishing method Download PDFInfo
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- CN104782574B CN104782574B CN201410750572.1A CN201410750572A CN104782574B CN 104782574 B CN104782574 B CN 104782574B CN 201410750572 A CN201410750572 A CN 201410750572A CN 104782574 B CN104782574 B CN 104782574B
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- frankliniella occidentalis
- caulis
- folium brassicae
- inbreeding
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- 241000927584 Frankliniella occidentalis Species 0.000 title claims abstract description 36
- 238000009399 inbreeding Methods 0.000 title claims abstract description 25
- 238000000034 method Methods 0.000 title claims abstract description 20
- 238000005286 illumination Methods 0.000 claims abstract description 10
- 238000009395 breeding Methods 0.000 claims abstract description 5
- 230000001488 breeding effect Effects 0.000 claims abstract description 5
- 230000008859 change Effects 0.000 claims description 9
- 238000012360 testing method Methods 0.000 claims description 5
- 241000382353 Pupa Species 0.000 claims description 4
- 230000012447 hatching Effects 0.000 claims description 3
- 241001249699 Capitata Species 0.000 claims 1
- 230000020509 sex determination Effects 0.000 abstract description 7
- 238000004458 analytical method Methods 0.000 abstract description 6
- 230000002068 genetic effect Effects 0.000 abstract description 3
- 108091092878 Microsatellite Proteins 0.000 abstract description 2
- 238000002474 experimental method Methods 0.000 abstract description 2
- 230000001776 parthenogenetic effect Effects 0.000 abstract 1
- 230000007246 mechanism Effects 0.000 description 5
- 240000007124 Brassica oleracea Species 0.000 description 4
- 235000003899 Brassica oleracea var acephala Nutrition 0.000 description 4
- 235000011301 Brassica oleracea var capitata Nutrition 0.000 description 4
- 235000001169 Brassica oleracea var oleracea Nutrition 0.000 description 4
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 230000018109 developmental process Effects 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 239000000463 material Substances 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 238000009396 hybridization Methods 0.000 description 2
- 229910052742 iron Inorganic materials 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 230000033001 locomotion Effects 0.000 description 2
- 230000008774 maternal effect Effects 0.000 description 2
- 230000008186 parthenogenesis Effects 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 230000001902 propagating effect Effects 0.000 description 2
- 230000001681 protective effect Effects 0.000 description 2
- 238000010010 raising Methods 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 241000238631 Hexapoda Species 0.000 description 1
- 108091030071 RNAI Proteins 0.000 description 1
- 241001414989 Thysanoptera Species 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000007622 bioinformatic analysis Methods 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 230000027326 copulation Effects 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000009368 gene silencing by RNA Effects 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 239000002917 insecticide Substances 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 230000017448 oviposition Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/033—Rearing or breeding invertebrates; New breeds of invertebrates
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- Life Sciences & Earth Sciences (AREA)
- Environmental Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Zoology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a frankliniella occidentalis arrhenotoky inbreeding population establishing method. According to the method, frankliniella occidentalis artificially bred is transferred into a centrifugal tube for single parthenogenetic breeding to establish an inbreeding population by controlling temperature, moisture and illumination manually. The method is characterized in that the frankliniella occidentalis arrhenotoky inbreeding population can be established, and obtained male offspring and multiple continuous generations of male and female offspring can be applied to karyotype analysis, microsatellite analysis and other experiments. The method has great significance for further learning the frankliniella occidentalis arrhenotoky features and establishing the inbreeding population and can further promote the analysis of genetic structure and sex determination mode.
Description
Technical field
The present invention relates to a kind of raising of insecticide and population foundation technology, particularly Frankliniella occidentalis arrhenotocia close relative
The method for building up of propagating population.
