Kit and method for performing Pelteobagrus vachelli paternity test based on four-base microsatellite marker

Abstract

The invention discloses a kit and a method for performing Pelteobagrus vachelli paternity test based on a four-base microsatellite marker, and relates to the technical field of aquatic animal breeding. Wherein the four-base microsatellite markers comprise (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, the application realizes accurate identification of the genetic relationship of the Pelteobagrus vachelli family based on the four-base microsatellite markers with high polymorphism and a multiple PCR genotyping technology, and provides a reliable and efficient detection platform for genetic breeding of Pelteobagrus vachelli and avoiding of inbreeding mating.

Description

Technical Field

[0001] The present application relates to the technical field of aquatic animal reproduction and breeding, and in particular to a kit and method for performing parentage identification of Pelteobagrus vachelli based on four-base microsatellite markers. Background Art

[0002] Pelteobagrus vachelli Pelteobagrus vachelli ) is commonly known as the Yellow Catfish of the Yangtze River. It belongs to the genus Pelteobagrus of the family Catfish of the order Siluriformes. It is the largest and fastest growing species in the genus, and can reach a maximum weight of more than 3,000 grams. It is favored by people because of its strong adaptability, delicious meat, and rich nutrition. At present, Pelteobagrus vachelli has become an important freshwater aquaculture species in my country. It is also the breeding parent of hybrid Yellow Catfish (Pelteobagrus vachelli ♀×Pelteobagrus vachelli ♂).

[0003] Inbreeding is common in Pelteobagrus vachelli, which not only affects its growth performance and disease resistance, but also may lead to a reduction in genetic diversity. When conducting family selection or comprehensive breeding, maintaining the integrity and accuracy of pedigree information is essential to guide the selection and pairing of breeding parents. Mixing of different families can more accurately evaluate the growth performance, hybrid vigor and genetic parameters of the families, but the distinction between families after mixed breeding is still indispensable.

[0004] Traditional physical marker methods have many shortcomings in distinguishing family lines, such as high cost, time-consuming and labor-intensive, easy loss of markers, difficulty in replenishment, and short duration. Microsatellite markers have been widely used in population structure research, pedigree tracing, and parentage testing. Among them, microsatellite parentage testing is widely used in germplasm resource protection, new variety breeding, and population genetic management. In particular, it can avoid inbreeding and guide production in variety breeding. At present, most microsatellite markers of Pelteobagrus vachelli are dinucleotide repeat sequences. During the polymerase chain reaction (PCR) amplification process, these sequences may produce non-specific bands that are 2 base pairs less than the target fragment due to chain slippage, resulting in "diffuse bands" during electrophoresis, making it difficult to accurately read alleles, resulting in unstable experimental results and increasing the uncertainty and misjudgment rate of genotype determination. Summary of the invention

[0005] In view of the above shortcomings, the purpose of the present invention is to provide a yellow catfish parentage identification kit based on four-base microsatellite markers, which can reduce the non-specific "diffuse bands" in PCR amplification, make the genotyping results more accurate, and increase the accuracy of parentage identification. To this end, the technical solution adopted in this application is as follows.

[0006] The first aspect of the present application provides the use of a detection reagent for a four-base microsatellite marker combination in the preparation of a kit for parentage identification of Pelteobagrus vachelli, wherein the four-base microsatellite marker combination includes (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 composed of repetitive sequences consisting of 2 to 6 nucleotide units and is widely distributed in the genomes of eukaryotes. Due to the advantages of high stability, rich polymorphism information, following Mendelian inheritance laws, co-dominant inheritance characteristics, and easy genotyping of microsatellite DNA, it is regarded as an ideal molecular marker.

[0008] In the present application, for example, (AGAA) 12 means that the four bases AGAA are repeated 12 times, that is, AGAAAGAAAGAAAGAAAGAAAGAAAGAAAGAAAGAAAGAAAGAAAGAA.

