LU504583B1 - Micropterus salmoides paternity identification kit and application thereof - Google Patents

Micropterus salmoides paternity identification kit and application thereof Download PDF

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Publication number
LU504583B1
LU504583B1 LU504583A LU504583A LU504583B1 LU 504583 B1 LU504583 B1 LU 504583B1 LU 504583 A LU504583 A LU 504583A LU 504583 A LU504583 A LU 504583A LU 504583 B1 LU504583 B1 LU 504583B1
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fam
dna
primer
note
qualifiers
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LU504583A
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French (fr)
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Jun Qiang
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Freshwater Fisheries Res Ct Cafs
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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
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • 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

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Abstract

The present disclosure discloses a Micropterus salmoides paternity identification kit and belongs to the genetic breeding technology of aquatic animals. The kit of the present disclosure comprises multiple pairs of fluorescent microsatellite primers, which can accurately and efficiently identify the parents and offspring of Micropterus salmoides, reducing the problem of unidentifiable parental sources during mixed breeding of offspring.

Description

BL-5712
MICROPTERUS SALMOIDES PATERNITY IDENTIFICATION KIT AND LU504583
APPLICATION THEREOF
TECHNICAL FIELD
[0001] The present disclosure belongs to the genetic breeding technology of aquatic animals, particularly relates to a Micropterus salmoides paternity identification kit and operating procedures thereof.
BACKGROUND
[0002] Micropterus salmoides, also known as largemouth bass and Perca americana, is a wide-temperate freshwater fish with the characteristics of fast growth, strong disease resistance and adaptability, and delicious taste, etc. Micropterus salmoides is introduced into China in the 1980s. After solving the key technology of large-scale fry breeding in the 1990s, the breeding scale has expanded rapidly; in 2021, the output has exceeded 700,000 tons, and has become into one of the freshwater cultured fishes with high single output value in China. The Micropterus salmoides seed industry system is relatively sound. The annual demand for Micropterus salmoides parent fish exceeds 15 million pairs and the demand for fish fries exceeds 100 billion in China. The selective breeding based on large-scale populations is currently the main technical system adopted by many Micropterus salmoides breeding institutions. By using the population breeding technology, new varieties with excellent traits and genetic stability can be quickly bred. However, if new germplasm cannot be supplemented, the breeding progress will gradually shrink, and inbreeding decline will increase from generation to generation.
[0003] Family breeding is also a commonly used fish breeding method, but presently it has not been applied in the breeding of Micropterus salmoides. In this system, genetic progress mainly depends on the heritability and selection intensity of the target trait.
Studies have shown that increasing the number of families, expanding the size of the breeding population, and increasing the selection intensity during the breeding process can effectively increase genetic progress when parameters such as heritability have been established in the founder population. Therefore, the use of family selection can effectively expand the breeding potential of selected offspring. Before carrying out 1
BL-5712 family selection, we shall establish accurate parent-child identification markers, which LU504583 can accurately distinguish between different parents and offspring. The routinely used markers mainly include RFLP, RAPD, microsatellite, SNP and so on. Microsatellite
DNA, also known as short tandem repeats or simple repeats, is a DNA segment widely distributed in eukaryotic genomes. As one of the rapidly developing and widely used molecular markers in recent years, microsatellite DNA has the advantages of multiple types, wide distribution, Mendelian co-dominant inheritance, high polymorphic information content, and relatively simple operation, etc, so it is an ideal molecular marker for studies such as population structure analysis and individual identification.
The present disclosure discloses a Micropterus salmoides paternity identification kit and operating procedures thereof. According to the present disclosure, different
Micropterus salmoides families are constructed according to the genetic distance between individuals, and then the genetic diversity of the family and the paternity identification with the offspring are carried out by using the developed multiple pairs of microsatellite markers. The technology of the present disclosure is accurate and efficient, has high operability, and can replace physical markers for the pedigree identification.
SUMMARY
[0004] In order to accurately identify parents and offspring of Micropterus salmoides, the present disclosure discloses a Micropterus salmoides paternity identification kit and operating procedures thereof, which can accurately and efficiently identify the parents and offspring of Micropterus salmoides, reducing the problem of unidentifiable parental sources during mixed breeding of offspring.
