CN106399530A - Spinibarbus dneticulatus microsatellite family identification method - Google Patents
Spinibarbus dneticulatus microsatellite family identification method Download PDFInfo
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Abstract
The method discloses a spinibarbus dneticulatus microsatellite family identification method. The method comprises carrying out artificial breeding on spinibarbus dneticulatus parents from the natural population to obtain two families, extracting a genomic DNA, designing and synthesizing a series of microsatellite primer sequences, carrying out PCR amplification on the genomic DNA, screening 9 pairs of the primers with high amplification stability, high polymorphism and high heterozygosity, combining the primers to form three multiplex PCR systems, respectively carrying out PCR amplification on the genomic DNA to obtain a multiplex PCR product, analyzing the multiplex PCR product, and determining the parents in a parent-child relationship with the individual to be detected. The method provides novel technological means for assessment of identification of the spinibarbus dneticulatus family, seed quality management and proliferation and release effects.
Description
Technical field
The present invention relates to fish breeding and molecular marking technique field are and in particular to a kind of reflected using microsatellite mark
The method determining hangnail family.
Background technology
Hangnail (Spinibarbus denticulatus denticulatus), is subordinate to Cypriniformes
(Cyprinfomres), Cyprinidae (Cyprindae), subfamily (Barbinae), hangnail belong to (Spinibarbus), are commonly called as green grass or young crops
Bamboo carp, is distributed mainly on the middle and upper reaches in Bei Jiang, the Xijiang River and the Dong Jiang of China's Pearl River system, and Chang Huajiang, the Nan Du in Hainan Island
River, is Pearl River system Quality and economy fish.Because it grows the features such as fast, build is big, feeding habits are miscellaneous, meat good, premunition is strong, it is one
Individual have the breed variety promoting future very much.
Hangnail breeds parent essentially from wild or culture number generation population at present, not through artificially breeding, leads to
Seed germplasm is degenerated and is aggravated.The conventional seed speed of growth is slow, and its speed of growth, resistance against diseases and other culture performance etc. are also not
Reach the degree of improved variety, and the inbred phenomenon of generally existing, increasingly highlight the problems such as disease breaks out；Simultaneously also one
Determine to affect hangnail popularization in a wider context in degree.Therefore, carry out the work of hangnail fine-variety breeding as early as possible and strengthen hangnail
Germ plasm resource detection and assessment, select and grow the fast, improved seeds of good stress resistance, for hangnail population conservation and satisfaction
Intensive culture industry in the urgent need to having great importance.
Family selective breeding is the important means obtaining improved seeds.During family selective breeding, keep complete, accurate pedigree letter
Breath can effectively instruct parent's seed selection, thus avoiding inbreeding depression phenomenon.In traditional aquatic livestock selection and use, typically will
Different familys is divided to support and is divided in foster pond to maintain family information in different.This method due to different points of foster ponds between environment because
Son has differences, and this may result in the related genetic parameter estimation of breeding and produces deviation, is unfavorable for the formulation of breeding plan.
In mixed breed colony, typically by physical markings such as：Cut fin, branding, listed, fluorescent dye etc. and keep house
It is information, but these physical markings equally exists problems there is complex operation, easy damaged fish body, mark using physical markings
Note existence time is not long and is not easy to the shortcomings of be marked on Larva and fry.
With scientific and technical development, the identification occurring making to raise together with affiliation of molecular labeling is possibly realized, with micro-
Paternity test technology based on satellite parting be during current aquatic livestock pedigree confirms most widely used most reliable means it
One.
Microsatellite marker has that polymorphism is high, stability is strong, specificity is high, codominant inheritance the features such as, be aquatic livestock
Keep family information in seed selection, confirm that affiliation, tracking pedigree provide useful instrument.At present grass carp, Pelteobagrus fulvidraco,
Confirm in the big aquatic economic animals such as squama flounder and Environment of Litopenaeus vannamei Low microsatellite in aquatic livestock the feasibility of Parentage determination and
Using value.Have not yet to see the report that microsatellite marker is applied to hangnail Parentage determination.
Content of the invention
In order to overcome the defect of prior art, it is an object of the invention to provide a kind of hangnail microsatellite Parentage determination side
Method, is applied to hangnail paternity test, germplasm management, family management and evaluates enhancement effect etc..