[0009] In some specific embodiments of the present application, the primer pair capable of specifically amplifying (AGAA) 12 includes the forward primer shown in SEQ ID No. 1 and the reverse primer shown in SEQ ID No. 2; the primer pair capable of specifically amplifying (AGAC)8 includes the forward primer shown in SEQ ID No. 3 and the reverse primer shown in SEQ ID No. 4; the primer pair capable of specifically amplifying (AGAC)9 includes the forward primer shown in SEQ ID No. 5 and the reverse primer shown in SEQ ID No. 6; the primer pair capable of specifically amplifying (TCAA)8 includes the forward primer shown in SEQ ID No. 9 and the reverse primer shown in SEQ ID No. 10; the primer pair capable of specifically amplifying (GATG) 10 includes the forward primer shown in SEQ ID No. 13 and the reverse primer shown in SEQ ID No. 14; the primer pair capable of specifically amplifying (TTTC) 18 includes the forward primer shown in SEQ ID No. 15 and the reverse primer shown in SEQ ID No. 16; the primer pair capable of specifically amplifying (ACAG)11 The primer pair includes the forward primer shown in SEQ ID No. 17 and the reverse primer shown in SEQ ID No. 18; the primer pair capable of specifically amplifying (AACT)5 includes the forward primer shown in SEQ ID No. 19 and the reverse primer shown in SEQ ID No. 20; the primer pair capable of specifically amplifying (ATAC) 11 The primer pair includes the forward primer shown in SEQ ID No. 27 and the reverse primer shown in SEQ ID No. 28; the primer pair capable of specifically amplifying (CAGT)7 includes the forward primer shown in SEQ ID No. 29 and the reverse primer shown in SEQ ID No. 30; the primer pair capable of specifically amplifying (TACA)7 includes the forward primer shown in SEQ ID No. 31 and the reverse primer shown in SEQ ID No. 32; the primer pair capable of specifically amplifying (CATC) 11 The primer pair includes the forward primer shown in SEQ ID No. 33 and the reverse primer shown in SEQ ID No. 34; the primer pair capable of specifically amplifying (ACAT) 16 The primer pair includes the forward primer shown in SEQ ID No. 37 and the reverse primer shown in SEQ ID No. 38.

[0010] In some embodiments of the present application, 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.

[0011] In some specific embodiments of the present application, the primer pairs capable of specifically amplifying (ACTT)8 include the forward primer shown in SEQ ID No. 7 and the reverse primer shown in SEQ ID No. 8; the primer pairs capable of specifically amplifying (GTAA)6 include the forward primer shown in SEQ ID No. 11 and the reverse primer shown in SEQ ID No. 12; the primer pairs capable of specifically amplifying (CATA)5 include the forward primer shown in SEQ ID No. 21 and the reverse primer shown in SEQ ID No. 22; the primer pairs capable of specifically amplifying (ACTA)6 include the forward primer shown in SEQ ID No. 23 and the reverse primer shown in SEQ ID No. 24; the primer pairs capable of specifically amplifying (GACT)5 include the forward primer shown in SEQ ID No. 25 and the reverse primer shown in SEQ ID No. 26; the primer pairs capable of specifically amplifying (AGTG)5 include the forward primer shown in SEQ ID No. 35 and the reverse primer shown in SEQ ID No. 36; the primer pairs capable of specifically amplifying (ATAA)5 include the forward primer shown in SEQ ID No. 39 and the reverse primer shown in SEQ ID No. 40.

[0012] The second aspect of the present application provides a kit for parentage identification of Pelteobagrus vachelli based on tetranucleotide microsatellite markers. The kit includes detection reagents for a tetranucleotide microsatellite marker combination, and the tetranucleotide microsatellite marker combination includes (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 . The detection reagents are primer pairs capable of specifically amplifying each tetranucleotide microsatellite marker in the tetranucleotide microsatellite marker combination.

[0013] In some specific embodiments of the present application, the primer pairs capable of specifically amplifying (AGAA) 12The primer pair includes the forward primer shown in SEQ ID No. 1 and the reverse primer shown in SEQ ID No. 2; the primer pair capable of specifically amplifying (AGAC)8 includes the forward primer shown in SEQ ID No. 3 and the reverse primer shown in SEQ ID No. 4; the primer pair capable of specifically amplifying (AGAC)9 includes the forward primer shown in SEQ ID No. 5 and the reverse primer shown in SEQ ID No. 6; the primer pair capable of specifically amplifying (TCAA)8 includes the forward primer shown in SEQ ID No. 9 and the reverse primer shown in SEQ ID No. 10; the primer pair 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; the primer pair capable of specifically amplifying (TTTC) 18 The primer pair includes the forward primer shown in SEQ ID No. 15 and the reverse primer shown in SEQ ID No. 16; the primer pair capable of specifically amplifying (ACAG) 11 The primer pair includes the forward primer shown in SEQ ID No. 17 and the reverse primer shown in SEQ ID No. 18; the primer pair capable of specifically amplifying (AACT)5 includes the forward primer shown in SEQ ID No. 19 and the reverse primer shown in SEQ ID No. 20; the primer pair capable of specifically amplifying (ATAC) 11 The primer pair includes the forward primer shown in SEQ ID No. 27 and the reverse primer shown in SEQ ID No. 28; the primer pair capable of specifically amplifying (CAGT)7 includes the forward primer shown in SEQ ID No. 29 and the reverse primer shown in SEQ ID No. 30; the primer pair capable of specifically amplifying (TACA)7 includes the forward primer shown in SEQ ID No. 31 and the reverse primer shown in SEQ ID No. 32; the primer pair capable of specifically amplifying (CATC) 11 The primer pair includes the forward primer shown in SEQ ID No. 33 and the reverse primer shown in SEQ ID No. 34; the primer pair capable of specifically amplifying (ACAT) 16 The primer pair includes the forward primer shown in SEQ ID No. 37 and the reverse primer shown in SEQ ID No. 38.

