CN112831570B - Black iso-striated lipocarp microsatellite marker locus and general application thereof in fishes of the genus and the near-source genus - Google Patents

Black iso-striated lipocarp microsatellite marker locus and general application thereof in fishes of the genus and the near-source genus Download PDF

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CN112831570B
CN112831570B CN202110173358.4A CN202110173358A CN112831570B CN 112831570 B CN112831570 B CN 112831570B CN 202110173358 A CN202110173358 A CN 202110173358A CN 112831570 B CN112831570 B CN 112831570B
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lipocarp
black
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cyprinus
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CN112831570A (en
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徐润枫
刘宏毅
许唯
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Nanjing Forestry University
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Abstract

The invention discloses a black iso-striated lipocarp microsatellite marker locus and application thereof in the universality of fishes belonging to the genus and the near source, belonging to the field of biotechnology detection. The invention provides black spot fatty carp 5 microsatellite molecular marker sites, a polymorphism primer thereof and universality thereof in part of species in the genus Cyprinus and the genus Cyprinus of the family Cyprinaceae, as shown in a nucleotide sequence table, an amplification result of the obtained primer has high polymorphism and stability, and technical data are provided for identification of genetic diversity and genetic relationship among populations of five species of the genus Cyprinus of the family Cyprinidae , including the genus Cyprinus, and the genus Cyprinus of the family Cyprinus, and make up for the defects of the prior art.

Description

Black iso-striated lipocarp microsatellite marker locus and general application thereof in fishes of the genus and the near-source genus
Technical Field
The invention belongs to the field of biotechnology detection, and particularly relates to a black iso-striated lipocarp microsatellite marker locus and a universal application thereof in lipocarp and linear lipocarp partial species.
Background
Lipofencus (Hyphesbycon) and Trichoprinus lineatus (Moenkhausia) belong to Cypriniformes (Cypriniformes), Cypriniformes (Characoidei) and Cyprinidae (Characidae) in classification, most of the Lipofencus lineatus are tropical aquarium fishes which are originally produced in Asian river in south America, African Congo river basin and the like and are easy to raise, wherein the smaller species are called 'Channa argus', and are introduced into China at the middle and later period of the last century and called popular aquarium fishes. Wherein Cyprinus carpioides are about 57, and the Linear Cyprinus carpioides are about 30. Well known ornamental fish species known as fatty carp, fatty carp, kosta's linear fatty carp and the like are rare.
Black Isodon lipocarp (Hyphyostauron herbertaxelrodi) belongs to Cypriniformes (Cypriniformes), lipocarp subgenus (Characoidei), lipocarp family (Characidae), lipocarp (Hyphyostauron) in classification. Distributed in the water area of the original forest land of the coastal tropical zone downstream of the Brazilian amazon river in south America. The black and different pattern lipocarp has smaller body, bright color, mild and smooth temperament and easy breeding, and is deeply favored by ornamental fish industry all over the world. Belongs to oviparous fishes on grass, the discharged eggs have viscosity, and fertilized eggs need to be attached to attachments such as aquatic weeds and the like for hatching.
Nowadays, commercial propagation of part of tropical ornamental fishes is started in many countries, so as to obtain a larger number of individuals and reduce the collection of wild resources, and development of microsatellite primers is particularly important in order to quickly and effectively identify families and sources of lipocarp and lypis species represented by the black allogama lipocarp.
Microsatellites (SSRs) are widely distributed in the genome of eukaryotes, conform to a Mendelian genetic pattern, and have the advantages of co-dominant expression, rich polymorphism information, strong interspecies universality, easiness in detection and the like. Therefore, the method is used as an advanced molecular marker means and widely applied to the production fields of fish population genetic diversity investigation, parent selection, genetic relationship identification, molecular marker assisted breeding and the like. Nowadays, commercial propagation of part of tropical ornamental fishes is started in many countries, so as to obtain a larger number of individuals and reduce the collection of wild resources, and development of microsatellite primers is particularly important in order to quickly and effectively identify families and sources of lipocarp and lypis species represented by the black allogama lipocarp. At present, the development and application of microsatellite molecular markers of the above species are not common.
