CN109486915A - One kind detecting Spawning of The Grass Carp, ctenopharyngodon Idellus field method based on environment DNA - Google Patents
One kind detecting Spawning of The Grass Carp, ctenopharyngodon Idellus field method based on environment DNA Download PDFInfo
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- CN109486915A CN109486915A CN201811640960.9A CN201811640960A CN109486915A CN 109486915 A CN109486915 A CN 109486915A CN 201811640960 A CN201811640960 A CN 201811640960A CN 109486915 A CN109486915 A CN 109486915A
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Abstract
The invention discloses one kind to detect Spawning of The Grass Carp, ctenopharyngodon Idellus field method based on environment DNA, step is: (1) according to grass carp reproductive habit, in annual 4-7 month grass carp mating period, screen Spawning of The Grass Carp, ctenopharyngodon Idellus field position range, in the Changjiang river cross section of each sampled point, sampling location is set, it is respectively left, left, intermediate, right in, it is right, in each sampling location when sampling, the Changjiang river surface layer, middle layer and lower water are taken respectively three times, and when sampling must be sampled simultaneously；(2) the Changjiang river water sample adopted back is filtered, and filtered filter membrane is subjected to DNA extraction；(3) filter membrane DNA is extracted into result and carries out quantitative fluorescent PCR analysis, establish standard curve, the CT value obtained according to quantitative fluorescent PCR, the experimental result of more each sampling position calculates that the highest sampling position of grass carp DNA concentration is the position in spawning ground.This method is easy, easy to operate, can quickly and accurately find out the oviposition place of the Changjiang river grass carp, has biggish application value.
Description
Technical field
The invention belongs to animal molecular genetics fields, are more particularly to a kind of based on environment DNA detection Spawning of The Grass Carp, ctenopharyngodon Idellus field
Method.
Background technique
Grass carp is unique kind also known as the ctenopharyngodon idellus that Cypriniformes Cyprinidae Leuciscinae grass carp belongs to, grass roots fish, thick fish, and black carp,
Bighead, silver carp simultaneously claim Chinese four freshwater fish.Body is slightly cylindrical, and head is slightly put down flat, and tail portion is flat-sided；Mouth is arc-shaped, without；On
Jaw is slightly longer than lower jaw；2 row of hypopharynx tooth, it is flat-sided, it is in comb shape, flank has traverse furrow line；Squama median size；Body is in shallow tea yellow, back
Steel gray, abdomen is greyish white, chest, abdomeinal fin slightly sallow, other each fins are light grey.For plains regions such as Eastern China Guangxi to Heilungkiang
Endemic fish.The color at grass carp back is dark brown, scale edge is dark brown, and chest, abdomeinal fin are lark, and side line is straight,
Meat is delicate, and spur is few, and suitable knife with shaped cutting edge makees the moulding dishes such as chrysanthemum fish.Grass carp inhabits the rivers and lakes of plains region, general to like
Occupy middle lower layer and the more water plant regions of offshore of water.Property it is active, swimming rapidly, often hunts in packs, and is usually being flooded not shallow
Beach meadow and flashing region and the attached water body of Heavenly Stems and Earthly Branches stream are ingested fattening.For typical herbivorous fishes.Grass carp is food with grass, therefore
Raising grass carp is also more in the north.It is overwintering at the deep water in mainstream or lake.Breeding season parent population, which is traced back, swims habit, in torrent river section
Oviposition.Egg-laying season is in 3~July.Ovum drift.The egg membrane water swelling of fertilized eggs, drifts about downstream.4 rheological properties are mature.Fish brood amount is
300000~1,380,000.Grass carp has moved many countries for growing Asia, Europe, the United States, non-each continent.Rapidly because of its growth, meat flavour is good, feed
Source is wide, is four large Chinese carps of CHINESE FRESHWATER cultivation.
The environmental sample of environment DNA (eDNA) is a very loose concept, may include soil, deposit, excretion
Object, air, water body or even bion itself (such as insect of geneva net capture).Animal lives in some environment, with it
Various traces meeting carrier itself DNA fall to surrounding.As a technology, the purpose for analyzing eDNA is exactly to obtain these environment
The taxonomic information and gene function information of DNA institute species in sample.Retell it is more popular, in scientist's extraction environment sample
EDNA, be exactly which species belonged in order to analyze these DNA respectively, to confirm that there are which objects in respective environment
Kind.The purpose of eDNA is consistent in fact with traditional animals and plants Classification Count.