[0014] In some embodiments of the present application, 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.

[0015] In some specific embodiments of the present application, the primer pairs capable of specifically amplifying (ACTT)8 include the forward primer shown in SEQ ID No. 7 and the reverse primer shown in SEQ ID No. 8; the primer pairs capable of specifically amplifying (GTAA)6 include the forward primer shown in SEQ ID No. 11 and the reverse primer shown in SEQ ID No. 12; the primer pairs capable of specifically amplifying (CATA)5 include the forward primer shown in SEQ ID No. 21 and the reverse primer shown in SEQ ID No. 22; the primer pairs capable of specifically amplifying (ACTA)6 include the forward primer shown in SEQ ID No. 23 and the reverse primer shown in SEQ ID No. 24; the primer pairs capable of specifically amplifying (GACT)5 include the forward primer shown in SEQ ID No. 25 and the reverse primer shown in SEQ ID No. 26; the primer pairs capable of specifically amplifying (AGTG)5 include the forward primer shown in SEQ ID No. 35 and the reverse primer shown in SEQ ID No. 36; the primer pairs capable of specifically amplifying (ATAA)5 include the forward primer shown in SEQ ID No. 39 and the reverse primer shown in SEQ ID No. 40.

[0016] In some embodiments of the present application, the 5' end of the forward primer of each primer pair in the primer pair combination is fluorescently labeled.

[0017] Those skilled in the art can perform multiplex PCR amplification on each tetranucleotide microsatellite based on cost and operability considerations, so different fluorescent labels are used for the forward primers of each primer pair.

[0018] In some feasible embodiments of the present application, fluorescent labeling and grouping are performed according to the following table:

[0019] For each group, for the primer pairs targeting different tetranucleotide microsatellite markers, only the forward primer and the reverse primer targeting the same tetranucleotide microsatellite marker can be mixed, or all the primers of each primer pair within the same group can be mixed.

[0020] In some embodiments of the present application, the kit further includes a DNA extraction reagent and / or a PCR amplification buffer. In particular, the PCR amplification buffer contains the enzymes and dNTPs necessary for PCR amplification.

[0021] The third aspect of the present application provides a method for parentage identification of Pelteobagrus vachelli based on tetranucleotide microsatellite markers, including the following steps: S1, obtaining DNA samples of the Pelteobagrus vachelli to be tested and candidate fathers and / or candidate mothers; S2. Use the kit according to any one of the second aspects of the present application to perform PCR amplification on the DNA sample to obtain the four-base microsatellite genotyping results of the to-be-detected Pelteobagrus vachelli and the candidate male parent and / or the candidate female parent; S3. Determine the parent-offspring relationship between the to-be-detected Pelteobagrus vachelli and the candidate male parent and / or the candidate female parent according to the genotyping results.

[0022] In some embodiments of the present application, as described in the second aspect of the present application, the 5'-end of the forward primer of each primer pair in the primer pair combination is fluorescently labeled.

[0023] In some feasible embodiments of the present application, in step S2, the PCR amplification is multiplex amplification, and in the same reaction system, the fluorescent labels of the forward primers for different four-base microsatellite markers are different.

[0024] In some feasible embodiments of the present application, as described in the second aspect of the present application, the multiplex PCR amplification can be carried out in groups, and a specific grouping situation is shown in the table in the second aspect of the present application.

[0025] In some embodiments of the present application, the PCR amplification system is calculated based on 25 μL, including 30 - 100 ng template DNA; 12.5 μL 2×Rapid Taq premix; 0.5 μL primer mixture, and the concentration of each primer in the primer mixture is 10 μM; add ddH2O to 25 μL. The PCR program is: pre-denaturation at 94 °C for 5 min; denaturation at 94 °C for 30 s, annealing for 40 s, extension at 72 °C for 50 s, for a total of 32 cycles; extension at 72 °C for 10 min.

[0026] In some embodiments of the present application, the annealing temperature is determined by gradient PCR. In some feasible embodiments of the present application, the annealing temperature is shown in Table 2. In some other feasible embodiments of the present application, the annealing temperature is 58 °C.