At present, a microsatellite fluorescent marking full-automatic genotyping technology is generally adopted, and microsatellite allele fragments can be accurately calculated by marking microsatellite DNA primers by fluorescent dyes with different colors. The method has the advantages of high yield, low cost, rapidness, simplicity and convenience and the like, and shows wide application prospect. The experiment lays a foundation for genetic detection of black allostriation lipocarp by screening highly polymorphic microsatellite loci of black allostriation lipocarp, selects part of universal microsatellite loci from the black allostriation lipocarp, and lays a foundation for genetic detection of five species of rose lipocarp, Eswimming lipocarp, Soxhlet lipocarp, big fin lipocarp and Kesta's linear lipocarp.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to provide fatty carp 5 microsatellite molecular marker loci and polymorphic primers thereof, provide data for identifying genetic diversity and genetic relationship among populations of fatty carp, and make up for the defects in the prior art. The invention aims to solve another technical problem of providing the universal application of the 5 microsatellite molecular marker loci in rose fatty carps, Eichhoretic fatty carps, Soxhlet fatty carps, big fin fatty carps and costas' linear fatty carps.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
the invention utilizes a DNA small fragment library sequencing technology to screen 5 microsatellite loci from the genome DNA of the black allogamy lipocarp, and the marker numbers are Hy-01, Hy-02, Hy-03, Hy-04 and Hy-05.
The black alloglyph lipocarp microsatellite molecular markers Hy-01, Hy-02, Hy-03, Hy-04, Hy-05 or the combination thereof are applied to population genetic diversity detection, individual identification or molecular assisted breeding of the black alloglyph lipocarp.
The black allogamy lipocarp microsatellite molecular markers Hy-01, Hy-02, Hy-04, Hy-05 or the combination thereof are applied to population genetic diversity detection, individual identification or molecular assisted breeding of the large fin lipocarp.
The black alloglyph fatty carp microsatellite molecular markers Hy-02, Hy-04, Hy-05 or the combination thereof are applied to population genetic diversity detection, individual identification or molecular assisted breeding of rose fatty carps.
The black alloglyph lipocarp microsatellite molecular markers Hy-02, Hy-03, Hy-04, Hy-05 or the combination thereof are applied to population genetic diversity detection, individual identification or molecular assisted breeding of the Ephoretic lipocarp.
The black spot fatty carp microsatellite molecular markers Hy-01, Hy-02, Hy-03, Hy-04, Hy-05 or the combination thereof are applied to population genetic diversity detection, individual identification or molecular assisted breeding of Soxhlet fatty carp.
The black alloglyph fatty carp microsatellite molecular markers Hy-02, Hy-04, Hy-05 or the combination thereof are applied to population genetic diversity detection, individual identification or molecular assisted breeding of kosta's fatty carp.
The microsatellite locus primers are forward (F) primers and reverse (R) primers which are designed from flanking sequences at two ends of a Hy01-05 microsatellite, and the sequences of the microsatellite primers are SEQ ID No.06-15 respectively.
The core repetitive sequences, primer sequences, fluorescent markers and optimal annealing temperature information of 5 universal microsatellite loci of the black allogamy lipocarp are shown in table 1:
TABLE 1 core repeat sequence, primer sequence, fluorescent marker and optimal annealing temperature of microsatellite locus of Cyprinus carpiovar nigricans
The microsatellite polymorphism primer is used for detecting the genetic diversity of black spot lipocarp and partial congeneric and closely-related species thereof, and comprises the following steps:
1) extraction of genomic DNA: adopting a DNA extraction kit of Nanjing NuoZan biotechnology limited;
2) micro-satellite PCR amplification: amplifying the genome DNA of the black alloglyph lipocarp by adopting a microsatellite primer fluorescently labeled by FAM, HEX and TAMRA to obtain an amplification product;
3) genetic diversity analysis: genotypes were determined based on the molecular weight of each individual microsatellite amplification product and genetic diversity parameters were calculated using Cervus 3.0.7.
4) Cross-species microsatellite PCR amplification: and amplifying genome DNA of rose lipocarp, Eicheng lipocarp, Soxhlet lipocarp, big fin lipocarp and costas' linear lipocarp by adopting FAM, HEX and TAMRA fluorescence labeled microsatellite primers to obtain partial amplification products of the genomic DNA.
