Kit and method for parentage identification of paralichthys dentatus based on four-base microsatellite markers
By using a tetrabase microsatellite marker-based paternity testing kit, the genome of the yellow catfish *Pelteobagrus vachelli* was specifically amplified, overcoming the shortcomings of traditional marker methods and achieving efficient and accurate paternity testing and genetic selection.
Patent Information
- Application Number
- CN202510407341.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-02
- Publication Date
- 2025-12-26
- Estimated Expiration
- 2045-04-02
AI Technical Summary
In existing technologies, traditional physical marker methods for parentage identification of yellow catfish (Pelteobagrus vachelli) are costly, time-consuming, labor-intensive, prone to label detachment, and difficult to sustain. Furthermore, microsatellite markers are prone to non-specific diffusion bands during PCR amplification, leading to inaccurate genotype determination.
A paternity testing kit based on tetrabase microsatellite markers was used. Primer pairs that specifically amplify tetrabase microsatellite marker combinations were combined with multiplex PCR technology. The specific labeling of fluorescent markers reduced nonspecific diffusion bands and improved the accuracy of genotyping.
It achieves highly accurate and efficient parentage testing, reduces experimental costs and time, and can accurately distinguish different families under mixed-culture conditions in the same pond, thereby improving breeding efficiency and the reliability of genetic selection.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of aquatic animal breeding, and particularly relates to a reagent kit and method for parentage identification of Pelteobagrus vishnui based on four-base microsatellite markers. BACKGROUND
[0002] Pelteobagrus vishnui (Richardson), commonly known as Jianghuangpufu, belongs to Siluriformes Bagridae Pelteobagrus, and is the largest and fastest-growing species in the genus, which can reach more than 3000 grams. Due to its strong adaptability, delicious meat, and rich nutrition, it is favored by people. At present, Pelteobagrus vishnui has become an important freshwater aquaculture species in China, and it is also the father of hybrid Pelteobagrus (Pelteobagrus ♀ x Pelteobagrus vishnui ♂). Pelteobagrus vachelli Pelteobagrus vishnui inbreeding is common, which not only affects the growth performance and disease resistance of Pelteobagrus vishnui, but also may lead to a decrease in genetic diversity. When carrying out family selection or comprehensive selection, maintaining the integrity and accuracy of pedigree information is crucial for guiding the selection and pairing of breeding parents. The implementation of mixed breeding of different families can more accurately evaluate the growth performance, hybrid advantage and genetic parameters of the families, but the differentiation of families after mixed breeding is still indispensable.
[0003] Traditional physical marking methods have many shortcomings in differentiating families, such as high cost, time-consuming and laborious, easy to fall off, difficult to supplement, and short duration. Microsatellite markers have been widely used in population structure research, pedigree tracing, parentage identification, etc. Among them, microsatellite parentage identification is widely used in germplasm resource protection, new variety breeding and population genetic management, etc., especially in variety breeding work, which can avoid inbreeding and guide production. At present, the microsatellite markers of Pelteobagrus vishnui are mainly dinucleotide repeats, which may produce non-specific bands with 2 base pairs less than the target fragment due to strand slippage during chain polymerase reaction (PCR) amplification, resulting in "diffusion bands" during electrophoresis, making it difficult to accurately read alleles, leading to unstable experimental results, increasing the uncertainty of genotype determination and misjudgment rate.
[0004] SUMMARY In view of the above problems, the purpose of the present application is to provide a Pelteobagrus vishnui parentage identification reagent kit based on four-base microsatellite markers, which can reduce non-specific "diffusion bands" in PCR amplification, make the genotyping results more accurate, and the parentage identification accuracy higher. Therefore, the technical solution adopted by the present application is as follows.
[0005]
[0006] The application provides application of a detection reagent of a four-base microsatellite marker combination in preparation of a kit for parentage identification of Parabramis veillardii, wherein the four-base microsatellite marker combination comprises (AGAA) 12 , (AGAC)8, (AGAC)9, (TCAA)8, (GATG) 10 , (TTTC) 18 , (ACAG) 11 , (AACT)5, (ATAC) 11 , (CAGT)7, (TACA)7, (CATC) 11 and (ACAT) 16 , and the detection reagent is a primer pair combination capable of specifically amplifying each four-base microsatellite marker in the four-base microsatellite marker combination.
[0007] Microsatellite DNA is a repeated sequence composed of 2 to 6 nucleotide units, and is widely distributed in the genomes of eukaryotes. Microsatellite DNA is considered as an ideal molecular marker due to the advantages of high stability, rich polymorphism information, Mendelian inheritance law, co-dominant inheritance characteristics and easy genotyping.
[0008] In the application, for example, (AGAA) 12 represents that the four-base AGAA is repeated 12 times, i.e., AGAAAGAAAGAAAGAAAGAAAGAAAGAAAGAAAGAAAGAAAGAAAGAA.