[0027] In some embodiments of the present application, in step S2, the PCR amplification products are mixed, mixed with the internal standard LIZ500, and the four-base microsatellite genotyping results of the to-be-detected Pelteobagrus vachelli and the candidate male parent and / or the candidate female parent are obtained by using an ABI 3730XL genetic analyzer.

[0028] In some embodiments of the present application, in step S3, software Cervus is used to calculate the number of alleles, observed heterozygosity, expected heterozygosity, polymorphic information content, null allele frequency, exclusion probability, and cumulative exclusion probability of the four-base microsatellite markers.

[0029] Compared with the prior art, the present application has the following beneficial effects: Each microsatellite marker in the microsatellite marker combination in the present application has high polymorphism, so that good identification results can be obtained in the parentage identification of Pelteobagrus vachelli using a small number of microsatellite markers.

[0030] Each microsatellite marker in the microsatellite marker combination in the present application is a four-base repeat unit, which can reduce the non-specific "smear band" in PCR amplification, making the genotyping results more accurate and the accuracy of parentage identification higher.

[0031] In some embodiments of the present application, primer pairs capable of specifically amplifying each four-base microsatellite marker in the four-base microsatellite marker combination are grouped, and four multiplex PCR systems are established and optimized. Compared with traditional single PCR, multiplex PCR can amplify multiple loci simultaneously, greatly reducing the time and cost of parentage identification.

[0032] Using the technical solution of the present application, the problem that different families cannot be cultured in the same pond can be solved, realizing the co-culture of multiple families in the same pond, eliminating the errors in growth, resistance, etc. caused by different breeding environments, and more accurately observing the differences in economic traits of different families. Moreover, co-culture in the same pond saves breeding space, improves breeding efficiency, and reduces breeding costs.

[0033] The kit and method of the present application overcome the limitations of traditional physical markers such as high cost, cumbersome operation, easy damage to fish bodies, and easy shedding of markers, providing a reliable and efficient detection platform for the genetic breeding of Pelteobagrus vachelli and avoiding inbreeding.

[0034] It should be understood that the content described in this part is not intended to identify the 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 easily understood through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] By reading the following detailed description with reference to the accompanying drawings, the above and other objects, features, and advantages of the exemplary embodiments of the present application will become easily understood. In the drawings, several embodiments of the present application are shown in an exemplary and non-limiting manner, wherein: Figure 1 Shows the correlation between the polymorphism information content (PIC) of Pelteobagrus vachelli loci and the single-parent exclusion probability (E-1P) when the parents are unknown in Example 1 of the present application; Figure 2 Shows the comprehensive exclusion probability curve of 20 SSR loci of Pelteobagrus vachelli in Example 1 of the present application; Figure 3 Shows the multiplex PCR genotyping maps of four groups of SSR loci in Example 3 of the present application; Figure 4 Shows the genotyping maps of microsatellite loci JHS1 (A) and JHS2 (B) in Example 3 of this application.

[0036] Figure 5 Shows the identification accuracy rates (95% confidence level) of 13 SSR loci based on the genotypes of 10,000 simulated offspring of Pelteobagrus vachelli in Example 3 of this application. Detailed implementation manners

[0037] In order to make the technical problems, technical solutions and beneficial effects solved by this application more clear and understandable, the following further details this application in conjunction with examples.

[0038] The following examples are used here to demonstrate the preferred implementation manners of this application. Those skilled in the art will understand that the technologies disclosed in the following examples represent the technologies that the inventors have found can be used to implement this application, and thus can be regarded as the preferred solutions for implementing this application. However, those skilled in the art should understand according to this specification that many modifications can be made to the specific embodiments disclosed here, and still obtain the same or similar results, without departing from the spirit or scope of this application.

[0039] Those skilled in the art will realize or can learn through routine experiments many equivalent technologies of many specific implementation manners of the inventions described here. These equivalents will be included in the claims.

[0040] Unless otherwise specified, the experimental methods in the following examples are all conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all conventional laboratory instruments and equipment; unless otherwise specified, the test materials used in the following examples are all obtained from regular biochemical reagent stores.

[0041] Example 1 Screening and verification of SSR markers 1. Sample collection The invention collected 120 caudal fin samples from 3 different Pelteobagrus vachelli breeding populations in Anhui Province and stored them in liquid nitrogen. Among them, 90 caudal fin samples were used for whole-genome resequencing and microsatellite marker screening. The remaining 30 caudal fin samples (including parents and reserve parents from different farming populations in different water systems of the Yangtze River and Huaihe River) were used for microsatellite marker verification.