[0005] In the present disclosure, in order to accurately identify parents and offspring of Micropterus salmoides, the present disclosure provides a Micropterus salmoides paternity identification kit and operating procedures thereof. The procedures mainly include the following steps:
[0006] I. Family construction. In the Yangcheng Lake Base of the Freshwater
Fisheries Research Center, Chinese Academy of Fishery Sciences (CAFS), 18 families are constructed based on the genetic distance (>0.32) between reserved individuals of a core group of the northern subspecies of generation F1 of Micropterus salmoides in 2
BL-5712 the United States, as core breeding families of generation F2 of Micropterus salmoides, 17006588 and the parent tail fin samples are retained at -20°C. Thirty individuals are randomly selected from each family in the generation F2, in a total of 540 individuals, together with 18 male parents and 18 female parents, as sample materials for this experiment.
[0007] II. Microsatellite typing. Sample genomic DNA was extracted with reference to the method described in the instruction manual of Tiangen Centrifuge Column
Genomic DNA Extraction Kit produced by Beijing Tiangen Biotechnology Co., Ltd.
The DNA quality and concentration were tested by 1% agarose gel electrophoresis and a spectrophotometer, and stored at -20°C for later use. Multiple pairs of fluorescent microsatellite primers (shown in Table 1) were synthesized, and FAM fluorophores were added to 5' end of the primers. Based on 10 uL of PCR reaction system, including:
Templ
System Buffer Ex Taq dNTP ddH,O Primer ate
Volume 1.5ul Tul 0.3 0.8ul 6.2 Tul
[0008] PCR amplification program: 95°C 2min—94°C 30s—TM°C 90s—72°C
Imin—72°C 10min
[0009] —"
[0010] 40 cycles
[0011] Note: TM represented the annealing temperature of microsatellite primers (Table 2)
[0012] With 3% agarose gel electrophoresis testing, the electrophoresis effect of PCR products was observed, and whether PCR amplification was successful was identified.
Then, fluorescence labeled PCR products were tested with a 3730XL sequencer to identify microsatellite typing results. The presence or absence of double peaks was verified, and double peaks present in an amplified product indicated that the locus has polymorphism within the population.
[0013] III. Data processing and analysis. The expected heterozygosity (He), observed heterozygosity (Ho), and polymorphic information content (PIC) of microsatellite loci were analyzed using the Cervus 3.0.7 software. Paternity identification was performed 3
BL-5712 . . _ . . LU504583 using an exclusion method, and the probability of excluding candidate parents who were not real parents was indicated by the cumulative probability of exclusion (CPE).
For identification of genetic relationship, the Cervus 3.0.7 software was used for determination and analysis through values of log likelihood ratio (LOD).
[0014] In the present disclosure, the results show that using highly polymorphic microsatellite markers instead of physical markers to identify families is a relatively accurate and feasible method in trait testing and pedigree tracing, which not only reduces the cumbersome operation and stress death during physical marker injection, but also reduces the problem of individual identification caused by marker loss.
[0015] Table 1 Microsatellite primer sequences of Micropterus salmoides
PCR
Primer name Upper Primer sequence (5’-3”) Lower Primer sequence (5’-3”) Probe type length
AGTTAACCCGCTTTGTGCTG GAAGGCGAAGAAGGGAGAGT 207—
JZL60 FAM (SEQ ID NO.1) (SEQ ID NO.2) 227
CCGCTAATGAGAGGGAGACA ACAGACTAGCGTCAGCAGCA (SEQ 253—
JZL67 FAM (SEQ ID NO.3) ID NO.4) 266
GAAAACAGCCTCGGGTGTAA CACTTGTTGCTGCGTCTGTT (SEQ 179—
JZL84 FAM (SEQ ID NO.5) ID NO.6) 209
GGGGCTCACTCACTGTGTTT GTGCGCAGACAGCTAGACAG (SEQ 199—