[0009] In some specific embodiments of the application, the primer pair capable of specifically amplifying (AGAA) 12 comprises a forward primer shown in SEQ ID No. 1 and a reverse primer shown in SEQ ID No. 2; the primer pair capable of specifically amplifying (AGAC)8comprises a forward primer shown in SEQ ID No. 3 and a reverse primer shown in SEQ ID No. 4; the primer pair capable of specifically amplifying (AGAC)9comprises a forward primer shown in SEQ ID No. 5 and a reverse primer shown in SEQ ID No. 6; the primer pair capable of specifically amplifying (TCAA)8comprises a forward primer shown in SEQ ID No. 9 and a reverse primer shown in SEQ ID No. 10; the primer pair capable of specifically amplifying (GATG) 10 comprises a forward primer shown in SEQ ID No. 13 and a reverse primer shown in SEQ ID No. 14; the primer pair capable of specifically amplifying (TTTC) 18 comprises a forward primer shown in SEQ ID No. 15 and a reverse primer shown in SEQ ID No. 16; the primer pair capable of specifically amplifying (ACAG)11 the forward primer set forth in SEQ ID No. 17 and the reverse primer set forth in SEQ ID No. 18; the primer pair capable of specifically amplifying (AACT)5comprises the forward primer set forth in SEQ ID No. 19 and the reverse primer set forth in SEQ ID No. 20; the primer pair capable of specifically amplifying (ATAC) 11 the forward primer set forth in SEQ ID No. 27 and the reverse primer set forth in SEQ ID No. 28; the primer pair capable of specifically amplifying (CAGT)7comprises the forward primer set forth in SEQ ID No. 29 and the reverse primer set forth in SEQ ID No. 30; the primer pair capable of specifically amplifying (TACA)7comprises the forward primer set forth in SEQ ID No. 31 and the reverse primer set forth in SEQ ID No. 32; the primer pair capable of specifically amplifying (CATC) 11 the forward primer set forth in SEQ ID No. 33 and the reverse primer set forth in SEQ ID No. 34; the primer pair capable of specifically amplifying (ACAT) 16 the forward primer set forth in SEQ ID No. 37 and the reverse primer set forth in SEQ ID No. 38.
[0010] In some embodiments of the application, the combination of four-base microsatellite markers further comprises one or more of (ACTT)8, (GTAA)6, (CATA)5, (ACTA)6, (GACT)5, (AGTG)5, and (ATAA)5.
[0011] In some embodiments of the present application, the primer pair capable of specifically amplifying (AGAA)8includes a forward primer as set forth in SEQ ID No. 7 and a reverse primer as set forth in SEQ ID No. 8; the primer pair capable of specifically amplifying (GTAA)6includes a forward primer as set forth in SEQ ID No. 11 and a reverse primer as set forth in SEQ ID No. 12; the primer pair capable of specifically amplifying (CATA)5includes a forward primer as set forth in SEQ ID No. 21 and a reverse primer as set forth in SEQ ID No. 22; the primer pair capable of specifically amplifying (ACTA)6includes a forward primer as set forth in SEQ ID No. 23 and a reverse primer as set forth in SEQ ID No. 24; the primer pair capable of specifically amplifying (GACT)5includes a forward primer as set forth in SEQ ID No. 25 and a reverse primer as set forth in SEQ ID No. 26; the primer pair capable of specifically amplifying (AGTG)5includes a forward primer as set forth in SEQ ID No. 35 and a reverse primer as set forth in SEQ ID No. 36; the primer pair capable of specifically amplifying (ATAA)5includes a forward primer as set forth in SEQ ID No. 39 and a reverse primer as set forth in SEQ ID No. 40.
[0012] The second aspect of the present application provides a kit for parentage identification of Wushibaorhynchus wuhsieni based on four-base microsatellite markers, which comprises detection reagents of a combination of four-base microsatellite markers, the combination of four-base microsatellite markers comprising (AGAA) 12 , (AGAC)8, (AGAC)9, (TCAA)8, (GATG) 10 , (TTTC) 18 , (ACAG) 11 , (AACT)5, (ATAC) 11 , (CAGT)7, (TACA)7, (CATC) 11 , and (ACAT) 16 , wherein the detection reagents are a combination of primer pairs capable of specifically amplifying each four-base microsatellite marker in the combination of four-base microsatellite markers.
[0013] In some embodiments of the present application, the primer pair capable of specifically amplifying (AGAA) 12the primer pair capable of specifically amplifying (AGAC)8includes a forward primer set forth in SEQ ID No. 3 and a reverse primer set forth in SEQ ID No. 4; the primer pair capable of specifically amplifying (AGAC)9includes a forward primer set forth in SEQ ID No. 5 and a reverse primer set forth in SEQ ID No. 6; the primer pair capable of specifically amplifying (TCAA)8includes a forward primer set forth in SEQ ID No. 9 and a reverse primer set forth in SEQ ID No. 10; the primer pair capable of specifically amplifying (GATG) 10 the primer pair capable of specifically amplifying (TTTC) 18 the primer pair capable of specifically amplifying (ACAG) 11 the primer pair capable of specifically amplifying (ACAG) 11 the primer pair capable of specifically amplifying (ACAG) 11 the primer pair capable of specifically amplifying (ACAG) 16 the primer pair capable of specifically amplifying (ACAG)
[0014] In some embodiments of the present application, the combination of four-base microsatellite markers further includes one or more of (ACTT)8, (GTAA)6, (CATA)5, (ACTA)6, (GACT)5, (AGTG)5, and (ATAA)5.
