CN116640856A - Macrobrachium rosenbergii molecular auxiliary breeding mark - Google Patents
Macrobrachium rosenbergii molecular auxiliary breeding mark Download PDFInfo
- Publication number
- CN116640856A CN116640856A CN202310541894.4A CN202310541894A CN116640856A CN 116640856 A CN116640856 A CN 116640856A CN 202310541894 A CN202310541894 A CN 202310541894A CN 116640856 A CN116640856 A CN 116640856A
- Authority
- CN
- China
- Prior art keywords
- macrobrachium rosenbergii
- ssr
- pcr
- primer
- genotype
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 241001327110 Macrobrachium rosenbergii Species 0.000 title claims abstract description 41
- 238000009395 breeding Methods 0.000 title abstract description 21
- 230000001488 breeding effect Effects 0.000 title abstract description 21
- 238000000034 method Methods 0.000 claims abstract description 19
- 238000012216 screening Methods 0.000 claims abstract description 15
- 230000002068 genetic effect Effects 0.000 claims abstract description 10
- 239000003550 marker Substances 0.000 claims abstract description 10
- 238000010276 construction Methods 0.000 claims abstract description 5
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 claims abstract description 5
- 238000001917 fluorescence detection Methods 0.000 claims abstract description 5
- 238000011144 upstream manufacturing Methods 0.000 claims description 10
- 238000004458 analytical method Methods 0.000 claims description 8
- 238000012408 PCR amplification Methods 0.000 claims description 4
- 238000012163 sequencing technique Methods 0.000 claims description 3
- 238000002372 labelling Methods 0.000 claims 1
- 238000012098 association analyses Methods 0.000 abstract description 6
- 238000012214 genetic breeding Methods 0.000 abstract description 3
- 108090000623 proteins and genes Proteins 0.000 abstract description 3
- 238000011160 research Methods 0.000 abstract description 3
- 238000010219 correlation analysis Methods 0.000 abstract description 2
- 108020004414 DNA Proteins 0.000 description 10
- 241000238557 Decapoda Species 0.000 description 8
- 108700028369 Alleles Proteins 0.000 description 7
- 230000037396 body weight Effects 0.000 description 7
- 239000003147 molecular marker Substances 0.000 description 6
- 108091092878 Microsatellite Proteins 0.000 description 5
- 239000002773 nucleotide Substances 0.000 description 3
- 125000003729 nucleotide group Chemical group 0.000 description 3
- 238000007400 DNA extraction Methods 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 108091081062 Repeated sequence (DNA) Proteins 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- 238000007405 data analysis Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 238000003205 genotyping method Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- 241000238424 Crustacea Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000276707 Tilapia Species 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 235000013601 eggs Nutrition 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000012165 high-throughput sequencing Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000013011 mating Effects 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 238000009394 selective breeding Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
Classifications
-
- 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
- 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
-
- 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
- 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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
-
- 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
-
- 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
- C12Q2600/156—Polymorphic or mutational markers
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a molecular auxiliary breeding SSR marker of macrobrachium rosenbergii, which is obtained by carrying out polymorphism screening on SSR loci in transcriptomes and then carrying out correlation analysis on the SSR markers and body length, and can be used for genetic breeding of macrobrachium rosenbergii. The SSR markers related to the length of the macrobrachium rosenbergii provided by the invention are LSZX302 and LSZX405. The method for screening high-body macrobrachium rosenbergii provided by the invention is characterized in that fluorescein is marked at the 5' end of a forward primer for PCR reaction, and the PCR product is automatically detected by fluorescence; generating a map file of the loci by using Genemapper4.0 software according to the fluorescence detection data, and measuring the length and fluorescence intensity peak value of PCR amplified products to obtain genotypes of each locus; and determining whether the macrobrachium rosenbergii is high-body macrobrachium rosenbergii according to the genotype. The SSR markers related to the body length of the macrobrachium rosenbergii obtained by screening are related to functional genes, and can be used for research on growth trait association analysis, genetic map construction, QTL positioning and the like.
Description
Technical Field
The invention belongs to the technical field of macrobrachium rosenbergii breeding, and particularly relates to a macrobrachium rosenbergii molecular auxiliary breeding marker.
