A SNP molecular marker for identifying a body color trait of cyprinus carpio, primers and application thereof

Abstract

The application relates to a SNP molecular marker for identifying a carp body color trait, primers and application, relates to the technical field of genes, and the SNP molecular marker is located at the 21286787th base of B19 chromosome, and the base is C or G. If the genotype of the SNP molecular marker is CC or CG, the corresponding carp body color trait is normal body color, and if the genotype of the SNP molecular marker is GG, the corresponding carp body color trait is a rainbow cell deletion trait. The hybridization of the rainbow cell deletion trait of the carp is identified, the utilization of the body color trait improvement of the carp is accelerated through molecular marker assisted breeding, and the breeding benefit is improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of genetic technology, in particular to a SNP molecular marker for identifying the body color trait of Cyprinus carpio, primers and application. BACKGROUND

[0002] Cyprinus carpio is one of the important aquaculture species in China, and its germplasm resources are rich and its body color diversity is high, such as Qinghui Cyprinus carpio, Oujian Cyprinus carpio, Hebao Cyprinus carpio, Xingguo Cyprinus carpio, etc. The iridescent cell deletion trait is another type of Cyprinus carpio body color, which is characterized by semi-transparent gill cover and abdominal muscle, and the gill and internal organs are visible. The typical Cyprinus carpio population with this trait is the rice field breeding population "Quanzhou Hehua Cyprinus carpio" in northern Guangxi. The iridescent cell deletion trait is the basis for judging the germplasm source and defining the economic value, and directly determines the breeding benefit. Genetic analysis shows that the iridescent cell deletion trait is a single gene controlled recessive trait. If the population is used for hybridization breeding or genetic research, the offspring carrying the related gene heterozygote individuals cannot be distinguished from the appearance, affecting the selection efficiency.

[0003] Single nucleotide polymorphism (SNP) marker is a genetic marker of single nucleotide mutation at a specific site in the genome, which has high richness, large density and is easy to genotype, and is widely used in plant and animal breeding, disease resistance gene marker, superior variety screening and disease related gene identification.

[0004] At present, there are few molecular markers for the iridescent cell deletion trait of Cyprinus carpio, and no reports on SNP molecular markers for identifying the trait or for assisted selection. Therefore, the present application provides a SNP molecular marker for identifying the body color trait of Cyprinus carpio, primers and application. SUMMARY

[0005] The technical problem to be solved by the present application is to provide a SNP molecular marker for identifying the body color trait of Cyprinus carpio, primers and application. The purpose is to establish a molecular marker assisted breeding method for identifying the heterozygote or purifying the trait of the iridescent cell deletion trait of Cyprinus carpio, to speed up the improvement and utilization of the body color trait of Cyprinus carpio, and to improve the breeding benefit.

[0006] On the basis of the above technical scheme, the present application can also be improved as follows.

[0007] In order to solve the above technical problem, the first purpose of the present application is to provide a SNP molecular marker for identifying the body color trait of Cyprinus carpio, which is located at the 21286787th base of B19 chromosome, and the base is C or G.

[0008] If the genotype of the SNP molecular marker is CC or CG, the corresponding body color trait of the carp is normal body color, and if the genotype of the SNP molecular marker is GG, the corresponding body color trait of the carp is iridescent cell deletion trait.

[0009] The present application has the beneficial effect that the SNP molecular marker is used to identify the heterozygote of the iridescent cell deletion trait of the carp, and the molecular marker assisted breeding is used to accelerate the improvement of the body color trait of the carp and improve the breeding efficiency.

[0010] The second object is to provide a primer of the SNP molecular marker for identifying the body color trait of the carp, which comprises an upstream typing primer F1, a downstream typing primer F2 and a downstream universal primer R, the nucleotide sequence of the upstream typing primer F1 is SEQ ID No: 1, the nucleotide sequence of the downstream typing primer F2 is SEQ ID No: 2, and the nucleotide sequence of the universal primer R is SEQ ID No: 3.

[0011] Further, the 5' end of the upstream typing primer F1 is connected with a fluorescent molecule FAM or a fluorescent molecule HEX; and the 5' end of the downstream typing primer F2 is connected with a fluorescent molecule FAM or a fluorescent molecule HEX.

[0012] Further, the 5' end of the upstream typing primer F1 is connected with a fluorescent molecule FAM; and the 5' end of the downstream typing primer F2 is connected with a fluorescent molecule HEX.

[0013] The beneficial effect of the above-mentioned scheme is that the primer connected with the fluorescent molecule is used to quickly identify the heterozygote of the iridescent cell deletion trait of the carp and perform genotyping.

[0014] The third object is to provide a kit of the SNP molecular marker for identifying the body color trait of the carp, which comprises the above-mentioned primer.

[0015] The beneficial effect of the above-mentioned scheme is to quickly identify the heterozygote of the iridescent cell deletion trait.

