A parentage identification method for procambarus clarkii based on four-base microsatellite fluorescence multiplex PCR

Abstract

The application provides a kind of based on four base microsatellite fluorescence multiplex PCR red claw crayfish parentage identification method, belong to biological engineering technical field, the application includes the following steps: (1) extracting the genome DNA of sample to be measured;(2) the screening of red claw crayfish microsatellite primer (3) the construction of multiplex PCR amplification system 20 pairs of primers in step (2) are grouped according to 120-200bp, 220-260bp, 280-340bp, 360-420bp, 440bp-500bp interval, select amplification combination, obtain multiple multiplex PCR amplification system;(4) parentage identification sample genome DNA amplification, obtain multiplex PCR product, typing, read parent and offspring genotype, judge parent-child relationship.The application can realize that there is no interaction between multiple primers, no non-specific amplification, do not make the amplified product overlap.In the process of PCR amplification, multiple target fragments are amplified simultaneously, which can save experimental samples, experimental cost, improve experimental efficiency, and achieve 100% parentage identification success rate for red claw crayfish.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of bioengineering, and particularly relates to a parent-offspring identification method for red claw crayfish based on four-base microsatellite fluorescence multiplex PCR. BACKGROUND

[0002] The red claw crayfish, also known as Australian freshwater lobster, is originally from Australia and was introduced into China in the 1990s. The red claw crayfish has the following characteristics: first, it is large and fat, with a weight of 100-200 g, and the largest individual in Australia weighs up to 500 g; second, it is rich in nutrients, with tender, delicious, sweet and refreshing meat; third, it has high ornamental value, with a brown-green body and a membrane-like bright red band on the top of the outer side, which is beautiful and attractive; fourth, it is suitable for long-distance transportation and will not die for a long time out of water; fifth, it has a varied diet, eating both animal feed and plant feed as well as artificial compound feed; sixth, it grows fast, has high yield and good economic benefits, and can be harvested in the same year after being released. However, since the red claw crayfish is an introduced species, it is important to monitor the germplasm during the breeding and propagation process to prevent germplasm degradation. Improving the germplasm of the red claw crayfish through genetic improvement and cultivating fast-growing and highly resistant superior varieties are particularly urgent for the stable development of the red claw crayfish aquaculture industry.

[0003] On the basis of traditional genetic selection, using molecular markers to study the genetic diversity of selected populations and assisting in parent selection can effectively improve the selection efficiency, especially for traits that are difficult to determine phenotypically, such as disease resistance. Microsatellites (also known as simple sequence repeats, SSR) have high polymorphism and good stability, and are co-dominant markers, making them an ideal molecular marker. Currently, microsatellite marker technology has been widely used in genetic map construction, family identification, variety purity detection, and target trait gene mapping research fields. Molecular markers are an important technical means in the research and application of germplasm resource identification, family selection, and release effect evaluation. In family selection, understanding the genetic diversity of the entire family, the correspondence between offspring and parents, and the relationship between offspring and offspring can guide parent selection, thereby avoiding inbreeding and preventing germplasm degradation.

[0004] Microsatellite DNA is a short tandem repeat sequence in eukaryotic genome. Microsatellite DNA has been widely used due to its large number of loci, uniform distribution in genome, co-dominant inheritance and easy detection. Microsatellite of Procambarus clarkii has been used to solve genetic diversity, genetic structure and other problems. At present, the number of microsatellite markers of Procambarus clarkii is small, and only 55 microsatellite sequences of Procambarus clarkii are contained in GenBank, and these microsatellites are all two-base microsatellites. Compared with two-base microsatellites, four-base microsatellites have the advantages of easy typing and not easy to produce shadow bands. The lack of genetic markers is one of the important reasons affecting the selection of Procambarus clarkii.

[0005] On the other hand, the above genetic experiments all need to use multiple microsatellite loci, and researchers need to spend a lot of time and experimental cost. More importantly, a large amount of DNA sample is consumed. Therefore, it is of great significance to develop a microsatellite experiment system with less sample requirement, low cost and high efficiency for the breeding research of Procambarus clarkii.

[0006] Microsatellite multiplex PCR refers to a PCR technology in which multiple pairs of microsatellite specific primers are added to a PCR system to simultaneously amplify different regions of the same template. Microsatellite multiplex PCR can simultaneously amplify multiple target fragments, which can save experimental samples, experimental cost and improve experimental efficiency. The core content of microsatellite multiplex PCR is how to design and optimize the primer combination so that there is no interaction between primers, no non-specific amplification and no overlapping of amplification products.

[0007] The Chinese patent with the published application number CN201510084807.2 discloses a primer set and method for identifying Procambarus clarkii family using microsatellite markers, which belongs to the technical field of breeding of Procambarus clarkii. The steps include: breeding Procambarus clarkii family by artificial breeding method; collecting Procambarus clarkii samples to be identified and extracting genomic DNA; taking the obtained genomic DNA as a template and selecting 12 pairs of microsatellite primers for PCR amplification; performing electrophoresis detection on the PCR amplification products; and digitizing the electrophoresis results and performing parentage identification by using genetic analysis software. The present application can quickly and accurately identify the parentage of Procambarus clarkii, distinguish different families, reduce the phenomenon of inbreeding in Procambarus clarkii population breeding, and is more conducive to realizing family selection, thereby accelerating the breeding process and improving the breeding effect.

[0008] Because multiple primers are easy to interact with each other or produce non-specific amplification, and the amplification products overlap during the process of PCR amplification, the primers in the PCR amplification system are usually single primers. Most of the existing PCR amplification technologies are to amplify single target fragment one by one, which wastes experimental samples, experimental cost and takes a long time to operate. SUMMARY

[0009] Therefore, the application provides a parentage identification method of Procambarus clarkii based on four-base microsatellite fluorescence multiplex PCR.

[0010] The application is a parentage identification method of Procambarus clarkii based on four-base microsatellite fluorescence multiplex PCR, comprising the following steps.

[0011] (1) DNA extraction of Procambarus clarkii samples

[0012] The appendages of Procambarus clarkii individuals are taken, and the genomic DNA of the sample to be tested is extracted by the phenol-chloroform method, and the concentration of the genomic DNA of the sample to be tested is diluted to 25 ng / μL.