Background
The molecular marker assisted breeding utilizes the characteristic that a molecular marker is closely linked with a gene for determining a target character, and the purpose of selecting the target character is achieved by detecting the molecular marker, so that the molecular marker assisted breeding has the advantages of being rapid, accurate and free from interference of environmental conditions. The microsatellite (microsatellites) is also called as a simple repeated sequence (simple sequence repeats, SSR) serving as a second generation molecular marker, has the advantages of high polymorphism, abundant information content, co-dominant inheritance and the like, and is widely applied to aspects of germplasm identification, genetic structure analysis, genetic linkage map construction, QTL positioning and the like of aquatic animals.
Macrobrachium rosenbergii (Macrobrachium rosenbergii) is an important economic shrimp in the world, and is a species which is popularized and cultivated to the world by the United nations grain and agriculture organization (FAO), particularly to the developing nations, after tilapia. In 1976, china introduced the shrimp from Japan to carry out cultivation popularization, with the breakthrough of artificial breeding technology and the development of cultivation technology, china rapidly developed into the biggest world of the global macrobrachium rosenbergii industry, according to incomplete statistics, to 2022, the Jiangzhe region of China owns 100 macrobrachium rosenbergii total artificial breeding farms, the annual production of seedlings reaches 400 hundred million, the cultivation area exceeds 3.5 ten thousand hectares, and the total yield exceeds 16 ten thousand tons (accounting for 68 percent of the total global cultivation yield). Meanwhile, links such as feed, finished shrimp processing and export are driven, the total yield of the industrial chain exceeds 200 hundred million yuan, and the seedlings and the cultivation blocks with special characteristics and definite division of labor are formed.
The improved variety breeding and popularization are the basis of stable and rapid development of industry, and the new varieties 'nan Taihu No. 2' and 'nan Taihu No. 3' obtained by traditional family breeding are the most extensive new varieties in industry at present. After genetic improvement, the growth speed of the macrobrachium rosenbergii No. 2 is improved by 36.87% and the survival rate is improved by 7.76% compared with the unselected seedlings. The growth rate of the 'nan Taihu No. 3' is improved by 23.36% on the basis of the 'nan Taihu No. 2'.
However, the traditional selective breeding method for improving the macrobrachium rosenbergii character is based on phenotypic value for breeding, and the breeding cycle is long and the efficiency is low. Typically in the collection of growth trait phenotype data for use in breeding, such as body weight and body length data. However, for crustaceans, especially macrobrachium rosenbergii, the measured individual weight data often have deviation, and the main reasons for the deviation are that the macrobrachium rosenbergii is fighting, the molting period is self-phase and is not smooth, the appendages and frontal thorns of the macrobrachium rosenbergii are broken, and especially the second step of foot is incomplete during measurement, so that the authenticity of the weight phenotype value is greatly influenced. In addition, female and non-female eggs are held during measurement, and the body weight phenotype value is directly influenced. Therefore, the acquired body length phenotype data can more truly reflect the growth condition of the body, and the molecular markers obtained by correlating the body length data are more reliable than the body weight data.
Disclosure of Invention
The invention provides a molecular auxiliary breeding SSR marker of macrobrachium rosenbergii, which is obtained by carrying out polymorphism screening on SSR loci in transcriptomes and then carrying out correlation analysis on the SSR markers and body length, and can be used for genetic breeding of macrobrachium rosenbergii.
The invention firstly provides SSR markers related to the length of macrobrachium rosenbergii, wherein the markers are LSZX302 and LSZX405, and the LSZX302 has the following sequence:
cgttgtcatcaccgtttcactttctgtgc(catt) n ctactcttctgtcttcgttcaccactcatcattcac acacaaaattgagcatctgtgggttgcttagaagaagaagaaccttttgaggaggtgaaatcactatcttc attctttagcttcgttctttttgaaatctgtataagtggttatgaattcccacgcccctcattcttctgtctcaatc gttagcacagccctcaggaac; wherein n is 8 to 9,
the sequence of LSZX405 is as follows:
aggctacgacatcatcccacacaccgcgcat(gctc) n tcgctgcgtcgctgtcgcttcgtttctgg cgcctctctttctctctttctctcttctttcggccaatcacttcgtccaccggaaaaggcagtcgtcaaaaggt tattacataggctcttttatggtttgtatgtttgcgca; wherein n is 1-4;
the LSZX302 and LSZX405 markers provided by the invention are related to the length of the macrobrachium rosenbergii, so that the primer pair for detecting the two markers can be used for screening parents with high body length of macrobrachium rosenbergii;
wherein the LSZX302 labeled core sequence is (CATT) n, the primer pair for detecting polymorphism of the core sequence has an upstream primer sequence of 5'-CGTTGTCATCACCGTTTCAC-3' and a downstream primer sequence of 5'-GTTCCTGAGGGCTGTGCTAA-3';
the LSZX405 core sequence is marked as (GCTC) n, and the polymorphism primer pair of the core sequence is detected, wherein the upstream primer sequence is 5'-AGGCTACGACATCATCCCAC-3', and the downstream primer sequence is 5'-TGCGCAAACATACAAACCAT-3'.