Background technology
The Frankliniella occidentalis adult stage is considerably longer than the immature phase, and the female adult life-span is considerably longer than male worm, is by setting up close relative
Propagating population studies its Sex Determination Mechanism there is provided good genetic prerequisite.Set up using Frankliniella occidentalis backcrossing breeding as starting point
Frankliniella occidentalis arrhenotocia inbreeding population, by chromosome karyotype analysis, microsatellite analysis, differentially expressed protein mirror
The technological means such as fixed and bioinformatic analysis and RNAi, carry out system to each stage of Frankliniella occidentalis inbreeding population development
Detection and relative analyses, judge Frankliniella occidentalis Sex Determination Mechanism, disclose the hereditary constitution of Frankliniella occidentalis inbreeding population, gram
The grand related gene with checking Sex determination.Result of study will tentatively disclose Frankliniella occidentalis arrhenotocia Sex Determination Mechanism
And its sex phenotype mechanism, enrich and develop arrhenotocia Sex determination Study on Evolution it is theoretical, for next step Frankliniella occidentalis
The research of adversity gene sex linked inheritance and expression provides solid theory and technology and supports, and the finally genetic control for Frankliniella occidentalis
The exploitation of technology provides rationale foundation.But because Frankliniella occidentalis individuality is little, motion is fast, and vitality is not strong, and single head is raised
Mortality rate is higher, if wanting to set up the inbred population of parthenogenesis even more extremely difficult, the method for breeding that there is no to this.
The content of the invention
It is an object of the invention to provide a kind of method for building up of Frankliniella occidentalis arrhenotocia inbreeding population, overcomes
The individual little motion of Frankliniella occidentalis is fast, and vitality is not strong, and single head raises the high shortcoming of mortality rate.
The purpose of the present invention is achieved through the following technical solutions：
Material requested and equipment:1.5ml centrifuge tubes, 10ml centrifuge tubes, pincet, filter paper, marking pen, shears, knife, hair
Pen, pallet (70cm × 50cm × 5cm), 200 mesh gauzes, fresh purple cabbage.
1st, the preparation of material：The gauze of 200 mesh is cut into into 2cm × 2cm, two kinds of specifications of 3cm × 3cm with standby；By awl
Burn red on alcohol burner, the hole of diameter 3mm is plucked out in the lid of 1.5ml centrifuge tubes, iron wire loop is burnt into red, in 10ml centrifuge tubes
Lid plucks out the hole of diameter 8mm, and all centrifuge tubes are will be through high temperature sterilize using before；Filter paper is cut into into bottom × a height of
The triangle filter paper of two kinds of specifications of 1.5cm × 0.5cm and 3cm × 1cm is with standby；Buy fresh purple cabbage and remove slender lobule, by blade
The fritter that volume is about 3mm × 3mm × 10mm is cut in middle plump part, and the Caulis et Folium Brassicae capitatae block of shape uniform rules is put into
In 10ml pipes；Illumination box, raises the environmental condition of Frankliniella occidentalis：Temperature 25+1 DEG C, humidity 60%-80%, photoperiod L:D
=14：8.
2nd, select the female adult of just emergence
Triangle filter paper and little Caulis et Folium Brassicae capitatae block are put in 1.5ml centrifuge tubes, with the pupa of brush pen picking Frankliniella occidentalis to 1.5ml's
In centrifuge tube, one layer of little gauze for cutting is spread in the mouth of pipe and cover lid.Caulis et Folium Brassicae capitatae and triangle filter paper are changed within regular two days with tweezers
Ensure one cephalont of a pipe, raise to turning into adult.
3rd, arrhenotocia
Treat that worm turns into adult, select female adult and number to worm in lid successively, change fresh Caulis et Folium Brassicae capitatae, afterwards timing daily
Caulis et Folium Brassicae capitatae is taken out with tweezers, is placed in the centrifuge tube of 10ml, one layer of big gauze for cutting is spread in the mouth of pipe, tighten lid, 10ml's
Centrifuge tube is put into bulk Caulis et Folium Brassicae capitatae and triangle filter paper, and the centrifuge tube numbering of the centrifuge tube correspondence 1.5ml of 10ml, record date are put
Raise in entering illumination box.The observed and recorded of timing daily, changes fresh Caulis et Folium Brassicae capitatae and be dried three in regular 2 days after worm hatching
Angle filter paper.
