CN111269992A - Saline-alkali-resistant molecular marker C1245 of portunus trituberculatus and application thereof - Google Patents
Saline-alkali-resistant molecular marker C1245 of portunus trituberculatus and application thereof Download PDFInfo
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- CN111269992A CN111269992A CN202010156786.1A CN202010156786A CN111269992A CN 111269992 A CN111269992 A CN 111269992A CN 202010156786 A CN202010156786 A CN 202010156786A CN 111269992 A CN111269992 A CN 111269992A
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/124—Animal traits, i.e. production traits, including athletic performance or the like
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
The invention provides a saline-alkali tolerant molecular marker C1245 of portunus trituberculatus and application thereof. The nucleotide sequence of the molecular marker C1245 is shown as SEQ ID No.1, and the nucleotide sequences of the primer pair for detecting the molecular marker C1245 are shown as SEQ ID No.2 and SEQ ID No. 3. The molecular marker C1245 is an Indel marker, and the alkali-resistant genotype is an insertion homozygous genotype. The molecular marker C1245 provided by the invention can not be limited by the growth stage of the portunus trituberculatus, obviously quickens the breeding process of the portunus trituberculatus and quickly breeds the crab species with excellent saline-alkali tolerance, and the molecular marker C1245 is accurate, reliable and simple to operate, is beneficial to the healthy breeding and development of the portunus trituberculatus, so that the molecular marker C1245 has wide application prospect.
Description
Technical Field
The invention belongs to the technical field of aquatic animal DNA molecular markers, and particularly relates to a saline-alkali-resistant molecular marker C1245 of portunus trituberculatus and application thereof.
Background
Portunus trituberculatus (Portugulus trituberculatus) belongs to Crustacea, decapod, Paramicidae, commonly called Portunus, and is an important large-scale marine economic crab in China. The swimming crabs are delicious in meat quality and rich in nutrition, and are deeply loved by consumers. The saline-alkali water area is a worldwide low-yield water resource, 4587 ten thousand hm2 saline-alkali water area exists in China, and due to the characteristics of high salinity, high alkalinity, high pH value, complex ion composition and the like, common aquatic animals cannot normally live and breed in the saline-alkali water area, so that the development and utilization of the water resource are greatly hindered. The portunus trituberculatus can affect the physiology and biochemistry of the portunus trituberculatus under the stimulation of high salt and alkali, and the high salt and alkali can reduce the ingestion rate, the metamorphosis rate and the survival rate of young crabs. Therefore, the saline-alkali tolerant character is one of important breeding characters of the portunus trituberculatus, and the method has important significance for improving the culture rate of the portunus trituberculatus and promoting the cultivation and popularization of the portunus trituberculatus in saline-alkali soil. However, the saline-alkali tolerant character has obvious low heritability characteristics, the inheritance of the traditional breeding method is slowly progressed, an advanced molecular marker-assisted breeding technology is urgently needed to accelerate the breeding process, and the identification and innovative application of the saline-alkali tolerant molecular marker are necessary preconditions and ways for developing molecular marker-assisted breeding.
At present, the research on the development of the saline-alkali tolerant molecular marker of the blue crab is less, and the industry lacks a marker which can be applied to molecular marker assisted breeding. Therefore, the development of the molecular marker related to the saline-alkali tolerant character has important significance for the healthy breeding and selective breeding of the portunid.
Disclosure of Invention
The invention provides a saline-alkali tolerant molecular marker C1245 of portunus trituberculatus and application thereof, the invention utilizes methods of polymorphic site filtration, comparison analysis and PCR sequencing of sequencing data to obtain SNP and InDel markers, and a new saline-alkali tolerant molecular marker C1245 of portunus trituberculatus is finally obtained through gradual screening and verification of the markers, and the molecular marker is beneficial to the breeding of the saline-alkali tolerant character of the portunus trituberculatus.
In order to realize the purpose of the invention, the invention adopts the following technical scheme to realize:
the invention provides a saline-alkali tolerant molecular marker C1245 of blue crab, and the nucleotide sequence of the molecular marker C1245 is shown in SEQ ID No. 1.
Further, the molecular marker C1245 is an Indel marker.
Further, the saline-alkali tolerance genotype of the molecular marker C1245 is an insertion homozygous genotype.
The invention also provides a primer pair for detecting the molecular marker C1245 as claimed in claim 1, wherein the nucleotide sequence of the forward primer in the primers is shown as SEQ ID No.2, and the nucleotide sequence of the reverse primer is shown as SEQ ID No. 3.