JZL85 FAM (SEQ ID NO.7) ID NO.8) 225
GTGTCCCTGACTGTATGGC TCTGATGAGGCTGTGAAAT (SEQ 284—
JZL105 FAM (SEQ ID NO.9) ID NO.10) 320
GCAGGCAGTGAACCCAGATT TATGTATTGACGAGCGAGCAG 250—
JZL106 FAM (SEQ ID NO.11) (SEQ ID NO.12) 286
CAACATGGACGCTACTAT (SEQ CAACCATCACATGCTTCT (SEQ ID 166—
MiSaTPW11 FAM
ID NO.13) NO.14) 357
CCAAGGTCAGGTTTAAC (SEQ ACCTTTGTGCTGTTCTGTC (SEQ ID 270—
MiSaTPW25 FAM
ID NO.15) NO.16) 300 4
BL-5712
CU504583
ACTTCGCAAAGGTATAAC (SEQ CCTCATGCAGAAGATGTAA (SEQ 282—
MiSaTPW70 FAM
ID NO.17) ID NO.18) 609
ACACAGTGTCAGTTCTGCA GTGAATACCTCAGCAAGCAT (SEQ 257—
MiSaTPW76 FAM (SEQ ID NO.19) ID NO.20) 303
CTTCTAAATGTGTGTAGGGTTG AGCTTAGCATAAAGACTGGGAAC 372—
MiSaTPW96 FAM
C (SEQ ID NO.21) (SEQ ID NO.22) 405
TGTGAAAGGCACAACACAGCC ATCGACCTGCAGACCAGCAACACT 209—
MiSaTPW117 FAM
TGC (SEQ ID NO.23) (SEQ ID NO.24) 242
CCACACAGTGACACAAACTGTG GCCATTGTGCTGCTGCAGAG (SEQ 195—
MiSaTPW173 FAM
C (SEQ ID NO.25) ID NO.26) 271
TTGTATACCAAGTGACCTGTGG GGGAGTGCATCTTTCTGAAGTGCC 219—
MiSaTPW184 FAM (SEQ ID NO.27) (SEQ ID NO.28) 253
GTGCTAATAAAGGCTACTGTC TGTTCCCTTAATTGTTTTGA (SEQ 124—
Lar7 FAM (SEQ ID NO.29) ID NO.30) 210
CGTGTAACTATGGTGATGTG GAAGCGAGAGTCAGAGTG (SEQ ID
LMB24 203 FAM (SEQ ID NO.31) NO.32)
TGGTGAACACAGCAGAAC (SEQ GGACAGCCTCAATACATCTT (SEQ
LMB28 236 FAM
ID NO.33) ID NO.34)
TAGTGTATTGCTCTGAAGGT AACATACTGCTGATGGAGAA (SEQ
LMB38 302 FAM (SEQ ID NO.35) ID NO.36)
GAAGGACACCATACAACACT CCTCTCCATCCTCCTCATC (SEQ ID
LMB39 327 FAM (SEQ ID NO.37) NO.38)
GCAGAGCAGAAGAATGACT TGAACACAACGACACCAC (SEQ ID
LMB41 336 FAM (SEQ ID NO.39) NO.40)
[0016] Table 2 TM value and amplified repetitive sequence of microsatellite primers
Primer ™ Repetitive sequence
JZL60 59°C (CA)
JZL67 62°C (CA)
JZL84 56°C (CA)»o
BL-5712
JZL85 59°C (CA)17 1006008
JZL105 65°C (GT)13
JZL106 59°C (GT)ss
MiSaTPW11 62°C (AGAT)13
MiSaTPW25 56°C (AGAT)ı1
MiSaTPW70 60°C (AGAT)a3
MiSaTPW76 56°C (AGAT)22(AGAC)10
MiSaTPW96 62°C (AGAT)15(AGAC)s
MiSaTPW117 62°C (AC)
MiSaTPW173 60°C (AC)ıs
MiSaTPW184 59°C (AC)14(CT)1o
Lar7 56°C (AC)1s
LMB24 58°C 58°C
LMB28 56°C 56°C
LMB38 56°C 56°C
LMB39 65°C 65°C
LMB41 62°C 62°C
DETAILED DESCRIPTION OF THE EMBODIMENTS
[0017] I. Family construction. In the Yangcheng Lake Base of the Freshwater
Fisheries Research Center, Chinese Academy of Fishery Sciences (CAFS), 18 families were constructed based on the genetic distance (>0.32) between reserved individuals of a core group of the northern subspecies of generation F1 of Micropterus salmoides in the United States, as core breeding families of generation F2 of Micropterus salmoides, and the parent tail fin samples were retained at -20°C. Thirty individuals were randomly selected from each family in the generation F2, in a total of 540 individuals, together with 18 male parents and 18 female parents, as sample materials for this experiment.
[0018] II. Microsatellite typing. Sample genomic DNA was extracted with reference to the method described in the instruction manual of Tiangen Centrifuge Column
Genomic DNA Extraction Kit produced by Beijing Tiangen Biotechnology Co., Ltd. 6
BL-5712
LU504583
The DNA quality and concentration were tested by 1% agarose gel electrophoresis and a spectrophotometer, and stored at -20°C for later use. Multiple pairs of fluorescent microsatellite primers (shown in Table 1) were synthesized, and FAM fluorophores were added to 5' end of the primers. Based on10 uL of PCR reaction system, including:
Templ
System Buffer Ex Taq dNTP ddH,O Primer ate
Volume 1.5ul Tul 0.3 0.8ul 6.2 Tul
[0019] PCR amplification program: 95°C 2min—94°C 30s—TM°C 90s—72°C
Imin—72°C 10min
[0020]
[0021] 40cycles
[0022] Note: TM represented the annealing temperature of microsatellite primers
[0023] With 3% agarose gel electrophoresis testing, the electrophoresis effect of PCR products was observed, and whether PCR amplification was successful was identified.