[0013] (2) Screening of microsatellite primers of Procambarus clarkii

[0014] According to the genomic data of Procambarus clarkii obtained by the laboratory, the MISA software is used to screen

[0015] four-base microsatellite sequences, the microsatellite site primers are designed by using the Primer Premier 6.0 software, and 20 pairs of primers are screened: Cqaa-48, Cqaa-191, Cqaa-218, Cqaa-197, Cqaa-24, Cqaa-181, Cqaa-121, Cqaa-227, Cqaa-22, Cqaa-163, Cqaa-106, Cqaa-185, Cqaa-115, Cqaa-182, Cqaa-240, Cqaa-228, Cqaa-132, Cqaa-211, Cqaa-43, and Cqaa-167.

[0016] (3) Construction of multiplex PCR amplification system

[0017] The 20 pairs of primers in step (2) are preliminarily grouped according to the intervals of 120-200 bp, 220-260 bp, 280-340 bp, 360-420 bp, and 440 bp-500 bp, then 3-5 pairs of primers are selected in different groups, a clear combination is selected, a multiplex PCR amplification system is constructed, the 5' end of the upstream primer of each site is modified by fluorescence, and a plurality of multiplex PCR amplification systems composed of 20 microsatellite sites are obtained.

[0018] The multiplex PCR amplification system is five groups, and the multiplex PCR amplification system is: 2x Multiplex PCR Mix 10 μL, the working concentration of the upstream and downstream primers is 10 mmol / L, 50 ng of genomic DNA, and the sterile ultrapure water is supplemented to the system to 20 μL, and the multiplex PCR amplification system includes a first group, a second group, a third group, a fourth group and a fifth group;

[0019] (4) Parentage test

[0020] The genomic DNA of the sample to be tested in step (1) is amplified, and the amplification conditions of the genomic DNA of the sample to be tested are as follows: total denaturation at 94℃ for 5 minutes, denaturation at 94℃ for 30 seconds, annealing at 59℃ for 90 seconds, extension at 72℃ for 30 seconds, a total of 35 cycles, and finally extension at 60℃ for 30 minutes, to obtain a multiplex PCR product, then the multiplex PCR product is typed on a sequencer, the sequencer is a model ABI 3730 XL, capillary electrophoresis detection typing is performed, and GeneMapper 4.0 is used to analyze the results, so as to obtain the genotype data of the parent and the offspring of each locus, read the size bp of the alleles of the individual, form a digital genotype matrix, read the genotype of the parent and the offspring, and judge the parent-child relationship according to Mendel's law.

[0021] The parentage test method for red-kneed crayfish based on four-base microsatellite fluorescence multiplex PCR of the application utilizes the combination of microsatellite markers and multiplex fluorescence PCR technology, screens out 20 pairs of highly polymorphic microsatellite loci, forms five groups of multiplex PCR systems, and performs individual identification and parentage test on the red-kneed crayfish after sequencing, is suitable for popularization and application in the aspects of family management, population genetic diversity, good seed selection and genetic structure evaluation of the red-kneed crayfish; the multiplex PCR reaction of the application is relatively simple for unit point detection, can significantly improve the experimental efficiency and save experimental costs; and the multiplex PCR reaction system of the application can achieve a 100% parentage test success rate for the red-kneed crayfish at a 95% confidence level. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 It is a PCR genotyping peak graph of the red-kneed crayfish in Example 1 of the application;

[0023] Figure 2 It is the actual cumulative correct identification rate of the parentage test in Example 1 of the application. DETAILED DESCRIPTION

[0024] The application will be described in detail below in combination with specific embodiments.

[0025] Example 1

[0026] A parentage identification method of Procambarus clarkii based on four-base microsatellite fluorescence multiplex PCR, comprising the following steps:

[0027] (1) DNA extraction of Procambarus clarkii samples

[0028] Select 1-year-old Procambarus clarkii with mature gonads for artificial induced parturition, and establish 12 full-sibling single-pair mating families; collect all parent individuals (a total of 24) and part of the offspring individuals (11-12 offspring individuals in each family, a total of 143) of each family, take the appendages of Procambarus clarkii individuals, and extract the genomic DNA of the to-be-tested samples by the phenol-chloroform method; and dilute the concentration of the to-be-tested sample genomic DNA to 25 ng / μL;

[0029] (2) Screening of microsatellite primers of Procambarus clarkii

[0030] According to the genomic data of Procambarus clarkii obtained by the pre-monitoring and analysis of the laboratory, four-base microsatellite sequences are screened by MISA software

[0031] The four-base microsatellite site primers are designed by using Primer Premier 6.0 software, and it is ensured that the lengths of the target fragments have large differences (100-500 bp); the primer sequences are synthesized by a biological company, and 20 pairs of primers with better amplification effects are screened by using 2.5% agarose gel electrophoresis detection; the primers include forward primers and reverse primers; and the 20 pairs of primers are Cqaa-48, Cqaa-191, Cqaa-218, Cqaa-197, Cqaa-24, Cqaa-181, Cqaa-121, Cqaa-227, Cqaa-22, Cqaa-163, Cqaa-106, Cqaa-185, Cqaa-115, Cqaa-182, Cqaa-240, Cqaa-228, Cqaa-132, Cqaa-211, Cqaa-43 and Cqaa-167;

[0032] (3) Construction of multiplex PCR amplification system

[0033] The 20 pairs of primers in step (2) are preliminarily grouped according to the intervals of 120-200 bp, 220-260 bp, 280-340 bp, 360-420 bp and 440-500 bp, then 3-5 pairs of primers are selected in different groups, a clear combination is selected, and a multiplex PCR amplification system is constructed; the 5' end of the upstream primer of each site is modified by fluorescence, and a plurality of multiplex PCR amplification systems composed of 20 microsatellite sites are obtained;

[0034] The multiplex PCR amplification system is five groups, and the multiplex PCR amplification system is: 2x Multiplex PCR Mix 10 μL, the working concentration of the upstream and downstream primers is 10 mmol / L, 50 ng of genomic DNA, and the system is supplemented with sterile ultrapure water to 20 μL, the multiplex PCR amplification system includes a first group, a second group, a third group, a fourth group, and a fifth group;