The invention also provides application of the molecular marker C1245 in screening salt and alkali tolerant varieties of portunus trituberculatus.
Further: the application steps are as follows: extracting DNA of a sample to be tested of the portunus trituberculatus, taking the DNA as a template, carrying out PCR amplification by using a molecular-labeled amplification primer, sequencing a PCR product, and selecting a sample to be tested as a parent for cultivating the saline-alkali tolerant variety of the portunus trituberculatus if the genotype of the molecular marker in a sequencing result is an insertion homozygous genotype.
Further: the PCR amplification system is as follows: template 1. mu.l, forward primer (10. mu.M) 0.2. mu.l, reverse primer (10. mu.M) 0.2. mu.l, Buffer 1. mu.l, dNTPs 0.8. mu.l, HiFi 0.2. mu.l, ddH2O 6.6μl。
Further: the PCR amplification procedure is as follows: pre-denaturation at 94 deg.C for 2-5 min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 1-2kb/min, repeating for 35 cycles; finally, the extension is carried out for 5-10min at 72 ℃.
The invention also provides application of the molecular marker C1245 in genetic diversity analysis, germplasm identification and genetic map construction of the blue crab.
Compared with the prior art, the invention has the following advantages and beneficial effects:
1. the saline-alkali tolerant molecular marker C1245 of the portunus trituberculatus provided by the invention can not be limited by the growth stage of the portunus trituberculatus, and can be used for breeding early crab seedlings of the portunus trituberculatus, so that the breeding process of the portunus trituberculatus is obviously accelerated, and crab species with excellent saline-alkali tolerant characters are rapidly bred.
2. The molecular marker C1245 provided by the invention is used for detecting the saline-alkali tolerance character of the portunus trituberculatus, the method is accurate and reliable, the operation is simple, the character meeting the requirement can be effectively and quickly screened, the short-time and low-cost early-stage breeding of the saline-alkali tolerance portunus trituberculatus is assisted, the number of the portunus trituberculatus with excellent quality is increased, the ingestion rate, the metamorphosis rate and the breeding rate of the portunus trituberculatus are improved, the yield of the portunus trituberculatus is improved, the healthy breeding of the portunus trituberculatus is promoted, and the application prospect.
Drawings
FIG. 1 shows the result of gel electrophoresis bands of the products of PCR amplification with two sets of primers with obvious position difference corresponding to the screened sensitive population mixed template and the screened resistant population mixed template.
Detailed Description
The technical solution of the present invention will be described in further detail with reference to specific examples.
The blue crabs used in the invention are all from the experimental base of Changyi Haifeng aquatic products Limited company of yellow sea aquatic product research institute of Chinese aquatic product science institute, healthy and active blue crabs are randomly obtained from a pond by a trawl fishing method, are temporarily arranged in a net basket, are paved with a layer of waterweeds to prevent the crabs from fighting, and finally 300 blue crabs with the weight of 35 +/-3 g are obtained and are placed in 4 culture ponds (500cm multiplied by 300cm multiplied by 150cm) for temporary culture for 7d, the water temperature is kept at 22 +/-1 ℃ during the temporary culture, water is added to 20cm, the carbonate alkalinity is 4mmol/L, the pH value is 8.2 +/-0.5, oxygen is continuously supplied, fresh seawater is replaced at 8 am every day, fresh trash fish is fed at 5 pm, and the feeding amount is about 1/3 of the total weight of the crabs. And 7d, selecting the portunids with better vitality and shape for subsequent experiments.
During formal experiments, crabs with vigorous activity and perfect body surface are placed in 4 cement pools, 50 crabs are placed in each pool, the salinity of seawater carbonate is firstly adjusted to 25mmol/L by using sodium bicarbonate, the pH value of a water body is 8.0 +/-0.5, the water temperature is kept at 22 +/-1 ℃, and the water depth is 20 cm. Feeding fresh trash fish at 5 pm with 1/4 of crab weight to ensure no bait residue on the bottom of the pond and influence water quality. Recording the death time, the weight, the shelling period and other data of the 20 crabs which die firstly, increasing the alkalinity of carbonate to 35mmol/L after 24 hours, wherein the pH value of the water body is 8.5 +/-0.5, and the water temperature is kept at 22 +/-1 ℃. And (4) continuously recording data such as death time, number and the like until the number of the surviving crabs in the 4 pools is 20, and stopping the experiment. The first 20 dead individuals were considered as saline-alkali sensitive group (denoted as Q), the last 20 surviving individuals were considered as saline-alkali tolerant group (denoted as H), and muscle tissue was dissected out and placed in a freezing tube and stored in liquid nitrogen.