The molecular marker provided by the invention can be used for growth trait association analysis, genetic map construction and QTL positioning analysis;
in a further aspect, the invention provides a method for screening high-body macrobrachium rosenbergii by detecting whether the genotype of the LSZX302 marker in an individual to be screened is 266/266; whether the genotype of the LSZX405 marker is 174/190.
The method is completed by amplifying by PCR primers, sequencing the amplified product and analyzing the genotype.
Wherein the LSZX302 labeled primer is detected, and the sequence information of the upstream primer and the downstream primer is as follows:
F：5′-CGTTGTCATCACCGTTTCAC-3′，
R：5′-GTTCCTGAGGGCTGTGCTAA-3′；
wherein the LSZX405 labeled primer is detected, and the sequence information of the upstream primer and the downstream primer is as follows:
F：5′-AGGCTACGACATCATCCCAC-3′，
R：5′-TGCGCAAACATACAAACCAT-3′。
the method for screening high-body macrobrachium rosenbergii provided by the invention is characterized in that fluorescein is marked at the 5' end of a forward primer for PCR reaction, and the PCR product is automatically detected by fluorescence; generating a map file of the loci by using Genemapper4.0 software according to the fluorescence detection data, and measuring the length and fluorescence intensity peak value of PCR amplified products to obtain genotypes of each locus; and determining whether the macrobrachium rosenbergii is high-body macrobrachium rosenbergii according to the genotype.
The SSR markers related to the body length of the macrobrachium rosenbergii obtained by screening are related to functional genes, and can be used for research on growth trait association analysis, genetic map construction, QTL positioning and the like.
Drawings
Fig. 1: results of SSR analysis of Unigenes in the macrobrachium rosenbergii transcriptome,
fig. 2: LSZX302 locus genotyping result diagram,
fig. 3: LSZX405 locus genotyping results.
Detailed Description
The present invention will be described in detail with reference to the following examples and the accompanying drawings.
Example 1: screening SSR markers
1. Obtaining experimental shrimp
The method comprises the steps of randomly mating, breeding and emergence of seedlings by a group, setting the breeding density to be 2 ten thousand per mu, breeding for 4 months, randomly catching 199 shrimps, and measuring the body length of each shrimp. Muscle tissue was cut and fixed in 95% ethanol for DNA extraction.
2. Polymorphic SSR primer screening
Transcriptome sequencing is carried out on the Macrobrachium rosenbergii ovary tissue by using an Illumina HiSeqTM 4000 high-throughput sequencing platform, 95379 Unigenes are obtained by splicing after quality control and assembly of raw data, and SSR detection is carried out on the Unigenes by using MISA software. Three-base, four-base or five-base repeated sequences with the repetition times of more than 5 times are selected, primer premier5.0 software is used for designing primers, 10 individuals are randomly selected for screening polymorphic SSR sites, and the primers are synthesized by the division of biological engineering (Shanghai).
3. Genomic DNA extraction
The method comprises extracting genomic DNA from two groups of samples by using Takara genome extraction kit, detecting DNA quality and concentration by 1% agarose gel electrophoresis and spectrophotometry according to specific operation method, and storing the DNA sample at-20deg.C for use.
4. PCR amplification and fluorescence detection
The total volume of the PCR reaction was 25. Mu.L, the template was 50ng of genomic DNA, and the other components were labeled with fluorescein FAM (blue) at the 5' end of the forward primer according to Taq enzyme (Takara) specifications. The PCR reaction conditions were: 94 ℃ for 2min;94 ℃ for 30s,60 ℃ for 45s,72 ℃ for 2min,30 cycles; extending at 72 ℃ for 10min; preserving at 4 ℃. The PCR products were automatically detected by fluorescence using an ABI3730XL (applied biosystems) DNA analyzer.
5. Data analysis
Generating a map file of each site by using Genemapper4.0 software, and measuring the length and fluorescence intensity peak value of PCR amplified products to obtain the genotype of each site. Popgene32 calculated allele (numbers of the alleles, na), effective allele (numbers of the effective alleles, ne), expected heterozygosity (expected heterozygosity, he), observed heterozygosity (observed heterozygosity, ho), and Hard-weinberg equilibrium test (P-value). The polymorphic information content (Polymorphism information content, PIC) was calculated by reference to the method of Botstein et al.