Then, fluorescence labeled PCR products were tested with a 3730XL sequencer to identify microsatellite typing results. The presence or absence of double peaks was verified, and double peaks present in an amplified product indicated that the locus has polymorphism within the population. Results showed that loci JZL106, MiSaTPW25,
MiSaTPW184 and Lar7 exhibited a single peak pattern. Excluding these loci, we used the typing results of the remaining 16 microsatellite loci for subsequent data analysis.
[0024] Table 2 Polymorphism test results of microsatellite loci
Size Size
Locus 1(bp) 2(bp) 202- 206-
JZL60 216 234 239- 250
JZL67 250 184- 189-
JZL84 197 197 7
BL-5712
LU504583 194- 210
JZL85 210 270 270-
JZL105 274
JZL106 246 246
Lar7 126 126 194- 202-
LMB24 206 206 202- 213-
LMB28 208 262 304- 307-
LMB38 313 327 310- 310-
LMB39 325 325 326- 340-
LMB41 366 366 158- 158-
MiSaTPW11 206 206
MiSaTPW25 271 271 246- 286-
MiSaTPW70 460 474 236- 236-
MiSaTPW76 253 286 349- 361-
MiSaTPW96 378 390 201- 204-
MiSaTPW117 213 213 216- 220-
MiSaTPW173 269 271 8
BL-5712
MiSaTPW184 241 241 7504589
[0025] Note: sizel and size2 represented the positions of fragment peaks, and the corresponding numbers in these two columns represented the fragment size of a PCR product at the locus.
[0026] III Data processing and analysis. The expected heterozygosity (He), observed heterozygosity (Ho), and polymorphic information content (PIC) of microsatellite loci were analyzed using the Cervus 3.0.7 software. Paternity identification was performed using an exclusion method, and the probability of excluding candidate parents who were not real parents was indicated by the cumulative probability of exclusion (CPE).
For identification of genetic relationship, the Cervus 3.0.7 software was used for determination and analysis through values of log likelihood ratio (LOD).
[0027] Table 3 Genetic diversity of 18 families at 20 microsatellite loci
H
Locus k Ho He PIC NE-1P NE-2P NE-PP
W
JZL60 7 0.621 0.8 0.767 0.576 0.398 0.215 Hk
JZL67 4 0.579 0.609 0.561 0.799 0.631 0.452 NS
JZL84 6 0.884 0.805 0.772 0.571 0.393 0.213 Hk
JZL85 6 0.674 0.668 0.624 0.742 0.564 0.372 NS
JZL105 6 0.441 0.379 0.348 0.927 0.799 0.664 ND
LMB24 5 0.625 0.535 0.495 0.846 0.685 0.509 NS
LMB28 12 0.484 0.766 0.741 0.599 0.415 0.211 Hk
LMB38 4 0.531 0.589 0.498 0.827 0.705 0.564 *
LMB39 3 0.181 0.471 0.417 0.89 0.758 0.619 Hk
LMB41 7 0.634 0.707 0.669 0.695 0.512 0.315 NS
MiSaTPW11 8 0.625 0.829 0.802 0.521 0.348 0.171 Hk
MiSaTPW70 13 0.895 0.883 0.866 0.398 0.247 0.094 ND
MiSaTPW76 6 0.417 0.647 0.593 0.768 0.602 0.423 *
MiSaTPW96 11 0.865 0.844 0.824 0.475 0.308 0.132 NS
MiSaTPW117 4 0.323 0.495 0.43 0.878 0.751 0.613 Hk 9
BL-5712
MiSaTPW173 15 0.698 0.872 0.855 0.416 0.261 0.101 NS 0506583 0.5187 0.6019 0.5638
Mean 6.55 0.7345 0.5901 0.437 - 5 5
Cumulative exclusion 0.99899 0.99999 0.9999999989 rate 3 4 27
[0028] Polymorphic Information Content (PIC) is one of the indicators for testing the degree of DNA variation in a population. According to the PIC classification rule first proposed by Botstein et al. (1980), the average PIC content in this study population is 0.56, belonging to a high polymorphism (PIC>0.5). Microsatellite loci with PIC>0.5 were recommended to be selected as paternity relationship identification loci.