4th, population is set up in inbreeding
By the first male offspring's random choose sprouted wings, one is put into backcrossing in same centrifuge tube, and labelling with corresponding female generation
For backcross parent, record date.Caulis et Folium Brassicae capitatae is periodically taken out daily to be put in 10ml centrifuge tubes, is equally put into more bulk Caulis et Folium Brassicae capitatae, correspondence
Numbering, record date, are placed in illumination box and raise.Observed and recorded, treats that worm hatches, changes fresh every timing in 2 days daily
Caulis et Folium Brassicae capitatae and be dried filter paper.
5th, sisters' inbreeding
Treat that the offspring of both sexes backcrossing turns into adult, a male offspring and a female descendant are chosen as second filial generation elder sister
The inbred object of younger sister, was continuously raised for ten generations according to the method described above, in case follow-up test needs.
6th, points for attention：
If the male parent of backcross parent is dead in feeding process, female parent is still survived, in maternal corresponding arrhenotocia
Male offspring in again random choose go out a male worm continue therewith backcrossing；If maternal dead, male parent is still survived, by female parent
Corpse is placed in 1.5ml centrifuge tubes, and male parent is also placed in 1.5ml centrifuge tubes, be placed on freezen protective in -80 DEG C of refrigerators in case after
Continuous test needs.
Treat that the male offspring of most of arrhenotocia turns into adult, appropriate thrips is measured according to worm and is placed in -80 DEG C
Freezen protective in refrigerator in case follow-up test needs, other normal raisings, observed and recorded its development duration.
The suitable worm activity of centrifugation tube space of the 1.5ml used by the present invention, beneficial to the growth and development state of observation worm.
Triangle filter paper used by the present invention can be dried in moisture absorption holding pipe.
Fresh Caulis et Folium Brassicae capitatae stem used by the present invention can provide Frankliniella occidentalis and take food, it is to avoid which is escaped or dead.
Play holding inner air tube circulation in the hole of 200 mesh gauzes and lid used by the present invention, it is to avoid its escape.
The present invention select the Frankliniella occidentalis in pupa state be in order to prevent it sprout wings after copulation with other worms,
It is ensured of parthenogenesis.
Banister bruss of the present invention gently chooses the accuracy that insect is that artificial injury ensures experimental data when reducing transfer, it is ensured that
Survival rate.
Worm number consecutively is in order to will be their offspring corresponding, in case follow-up experiment needs by the present invention.
It is that, in order to set up population, it is convenient observation to be placed in 10ml centrifuge tubes and raise that the present invention takes out Caulis et Folium Brassicae capitatae every 24h timings
Its growth and development state and the accuracy for ensureing experimental data to its artificial disturbance can be reduced to greatest extent, be easy to observation note
Record.
Present invention tweezers change purple cabbage and triangle filter paper it is not only easy to operate and also can reduce artificial disturbance guarantee
The accuracy of experimental data.
In addition, Frankliniella occidentalis population growth in terms of whether there occurs change after close relative is returned in order to study, we will return
Female adult worm after friendship, and is not returned and non-closed crossing (♀：♂=1：3) female adult worm under two states has carried out a series of
The contrast (table 1) of life parameterses.As a result show, female adult worm after backcrossing, be not returned and normal hybridisation compared with, its spawning before
All there is no significant change in phase and total preoviposition period, but the egg-laying season substantially shortens than other states after backcrossing, per female spawning
Amount is decreased obviously, and the female adult life-span substantially shortens, while the post-egg stage after backcrossing is considerably longer than the state of backcrossing, but it is also bright
It is aobvious to be shorter than normal hybridisation state.The change of above life parameterses, show this method set up inbreeding population it is female into
Significant change is occurred in that in the performance of worm key life parameterses, this is next step with Frankliniella occidentalis inbreeding population as carrier depth
Enter to study the aspects such as Frankliniella occidentalis Sex determination, population differentiation and invasion mechanism to lay a solid foundation.