[0015] In some embodiments of the present application, the primer pair capable of specifically amplifying (ACTT)8 comprises a forward primer as set forth in SEQ ID No. 7 and a reverse primer as set forth in SEQ ID No. 8; the primer pair capable of specifically amplifying (GTAA)6 comprises a forward primer as set forth in SEQ ID No. 11 and a reverse primer as set forth in SEQ ID No. 12; the primer pair capable of specifically amplifying (CATA)5 comprises a forward primer as set forth in SEQ ID No. 21 and a reverse primer as set forth in SEQ ID No. 22; the primer pair capable of specifically amplifying (ACTA)6 comprises a forward primer as set forth in SEQ ID No. 23 and a reverse primer as set forth in SEQ ID No. 24; the primer pair capable of specifically amplifying (GACT)5 comprises a forward primer as set forth in SEQ ID No. 25 and a reverse primer as set forth in SEQ ID No. 26; the primer pair capable of specifically amplifying (AGTG)5 comprises a forward primer as set forth in SEQ ID No. 35 and a reverse primer as set forth in SEQ ID No. 36; the primer pair capable of specifically amplifying (ATAA)5 comprises a forward primer as set forth in SEQ ID No. 39 and a reverse primer as set forth in SEQ ID No. 40.
[0016] In some embodiments of the present application, the forward primer of each primer pair in the primer pair combination is fluorescently labeled at the 5' end.
[0017] Based on cost and operability considerations, one of skill in the art can multiplex PCR amplification for each four-base microsatellite marker, and thus different fluorescent labels can be used for the forward primers of each primer pair.
[0018] In some embodiments of the present application, the fluorescent labeling and grouping are as shown in the following table:
[0019]
[0020] For each group, the forward and reverse primers for each four-base microsatellite marker can be mixed together, or all primers in the same group can be mixed together.
[0021] In some embodiments of the present application, the kit further comprises a DNA extraction reagent and / or a PCR amplification buffer, and in particular, the PCR amplification buffer comprises the enzymes and dNTPs necessary for PCR amplification.
[0022] The third aspect of the present application provides a method for parentage identification of Wushiba fish based on four-base microsatellite markers, comprising the following steps:
[0023] S1, obtaining DNA samples of the to-be-tested Pseudobagrus vachelli, a candidate male parent and / or a candidate female parent;
[0024] S2, performing PCR amplification on the DNA samples by using the kit according to any one of the second aspect of the present application, to obtain four-base microsatellite typing results of the to-be-tested Pseudobagrus vachelli, the candidate male parent and / or the candidate female parent;
[0025] S3, determining the parent-offspring relationship between the to-be-tested Pseudobagrus vachelli and the candidate male parent and / or the candidate female parent according to the typing results.
[0026] In some embodiments of the present application, the forward primers of each primer pair in the primer pair combination are labeled with fluorescence at the 5' end, as described in the second aspect of the present application.
[0027] In some embodiments of the present application, the PCR amplification in step S2 is multiplex amplification, and the fluorescence labels of the forward primers of different four-base microsatellite markers are different in the same reaction system.
[0028] In some embodiments of the present application, the multiplex PCR amplification can be performed in groups, and a specific grouping case is shown in the table of the second aspect of the present application.
[0029] In some embodiments of the present application, the PCR amplification system is 25 μL, including 30-100 ng of template DNA; 12.5 μL of 2x Rapid Taq premix; 0.5 μL of primer mixture, the concentration of each primer in the primer mixture is 10 μM; and ddH2O is added to 25 μL.
[0030] The PCR program is as follows: 94 ℃ pre-denaturation for 5 min; 94 ℃ denaturation for 30 s, annealing for 40 s, 72 ℃ extension for 50 s, a total of 32 cycles; and 72 ℃ extension for 10 min.
[0031] In some embodiments of the present application, the annealing temperature is determined by gradient PCR, and in some embodiments of the present application, the annealing temperature is shown in Table 2, and in another embodiments of the present application, the annealing temperature is 58 ℃.
[0032] In some embodiments of the present application, in step S2, the PCR amplification product is mixed with an internal standard LIZ500, and the four-base microsatellite typing results of the to-be-tested Pseudobagrus vachelli, the candidate male parent and / or the candidate female parent are obtained by using an ABI 3730XL gene analyzer.
[0033] In some embodiments of the present application, in step S3, the allele number, observed heterozygosity, expected heterozygosity, polymorphic information content, frequency of null alleles, exclusion probability and cumulative exclusion probability of the four-base microsatellite marker are calculated by using software Cervus.
[0034] Compared with the prior art, the present application has the following beneficial effects:
[0035] Each microsatellite marker in the microsatellite marker combination in the present application has high polymorphism, so that a few microsatellite markers can achieve good identification effect in parentage identification of Wushihuangpuyu.
[0036] Each microsatellite marker in the microsatellite marker combination in the present application is a four-base repeat unit, which can reduce the non-specific "diffusion band" in PCR amplification, so that the genotyping result is more accurate and the accuracy of parentage identification is higher.
[0037] In some embodiments of the present application, the primer pair combination capable of specifically amplifying each four-base microsatellite marker in the four-base microsatellite marker combination is grouped, and four sets of multiplex PCR systems are established and optimized. Compared with traditional single PCR, multiplex PCR can amplify multiple loci at the same time, greatly reducing the time and cost of parentage identification.
[0038] By using the technical solutions of the present application, the problem of different families being unable to be cultured in the same pond can be solved, multiple families can be cultured in the same pond, errors in growth and resistance caused by different breeding environments can be eliminated, and the differences in economic traits of different families can be more accurately observed. In addition, the same pond culture saves breeding space, improves breeding efficiency, and reduces breeding cost.