Statistical analysis was performed using SPSS17.0 software, first to perform a normal test on the phenotypic data, a general linear model (General Linear Model, GLM) was applied to perform microsatellite locus and length association analysis, and multiple comparisons (LSD) of different genotypes at the same locus.
18592 SSR sites consisting of one to six nucleotide repeats were found in total, accounting for 19.49% of the total number of Unigenes. The proportion of single nucleotide repeat types is highest, up to 52.71%, and then the single nucleotide repeat types are sequentially two, three, four, five and six, and the proportion is 32.61%, 15.90%, 0.80%, 0.05% and 0.04% respectively. Among the mononucleotide, dinucleotide and trinucleotide repeat types, the most number of repeat motifs are A/T (9407), AG/GA (2244), AAT/ATA/TAA (344), and the least number of repeat motifs are C/G (392), CG/GC (12), CGG/GGC/GCG (15), respectively. The polymorphism screening was performed by randomly selecting 63 sites, and as a result 31 sites showed polymorphism, 32 sites had no polymorphism, and the proportion of polymorphic sites was 49.2% (FIG. 1).
Population genetic diversity analysis
Of the 31 polymorphic sites, 19 sites were selected for genetic diversity analysis. The results showed that 65 alleles were detected in total, an average allele factor of 3.4211, an effective allele factor of 1.0901-4.2309, an average of 2.0941, an observed heterozygosity of 0.0854-0.6734, an average of 0.4105, a desired heterozygosity of 0.0826-0.7656, an average of 0.4496, a polymorphic information content of 0.0805-0.7267, an average of 0.3882,6 loci PIC value of greater than 0.5,4 loci PIC value of less than 0.25,9 loci PIC value of between 0.25 and 0.5, and a balance of Hard-weinberg for the remaining 10 loci (P < 0.05) or very significant (P < 0.01) of 19 loci, LSZX008, LSZX100, LSZX107, LSZX202, LSZX207, LSZX300, LSZX304, LSZX406 and LSZX605 loci conforming to the balance (Table 1).
Table 1: population genetic diversity analysis
Example 2: SSR sites associated with body length
The SSR sites screened by using the GLM model and the macrobrachium rosenbergii body length association analysis show that the LSZX302 and LSZX405 are obviously related to the body length (P is less than 0.05) (Table 2).
Table 2: correlation of LSZX302 and LSZX405 loci and body length characters
Note that: the numerical values in the table are probability values of microsatellite loci and body length association analysis, and superscript ". Times.represents that body length characters are obviously related to marks (P is less than 0.05).
Multiple comparisons of different genotypes and body lengths were performed for SSR sites with significant differences, where the average of body lengths for individuals with LSZX302 locus 266/266 genotypes was significantly higher than for individuals with 270/270 genotypes (P < 0.05); the average body length of individuals with the LSZX405 locus 174/190 genotype was significantly higher than that of individuals with the 182/190 genotype (P < 0.05) (Table 3). While individuals enriched for the LSZX302 site 266/266 and LSZX405 site 174/190 genotypes had 26 tails, all individuals with the LSZX405 site 174/190 genotypes contained the LSZX302 site 266/266 genotype at the same time.
Table 3: genotype and length multiple comparison
Comparison of individuals containing both 266/266 and 174/190 genotypes with individuals containing only 266/266 showed that individuals enriched for both genotypes had an average body length of 9.02.+ -. 0.29cm and an average body weight of 19.83.+ -. 1.90g, whereas individuals of only 266/266 had an average body length of 8.51.+ -. 1.52cm and an average body weight of 17.21.+ -. 0.74g. I.e., individuals having both genotypes 266/266 and 174/190 had a mean length of 5.99% higher and a mean weight of 15.22% higher than individuals comprising only a single subject.
Example 3: verification of two loci in a breeding population
The body length related markers screened by the method can be used for rapidly and accurately screening the parents with excellent growth characteristics for breeding macrobrachium rosenbergii, and the method comprises the following steps:
1. the Takara genome extraction kit extracts genome DNA of samples of two populations, the specific operation method is referred to the specification, 1% agarose gel electrophoresis and a spectrophotometer detect the quality and concentration of the DNA, and the DNA sample is stored at-20 ℃ for standby.
