A molecular marker of the SNP locus of the rrp44 gene related to the growth of channel catfish, and a detection method and application thereof

Abstract

The present invention provides a molecular marker of an rrp44 gene SNP locus related to the growth of channel catfish, and a detection method and application thereof, and designs primers covering rrp44 the entire gene region and obtains 20 haplotype SNP molecular markers for screening fast-growing channel catfish individuals. Individuals with genotypes A / A and G / G are screened as parents to produce individuals with the A / G haplotype. The method of the present invention is simple to operate and rapid in detection, improves the accuracy of screening channel catfish parents, and can quickly obtain channel catfish parents with fast growth rate and genetic stability.

Description

Technical Field

[0001] This invention patent belongs to the field of molecular marker-assisted breeding of aquatic animals, and specifically relates to a molecular marker of SNP loci of the rrp44 gene related to the growth of channel catfish, and its detection method and application. Background Art

[0002] Channel catfish (Ictalures punctatu) is an important aquaculture variety in the world, accounting for more than 60% of the annual aquaculture production in the United States. Since it was introduced into China in 1984, channel catfish has been cultured in many provinces in China, with an annual output of more than 2 million tons. With the rapid development of the aquaculture industry, problems such as slow growth and uneven specifications have occurred during the cultivation of channel catfish, showing the phenomenon of germplasm degradation. Recently, genomic resources have been applied to channel catfish. In addition, some SNPs related to growth have been discovered in channel catfish. These will be conducive to improving the germplasm quality of channel catfish and cultivating new varieties of channel catfish with fast growth and strong stress resistance.

[0003] The Rrp44 gene encodes a type II RNase. In channel catfish, the rrp44 gene is located on chromosome 20. The Rrp44 gene was first discovered in a mutant yeast strain in which sister chromatids could not separate. The catalytic activity of RRP44 consists of a PINc domain responsible for endonuclease activity and an RNB domain responsible for 3'-5' exoribonuclease activity.

[0004] As a multifunctional enzyme, RRP44 is involved in almost all aspects of RNA metabolism and shows activity in the cell cycle. It participates in the kinetochore assembly checkpoint; forms a stable kinetochore-microtubule structure; regulates the expression of cyclins. In humans, studies have shown that abnormal expression of RRP44 leads to Perlman syndrome, resulting in fetal overgrowth. In addition, in a study on human height, it was found that the human homolog DIS3L2 (rs61033296) of RRP44 is one of 42 variants closely related to human height. Therefore, it is of great significance to determine SNPs and haplotype molecular markers related to growth traits of the rrp44 gene in channel catfish for breeding channel catfish parents with fast growth rate and stable genetic traits. Summary of the Invention

[0005] In order to overcome the deficiencies in the prior art, the present invention provides a rapid growth haplotype SNP molecular marker for channel catfish, and its detection method and application.

[0006] To achieve the above object, the present invention is based on a molecular marker of SNP loci of the rrp44 gene in channel catfish, including one or more of the following SNPs markers:

[0007] The first SNPs marker is located at the 1157th base in the coding region of the rrp44 gene. The mutation type is C / T, named g.rrp44 1157T>C, and its nucleotide sequence is shown in Sequence Listing SEQ ID NO:131;

[0008] The second SNPs marker is located at the 1708th base in the coding region of the rrp44 gene. The mutation type is G / A, named g.rrp44 1708C>T, and its nucleotide sequence is shown in Sequence Listing SEQ ID NO:132;

[0009] The third SNPs marker is located at the 1998th base in the coding region of the rrp44 gene. The mutation type is G / C, named g.rrp44 1998A>G, and its nucleotide sequence is shown in Sequence Listing SEQ ID NO:133;

[0010] The fourth SNPs marker is located at the 2045th base in the coding region of the rrp44 gene. The mutation type is G / C, named g.rrp44 2045C>T, and its nucleotide sequence is shown in Sequence Listing SEQ ID NO:134;