[0039] The kit and method of the present application overcome the limitations of traditional physical markers, such as high cost, complicated operation, easy damage to fish body, and easy falling off of the marker, and provide a reliable and efficient detection platform for genetic selection and breeding of Wushihuangpuyu and avoiding inbreeding.
[0040] It should be understood that the content described in this section is not intended to identify key or important features of the embodiments of the present application, nor is it used to limit the scope of the present application. Other features of the present application will become apparent through the following description. BRIEF DESCRIPTION OF DRAWINGS
[0041] The above and other objects, features and advantages of the example embodiments of the present application will be more apparent from the following detailed description taken in conjunction with the accompanying drawings, in which several embodiments of the present application are shown by way of example, and not limitation, in which:
[0042] Figure 1The correlation between PIC and E-1P of Parabramis pekinensis in Example 1 of the present application is shown.
[0043] Figure 2 The comprehensive exclusion probability curve of 20 SSR loci of Parabramis pekinensis in Example 1 of the present application is shown.
[0044] Figure 3 The multiplex PCR typing map of four groups of SSR loci in Example 3 of the present application is shown.
[0045] Figure 4 The genotyping map of microsatellite loci JHS1 (A) and JHS2 (B) in Example 3 of the present application is shown.
[0046] Figure 5 The identification accuracy (95% confidence level) of 13 SSR loci based on the genotypes of 10,000 simulated Parabramis pekinensis offspring in Example 3 of the present application is shown. DETAILED DESCRIPTION
[0047] In order to make the technical problems, technical solutions and beneficial effects solved by the present application clearer, the present application will be further described in detail below in combination with examples.
[0048] The following examples are presented herein for the purpose of demonstrating preferred embodiments of the present application. Those skilled in the art will appreciate that the technology disclosed in the following examples represents the inventors' knowledge of technology that can be used to practice the present application and, as such, is considered to be preferred embodiments of the present application. However, those skilled in the art will further appreciate, in view of the present specification, that modifications can be made to the particular embodiments disclosed herein, and still obtain a like or similar result, without departing from the spirit and scope of the present application.
[0049] Those skilled in the art will appreciate that or, by routine testing, a number of equivalents to the specific embodiments of the application described herein will be recognized. Such equivalents are included herein within the scope of the claims.
[0050] The experimental methods in the following examples are all routine methods unless otherwise specified. The instruments and equipment used in the following examples are all routine laboratory instruments and equipment unless otherwise specified. The test materials used in the following examples are all purchased from routine biochemical reagent stores unless otherwise specified.
[0051] Example 1 Screening and verification of SSR markers
[0052] 1. Sample collection
[0053] The invention collected 120 tail fin samples of 3 different breeding populations of Pseudobagrus vachelli in Anhui Province, which were preserved in liquid nitrogen. Among them, 90 tail fin samples were used for whole genome resequencing and microsatellite marker screening. The remaining 30 tail fin samples (including parents and backup parents from different breeding populations in the Yangtze River and Huaihe River) were used for microsatellite marker verification.
[0054] 2. Whole genome resequencing
[0055] DNA was extracted from samples using blood / cell / tissue genomic DNA extraction kit (Tiangen). The DNA concentration was quantified by Qubit dsDNA HS assay kit (Shenguo), and the DNA integrity was verified by 1% agarose gel electrophoresis. The DNA of different Pseudobagrus vachelli individuals was mixed in equal amounts to construct a library for next-generation sequencing (NGS).
[0056] The library preparation and sequencing process is as follows:
[0057] DNA fragmentation: 500 ng of genomic DNA was randomly fragmented using Covaris disruptor (Woburn);
[0058] End repair and A tail addition: Hieff NGS MaxUp II DNA library preparation kit (Yixing) was used for end repair, and A tail was added at the 3' end; ®
[0059] Adapter addition: Fast T4 DNA ligase was used to connect sequencing adapters to fragmented DNA, and enhancers were added to improve ligation efficiency;
[0060] PCR amplification and purification: Index primers were introduced during PCR, and the amplification product (about 400 bp) was purified by magnetic beads;
[0061] Library quality control: Qubit 4.0 (Thermo) was used to quantify the library concentration, and 2% agarose gel electrophoresis was used to verify the library fragment size;
[0062] High-throughput sequencing: After mixing the library, Illumina NovaSeq 6000 sequencing platform was used for double-end sequencing (2x150 bp).
[0063] 3. Sequence assembly, SSR motif detection and primer design
[0064] 3.1 Data preprocessing
[0065] Raw sequencing reads were quality controlled using Fastp (default parameters) to remove reads containing adapter sequences and low-quality / ambiguous bases at 5' or 3' ends, and to obtain clean reads.
[0066] 3.2 Sequence assembly
[0067] Filtered clean reads were assembled into contigs using SOAP denovo software.
[0068] 3.3 Simple sequence repeat (SSR) detection
[0069] MISA tool was used to scan SSR motifs in contigs, with the following detection thresholds:
[0070] Repeat unit length and minimum repeat number:
[0071] (1) Single-base (1-mer) repeat ≥ 10 times;
[0072] (2) Two-base (2-mer) repeat ≥ 6 times;
[0073] (3) Three to six-base (3-6-mer) repeat ≥ 5 times.
[0074] SSR spacing requirement: minimum distance between adjacent SSRs is 200 bp.