[0035] (4) Parentage test

[0036] The genomic DNA of the sample to be tested in step (1) is amplified, and the amplification conditions of the genomic DNA of the sample to be tested are as follows: total denaturation at 94°C for 5 minutes, denaturation at 94°C for 30 seconds, annealing at 59°C for 90 seconds, extension at 72°C for 30 seconds, a total of 35 cycles, and finally extension at 60°C for 30 minutes, to obtain a multiplex PCR product. Capillary electrophoresis analysis is performed on an ABI 3130 sequencer, and after detection, the multiplex PCR product is typed on a sequencer. The sequencer used for typing is an ABI 3730 XL sequencer for capillary electrophoresis detection typing, and the results are analyzed using GeneMapper 4.0, so as to obtain the genotype data of the parent and offspring of each locus. Read the size bp of the alleles of the individual, and arrange them into a digital genotype matrix. Read the genotype of the parent and offspring, and determine the parent-child relationship according to Mendel's law. Figure 1 It is a result chart of capillary electrophoresis detection typing of a certain red claw crayfish in this embodiment using an ABI 3730 XL. The peak chart is clean and clear, without impurity DNA.

[0037] When the multiplex PCR amplification system contains only the second group as combination 1, contains the first group and the second group as combination 2, contains the first group, the second group, and the third group as combination 3, contains the first group, the second group, the third group, and the fifth group as combination 4, and contains the first group, the second group, the third group, the fourth group, and the fifth group as combination 5, the correctness of the parentage test is tested, and the accuracy is as follows Figure 2 When a single combination is used for kinship identification, the actual identification accuracy of the second group is the highest, so it is placed in the front. Among the total of 24 parents and 143 offspring, at a confidence level of 95%, the five-group multiplex PCR (20 loci) reaction system described in the application can achieve a 100% success rate of parentage test.

[0038] Example 2

[0039] A red claw crayfish parentage test method based on four-base microsatellite fluorescence multiplex PCR, comprising the following steps:

[0040] (1) Collect 32 individuals of Procambarus clarkii in a certain breeding farm in Huzhou, extract the genomic DNA of each sample to be tested by using the phenol-chloroform method, and dilute the obtained genomic DNA of the sample to be tested to 25 ng / μL after determining the concentration;

[0041] The remaining steps refer to steps (2), (3) and (4) in Example 1, and the genotype data of the 32 individuals of the microsatellite marker are analyzed by using Genemapper v4.0 software, and the results are as follows:

[0042]

[0043] Among them, the sample size is 32; Na is the number of alleles; Ho is the observed heterozygosity; He is the expected heterozygosity; PIC is the polymorphic information content; NE-1P is the exclusion rate when the parents are unknown; NE-2P is the exclusion rate when one of the parents is known; and HW is the Hardy-Weinberg equilibrium. Significant indicates significant, NS indicates not significant, and ND indicates uncertain.

[0044] It is shown that the microsatellite multiplex fluorescence PCR method of the application can efficiently and quickly realize the parentage identification and analysis of the family of Procambarus clarkii, with an accuracy of about 100%, which meets the requirements of Procambarus clarkii germplasm identification and family management.

