CN109880919A - A kind of specific recognition method for Patinopecten yessoensis telocentric chromosome - Google Patents
A kind of specific recognition method for Patinopecten yessoensis telocentric chromosome Download PDFInfo
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Abstract
The present invention provides a kind of two SNP markers of recognition methods identification a pair of end portions centromere chromosome of Patinopecten yessoensis telocentric chromosome, and nucleotides sequence is classified as SEQ ID NO:1 and SEQ ID NO:2；Identify two SNP markers of another pair telocentric chromosome, nucleotides sequence is classified as SEQ ID NO:3 and SEQ ID NO:4；Third is identified to two SNP markers of telocentric chromosome, nucleotides sequence is classified as SEQ ID NO:5 and SEQ ID NO:6.The probe provided through the invention and chromosome recognition methods can quickly navigate to the every a pair of end portions centromere chromosome of Patinopecten yessoensis, compared to traditional form and karyotyping, the method discrimination is higher, and efficient stable, this has the meaning of positive important to the cytogenetics research of Patinopecten yessoensis.
Description
Technical field
The invention belongs to shellfish chromosome research technical fields, and in particular to a kind of Patinopecten yessoensis telocentric chromosome
Specific recognition method.
Background technique
Chromosome is the carrier of inhereditary material, and the specific recognition of chromosome is cytogenetic important research content,
It is also the Important Theoretic Foundation for carrying out genetic breeding work.Traditional chromosome is distinguished mainly by means of chromosome number and shape
State, but the chromosome of shell-fish is smaller, it is large number of, it is difficult to intuitive to distinguish.Chromosome banding pattern can show the interior of chromosome
Portion's structure, provides more information with mirror qualitative features, but existing research shows that the technology in shellfish chromosome research
In there are unstable result, the problems such as banding pattern is unintelligible, these seriously limit the identification of shellfish chromosome.
Patinopecten yessoensis is the important sea-farming shellfish of northern China, because it is with high economic value and good cultivation
Basis, Patinopecten yessoensis cultivation have become the very important pillar industry in China's Coastal Areas.Carry out the genetic breeding of Patinopecten yessoensis
The circulating benignly for prawns and scallops cultivation that work has the meaning of positive important.Patinopecten yessoensis high density genetic linkage maps in recent years
Building and gene order-checking are successively performed, and provide the guidance of molecular level for the genetic breeding work of Patinopecten yessoensis.But it will
Group is learned data and is further applicable in specific breeding work, it is desired nonetheless to the integration of chromosome and genomic information is carried out,
In important previous work be exactly chromosome identification.Existing research shows that Patinopecten yessoensis has 19 pairs of chromosomes, and karyotype formulas is
3m+5sm+8st+3t, but since chromosome quantitative is numerous, form is close, is difficult to accomplish that chromosome is identified by morphology.Cause
This, we carry out specific marker to chromosome by screening specific marker from genome, by means of FISH technology, and then solve
Certainly chromosome accurately identifies problem, this also will be helpful to push Patinopecten yessoensis cytogenetical study.
Summary of the invention
Present invention firstly provides a kind of specific recognition methods of Patinopecten yessoensis telocentric chromosome, and for spy
Six kinds of probes containing SNP marker of the opposite sex three pairs of telocentric chromosomes of positioning, to make up the deficiencies in the prior art.
