A SNP molecular marker for identifying low dissolved oxygen tolerance in Chinese mitten crab and its application

Abstract

This invention discloses a SNP molecular marker associated with the hypoxia tolerance trait of the Chinese mitten crab (Eriocheir sinensis) and its application. The SNP molecular marker originates from the SMO gene sequence of the Chinese mitten crab, as shown in SEQ ID NO:1. The SNP molecular marker disclosed in this invention can be used for marker-assisted breeding, allowing for selection during the juvenile stage of the Chinese mitten crab, thus shortening the selection process and accelerating the breeding speed of low dissolved oxygen tolerance varieties. This method is simple to operate and is accurate and reliable.

Description

Technical Field

[0001] This invention belongs to the field of aquatic animal genetics and breeding, specifically relating to an SNP molecular marker for identifying the low dissolved oxygen tolerance of the Chinese mitten crab and its application. Background Technology

[0002] Chinese mitten crab ( Eriocheir sinensis Chinese mitten crab (Eriocheir sinensis) is an important aquatic economic animal in my country. Low oxygen levels in densely stocked environments are a key environmental factor restricting the sustainable development of its industry. Currently, breeding for hypoxia tolerance in Chinese mitten crabs relies heavily on family phenotypic measurements and survival rate statistics, which is not only time-consuming and labor-intensive but also highly susceptible to environmental interference, making it difficult to guarantee the accuracy of selection. Molecular marker-assisted breeding is an effective means to improve breeding efficiency; however, current research on molecular marker-assisted selection of hypoxia tolerance in Chinese mitten crabs and the development of related reagent kits are severely lacking, hindering the rapid cultivation of superior stress-resistant strains.

[0003] SMO is a class-F G protein-coupled receptor (GPCR) and a key receptor in the Hedgehog signaling pathway. Studies have shown that the Hedgehog signaling pathway plays a crucial regulatory role in animal tissue homeostasis and hypoxic stress response. However, there are currently no reports on SNP markers linking the SMO gene in *Eriocheir sinensis* (Chinese mitten crab) to hypoxia tolerance. Therefore, developing an accurate and efficient SNP molecular marker linked to hypoxia tolerance is of great significance for accelerating the breeding of stress-resistant superior varieties of *Eriocheir sinensis*. Summary of the Invention

[0004] Purpose of the invention: This invention provides an SNP molecular marker for identifying the low dissolved oxygen tolerance of Chinese mitten crab and its application, solving the problems of traditional breeding relying on phenotypic selection, which is time-consuming and inefficient.

[0005] Technical solution: The SNP molecular marker for identifying the low dissolved oxygen tolerance of the Chinese mitten crab described in this invention is at least one of the following SNP sites: (1) The base located at position 1850 from the 5' end of the nucleic acid sequence of SEQ ID NO: 1 is A or C; (2) The base at position 2779 from the 5' end of the nucleic acid sequence of SEQ ID NO: 1 is G or A.

[0006] The dominant genotypes for the SNP molecular markers are: the AA genotype at position 1850 and / or the GG genotype at position 2779.

[0007] The primer set described in this invention for detecting the SNP molecular markers used to identify the low dissolved oxygen tolerance of the Chinese mitten crab includes a first primer set for detecting the 1850th site and / or a second primer set for detecting the 2779th site.

[0008] The first primer set is used to detect the 1850th site and consists of two allele-specific upstream primers and one universal downstream primer, the nucleotide sequences of which are shown in SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4, respectively. Upstream primer F1: 5'-ATGTTGATAATGTTCCAGGGCTGAA-3' (SEQ ID NO: 2); Upstream primer F2: 5'-ATGTTGATAATGTTCCAGGGCTGAC-3' (SEQ ID NO: 3); Downstream primer R1: 5'-TCCACTAACCTTCTGCTAATACTGT-3' (SEQ ID NO: 4).

[0009] The second primer set, used to detect the 2779th site, consists of two allele-specific upstream primers and one universal downstream primer, with nucleotide sequences shown in SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7, respectively. Upstream primer F3: 5'-TCTGGTGATGACAACAGTAATTCAA-3' (SEQ ID NO: 5); Upstream primer F4: 5'-TCTGGTGATGACAACAGTAATTCAG-3' (SEQ ID NO: 6); Downstream primer R2: 5'-TGATTTTACTCACAGGTGTTCCAGG-3' (SEQ ID NO: 7).