[0045] The parentage identification method of the application combines microsatellite markers with multiplex fluorescence PCR technology, screens 20 pairs of highly polymorphic microsatellite loci, establishes 5 groups of multiplex PCR amplification systems, and identifies individuals and parentage of Procambarus clarkii after sequencing, which is suitable for popularization and application in the management of Procambarus clarkii family, population genetic diversity, selection of fine breeds and evaluation of genetic structure; the multiplex PCR reaction of the application is relatively simple unit point detection, which can simultaneously amplify multiple target fragments, save experimental samples and experimental costs, improve experimental efficiency, and there is no interaction between primers, no non-specific amplification, and no overlapping of amplification products; and the multiplex PCR reaction system of the application can achieve a 100% parentage identification success rate for Procambarus clarkii at a 95% confidence level. SEQUENCE LISTING <110> Zhejiang Institute of Freshwater Aquatic Products <120> A parentage identification method for Procambarus clarkii based on four-base microsatellite fluorescence multiplex PCR <130> 2021 <160> 20 <170> SIPOSequenceListing 1.0 <210> 1 <211> 547 <212> DNA <213> Redclaw crayfish (Cherax quadricarinatus) <400> 1 atccaccagg aggcctggtc tcagaccggg ccgcgggggc gttgaccccc ggaactctct 60 ccaggtaaac aacattgtat tgtactgtat tgcattgtac tgtattgttc tgtattgtat 120 tgtattatat tgtactatat tgtgatgtat cgtactgtat tgtatcgtag tgtattgtat 180 aataaattaa taagttaata aataaataaa taaataaata aataaataaa tataattata 240 tggggtaggt tgtagagtaa gttagaagag agatgtttgt ttatgttgat ggttgctatg 300 gtgttgctag ggaagcgtca cactcactat tatggtcctc tgccaccatg acaaatattc 360 taccacattc tgacactggt taaagttagt aacttgccta cagttaatgg aaccatgtta 420 aaaaaaagaa aatgttgtat tttttttaag agttgtattg tgtacttgtg tacttcgtat 480 tttgttcata ggatcccaat ggaaataagt cactctgacc tttttgatag cccttgtggt 540 ttagcgc 547 <210> 2 <211> 700 <212> DNA <213> Redclaw crayfish (Cherax quadricarinatus) <400> 2 GAGAATAGTA ATAGTAATAG TAATAGTAAT AGTAATAGTA ATAGTAATAG TAATAGTATG TATGTA 56 AGTAATAGTA ATAGTAATAG TAATAGTAAT AGTAATAGTA ATAGTAATAG TAATAGTATG TATGTA 112 CAGAATAGTA ATAGTAATAG TAATAGTAAT AGTAATAGTA ATAGTAATAG TAATAGTATG TATGTA 168 GTATGTAATG TATGTAATGT ATGTAATAGTA TATAGTAATG TATGTAATAG TAATAGTAAT AGTAATAGTA 228 GCATAGTAAT AGTAATAGTA TATAGTAATG TATGTAATAG TAATAGTAAT AGTAATAGTA TATAGTAATG 288 TATAGTAATG TATGTAATAG TAATAGTAAT AGTAATAGTA TATAGTAATG TATGTAATAG TAATAGTAAT AGTAATAGTA 348 TATAGTAATG TATGTAATAG TAATAGTAAT AGTAATAGTA TATAGTAATG TATGTAATAG TAATAGTAAT AGTAATAGTA 408 TATAGTAATG TATGTAATAG TAATAGTAAT AGTAATAGTA TATAGTAATG TATGTAATAG TAATAGTAAT AGTAATAGTA 468 TATAGTAATG TATGTAATAG TAATAGTAAT AGTAATAGTA TATAGTAATG TATGTAATAG TAATAGTAAT AGTAATAGTA 528 TATAGTAATG TATGTAATAG TAATAGTAAT AGTAATAGTA TATAGTAATG TATGTAATAG TAATAGTAAT AGTAATAGTA 588 TATAGTAATG TATGTAATAG TAATAGTAAT AGTAATAGTA TATAGTAATG TATGTAATAG TAATAGTAAT AGTAATAGTA 648 TATAGTAATG TATGTAATAG TAATAGTAAT AGTAATAGTA TATAGTAATG TATGTAATAG TAATAGTAAT AGTAATAGTA 708 <210> 3 <211> 616 <212> DNA <213> Redclaw crayfish (Cherax quadricarinatus) <400> 3 cactaattac tctgctgatc ctgccattaa agtagtcaga gttgccatct ttgttccttt 60 atttaatgga tttctctcct tttcctcctc tatcatcttc cattatcctt tatcccttca 120 tacactatcc tctcctctca aaaagtaatc agtttccaca ctacatcgtc cactaatctc 180 tcatttcctt ctttttacca gtgtttctla gcttatcgcc gattcgtttg tttttgccac 240 agataatatc aggtggtcat ggaccgggcc gcgggggcgc tgacccctct gaaggtcctc 300 caggtgtact ccggttatca ctagttagat atttgtttct cttgttcttt cgtgagagac 360 acattacaga ctttgagagt cttcgctctc atgattagtc ttccttctct ccctctttcc 420 ttctttctgt ctgtctgtct gtctgtctgt ctgtctgtct gtctgtctct ctccctctct 480 cccattggcc tctaccatgc attttccatt actattttgt ttccatttca ctaatggttc 540 atcttttttt ttctctgtga tccttctgat gcttgctgcc agtcacagca tcctacattt 600 ttactttatc tctctc 616 <210> 4 <211> 776 <212> DNA <213> Redclaw crayfish (Cherax quadricarinatus) <400> 4 ctattgtaaa agttttttta attaaatgat agagccctta aaaccattat atgtaaagcg 60 gtttgtttta tttttcgaaa tttcgctttt gagtcgaaaa atggaaattt tatggtgatt 120 agaaatgatt aaagtgcaat gagcgttgcc agtataggga aatgtctacc tggagtatac 180 ctggagggtg ttctgggggt caacgccccc gcggtccgat ccatgaccaa acctcgctgt 240 gcatcagggc ctgatcaacc aggttcctgc taaacggaag cacgtaatcc aaagtgcgaa 300 ccgtctggtc aggtaaccac tttaaatatc tatctatttg tctcttaggc tcttttgggt 360 acttttattt atttatttat ttatttattt atttatttat ttatctgcat tcctcgccta 420 ccagttctat ctgtaggcgc gagatacaca cgttcaatgt aattccagcc tattttagcc 480 taaatccttt tctttctgcc cttcttcctc tggcaagatg gagaattaga cacaagcaac 540 atctgggtat ctttatttgt agacgtttcg