[0029] Botstein D, White RL, Skolnick M, et al. Construction of a genetic linkage map in man using restriction fragment length polymorphisms. American Journal of
Human Genetics, 1980, 32(3): 314-331.
[0030] The software Cervus 3.0.7 was used to calculate allele frequency, simulate paternity relationship, carry out paternity identification and evaluate the accuracy of paternity identification with information of 18 pairs of parental and offspring microsatellite genotyping, gender, etc. The accuracy of paternity relationship identification in 540 offspring is shown in Table 3. When the gender of a single parent is known, the cumulative exclusion rate value ranges from 99.89% to 99.99%, and the observation identification rate is around 60%; and when the genders of both parents are known, the cumulative exclusion rate value exceeds 99.99%, the observed identification rate is 100%, and the actual identification rate is 79%.
[0031] Table 4 Accuracy of paternity relationship identification in 540 offspring
Genotype Observation Expected Actual identification rate | identification rate | identification rate
Only the female 62% 84% parent on we wn we
BL-5712
LU504583 ma [TT
Parents (known 100% 100% 79% gender)
[0032] The results of the present disclosure indicate that, when the genders of both parents are known, the cumulative exclusion rate between parents and offspring is 99.99%, and the actual identification rate of offspring is 79%. Therefore, using highly polymorphic microsatellite markers instead of physical markers to identify families is a relatively accurate and feasible method in trait testing and pedigree tracing. 11
BL-5712
SEQUENCE LISTING LU504583
Sequence Listing Information:
DTD Version: V1_3
File Name: HKJU20230402507-sequence listing. xml
Software Name: WIPO Sequence
Software Version: 2.3.0
Production Date: 2023-06-25
General Information:
Current application / Applicant file reference: HKJU20230402507
Applicant name: Freshwater Fisheries Research Center of Chinese Academy of Fishery
Sciences
Applicant name / Language: en
Invention title: MICROPTERUS SALMOIDES PATERNITY IDENTIFICATION KIT AND
APPLICATION THEREOF (en)
Sequence Total Quantity: 40
Sequences:
Sequence Number (ID): 1
Length: 20
Molecule Type: DNA
Features Location/Qualifiers: - misc feature, 1..20 > note, Upper Primer sequence of primer JZL60 - source, 1..20 > mol type, other DNA > organism, synthetic construct
Residues: agttaacccg ctttgtgetg 20
Sequence Number (ID): 2
Length: 20
Molecule Type: DNA
Features Location/Qualifiers: - misc feature, 1.20 > note, Lower primer sequence of primer JZL60 - source, 1..20 > mol type, other DNA > organism, synthetic construct
Residues: gaaggcgaag aagggagagt 20
Sequence Number (ID): 3
Length: 20
Molecule Type: DNA
Features Location/Qualifiers: 12
BL-5712 - misc feature, 1..20 LU504583 > note, Upper Primer sequence of primer JZL67 - source, 1..20 > mol type, other DNA > organism, synthetic construct
Residues: ccectaatga gagggagaca 20
Sequence Number (ID): 4
Length: 20
Molecule Type: DNA
Features Location/Qualifiers: - misc feature, 1.20 > note, Lower primer sequence of primer JZL67 - source, 1..20 > mol type, other DNA > organism, synthetic construct
Residues: acagactagc gtcagcagca 20
Sequence Number (ID): 5
Length: 20
Molecule Type: DNA
Features Location/Qualifiers: - misc feature, 1..20 > note, Upper Primer sequence of primer JZL84 - source, 1..20 > mol type, other DNA > organism, synthetic construct
Residues: gaaaacagec tcgggtgtaa 20
Sequence Number (ID): 6
Length: 20
Molecule Type: DNA
Features Location/Qualifiers: - misc feature, 1.20 > note, Lower primer sequence of primer JZL84 - source, 1.20 > mol type, other DNA > organism, synthetic construct