1 inbreeding population of table is returned and Frankliniella occidentalis female adult breeding relevant parameter under non-backcrossing state
Specific embodiment
Embodiment 1：
1st, the preparation of material：The gauze of 200 mesh is cut into into 2cm × 2cm, two kinds of specifications of 3cm × 3cm with standby；By awl
Burn red on alcohol burner, the hole of diameter 3mm is plucked out in the lid of 1.5ml centrifuge tubes, iron wire loop is burnt into red, in 10ml centrifuge tubes
Lid plucks out the hole of diameter 8mm, and all centrifuge tubes are will be through high temperature sterilize using before；Filter paper is cut into into bottom × a height of
The triangle filter paper of two kinds of specifications of 1.5cm × 0.5cm and 3cm × 1cm is with standby；Buy fresh purple cabbage and remove slender lobule, by blade
The fritter that volume is about 3mm × 3mm × 10mm is cut in middle plump part, and the Caulis et Folium Brassicae capitatae block of shape uniform rules is put into
In 10ml pipes；Illumination box, raises the environmental condition of Frankliniella occidentalis：25 DEG C of temperature, humidity 60%-80%, photoperiod L:D=
14：8.
2nd, select the female adult of just emergence
Triangle filter paper and little Caulis et Folium Brassicae capitatae block are put in 1.5ml centrifuge tubes, with the pupa of brush pen picking Frankliniella occidentalis to 1.5ml's
In centrifuge tube, one layer of little gauze for cutting is spread in the mouth of pipe and cover lid.Caulis et Folium Brassicae capitatae and triangle filter paper are changed within regular two days with tweezers
Ensure one cephalont of a pipe, raise to turning into adult.
3rd, arrhenotocia
Treat that worm turns into adult, select female adult and number to worm in lid successively, change fresh Caulis et Folium Brassicae capitatae, afterwards timing daily
Caulis et Folium Brassicae capitatae is taken out with tweezers, is placed in the centrifuge tube of 10ml, one layer of big gauze for cutting is spread in the mouth of pipe, tighten lid, 10ml's
Centrifuge tube is put into bulk Caulis et Folium Brassicae capitatae and triangle filter paper, and the centrifuge tube numbering of the centrifuge tube correspondence 1.5ml of 10ml, record date are put
Raise in entering illumination box.The observed and recorded of timing daily, changes fresh Caulis et Folium Brassicae capitatae and be dried three in regular 2 days after worm hatching
Angle filter paper.
4th, population is set up in inbreeding
By the first male offspring's random choose sprouted wings, one is put into backcrossing in same centrifuge tube, and labelling with corresponding female generation
For backcross parent, record date.Caulis et Folium Brassicae capitatae is periodically taken out daily to be put in 10ml centrifuge tubes, is equally put into more bulk Caulis et Folium Brassicae capitatae, correspondence
Numbering, record date, are placed in illumination box and raise.Observed and recorded, treats that worm hatches, changes fresh every timing in 2 days daily
Caulis et Folium Brassicae capitatae and be dried filter paper.
5th, sisters' inbreeding
Treat that the offspring of both sexes backcrossing turns into adult, a male offspring and a female descendant are chosen as second filial generation elder sister
The inbred object of younger sister, was continuously raised for ten generations according to the method described above, in case follow-up test needs.
The above, the only present invention preferably specific embodiment, but protection scope of the present invention is not limited thereto,
Any those familiar with the art the invention discloses technical scope in, the change or replacement that can be readily occurred in,
Should all be included within the scope of the present invention.Therefore, protection scope of the present invention should be with the protection model of claims
Enclose and be defined.