[0042] 2. Whole-genome resequencing DNA was extracted from the samples using a Blood / Cell / Tissue Genomic DNA Extraction Kit (Tiangen Biotech), and the operation was performed according to the manufacturer's instructions. The DNA concentration was quantified using the Qubit dsDNA HS Assay Kit (Sangon Biotech), and the DNA integrity was verified by 1% agarose gel electrophoresis. After equal mixing of the DNA from different Pelteobagrus vachelli individuals, a library was constructed for next-generation sequencing (NGS).

[0043] The library preparation and sequencing procedures are as follows: DNA fragmentation: 500 ng of genomic DNA was randomly fragmented using a Covaris shearing instrument (Woburn); End repair and A-tailing: Hieff NGS ® MaxUp II DNA Library Prep Kit (Yeasen Biotech) was used for end repair and addition of an A-tail at the 3' end; Adapter addition: The sequencing adapter was ligated to the fragmented DNA using Fast T4 DNA ligase, and an enhancer was added simultaneously to improve the ligation efficiency; PCR amplification and purification: Index primers were introduced during PCR, and the amplification products (approx. 400 bp) were purified using magnetic beads; Library quality control: The library concentration was quantified using Qubit 4.0 (Thermo), and the library fragment size was verified by 2% agarose gel electrophoresis; High-throughput sequencing: After mixing the libraries, paired-end sequencing (2 × 150 bp) was performed using the Illumina NovaSeq 6000 sequencing platform.

[0044] 3. Sequence assembly, SSR motif detection, and primer design 3.1 Data preprocessing Fastp (default parameters) was used to perform quality control on the raw sequencing reads, removing reads containing adapter sequences and low-quality / ambiguous bases at the 5' or 3' ends to obtain high-quality clean reads.

[0045] 3.2 Sequence assembly The filtered clean reads were assembled into contigs using the SOAP denovo software.

[0046] 3.3 Simple sequence repeat (SSR) detection The MISA tool was used to scan for SSR motifs in the contigs, and the detection thresholds were set as follows: Repeat unit length and minimum repeat number: (1) Mononucleotide (1-mer) repeats ≥ 10 times; (2) The dinucleotide (2-mer) repeat is ≥ 6 times; (3) The trinucleotide to hexanucleotide (3-6-mer) repeat is ≥ 5 times.

[0047] Requirement for SSR spacing: The minimum distance between adjacent SSRs is 200 bp.

[0048] A total of 588,080 sequences were obtained from the whole-genome resequencing of Pelteobagrus vachelli, and 1,317,233 SSRs with a repeat number ≥ 5 times were identified from them (see Table 1 for details). The distribution of SSR types is as follows: (1) Dinucleotide repeat: The highest proportion, reaching 67.10%; (2) Mononucleotide repeat: The second highest, accounting for 17.45%; (3) Tetranucleotide repeat: Accounting for 8.40%; (4) Trinucleotide repeat: Accounting for 6.17%; (5) Pentanucleotide and hexanucleotide repeats: Relatively rare, accounting for only 0.78% and 0.09% respectively.

[0049] Table 1 Statistical distribution of SSRs in the whole genome of Pelteobagrus vachelli

[0050] Thus, it can be seen that SSRs in the genome of Pelteobagrus vachelli are mainly short repeat units (dinucleotide, mononucleotide), and the proportion of long repeat units (pentanucleotide, hexanucleotide) is extremely low.

[0051] 3.4 Primer design Based on the upstream and downstream sequences of the SSR motif, forward and reverse primers were designed using Primer3 (v4.1.0), and the parameter settings are as follows: (1) Product size range: 100 - 280 bp; (2) Maximum terminal stability: 250; (3) The remaining parameters were set to default values.

[0052] 4. Characterization of tetrameric SSR markers Primer pairs were designed for four-base microsatellite loci and PCR amplification was carried out in 10 samples respectively. The PCR reaction system was 25 μL, including 1 μL of genomic template DNA of Pelteobagrus vachelli, with a concentration of 50 ng / μL; 12.5 μL of 2×RapidTaq premix (Novoprotein; product number P222-03); 0.5 μL of the forward and reverse primer mixture (each primer concentration was 10 μM); ddH2O was added to 25 μL. The PCR program was pre-denaturation at 94 °C for 5 min; denaturation at 94 °C for 30 s, annealing for 40 s, extension at 72 °C for 50 s, for a total of 32 cycles; finally, extension at 72 °C 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.