5) Cross-species genetic diversity analysis: genotyping the size of the microsatellite amplification products of the five species individual in step 4), and calculating the genetic diversity parameter by using Cervus 3.0.7.
Compared with the prior art, the invention has the beneficial effects that:
the method utilizes a DNA small fragment library sequencing technology to obtain the DNA small fragment of the black allogynic lipocarp, utilizes the Geneius Prime software to splice according to a template sequence with high similarity to obtain a DNA large fragment, and finds a proper amount of potential microsatellite loci by searching simple repetitive sequences. Compared with other high-throughput sequencing technologies, the DNA small fragment library sequencing technology is low in price, the obtained small fragments can be used for searching a proper amount of microsatellite loci, meanwhile, large DNA fragments such as mitochondrial rings and the like of the species can be spliced, the application range is wide, data can be recycled, and the experiment cost is reduced. 5 microsatellite loci are screened from the potential microsatellite loci, specific primers are designed according to flanking sequences of the loci at two ends of the microsatellite loci, and amplification results of the obtained primers have polymorphism and stability, so that the primers can be applied to the fields of genetic diversity detection of lipocarp populations of Heiyi striatus, individual identification and molecular assisted breeding, and a solid foundation is laid for the microsatellite polymorphism detection of lipocarp of Heiyi striatus.
Meanwhile, after PCR amplification is carried out on 5 populations of roses lipocarp (Hyphobryonycon rosaceus), Epimeris lipocarp (Hyphobryonycon amandae), Soxhlet lipocarp (Hyphobryon socolifi), large fins lipocarp (Hyphobryon megalopterus) and Korstaki linears (Moenkhausia costae) by specific primers designed according to 5 microsatellite loci screened from genomic DNA of the Heihei lipocarp, the result shows that 3 pairs of primers, namely Hy-02, Hy-04 and Hy-05 are suitable for the above five populations, and 1 pair of primers, namely Hy-01 is suitable for the large fins lipocarp and the Soxhlet lipocarp, and 1 pair of primers, namely Hy- and Hy- are suitable for the Soxhlet.
Detailed Description
The invention is further described with reference to specific examples.
Example 1:
(1) extracting DNA of black allostriata lipocarp: 26 black allostriation lipocarp samples are collected from the Nanjing Temple flower and bird market and the Tianjin Baojidao flower and bird market, stored in absolute ethyl alcohol, and subjected to DNA extraction by adopting a DNA extraction kit of Nanjing Nuojingzan biotechnology limited company, and the specific steps are as follows:
the tail fin is cut into pieces by using clean scissors and transferred into a centrifuge tube, 230 mu L of Buffer GA and 20 mu L of PK working solution are added, and vortex mixing is carried out. The tissue is completely enzymolyzed by water bath at 55 deg.C. Add 250. mu.L Buffer GB to the digest, vortex at maximum speed and mix well for 20sec, water bath at 70 ℃ for 10 min. Then 250. mu.L of absolute ethanol was added to the digestion solution and centrifuged manually for 15-20 sec. The gDNA Columns were placed in a Collection tube of 2 mL. Transferring the mixed liquid (including precipitate) obtained in the last step to an adsorption column. Centrifuge at 12,000 Xg for 1 min. The filtrate was discarded and the adsorption column was placed in the collection tube. Add 500. mu.L of Washing Buffer A to the adsorption tube. Centrifuge at 12,000 Xg for 1 min. The filtrate was discarded and the adsorption column was placed in the collection tube. 650. mu.L of Washing Buffer B was added to the adsorption column. Centrifuge at 12,000 Xg for 1 min. Repeating the steps once, discarding the filtrate, and placing the adsorption column in a collection tube. Centrifuge in empty tube at 12,000 Xg for 2 min. The column was placed in a new 1.5 μ L centrifuge tube. Adding 30-100 μ L of waste heat to 70 deg.C Elution Buffer to the center of the adsorption column membrane, and standing at room temperature for 3 min. Centrifuge at 12,000 Xg for 1 min. The adsorption column was discarded and the DNA was stored at 2-8 ℃.