[0011] The fifth SNPs marker is located at the 2594th base in the coding region of the rrp44 gene. The mutation type is G / A, named g.rrp44 2594G>A, and its nucleotide sequence is shown in Sequence Listing SEQ ID NO:135;

[0012] The sixth SNPs marker is located at the 2596th base in the coding region of the rrp44 gene. The mutation type is T / C, named g.rrp44 2596C>G, and its nucleotide sequence is shown in Sequence Listing SEQ ID NO:136;

[0013] The seventh SNPs marker is located at the 2816th base in the coding region of the rrp44 gene. The mutation type is A / G, named g.rrp44 2816C>T, and its nucleotide sequence is shown in Sequence Listing SEQ ID NO:137;

[0014] The eighth SNPs marker is located at the 3226th base in the coding region of the rrp44 gene. The mutation type is C / T, named g.rrp44 3226G>A, and its nucleotide sequence is shown in Sequence Listing SEQ ID NO:138;

[0015] The ninth SNPs marker is located at the 4666th base in the coding region of the rrp44 gene. The mutation type is C / T, named g.rrp44 4666A>T, and its nucleotide sequence is shown in Sequence Listing SEQ ID NO:139;

[0016] The tenth SNPs marker is located at the 4712th base in the coding region of the rrp44 gene. The mutation type is C / T, named g.rrp44 4712C>G, and its nucleotide sequence is shown in Sequence Listing SEQ ID NO:140;

[0017] The eleventh SNPs marker is located at the 4713th base in the coding region of the rrp44 gene. The mutation type is C / T, named g.rrp44 4713A>G, and its nucleotide sequence is shown in Sequence Listing SEQ ID NO:141;

[0018] The twelfth SNPs marker is located at the 5254th base in the coding region of the rrp44 gene. The mutation type is C / T, named g.rrp44 5254G>A, and its nucleotide sequence is shown in Sequence Listing SEQ ID NO:142;

[0019] The thirteenth SNPs marker is located at the 5514th base in the coding region of the rrp44 gene. The mutation type is C / T, named g.rrp44 5514G>C, and its nucleotide sequence is shown in Sequence Listing SEQ ID NO:143;

[0020] The fourteenth SNPs marker is located at the 5854th base in the coding region of the rrp44 gene. The mutation type is C / T, named g.rrp44 5854A>G, and its nucleotide sequence is shown in Sequence Listing SEQ ID NO:144;

[0021] The fifteenth SNPs marker is located at the 6545th base in the coding region of the rrp44 gene. The mutation type is C / T, named g.rrp44 6545A>T, and its nucleotide sequence is shown in Sequence Listing SEQ ID NO:145;

[0022] The sixteenth SNPs marker is located at the 6587th base in the coding region of the rrp44 gene. The mutation type is C / T, named g.rrp44 6587A>T, and its nucleotide sequence is shown in Sequence Listing SEQ ID NO:146;

[0023] The seventeenth SNPs marker is located at the 6920th base in the coding region of the rrp44 gene. The mutation type is C / T, named g.rrp44 6920A>G, and its nucleotide sequence is shown in Sequence Listing SEQ ID NO:147;

[0024] The 18th SNPs marker is located at the 7025th base in the coding region of the rrp44 gene, with the mutation type of C / T, named g.rrp44 7025A>G, and its nucleotide sequence is shown in Sequence Listing SEQ ID NO:148;

[0025] The 19th SNPs marker is located at the 7499th base in the coding region of the rrp44 gene, with the mutation type of C / T, named g.rrp44 7499T>C, and its nucleotide sequence is shown in Sequence Listing SEQ ID NO:149;

[0026] The 20th SNPs marker is located at the 8803rd base in the coding region of the rrp44 gene, with the mutation type of C / T, named g.rrp44 8803A>G, and its nucleotide sequence is shown in Sequence Listing SEQ ID NO:150.