2. PCR amplification and fluorescence detection
The total volume of the PCR reaction was 25. Mu.L, the template was 50ng of genomic DNA, and the other components were labeled with fluorescein FAM (blue) at the 5' end of the forward primer according to Taq enzyme (Takara) specifications. The PCR reaction conditions were: 94 ℃ for 2min;94 ℃ for 30s,60 ℃ for 45s,72 ℃ for 2min,30 cycles; extending at 72 ℃ for 10min; preserving at 4 ℃. The PCR products were automatically detected by fluorescence using an ABI3730XL (applied biosystems) DNA analyzer.
3. Data analysis
Generating a map file of each site by using Genemapper4.0 software, and measuring the length and fluorescence intensity peak value of PCR amplified products to obtain the genotype of each site.
Wherein the LSZX302 labeled primer is detected, and the sequence information of the upstream primer and the downstream primer is as follows:
F:5’-CGTTGTCATCACCGTTTCAC-3’、
R:5’-GTTCCTGAGGGCTGTGCTAA-3’；
wherein the LSZX405 labeled primer is detected, and the sequence information of the upstream primer and the downstream primer is as follows:
F:5’-AGGCTACGACATCATCCCAC-3’、
R:5’-TGCGCAAACATACAAACCAT-3’。
genotypes amplified at two loci are shown in Table 4 below
Table 4: genotype information for LSZX001 and LSZX304
And randomly extracting 56 shrimps of one culture population cultured in the same pond, measuring the body length and the body weight of each shrimp, and verifying the screened SSR markers. The results showed that the 266/266 genotype individuals at the LSZX302 locus were significantly longer than those of other genotypes (P < 0.05), with an average length of 13.15% higher, and the 266/266 genotype individuals were significantly higher in weight than those of other genotypes, with an average weight of 32.86% higher (table 5). The 174/190 genotype individuals at the LSZX405 locus had significantly higher body lengths than those of the other genotypes (P < 0.05), with an average height of 12.61%, and the 174/190 genotype high body length individuals had significantly higher body weights than those of the other genotypes, with an average height of 34.48% (table 5).
Table 5: verification result table of two sites in breeding population
The research results show that the two markers can be used for rapidly and accurately screening the parent with excellent growth characteristics for genetic breeding of macrobrachium rosenbergii.
Claims (9)
1. An SSR marker associated with macrobrachium rosenbergii body length, wherein the marker is LSZX302 or LSZX405, and wherein the LSZX302 sequence is as follows:
cgttgtcatcaccgtttcactttctgtgc(catt) n ctactcttctgtcttcgttcaccactcatcattcacacacaaaattg agcatctgtgggttgcttagaagaagaagaaccttttgaggaggtgaaatcactatcttcattctttagcttcgttctttttgaaat ctgtataagtggttatgaattcccacgcccctcattcttctgtctcaatcgttagcacagccctcaggaac; wherein n is 8-9;
the sequence of LSZX405 is as follows:
aggctacgacatcatcccacacaccgcgcat(gctc) n tcgctgcgtcgctgtcgcttcgtttctggcgcctctctttc tctctttctctcttctttcggccaatcacttcgtccaccggaaaaggcagtcgtcaaaaggttattacataggctcttttatggttt gtatgtttgcgca; wherein n is 1-4.
2. A primer pair, wherein the primer pair detects the core region of the SSR marker of claim 1.
3. A primer pair for detecting the SSR-tagged core region of claim 2, wherein the primer pair for detecting the LSZX 302-tagged core region has an upstream primer sequence of 5'-CGTTGTC ATCACCGTTTCAC-3' and a downstream primer sequence of 5'-GTTCCTGAGGGCTGT GCTAA-3';
primer pair for detecting LSZX405 core sequence, wherein the upstream primer sequence is 5'-AGGCTAC GACATCATCCCAC-3' and the downstream primer sequence is 5'-TGCGCAAACATACAAACCAT-3'.
4. The application of the SSR marker in the analysis of the growth trait association of macrobrachium rosenbergii, genetic map construction and analysis of QTL positioning.
5. A method for screening high-body macrobrachium rosenbergii, which is characterized in that whether the genotype of the LSZX302 marker of claim 1 in an individual to be screened is 266/266 is detected; whether the LSZX405 marked genotype is 174/190 was used to screen high-body macrobrachium rosenbergii.
6. The method of claim 5, wherein the method is performed by amplifying the PCR primer and sequencing the amplified product to analyze the genotype.