ccagccagtg gctttatcaa cacaaattca 600 aggaaataat tggaatattg taaagctatg tagaaaggat tgaggtaatg agtccctcag 660 GAGAATTCGAGATCAAATTCACCATGATTTCGGCGTGATTATGATATTTGGCG ATGGCGTACGCGTTAATCGATGCCCGAGTTCAAATTCGTGTTTAA 120 CTCATGTTTTGTATATAGTTCTACTGTCCTGCCATTATGTC TTGAATCTGTATTGA 120 <210> 5 <211> 547 <212> DNA <213> Redclaw crayfish (Cherax quadricarinatus) <400> 5 CATAACGACCT GTTTTTTAAA TACTCATTTC ATGCTTAATT GTTATCTGTT TACAATAATA T 60 TTTACCACCG AATACTTCAT TGCTTAGTTG ATCTTAAGTT AATTCTTAAG CCTGCCCATA A 120 ATAAATAAAA TAAATAAATA AAATAAATCA ATAAATCACT GTCTTGCCAG 180 AGGCGTGCTT ACACACAGTT ATAAACCTGC AACATTAAC ACCCCTCCTT TAGAGTACAG 240 ACACTGCACA TCCCATCTCC AGGACTCAAG TCCGGCCTGC CGGTTCCTCT GAATCCCTTC 300 ATAAATGTTC CCTGCTCACA CTCCAACAGC ATTTCAAGTT CTAAAAGCCA TTTGTCTCC 360 ATTCAATCCT ATCAATATGC TATGCTTGCT GGAAATCCAA GCCCTTGTAC ACA 420 AAATTTCCCT TACCCTCTCC CTCCAGCTTT TCCTAGGCTG ACCCCTACCC TGCCTTGCCT 480 ccactacaga tttatacact ctcaatgtca tcctattttg tttcagcctc tctacatgtc 540 cgaacca 547 <210> 6 <211> 635 <212> DNA <213> Redclaw crayfish (Cherax quadricarinatus) <400> 6 tccaggcgac gttgtcacgt gtcacccaag aaagttaaca aagtgaaaca aatgtgtgac 60 cagtgaaacg aatatagcga atagggtaca ttatatatcg agaagaaacg gaggtggatc 120 tacgccagga catcacaatc agtgaagagg cggggccagg agctatgaat cgacccctgc 180 aatcacaaat aggtgagtac acatacactc tcactcactc actcactcac tcactcactc 240 actcactcac tcactcactc actctcctcc ttgtatcatt actgcctttt ctcggaaaat 300 ttgacgaatg atgaaattga attatggctt aggtagtaga attgaagaag gatgaagtga 360 aggcaaggtc agggagagtg tgatgtagag ggagcagagt ggcgtagagg gagcagagtg 420 gtgtagaggc agcagagtgg tgtagaggca gcagagtgat ttagaggaag cagagtgatt 480 tagagggagc agagtgattt agatggagca gtgtgattta gagggagcag agtgatttag 540 agggagcaga gtgattcaga gggagcagag tgattcagag ggagcagagt gattcagagg 600 gagcagagtg atgtagaggg agcagagtga tgtag 635 <210> 7 <211> 846 <212> DNA <213> Redclaw crayfish (Cherax quadricarinatus) <400> 7 ataagtttac aataatttaa taataaataa acacaatgaa atatattttt ttcgttaggt 60 tgaaaatgat ttttgagaaa ttattgcata cacaaatttt cgcttgcctt attcggcaag 120 aagagcattg ctatttaagc caaaaatctc aagtttgacc tattcggcac gacatgtata 180 tatgtagcaa aatttagcta aaaataaatt acgttaaatt gtaaggttag gtgagatgtc 240 taaattagag tagtccccat tttttttaca ccattgtcaa tgaaaaaatt acatccatct 300 gtaatttcaa tttttgcggg gaagttaatt ttttaaggta gttcggcctg ttctgcaagt 360 tcgacataat acatacatac atacatacat acatacatac atacatacat acatacatac 420 atacacaccc acataggaat caagaaatat tgcagtgatg ggtaggaaac aattcagtat 480 tgtgagcgag aatggaactt gatcctggtg attccaggat caccaggatc tgaactattt 540 actttgtgct gtgagctgaa gtgtcagcca tagtaattcc tatacactgg gtaagctaag 600 gtgtcagccg gtgtagctcc aatacactgg gtaaactgtt gtgtcagcta gtgtagtttg 660 aaatcagtgg ctgagctgat gtgtcagcta gggtagttcc cacactctag aaaagttgat 720 gtgtcagcca ggggagtcgt tacacacttg atgaattgat gtgtcagcca gggtaataac 780 tagaacagag tatactacag actctggcca tacaatagag cggaaaaata atgtaaggga 840 cctggg 846 <210> 8 <211> 392 <212> DNA <213> Redclaw crayfish (Cherax quadricarinatus) <400> 8 tcagacctgt cttgattatc ctaattgcat gagcctcctt taatgactga tgttcatcat 60 catcatcatt atttgataat gtgctccgat acatgcaact ttcttccatt tgaaaataaa 120 aatatttatg attctccgcc ctctcactct ctctatctcg agattatata tatatatatc 180 tcgagataga cagacagaca gacagacaga cagacagaca gacagacagg aacaaagaaa 240 gacaagcaca gagagagaga gagagagaga gagtgagagg gaggggattc ataaatattt 300 tttttcactc gctccagttt ctgaaaaaaa tatgctaatg acaggagact gggaaagttc 360 atgtgaaggg aaggaagttt gaatcttcat ta 392 <210> 9 <211> 604 <212> DNA <213> Redclaw crayfish (Cherax quadricarinatus) <400> 9 tttttttcat taggttcaga gtgatttttg cgaaattatt gcatacacaa attttcgctt 60 actcttattg ggaaagaaga gcgttgctat ttaagccaat attgcaagtt ttaccagtga 120 tgtaatgata tatcatacag acagataatt caagttaaaa attacttata tatgtacata 180 catacataca tacatacata catacataca tacatacata catacataca tacatacata 240 cacataaaat atgaatggga cgaaatactc aagtaaacga tcccaaacgc atttgaaaaa 300 ttggttaagt ctcctgaagc agtgttcatc tctttcgtaa tttgatggtt gagtggaagg 360 gtgctaacca ggcagtagtt cgacccccgg gggaggtgtg ggtggtgaaa ctaatgacac 420 agtccacttg taagatgcgg aagaaaaatt gtagaagtta atggaagatt ttggaagaga 480 gtgtgagata atgaaactga acgtgaacac agaaaagagc aaggggagag aatgtgtatt 540 tagttggttt tttttttttt gtggttttat tttcattttt catttgtgtg tacctactga 600 tttg 604 <210> 10 <211> 847 <212> DNA <213> Redclaw crayfish (Cherax quadricarinatus) <400> 10 acctaacatc aaatcataac accagcatca ataatgatct tttagctcca ctcacaacat 60 ttctgtttat ataatttatg ttcccactgg taccttgaac tctctctcac cccctctctc 120 actctctctc atcccctctc attgtttctc acagtaacca ctgccagaac atcggtaatt 180 gacatgttta cacatcttga tttgcagaag tttccgtctt tgtgattctc actaacaatt 240 cccattttgg caaatctcta aatatttatc caggggtgga aatttaaggt tcatttgtct 300 ttcattataa attataacaa caatggaaat aagtcactct