[0027] Preferably, the mutation of the 20th SNPs marker causes the amino acid at the 757th position of the RRP44 protein to change from serine to glutamine.

[0028] Preferably, the amino acid at the 757th position of the RRP44 protein changes from serine to asparagine.

[0029] The present invention provides primers for obtaining molecular markers of SNP sites of the rrp44 gene of channel catfish, and their sequences include those shown in SEQ ID NO:1-SEQ ID NO:130.

[0030] The present invention provides a method for obtaining molecular markers of SNP sites of the rrp44 gene of channel catfish, which comprises the following steps:

[0031] (1) Cut fin samples of the channel catfish to be tested from the core breeding population, and extract genomic DNA;

[0032] (2) Design specific primers for the rrp44 gene of channel catfish, and the sequences of the specific primers are as shown in SEQ ID NO:1-SEQ ID NO:130;

[0033] (3) Perform the first-round PCR amplification with channel catfish genomic DNA and the specific primers to obtain the first-round product;

[0034] (4) Perform the second-round PCR amplification with the first-round product in step (3) and barcode to obtain the second-round product;

[0035] (5) Mix the second-round amplification products in step (4) to construct a sequencing library, and perform 150bp paired-end sequencing on the Illumina HiSeqX-ten sequencing platform;

[0036] (6) After the raw sequencing data is filtered, it is aligned with the rrp44 gene, and SNPs markers related to body weight are identified in samples with an alignment rate greater than 75%.

[0037] (7) Perform linkage analysis on all SNPs loci using the SHEsis software, and determine the SNPs markers related to the growth of channel catfish according to the results of the linkage analysis.

[0038] The present invention provides an application of SNP locus molecular markers in the screening and identification of fast-growing traits of channel catfish.

[0039] The present invention provides an application of a primer in the screening and identification of fast-growing traits of channel catfish.

[0040] The beneficial effects of the present invention are as follows:

[0041] The purpose of the present invention is to provide a SNP molecular marker related to the fast growth of channel catfish and its application, that is, to use a haplotype SNP molecular marker related to the growth traits of channel catfish in the RRP44 gene of channel catfish for the identification and screening of fast-growing parents and subsequent breeding methods, screen individuals with genotypes A / A and G / G as parents, and produce individuals with the A / G haplotype. The method of the present invention is simple to operate, rapid in detection, improves the accuracy of screening channel catfish parents, and can quickly obtain channel catfish parents with fast growth speed and genetic stability. Description of the Drawings

[0042] Figure 1 It shows the amino acid change caused by the SNP locus g.8803 mutation. The upper figure is the enlarged view of the 3D structure and encoded amino acids of the RRP44 gene before mutation; the lower figure is the enlarged view of the 3D structure and encoded amino acids of the RRP44 gene after mutation. Detailed Embodiments

[0043] To better illustrate the purpose, technical solutions and advantages of the present invention, the present application will be further described below in conjunction with specific embodiments.

[0044] Example 1:

[0045] The purpose of the present invention is to provide a haplotype SNP molecular marker related to the fast growth of channel catfish and its application, that is, to use a haplotype SNP molecular marker related to the growth traits of channel catfish in the RRP44 gene of channel catfish for the identification and screening of fast-growing parents and subsequent breeding methods.

[0046] The technical solution adopted by this invention patent is:

[0047] The present invention provides seven haplotype SNP molecular markers related to the fast growth trait of channel catfish. The first SNPs marker is located at the 1708th base in the coding region of the rrp44 gene, with a mutation type of C / T, named g.rrp44 1708C>T, and its nucleotide sequence is shown in SEQ ID NO:132 of the sequence listing; the second SNPs marker is located at the 2594th base in the coding region of the rrp44 gene, with a mutation type of G / A, named g.rrp44 2594G>A, and its nucleotide sequence is shown in SEQ ID NO:135 of the sequence listing; the third SNPs marker is located at the 2596th base in the coding region of the rrp44 gene, with a mutation type of G / C, named g.rrp44 2596G>C, and its nucleotide sequence is shown in SEQ ID NO:136 of the sequence listing; the fourth SNPs marker is located at the 5514th base in the coding region of the rrp44 gene, with a mutation type of G / C, named g.rrp44 5514G>C, and its nucleotide sequence is shown in SEQ ID NO:143 of the sequence listing; the fifth SNPs marker is located at the 6920th base in the coding region of the rrp44 gene, with a mutation type of G / A, named g.rrp44 6920G>A, and its nucleotide sequence is shown in SEQ ID NO:147 of the sequence listing; the sixth SNPs marker is located at the 7499th base in the coding region of the rrp44 gene, with a mutation type of T / C, named g.rrp44 7499T>C, and its nucleotide sequence is shown in SEQ ID NO:149 of the sequence listing; the seventh SNPs marker is located at the 8803rd base in the coding region of the rrp44 gene, with a mutation type of A / G, named g.rrp44 8803A>G, and its nucleotide sequence is shown in SEQ ID NO:150 of the sequence listing. This mutation causes the amino acid at position 757 of the RRP44 protein to mutate from serine to asparagine, as Figure 1 shown.

[0048] Specifically, it includes the following steps:

[0049] (1) The G2 generation breeding population of channel catfish is the core selected population. Fin samples of the channel catfish to be tested are cut from it, and genomic DNA is extracted. (2) Specific primers are designed for the rrp44 gene of channel catfish, and the sequences of the specific primers are shown in SEQ ID NO.1 - SEQ ID NO.130. (3) The first round of PCR is carried out using the genomic DNA of channel catfish and the specific primers to obtain the first-round product. (4) The second round of PCR is carried out using the first-round product in step (3) and barcode to obtain the second-round product. (5) After the second-round PCR products of each sample are mixed in equal amounts, they are sequenced on the Illumina HiSeqX-ten sequencing platform using a 150bp paired-end sequencing strategy. (6) After the raw sequencing data is filtered, it is aligned with the rrp44 gene of channel catfish. The SNPs markers significantly related to body weight or body length in the samples with an alignment rate greater than 75% are the fast-growing SNPs markers.

[0050] Example 1: Screening of growth-related SNP markers based on the rrp44 gene of channel catfish

[0051] 1. Primer design

[0052] 65 pairs of specific primers are designed for the coding region sequence of the rrp44 gene of channel catfish to ensure that the amplification products can intersect with each other and cover the entire gene. The specific primer information is shown in Table 1.1 (SEQ ID NO:1 - SEQ ID NO:130 in sequence), and the primers are entrusted to Sangon Biotech (Shanghai) Co., Ltd. for synthesis.

[0053] Table 1.1 Information of multiplex PCR amplification primers for the coding region of the rrp44 gene of channel catfish

[0054]

[0055]

[0056]

[0057]

[0058] 2. Library construction and sequencing

[0059] Tail fin samples of 201 channel catfish individuals with known body lengths (34 - 54 cm) and body weights (303.1 - 1968.7 g) are collected, and their genomic DNA is extracted using an animal tissue DNA extraction kit (Nanjing Novoprotein Scientific Co., Ltd.).

[0060] First-round PCR: After dissolving the synthesized primers of SEQ ID NO.1 - SEQ ID NO.130, 10 μL was pipetted from each primer solution to prepare a Primer mix working solution, which was then aliquoted into a 96-well plate. The sample plate was fully thawed, shaken, and centrifuged at 1000 rpm for 1 s, and then loaded in a DNA automated workstation. The PCR amplification system and components are shown in Table 2.1.

[0061] PCR program: Pre-denaturation at 95°C for 15 min; denaturation at 94°C for 30 s, annealing at 60°C for 10 min, extension at 72°C for 30 s, for 4 cycles; denaturation at 94°C for 30 s, annealing at 60°C for 1 min, extension at 72°C for 30 s, for 24 cycles.