Claims (6)
1. a kind of method for building up of Frankliniella occidentalis arrhenotocia inbreeding population, comprises the steps of：
1) select the female adult of just emergence
The hole of diameter 3mm is plucked out in the lid of 1.5ml centrifuge tubes, after high temperature sterilize, be put in centrifuge tube little triangle filter paper and
Little Caulis et Folium Brassicae capitatae block, with the pupa of brush pen picking Frankliniella occidentalis in the centrifuge tube of 1.5ml, spreads one layer of little gauze lid for cutting in the mouth of pipe
Upper tube cap, changes Caulis et Folium Brassicae capitataes with regular two days of tweezers and little triangle filter paper ensures one cephalont of a pipe, raises to turning into adult；
2) arrhenotocia
Treat that worm turns into adult, select female adult and number to worm in lid successively, change fresh Caulis et Folium Brassicae capitatae, regularly use tweezer daily afterwards
Son takes out Caulis et Folium Brassicae capitatae, is placed in the centrifuge tube of 10ml, and the lid of centrifuge tube plucks out the hole of diameter 8mm, and centrifuge tube is using front wanting Jing
High temperature sterilize being crossed, one layer of big gauze for cutting being spread in the mouth of pipe, is tightened lid, the centrifuge tube of 10ml is put into bulk Caulis et Folium Brassicae capitatae and big by three
Angle filter paper, the centrifuge tube numbering of the centrifuge tube correspondence 1.5ml of 10ml, record date are raised in being put into illumination box, daily
Timing observed and recorded, changes fresh Caulis et Folium Brassicae capitatae and the big triangle filter paper being dried in regular 2 days after worm hatching；
3) population is set up in inbreeding
By the first male offspring's random choose sprouted wings, one is put into backcrossing in same centrifuge tube with corresponding female generation, and is labeled as back
Hand over parent, record date to take out daily periodically Caulis et Folium Brassicae capitatae and be put in 10ml centrifuge tubes, be equally put into more bulk Caulis et Folium Brassicae capitatae, reference numeral,
Record date, is placed in illumination box and raises, daily observed and recorded, treats that worm hatches, changes fresh Caulis et Folium Brassicae capitatae every timing in 2 days
With dry filter paper；
4) sisters' inbreeding
Treat that the offspring of both sexes backcrossing turns into adult, choose a male offspring and a female descendant is near as second filial generation sisters
The object of parent's breeding, was continuously raised for ten generations according to the method described above, in case follow-up test needs.
2. the method for building up of Frankliniella occidentalis arrhenotocia inbreeding population according to claim 1, its feature exist
In the condition setting of described illumination box it is：25 ± 1 DEG C of temperature, humidity 60%-80%, photoperiod L:D=14：8.
3. the method for building up of Frankliniella occidentalis arrhenotocia inbreeding population according to claim 1, its feature exist
In described little gauze is 200 mesh, and specification is 2cm × 2cm.
4. the method for building up of Frankliniella occidentalis arrhenotocia inbreeding population according to claim 1, its feature exist
In described big gauze is 200 mesh, and big gauze specification is 3cm × 3cm.
5. the method for building up of Frankliniella occidentalis arrhenotocia inbreeding population according to claim 1, its feature exist
In bottom × a height of 1.5cm × 0.5cm of described little triangle filter paper.
6. the method for building up of Frankliniella occidentalis arrhenotocia inbreeding population according to claim 1, its feature exist
In bottom × a height of 3cm × 1cm of described big triangle filter paper.
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| CN105660546B (en) * | 2016-04-01 | 2018-02-06 | 云南省农业科学院花卉研究所 | A kind of Frankliniella occidentalis indoor feeding method |
| CN105994176B (en) * | 2016-06-14 | 2018-08-31 | 中国农业科学院蔬菜花卉研究所 | Single-female generation Insectile stress-tolerant border near isogenic lines and its construction method and application |
| CN109077028B (en) * | 2018-08-28 | 2023-07-18 | 中国农业科学院蔬菜花卉研究所 | Method and device for carrying out RNA interference on frankliniella occidentalis |
| CN112931417A (en) * | 2021-02-26 | 2021-06-11 | 中国农业科学院植物保护研究所 | Method for group breeding of frankliniella occidentalis and application thereof |
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| CN104012472A (en) * | 2014-03-12 | 2014-09-03 | 云南农业大学 | Indoor breeding method of thrips |
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| JP4944289B2 (en) * | 2000-05-16 | 2012-05-30 | 大洋香料株式会社 | Citrus thrips control agent and control method |
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