[0075] A total of 588,080 sequences were obtained from the whole-genome resequencing of W. wui, and 1,317,233 SSRs with repeat number ≥ 5 were identified (see Table 1). The distribution of SSR types was as follows:
[0076] (1) Two-base repeat: highest proportion, up to 67.10%;
[0077] (2) Single-base repeat: second, accounting for 17.45%;
[0078] (3) Four-base repeat: accounting for 8.40%;
[0079] (4) Three-base repeat: accounting for 6.17%;
[0080] (5) Five-base and six-base repeat: relatively rare, accounting for only 0.78% and 0.09%, respectively.
[0081] Table 1. SSR distribution statistics of W. wui whole genome
[0082]
[0083] Therefore, the SSR in the genome of P. walsinghami is mainly composed of short repeat units (two bases, one base), and the proportion of long repeat units (five, six bases) is very low.
[0084] 3.4 Primer design
[0085] Based on the upstream and downstream sequences of the SSR motif, the forward and reverse primers were designed using Primer3 (v4.1.0), with the following parameter settings:
[0086] (1) Product size range: 100-280 bp;
[0087] (2) Maximum terminal stability: 250;
[0088] (3) The remaining parameters use the default values.
[0089] 4. Characterization of tetrameric SSR markers
[0090] For the four-base microsatellite site, primer pairs were designed and PCR amplification was performed in 10 samples. The PCR reaction system was 25 μL, including 1 μL of P. walsinghami genomic template DNA with a concentration of 50 ng / μL; 12.5 μL of 2×RapidTaq premix (Novozyme; Catalog No. P222-03); 0.5 μL of forward and reverse primer mixture (each primer concentration was 10 μM); and ddH2O to 25 μL. The PCR program was 94 ℃ pre-denaturation for 5 min; 94 ℃ denaturation for 30 s, annealing for 40 s, 72 ℃ extension for 50 s, for a total of 32 cycles; and finally 72 ℃ extension for 10 min. Among them, the annealing temperature was determined by gradient PCR. The PCR products were detected by 1.5% agarose gel electrophoresis to judge the amplification stability.
[0091] The results showed that the primer pairs of 23 four-base microsatellite sites were stable, located on 20 chromosomes, and one four-base microsatellite site was retained on the same chromosome. A total of 20 four-base microsatellite sites were obtained, and the corresponding primer information is shown in Table 2.
[0092] Table 2 Primer information of four-base microsatellite sites
[0093]
[0094] The PCR amplification products were wrapped with tin foil paper to avoid light and stored at low temperature, mixed with internal standard LIZ500, and subjected to SSR typing using ABI 3730XL gene analyzer. The SSR typing results were read by Genemarker software, and the number of alleles (Na), observed heterozygosity (Ho), expected heterozygosity (He), polymorphism information content (PIC), null allele frequency (FN), exclusion probability (E-P), and combined exclusion probability (CE-P) of the microsatellite loci were calculated using software Cervus 3.0.
[0095] The 20 screened four-base microsatellite loci were successfully amplified and had high specificity. Genetic data analysis of 30 samples showed (see Table 3 for details):
[0096] The Na of the four-base microsatellite loci ranged from 2 to 12 (average 5.60), and only the allele number of JHS-18 locus was 2; the Ho ranged from 0.233 to 1.000 (average 0.650); and the He ranged from 0.216 to 0.894 (average 0.602).
[0097] Among the 20 four-base microsatellite loci, 14 showed high polymorphism (PIC>0.5), 4 showed moderate polymorphism (PIC: 0.25-0.5), and only two loci, JHS-4 and JHS-18, showed low polymorphism (PIC<0.25).
[0098] In addition, Hardy-Weinberg equilibrium (HWE) test showed that after Bonferroni correction, 6 loci significantly deviated from HWE (P<0.01), and the FN ranged from -0.193 to 0.131.
[0099] Table 3 Genetic analysis results of four-base microsatellite loci in 30 samples
[0100]
[0101] The parent exclusion probability analysis results are shown in Table 4.
[0102] Table 4 Parental exclusion probability and cumulative of four-base microsatellite loci
[0103]
[0104] From Table 4, we can know that:
[0105] (1) Single-site exclusion probability range
[0106] Single parent exclusion probability (E-1P) when both parents are unknown: 0.023-0.606;
[0107] Single parent exclusion probability (E-2P) when one of the parents is known: 0.109-0.756;
[0108] Parent pair exclusion probability (E-PP) when both parents are unknown: 0.186-0.909.
[0109] (2) Cumulative exclusion probability
[0110] The 20 SSR loci were ranked in descending order according to the polymorphism information content (PIC) value, and the cumulative exclusion probability was calculated by accumulating high PIC loci.
[0111] The results showed that the PIC value of the locus was significantly positively correlated with the single parent exclusion probability (E-1P) when both parents were unknown (R²=0.9125, see Figure 1 ).
[0112] (3) Cumulative exclusion probability threshold
[0113] When using 13 high PIC loci, the cumulative single parent exclusion probability (CE-1P) when both parents are unknown reached 99.48%, and the cumulative single parent exclusion probability (CE-2P) when one of the parents is known and the cumulative parent pair exclusion probability (CE-PP) when both parents are unknown both reached 99.99%. Further increasing the number of loci (14-20) did not significantly improve the exclusion probability (see Figure 2 ).