The purpose of the present invention is achieved through the following technical solutions：
A kind of hangnail microsatellite Parentage determination method, comprises the following steps：
(1) the hangnail parent from natural population is carried out artificial breeding, obtain 2 familys, treat that fry hatching goes out three
The family colony as paternity test for the fry of more than 30 tails is taken respectively after individual month；
(2) randomly select the fry of parent and progeny population in step (1), genome is extracted with their isozyme
DNA；
(3) design and synthesize a series of hangnail micro-satellite primers sequences, and performing PCR is entered to the genomic DNA of step (2)
Amplification, filters out the primer that amplification is stable, polymorphism is high, heterozygosity is high；Filter out following 9 pairs of hangnail micro-satellite primers altogether：
SPMi1、SPMi7、SPMi15、SPMi18、SPMi23、SPMi24、SPMi31、SPMi33、SPMi36；
The sequence of SPMi1 is respectively as shown in SEQ.ID.NO.1 and SEQ.ID.NO.2；
The sequence of SPMi7 is respectively as shown in SEQ.ID.NO.7 and SEQ.ID.NO.8；
The sequence of SPMi15 is respectively as shown in SEQ.ID.NO.13 and SEQ.ID.NO.14；
The sequence of SPMi18 is respectively as shown in SEQ.ID.NO.9 and SEQ.ID.NO.10；
The sequence of SPMi23 is respectively as shown in SEQ.ID.NO.3 and SEQ.ID.NO.4；
The sequence of SPMi24 is respectively as shown in SEQ.ID.NO.11 and SEQ.ID.NO.12；
The sequence of SPMi31 is respectively as shown in SEQ.ID.NO.15 and SEQ.ID.NO.16；
The sequence of SPMi33 is respectively as shown in SEQ.ID.NO.17 and SEQ.ID.NO.18；
The sequence of SPMi36 is respectively as shown in SEQ.ID.NO.5 and SEQ.ID.NO.6；
(4) 9 pairs of hangnail micro-satellite primers that step (3) is sieved are combined into 3 multiplex PCR systems, multiplex PCR system
1 includes SPMi1, SPMi23, SPMi36；Multiplex PCR system 2 includes SPMi7, SPMi18, SPMi24；Multiplex PCR system 3 includes
SPMi15, SPMi31, SPMi33, the upper fluorescence labeling of 5 ' end marks of the forward primer to primer for each of which；Then with more than 3
Weight PCR system enters performing PCR amplification respectively to the genomic DNA of step (2), obtains multiple PCR products；
Described primer SPMi1, SPMi7, SPMi15, SPMi18, SPMi23, SPMi24, SPMi31, SPMi33, SPMi36
Forward primer 5 ' end mark fluorescence labeling be TAMRA, HEX, HEX, FAM, FAM, TAMRA, TAMRA, FAM, HEX respectively；
The cumulative volume 20 μ l of multiplex PCR system 1, including genomic DNA 20ng, PCR Mix10 μ l, the positive anti-primer of SPMi1
Each 0.2 μ l, each 0.4 μ l of the positive anti-primer of SPM23, SPMi36, supplement sterilizing distilled water to 20 μ l, PCR reaction condition is：94 DEG C pre-
Denaturation 4min；94 DEG C of denaturation 30s, 60 DEG C of annealing 45s, 72 DEG C of extension 40s, totally 28 circulations；72 DEG C of extension 10min, last 4 DEG C
Preserve；
The cumulative volume 20 μ l of multiplex PCR system 2, including genomic DNA 20ng, PCR Mix10 μ l, SPMi7, SPMi18 are just
The each 0.2 μ l of anti-primer, each 0.4 μ l of the positive anti-primer of SPMi24, supplement sterilizing distilled water to 20 μ l, PCR reaction condition and polyad
Be 1 identical；
PCR system in addition to primer for the multiplex PCR system 3 is identical with multiplex PCR system 1 with reaction condition；
(5) 3 groups of multiple PCR products are carried out Genotyping on ABI3730xl analyzer, read individual allele big
Little, and data is analyzed, according to the correlation between test individual and parent genotype, determined test individual and which parent
Originally there is parent child relationship；
Described data is analyzed is to be analyzed with software CERVUS3.0.
The present invention has such advantages as with respect to prior art and effect：
1st, the present invention is combined with multiple fluorescence PCR technology using microsatellite marker, has screened 9 high polymorphism microsatellites
Site, sets up 3 multiple fluorescence PCR systems, by ABI3730xl analyzer parting, using CERVUS3.0 software to hangnail
Family carries out individual identification and parent child relationship analysis.
2nd, PCR of present invention reaction can detect 3 microsatellite locus, efficiency than improve three times in the past, expense
Drop to original 1/3rd about.