[0010] The kit described in this invention includes the primer set.

[0011] The method for identifying the low dissolved oxygen tolerance of Chinese mitten crabs described in this invention utilizes the aforementioned SNP molecular markers for detection. If a dominant genotype is detected at the corresponding locus in the tested individual, the individual is determined to possess strong low dissolved oxygen tolerance. It was found that the frequency of the AA genotype was significantly higher in the low dissolved oxygen tolerance group than in the hypoxia-sensitive group, and the frequency of the GG genotype was significantly higher in the low dissolved oxygen tolerance group than in the hypoxia-sensitive group.

[0012] Specifically, the method for identifying the low dissolved oxygen tolerance of the Chinese mitten crab includes the following steps: (1) Take tissue from Chinese mitten crab and extract its genomic DNA; (2) PCR amplification was performed using the primer set for the genomic DNA of the Chinese mitten crab; (3) Amplification was performed using KASP real-time PCR and fluorescence signals were read. The genotype of the SNP molecular marker was determined based on the fluorescence signal results. (4) Correlation analysis between different genotypes and low dissolved oxygen tolerance of Chinese mitten crab.

[0013] The application of the SNP molecular marker and primer set described in this invention in identifying the low dissolved oxygen tolerance of Chinese mitten crab.

[0014] This invention targets the single nucleotide polymorphism (SNP) site in SEQ NO: 1 of the Chinese mitten crab (Eriocheir sinensis), and finds that the dominant genotypes located at the SNP molecular marker are the AA and GG genotypes. In genetic breeding research of Chinese mitten crabs using low dissolved oxygen tolerance as a selection indicator, individuals with the AA genotype at the SNP g.1850A>C site and / or the GG genotype at the SNP g.2779G>A site of this sequence can be preferentially selected as breeding parents. This has important guiding significance for the breeding of new strains of Chinese mitten crabs with excellent stress resistance traits.

[0015] Technical Effects: Compared with existing technologies, this invention has the following advantages: Through molecular-assisted breeding, individuals carrying hypoxia-tolerant genes can be rapidly screened, optimizing germplasm resources and significantly improving the survival rate and farming efficiency of crabs in hypoxic environments. By systematically screening hypoxia-tolerant SNP molecular markers and combining them with functional gene analysis, a molecular-level theoretical basis can be provided for the genetic mechanism of hypoxia tolerance traits. The method of detecting SNP sites at positions 1850 and 2779 (5' end) of the nucleotide sequence shown in SEQ ID NO: 1 using two sets of primers is accurate, reliable, and easy to operate. The SNP molecular markers provided by this invention can be used alone for low dissolved oxygen tolerance identification, or two sites can be used in combination to improve screening accuracy, exhibiting flexibility and high efficiency. Attached Figure Description

[0016] Figure 1 KASP genotyping map of SNP sites. Detailed Implementation

[0017] Example 1: Establishment of a Chinese mitten crab population and hypoxia stress experiment The Chinese mitten crabs used in the experiment were obtained from Qingsong Aquatic Products Professional Cooperative in Gaochun District, Nanjing, Jiangsu Province. Two hundred healthy individuals were used, with an average weight of 16.2 ± 3.1 g. They were temporarily housed in the laboratory for one week prior to the experiment. The crabs were evenly divided and temporarily housed in 10 rearing boxes (620 mm × 430 mm × 230 mm), with 20 individuals per box. Dissolved oxygen was maintained at 7.0 ± 0.3 mg / L, pH at 7.5 ± 0.1, and temperature at 24.3 ± 0.6 ℃. During the temporary housing period, they were fed commercial feed every morning (8:00 AM), and one-third of the water volume was changed every two days. They were fasted for 24 hours before the experiment began.

[0018] At the onset of hypoxic stress, the initial dissolved oxygen concentration and saturation were recorded. Nitrogen gas was then continuously introduced into the water to gradually decrease the dissolved oxygen concentration to 2.0 mg / L at a rate of 0.5 mg / L / h, followed by a further decrease at 0.3 mg / L / h until the DO concentration reached 0 or all individuals died. Throughout this process, a dissolved oxygen meter (LDO, HACH) was used to monitor water temperature, dissolved oxygen concentration, and saturation in real time. The mitten crab was considered to be in a hypoxic state when it lost balance (LOE) and could not return to a normal posture within 10 seconds. The time of the first hypoxic individual was recorded as the starting point (LOE = 0 h), and muscle tissue was immediately collected and preserved in a -80°C cryogenic freezer for subsequent analysis.