[0016] The fourth object is to provide an application of the SNP molecular marker for identifying the body color trait of the carp, the application of the above-mentioned SNP molecular marker in identifying the body color trait of the carp; or the application of the above-mentioned SNP molecular marker in assisted breeding of the carp.

[0017] The fifth object is to provide an application of the primer of the SNP molecular marker for identifying the body color trait of the carp, the application of the above-mentioned primer in identifying the body color trait of the carp; or the application of the above-mentioned primer in assisted breeding of the carp.

[0018] The sixth object is to provide an application of a kit for identifying a SNP molecular marker of a body color trait of a common carp, the application of the kit in identifying the body color trait of the common carp, or the application of the kit in assisted breeding of the common carp.

[0019] The seventh object is a method for identifying a body color trait of a common carp, comprising the following steps:

[0020] Step 1: extracting DNA of a common carp to be tested as a DNA template,

[0021] Step 2: performing PCR amplification on the DNA template by using the primer or the kit, to obtain a PCR amplification product;

[0022] Step 3: performing fluorescence scanning on the PCR amplification product, and performing genotype analysis according to different colors, if the genotype is CC or CG, the corresponding body color trait of the common carp is a normal body color, and if the genotype is GG, the corresponding body color trait of the common carp is a rainbow cell deletion trait.

[0023] Further, denaturation is performed at 95℃ for 10 min for 1 cycle, then denaturation is performed at 95℃ for 20 s, annealing and extension are performed at 61-55℃ for 60 s for 10 cycles, denaturation is performed at 95℃ for 20 s, annealing and extension are performed at 55℃ for 60 s for 28-45 cycles, and finally data reading is performed at 25℃ for 30 s. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 Fig. 1 is a diagram of separation of a body color trait of an F2 generation family according to the present application;

[0025] Figure 2 Fig. 4 is a Manhattan plot of G statistics according to the present application;

[0026] Figure 3 Fig. 5 is a Manhattan plot of Euclidean distance algorithm according to the present application;

[0027] Figure 4 Fig. 6 is a KASP typing result diagram of sample numbers 1-92 of individuals according to the present application;

[0028] Figure 5 Fig. 7 is a KASP typing result diagram of sample numbers 93-183 of individuals according to the present application. DETAILED DESCRIPTION

[0029] The principles and characteristics of the present application are described below, and the examples are only used to explain the present application, and are not used to limit the scope of the present application.

[0030] Experimental reagents and instruments:

[0031] (1) Reagents and consumables

[0032] KASP 2X PCR mix, STO Rox, 96-well PCR tape, sealing film (or paraffin oil)

[0033] (2) Main experimental instruments

[0034] Bio-Rad CFX Connect Real-Time System, BIO-RAD; pipettes, Eppendorf, Germany.

[0035] Example 1: Positioning of SNP molecular markers closely related to the iridophore deficiency trait in common carp

[0036] 1.1 Construction of a population segregating for iridophore-deficient traits

[0037] F1 generation family construction: Quanzhou Hehua carp was selected as the male parent and Jian carp as the female parent, artificial induction hatching technology was used for cross breeding, and more than 2,000 F1 generation families were obtained. 2 The fry were reared in a pond, and after 30 days of age, 200 were randomly retained and continued to be reared until sexual maturity. The results showed that the body color traits of all F1 generation individuals were consistent with those of the mother.

[0038] F2 generation family construction: Select mature individuals in the F1 generation family as parents, and use artificial induction technology to inbreed within the family to obtain more than 5,000 F2 generation tails in an area of ​​1,000m 2 The F2 generation was cultured in the above ponds until 100 days old. The characterization of 1100 individual fish showed that the F2 generation had segregation of traits, with the ratio of normal body color to iridescent cell deficiency individuals being 3:1 (845:255), which is consistent with the inheritance law of single gene control of quality traits (see Appendix for details). Figure 1 ).

[0039] 1.2 Genome resequencing of parental and different color groups

[0040] DNA was extracted from 30 individuals of each of the F1 paternal, F1 maternal, and F2 generation two-color individual pools using the CTAB (cetyltrimethylammonium bromide) method. After the genomic DNA was randomly broken into short DNA fragments with enzymes, the ends were repaired. Then dA tails were added to the ends of the DNA fragments, and sequencing adapters were ligated. The adapter-ligated DNA fragments were purified using AMPure XP magnetic beads, and fragments in the 300-400 bp range were selected for PCR amplification to construct the library. The constructed library was purified, library checked, and sequenced on the HiSEQ X10PE150. The results showed that the genomic resequencing of the two parent and two offspring pools yielded clear data of 48582261300-50735146500 bp, Q20 values of 95.30%-95.94%, and alignment rates of more than 95% with the reference genome (GCA_905221575.1; https: / / www.ncbi.nlm.nih.gov / assembly / GCA_905221575.1), as shown in Table 1. In Table 1, FB represents the paternal parent (normal phenotype), GA represents the maternal parent (iridescent cell deletion), GB is the F2 generation pool consistent with the paternal parent trait, and MA is the F2 generation pool consistent with the maternal parent trait.