[0053] The results showed that the primer pairs of 23 four-base microsatellite loci were amplified stably, which were located on 20 chromosomes respectively. One four-base microsatellite locus was retained on the same chromosome, and 20 four-base microsatellite loci were obtained. The corresponding primer information is shown in Table 2.

[0054] Table 2 Primer information of four-base microsatellite loci

[0055] The PCR amplification products were wrapped with tin foil and stored in the dark at low temperature. They were mixed with the internal standard LIZ500, and SSR genotyping was carried out using an ABI 3730XL genetic analyzer. The SSR genotyping 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 microsatellite loci were calculated using software Cervus 3.0.

[0056] The 20 four-base microsatellite loci screened were successfully amplified and had high specificity. The genetic data analysis of 30 samples showed (see Table 3 for details): The range of the four-base microsatellite locus Na was 2 - 12 (average 5.60), and the number of alleles at only the JHS-18 locus was 2; the range of Ho was 0.233 - 1.000 (average 0.650); the range of He was 0.216 - 0.894 (average 0.602).

[0057] Among the 20 four-base microsatellite loci, 14 showed high polymorphism (PIC > 0.5), 4 loci were moderately polymorphic (PIC: 0.25 - 0.5), and only two loci, JHS-4 and JHS-18, were low polymorphic (PIC < 0.25).

[0058] In addition, the Hardy-Weinberg equilibrium (HWE) test showed that after Bonferroni correction, 6 loci significantly deviated from HWE (P < 0.01), and the range of FN was -0.193 - 0.131.

[0059] Table 3 Genetic analysis results of four-base microsatellite loci in 30 samples

[0060] The results of the parental exclusion probability analysis are shown in Table 4.

[0061] Table 4 Parental exclusion probability and accumulation of four-base microsatellite loci

[0062] As can be seen from Table 4: (1) Range of single-locus exclusion probability Probability of single-parent exclusion when both parents are unknown (E-1P): 0.023 - 0.606; Probability of single-parent exclusion when one parent is known (E-2P): 0.109 - 0.756; Probability of parental pair exclusion when both parents are unknown (E-PP): 0.186 - 0.909.

[0063] (2) Cumulative exclusion probability Arrange the 20 SSR loci in descending order of polymorphism information content (PIC) value, and calculate the cumulative exclusion probability by accumulating high PIC loci.

[0064] The results showed that there was a significant positive correlation between the PIC value of the locus and the probability of single-parent exclusion when both parents are unknown (E-1P) (R² = 0.9125, see Figure 1 ).

[0065] (3) Cumulative exclusion probability threshold When using 13 highly polymorphic PIC loci, the cumulative exclusion probability for a single parent (CE-1P) reaches 99.48% when both parents are unknown, and both the cumulative exclusion probability for a single parent (CE-2P) when one of the parents is known and the cumulative exclusion probability for a pair of parents (CE-PP) when both parents are unknown reach 99.99%. Further increasing the number of loci (14 - 20) does not significantly improve the exclusion probability (see Figure 2 )

[0066] Based on the balance between genotyping cost and identification efficiency, the inventors selected 13 highly 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.

[0067] Example 2 Multiplex PCR Amplification Kit and Its Usage Method The 5' ends of the forward primers of the primer pairs for the 13 microsatellite loci screened in Example 1 were all fluorescently modified, and the following fluorescent labels were used for the fluorescent modification: hexachlorofluorescein (HEX, green), 6-carboxyfluorescein (FAM, blue), n-ethylmaleimide derivative (NED, black), and carboxy-x-rhodamine (ROX, red).

[0068] The primer sequences, annealing temperatures, product sizes, and fluorescent label information were imported into Multiplex Manager 1.0 software to generate a multiplex PCR combination scheme. Based on the simulation analysis results, the final concentrations of the primers and the annealing temperatures were optimized, and finally 4 groups of optimal multiplex PCR combinations were determined, as shown in Table 5.

[0069] Table 5 Multiplex PCR Primer Combination Information

[0070] For each locus, the concentrations of the forward primer and the reverse primer are both 10 μM, and the added amounts are 0.3 μL, 0.5 μL, or 0.8 μL respectively, as shown in Table 5.

[0071] The forward primer and the reverse primer for the same locus can be mixed to obtain a primer mixture (the added amount becomes 1 μL after mixing). Further, for each group, the primer mixtures for different loci can be further mixed to facilitate addition, and of course, they can also be mixed before use. The kit also includes 2×Rapid Taq premix (Novoprotein; product number P222 - 03) and ddH2O.

[0072] When the kit is in use, the multiplex PCR reaction system (25 μL) contains: 1 μL of DNA template (50 ng / μL); 12.5 μL of 2×Rapid Taq premix; add primers or primer mixture according to the primer addition volume in Table 5, and make up to a final volume of 25 μL with ddH2O.