Due to the aggravation of the Changjiang river heavy contamination and mankind's activity in recent years, Spawning of The Grass Carp, ctenopharyngodon Idellus field and habitat are destroyed,
Wild grass carp quantity straight line decline.So it is very necessary for being looked for the spawning ground of grass carp and carrying out protection.However
Traditional method for looking for Spawning of The Grass Carp, ctenopharyngodon Idellus field needs a large amount of human and material resources and financial resources, including fishing to grass carp ovum seedling and right
The fishing of food ovum fish is dissected.Looking for for the Changjiang river Spawning of The Grass Carp, ctenopharyngodon Idellus field is had not been reported currently with the method for environment DNA.
Summary of the invention
Spawning of The Grass Carp, ctenopharyngodon Idellus field method is detected based on environment DNA the object of the present invention is to provide a kind of, this method is easy, operation
Simplicity can quickly and accurately find out the oviposition place of the Changjiang river grass carp.
In order to achieve the above purpose, the present invention uses following technical measures:
One kind detecting Spawning of The Grass Carp, ctenopharyngodon Idellus field method based on environment DNA comprising following steps:
(1) Spawning of The Grass Carp, ctenopharyngodon Idellus field position is screened in the season that annual 4-7 month grass carp breeds according to grass carp reproductive habit
4-6 sample bits are arranged in the Changjiang river cross section of each sampled point in range, 1-3 sampled point of every 2.5-3.5 kilometers of setting
Set, it is respectively left, left, intermediate, right in, it is right.When each sampling location samples, the Changjiang river surface layer, middle layer and lower layer are taken respectively
(not having specific requirement, can deeply can be shallow because the fertilized eggs of grass carp are randomly dispersed in the water in the Changjiang river) water is three times.It must be same when sampling
Shi Jinhang sampling.Each sampled point must continuous several times (3-8 times) sampling.
(2) the Changjiang river water sample adopted back is filtered, and filtered filter membrane is subjected to DNA extraction.
(3) filter membrane DNA is extracted into result and carries out quantitative fluorescent PCR analysis, standard curve is established, according to quantitative fluorescent PCR
The CT value obtained, the experimental result of more each sampling position calculate that (concentration is higher to illustrate that grass carp is living to grass carp DNA concentration highest
It is dynamic more active) sampling position be spawning ground position.
The primer of the quantitative fluorescent PCR are as follows:
Forward primer: 5 '-TGAGATGTGCGCTATAAAA-3 '；
Reverse primer: 5 '-CTACGCTCAGCAAATCCT-3 '；
The filter membrane of the filtering water sample is the nylon material filter membrane that aperture is 10 μm.
The quantitative fluorescent PCR response procedures are as follows: 94 DEG C of initial denaturation 3min；94 DEG C of denaturation 30s, annealing temperature renaturation
30s, 72 DEG C of extension 30s, 40 circulations；72 DEG C of extension 5min；4 DEG C of preservations.Solubility curve is to be passed with 0.2 DEG C of amplification from 65 DEG C
Increase to 95 DEG C.The reaction system of quantitative fluorescent PCR are as follows: each 1ul of DNA 2ul, primer, ultrapure water 6ul, SYBR Taq are
10ul。
One kind detecting Spawning of The Grass Carp, ctenopharyngodon Idellus field method based on environment DNA, and method is used for the detection of Spawning of The Grass Carp, ctenopharyngodon Idellus field position, is adopted
The highest place of water sample grass carp DNA concentration is the position in spawning ground, the time sampled can grass carp bred in spawning ground
Time.
Pass through above-mentioned technical measures: the step of most critical is to carry out doing fluorescence using primer pair water sample provided by the invention
Quantitative pcr amplification judges the mrna concentration of the purpose sample of each sampled point.The present invention thoroughly gets rid of traditional spawning ground inspection
Survey method does not need to carry out the fishing and dissection of food ovum fish, does not need to catch the ovum seedling of grass carp yet and can determine grass carp
The specific location in spawning ground.The present invention, which only need to acquire water sample from place to be measured, can determine that whether the place is Spawning of The Grass Carp, ctenopharyngodon Idellus field,
With biggish application value.
Compared with prior art, the present invention having the following advantages that and effect:
Spawning of The Grass Carp, ctenopharyngodon Idellus field method is detected based on environment DNA using one kind provided by the invention, is not needed to Grass carps' fries
Fishing does not need to catch food ovum fish yet, does not need more only the Changjiang river water sample need to be taken to be detected by large-scale detection instrument
It can determine whether the position of Spawning of The Grass Carp, ctenopharyngodon Idellus field.The present invention saves a large amount of material resources and wealth than traditional detection Spawning of The Grass Carp, ctenopharyngodon Idellus field position
Power, and it is more accurate, there is huge application value.