3rd, the present invention utilizes sequenator parting by Capillary Electrophoresis, overcomes using polyacrylamide gel electrophoresis parting
The problem of allelic size parallax error, substantially increases the accuracy of genotype data.
4th, the family management, germplasm management and the enhancement effect assessment that are established as hangnail of the inventive method provides
A kind of new technological means.
Specific embodiment
With reference to embodiment, the present invention is described in further detail, but embodiments of the present invention not limited to this.
Embodiment
A kind of hangnail microsatellite Parentage determination method, comprises the following steps：
1) structure of hangnail family
Select excellent Parent from hangnail natural population (northern Jiang Shaoguan section), pedestrian is entered by the method injecting hormone
Work is hastened parturition.1 female method is joined by 1 hero and constructs two hangnail family full-sibses.Clip breeds the anal fin fin ray of parent
40mg about, it is stored in 95% alcohol, be stored in -20 DEG C.Different familys are divided and supports in different points of foster ponds, wait to feed three
After individual month, each family randomly selected for 30 odd amount in addition to the round number generations, clip hangnail 40mg about tail fin tissue, be stored in 95% alcohol
Interior, it is stored in -20 DEG C.
2) hangnail parent and progeny genome DNA are extracted
" Ezup pillar Animal genome DNA extraction agent box " using Shanghai Sheng Gong bio-engineering corporation extracts hangnail
Parent and progeny genome DNA.Detect DNA concentration and quality with NanoDrop-1000 UV detector, will after detection
DNA sample is diluted to 20ng/ μ l.
3) screening of polymorphic micro-satellite markers
Design and synthesize serial primer, and respectively 10 hangnail individualities (with the genomic DNA of step (2)) are carried out
PCR expands, and filters out the primer that amplification is stable, polymorphism is high, heterozygosity is high；
Filter out 9 pairs of hangnail micro-satellite primers altogether：SPMi1、SPMi7、SPMi15、SPMi18、SPMi23、SPMi24、
SPMi31、SPMi33、SPMi36.
4) optimization of hangnail microsatellite multiplex PCR condition and amplification
By the above-mentioned 9 pairs of primers screening, be combined into 3 multiplex PCRs, multiplex PCR system 1 include primer SPMi1,
SPMi23、SPMi36；Multiplex PCR system 2 includes primer SPMi7, SPMi18, SPMi24；Multiplex PCR system 3 includes primer
SPMi15, SPMi31, SPMi33, the upper fluorescence labeling of 5 ' end marks of the forward primer to primer for each of which；SPMi1、
The fluorescence labeling of SPMi7, SPMi15, SPMi18, SPMi23, SPMi24, SPMi31, SPMi33, SPMi36 is respectively：TAMRA、
HEX, HEX, FAM, FAM, TAMRA, TAMRA, FAM, HEX, are shown in Table 1.
Then with 3 multiplex PCR systems, respectively the genomic DNA of step (2) is entered with performing PCR amplification, obtain multiplex PCR and produce
Thing；
The primer sequence of table 13 multiple fluorescence PCR systems of hangnail and fluorescence labeling
Note：F represents forward primer, and R represents reverse primer, and fluorescence labeling is all marked at 5 ' ends of forward primer.
The cumulative volume of multiplex PCR system 1 is 20 μ l, and including genomic DNA 20ng, PCR Mix10 μ l, SPMi1 is positive and negative to be drawn
The each 0.2 μ l of thing, each 0.4 μ l of the positive anti-primer of SPM23, SPMi36, supplement sterilizing distilled water to 20 μ l, PCR reaction condition is：94℃
Denaturation 4min；94 DEG C of denaturation 30s, 60 DEG C of annealing 45s, 72 DEG C of extension 40s, totally 28 circulations；72 DEG C of extension 10min, last 4
DEG C preserve.
The cumulative volume of multiplex PCR system 2 is 20 μ l, including genomic DNA 20ng, PCR Mix10 μ l, SPMi7, SPMi18
The positive each 0.2 μ l of anti-primer, each 0.4 μ l of the positive anti-primer of SPMi24, supplement sterilizing distilled water to 20 μ l, PCR reaction condition is：94℃
Denaturation 4min；94 DEG C of denaturation 30s, 60 DEG C of annealing 45s, 72 DEG C of extension 40s, totally 28 circulations；72 DEG C of extension 10min, last 4
DEG C preserve.