[0114] Based on the balance between genotyping cost and identification efficiency, the inventors selected 13 high polymorphic four-base microsatellite loci (JHS-1, JHS-2, JHS-3, JHS-5, JHS-7, JHS-8, JHS-9, JHS-10, JHS-14, JHS-15, JHS-16, JHS-17, and JHS-19) for subsequent kinship identification.
[0115] Example 2 Multiplex PCR amplification kit and use method
[0116] The forward primers of the primer pairs for the 13 microsatellite loci screened in Example 1 were modified at the 5' end with fluorescence modification, and the fluorescence modification was labeled with hexachlorofluorescein (HEX, green), 6-carboxyfluorescein (FAM, blue), n-ethylmaleimide derivative (NED, black), and carboxy-x-rhodamine (ROX, red).
[0117] The primer sequence, annealing temperature, product size and fluorescence label information were introduced into Multiplex Manager 1.0 software to generate a multiplex PCR combination scheme. Based on the simulation analysis results, the primer final concentration and annealing temperature were optimized, and finally four groups of optimal multiplex PCR combinations were determined, as shown in Table 5.
[0118] Table 5 Multiplex PCR primer combination information
[0119]
[0120] For each site, the concentration of the forward primer and the reverse primer was 10 μM, and the added amount was 0.3 μL, 0.5 μL or 0.8 μL, as shown in Table 5.
[0121] The forward primer and the reverse primer for the same site can be mixed to obtain a primer mixture (the added amount after mixing is 1 μL), and further, for each group, the primer mixtures for different sites can be further mixed for convenient addition, and of course, mixing can also be performed before use. The kit also includes 2×Rapid Taq premix (Novozyme; Catalog No. P222-03) and ddH2O.
[0122] When the kit is used, the multiplex PCR reaction system (25 μL) contains: 1 μL DNA template (50 ng / μL); 12.5 μL 2×Rapid Taq premix; the primer or primer mixture is added according to the primer addition volume in Table 5, and ddH2O is added to a final volume of 25 μL.
[0123] The PCR program is: 94 ℃ pre-denaturation for 5 min; 94 ℃ denaturation for 30 s, 58 ℃ annealing for 40 s, 72 ℃ extension for 50 s, a total of 32 cycles; finally 72 ℃ extension for 10 min.
[0124] It is worth noting that for group D, when the primer addition amount is also 0.5 μL, the ROX signal (red) value is low, as shown in Figure 3 Therefore, the addition amount of the primer pair for JHS-14 site is increased (0.8 μL for each of the forward primer and the reverse primer), and the addition amount of the primer pair for JHS-15 and JHS-19 is reduced (0.3 μL for each of the forward primer and the reverse primer).
[0125] The kit realizes high-throughput and high-precision kinship identification analysis by balancing detection efficiency and cost.
[0126] Example 3 Application of the kit in Wasi yellow catfish parentage identification
[0127] (1) Establishment of full-sibling family of Wasi yellow catfish
[0128] In the breeding season, the individuals of Pseudobagrus vachelli with strong constitution and mature gonad were selected as the breeding parents for artificial propagation, and the ratio of female to male was 1:1. The fertilized eggs of each pair of Pseudobagrus vachelli were hatched and cultured in a separate plastic bucket. A total of 7 full-sibling families were established, and the tail fins of each parent were cut off and preserved in anhydrous ethanol for DNA extraction. From each family, 20-30 tail fins of the offspring were randomly collected, a total of 190, and preserved in anhydrous ethanol as samples for parentage identification, and the female parent was marked as X and the male parent was marked as Y.
[0129] (2) DNA extraction
[0130] The genomic DNA of the parents and offspring of Pseudobagrus vachelli was extracted, as in Example 1.
[0131] (3) Multiplex PCR amplification
[0132] Using the extracted DNA as a template, the kit and method of Example 2 were used for multiplex PCR amplification, and the multiplex PCR typing map of the four groups of microsatellite loci is shown in Figure 4 .
[0133] (4) Genotyping
[0134] The PCR amplification products were separated on an ABI 3730XL genetic analyzer, and the genotypes of each individual at each microsatellite locus were read using GeneMarker software.
[0135] (5) Parentage identification
[0136] The software Cervus 3.0 was used for allele frequency analysis, simulation analysis and parentage identification analysis. The simulation analysis was set to 10,000 simulated offspring, and the number of candidate parents was set according to the actual situation. The simulation analysis was performed on 100% of the candidate parents, with a 1% typing error allowed and a confidence level of 95%. The correlation between the genotypes of the tested individuals and the parents was tested by likelihood ratio (LOD), to determine the parentage relationship between the tested individuals and the candidate parents. When the LOD value is greater than 0, the candidate parent may be the true parent, and the individual with the highest LOD value is the most likely parent; when the LOD value is less than 0, the candidate parent is unlikely to be the true parent.
[0137] As shown in Figure 5 , when the size of the candidate parent pool is ≤60, the identification accuracy of the individual female parent, the individual male parent and the parent pair is 100%; when the size of the candidate parent pool increases to 100, the identification accuracy of the individual female parent or the individual male parent decreases to about 90%, while the accuracy of the parent pair remains 100%; when the size of the candidate parent pool expands to 110: the identification accuracy of the individual female parent or the individual male parent further decreases to 88%, but the accuracy of the parent pair remains stable at 100%.