Residues: cacttettec tecetctett 20 13
BL-5712
Sequence Number (ID): 7 LU504583
Length: 20
Molecule Type: DNA
Features Location/Qualifiers: - misc feature, 1..20 > note, Upper Primer sequence of primer JZL85 - source, 1..20 > mol type, other DNA > organism, synthetic construct
Residues: ggggetcact cactgtgttt 20
Sequence Number (ID): 8
Length: 20
Molecule Type: DNA
Features Location/Qualifiers: - misc_feature, 1..20 > note, Lower primer sequence of primer JZL85 - source, 1..20 > mol_type, other DNA > organism, synthetic construct
Residues: gtgcgcagac agctagacag 20
Sequence Number (ID): 9
Length: 19
Molecule Type: DNA
Features Location/Qualifiers: - misc_feature, 1..19 > note, Upper Primer sequence of primer JZL105 - source, 1..19 > mol type, other DNA > organism, synthetic construct
Residues: gteteccctga ctgtatggc 19
Sequence Number (ID): 10
Length: 19
Molecule Type: DNA
Features Location/Qualifiers: - misc feature, 1.19 > note, Lower primer sequence of primer JZL105 - source, 1..19 > mol type, other DNA 14
BL-5712 > organism, synthetic construct LUS04583
Residues: tctgatgagg ctetgaaat 19
Sequence Number (ID): 11
Length: 20
Molecule Type: DNA
Features Location/Qualifiers: - misc feature, 1.20 > note, Upper Primer sequence of primer JZL106 - source, 1..20 > mol type, other DNA > organism, synthetic construct
Residues: gcaggcagtg aacccagatt 20
Sequence Number (ID): 12
Length: 21
Molecule Type: DNA
Features Location/Qualifiers: - misc feature, 1.21 > note, Lower primer sequence of primer JZL106 - source, 1.21 > mol type, other DNA > organism, synthetic construct
Residues: tatgtattga cgagcgagca g 21
Sequence Number (ID): 13
Length: 18
Molecule Type: DNA
Features Location/Qualifiers: - misc feature, 1..18 > note, Upper Primer sequence of primer MiSaTPW11 - source, 1.18 > mol type, other DNA > organism, synthetic construct
Residues: caacatggac gctactat 18
Sequence Number (ID): 14
Length: 18
Molecule Type: DNA
Features Location/Qualifiers:
BL-5712 - misc feature, 1..18 LU504583 > note, Lower primer sequence of primer MiSaTPW11 - source, 1..18 > mol type, other DNA > organism, synthetic construct
Residues: caaccatcac atecttct 18
Sequence Number (ID): 15
Length: 17
Molecule Type: DNA
Features Location/Qualifiers: - misc_ feature, 1.17 > note, Upper Primer sequence of primer M1SaTPW25 - source, 1..17 > mol type, other DNA > organism, synthetic construct
Residues: ccaaggtcag gtttaac 17
Sequence Number (ID): 16
Length: 19
Molecule Type: DNA
Features Location/Qualifiers: - misc feature, 1.19 > note, Lower primer sequence of primer MiSaTPW25 - source, 1..19 > mol type, other DNA > organism, synthetic construct
Residues: acctttgtge tettctetc 19
Sequence Number (ID): 17
Length: 18
Molecule Type: DNA
Features Location/Qualifiers: - misc feature, 1.18 > note, Upper Primer sequence of primer MiSaTPW70 - source, 1.18 > mol type, other DNA > organism, synthetic construct
Residues: acttcgcaaa ggtataac 18 16
BL-5712
Sequence Number (ID): 18 LU504583
Length: 19
Molecule Type: DNA
Features Location/Qualifiers: - misc feature, 1..19 > note, Lower primer sequence of primer MiSaTPW70 - source, 1..19 > mol type, other DNA > organism, synthetic construct
Residues: cctcatgcag aagatgtaa 19
Sequence Number (ID): 19
Length: 19
Molecule Type: DNA
Features Location/Qualifiers: - misc feature, 1..19 > note, Upper Primer sequence of primer MiSaTPW76 - source, 1..19 > mol type, other DNA > organism, synthetic construct
Residues: acacagtgtc agttcteca 19
Sequence Number (ID): 20
Length: 20
Molecule Type: DNA
Features Location/Qualifiers: - misc feature, 1.20 > note, Lower primer sequence of primer MiSaTPW76 - source, 1..20 > mol type, other DNA > organism, synthetic construct
Residues: gtgaatacct cagcaagcat 20