[0041] Table 1 Genomic resequencing of different color samples of G. elegans

[0042] Sample Name Data Amount (bp) Q20(％) Matching Rate (%) FB 50735146500 95.93 95.43 GA 50618450100 95.94 95.44 GB 49517571600 95.73 95.28 MA 48582261300 95.30 95.32

[0043] 1.3 SNP analysis and screening

[0044] GATK software was used to detect SNP sites, and the results showed that the two parent and two offspring pools cumulatively obtained 15800497 SNP sites. To control background noise as much as possible, SNP / Indel sites were filtered according to the following standards, and sites meeting the following conditions were used for subsequent BSA analysis: 1) when both parents are present, there is a difference between the parents and the segregation form meets the population type (the segregation form of the markers retained by the F1 population is nnxnp, lmxll, hkxhk, and the segregation form of the markers retained by other populations is aaxbb); 2) when the parent is present, the sequencing depth of the parent(s) is greater than or equal to the given threshold, and the threshold used in this analysis is 5X; 3) both offspring pools are not missing; 4) the sequencing depth of each offspring pool is greater than 10X and less than 500X; 5) the SNP-index of at least one offspring pool is greater than 0.3; 6) the SNP of at least one offspring pool is less than 0.7; the number of marker sites before and after filtering is 1233418.

[0045] Table 2 SNP and InDel screening of color segregation populations of G. elegans

[0046]

[0047]

[0048] 1.4 BSA positioning analysis

[0049] The BSA analysis was performed by using SNP-index, G statistic, Euclidean Distance (ED) algorithm and Fisher's exact test, respectively. The results showed that no significant interval was obtained on the chromosome by using SNP-index method and Fisher's exact test. G statistic and Euclidean Distance (ED) algorithm obtained a relatively unified positioning interval, which was located in the interval of 20200001bp to 22200000bp on chromosome B19 (see Appendix Figure 2 and 3 ).

[0050] 1.5 SNP site search positioning

[0051] According to the whole genome resequencing data of two parents, the SNP variation site in the interval of 20200001bp to 22200000bp on chromosome B19 was searched. A SNP site variation T / G was found at the position of 21286787bp.

[0052] Example 2: Verification experiment of SNP molecular marker and KASP primer

[0053] 2.1 Experimental materials

[0054] (1) Experimental fish

[0055] 149 normal body color individuals and 43 iridophore cell deletion individuals in the F2 population in Example 1 were collected as experimental materials, and the genomic DNA was extracted.

[0056] 2.2 Primer design and synthesis and KASP typing

[0057] According to the sequence of 300bp upstream and downstream of the SNP site at the position of 37371324bp, KASP primer was designed by using primer premier 5.0 software. The primer sequence is shown in Table 3, wherein the 5' end of the upstream typing primer F1 (21286787-F) is connected with the fluorescent molecule FAM, the 5' end of the downstream typing primer F2 (21286787-V) is connected with the fluorescent molecule HEX, and the downstream universal primer R (21286787-R).

[0058] Table 3 Sequence list of primers

[0059]

[0060] Synthetic primers were dissolved in TE (pH 8.0) to 50 μM, and then mixed according to the ratio of 1:1:3 of upstream typing primer F1: downstream typing primer F2: downstream universal primer R, and then loaded onto the machine, with 0.5 μL of primer mixture added to each 10 μL reaction system.

[0061] PCR reaction: the total reaction volume of the 96-well plate reaction system was 10 ul (see Table 4 for details), and the DNA was 2.5 μL in the reaction system (the concentration was about 10 ng / μl). The 96-well PCR reaction plate was sealed, shaken, centrifuged, and the reaction system was mixed uniformly. After centrifugation, PCR reaction was carried out, and the reaction conditions were as follows in Table 5.

[0062] Table 4 PCR reaction system

[0063]

[0064] Table 5 PCR reaction conditions

[0065]

[0066] The fluorescence signal was read using a TECAN infinite M1000 enzyme marker, and three fluorescence signals appeared. The fluorescence signal was converted and analyzed online using snpdecoder software http: / / www.snpway.com / snpdecoder / , and clear and intuitive typing figures were obtained, and genotype results were output according to different colors.