gtctgacttt tttgggttat 360 cccaggttct ctacacatat gctgctatgt atgataattc tatgtaacta tatttgtgta 420 tacctgaata aacttactta cttacttact tacttactta cttacttact tatcatataa 480 caggtgatga acggggttat atcatgttat attacgattt gacatactga gtagctaaag 540 atatatcgat atgacgacag gaaaacggaa gacaggtgct gtagagcacg ctcgtgttgg 600 caagaaataa ttcgctctgt gtagagcttt attaggactt gataaagctc tatagagctt 660 cactaggatt tgataaagct ctacagagct ttactatgac ttgataaagc gctacagact 720 cacagagctt cactaggact tgataagact ctatagagct tcactaggac ttggtaaagc 780 tctatagagc ttcactggaa ctcgataaag ctccacgagc ttcactagaa cttgataaag 840 ctctacg 847 <210> 11 <211> 604 <212> DNA <213> Redclaw crayfish (Cherax quadricarinatus) <400> 11 tttctactct ggctgggaat cgaacccagg ccctcaccgt gtgaagcgag agcgttaacc 60 accaggccac ctaacataat acaccattct agtgattaca cagcaacaca gtaatgacca 120 cataacctgt ctttgtaata ctcatttgta ctaaattgtt atctgtttac aataattttt 180 gtaccactga atatatcatt gcttagttaa tcttaagtta atgttaatcc tgcccataat 240 gccctgcata caagtggctt tggcatgttg cactttaaaa attgtattcc ttgtacttct 300 ctgtatcatg ttcaaataaa taaataaata aataaataaa taaataaata aataaataaa 360 ggaagtgtca gtagcccctc aacagtcata ccaacacatt aaggcaaggc ggtctgatta 420 ttactgtggt gtcatgtacg gtgtcggatg tatatatctc gtattaacat tacataccac 480 ggattttcgt gctcgttagt acataaacga atgtctttca aacacttctt atatcgtatt 540 aaataacatt gaactgtaca ggatgaacat ttacatcagc agagttggca acactgtctc 600 agcc 604 <210> 12 <211> 560 <2​​​​​​​​​​​​​​​GAGAATAGTA TATTTGTTCC ATTTGTTTCT GATGTTGTTG ATGTTGTTGT GATGTTGTT 60 GAGAATAGTA TATTTGTTCC ATTTGTTTCT GATGTTGTTG ATGTTGTTGT GATGTTGTT 60 GAGAATAGTA TATTTGTTCC ATTTGTTTCT GATGTTGTTG ATGTTGTTGT GATGTTGTT 60 GAGAATAGTA TATTTGTTCC ATTTGTTTCT GATGTTGTTG ATGTTGTTGT GATGTTGTT 60 GAGAATAGTA TATTTGTTCC ATTTGTTTCT GATGTTGTTG ATGTTGTTGT GATGTTGTT 60 <210> 13 <211> 459 <212> DNA <213> Redclaw crayfish (Cherax quadricarinatus) <400> 13 GAGAATAGTA TATTTGTTCC ATTTGTTTCT GATGTTGTTG ATGTTGTTGT GATGTTGTT 60 GAGAATAGTA TATTTGTTCC ATTTGTTTCT GATGTTGTTG ATGTTGTTGT GATGTTGTT 60 GAGAATAGTA TATTTGTTCC ATTTGTTTCT GATGTTGTTG ATGTTGTTGT GATGTTGTT 60 GAGAATAGTA TATTTGTTCC ATTTGTTTCT GATGTTGTTG ATGTTGTTGT GATGTTGTT 60 GAGAATAGTA TATTTGTTCC ATTTGTTTCT GATGTTGTTG ATGTTGTTGT GATGTTGTT 60 GAGAATAGTA TATTTGTTCC ATTTGTTTCT GATGTTGTTG ATGTTGTTGT GATGTTGTT 60 taagctttat gagatcaaat aatttacttt ttgaatctct gtttggcctc aaagtcgcag 420 ccgctacaac atcgtggtga tgcgtcacta gatatgtgt 459 <210> 14 <211> 547 <212> DNA <213> Redclaw crayfish (Cherax quadricarinatus) <400> 14 agtactggtc tcagtactga cccttgtgga acactgtgac ctcataccca cacctcttct 60 ctggtagttt ctcgttaaat attccatgat atactgtagt gccatccctg ttattcctgc 120 cacctcctct agcttttgat tcagtctctt ttgaggcgct gtattcatca tgcatgatag 180 actgatggaa tttctggtgt cagtctctct agtatcaagt caggttcatt tgaagatctt 240 ttcgcattat caaagtgctt gtgcacactc ggagttgtaa tttacttaag taagtaagta 300 agtaagtaag taagtaagta agtaagtaag taagtaagta attttattca ggtatatcag 360 ttttatcatg ctattgaagg tcaatgtgaa agagtatcca caagtgaagc actttgactt 420 tactgtctac gatactacca tacctgtctc tggcttttaa aacggtttag tttaatatct 480 tctaatgcac cccataccca tcctgtgggc ggtagtcacc ccatacccat cctgtgggcg 540 gtagtca 547 <210> 15 <211> 752 <212> DNA <213> Redclaw crayfish (Cherax quadricarinatus) <400> 15 gttaagggaa attggcctga cgatcctgaa ggacaggaca gttaggggag acatgataaa 60 gacatacaaa atactgccaa gaattgacaa ggtggacaga gacaggatgt tccagagatg 120 gaacacggaa acaaggggtc tcaattggaa gatgatgagc cagatgagtc aaatggatgt 180 taggaagtat atctttagcc acagagttgt caggaagtgg aatagtctga caagtgacat 240 aatggaggca ggaaccctac atagttttaa gacgaggtat gatgaaggtc atagaacaag 300 gagagagagg cccaagtagc gatcagtgaa cagccggggc caggggctat gaatcgaccc 360 ctgcagccac aaataggtga gtacatacat acatgaagtt gaataatcta gagtgatatg 420 gtggaggcaa gatccataca aagctttaag aagaggtaca ataaagttca tggagcaggg 480 agagagtgaa cctagtagcg accagcgaag agacggggcc aggagctgtg attcgactcc 540 tgaaaattac aaacaggtga gtacatacat acatacatac acatacatac acatatacat 600 acatacatac atacatacat acatacatac atacatatac atacataccg ttttgcgtgc 660 cgatctgcct tcctctctac cttcatacct ggtcaccttc ccatgtgcct gccagaaaac 720 ttgcctgcat tcatgaatac ctacctgctt gc 752 <210> 16 <211> 680 <212> DNA <213> Redclaw crayfish (Cherax quadricarinatus) <400> 16 taaacaggaa taaacagcac accgtcacac aagaactcaa gaacaacaaa acgaccctaa 60 ctggagacct tcagcagatc ctcccgaaaa ggctagactc ccaatttact gaaactgagt 120 tcagcaattc cagtctttaa acgttgatag tgtgtagatg ccaaaacaat gtaggtcgag 180 agatgaactc gggaccagag atatagagag tgtctccacg acacataacc tctgaaaaag 240 ttaaatatca ctaagtctaa ataatgcttt gttaagcaag taacacaaag ttacgagtgt 300 tttgtcccag atcaccattg gagacattct cttccaaaac agactctaaa caacatgagc 360 tgaggagaac agcattctcc cagacaaagc agagatgaag gtgtttccac cacgagagag 420 agagagacag acagacagac agacagacag acagacagac agacacagac