[0073] The PCR program is: pre-denaturation at 94 °C for 5 min; denaturation at 94 °C for 30 s, annealing at 58 °C for 40 s, extension at 72 °C for 50 s, for a total of 32 cycles; finally, extension at 72 °C for 10 min.

[0074] It should be noted that for group D, when the primer addition amount is also 0.5 μL, the ROX signal (red) value is relatively low, as Figure 3 shown. Therefore, increase the addition amount of the primer pair targeting the JHS-14 locus (0.8 μL each for the forward primer and the reverse primer), and at the same time reduce the addition amount of the primer pairs of JHS-15 and JHS-19 (0.3 μL each for the forward primer and the reverse primer).

[0075] This kit achieves high-throughput and high-precision kinship identification analysis by balancing detection efficiency and cost.

[0076] Example 3 Application of the kit in parentage identification of Pelteobagrus vachelli (1) Establishment of full-sib families of Pelteobagrus vachelli Select healthy and gonadally mature Pelteobagrus vachelli individuals as breeding parents for artificial reproduction during the breeding season, with a male-female ratio of 1:1. The fertilized eggs of each pair of Pelteobagrus vachelli are hatched and cultivated in a separate plastic bucket. A total of 7 full-sib families are established, and at the same time, the caudal fins of each parent are cut and stored in absolute ethanol for DNA extraction. Randomly collect 20 - 30 fin rays of juvenile fish from each family's offspring, a total of 190, and store them in absolute ethanol as samples for parentage identification. The female parent is denoted as X, and the male parent is denoted as Y.

[0077] (2) DNA extraction Extract the genomic DNA of Pelteobagrus vachelli parents and offspring in the same way as in Example 1.

[0078] (3) Multiplex PCR amplification Using the extracted DNA as a template, perform multiplex PCR amplification using the kit and method in Example 2. The multiplex PCR genotyping maps of the four groups of microsatellite loci are shown in Figure 4 .

[0079] (4) Genotyping Separate the PCR amplification products on an ABI 3730XL genetic analyzer, and use GeneMarker software to read the genotypes of each individual at each microsatellite locus.

[0080] (5)Parentage testing Allele frequency analysis, simulation analysis and parentage testing analysis were performed using the software Cervus 3.0. In the simulation analysis, the number of simulated offspring was set to 10,000, the number of candidate parents was set according to the actual situation, sampling simulation analysis was performed on 100% of the candidate parents, allowing a genotyping error of 1%, and the confidence level was 95%. The relationship between the genotype of the individual to be tested and the parental genotype was tested by the likelihood ratio (LOD) to determine the parent-child relationship between the individual to be tested 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 cannot be the true parent.

[0081] As Figure 5 shown, when the size of the candidate parent pool ≤ 60, the identification accuracy rates of the single mother, single father and parent pairs are all 100%; when the candidate parent pool increases to 100, the identification accuracy rate of the single mother or father drops to about 90%, while the accuracy rate of the parent pair remains 100%; when the candidate parent pool expands to 110: the identification accuracy rate of the single mother or single father further drops to 88%, but the accuracy rate of the parent pair stabilizes at 100%.

[0082] Parentage testing analysis was performed on 190 offspring and 7 full-sib families based on the Cervus software. The detection rate of candidate parents was 100%, the locus genotyping success rate was set to 0.99, the analysis error rate was 0.01, and the critical values of the confidence level were set to 80% and 95%. The LOD of all 190 offspring individuals was greater than 0 (Table 6), and the simulated identification rate was 100%. Compared with the actual recorded information, two offspring could not be matched to the correct parents, and the actual identification accuracy rate was 98.94%.

[0083] Table 6 Results of parentage testing of full-sib families

[0084] In addition, it should be understood that after reading the above teachings of the present application, those skilled in the art can make various changes or modifications to the present application, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims

1. Use of a detection reagent of a combination of four-base microsatellite markers in the preparation of a kit for parentage identification of Pelteobagrus vachelli, characterized in that: The four-base microsatellite marker combination includes (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 The detection reagent is a primer pair combination that can specifically amplify each four-base microsatellite marker in the four-base microsatellite marker combination.