Present invention firstly provides the SNP markers for being used to prepare detection end centromere chromosome probe, include:
Two SNP markers for identifying a pair of end portions centromere chromosome are M3611 and M3897, and nucleotides sequence is classified as
ACAACCAGGAGTTGGAGTCTGGTGAAA (SEQ ID NO:1) and TGCGCATGTACGTCACCTCCGCATCAT (SEQ ID
NO:2)；
Two SNP markers for identifying second pair of telocentric chromosome are M1444 and M6119, and nucleotides sequence is classified as
ACTCCACCAACACAGCCTCCAGGGGTC (SEQ ID NO:3) and GTATCAATAACATAAGCTCCTTTTCCT (SEQ ID
NO:4)；
Identify that third is M4445 and M4162 to two SNP markers of telocentric chromosome, nucleotides sequence is classified as
ATAGAAAGGAGTATATGTATTTCATAC (SEQ ID NO:5) and GATGACCGGAGGCAAAGTGACCAAGAC (SEQ ID
NO:6)；
Above-mentioned SNP marker is used to prepare from Fosmid monoclonal to position Patinopecten yessoensis three to telocentric chromosome
Probe,
Its it is a kind of specific steps are as follows:
1) screening of Fosmid monoclonal: SNP marker sequence and Patinopecten yessoensis fosmid monoclonal decoded information are carried out
Intersect and compare, positioning obtains fosmid monoclonal containing SNP homologous sequence；
2) Fosmid cloned plasmids extract: expanding numerous Escherichia coli comprising clone by LB culture medium, use phenol chloroform/alkali
The method of cracking extracts the fosmid monoclonal Plasmid DNA containing SNP homologous sequence；
3) prepare probe: taking the Plasmid DNA of 3ug, under the conditions of 16 DEG C using with fluorescent marker incising translation enzyme into
70~90min of row digestion come prepare size be 200~500bp fluorescence labeling probe；
Above-mentioned probe is for positioning Patinopecten yessoensis three to telocentric chromosome；
The present invention also provides a kind of specific recognition methods of Patinopecten yessoensis telocentric chromosome, are using above-mentioned
Probe includes following step come what is carried out:
1) prepared by chromosome: Patinopecten yessoensis trochophore is collected, is handled using 0.01% colchicine and 0.075M KCL,
Then it is stored in Carnot's reagent, is then dissociated using 50% acetic acid, film-making is carried out using hot drop method,
2) FISH and cohybridization: chromosome prepared by step 1) being placed in the denaturing liquid of 76 DEG C of preheatings and be denaturalized, and simultaneously will
Probe mixed liquor is denaturalized under the conditions of 90 DEG C, then the probe mixed liquor being denaturalized is added rapidly on chromosome, close the lid glass
Piece sealing is incubated for 10~12h, is then blockaded using bovine serum albumin and is dyed with rhodamine；Living through elution
Afterwards, redying for chromosome is carried out using DAPI, then the micro- sem observation fluorescence signal of mounting；
3) chromosome identifies: being dyed by the FISH positioning result of probe and different probe common location interpretation of result label
Position and karyological character on body, the results showed that label is successfully located in respectively on the chromosome of a pair of end portions centromere, and
Gradually compare the chromosome morphology and length characteristic that probe is positioned by label common location result, it was demonstrated that three pairs of probe signals
It is distributed on three pairs of different telocentric chromosomes, and completes the core of three pairs of telocentric chromosomes based on probe signals
Type analysis and Division identification to every a pair of end portions centromere chromosome.
The probe provided through the invention and chromosome recognition methods can quickly navigate to the every a pair of end portions of Patinopecten yessoensis
Centromere chromosome, compared to traditional form and karyotyping, the method discrimination is higher, and efficient stable, this prawn
The cytogenetics research of smooth scallop has the meaning of positive important.
Detailed description of the invention
Fig. 1: FISH positioning result figure of the probe containing SNP marker on Patinopecten yessoensis chromosome, in which:
A: for probe PF1003E16 specific localization on the chromosome of a pair of end portions centromere, fluorescence signal is located in end
The centromere position of portion centromere chromosome；
B: for probe PF1003D11 specific localization on the chromosome of a pair of end portions centromere, fluorescence signal is located in end
The long-armed end of portion centromere chromosome；
C: for probe PF10D8 specific localization on the chromosome of a pair of end portions centromere, fluorescence signal is located in end
The centromere position of silk grain chromosome；
D: for probe PF119E24 specific localization on the chromosome of a pair of end portions centromere, fluorescence signal is located in end
The long-armed end of centromere chromosome；
E: for probe PF12L15 specific localization on the chromosome of a pair of end portions centromere, fluorescence signal is located in end
The centromere position of silk grain chromosome；
F: for probe PF124E19 specific localization on the chromosome of a pair of end portions centromere, fluorescence signal is located in end
The long-armed end of silk grain chromosome；
G:PF1003E16 and PF1003D11 clone-specific is located on the chromosome of identical a pair of end portions centromere, glimmering
Optical signal be respectively positioned at telocentric chromosome centromere position and long-armed end；
H:PF10D8 and PF119E24 specific localization is on the chromosome of identical a pair of end portions centromere, fluorescence signal point
Be not located in telocentric chromosome centromere position and long-armed end；
I::PF12L15 and PF124E19 specific localization is on the chromosome of identical a pair of end portions centromere, fluorescence signal point
Be not located in telocentric chromosome centromere position and long-armed end；
J:PF1003D11 and PF10D8 is respectively positioned at difference on telocentric chromosome, and wherein PF1003D11 is marked
The telocentric chromosome of note is longer than the telocentric chromosome of PF10D8 label；
K:PF1003D11 and PF12L15 is respectively positioned at different on telocentric chromosome, wherein PF1003D11
The telocentric chromosome of label is longer than the telocentric chromosome of PF12L15 label；
L:PF124E19 and PF10D8 is respectively positioned at difference on telocentric chromosome, and wherein PF10D8 is marked
Telocentric chromosome is longer than the telocentric chromosome of PF124E19 label.
Concrete operations mode
The present invention is based on the library Patinopecten yessoensis genome large fragment fosmid, SNP high density linkage map and FISH technologies
It develops specific probe to mark as Patinopecten yessoensis telocentric chromosome specific recognition, to realize that Patinopecten yessoensis three is right
The differentiation identification of telocentric chromosome and specific recognition have further pushed Patinopecten yessoensis cytogenetical study.
The following further describes the technical solution of the present invention for concrete operations by the following examples.
Embodiment 1: the preparation of probe
1) Fosmid monoclonal screens: according to the integration work of Patinopecten yessoensis SNP linkage map and chromosome map, wherein
LG3, LG15 and LG18 correspond to Patinopecten yessoensis telocentric chromosome.
Two SNP markers for wherein identifying a pair of end portions centromere chromosome are M3611 and M3897, nucleotides sequence
It is classified as ACAACCAGGAGTTGGAGTCTGGTGAAA (SEQ ID NO:1) and TGCGCATGTACGTCACCTCCGCATCAT (SEQ
ID NO:2)；
Two SNP markers for identifying second pair of telocentric chromosome are M1444 and M6119, and nucleotides sequence is classified as
ACTCCACCAACACAGCCTCCAGGGGTC (SEQ ID NO:3) and GTATCAATAACATAAGCTCCTTTTCCT (SEQ ID
NO:4)；
Identify that third is M4445 and M4162 to two SNP markers of telocentric chromosome, nucleotides sequence is classified as
ATAGAAAGGAGTATATGTATTTCATAC (SEQ ID NO:5) and GATGACCGGAGGCAAAGTGACCAAGAC (SEQ ID
NO:6)；
Fosmid monoclonal is positioned by above-mentioned SNP marker, and probe is prepared by Fosmid monoclonal.
It is restricted using BsaXI and two kinds of FspEI by mixing building row pond, column pond, Ban Chi first with WGP method
Restriction endonuclease is decoded the monoclonal of 40 384 orifice plates according to the mixed pond strategy of three-dimensional to obtain digestion label.Monoclonal solution
Code message identification go out each digestion sequence label in Patinopecten yessoensis genome where scaffold serial number and at this
Position on scaffold.Digestion sequence label all in the same monoclonal count and then available fosmid is mono-
Sequence information included in clone, sequence scaffold serial number in the genome and sequence section start-stop location information.
