WO2007068774A2 - Procédé moléculaire destiné à l'étude génétique de populations et à l'analyse du pedigree de la daurade royale (sparus aurata) et nécessaire correspondant - Google Patents

Procédé moléculaire destiné à l'étude génétique de populations et à l'analyse du pedigree de la daurade royale (sparus aurata) et nécessaire correspondant Download PDF

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Publication number
WO2007068774A2
WO2007068774A2 PCT/ES2006/000679 ES2006000679W WO2007068774A2 WO 2007068774 A2 WO2007068774 A2 WO 2007068774A2 ES 2006000679 W ES2006000679 W ES 2006000679W WO 2007068774 A2 WO2007068774 A2 WO 2007068774A2
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WIPO (PCT)
Prior art keywords
sau
sal
pmol
reaction
analysis
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PCT/ES2006/000679
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English (en)
Spanish (es)
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WO2007068774A3 (fr
Inventor
Javier Porta Pelayo
José María PORTA PELAYO
María del Carmen ALVAREZ HERRERO
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Universidad De Málaga
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Publication of WO2007068774A2 publication Critical patent/WO2007068774A2/fr
Publication of WO2007068774A3 publication Critical patent/WO2007068774A3/fr

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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
    • 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
    • C12Q1/6858—Allele-specific amplification
    • 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
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the golden (Sparus aurata), is a species widely distributed by the Mediterranean Sea and the Atlantic coast from the British Isles to the Island of Cape Verde and the Canary Islands.
  • sea bream represents one of the most important species in marine aquaculture, especially in the Mediterranean area.
  • the main producers are Greece, Turkey, Spain, Italy and France.
  • Other minor productions are in Portugal, Weg, Cyprus, Israel, Malta, Egypt, Tunisia and Morocco, while there are incipient productions in Bulgaria, Norway and republic.
  • a new molecular tool based on microsatellite markers is presented.
  • This tool consists of co-amplification using the polymerase chain reaction (PCR) technique and analysis of 10 loci together, so that, in a fast and reliable way, sufficient data from an individual or group of individuals is obtained to carry
  • PCR polymerase chain reaction
  • 50 ng which can be obtained from any type of sample containing nucleated cells, without causing damage to the animal, such as eggs, larvae, fin, blood, pieces of dead or awakened and even processed fish tissues - Standardization of Loci employees and possible comparison of results of different works carried out with this tool.
  • loci employees are highly informative and have been tested in the identification of individuals, in pedigree studies and characterization of populations with very satisfactory results. Although genotyping methods developed for other organisms have been described
  • microsatellite molecular markers are repeated DNA sequences that are randomly distributed throughout the genome of organisms and that constitute highly variable areas and therefore, distinct from one individual to another. By studying these regions we can distinguish an individual from the rest of individuals of the same species with the highest precision. This is what the genetic typing or genotyping of an individual means.
  • the proposed tool also allows pedigree studies to identify the biological parents of a specific individual. The more numerous and variable these regions we study, the greater accuracy is obtained.
  • the invention consists of an optimized protocol for the study of 10 of these markers in the sea bream, Sparus aurata (Sparus aurata genotyping and paternity tool).
  • Co-amplification multiplex PCR
  • a and B simultaneous reactions
  • loci microsatellite markers
  • PCR polymerase chain reaction
  • the final objectives of automatic detection are to optimize the conditions for the analysis in automatic sequencers of type AB 310, 3100, 3130 and 3130 Advance (Applied Biosystem).
  • this tool could also be used with other genetic marker analysis equipment.
  • This method can be carried out through a marketable product consisting of a Mt with three possible presentations that allow the standardization and automation of the genotyping technique in a simple and fully reproducible way.
  • This method has been tested with excellent results, in the identification of individuals and determination of the pedigree in individuals born in a gilthead farm, an aspect of singular importance in the cultivation of fish in which it is not possible to track the breeders that really they participate in the laying, unless artificial fertilization is done.
  • this tool has been tested in the genetic characterization of a population of 50 individuals from the natural environment.
  • This method can be used to control the genetic structure of stocks and thus maintain levels of variability consistent with optimal biological efficiency, apply selection methods and / or facilitate accurate monitoring of the processes of reproduction, breeding, marketing and traceability.
  • This control of the cultivated stocks would allow to avoid possible ecological disasters in the natural populations, after possible escapes or repopulation.
  • the conditions are the general ones of any PCR amplification protocol, including the presence of thermostable polymerase, dNTPs (deoxyribonucleoside triphosphate); ionic strength conditions [ClMg + 2] and a pH suitable for enzyme activity.
  • thermostable polymerase deoxyribonucleoside triphosphate
  • ionic strength conditions [ClMg + 2]
  • pH suitable for enzyme activity for the optimal and balanced amplification of the different loci, very specific cycle conditions and proportions of reagents and primers are necessary.
  • the probability of identity represents the inverse of the number of individuals that must be analyzed before finding the same genotype in a randomly selected sample.
  • the power of exclusion refers to the probability that a randomly chosen adult will be excluded as a parent of a descendant.
  • Sparus aurata genotyping and paternity tool kit A + B allows pedigree studies to be carried out using molecular exclusion methods.
  • the direct exclusion of paternity means that when a child has genetic information that neither the mother nor the presumed father has, both should be excluded as biological parents.
  • the exclusion power of these tools can be measured by exclusion estimators (Exclusion Value 1 when neither parent is known and Exclusion Value 2 when at least one parent knows).
  • Sparus aurata genotyping and paternity tool kit provides exclusion values 1 and 2 of 0.999789 and 0.999998, respectively, which evidences. In the same way the following table shows the sensitivity of reactions A, B and A + B.
  • kits developed are based on the methodology described above, with the particularity that the primers are marked with specific fluorescent labels for analysis in automatic sequencers type AB 310, 3100, 3130 and 3130 Advance (Applied Biosystem).
  • the fluorescent labels described above are the following: 1 corresponds to 6- FAM (Applied Biosystem), 2 corresponds to NED (Applied Biosystem), 3 corresponds to PET (Applied Biosystem) and 4 corresponds to VIC (Applied Biosystem).
  • Fig.l Simplified scheme of the method of protection.
  • Fig. 2. Simplified scheme of a preferred embodiment (pedigree determination). Contrast of an individual's DNA footprint with that of their supposed parents (exclusion method). These analyzes allow to determine the paternity of an individual with a practically 100% reliability. Circles indicate exclusive loci.
  • Reaction A Preparation of Reaction A; Add 0.5 ⁇ l of Reaction A, 0.5 ⁇ l of 500 LIZ TM Size Standard (Applied Biosystem) and 10 ⁇ l of deionized formamide. Denature 10 minutes at 94 0 C and put cold until loading in the device.
  • Reaction B Preparation of Reaction B; Add 2 ⁇ l of Reaction B, 0.5 ⁇ l of 500 LIZ TM Size Standard (Applied Biosystem) and 10 ⁇ l of deionized formamide. Denature 10 minutes at 94 0 C and put cold until loading in the device.
  • Matrix Matrix standard set DS 33