2. The use according to claim 1, characterized in that: Capable of specific amplification (AGAA) 12 The primer pair includes 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)8 includes 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)9 includes 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)8 includes a forward primer shown in SEQ ID No. 9 and a reverse primer shown in SEQ ID No. 10; Capable of specific amplification (GATG) 10 The primer pair includes a forward primer shown in SEQ ID No. 13 and a reverse primer shown in SEQ ID No. 14; Capable of specific amplification (TTTC) 18 The primer pair includes a forward primer shown in SEQ ID No. 15 and a reverse primer shown in SEQ ID No. 16; Capable of specific amplification (ACAG) 11 The primer pair includes a forward primer shown in SEQ ID No. 17 and a reverse primer shown in SEQ ID No. 18; The primer pair capable of specifically amplifying (AACT) 5 includes a forward primer shown in SEQ ID No. 19 and a reverse primer shown in SEQ ID No. 20; Able to amplify specificity (ATAC) 11 The primer pair includes a forward primer shown in SEQ ID No. 27 and a reverse primer shown in SEQ ID No. 28; The primer pair capable of specifically amplifying (CAGT)7 includes a forward primer shown in SEQ ID No. 29 and a reverse primer shown in SEQ ID No. 30; The primer pair capable of specifically amplifying (TACA)7 includes a forward primer shown in SEQ ID No. 31 and a reverse primer shown in SEQ ID No. 32; Capable of specific amplification (CATC) 11 The primer pair includes a forward primer shown in SEQ ID No. 33 and a reverse primer shown in SEQ ID No. 34; Capable of specific amplification (ACAT) 16 The primer pair includes a forward primer shown in SEQ ID No. 37 and a reverse primer shown in SEQ ID No.

38.

3. The use according to claim 1 or 2, characterized in that: The four-base microsatellite marker combination also includes one or more of (ACTT)8, (GTAA)6, (CATA)5, (ACTA)6, (GACT)5, (AGTG)5 and (ATAA)5.

4. The use according to claim 3, characterized in that: The primer pair capable of specifically amplifying (ACTT) 8 includes a forward primer shown in SEQ ID No. 7 and a reverse primer shown in SEQ ID No. 8; The primer pair capable of specifically amplifying (GTAA)6 includes a forward primer shown in SEQ ID No. 11 and a reverse primer shown in SEQ ID No. 12; The primer pair capable of specifically amplifying (CATA) 5 includes a forward primer shown in SEQ ID No. 21 and a reverse primer shown in SEQ ID No. 22; The primer pair capable of specifically amplifying (ACTA) 6 includes a forward primer shown in SEQ ID No. 23 and a reverse primer shown in SEQ ID No. 24; The primer pair capable of specifically amplifying (GACT) 5 includes a forward primer shown in SEQ ID No. 25 and a reverse primer shown in SEQ ID No. 26; The primer pair capable of specifically amplifying (AGTG)5 includes a forward primer shown in SEQ ID No. 35 and a reverse primer shown in SEQ ID No. 36; The primer pair capable of specifically amplifying (ATAA)5 includes a forward primer represented by SEQ ID No. 39 and a reverse primer represented by SEQ ID No.

40.

5. A kit for parentage identification of Pelteobagrus vachelli based on four-base microsatellite markers, characterized in that: The kit includes a detection reagent for a four-base microsatellite marker combination, and the four-base microsatellite marker combination includes (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 The detection reagent is a primer pair combination that can specifically amplify each four-base microsatellite marker in the four-base microsatellite marker combination.

6. The kit according to claim 5, characterized in that The four-base microsatellite marker combination also includes one or more of (ACTT)8, (GTAA)6, (CATA)5, (ACTA)6, (GACT)5, (AGTG)5 and (ATAA)5.

7. The kit according to claim 5 or 6, characterized in that The 5' end of the forward primer of each primer pair in the primer pair combination is fluorescently labeled.

8. A method for parentage identification of Pelteobagrus vachelli based on four-base microsatellite markers, characterized in that: The following steps are involved: S1, obtaining DNA samples of the tested Pelteobagrus vachelli and the candidate father and / or candidate mother; S2, using the kit described in claim 7 to perform PCR amplification on the DNA sample to obtain the four-base microsatellite typing results of the tested P. vachelli and the candidate father and / or the candidate mother; S3, determining the parent-offspring relationship between the tested Pelteobagrus vachelli and the candidate sire and / or the candidate dam according to the typing result.

9. The method according to claim 8, characterized in that In step S2, the PCR amplification is multiplex amplification, and in the same reaction system, the forward primers for different four-base microsatellite markers have different fluorescent labels.

10. The method according to claim 9, characterized in that The PCR amplification system is based on 25 μL, including 30-100 ng of 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, The PCR program was as follows: pre-denaturation at 94 °C for 5 min; denaturation at 94 °C for 30 s, annealing for 40 s, and extension at 72 °C for 50 s, for a total of 32 cycles; and extension at 72 °C for 10 min.

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