Detailed description of the invention
Fig. 1 is a kind of fluorescent quantitation expression of results schematic diagram
It can be seen that the fluorescent quantitation expression of results of the first experimental group is apparently higher than second and third experimental group fluorescent quantitation
Expression of results, the grass carp DNA concentration for illustrating that the first experimental group detects is higher, the grass carp DNA concentration that third experimental group detects
It is lower.
Specific embodiment
Embodiment 1:
One kind detecting Spawning of The Grass Carp, ctenopharyngodon Idellus field method based on environment DNA, the steps include:
(1) Spawning of The Grass Carp, ctenopharyngodon Idellus field position is screened in the season that 4 or 5 or 6 or grass carp in July are bred according to grass carp reproductive habit
4 or 5 or 6 sample bits are arranged in the Changjiang river cross section of each sampled point in range, 1 or 2 or 3 sampled point of every 3 kilometers of settings
Set, it is respectively left, left, intermediate, right in, it is right.When each sampling location samples, the Changjiang river surface layer, middle layer and lower layer are taken respectively
Water is three times.It must be sampled simultaneously when sampling.Each sampled point must continuous 3 or 4 or 5 or 6 or 7 or 8 samplings.
(2) the Changjiang river water sample adopted back is filtered and (according to machine, the air pressure in bottle,suction is lower, water sample passes through pumping
Filter paper is intake in bottle,suction, but the DNA in water sample is stayed on paper), and filtered filter membrane is subjected to DNA extraction.
(3) filter membrane DNA is extracted into result and carries out quantitative fluorescent PCR analysis, standard curve is established, according to quantitative fluorescent PCR
The CT value obtained, the experimental result of more each sampling position calculate that grass carp DNA concentration highest (does not have specific requirement, concentration is got over
High grass carp activity is more active) sampling position be spawning ground position.
(4) response procedures: the quantitative fluorescent PCR response procedures are as follows: 94 DEG C of initial denaturation 3min；94 DEG C of denaturation 30s, are moved back
Fiery temperature renaturation 30s, 72 DEG C of extension 30s, 40 circulations；72 DEG C of extension 5min；4 DEG C of preservations.Solubility curve is with 0.2 DEG C of increasing
Width is incremented to 95 DEG C from 65 DEG C.The reaction system of quantitative fluorescent PCR are as follows: each 1ul of DNA 2ul, primer, ultrapure water 6ul,
SYBR Taq is 10ul.
The primer of the quantitative fluorescent PCR are as follows:
Forward primer: 5 '-TGAGATGTGCGCTATAAAA-3 '；
Reverse primer: 5 '-CTACGCTCAGCAAATCCT-3 '；
The filter membrane of the filtering water sample is the nylon material filter membrane that aperture is 10 μm.
It, can be with the position in accurate judgement spawning ground by above-mentioned measure.
Embodiment 2:
One kind detecting Spawning of The Grass Carp, ctenopharyngodon Idellus field method based on environment DNA, the steps include:
A, below the Ge Zhou Ba of the Changjiang river be starting point, until Yidu City river section be terminal, one sampled point of every 3 kilometers of settings,
Each sampled point the Changjiang river cross section be arranged 5 sampling locations, it is respectively left, left, intermediate, right in, the right side.In each sampling
When position samples, the Changjiang river surface layer, middle layer and lower water are taken respectively three times.It must be sampled simultaneously when sampling.Each sampled point
It is primary in the sampling of every two hour mating period.
B, it transports the water sample of acquisition back laboratory, and water sample is filtered, DNA is extracted to filtered filter membrane.
C, filter membrane DNA is extracted into result and carries out quantitative fluorescent PCR analysis, the primer are as follows: 5 '-
TGAGATGTGCGCTATAAAA-3'；5'-CTACGCTCAGCAAATCCT-3'.
D, quantitative fluorescent PCR response procedures are as follows: 94 DEG C of initial denaturation 3min；94 DEG C of denaturation 30s, annealing temperature renaturation 30s, 72
DEG C extend 30s, 40 circulation；72 DEG C of extension 5min；4 DEG C of preservations.Solubility curve is to be incremented to 95 from 65 DEG C with 0.2 DEG C of amplification
℃.The reaction system of quantitative fluorescent PCR are as follows: each 1ul of DNA 2ul, primer, ultrapure water 6ul, SYBR Taq are 10ul.It establishes
Standard curve, the CT value obtained according to quantitative fluorescent PCR, the experimental result of more each sampling position calculate grass carp DNA concentration
Highest sampling position is the position in spawning ground.