[0062] Table 2.1 First-round PCR amplification system for constructing a targeted resequencing library

[0063]

[0064] Second-round PCR: 90 μL of ddH2O was added to the first-round PCR product for 10-fold dilution, followed by instantaneous centrifugation and then standing at room temperature for 10 min. The diluted first-round PCR product was used as the amplification template for the second-round PCR reaction. The amplification system and components are shown in Table 2.2.

[0065] PCR program: Pre-denaturation at 95°C for 15 min; denaturation at 94°C for 30 s, annealing at 60°C for 4 min, extension at 72°C for 30 s, for 5 cycles; denaturation at 94°C for 30 s, annealing at 65°C for 1 min, extension at 72°C for 30 s, for 10 cycles.

[0066] Table 2.2 Second-round PCR amplification system for constructing a targeted resequencing library

[0067]

[0068] After PCR amplification, all PCR products were detected by 3% agarose gel electrophoresis and qRT-PCR quality control. After passing the quality control, all the second-round PCR amplification products were mixed to construct a sequencing library. The sequencing library was sequenced on the Illumina HiSeqX-ten sequencing platform using a 150 bp paired-end sequencing strategy. Finally, approximately 5.79 Gb of raw data was obtained, including 175.09 million 150 bp paired-end reads, with an average sequencing depth of 8,102×. The average Q30 of the sequencing data for all samples was ~95.78%.

[0069] 3. Data filtering and variant screening

[0070] After classifying the sequencing reads of each sample according to the barcode sequence information, the reads containing more than 10% poly-N sequences, low-quality reads (more than 50% of the bases with Phred score below 5%), and reads with incorrect barcode sequences were removed using the SOAPnuke software. Approximately 167M clean reads were obtained for subsequent analysis.

[0071] These high-quality paired-end sequencing reads were aligned with the reference gene sequence of Ictalurus punctatus rrp44 using the SOAP2.22 software. The average alignment rate of 201 samples was 95.78% (94.09% - 97.52%). Based on the SOAP alignment results, SOAPsnp v1.05 and SOAPindel (v2.1) were used to retrieve SNP and InDel (Insertion-deletion) variant information in all sequencing samples, respectively. To ensure the accuracy of SNP genotyping, samples with an alignment rate below 75% were removed. After statistics, a total of 20 SNP markers were identified in the Ictalurus punctatus rrp44 gene, and no InDel markers were detected in all samples. The 20 SNP markers were located at the 1157th, 1708th, 1998th, 2045th, 2594th, 2596th, 2816th, 3226th, 4666th, 4712th, 4713th, 5254th, 5514th, 5854th, 6545th, 6587th, 6920th, 7025th, 7499th, and 8803rd bases in the coding region of the rrp44 gene, and were named g.rrp44 1157 T>C, g.rrp44 1708 C>T, g.rrp44 1998 A>G, g.rrp44 2045 C>T, g.rrp44 2594 G>A, g.rrp44 2596 C>G, g.rrp44 2816 C>T, g.rrp443226 G>A, g.rrp44 4666 A>T, g.rrp44 4712 C>G, g.rrp44 4713 A>G, g.rrp44 5254 G>A, g.rrp44 5514 G>C, g.rrp44 5854 A>G, g.rrp44 6545 A>T, g.rrp44 6587 A>T, g.rrp446920 A>G, g.rrp44 7025 A>G, g.rrp44 7499 T>C, g.rrp44 8803 A>G, respectively. The characteristics of the 20 SNP loci identified in the rrp44 gene are shown in Table 3.1, and the sequence information of the SNP loci corresponds to the sequence information in Table 3.2 in turn.

[0072] Table 3.1 Characteristics of 20 SNP markers in the rrp44 gene

[0073]

[0074]

[0075] Table 3.2 Sequence characteristics of 20 growth-related SNPs of the rrp44 gene

[0076]

[0077]

[0078] Y, R, W, and S in the sequence are all degenerate bases.