(2) Screening of polymorphism microsatellite locus primers of the cyprinus carpio heiloides with black striae :
according to the whole genome sequence of Mexico Li fat carp (Astyanax mexicanus), the Geneius Prime software is utilized to splice the small DNA segments of the black iso-striated fat carp obtained by the DNA small segment library sequencing technology into large DNA segments, a simple repeated sequence is searched through a Gramene website to obtain a proper amount of microsatellite loci, and the available microsatellite loci are screened after one-generation amplification. The microsatellite screening is mainly based on the following steps: whether the microsatellite locus can obtain an amplification result; whether the amplification result is the original microsatellite sequence. Finally, 5 microsatellite loci are screened out, and specific primers are designed on flanking sequences at two ends of the microsatellite loci to obtain 5 pairs of microsatellite primers.
(3) Black striae lipocarp fluorescence labeling microsatellite PCR amplification:
and (3) marking 5 pairs of microsatellite primers and forward primers obtained by screening in the step (2) by using different fluorescent substances, wherein the fluorescent label of Hy-01 is HEX, the fluorescent labels of Hy-02, Hy-04 and Hy-05 are FAM, the fluorescent label of Hy-03 is TAMRA, and carrying out PCR amplification on the DNA sample of the black allochroic lipocarp obtained in the step (1) by using the fluorescent primers. The PCR reaction system is as follows: 7.5 μ L2 × Premix TaqTM1. mu.L of template genomic DNA, 0.5. mu.L of each of the upstream and downstream primers, and 5.5. mu.L of ddH2O, the PCR amplification program is as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 55-60 ℃ for 30s, and extension at 72 ℃ for 30s, and performing 30 cycles; most preferablyAnd then further extension at 72 ℃ for 5 min. The Allele number (Na), the expected heterozygosity (He) and the Polymorphic Information Content (PIC) of each microsatellite marker were calculated using the allee frequency analysis in the software Cervus v.3.0.7, and the results showed that the number of alleles per microsatellite locus was 3, the expected heterozygosity (He) ranged from 0.242 to 0.541 and the Polymorphic Information Content (PIC) ranged from 0.217 to 0.438, with the detailed results shown in table 2.
(4) Analysis of universality of pleochromorphic lipocarp polymorphic microsatellite primers in other species
Extracting genome DNA of rose lipocarp, Eichhoro lipocarp, Soxhlet lipocarp, big fin lipocarp and Costis lineate lipocarp according to the method of the step (1). And (3) carrying out PCR amplification on the genomic DNA of the five species by using the 5 pairs of black striae lipocarp microsatellite primers obtained by screening in the step (2) through the method in the step (3). Allele factors (Na), desired heterozygosity (He) and Polymorphic Information Content (PIC) of each microsatellite marker were calculated using the allee frequency analysis in the software Cervus v.3.0.7 and the results showed:
A. the total number of microsatellite sites which can be amplified in the genomic DNA of Cyprinus carpiod (Hyphyostrebrycon megalopterus) of large fins is 4, namely Hy-01, Hy-02, Hy-04, Hy-05, the number of alleles per microsatellite site varies from 2 to 7, the desired heterozygosity (He) ranges from 0.216 to 0.837, the Polymorphism Information Content (PIC) ranges from 0.194 to 0.762, and the detailed results are shown in Table 3.
B. The total of 3 microsatellite loci which can be amplified in genomic DNA of rose lipocarp (Hyphyostauromyconosaceus) was 3, namely Hy-02, Hy-04, Hy-05, the number of alleles per microsatellite locus varied from 4 to 5, the desired heterozygosity (He) ranged from 0.350 to 0.725, and the Polymorphism Information Content (PIC) ranged from 0.313 to 0.641, and the detailed results are shown in Table 4.
C. The total of 4 microsatellite sites which can be amplified in the genomic DNA of Epimedium lipocarp (Hyphobrycon amandae) were Hy-02, Hy-03, Hy-04, Hy-05, the number of alleles per microsatellite site varied from 2 to 6, the desired heterozygosity (He) ranged from 0.523 to 0.837, and the Polymorphic Information Content (PIC) ranged from 0.372 to 0.759, and the detailed results are shown in Table 5.