The cumulative volume of multiplex PCR system 3 is 20 μ l, and including genomic DNA 20ng, PCR Mix10 μ l, SPMi15 is positive and negative to be drawn
The each 0.2 μ l of thing, each 0.4 μ l of the positive anti-primer of SPM31, SPMi33, supplement sterilizing distilled water to 20 μ l, PCR reaction condition is：94℃
Denaturation 4min；94 DEG C of denaturation 30s, 60 DEG C of annealing 45s, 72 DEG C of extension 40s, totally 28 circulations；72 DEG C of extension 10min, last 4
DEG C preserve.
The multiple microsatellite fluorescence labeling PCR reaction system of table 2 hangnail paternity test
5) microsatellite locus Genotyping and paternity test
Three groups of PCR samples that upper step is obtained are utilized respectively ABI3730xl analyzer and carry out microsatellite parting, LIZ500
Fluorescence molecule is as internal standard.Using GeneMapper V1.75 software, microsatellite typing data is read out, adds in conjunction with artificial
To correct, line up digital gene type matrix.
The equipotential base that gene frequency analysis includes each microsatellite locus is carried out using CERVUS 3.0 to gene data
Because of frequency, observation heterozygosity, expectation heterozygosity, polymorphism information content, Hardy-Weinberg balance and amorph frequency
Rate, is then simulated analysis and paternity test analysis.By likelihood ratio (LOD) check test individual and parent genotype it
Between relevance, and under 95% confidence level, determine that test individual and which parent have parent child relationship.
6) result
Using 9 microsatellite locus, in 58 odd amount in addition to the round number generations and 4 tail parents, are expanded and Genotyping, genotype is applied
CERVUS 3.0 software analysis result such as table 3.For ensureing qualification result accurately, when candidate's Parent is identified according to LOD value, only
More consistent than family record data, and LOD is more than 0 just confirmation parent child relationship.Finally confirmed for 57 odd amount in addition to the round number generations, identify accuracy rate
For 98.3%.Result above shows, the microsatellite multiple fluorescence PCR method energy efficient quick using the present invention realizes hangnail man
The requirement of the paternity test analysis of system, germplasm management and enhancement effect assessment.
Detection parameter in hangnail family for 39 microsatellite locus of table
Note：Na is allele number；Ho is observation heterozygosity；He is expectation heterozygosity；PIC is polymorphism information content；
HW is to breathe out temperature
Balance；Fn is amorph frequency；Ns represents not notable, and * represents P<0.01, through Bonferroni correction.
Above-described embodiment is the present invention preferably embodiment, but embodiments of the present invention are not subject to above-described embodiment
Limit, other any Spirit Essences without departing from the present invention and the change made under principle, modification, replacement, combine, simplify,
All should be equivalent substitute mode, be included within protection scope of the present invention.
Claims (6)
1. a kind of hangnail microsatellite Parentage determination method is it is characterised in that comprise the following steps：
(1) the hangnail parent from natural population is carried out artificial breeding, obtain 2 familys, treat that fry hatching goes out three months
Take the family colony as paternity test for the fry of more than 30 tails afterwards respectively；
(2) randomly select the fry of parent and progeny population in step (1), genomic DNA is extracted with their isozyme；
(3) design and synthesize a series of hangnail micro-satellite primers sequence, and the genomic DNA of step (2) is entered with performing PCR and expand
Increase, filter out the primer that amplification is stable, polymorphism is high, heterozygosity is high；Filter out following 9 pairs of hangnail micro-satellite primers altogether：
SPMi1、SPMi7、SPMi15、SPMi18、SPMi23、SPMi24、SPMi31、SPMi33、SPMi36；
The sequence of SPMi1 is respectively as shown in SEQ.ID.NO.1 and SEQ.ID.NO.2；
The sequence of SPMi7 is respectively as shown in SEQ.ID.NO.7 and SEQ.ID.NO.8；
The sequence of SPMi15 is respectively as shown in SEQ.ID.NO.13 and SEQ.ID.NO.14；
The sequence of SPMi18 is respectively as shown in SEQ.ID.NO.9 and SEQ.ID.NO.10；
The sequence of SPMi23 is respectively as shown in SEQ.ID.NO.3 and SEQ.ID.NO.4；
The sequence of SPMi24 is respectively as shown in SEQ.ID.NO.11 and SEQ.ID.NO.12；
The sequence of SPMi31 is respectively as shown in SEQ.ID.NO.15 and SEQ.ID.NO.16；
The sequence of SPMi33 is respectively as shown in SEQ.ID.NO.17 and SEQ.ID.NO.18；
The sequence of SPMi36 is respectively as shown in SEQ.ID.NO.5 and SEQ.ID.NO.6；
(4) 9 pairs of hangnail micro-satellite primers that step (3) is sieved are combined into 3 multiplex PCR systems, multiplex PCR system 1 is wrapped
Include SPMi1, SPMi23, SPMi36；Multiplex PCR system 2 includes SPMi7, SPMi18, SPMi24；Multiplex PCR system 3 includes
SPMi15, SPMi31, SPMi33, the upper fluorescence labeling of 5 ' end marks of the forward primer to primer for each of which；Then with more than 3
Weight PCR system enters performing PCR amplification respectively to the genomic DNA of step (2), obtains multiple PCR products；
(5) 3 groups of multiple PCR products are carried out Genotyping on ABI3730xl analyzer, read individual allele size,
And data is analyzed, according to the correlation between test individual and parent genotype, determined test individual and which parent
There is parent child relationship.