acagacagag 480 aaagacagat agagacaga agaagcaagg cctgcagata caacgtggag ttcttgtacc 540 agctggagcc tggaatgaat tacaaaccaa gcaagcaggt agttagcagc gttggcagcc ttcagaactc tccaccaacg agcacaatga gagatgtagt tgctacctcc tacttactta cattagtaa 680 sq. ft <210> 17 <211> 616 <212> DNA <213> Cherax quadricarinatus (Cherax quadricarinatus) <400> 17 atttcctctc tcacagtttt gttgtttttc tcttcataaa acctgttatt ctctgtttat cttatttatt ttccccttcc tttaattcct ccctcccatc ttctctctca cttttacaca gggtttgaca aggttaggtt aatgatccct agctttattg acaagctatc tatctatcta tctatctatc tatctatc tctatctatc tattgtc tcaatttatc tctctccctc tctccctttc tccatcacac tttcctcctt cccctctcct tctctacttc cctctttccg 300 ccctcctttc cttcccttca tttattcata tgcttttttt tttaacaggg tttgacaagg 360 ttaggttag gatctctagc tatttacagg taaaggattt ctaactttat tgagaagcta agagctgtta cctacatcag ctcatttgaa agcatttgaa agtaccattt gtggcaccag 480 ggcgtcttag caaaacttca agctctggga attgcaggct ctacgctatg acgcctcagt 540 gactaccttc atggtagatc tctaagggta gttttcaatg gaacagaatc agaaagacat 600 cctattgggg caagtg 616 <210> 18 <211> 760 <212> DNA <213> Redclaw crayfish (Cherax quadricarinatus) <400> 18 gagttgtact cagagagttg tactcacata gttattgttc acagagttgt gctcactgag 60 atgtacccac gtacctgcat ctcagttcct ggccccatct cttaacttgt aactgacgga 120 tgttcatggc tcccaacctc ttgggcttga tcatacttac tcgtcaaact gtgcaggaaa 180 tttgcttcac gctactttat tcaggtcacc cagcttcttt tttctcgttg tatatacctt 240 tcctaaagaa gtatttcatg aagtccctgt actttttctt cggtgtttat ctttctctgc 300 ttctttcttc tcgtccttcc catgtcactt tctccttaac taccttgtta actcattgaa 360 gtattttgca tggtgtggtc atatctcctc ctgaattgat tctcatacat ctcatatgtc 420 tctgtctgtc tgtctgtctg tctgtctgtc tgtctgtctg tctctcgctc tctctctctc 480 tctctcttca taataacgcc ataaattagg cacaatgtat aaaatatacg cctatatttt 540 tatcagatac ctggaaatac atccacagta tttctgccgc ccaggtgaag ctgcttcacc 600 acataaaaat tccatacaca aagggttgga gaattttttt ttgcactgga gtgatcaaaa 660 cagaggatcc gatacccttt tagggtaacg gactgaggca gagtctcagt gttagagtct 720 ctctctctct ctctctctct ctctctctct ctctctctct 760 <210> 19 <211> 401 <212> DNA <213> Redclaw crayfish (Cherax quadricarinatus) <400> 19 gatcctctca gaatagatca atcccaagca ggtgttgggg aaggcgaggc acccacggtg 60 atcactagac ctaagctgta aaaactgacc cctctagtgg cagaatactt taacgtaaaa 120 tgcttcacag aatgtagagg taaatagata aatagataga cagatagata gatagataga 180 tagatagata gatacataca tacatacata catacataca tacatacata catacataca 240 tagagagaga gagagaaaga gagaaataca tgagataatg taactagcac atgagataaa 300 tgtaactacc ataggaggga aatacaaaga gtgaaaaaga gacgcctcag taaactcaga 360 tgtaactacc ataggaggga aatacaaaga gtgaaaaaga gacgcctcag taaactcaga 360 gatctctagc acaagactcg tctccctcac aactgatctc t 401 gatctctagc acaagactcg tctccctcac aactgatctc t 401 <210> 20<210> 20 <211> 1032<211> ~1032 <212> DNA<212> DNA <213> 红螯螯虾(Cherax quadricarinatus)<213> Redclaw crayfish (Cherax quadricarinatus) <400> 20<400> 20 tgtaggcgaa acgtttcata ataaagatac ctaactgttg catatgtgtc ttacctaaca 60 tgtaggcgaa acgtttcata ataaagatac ctaactgttg catatgtgtc ttacctaaca 60 aggttaggta agtttcctaa ggttttcttg gtaaaaaatc attaattttt atattaacat 120 aggttaggta agtttcctaa ggttttcttg gtaaaaaatc attaattttt atattaacat 120 attcagacat acctaattca cctaactgtg attgcaggcg tcatagctcc tggccctgcc 180 attcagacat acctaattca cctaactgtg attgcaggcg tcatagctcc tggccctgcc 180 ttttcattgg tcgctactat gtccactctt tccctgctct atgagcttta tcccctcaag 240 ttttcattgg tcgctactat gtccactctt tccctgctct atgagcttta tcccctcaag 240 gaaggttcct tgatgttggt gaggggctct tgatataggg aattggatct gtgctccagt 300 gaaggttcct tgatgttggt gaggggctct tgatataggg aattggatct gtgctccagt 300 tccccgaatt aagcctgaat gccttccacc ccccccaggc gctgtataat cctccgggtt 360 tccccgaatt aagcctgaat gccttccacc ccccccaggc gctgtataat cctccgggtt 360 tagcgcttcc cccttgatta taataataat aatctatgag ctttatcata gctcttctta 420 tagcgcttcc cccttgatta taataataat aatctatgag ctttatcata gctcttctta 420 aatctatgtg tggatcctgc ctccattaca tcactctcca gactattcca tttcttgaca 480 aatctatgtg tggatcctgc ctccattaca tcactctcca gactattcca tttcttgaca 480 GAGAATAGTA ATAGTAATAG TAATAGTAAT AGTAATAGTA ATAGTAATAG TAATAGTATG TATGTAATAA TATATATAAT ATATATAATAT ATATATATAT ATAT 55 CCTCTGTGAC TGAAGAAATA CTTCCTAATA TCCCTTTGAC TCATCTGCGT CTTCACTTTC 540 CCTCTGTGAC TGAAGAAATA CTTCCTAATA TCCCTTTGAC TCATCTGCGT CTTCACTTTC 540 CCTCTGTGAC TGAAGAAATA CTTCCTAATA TCCCTTTGAC TCATCTGCGT CTTCACTTTC 540 CCTCTGTGAC TGAAGAAATA CTTCCTAATA TCCCTTTGAC TCATCTGCGT CTTCACTTTC 540 CCTCTGTGAC TGAAGAAATA CTTCCTAATA TCCCTTTGAC TCATCTGCGT CTTCACTTTC 540 CCTCTGTGAC TGAAGAAATA CTTCCTAATA TCCCTTTGAC TCATCTGCGT CTTCACTTTC 540 CCTCTGTGAC TGAAGAAATA CTTCCTAATA TCCCTTTGAC TCATCTGCGT CTTCACTTTC 540 CCTCTGTGAC TGAAGAAATA CTTCCTAATA TCCCTTTGAC TCATCTGCGT CTTCACTTTC 540 CCTCTGTGAC TGAAGAAATA CTTCCTAATA TCCCTTTGAC TCATCTGCGT CTTCACTTTC 540