Sequence Number (ID): 21
Length: 23
Molecule Type: DNA
Features Location/Qualifiers: - misc feature, 1..23 > note, Upper Primer sequence of primer MiSaTPW96 - source, 1.23 > mol type, other DNA 17
BL-5712 > organism, synthetic construct LUS04583
Residues: cttctaaatg tgtgtagget tgc 23
Sequence Number (ID): 22
Length: 23
Molecule Type: DNA
Features Location/Qualifiers: - misc_feature, 1..23 > note, Lower primer sequence of primer MiSaTPW96 - source, 1..23 > mol type, other DNA > organism, synthetic construct
Residues: agcttagcat aaagactggg aac 23
Sequence Number (ID): 23
Length: 24
Molecule Type: DNA
Features Location/Qualifiers: - misc feature, 1..24 > note, Upper Primer sequence of primer MiSaTPW117 - source, 1.24 > mol type, other DNA > organism, synthetic construct
Residues: totgaaagec acaacacage ctgc 24
Sequence Number (ID): 24
Length: 24
Molecule Type: DNA
Features Location/Qualifiers: - misc feature, 1.24 > note, Lower primer sequence of primer MiSaTPW117 - source, 1.24 > mol type, other DNA > organism, synthetic construct
Residues: atcgacctgc agaccagcaa cact 24
Sequence Number (ID): 25
Length: 23
Molecule Type: DNA
Features Location/Qualifiers: 18
BL-5712 - misc_feature, 1..23 LU504583 > note, Upper Primer sequence of primer MiSaTPW173 - source, 1..23 > mol type, other DNA > organism, synthetic construct
Residues: ccacacagtg acacaaactg tec 23
Sequence Number (ID): 26
Length: 20
Molecule Type: DNA
Features Location/Qualifiers: - misc feature, 1..20 > note, Lower primer sequence of primer MiSaTPW173 - source, 1..20 > mol type, other DNA > organism, synthetic construct
Residues: eccattetec tectecagag 20
Sequence Number (ID): 27
Length: 22
Molecule Type: DNA
Features Location/Qualifiers: - misc feature, 1.22 > note, Upper Primer sequence of primer MiSaTPW184 - source, 1..22 > mol type, other DNA > organism, synthetic construct
Residues: ttgtatacca agtgacctgt gg 22
Sequence Number (ID): 28
Length: 24
Molecule Type: DNA
Features Location/Qualifiers: - misc feature, 1..24 > note, Lower primer sequence of primer MiSaTPW184 - source, 1.24 > mol type, other DNA > organism, synthetic construct
Residues: gggagtecat ctttctgaag tgcc 24 19
BL-5712
Sequence Number (ID): 29 LU504583
Length: 21
Molecule Type: DNA
Features Location/Qualifiers: - misc feature, 1..21 > note, Upper Primer sequence of primer Lar7 - source, 1.21 > mol type, other DNA > organism, synthetic construct
Residues: gtgctaataa aggctactet c 21
Sequence Number (ID): 30
Length: 20
Molecule Type: DNA
Features Location/Qualifiers: - misc feature, 1.20 > note, Lower primer sequence of primer Lar7 - source, 1..20 > mol type, other DNA > organism, synthetic construct
Residues: tettccctta attettttga 20
Sequence Number (ID): 31
Length: 20
Molecule Type: DNA
Features Location/Qualifiers: - misc feature, 1.20 > note, Upper Primer sequence of primer LMB24 - source, 1..20 > mol type, other DNA > organism, synthetic construct
Residues: cgtgtaacta tggtgatgte 20
Sequence Number (ID): 32
Length: 18
Molecule Type: DNA
Features Location/Qualifiers: - misc feature, 1..18 > note, Lower primer sequence of primer LMB24 - source, 1.18 > mol type, other DNA
BL-5712 > organism, synthetic construct LUS04583
Residues: gaagcgagag tcagagtg 18
Sequence Number (ID): 33
Length: 18
Molecule Type: DNA
Features Location/Qualifiers: - misc feature, 1.18 > note, Upper Primer sequence of primer LMB28 - source, 1.18 > mol type, other DNA > organism, synthetic construct
Residues: tegtgaacac agcagaac 18
Sequence Number (ID): 34
Length: 20
Molecule Type: DNA
Features Location/Qualifiers: - misc feature, 1.20 > note, Lower primer sequence of primer LMB28 - source, 1..20 > mol type, other DNA > organism, synthetic construct