Example 1
Screening of candidate molecular markers related to saline-alkali tolerance character
1. Sequencing data filtering and alignment
DNA extraction is carried out by adopting a kit of the whole gold company and by utilizing the principle that silica gel membrane centrifugal columns specifically adsorb DNA. First, approximately 30mg of a tissue sample was put into a sterile 1.5ml centrifuge tube, 200. mu.l of Lysis Buffer 8(LB8) and 20. mu.l of RNaseA (10mg/ml) were added, the mixture was incubated at room temperature for about 10 seconds with shaking for 2min, 20. mu.l of protease K (20mg/ml) was added, the mixture was thoroughly mixed with shaking, incubated at 55 ℃ until complete lysis, 1.5 times the volume of Binding Buffer 8(BB8) was added, the mixture was added to a centrifugal column, centrifuged at 12000rpm in a high-speed low-temperature refrigerated centrifuge (model Eppendorf58 5804R) for 30 seconds, and the waste liquid was discarded. Then 500. mu.l of Clean Buffer 8(CB8) was added, centrifuged at 12000rpm for 30s, the waste liquid was discarded (repeated), 500. mu.l of Wash Buffer 8(WB8) was added, centrifuged at 12000rpm for 30s, the waste liquid was discarded (repeated), and the mixture was left to stand at 12000rpm for 2min to completely remove the remaining WB 8. The column was placed in a clean centrifuge tube, 50. mu.l of Elution Buffer (EB) was added to the center of the column, and the column was allowed to stand at room temperature for 2min, centrifuged at 12000rpm for 1min, and the DNA was eluted. DNA purity and integrity was analyzed by agarose gel electrophoresis; the purity of the DNA (OD260/280 ratio) was measured by Nanodrop, and the DNA concentration was precisely quantified by Qubit.
Equivalently mixing the DNA samples qualified by the test into two mixing pools which are respectively named as a saline-alkali sensitive DNA mixing pool (SG) and a saline-alkali tolerant DNA mixing pool (TG). Randomly breaking a mixed DNA sample into fragments with the length of 350bp by a Covaris crusher, constructing a Library by adopting a TruSeq Library Construction Kit, and completing the preparation of the whole Library by the steps of end repair, ployA tail addition, sequencing joint addition, purification, PCR amplification and the like of the DNA fragments. The constructed library was sequenced by illumina hiseq PE 150. And filtering Raw reads obtained by sequencing to obtain Clean reads for subsequent analysis, wherein the sequencing data result is shown in table 1.
TABLE 1 summary of sequencing data quality
The filtered effective data are compared by Burrows-Wheeler alignment tool (BWA) software, and the comparison result is subjected to SAMTOOLS to remove duplication. The alignment rate of all samples is between 84.94% and 86.21%, the average coverage depth is between 44.65X and 48.31X, and the 1X coverage (coverage of at least one base) is more than 80.37%. The comparison result is normal, and can be used for subsequent variation detection and related analysis.
2. Marker detection and annotation
SNP and InDel are detected by a UnifiedGenottyper module in Genome analysis toolkit 3.8(GATK) software, the filtration parameters of the InDel are set to be QD < 4, FS > 200, and the total number of finally obtained InDel markers is 1,427,108.
3. Indel frequency difference analysis
The analysis calculates the Indel-index of each site of two groups of individuals, and filters the polymorphic sites, with the following filter criteria:
(1) the Indel-index frequencies in both groups were less than 0.3;
(2) the sites of Indel deletion in one individual were filtered out.
And meanwhile, calculating the frequency difference distribution of the indels in the following direction of △ (index) -index (saline and alkaline tolerance property), filtering out sites with △ index smaller than 0.3, and finally obtaining indels 1051 with difference among groups, wherein the indels are saline and alkaline tolerance related candidate molecular markers, and the statistics of the results of the candidate InDel are shown in Table 2.
TABLE 2 Indel test and annotation statistics
4. Marker screening
And screening candidate Indel markers, and selecting a site with the All-index close to 0 in the individual or selecting a site with the All-index close to 1 in the individual as a preferential selection site for next verification. The screening criteria were as follows:
based on the annotation information of InDel sites, sites with more than 5 inserted or deleted bases are preferably selected based on the high-to-low ordering by △ index.