[0019] Example 2: Extraction of Genomic DNA Genomic DNA was extracted from the Chinese mitten crab (Eriocheir sinensis) using either the conventional phenol-chloroform extraction method or a commercially available animal tissue genomic DNA extraction kit. The specific procedures are as follows: 1. Place muscle tissue in a 1.5 mL centrifuge tube, add 550 µL of TNES lysis buffer, and grind thoroughly; add 10 µL of proteinase K; incubate at 55°C for 3 h or at 37°C overnight. 2. Add 300 µL of saturated phenol (pH > 7.8) to each of the above solutions; add 300 µL of chloroform / isoamyl alcohol mixed solution (24:1), gently shake for 5-10 min; centrifuge at 12000 r / min for 15 min at 4℃; and collect the supernatant. 3. Add 600 µL of chloroform / isoamyl alcohol mixture (24:1) to the supernatant, gently shake for 20 min; centrifuge at 12000 r / min for 20 min at 4℃. 4. Take the supernatant, add 50 µL NaAC (pH: 5.2) and shake slowly; add 700 µL isopropanol (more than one volume of the supernatant); freeze at -20℃ for 2-3 h to precipitate DNA; centrifuge at 4℃, 12800 r / min for 15-20 min. 5. Discard the supernatant, wash the precipitate with 700 µL of 70% ethanol; centrifuge at 12800 r / min for 5-20 min at 4℃ to remove excess salt ions; 6. Discard the supernatant and wash the precipitate with 700 µL of anhydrous ethanol; centrifuge at 12800 r / min for 5-10 min at 4℃ to remove excess water; discard the supernatant and dry for 5 min. 7. Add 30-100 µL of TE or sterile water and dissolve by pipetting; dry in an oven at 37°C for 1 hour; then incubate at 4°C overnight to completely dissolve the DNA. 8. DNA integrity, concentration, and OD value were determined using 1% agarose gel electrophoresis and a Nanodrop UV spectrophotometer; Example 3: Primer design and PCR amplification targeting SNP sites in the SMO gene The PCR primer set consists of an upstream primer and a downstream primer; the upstream primer and the downstream primer are shown in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7.

[0020] The PCR reaction system consisted of: 5 μL of 2× KASP Master Mix, 0.14 μL of KASP Primer Assaymix containing three primers (two specific upstream primers with different fluorescent tag sequences and one universal downstream primer), 1 μL of DNA template, and sterile water to a final volume of 10 μL. The thermal cycling conditions were: 94℃ pre-denaturation for 15 min; Touchdown phase: 94℃ denaturation for 20 s, 61℃ annealing / extension for 60 s (decreasing by 0.6℃ per cycle), for a total of 10 cycles; Conventional amplification phase: 94℃ denaturation for 20 s, 55℃ annealing / extension for 60 s, for a total of 26 cycles; fluorescence signals were read at 37℃ after the reaction. The amplified products were detected using a 7500 fluorescence quantitative PCR instrument. Based on the amplification results, the SNP sites of the Chinese mitten crab were genotyped, as follows: Figure 1 As shown.

[0021] Example 4: Correlation analysis between SNP locus genotype and hypoxia tolerance in Chinese mitten crab The SNP genotyping of 200 individuals of *Eriocheir sinensis* is shown in Table 1. Based on the data, a linear analysis model was constructed using SPSS software, taking into account the characteristics of the traits and the experimental population, to conduct association analysis between gene polymorphism and traits, as shown in Table 2. The KASP genotyping diagrams for the two loci are shown below. Figure 1 As shown.