Claims

1. A method for parentage identification of Procambarus clarkia based on four-base microsatellite fluorescence multiplex PCR, characterized in that, Comprising the following steps; Step (1) DNA extraction of the sample of the Astacus leptodactylus: Take the appendages of Astacus leptodactylus individuals, and extract the genomic DNA of the sample to be tested by the phenol-chloroform method; Step (2) Screening of microsatellite primers of Astacus leptodactylus: According to the genomic sequence of Astacus leptodactylus preserved in the laboratory, 20 four-base microsatellite sequences are screened by MISA software: Cqaa-48, the nucleotide sequence of which is shown as sequence 1; Cqaa-191, the nucleotide sequence of which is shown as sequence 2; Cqaa-218, the nucleotide sequence of which is shown as sequence 3; Cqaa-197, the nucleotide sequence of which is shown as sequence 4; Cqaa-24, the nucleotide sequence of which is shown as sequence 5; Cqaa-181, the nucleotide sequence of which is shown as sequence 6; Cqaa-121, the nucleotide sequence of which is shown as sequence 7; Cqaa-227, the nucleotide sequence of which is shown as sequence 8; Cqaa-22, the nucleotide sequence of which is shown as sequence 9; Cqaa-163, the nucleotide sequence of which is shown as sequence 10; Cqaa-106, the nucleotide sequence of which is shown as sequence 11; Cqaa-185, the nucleotide sequence of which is shown as sequence 12; Cqaa-115, the nucleotide sequence of which is shown as sequence 13; Cqaa-182, the nucleotide sequence of which is shown as sequence 14; Cqaa-240, the nucleotide sequence of which is shown as sequence 15; Cqaa-228, the nucleotide sequence of which is shown as sequence 16; Cqaa-132, the nucleotide sequence of which is shown as sequence 17; Cqaa-211, the nucleotide sequence of which is shown as sequence 18; Cqaa-43, the nucleotide sequence of which is shown as sequence 19; Cqaa-167, the nucleotide sequence of which is shown as sequence 20; Primers for microsatellite loci are designed for the 20 four-base microsatellite sequences by using Primer Premier 6.0 software, and 20 pairs of primers are screened; Step (3) Construction of multiplex PCR amplification system: The 20 pairs of primers designed for the four-base microsatellite sequences in step (2) are grouped according to the intervals of 120-200bp, 220-260bp, 280-340bp, 360-420bp and 440bp-500bp, then 3-5 pairs of primers are selected in different groups, and the amplification combination is selected, the 5' end of the upstream primer of each locus is modified by fluorescence, and a plurality of multiplex PCR amplification systems are obtained; Step (4) Parentage identification: The genomic DNA of the sample to be tested described in step (1) is amplified based on the plurality of multiplex PCR amplification systems constructed in step (3) to obtain multiplex PCR products, then the multiplex PCR products are typed on a sequencer, the individual allele size bp is read, a digital genotype matrix is formed, the genotype of the parent and offspring is read, and the parent-offspring relationship is judged according to Mendel's law; Wherein, In step (3), the primer combination of the multiple PCR amplification system consists of a first group, a second group, a third group, a fourth group and a fifth group of primers; the first group of primers consists of primers designed for four-base microsatellite loci Cqaa-48, Cqaa-191, Cqaa-218, Cqaa-197 and Cqaa-24, the second group of primers consists of primers designed for four-base microsatellite loci Cqaa-181, Cqaa-121, Cqaa-227 and Cqaa-22, the third group of primers consists of primers designed for four-base microsatellite loci Cqaa-163, Cqaa-106, Cqaa-185 and Cqaa-115, the fourth group of primers consists of primers designed for four-base microsatellite loci Cqaa-182, Cqaa-240, Cqaa-228 and Cqaa-132, and the fifth group of primers consists of primers designed for four-base microsatellite loci Cqaa-211, Cqaa-43 and Cqaa-167; In step (4), the amplification conditions of the test sample genomic DNA are as follows: total denaturation at 94℃ for 5 minutes, denaturation at 94℃ for 30 seconds, annealing at 59℃ for 90 seconds, extension at 72℃ for 30 seconds, a total of 35 cycles, and finally extension at 60℃ for 30 minutes.

2. The method for parentage identification of Procambarus clarkia based on four-base microsatellite fluorescence multiplex PCR according to claim 1, characterized in that, Step (1) further comprises diluting the concentration of the test sample genomic DNA to 25 ng / μL.

3. The method according to claim 2, wherein the method is characterized in that, In step (4), the typing is performed by capillary electrophoresis detection typing using a sequencer with a model number of ABI 3730 XL, and the results are analyzed by using GeneMapper 4.0 to obtain the genotype data of each locus of the parent and the offspring.

4. The method according to claim 3, wherein the method is characterized in that, The multiple PCR amplification system is as follows: 2×Multiplex PCR Mix 10 μL, working concentration of the upper and lower primers is 10 mmol / L, 50 ng of genomic DNA, and sterilized ultrapure water is added to the system to a total volume of 20 μL.

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