2. hangnail microsatellite Parentage determination method according to claim 1 it is characterised in that：Described primer SPMi1,
It is glimmering that the forward primer 5 ' end of SPMi7, SPMi15, SPMi18, SPMi23, SPMi24, SPMi31, SPMi33, SPMi36 marks
Signal is TAMRA, HEX, HEX, FAM, FAM, TAMRA, TAMRA, FAM, HEX respectively.
3. hangnail microsatellite Parentage determination method according to claim 1 it is characterised in that：Step (4) is described multiple
The cumulative volume 20 μ l of PCR system 1, including genomic DNA 20ng, PCR Mix10 μ l, each 0.2 μ l of the positive anti-primer of SPMi1,
The each 0.4 μ l of the positive anti-primer of SPM23, SPMi36, supplements sterilizing distilled water to 20 μ l, PCR reaction condition is：94 DEG C of denaturations
4min；94 DEG C of denaturation 30s, 60 DEG C of annealing 45s, 72 DEG C of extension 40s, totally 28 circulations；72 DEG C of extension 10min, last 4 DEG C of guarantors
Deposit.
4. hangnail microsatellite Parentage determination method according to claim 1 it is characterised in that：Step (4) is described multiple
The cumulative volume 20 μ l of PCR system 2, including genomic DNA 20ng, PCR Mix10 μ l, each 0.2 μ of the positive anti-primer of SPMi7, SPMi18
The each 0.4 μ l of the positive anti-primer of l, SPMi24, supplements sterilizing distilled water to 20 μ l, the complete phase of PCR reaction condition and multiple system 1
With.
5. hangnail microsatellite Parentage determination method according to claim 1 it is characterised in that：Step (4) is described multiple
PCR system in addition to primer for the PCR system 3 is identical with multiplex PCR system 1 with reaction condition.
6. hangnail microsatellite Parentage determination method according to claim 1 it is characterised in that：Right described in step (5)
Data is analyzed being to be analyzed with software CERVUS3.0.
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| CN108998547A (en) * | 2018-09-18 | 2018-12-14 | 中国水产科学研究院长江水产研究所 | A kind of microsatellite marking method for C. guichenoti paternity test |
| CN112226519A (en) * | 2020-10-10 | 2021-01-15 | 水利部中国科学院水工程生态研究所 | Sinocyclocheilus sinensis paternity test kit based on microsatellite markers and method thereof |
| CN112795662A (en) * | 2021-01-08 | 2021-05-14 | 广州大学 | Identification method of barbel grahami and application thereof |
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| CN107012224A (en) * | 2017-04-20 | 2017-08-04 | 广州大学 | A kind of method of quick screening light hangnail Barb fast-growths individual and kit and application |
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| CN106939348A (en) * | 2017-04-28 | 2017-07-11 | 四川省农业科学院水产研究所 | A kind of microsatellite marker primer and its authentication method for acipenser dabryanus Parentage determination |
| CN108998547A (en) * | 2018-09-18 | 2018-12-14 | 中国水产科学研究院长江水产研究所 | A kind of microsatellite marking method for C. guichenoti paternity test |
| CN108998547B (en) * | 2018-09-18 | 2021-06-29 | 中国水产科学研究院长江水产研究所 | Microsatellite marking method for paternity test of cupfish |
| CN112226519A (en) * | 2020-10-10 | 2021-01-15 | 水利部中国科学院水工程生态研究所 | Sinocyclocheilus sinensis paternity test kit based on microsatellite markers and method thereof |
| CN112226519B (en) * | 2020-10-10 | 2022-07-05 | 水利部中国科学院水工程生态研究所 | Sinocyclocheilus sinensis paternity test kit based on microsatellite marker and method thereof |
| CN112795662A (en) * | 2021-01-08 | 2021-05-14 | 广州大学 | Identification method of barbel grahami and application thereof |
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Application publication date: 20170215 |