D. The total of 5 microsatellite sites which can be amplified in the genomic DNA of lipocarp (Hyphessbergyon Socoifi) are Hy-01, Hy-02, Hy-03, Hy-04, Hy-05, the number of alleles per microsatellite site varies from 2 to 4, the desired heterozygosity (He) ranges from 0.209 to 0.703, the Polymorphism Information Content (PIC) ranges from 0.178 to 0.580, and the detailed results are shown in Table 6.
E. The total number of microsatellite loci which can be amplified in the genomic DNA of kosta rectilinearly-charged Cyprinus carpioi (Moenkhausia costae) is 3, namely Hy-02, Hy-04, Hy-05, the number of alleles per microsatellite locus varies from 2 to 8, the desired heterozygosity (He) ranges from 0.523 to 0.908, and the Polymorphism Information Content (PIC) ranges from 0.375 to 0.842, and the detailed results are shown in Table 7.
TABLE 2 detection values of microsatellite loci of Cyprinus carpioides of Heterophyllus nigricans
TABLE 3 detection values of microsatellite loci of Cyprinus carpioides of large fin
TABLE 4 detection values of microsatellite loci of rose lipocarp
TABLE 5 Everophore detection value of microsatellite locus of lipocarp
TABLE 6 microsatellite locus detection values of Soxhlet lipocarp
TABLE 7 detection values of microsatellite loci of kosta's linear lipocarp
Sequence listing
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<120> Heterophylla nigricans lipocarp microsatellite marker locus and general application thereof in fishes of the genus and the genus of the near source
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ccgagccttc gacacctcac ctcctacaca cacacacaca atgttggtta ttctgttatt 180
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<210> 5
<211> 183
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Claims (10)

1. The black iso-striated lipocarp microsatellite molecular marker is characterized in that the nucleotide sequence of the microsatellite molecular marker is shown as any one of SEQ ID No.1 to SEQ ID No 5 in a sequence table, and the microsatellite molecular marker is respectively marked with numbers of Hy-01, Hy-02, Hy-03, Hy-04 and Hy-05.
2. The combination of microsatellite molecular markers of Cyprinus carpioi with black soxhlet pattern 1, wherein two or more of the microsatellite molecular markers Hy-01, Hy-02, Hy-03, Hy-04 and Hy-05 are selected from the group consisting of Cyprinus carpiovar nigrioides .
3. The black alloline lipocarp microsatellite molecular marker primer according to claim 1, characterized in that: the primer sequence is SEQ ID No.6-15, wherein the sequence SEQ.ID No.6-7 is used for amplifying Hy-01, and the like.
4. The combination of black soychnos lipocarp microsatellite molecular marker primers according to claim 3 selected from two or more sets of amplified Hy-01 to Hy-05 primer pairs.
5. Use of the black soychniki lipocarp microsatellite molecular marker of claim 1 or the combination of the black soychniki lipocarp microsatellite molecular markers of claim 2 in population genetic diversity detection, individual identification or molecular assisted breeding of black soychniki lipocarp.
6. The use of Hy-01, Hy-02, Hy-04, Hy-05 or a combination thereof in the microsatellite molecular marker of the black allogamy lipocarp of claim 1 for population genetic diversity detection, individual identification or molecular assisted breeding of the large fin lipocarp.
7. The use of Hy-02, Hy-04, Hy-05 or a combination thereof in microsatellite molecular markers of Cyprinus carpiod of Hemisalanx in claim 1 for population genetic diversity detection, individual identification or molecular assisted breeding of rose Cyprinus carpiod.
8. The use of Hy-02, Hy-03, Hy-04, Hy-05 or a combination thereof in the microsatellite molecular markers of the black allogamy lipocarp of claim 1 for population genetic diversity detection, individual identification or molecular assisted breeding of the eifeng lipocarp.
9. Use of the black soxhlet lipocarp microsatellite molecular marker of claim 1 or the combination of the black soxhlet lipocarp microsatellite molecular markers of claim 2 for population genetic diversity detection, individual identification or molecular assisted breeding of lycopi lipocarp.
10. The use of Hy-02, Hy-04, Hy-05 or a combination thereof in the microsatellite molecular markers of the black allogamy lipocarp of claim 1 for population genetic diversity detection, individual identification or molecular assisted breeding of kosta's lipocarp.
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