[0138] Based on Cervus software, 190 offspring and 7 full-sib families were analyzed by parentage identification. The detection rate of candidate parents was 100%, the success rate of site typing was set to 0.99, the error rate of analysis was 0.01, and the critical value of confidence level was set to 80% and 95%. The LOD of 190 offspring individuals was all greater than 0 (Table 6), and the simulation identification rate was 100%. Compared with the actual recorded information, two offspring failed to match the correct parents, and the actual identification accuracy was 98.94%.
[0139] Table 6 Parentage identification results of full-sib families
[0140]
[0141] It should also be understood that various changes and modifications to the application described herein will become apparent to those skilled in the art after reviewing the above description, and these changes and modifications can be made. Such changes and modifications can be made without departing from the scope of the application and the following claims and are therefore intended to be within the scope of the application.
Claims
1. The use of a detection reagent of a four-base microsatellite marker combination in the preparation of a kit for parentage identification of Procyprinus paluni, characterized in that, the combination of tetranucleotide microsatellite markers comprises (AGAA) 12 , (AGAC)8, (AGAC)9, (TCAA)8, (GATG) 10 , (TTTC) 18 , (ACAG) 11 , (AACT)5, (ATAC) 11 , (CAGT)7, (TACA)7, (CATC) 11 and (ACAT) 16 , and the detection reagent is a combination of primer pairs capable of specifically amplifying each of the tetranucleotide microsatellite markers of the combination of tetranucleotide microsatellite markers, wherein: The primer pair capable of specifically amplifying (AGAA) 12 includes a forward primer as set forth in SEQ ID No. 1 and a reverse primer as set forth in SEQ ID No. 2; The primer pair capable of specifically amplifying (AGAC)8 includes a forward primer as set forth in SEQ ID No. 3 and a reverse primer as set forth in SEQ ID No. 4; The primer pair capable of specifically amplifying (AGAC)9 includes a forward primer as set forth in SEQ ID No. 5 and a reverse primer as set forth in SEQ ID No. 6; The primer pair capable of specifically amplifying (TCAA)8 includes a forward primer as set forth in SEQ ID No. 9 and a reverse primer as set forth in SEQ ID No. 10; Capable of specifically amplifying (GATG) 10 The primer pair includes the forward primer shown in SEQ ID No. 13 and the reverse primer shown in SEQ ID No. 14; A primer pair capable of specifically amplifying (TTTC) 18 comprises a forward primer as set forth in SEQ ID No. 15 and a reverse primer as set forth in SEQ ID No. 16; A primer pair capable of specifically amplifying (ACAG) 11 comprises a forward primer set forth in SEQ ID No. 17 and a reverse primer set forth in SEQ ID No. 18; The primer pair capable of specifically amplifying (AGAC)8 includes a forward primer as set forth in SEQ ID No. 3 and a reverse primer as set forth in SEQ ID No. 4; A primer pair capable of specifically amplifying (ATAC) 11 includes a forward primer set forth in SEQ ID No. 27 and a reverse primer set forth in SEQ ID No. 28; The primer pair capable of specifically amplifying (CAGT)7 includes a forward primer as set forth in SEQ ID No. 29 and a reverse primer as set forth in SEQ ID No. 30; The primer pair capable of specifically amplifying (TACA)7 includes a forward primer as set forth in SEQ ID No. 31 and a reverse primer as set forth in SEQ ID No. 32; A primer pair capable of specifically amplifying (CATC) 11 includes a forward primer set forth in SEQ ID No. 33 and a reverse primer set forth in SEQ ID No. 34; A primer pair capable of specifically amplifying (ACAT) 16 includes a forward primer set forth in SEQ ID No. 37 and a reverse primer set forth in SEQ ID No.
38.
2. Use according to claim 1, characterized in that, The four-base microsatellite marker combination further includes one or more of (ACTT)8, (GTAA)6, (CATA)5, (ACTA)6, (GACT)5, (AGTG)5, and (ATAA)5, wherein: The primer pair capable of specifically amplifying (AGAC)8 includes a forward primer as set forth in SEQ ID No. 3 and a reverse primer as set forth in SEQ ID No. 4; The primer pair capable of specifically amplifying (GTAA)6 includes a forward primer as set forth in SEQ ID No. 11 and a reverse primer as set forth in SEQ ID No. 12; The primer pair capable of specifically amplifying (CATA)5 includes a forward primer as set forth in SEQ ID No. 21 and a reverse primer as set forth in SEQ ID No. 22; The primer pair capable of specifically amplifying (AGAC)8 includes a forward primer as set forth in SEQ ID No. 3 and a reverse primer as set forth in SEQ ID No. 4; The primer pair capable of specifically amplifying (AGAC)8 includes a forward primer as set forth in SEQ ID No. 3 and a reverse primer as set forth in SEQ ID No. 4; The primer pair capable of specifically amplifying (AGAC)8 includes a forward primer as set forth in SEQ ID No. 3 and a reverse primer as set forth in SEQ ID No. 4; The primer pair capable of specifically amplifying (AGAC)8 includes a forward primer as set forth in SEQ ID No. 3 and a reverse primer as set forth in SEQ ID No. 4; 3. A kit for parentage testing of Wushin Yellow Catfish based on four-base microsatellite markers, characterized