[0079] 4. Identification of sex-linked SNPs markers

[0080] Use the SHEsis software to perform association analysis on all SNP loci. SNP loci with a significant P value < 0.05 are considered relevant. Determine the growth-related SNPs markers of channel catfish according to the results of the association analysis. The results are shown in Table 4.1. The association analysis shows that 7 out of 20 SNPs markers are weight-related SNPs markers. The 7 weight-related SNPs markers are located at the 1708th, 2594th, 2596th, 5514th, 6920th, 7499th, and 8803rd bases of the rrp44 gene respectively. The association analysis of these SNPs with weight is shown in Table 4.1. There are a total of 2 SNPs in the coding region of the rrp44 gene. Among them, the mutation at the g.rrp44 8803 locus causes an amino acid change in the rrp44 gene-encoded amino acid( Figure 1 ), that is, Ser757Asn. The single-base substitution at g.rrp44 6920 does not cause a change in amino acid coding. The remaining 6 SNPs are located in the non-coding region and do not cause a change in amino acid coding.

[0081] Table 4.1 Characteristics of 7 growth-related SNPs markers of the rrp44 gene

[0082]

[0083]

[0084] The present invention relates to the field of molecular marker-assisted breeding of aquatic animals. The SNP molecular marker is obtained by resequencing the rrp44 gene, and the nucleotide sequences of the above-mentioned molecular markers are shown in Sequence Listing SEQ ID NO: 131 and SEQ ID NO. 150. The present invention also discloses the application of the above-mentioned molecular markers in the molecular detection of the growth traits of channel catfish. Individuals with genotypes A / A and G / G are selected as parents to produce individuals with the A / G haplotype. The method of the present invention is simple to operate and rapid in detection, improves the accuracy of screening channel catfish parents, and can quickly obtain channel catfish parents with fast growth speed and genetic stability.

[0085] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. Detection of channel catfish rrp44 A primer composition for a haplotype SNP molecular marker of a gene, characterized in that: The primer composition includes those shown in SEQ ID NO:19 and SEQ ID NO:20, SEQ ID NO:27 and SEQ ID NO:28, SEQ ID NO:56 and SEQ ID NO:60, SEQ ID NO:75 and SEQ ID NO:76, SEQ ID NO:81 and SEQ ID NO:82, SEQ ID NO:95 and SEQ ID NO:

96. The haplotype SNP molecular markers include: Located at rrp44 the 1708th base of the gene coding region, the mutation type is G / A, named g.rrp44 1708 C>T, and its nucleotide sequence is shown in Sequence Listing SEQ ID NO:132 Located at rrp44 the 2594th base of the gene coding region, the mutation type is G / A, named g.rrp44 2594 G>A, and its nucleotide sequence is shown in Sequence Listing SEQ ID NO:135, Located at rrp44 the 2596th base in the gene coding region, the mutation type is T / C, named g.rrp44 2596 C>G, and its nucleotide sequence is shown in Sequence Listing SEQ ID NO:136, Located at rrp44 the 5514th base in the gene coding region, the mutation type is C / T, named g.rrp44 5514 G>C, and its nucleotide sequence is shown in Sequence Listing SEQ ID NO:143, Located at rrp44 the 6920th base of the gene coding region, the mutation type is C / T, named g.rrp44 6920 A>G, and its nucleotide sequence is shown in Sequence Listing SEQ ID NO:147 Located at rrp44 the 7499th base of the gene coding region, the mutation type is C / T, named g.rrp44 7499 T>C, and its nucleotide sequence is shown in Sequence Listing SEQ ID NO:149, and Located at rrp44 the 8803rd base in the gene coding region, the mutation type is C / T, named g.rrp44 8803 A>G, and its nucleotide sequence is shown in Sequence Listing SEQ ID NO:

150.

2. Use of the primer composition according to claim 1 in screening and identifying the fast growth trait of channel catfish: characterized in that: The fast growth trait is body weight.

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