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Analytical Chemistry (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

L'invention concerne un procédé moléculaire destiné à l'étude génétique de populations et à l'analyse du pedigree de la daurade (Sparus aurata) et le nécessaire correspondant (trousse d'outils de génotypage et de paternité du Sparus aurata). L'invention concerne également un héritage moléculaire basé sur des marqueurs microsatellites, qui consiste en une co-amplification par le biais de la technique de réaction en chaîne de la polymérase et l'analyse de 10 loci, permettant l'obtention, rapide et fiable, des données d'un individu ou d'un groupe d'individus suffisantes pour réaliser des études de populations, établir des relations de parenté ou de pedigree de grande précision. Cette information permet de contrôler et d'améliorer les caractéristiques des populations de reproducteurs de daurade. En outre, ce protocole s'est révélé adéquat pour la détection et l'analyse automatique dans des équipes de séquençage AB (Applied Biosystem). Elle concerne enfin un nécessaire ayant trois formats possibles qui permet la standardisation et l'automatisation du procédé de manière aisée et complètement reproductible.
PCT/ES2006/000679 2005-12-13 2006-12-12 Procédé moléculaire destiné à l'étude génétique de populations et à l'analyse du pedigree de la daurade royale (sparus aurata) et nécessaire correspondant WO2007068774A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
ES200502458A ES2277536B1 (es) 2005-12-13 2005-12-13 Metodo molecular para el estudio genetico de poblaciones y el analisis de pedigri de la dorada (sparus aurata) y kit correspondiente (sparus aurata genotyping and paternity tool kit).
ESP200502458 2005-12-13