Select two SNP markers from every linkage group end, using SNP marker sequence and Patinopecten yessoensis fosmid library sequence information into
The homologous comparison of row, i.e. flag sequence must be included in the sequence of fosmid monoclonal, and label is with fosmid monoclonal
It is unique to compare, the monoclonal comprising SNP marker is sifted out with each linkage group of the method to be used as chromosome identification probe.
Table 1: the fosmid monoclonal information containing SNP marker
2) Fosmid cloned plasmids extract: in the superclean bench of ultraviolet sterilization, using sterilizing toothpick from corresponding 384 hole
Plate picking contains the Escherichia coli of monoclonal, is placed in LB culture medium, 8~12h of activation culture in 37 DEG C of constant incubators.To
After muddiness occurs in bacterium solution, the extraction of Plasmid DNA is carried out using phenol chloroform method, the Plasmid DNA for drawing 1ul dilutes 10 times, and 4ul is taken to run
1% agarose gel electrophoresis, electrophoresis result show complete single DNA band, draw 1ul dilution by nano and measure plasmid
Quality and concentration, A260/A230 is greater than 1.8, A260/A280 between 1.8~2.0, show the Plasmid DNA of extraction compared with
To be pure, Concentration Testing shows that final concentration is greater than 200ng/ul, meets the requirement of probe preparation.
3) preparation of probe: taking the Plasmid DNA of 3ug, and it is right under the conditions of 16 DEG C to translate enzyme using incising with fluorescent marker
Plasmid DNA carries out 70~90min of digestion, then takes the digestion products of 1ul to be detected with 1% agarose gel electrophoresis, piece segment length
Degree then terminates endonuclease reaction to dropwise addition 1ul 0.5M EDTA in reaction system, finally uses DNA product in 200~500bp
Probe is placed in -20 DEG C of preservations by Purification Kit probe.
Embodiment 2: probe positioning dyeing body is used
1) prepared by chromosome: Patinopecten yessoensis trochophore is collected using 500 mesh thin,tough silk, at 0.01% colchicine
2.5h is managed to carry out the fixation of larva, Hypotonic treatment 30min then is carried out to larva material with 0.075M KCL, then by material
It is placed in the Carnot's reagent of Fresh, and replaces Carnot's reagent three times, can be protected herein place the material in -20 DEG C long-term
It deposits, is then dissociated using 50% acetic acid of Fresh, 5~10min, film-making is carried out using hot drop method, will be dyed
Body material is fixed on glass slide, and 2~3h is placed in 60 DEG C of baking ovens is attached to chromosome material closely on slide, is prevented miscellaneous
Friendship process chromosome material falls off, and chromosome prepares bibliography (Huang et al.2007.Mapping of
ribosomal DNA and(TTAGGG)n telomeric sequence by FISH in the bivalve
Patinopecten yessoensis(Jay,1857))。
2) FISH and cohybridization: the chromosome material of preparation is placed in the formamide denaturation liquid of 76 DEG C of preheatings and is become in advance
Property, while the mixed liquor of probe, sodium citrate buffer solution and formamide being denaturalized to 5min under the conditions of 90 DEG C, then it will be denaturalized
Probe mixed liquor be added in rapidly through denaturation and Gradient elution using ethanol chromosome on, be covered with coverslip and sealed membrane, be placed in
In the wet box being protected from light, 10~12h is incubated overnight under the conditions of 37 DEG C.Extra spy is washed in 37 DEG C of sodium citrate buffer solutions
Needle is then blockaded with bovine serum albumin(BSA) and anti-digoxin rhodamine carries out dyeing 1h.Then sodium citrate is reused
Extra dye liquor is removed in buffer elution, redyes 20~30min of chromosome using DAPI nucleic acid dye liquor, then mounting is fixed, finally
The observation of fluorescence signal, FISH experimentation bibliography (Huang et are carried out using fluorescence microscope
al.2007.Mapping of ribosomal DNA and(TTAGGG)n telomeric sequence by FISH in
the bivalve Patinopecten yessoensis(Jay,1857))。
3) chromosome identifies: under the microscope it can be observed that probe PF1003E16 specific localization is in a pair of end portions
On silk grain chromosome, fluorescence signal is located in the centromere position of telocentric chromosome (shown in Figure 1A)；Probe
For PF1003D11 specific localization on the chromosome of a pair of end portions centromere, fluorescence signal is located in telocentric chromosome
Long-armed end (shown in Figure 1B)；Probe PF10D8 specific localization is on the chromosome of a pair of end portions centromere, fluorescence signal
It is located in the centromere position of telocentric chromosome (shown in Fig. 1 C)；Probe PF119E24 specific localization is in a pair of end portions
On the chromosome of centromere, fluorescence signal is located in the long-armed end of telocentric chromosome (shown in Fig. 1 D)；Probe
PF12L15 specific localization on the chromosome of a pair of end portions centromere, fluorescence signal be located in telocentric chromosome
Silk grain position (shown in Fig. 1 E)；For probe PF124E19 specific localization on the chromosome of a pair of end portions centromere, fluorescence signal is fixed
In the long-armed end (shown in Fig. 1 F) of telocentric chromosome, the above results show that six pairs of probes can identify a pair respectively for position
Telocentric chromosome.But two from same linkage group mark whether to be located in same a pair of end portions centromere chromosome
On also not it is found that therefore we have first carried out cohybridization experiment to two SNP markers from same linkage group, the results showed that visit
Needle PF1003E16 and PF1003D11 clone-specific is located on the chromosome of identical a pair of end portions centromere, fluorescence signal point
It is not located in the centromere position and long-armed end of telocentric chromosome (shown in Fig. 1 G)；Probe PF10D8 and PF119E24
For specific localization on the chromosome of identical a pair of end portions centromere, fluorescence signal is respectively positioned at telocentric chromosome
Centromere position and long-armed end (shown in Fig. 1 H)；Probe PF12L15 and PF124E19 specific localization are in identical a pair of end portions
On the chromosome of centromere, fluorescence signal be respectively positioned at telocentric chromosome centromere position and long-armed end (Fig. 1's
Shown in I), the above results show that six labels from three linkage groups are located in same a pair of end portions centromere dyeing two-by-two respectively
On body.
Cohybridization experiment from different chain group marks, the results showed that probe PF1003D11 and PF10D8 are positioned respectively
In difference on telocentric chromosome, wherein the telocentric chromosome of PF1003D11 label is longer than PF10D8 label
Telocentric chromosome (shown in the J of Fig. 1)；Probe PF1003D11 and PF12L15 are respectively positioned at difference to end silk
On grain chromosome, wherein the telocentric chromosome of PF1003D11 label is longer than the end centromere dyeing of PF12L15 label
Body (shown in the K of Fig. 1)；Probe PF124E19 and PF10D8 be respectively positioned at it is different on telocentric chromosome, wherein
The telocentric chromosome of PF10D8 label is longer than the telocentric chromosome of PF124E19 label (shown in the L of Fig. 1).On
Result is stated it is found that three pairs of probes are respectively positioned on different telocentric chromosomes, at the same probe PF1003E16 and
PF1003D11 specific localization is on the chromosome of longest a pair of end portions centromere, probe PF12L15 and PF124E19 specificity
Be located on most short a pair of end portions centromere chromosome, the chromosome that probe PF10D8 and PF119E24 are positioned between the two it
Between.According to chromosome according to the ordering rule of form length, i.e. probe PF1003E16 and PF1003D11 specific recognition first
To telocentric chromosome, probe PF10D8 and PF119E24 specific recognition second is to telocentric chromosome, probe
PF12L15 and PF124E19 specific recognition third is to telocentric chromosome.
Sequence table
<110>Chinese Marine University
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gtatcaataa cataagctcc ttttcct 27
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gatgaccgga ggcaaagtga ccaagac 27
Claims (7)
1. a kind of SNP marker for being used to prepare detection end centromere chromosome probe, which is characterized in that the SNP marker
Include:
Identify two SNP markers of a pair of end portions centromere chromosome, nucleotides sequence is classified as SEQ ID NO:1 and SEQ ID
NO:2；
Identify two SNP markers of second pair of telocentric chromosome, nucleotides sequence is classified as SEQ ID NO:3 and SEQ ID
NO:4；
Third is identified to two SNP markers of telocentric chromosome, nucleotides sequence is classified as SEQ ID NO:5 and SEQ ID
NO:6。
2. SNP marker described in claim 1 is in the preparation positioning Patinopecten yessoensis end centromere dyeing from Fosmid monoclonal
Application in the probe of body.
3. a kind of probe for positioning Patinopecten yessoensis telocentric chromosome, which is characterized in that the probe is using right
It is required that SNP marker described in 1 is screened from Patinopecten yessoensis Fosmid monoclonal and to be obtained.
4. the preparation method of probe as claimed in claim 3, which is characterized in that the step of described method is as follows:
1) screening of Fosmid monoclonal: by the sequence of SNP marker described in claim 1 and Patinopecten yessoensis fosmid monoclonal
Decoded information carries out intersection comparison, and positioning obtains the fosmid monoclonal containing SNP homologous sequence；
2) Fosmid cloned plasmids extract: extracting the fosmid monoclonal Plasmid DNA containing SNP homologous sequence；
3) it prepares probe: translating enzyme to the progress digestion of fosmid monoclonal Plasmid DNA using incising with fluorescent marker to make
The fluorescence labeling probe that standby size is 200~500bp.
5. application of the probe as claimed in claim 3 in positioning Patinopecten yessoensis telocentric chromosome.
6. a kind of specific recognition method of Patinopecten yessoensis telocentric chromosome, which is characterized in that the method is to make
It is positioned with probe as claimed in claim 3.
7. method as claimed in claim 6, which is characterized in that the step of described method is as follows:
1) prepared by chromosome: collecting Patinopecten yessoensis trochophore, is handled using 0.01% colchicine and 0.075M KCL, then
It is stored in Carnot's reagent, is then dissociated using 50% acetic acid, film-making is carried out using hot drop method,
2) FISH and cohybridization: chromosome prepared by step 1) is placed in the denaturing liquid of 76 DEG C of preheatings and is denaturalized, while by probe
Mixed liquor is denaturalized under the conditions of 90 DEG C, then the probe mixed liquor being denaturalized is added rapidly on chromosome, covered is close
Envelope is incubated for 10~12h, is then blockaded using bovine serum albumin and is dyed with rhodamine；After living through elution, make
Redying for chromosome is carried out with DAPI, then the micro- sem observation fluorescence signal of mounting；
3) chromosome identifies: on chromosome by the FISH positioning result of probe and different probe common location interpretation of result label
Position and karyological character.
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|---|---|---|---|---|
| CN103740729A (en) * | 2014-01-25 | 2014-04-23 | 中国海洋大学 | SNP locus related to growth characteristics of patinopecten yessoensis and detection and application thereof |
| CN105087815A (en) * | 2015-09-23 | 2015-11-25 | 黑龙江省畜牧研究所 | Primers for comb shell EST-SSR detection and molecular marking method thereof |
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| CN103740729A (en) * | 2014-01-25 | 2014-04-23 | 中国海洋大学 | SNP locus related to growth characteristics of patinopecten yessoensis and detection and application thereof |
| CN105087815A (en) * | 2015-09-23 | 2015-11-25 | 黑龙江省畜牧研究所 | Primers for comb shell EST-SSR detection and molecular marking method thereof |
| CN105543342A (en) * | 2015-11-26 | 2016-05-04 | 集美大学 | Method for displaying centromeres and short arms of Larimichthys crocea |
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| LI-PING HU等: "Comparative Cytogenetics Analysis of Chlamys farreri,Patinopecten yessoensis, and Argopecten irradians with C0t-1 DNA by Fluorescence In Situ Hybridization", 《EVIDENCE-BASED COMPLEMENTARY AND ALTERNATIVE MEDICINE》 * |
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