Other implementation steps are same as Example 1.
Embodiment 3:
One kind detecting Spawning of The Grass Carp, ctenopharyngodon Idellus field method based on environment DNA, and process is: taking 9 identical buckets and 93 sizes
Identical grass carp is tested, and three buckets are taken, and each bucket cultivates 20 grass carps, is first experiment group, is then taken other three
A bucket, each bucket cultivate 10 grass carps, are second experimental group, and last three buckets are that each bucket cultivates 1 grass carp,
For third experimental group, culturing time is 1 day.Water sample is acquired from each bucket respectively, measures and is acquired according to this method
Water sample grass carp DNA content size.As a result as shown in Figure 1, the grass carp DNA content highest of first experiment group, third
The grass carp DNA content of experimental group is minimum, and applicant may determine that the water sample of first experiment group acquisition has more grass carp
DNA, first experiment group grass carp activity is relatively more, and repeating detection has equivalent effect.
Other implementation steps are same as Example 1.
Traditional spawning ground detection method can be preferably replaced using the present invention, can be produced using the present invention with accurate judgement
The oviposition place of ovum field and time.
Sequence table
<110>Changjiang Aquatic Products Inst., Chinese Academy of Aquatic Products Sciences
<120>a kind of that Spawning of The Grass Carp, ctenopharyngodon Idellus field method is detected based on environment DNA
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
tgagatgtgc gctataaaa 19
<210> 2
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
ctacgctcag caaatcct 18
Claims (1)
1. one kind detects Spawning of The Grass Carp, ctenopharyngodon Idellus field method based on environment DNA, which is characterized in that the steps include:
(1) Spawning of The Grass Carp, ctenopharyngodon Idellus field position range is screened, often in annual 4-7 month grass carp mating period according to grass carp reproductive habit
4-6 sampling location is arranged in the Changjiang river cross section of each sampled point, respectively in 1-3 sampled point of 2.5-3.5 kilometers of settings
For in left, left, intermediate, right, it is right, when each sampling location samples, take the Changjiang river surface layer, middle layer and lower water respectively three times,
It must be sampled simultaneously when sampling, each sampled point must continuous 3-8 sampling；
(2) the Changjiang river water sample adopted back is filtered, and filtered filter membrane is subjected to DNA extraction；
(3) filter membrane DNA is extracted into result and carries out quantitative fluorescent PCR analysis, established standard curve, obtained according to quantitative fluorescent PCR
CT value, the experimental result of more each sampling position, calculate the highest sampling position of grass carp DNA concentration be spawning ground position
It sets；
The primer of the quantitative fluorescent PCR are as follows:
Forward primer: 5 '-TGAGATGTGCGCTATAAAA-3 '；
Reverse primer: 5 '-CTACGCTCAGCAAATCCT-3 '；
The filter membrane of the filtering water sample is the nylon material filter membrane that aperture is 10 μm；
The quantitative fluorescent PCR response procedures are as follows: 94 DEG C of initial denaturation 3min；94 DEG C of denaturation 30s, annealing temperature renaturation 30s, 72
DEG C extend 30s, 40 circulation；72 DEG C of extension 5min；4 DEG C of preservations, solubility curve are to be incremented to 95 from 65 DEG C with 0.2 DEG C of amplification
DEG C, the reaction system of quantitative fluorescent PCR are as follows: each 1ul of DNA 2ul, primer, ultrapure water 6ul, SYBR Taq are 10ul.
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Cited By (2)
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| CN110172518A (en) * | 2019-05-31 | 2019-08-27 | 西安理工大学 | A kind of method in quick detection field Lanzhou Huanghe catfish spawning ground |
| CN110229873A (en) * | 2019-05-31 | 2019-09-13 | 西安理工大学 | A kind of method in quick detection field the Yellow River carp spawning ground |
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| CN104531879A (en) * | 2015-01-06 | 2015-04-22 | 上海海洋大学 | Environment DNA identification method for fish community structure researching |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110172518A (en) * | 2019-05-31 | 2019-08-27 | 西安理工大学 | A kind of method in quick detection field Lanzhou Huanghe catfish spawning ground |
| CN110229873A (en) * | 2019-05-31 | 2019-09-13 | 西安理工大学 | A kind of method in quick detection field the Yellow River carp spawning ground |
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