Finally, screening out the marks with large frequency difference before and after salt and alkali stress, and screening out 11 Indel marks in total
II, salt and alkali tolerance related molecular marker verification
The method for sequencing the PCR product is adopted to verify the salt and alkali tolerance character related candidate molecular markers in SG and TG populations:
(1) firstly, designing primers on flanking sequences of marker sites, wherein at least one primer is more than 70bp away from the marker sites;
(2) carrying out PCR amplification by using designed primers and SG and TG group individual DNA materials as templates respectively, and carrying out 2% agarose gel electrophoresis analysis on PCR products successfully amplified;
(3) and (4) counting the genotype of each individual according to the electrophoresis result, and analyzing whether the marker is related to the saline-alkali tolerance character or not by SPSS software.
The specific operation steps are as follows:
1. PCR amplification
The PCR amplification system of the invention is as follows: template 1. mu.l, forward primer (10. mu.M) 0.2. mu.l, reverse primer (10. mu.M) 0.2. mu.l, Buffer 1. mu.l, dNTPs 0.8. mu.l, HiFi 0.2. mu.l, ddH2O 6.6μl。
After the sample is added according to the system, PCR amplification is carried out according to the following reaction conditions: pre-denaturation at 94 deg.C for 2-5 min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 1-2kb/min, repeating for 35 cycles; finally, extending the temperature of 72 ℃ for 5-10 min; storing at 4 ℃.
2. Electrophoretic detection
Preparing 2% of electrophoresis gel by using agarose, mixing the agarose and TAE in a certain ratio, heating the mixture in a microwave oven until the mixture is dissolved into colorless transparent liquid, pouring the colorless transparent liquid into a gel preparation mould, inserting a comb, standing for 20min, solidifying, then pulling out the comb, putting the prepared agarose gel into a horizontal electrophoresis tank, arranging a sample application hole at a negative electrode, taking 0.5% TAE as a buffer solution, selecting Genegeen as a nucleic acid coloring agent, sucking 3ul of PCR products of a mixed template out of the sample hole by using a liquid transfer gun, adjusting the voltage and the current to 120V and 60mA respectively, setting the time to be 30min for carrying out gel electrophoresis, stopping when a dyeing zone reaches 2/3, observing by using a gel imaging system after the electrophoresis is finished, and taking a picture and recording.
3. Statistical analysis
Marking and typing individuals according to the electrophoresis bands, counting the number of each genotype, introducing the genotype information of the individuals into SPSS software, calculating a P value by using a chi-square test method, selecting primers with the P value less than 0.05, and finally verifying 7 Indel marks in total.
As shown in table 3, it can be seen that the genotype insertion in C1245 accounts for 65% of the live group, while the deletion homozygous genotype accounts for 75% of the early dead individuals, and the group data P value is 0.000, which is significantly different, and therefore, the genotype insertion at this site is considered to be homozygous for the salt and alkali tolerant genotype. The nucleotide sequence of the C1245 molecular marker is shown as SEQ ID No.1, wherein the amplification primers for developing the molecular marker are shown as SEQ ID No.2 and SEQ ID No.3 (Table 4).
TABLE 3 genotype results for C1245 molecular markers
TABLE 4 amplification primers for molecular markers
The molecular marker C1245 obtained by the invention can be used for assisting in breeding the salt and alkali tolerant varieties of portunus trituberculatus, and the application steps are simply as follows: extracting DNA of a sample to be tested of the blue crab, taking the DNA as a template, carrying out PCR amplification by using an amplification primer of a molecular marker C1245, sequencing a PCR product, and selecting the sample to be tested as a parent for cultivating the saline-alkali tolerant variety of the blue crab if the genotype of the molecular marker C1245 in a sequencing result is an insertion homozygous genotype. In addition, the molecular marker can be used for analyzing the genetic diversity of the blue crab, identifying the germplasm and constructing the genetic map of the blue crab.
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions.
Sequence listing
<110> research institute for aquatic products in yellow sea of China institute for aquatic science
<120> saline-alkali-tolerant molecular marker C1245 of portunus trituberculatus and application thereof
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tagctgaatg aaccacactt tattatttat ttatttattt tattttatta ttatttttat 180
cactatttta atccccacgt aagtttctga aggtgtataa aatcaccaaa taataagcgg 240
agtgaatatg aaagcgtgtc gtggcagtga agggtttaaa gcggcaggat caacatgtgc 300
tcttcacctt cgcgtccagg ccttcctttc cgccgcaaac agcaaagact cccgctaatg 360
tcctgtaatg atataatggt aatgtccggt aagcaccttt tggtaagctt cattcatgca 420
ctctccgcca gcccgccatt aatgctgaaa atgatgcagc ttcataaatt tcctctttgc 480
cctgatgaaa agataatggc agtgacctca gatgaccttc actgtgacct cagatgacct 540
tcacttacgc tgcttattct tattcgtggc tgatggatgg aaagtaattg caacaaactc 600
cattggaatt agtttgttgg tatttcatta ggaagtccat attcaaccca acccaactta 660
acctaatcca acgtagccaa acccaatcca acataacata gtccagccca gcctaaccta 720
acccaaccca ttatgatcct atctaaccta acccagccta accaaaccca acctagctct 780
caatcatgtg caggtaaccc actgtaagcc ttacctaacc ctatctaacc tcactaaccc 840
aagcaaacca aactcgaccg aaccctttct taattacata cagataatcc acgagttata 900
taattcgctt ctttattcta gtgtatttat ccagcgagac tattacttct tattaccata 960
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Claims (9)
1. A saline-alkali-resistant molecular marker C1245 of blue crab is characterized in that the nucleotide sequence of the molecular marker C1245 is shown in SEQ ID No. 1.