Publications (2)

Publication Number Publication Date
WO2007068774A2 true WO2007068774A2 (fr) 2007-06-21
WO2007068774A3 WO2007068774A3 (fr) 2007-08-02

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104263840A (zh) * 2014-10-13 2015-01-07 青岛农业大学 一种利用est微卫星标记鉴别韭菜迟眼蕈蚊与异迟眼蕈蚊幼虫的引物及方法
CN106520939A (zh) * 2016-11-04 2017-03-22 中国水产科学研究院淡水渔业研究中心 一种鲷鱼种质鉴定的方法及其应用
CN106947816A (zh) * 2016-10-28 2017-07-14 中山大学 一种斜带石斑鱼亲子鉴定微卫星荧光多重pcr的方法
CN113186302A (zh) * 2021-06-04 2021-07-30 华中农业大学 鲌鲂杂交种先锋2号的性别特异分子标记及应用

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003060160A2 (fr) * 2002-01-18 2003-07-24 Genomar Asa Verification des origines alimentaires fondees sur la reconnaissance du motif d'acide nucleique

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003060160A2 (fr) * 2002-01-18 2003-07-24 Genomar Asa Verification des origines alimentaires fondees sur la reconnaissance du motif d'acide nucleique

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
BROWN R.C.: 'Additional microsatellites for Sparus aurata and cross-species amplification within the Sparidae family' MOLECULAR ECOLOGY NOTES vol. 5, no. 3, September 2005, pages 605 - 607, XP003016139 *
BROWN R.C.: 'Factors influencing effective population size in commercial populations of gilthead seabream, Sparus aurata' AQUACULTURE vol. 247, no. 1-4, June 2005, pages 219 - 225, XP004912492 *
CHISTIAKOV D. ET AL.: 'Microsatellites and their genomic distribution, evolution, function and applications: A review with special reference to fish genetics' AQUACULTURE vol. 255, no. 1-4, June 2006, pages 1 - 29, XP005464119 *
DE INNOCENTIIS S. ET AL.: 'Microsatellite markers reveal population structure in gilthead sea bream Spartus auratus from the Atlantic Ocean and Mediterranean Sea' FISHERIES SCIENCE vol. 70, no. 5, October 2004, pages 852 - 859, XP003016138 *
LAUNEY S. ET AL.: 'Twelve new microsatellite markers for gilted seabream (Sparus aurata L.): Characterization, polymorphism and linkage' MOLECULAR ECOLOGY NOTES vol. 3, no. 3, September 2003, pages 457 - 459, XP003016137 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104263840A (zh) * 2014-10-13 2015-01-07 青岛农业大学 一种利用est微卫星标记鉴别韭菜迟眼蕈蚊与异迟眼蕈蚊幼虫的引物及方法
CN106947816A (zh) * 2016-10-28 2017-07-14 中山大学 一种斜带石斑鱼亲子鉴定微卫星荧光多重pcr的方法
CN106947816B (zh) * 2016-10-28 2020-08-04 中山大学 一种斜带石斑鱼亲子鉴定微卫星荧光多重pcr的方法
CN106520939A (zh) * 2016-11-04 2017-03-22 中国水产科学研究院淡水渔业研究中心 一种鲷鱼种质鉴定的方法及其应用
CN106520939B (zh) * 2016-11-04 2019-09-13 中国水产科学研究院淡水渔业研究中心 一种鲷鱼种质鉴定的方法及其应用
CN113186302A (zh) * 2021-06-04 2021-07-30 华中农业大学 鲌鲂杂交种先锋2号的性别特异分子标记及应用
CN113186302B (zh) * 2021-06-04 2022-02-01 华中农业大学 鲌鲂杂交种先锋2号的性别特异分子标记及应用

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Publication number Publication date
ES2277536A1 (es) 2007-07-01
WO2007068774A3 (fr) 2007-08-02
ES2277536B1 (es) 2008-06-16

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