in that, The kit comprises a detection reagent for a four-base microsatellite marker combination comprising (AGAA) 12 , (AGAC)8, (AGAC)9, (TCAA)8, (GATG) 10 , (TTTC) 18 , (ACAG) 11 , (AACT)5, (ATAC) 11 , (CAGT)7, (TACA)7, (CATC) 11 , and (ACAT) 16 , which is a primer pair combination capable of specifically amplifying each four-base microsatellite marker in the four-base microsatellite marker combination. The primer pair capable of specifically amplifying (AGAA) 12 includes a forward primer as set forth in SEQ ID No. 1 and a reverse primer as set forth in SEQ ID No. 2; The primer pair capable of specifically amplifying (AGAC)8 includes a forward primer as set forth in SEQ ID No. 3 and a reverse primer as set forth in SEQ ID No. 4; The primer pair capable of specifically amplifying (AGAC)8 includes a forward primer as set forth in SEQ ID No. 3 and a reverse primer as set forth in SEQ ID No. 4; The primer pair capable of specifically amplifying (TCAA)8 comprises a forward primer shown as SEQ ID No. 9 and a reverse primer shown as SEQ ID No. 10; Capable of specifically amplifying (GATG) 10 The primer pair includes the forward primer shown in SEQ ID No. 13 and the reverse primer shown in SEQ ID No. 14; A primer pair capable of specifically amplifying (TTTC) 18 comprises a forward primer as set forth in SEQ ID No. 15 and a reverse primer as set forth in SEQ ID No. 16; The primer pair capable of specifically amplifying (ACAG) 11 includes a forward primer set forth in SEQ ID No. 17 and a reverse primer set forth in SEQ ID No. 18; The primer pair capable of specifically amplifying (AACT)5 comprises a forward primer shown as SEQ ID No. 19 and a reverse primer shown as SEQ ID No. 20; A primer pair capable of specifically amplifying (ATAC) 11 includes a forward primer set forth in SEQ ID No. 27 and a reverse primer set forth in SEQ ID No. 28; The primer pair capable of specifically amplifying (CAGT)7 comprises a forward primer shown as SEQ ID No. 29 and a reverse primer shown as SEQ ID No. 30; The primer pair capable of specifically amplifying (TACA)7 comprises a forward primer shown as SEQ ID No. 31 and a reverse primer shown as SEQ ID No. 32; A primer pair capable of specifically amplifying (CATC) 11 includes a forward primer set forth in SEQ ID No. 33 and a reverse primer set forth in SEQ ID No. 34; A primer pair capable of specifically amplifying (ACAT) 16 includes a forward primer set forth in SEQ ID No. 37 and a reverse primer set forth in SEQ ID No.
38.
4. The kit of claim 3, wherein The four-base microsatellite marker combination further comprises one or more of (ACTT)8, (GTAA)6, (CATA)5, (ACTA)6, (GACT)5, (AGTG)5 and (ATAA)5, wherein: The primer pair capable of specifically amplifying (ACTT)8 comprises a forward primer shown as SEQ ID No. 7 and a reverse primer shown as SEQ ID No. 8; The primer pair capable of specifically amplifying (GTAA)6 comprises a forward primer shown as SEQ ID No. 11 and a reverse primer shown as SEQ ID No. 12; The primer pair capable of specifically amplifying (CATA)5 comprises a forward primer shown as SEQ ID No. 21 and a reverse primer shown as SEQ ID No. 22; The primer pair capable of specifically amplifying (ACTA)6 comprises a forward primer shown as SEQ ID No. 23 and a reverse primer shown as SEQ ID No. 24; The primer pair capable of specifically amplifying (GACT)5 comprises a forward primer shown as SEQ ID No. 25 and a reverse primer shown as SEQ ID No. 26; The primer pair capable of specifically amplifying (AGTG)5 comprises a forward primer shown as SEQ ID No. 35 and a reverse primer shown as SEQ ID No. 36; The primer pair capable of specifically amplifying (ATAA)5 comprises a forward primer shown as SEQ ID No. 39 and a reverse primer shown as SEQ ID No.
40.
5. The kit according to claim 3 or 4, characterized in that, The forward primer of each primer pair in the primer pair combination is fluorescently labeled at the 5' end.
6. A method for parentage testing of Wushin Yellow Catfish based on four-base microsatellite markers, characterized in that, The method comprises the following steps: S1, obtaining DNA samples of a to-be-tested Pseudobagrus vachliopterus, a candidate male parent and / or a candidate female parent; S2, performing PCR amplification on the DNA samples by using the kit according to claim 5 to obtain four-base microsatellite typing results of the to-be-tested Pseudobagrus vachliopterus, the candidate male parent and / or the candidate female parent; S3, determining the parent-offspring relationship between the to-be-tested Pseudobagrus vachliopterus and the candidate male parent and / or the candidate female parent according to the typing results.
7. The method of claim 6, wherein, In step S2, the PCR amplification is multiplex amplification, and the fluorescent labels of the forward primers for different four-base microsatellite markers are different in the same reaction system.
8. The method of claim 7, wherein, The PCR amplification system is 25 μL, including 30-100 ng template DNA; 12.5 μL 2×Rapid Taq premix; 0.5 μL primer mixture, the concentration of each primer in the primer mixture is 10 μM; add ddH2O to 25 μL, PCR program: 94 ℃ pre-denaturation for 5 min; 94 ℃ denaturation for 30 s, annealing for 40 s, 72 ℃ extension for 50 s, a total of 32 cycles; 72 ℃ extension for 10 min.