[0067] 2.3 Results and analysis

[0068] Using the SNP molecular marker of Example 1 and the KASP primer designed and synthesized, the F2 separation population (183 tails) was detected, and the results are shown in Table 6. Among them, the fluorescence signal of 48 tails of C:C, the fluorescence signal of 83 tails of G:C, and the fluorescence signal of 51 tails of G:G. Among them, the individuals with C:C fluorescence signal were all normal traits, 6 tails (3.28%) of individuals with G:C fluorescence signal were iridescence cell deletion traits, and the rest were normal traits. 9 tails (4.92%) of individuals with G:G fluorescence signal were normal traits, and the rest were iridescence cell deletion traits. It can be seen that the accuracy rate of the detection results consistent with the phenotype is 91.8 (168 / 183). Among the 183 tails, the individual numbered 124 failed to amplify by PCR, and no fluorescence signal was detected, and Undetermined indicates that no fluorescence signal was detected.

[0069] Table 6 KASP typing results

[0070]

[0071]

[0072]

[0073] KASP genotyping results are shown in Figure 4 and 5 Figure 4 and 5 The genotyping result in the upper left corner of Figure 4 and 5 The genotyping result in the middle part of Figure 4 and 5 The genotyping result in the lower right corner of

[0074] Example 3: Method for identifying the body color trait of common carp using SNP molecular markers

[0075] A method for identifying the body color trait of common carp, comprising the following steps:

[0076] Step 1: Extract the DNA of the common carp to be tested as a DNA template; the extraction method can refer to the DNA extraction in Example 1.

[0077] Step 2: Use the primer design and synthesis method and KASP genotyping method described in Example 2 to perform PCR amplification on the DNA template to obtain an amplification product;

[0078] Step 3: Perform fluorescence scanning on the PCR amplification product, and then perform genotype analysis; if the genotype is CC or CG, the corresponding body color trait of the common carp is normal body color, and if the genotype is GG, the corresponding body color trait of the common carp is iridescent cell deletion trait.

[0079] In summary, the SNP molecular marker of Example 1, the variation site C is closely linked to the normal body color trait of common carp, and G is closely linked to the iridescent cell deletion trait. By using this marker to detect the parent material, it can be determined whether the parent with normal body color carries the gene related to the iridescent cell deletion trait, so as to select the target individual, reduce the workload of later screening and identification, and speed up the breeding process.

[0080] Although the embodiments of the present application have been shown and described above, it should be understood that the above embodiments are exemplary and should not be construed as limiting the present application, and those skilled in the art can make changes, modifications, replacements and variations to the above embodiments within the scope of the present application.​

Claims

1. A primer for identifying a SNP molecular marker of a body color trait of a carp, characterized by, The primers include an upstream typing primer F1, a downstream typing primer F2 and a downstream universal primer R, wherein the nucleotide sequence of the upstream typing primer F1 is SEQ ID No: 1, the nucleotide sequence of the downstream typing primer F2 is SEQ ID No: 2, and the nucleotide sequence of the downstream universal primer R is SEQ ID No: 3; The SNP molecular marker is located at base 21286787 of chromosome B19, and the base is C or G.

2. The primer of claim 1, wherein The 5' end of the upstream typing primer F1 is connected to a fluorescent molecule FAM or a fluorescent molecule HEX; the 5' end of the downstream typing primer F2 is connected to a fluorescent molecule FAM or a fluorescent molecule HEX.

3. The primer of claim 1, wherein The 5' end of the upstream typing primer F1 is connected to a fluorescent molecule FAM; the 5' end of the downstream typing primer F2 is connected to a fluorescent molecule HEX.

4. A kit for identifying a SNP molecular marker of the body color trait in Cyprinus carpio L., characterized by, Comprising the primer according to any one of claims 1 to 3.

5. Use of a primer of a SNP molecular marker for identifying a body color trait of a carp, characterized in that, Use of the primer according to any one of claims 1 to 3 in identifying body color traits of carp.

6. Use of a kit of SNP molecular markers for the identification of the body color trait in Cyprinus carpio L., characterized by, Use of the kit according to claim 4 in identifying carp body color traits.

7. A method for identifying a body color trait in a carp, characterized by, The steps include: Step 1: Extract DNA from the sample carp as a DNA template. Step 2: performing PCR amplification on the DNA template using the primers according to any one of claims 1 to 3 or the kit according to claim 4 to obtain a PCR amplification product; Step 3: Perform fluorescence scanning on the PCR amplification product and perform genotype analysis based on the color. If the genotype is CC or CG, the corresponding carp body color trait is normal body color. If the genotype is GG, the corresponding carp body color trait is iridophore deficiency trait.

8. A method for identifying a body color trait in Cyprinus carpio according to claim 7, characterized in that, The PCR amplification reaction steps are as follows: first, denaturation at 95°C for 10 minutes, one cycle, then denaturation at 95°C for 20 seconds, annealing and extension at 61-55°C for 60 seconds, 10 cycles, then denaturation at 95°C for 20 seconds, annealing and extension at 55°C for 60 seconds, 28-45 cycles, and finally data reading at 25°C for 30 seconds.

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