Residues: ggacagcete aatacatett 20
Sequence Number (ID): 35
Length: 20
Molecule Type: DNA
Features Location/Qualifiers: - misc feature, 1.20 > note, Upper Primer sequence of primer LMB38 - source, 1..20 > mol type, other DNA > organism, synthetic construct
Residues: tagtgtattg ctctgaaget 20
Sequence Number (ID): 36
Length: 20
Molecule Type: DNA
Features Location/Qualifiers: 21
BL-5712 - misc feature, 1..20 LU504583 > note, Lower primer sequence of primer LMB38 - source, 1..20 > mol type, other DNA > organism, synthetic construct
Residues: aacatactgc tgatggagaa 20
Sequence Number (ID): 37
Length: 20
Molecule Type: DNA
Features Location/Qualifiers: - misc feature, 1..20 > note, Upper Primer sequence of primer LMB39 - source, 1..20 > mol type, other DNA > organism, synthetic construct
Residues: gaaggacacc atacaacact 20
Sequence Number (ID): 38
Length: 19
Molecule Type: DNA
Features Location/Qualifiers: - misc feature, 1..19 > note, Lower primer sequence of primer LMB39 - source, 1..19 > mol type, other DNA > organism, synthetic construct
Residues: cetetecatc ctecctcatc 19
Sequence Number (ID): 39
Length: 19
Molecule Type: DNA
Features Location/Qualifiers: - misc feature, 1.19 > note, Upper Primer sequence of primer LMB41 - source, 1..19 > mol type, other DNA > organism, synthetic construct
Residues: gcagagcaga agaatgact 19 22
BL-5712
Sequence Number (ID): 40 LUS04583
Length: 18
Molecule Type: DNA
Features Location/Qualifiers: - misc feature, 1.18 > note, Lower primer sequence of primer LMB41 - source, 1.18 > mol type, other DNA > organism, synthetic construct
Residues: tgaacacaac gacaccac 18
END
23

Claims (2)

BL-5712 WHAT IS CLAIMED IS: 7504589
1. A Micropterus salmoides paternity identification kit, comprising multiple pairs of fluorescent microsatellite primers as shown in Table 1: Table 1 PCR Primer name Upper Primer sequence (5-3) Lower Primer sequence (5’-3’) Probe type length 207— JZL60 AGTTAACCCGCTTTGTGCTG GAAGGCGAAGAAGGGAGAGT FAM 227 253— JZL67 CCGCTAATGAGAGGGAGACA ACAGACTAGCGTCAGCAGCA FAM 266 179— JZL84 GAAAACAGCCTCGGGTGTAA CACTTGTTGCTGCGTCTGTT FAM 209 199— JZL85 GGGGCTCACTCACTGTGTTT GTGCGCAGACAGCTAGACAG FAM 225 284— JZL105 GTGTCCCTGACTGTATGGC TCTGATGAGGCTGTGAAAT FAM 320 250— JZL106 GCAGGCAGTGAACCCAGATT TATGTATTGACGAGCGAGCAG FAM 286 166— MiSaTPW11 CAACATGGACGCTACTAT CAACCATCACATGCTTCT FAM 357 270— MiSaTPW25 CCAAGGTCAGGTTTAAC ACCTTTGTGCTGTTCTGTC FAM 300 282— MiSaTPW70 ACTTCGCAAAGGTATAAC CCTCATGCAGAAGATGTAA FAM 609 257— MiSaTPW76 ACACAGTGTCAGTTCTGCA GTGAATACCTCAGCAAGCAT FAM 303 24
BL-5712 CUS04583 CTTCTAAATGTGTGTAGGGTT AGCTTAGCATAAAGACTGGGAA 372— MiSaTPW96 FAM GC C 405 TGTGAAAGGCACAACACAGC ATCGACCTGCAGACCAGCAACA 209— MiSaTPW117 FAM CTGC CT 242 CCACACAGTGACACAAACTGT 195— MiSaTPW173 GCCATTGTGCTGCTGCAGAG FAM GC 271 TTGTATACCAAGTGACCTGTG GGGAGTGCATCTTTCTGAAGTGC 219— MiSaTPW184 FAM G C 253 124— Lar7 GTGCTAATAAAGGCTACTGTC TGTTCCCTTAATTGTTTTGA FAM 210 LMB24 CGTGTAACTATGGTGATGTG GAAGCGAGAGTCAGAGTG 203 FAM LMB28 TGGTGAACACAGCAGAAC GGACAGCCTCAATACATCTT 236 FAM LMB38 TAGTGTATTGCTCTGAAGGT AACATACTGCTGATGGAGAA 302 FAM LMB39 GAAGGACACCATACAACACT CCTCTCCATCCTCCTCATC 327 FAM LMB41 GCAGAGCAGAAGAATGACT TGAACACAACGACACCAC 336 FAM
2. The kit according to claim 1, wherein the kit is used for paternity identification.
LU504583A 2023-06-27 2023-06-27 Micropterus salmoides paternity identification kit and application thereof LU504583B1 (en)

Priority Applications (1)

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Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
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Publications (1)

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LU504583B1 true LU504583B1 (en) 2024-01-11

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