2. The portunus trituberculatus saline-alkali tolerant molecular marker C1245 of claim 1, wherein the molecular marker C1245 is an Indel marker.
3. The portunus trituberculatus saline-alkali tolerant molecular marker C1245 of claim 1 or 2, wherein the saline-alkali tolerant genotype of the molecular marker C1245 is an insertion homozygous genotype.
4. The primer pair for detecting the molecular marker C1245 as claimed in claim 1, wherein the nucleotide sequence of the forward primer in the primers is shown as SEQ ID No.2, and the nucleotide sequence of the reverse primer is shown as SEQ ID No. 3.
5. The application of the molecular marker C1245 of claim 1 in screening of salt and alkali tolerant varieties of portunus trituberculatus.
6. The application of the molecular marker C1245 in screening the salt and alkali tolerant character varieties of blue crabs according to claim 5, wherein the molecular marker C1245 is characterized in that: the application steps are as follows: extracting DNA of a sample to be tested of the blue crab, taking the DNA as a template, carrying out PCR amplification by using an amplification primer of a molecular marker C1245, sequencing a PCR product, and selecting the sample to be tested as a parent for cultivating the saline-alkali tolerant variety of the blue crab if the genotype of the molecular marker C1245 in a sequencing result is an insertion homozygous genotype.
7. The application of the molecular marker C1245 in screening the salt and alkali tolerant character varieties of blue crabs according to claim 6, wherein the molecular marker C1245 is characterized in that: the PCR amplification system is as follows: template 1. mu.l, forward primer (10. mu.M) 0.2. mu.l, reverse primer (10. mu.M) 0.2. mu.l, Buffer 1. mu.l, dNTPs 0.8. mu.l, HiFi 0.2. mu.l, ddH2O 6.6μl。
8. The application of the molecular marker C1245 in screening the salt and alkali tolerant character varieties of blue crabs according to claim 6, wherein the molecular marker C1245 is characterized in that: the PCR amplification procedure is as follows: pre-denaturation at 94 deg.C for 2-5 min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 1-2kb/min, repeating for 35 cycles; finally, the extension is carried out for 5-10min at 72 ℃.
9. The application of the molecular marker C1245 of claim 1 in the genetic diversity analysis, germplasm identification and genetic map construction of blue crab.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103387980A (en) * | 2013-08-22 | 2013-11-13 | 宁波大学 | Microsatellite loci of portunus trituberculatus and primer |
| CN103789301A (en) * | 2013-06-05 | 2014-05-14 | 浙江海洋学院 | Specific primers of portunus trituberculatus microsatellite marker and screening method |
| CN108208318A (en) * | 2018-01-05 | 2018-06-29 | 浙江新篁生物技术有限公司 | A kind of sea crab feed addictive and its manufacturing method |
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2020
- 2020-03-06 CN CN202010156786.1A patent/CN111269992A/en not_active Withdrawn
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103789301A (en) * | 2013-06-05 | 2014-05-14 | 浙江海洋学院 | Specific primers of portunus trituberculatus microsatellite marker and screening method |
| CN103387980A (en) * | 2013-08-22 | 2013-11-13 | 宁波大学 | Microsatellite loci of portunus trituberculatus and primer |
| CN108208318A (en) * | 2018-01-05 | 2018-06-29 | 浙江新篁生物技术有限公司 | A kind of sea crab feed addictive and its manufacturing method |
Non-Patent Citations (2)
| Title |
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| JEONG,J.-H.等: "Portunus trituberculatus scaffold282094,whole genome shotgun sequence", 《GENBANK》 * |
| 高焕等: "逆环境因素对三疣梭子蟹幼蟹致死效应分析", 《海洋科学》 * |
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