7. The method of claim 6, wherein the PCR amplification is performed wherein LSZX 302-labeled primers are detected with the following upstream and downstream primer sequence information:
F：5′-CGTTGTCATCACCGTTTCAC-3′，
R：5′-GTTCCTGAGGGCTGTGCTAA-3′。
8. the method of claim 6, wherein the PCR amplification is performed wherein LSZX 405-labeled primers are detected with the following upstream and downstream primer sequence information:
F：5′-AGGCTACGACATCATCCCAC-3′，
R：5′-TGCGCAAACATACAAACCAT-3′。
9. the method of claim 6, wherein the method comprises labeling fluorescein at the 5' end of the forward primer for PCR reaction, and automatically detecting the PCR product by fluorescence; generating a map file of the loci by using Genemapper4.0 software according to the fluorescence detection data, and measuring the length and fluorescence intensity peak value of PCR amplified products to obtain genotypes of each locus; and determining whether the macrobrachium rosenbergii is high-body macrobrachium rosenbergii according to the genotype.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310541894.4A CN116640856A (en) | 2023-05-15 | 2023-05-15 | Macrobrachium rosenbergii molecular auxiliary breeding mark |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310541894.4A CN116640856A (en) | 2023-05-15 | 2023-05-15 | Macrobrachium rosenbergii molecular auxiliary breeding mark |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116640856A true CN116640856A (en) | 2023-08-25 |
Family
ID=87639104
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310541894.4A Pending CN116640856A (en) | 2023-05-15 | 2023-05-15 | Macrobrachium rosenbergii molecular auxiliary breeding mark |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116640856A (en) |
-
2023
- 2023-05-15 CN CN202310541894.4A patent/CN116640856A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109971865B (en) | SNP marker significantly related to weight traits of litopenaeus vannamei and application | |
| CN105936937B (en) | A kind of relevant SNP marker of dissolved oxygen tolerance low to litopenaeus vannamei and its screening and application | |
| CN108192990B (en) | SNP molecular marker related to watermelon peel background color and application thereof | |
| CN109182556B (en) | SNP molecular marker related to growth traits of pelteobagrus vachelli and application | |
| CN107858447B (en) | Single nucleotide polymorphism marker site, primer pair, kit and application for identifying peach blossom single-petal/double-petal character | |
| CN116356038A (en) | Breeding method for screening Fugu rubripes individuals with rapid growth performance | |
| CN111304337A (en) | SRAP molecular marker, kit and method for identifying first filial generation of pelteobagrus fulvidraco, pelteobagrus vachelli and hybride and application | |
| CN109609687B (en) | KASP marker primer combination for detecting watermelon fusarium wilt resistance and application thereof | |
| CN110029174B (en) | SSR (simple sequence repeat) marker related to quality of macrobrachium rosenbergii bodies | |
| CN112011640B (en) | KASP molecular marker, primer and application for identifying pH of watermelon fruit | |
| CN113528703A (en) | Development and application of KASP molecular marker of rice blast resistance gene Pid3-A4 | |
| CN109706231B (en) | High-throughput SNP (single nucleotide polymorphism) typing method for molecular breeding of litopenaeus vannamei | |
| CN110016510B (en) | Molecular marker for genetic breeding of macrobrachium rosenbergii | |
| CN112375827A (en) | SNP marker related to growth of macrobrachium rosenbergii as well as typing method and application thereof | |
| CN106167825A (en) | Microsatellite marker that a kind of yellow catfish growing characteristic is relevant and detection thereof and application | |
| CN107619875B (en) | Insertion deletion marker locus for identifying watermelon fruit shape, primer and application | |
| CN116640856A (en) | Macrobrachium rosenbergii molecular auxiliary breeding mark | |
| CN116622855A (en) | SSR (simple sequence repeat) marker related to macrobrachium rosenbergii body length and application thereof | |
| CN106636427B (en) | Microsatellite marker primer and method for identifying inbred families of exopalaemon carinicauda | |
| CN108588242B (en) | SNP locus of crassostrea gigas AHR gene | |
| CN113981103A (en) | Microsatellite primer pair for parent-child identification of macrobrachium rosenbergii microsatellites, detection kit and identification method | |
| CN108531621B (en) | SNP locus related to rapid growth of crassostrea gigas | |
| CN107858448B (en) | Single nucleotide polymorphism marker site, primer pair, kit and application for identifying peach pollen fertility character | |
| CN112322755B (en) | SNP locus related to growth traits of fugu rubripes and application of SNP locus in genetic breeding | |
| CN117683938B (en) | KASP molecular marker closely linked with tomato fruit width and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |