CN109880919B - Specific identification method for patinopecten yessoensis end centromere chromosome - Google Patents
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Abstract
The invention provides a method for identifying patinopecten yessoensis end centromere chromosomes, which identifies two SNP markers of a first pair of end centromere chromosomes, wherein the nucleotide sequences of the SNP markers are SEQ ID NO. 1 and SEQ ID NO. 2; two SNP markers for identifying another pair of centromere chromosomes at the ends, the nucleotide sequences of which are SEQ ID NO. 3 and SEQ ID NO. 4; two SNP markers recognizing the centromere chromosome at the third pair of ends have the nucleotide sequences of SEQ ID NO. 5 and SEQ ID NO. 6. The probe and the chromosome recognition method provided by the invention can be used for rapidly positioning each pair of end centromere chromosomes of the patinopecten yessoensis, compared with the traditional morphology and karyotype analysis, the method has higher discrimination, is efficient and stable, and has positive and important significance for cytogenetic research of the patinopecten yessoensis.
Description
Technical Field
The invention belongs to the technical field of shellfish chromosome research, and particularly relates to a specific identification method of patinopecten yessoensis end centromere chromosomes.
Background
Chromosomes are carriers of genetic materials, and specific identification of chromosomes is important research content of cytogenetics and also an important theoretical basis for developing genetic breeding work. The traditional chromosome differentiation mainly depends on the chromosome number and the chromosome morphology, but the chromosomes of shellfish are small and numerous, and are difficult to distinguish visually. The chromosome banding pattern can show the internal structure of the chromosome and provide more information with identifying characteristics, but the existing research shows that the technology has the problems of unstable result, unclear banding pattern and the like in the shellfish chromosome research, and the problems seriously limit the shellfish chromosome recognition.
Patinopecten yessoensis is an important seawater culture shellfish in the north of China, and the patinopecten yessoensis culture becomes a very important support industry in coastal areas of China due to high economic value and good culture foundation. The development of the virtuous cycle of the genetic breeding work of the patinopecten yessoensis has positive and important significance. In recent years, the construction of high-density genetic linkage maps of Japanese scallops and the genome sequencing are completed successively, and molecular level guidance is provided for the genetic breeding work of the Japanese scallops. However, the further application of omics data to specific breeding work still requires the integration of chromosome and genome information, wherein the important early work is chromosome identification. The existing research shows that the Japanese scallop has 19 pairs of chromosomes, the karyotype formula is 3m +5sm + 8st +3t, but the chromosome number is large, the form is close, and the chromosome recognition is difficult to realize through morphology. Therefore, we can solve the problem of accurate identification of the chromosome by screening specific markers from the genome and specifically marking the chromosome by means of FISH technology, which will also help to promote the cytogenetic research of the Japanese scallop.
Disclosure of Invention
The invention firstly provides a specific identification method of patinopecten yessoensis end centromere chromosomes and six probes containing SNP markers for specifically positioning three pairs of end centromere chromosomes, thereby making up the defects of the prior art.
The present invention firstly provides an SNP marker for preparing a probe for detecting an end centromere chromosome, comprising:
two SNP markers, M3611 and M3897, whose nucleotide sequences are ACAACCAGGAGTTGGAGTCTGGTGAAA (SEQ ID NO:1) and TGCGCATGTACGTCACCTC CGCATCAT (SEQ ID NO:2), that recognize the first pair of terminal centromeric chromosomes;
the two SNP markers that recognize the second opposite terminal centromere chromosome are M1444 and M6119, whose nucleotide sequences are ACTCCACCAACACAGCCTCCAGGGGTC (SEQ ID NO:3) and GTATCAATAACATAAGCTC CTTTTCCT (SEQ ID NO: 4);
the two SNP markers that identify the third pair of terminal centromere chromosomes are M4445 and M4162, whose nucleotide sequences are ATAGAAAGGAGTATATGTATTTCATAC (SEQ ID NO:5) and GATGACCGGAGGCAAAGTG ACCAAGAC (SEQ ID NO: 6);
the SNP marker is used for preparing a probe for positioning the centromere chromosomes at the three pairs of end parts of the comb shell from Fosmid single clone.
One specific step is as follows:
1) screening of Fosmid monoclonals: carrying out cross comparison on the SNP marker sequence and patinopecten yessoensis fosmid monoclonal decoding information, and positioning to obtain fosmid monoclonal containing SNP homologous sequences;
2) extracting Fosmid clone plasmid: amplifying Escherichia coli containing clones by LB culture medium, and extracting fosmid monoclonal plasmid DNA containing SNP homologous sequences by phenol chloroform/alkali lysis method;
3) preparing a probe: taking 3ug of plasmid DNA, and performing enzyme digestion for 70-90 min at 16 ℃ by using nicking translation enzyme with a fluorescent label to prepare a fluorescent label probe with the size of 200-500 bp;
the probes are used for positioning centromere chromosomes at the three pairs of end parts of the comb shells;
the invention also provides a specific identification method of the patinopecten yessoensis end centromere chromosome, which is carried out by using the probe and comprises the following steps:
1) preparing a chromosome: collecting Japanese scallop trochophore, treating the Japanese scallop trochophore by adopting 0.01 percent colchicine and 0.075M KCL, then storing the Japanese scallop trochophore in a carnot reagent, dissociating the Japanese scallop trochophore by using 50 percent acetic acid, and making a sheet by adopting a hot drop method;
2) FISH and co-hybridization: placing the chromosome prepared in the step 1) in a preheated denaturing solution at 76 ℃ for denaturation, simultaneously denaturing the probe mixed solution at 90 ℃, then rapidly adding the denatured probe mixed solution to the chromosome, covering a cover glass, sealing, incubating for 10-12 h, and then using bovine serum albumin for blocking and rhodamine for dyeing; after undergoing elution, counterstaining of chromosomes was performed using DAPI, and then mounting was observed for a fluorescent signal with a microscope;
3) chromosome recognition: the position and chromosome characteristics of the marker on the chromosome are analyzed through the FISH positioning result of the probe and the co-positioning result of different probes, the result shows that the marker is successfully positioned on a pair of terminal centromere chromosomes respectively, the morphology and length characteristics of the chromosome positioned by the probe are gradually compared through the co-positioning result of the marker, the signals of the three pairs of probes are proved to be distributed on the three pairs of terminal centromere chromosomes, and the karyotype analysis of the three pairs of terminal centromere chromosomes and the distinguishing and identification of each pair of terminal centromere chromosomes are completed based on the signals of the probes.
The probe and the chromosome recognition method provided by the invention can be used for rapidly positioning each pair of end centromere chromosomes of the patinopecten yessoensis, compared with the traditional morphology and karyotype analysis, the method has higher discrimination, is efficient and stable, and has positive and important significance for cytogenetic research of the patinopecten yessoensis.
Drawings
FIG. 1: FISH positioning result graph of the probe containing the SNP marker on the chromosome of the Japanese scallop, wherein:
a: the probe PF1003E16 is specifically positioned on a pair of terminal centromere chromosomes, and the fluorescence signal of the probe is positioned at the centromere position of the terminal centromere chromosomes;
b: the probe PF1003D11 is specifically positioned on a pair of terminal centromere chromosomes, and the fluorescence signal of the probe is positioned at the tail ends of the long arms of the terminal centromere chromosomes;
c: the probe PF10D8 is specifically positioned on a pair of terminal centromere chromosomes, and the fluorescence signal of the probe is positioned at the centromere position of the terminal centromere chromosomes;
d: the probe PF119E24 is specifically positioned on a pair of terminal centromere chromosomes, and the fluorescence signal of the probe is positioned at the tail ends of the long arms of the terminal centromere chromosomes;
e: the probe PF12L15 is specifically positioned on a pair of terminal centromere chromosomes, and the fluorescent signal is positioned at the centromere position of the terminal centromere chromosomes;
f: the probe PF124E19 is specifically positioned on a pair of terminal centromere chromosomes, and the fluorescence signal is positioned at the tail ends of the long arms of the terminal centromere chromosomes;
g: clones PF1003E16 and PF1003D11 were specifically located on the same pair of terminal centromere chromosomes, with their fluorescent signals located at the centromere position and long arm end of the terminal centromere chromosome, respectively;
h: PF10D8 and PF119E24 were specifically located on the same pair of terminal centromeric chromosomes, with their fluorescent signals located at the centromeric position and the long arm ends, respectively, of the terminal centromeric chromosomes;
i: PF12L15 and PF124E19 were specifically located on the same pair of terminal centromeric chromosomes, with the fluorescence signal located at the centromeric position and the long arm end of the terminal centromeric chromosomes, respectively;
j: PF1003D11 and PF10D8 were located on different opposite terminal centromere chromosomes, respectively, wherein the PF1003D 11-labeled terminal centromere chromosome was longer than the PF10D 8-labeled terminal centromere chromosome;
k: PF1003D11 and PF12L15 were located on different opposite terminal centromere chromosomes, respectively, wherein the PF1003D 11-labeled terminal centromere chromosome was longer than the PF12L 15-labeled terminal centromere chromosome;
l: PF124E19 and PF10D8 were located on different opposite terminal centromere chromosomes, respectively, wherein the PF10D 8-labeled terminal centromere chromosome was longer than the PF124E 19-labeled terminal centromere chromosome.
Detailed description of the preferred embodiment
The specific probe is developed based on a large fragment fosmid library of a comb shell genome, an SNP high-density linkage map and a FISH technology and is used as a specific identification marker of the end centromere chromosome of the comb shell, so that the distinguishing identification and the specific identification of three pairs of end centromere chromosomes of the comb shell are realized, and the cytogenetic research of the comb shell is further promoted.
The technical solution of the present invention is further described by the specific operations of the examples below.
Example 1: preparation of Probe
1) Fosmid monoclonal screening: according to the integration work of the SNP linkage map and the chromosome map of the Japanese scallop, LG3, LG15 and LG18 correspond to the centromere chromosome of the end part of the Japanese scallop.
Wherein the two SNP markers which recognize the first pair of terminal centromeric chromosomes are M3611 and M3897, the nucleotide sequences of which are ACAACCAGGAGTTGGAGTCTGGTGAAA (SEQ ID NO:1) and TGCGCATGTACGTCACCTC CGCATCAT (SEQ ID NO: 2);
the two SNP markers that recognize the second opposite terminal centromere chromosome are M1444 and M6119, whose nucleotide sequences are ACTCCACCAACACAGCCTCCAGGGGTC (SEQ ID NO:3) and GTATCAATAACATAAGCTC CTTTTCCT (SEQ ID NO: 4);
the two SNP markers that identify the third pair of terminal centromere chromosomes are M4445 and M4162, whose nucleotide sequences are ATAGAAAGGAGTATATGTATTTCATAC (SEQ ID NO:5) and GATGACCGGAGGCAAAGTG ACCAAGAC (SEQ ID NO: 6);
fosmid monoclonals are located by the SNP markers described above, and probes are prepared by the Fosmid monoclonals.
Firstly, a WGP method is utilized, a row pool, a column pool and a plate pool are constructed by mixing, restriction enzymes BsaXI and FspEI are used for obtaining enzyme cutting labels, and the single clone of 40 pieces of 384-hole plates is decoded according to a three-dimensional pool mixing strategy. The monoclonal decoding information identifies the scaffold number of each restriction tag sequence in the patinopecten yessoensis genome and the position on the scaffold. All enzyme cutting label sequences in the same monoclonal are counted to further obtain sequence information contained in the fosmid monoclonal, a scaffold serial number of the sequence on a genome and start and stop position information of a sequence section. Two SNP markers are selected from the end part of each linkage group, and homologous alignment is carried out by utilizing the SNP marker sequence and comb scallop fosmid library sequence information, namely the marker sequence must be contained in the sequence of fosmid monoclonal, and the marker and the fosmid monoclonal are uniquely aligned, and each linkage group is screened out a single clone containing the SNP markers by the method to be used as a chromosome recognition probe.
Table 1: fosmid monoclonal information containing SNP marker
2) Extracting Fosmid clone plasmid: and (3) selecting Escherichia coli containing monoclone from a corresponding 384-well plate by using a sterilization toothpick on an ultra-clean workbench subjected to ultraviolet sterilization, placing the Escherichia coli in an LB culture medium, and performing activated culture in a constant-temperature incubator at 37 ℃ for 8-12 h. After the bacterial liquid is turbid, extracting plasmid DNA by using a phenol chloroform method, sucking 1ul of plasmid DNA for diluting by 10 times, taking 4ul of the plasmid, running 1% agarose gel for electrophoresis, wherein an electrophoresis result shows a complete single DNA band, sucking 1ul of diluent, and determining the quality and the concentration of the plasmid through nano, wherein A260/A230 is more than 1.8, A260/A280 is between 1.8 and 2.0, which shows that the extracted plasmid DNA is purer, and the concentration detection shows that the final concentration is more than 200ng/ul, thereby meeting the requirement of probe preparation.
3) Preparing a probe: taking 3ug of plasmid DNA, carrying out enzyme digestion on the plasmid DNA for 70-90 min at 16 ℃ by using nicking translation enzyme with a fluorescent marker, then taking 1ul of enzyme digestion product, detecting by using 1% agarose gel electrophoresis, dropping 1ul of 0.5M EDTA into a reaction system to terminate the enzyme digestion reaction, finally purifying the probe by using a DNA product purification kit, and storing the probe at-20 ℃.
Example 2: mapping chromosomes using probes
1) Preparing a chromosome: collecting Patinopecten yessoensis trochophore by using 500-mesh bolting silk, treating for 2.5h by using 0.01% colchicine to fix the larva, then carrying out hypotonic treatment on the larva material for 30min by using 0.075M KCL, then placing the material in a freshly prepared Carnot reagent, and replacing the Carnot reagent for three times, wherein the material can be placed at-20 ℃ for long-term storage, then dissociating by using 50% acetic acid which is freshly prepared for 5-10 min, then carrying out slide making by using a hot drop method, fixing the chromosome material on a glass slide, placing in an oven at 60 ℃ for 2-3 h to make the chromosome material tightly attached to the glass slide, and preventing the chromosome material from falling off in the hybridization process, and preparing a reference document of chromosomes (Huang et al.2007.mapping of chromosomal FISCE. by the same DNA and (TTAGGG) n-transcriptional sequence by the same FISH H in a bivalopic Patinopyeensis (Jay, 1857)).
2) FISH and co-hybridization: the prepared chromosome material is placed in formamide denaturation liquid preheated at 76 ℃ for pre-denaturation, meanwhile, the mixed liquid of the probe, the sodium citrate buffer solution and the formamide is denatured for 5min at 90 ℃, then the denatured probe mixed liquid is rapidly added on the chromosome which is subjected to denaturation and ethanol gradient dehydration, a cover glass and a sealing film are covered, the chromosome is placed in a light-proof wet box, and the chromosome is incubated overnight at 37 ℃ for 10-12 h. Excess probe was washed out with sodium citrate buffer at 37 ℃ followed by staining with bovine serum albumin blocking and anti-digoxigenin rhodamine dye for 1 h. Then, the excess dye solution is eluted again by using sodium citrate buffer solution, the chromosome is counterstained by using DAPI nucleic acid dye solution for 20-30 min, then mounting and fixing are carried out, and finally, the observation of fluorescence signal is carried out by using a fluorescence microscope, FISH experimental process reference (Huang et al 2007.mapping of nucleic acid sequence by FISH in the bivalve Patinopecten yessoensis (Jay, 1857)).
3) Chromosome recognition: under a microscope, the probe PF1003E16 is observed to be specifically positioned on a pair of end centromere chromosomes, and the fluorescence signal of the probe is positioned at the centromere position of the end centromere chromosomes (shown in FIG. 1A); the probe PF1003D11 is specifically positioned on a pair of terminal centromere chromosomes, and the fluorescence signal of the probe is positioned at the tail ends of the long arms of the terminal centromere chromosomes (shown in FIG. 1B); probe PF10D8 was specifically localized on a pair of terminal centromere chromosomes, with its fluorescent signal localized at the centromere position of the terminal centromere chromosome (shown in fig. 1C); probe PF119E24 was specifically localized on a pair of terminal centromere chromosomes, with its fluorescent signal localized at the long arm ends of the terminal centromere chromosomes (shown in fig. 1D); probe PF12L15 was specifically localized on a pair of terminal centromere chromosomes, and the fluorescence signal was localized at the centromere position of the terminal centromere chromosomes (shown in fig. 1E); the results of probes PF124E19, which specifically mapped to a pair of terminal centromere chromosomes and fluorescence signals mapped to the ends of the long arms of the terminal centromere chromosomes (FIG. 1F), indicate that six pairs of probes can recognize a pair of terminal centromere chromosomes, respectively. However, it was not known whether two markers from the same linkage group are located on the same pair of terminal centromere chromosomes, and therefore, we performed a co-hybridization experiment on two SNP markers from the same linkage group, and the results showed that the clones of probes PF1003E16 and PF1003D11 are specifically located on the same pair of terminal centromere chromosomes, and their fluorescence signals are located at the centromere position and the long-arm end of the terminal centromere chromosomes, respectively (FIG. 1G); probes PF10D8 and PF119E24 were specifically located on the same pair of terminal centromere chromosomes, with their fluorescent signals located at the centromere position and the long arm end of the terminal centromere chromosome, respectively (FIG. 1H); the results of probes PF12L15 and PF124E19, which specifically mapped on the same pair of terminal centromere chromosomes, and fluorescence signals mapped on the centromere position and the long-arm end of the terminal centromere chromosomes, respectively (shown in FIG. 1I), indicate that six markers from three linkage groups are mapped two by two on the same pair of terminal centromere chromosomes, respectively.
From co-hybridization experiments with different linkage group markers, the results showed that probes PF1003D11 and PF10D8 were located on different opposite terminal centromere chromosomes, respectively, wherein the terminal centromere chromosome labeled PF1003D11 was longer than the terminal centromere chromosome labeled PF10D8 (shown in J of fig. 1); probes PF1003D11 and PF12L15 were located on different opposite terminal centromere chromosomes, respectively, wherein the PF1003D 11-labeled terminal centromere chromosome was longer than the PF12L 15-labeled terminal centromere chromosome (shown in K of fig. 1); probes PF124E19 and PF10D8 were located on different opposite terminal centromere chromosomes, respectively, wherein the PF10D 8-labeled terminal centromere chromosome was longer than the PF124E 19-labeled terminal centromere chromosome (shown in L of FIG. 1). From the above results, it was found that three pairs of probes were located on different terminal centromere chromosomes, while probes PF1003E16 and PF1003D11 were located on the longest pair of terminal centromere chromosomes, probes PF12L15 and PF124E19 were located on the shortest pair of terminal centromere chromosomes, and probes PF10D8 and PF119E24 were located between the two chromosomes. According to the sorting rule of chromosomes according to morphological length, namely probes PF1003E16 and PF1003D11 specifically recognize a first pair of terminal centromere chromosomes, probes PF10D8 and PF119E24 specifically recognize a second pair of terminal centromere chromosomes, and probes PF12L15 and PF124E19 specifically recognize a third pair of terminal centromere chromosomes.
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Claims (1)
1. The application of the molecular marker in preparing a probe for positioning the centromere chromosome at the end part of the patinopecten yessoensis from the Fosmid monoclonal; the molecular marker comprises:
two molecular markers for identifying the first paired end centromere chromosome, the nucleotide sequences of which are SEQ ID NO. 1 and SEQ ID NO. 2;
two molecular markers for identifying the centromere chromosome at the second opposite end, the nucleotide sequences of which are SEQ ID NO. 3 and SEQ ID NO. 4;
two molecular markers recognizing the third paired end centromere chromosome, the nucleotide sequences of which are SEQ ID NO. 5 and SEQ ID NO. 6.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Non-Patent Citations (2)
| Title |
|---|
| Chromosomal mapping of tandem repeats in the Yesso Scallop, Patinopecten yessoensis (Jay, 1857), utilizing fluorescence in situ hybridization;Xuan Li等;《ComCytogen》;20160321;第10卷(第1期);157-169 * |
| Comparative Cytogenetics Analysis of Chlamys farreri,Patinopecten yessoensis, and Argopecten irradians with C0t-1 DNA by Fluorescence In Situ Hybridization;Li-Ping Hu等;《Evidence-Based Complementary and Alternative Medicine》;20110707;第2011卷;1-7 * |
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