[0022] Table 1 Genotyping of SNP loci in Chinese mitten crab 1 AC GA 0 2 CC AA 1 3 AC GA 2 4 CC AA 3 5 CC AA 4 6 CC AA 4 7 AA GG 6 8 AC GA 6 9 CC AA 7 10 CC AA 9 11 CC AA 9 12 CC AA 9 13 AC GA 10 14 AA GG 10 15 AA GG 10 16 CC AA 10 17 AA GG 11 18 AC GA 11 19 CC AA 11 20 AC GA 11 21 CC AA 12 22 AA GG 12 23 CC AA 12 24 AC GA 12 25 CC AA 12 26 CC AA 13 27 CC GA 13 28 CC GA 13 29 AC GA 13 30 CC AA 13 31 CC AA 14 32 AC ​ 14 33 ​ ​ 14 34 ​ ​ 15 35 ​ ​ 15 36 ​ ​ 15 37 ​ ​ 15 38 ​ ​ 16 39 ​ ​ 16 40 ​ ​ 16 41 ​ ​ 16 42 ​ ​ 17 43 ​ ​ 17 44 ​ ​ 17 45 ​ ​ 18 46 ​ ​ 18 47 ​ ​ 18 48 ​ ​ 19 49 ​ ​ 19 50 ​ ​ 19 51 ​ ​ 19 52 ​ ​ 19 53 ​ ​ 20 54 ​ ​ 21 55 ​ ​ 22 56 ​ ​ 22 57 ​ ​ 22 58 ​ ​ 22 59 ​ ​ 23 60 ​ ​ 23 61 ​ ​ 25 62 ​ ​ 26 63 ​ ​ 26 64 ​ ​ 26 65 ​ ​ 27 66 ​ ​ 28 67 ​ ​ 28 68 ​ ​ 28 69 ​ ​ 29 70 ​ ​ 29 71 ​ ​ 29 72 ​ ​ 31 73 ​ ​ 31 74 ​ ​ 31 75 ​ ​ 31 76 ​ ​ 31 77 ​ ​ 32 78 ​ ​ 32 79 ​ ​ 32 80 ​ ​ 32 81 ​ ​ 32 82 ​ ​ 33 83 ​ ​ 33 84 ​ ​ 34 85 ​ ​ 34 86 ​ ​ 35 87 ​ ​ 35 88 ​ ​ 35 89 ​ ​ 36 90 ​ ​ 38 91 ​ ​ 38 92 ​ ​ 39 93 ​ ​ 39 94 ​ ​ 39 95 ​ ​ 40 96 ​ ​ 41 97 ​ ​ 41 98 ​ ​ 42 99 ​ ​ 44 100 ​ ​ 44 101 ​ ​ 45 102 ​ ​ 45 103 ​ ​ 45 104 ​ ​ 46 105 ​ ​ 46 106 ​ ​ 47 107 ​ ​ 48 108 ​ ​ 48 109 ​ ​ 49 110 ​ ​ 50 111 ​ ​ 51 112 ​ ​ 52 113 ​ ​ 52 114 ​ ​ 53 115 ​ ​ 53 116 ​ ​ 53 117 ​ ​ 54 118 ​ ​ 54 119 ​ ​ 56 120 ​ ​ 56 121 ​ ​ 57 122 ​ ​ 58 123 ​ ​ 59 124 ​ ​ 60 125 ​ ​ 61 126 ​ ​ 63 127 ​ ​ 63 128 ​ ​ 64 129 ​ ​ 65 130 ​ ​ 66 131 ​ ​ 67 132 ​ ​ 68 133 ​ ​ 73 134 ​ ​ 74 135 ​ ​ 76 136 ​ ​ 76 137 ​ ​ 77 138 ​ ​ 78 139 ​ ​ 78 140 ​ ​ 80 141 ​ ​ 82 142 ​ ​ 82 143 ​ ​ 83 144 ​ ​ 85 145 ​ ​ 86 146 ​ ​ 87 147 ​ ​ 87 148 ​ ​ 91 149 ​ ​ 91 150 ​ ​ 92 151 ​ ​ 94 152 ​ ​ 94 153 ​ ​ 95 154 ​ ​ 95 155 ​ ​ 96 156 ​ ​ 97 157 ​ ​ 97 158 ​ ​ 97 159 ​ ​ 98 160 ​ ​ 98 161 ​ ​ 98 162 ​ ​ 99 163 ​ ​ 99 164 ​ ​ 100 165 ​ ​ 101 166 ​ ​ 101 167 ​ ​ 102 168 ​ ​ 102 169 ​ ​ 104 170 ​ ​ 105 171 ​ ​ 107 172 ​ ​ 107 173 ​ ​ 107 174 ​ ​ 109 175 ​ ​ 109 176 ​ ​ 109 177 ​ ​ 110 178 ​ ​ 110 179 ​ ​ 111 180 ​ ​ 111 181 ​ ​ 111 182 ​ ​ 112 183 ​ ​ 112 184 ​ ​ 112 185 ​ ​ 114 186 ​ ​ 115 187 ​ ​ 115 188 ​ ​ 115 189 ​ ​ 115 190 ​ ​ 115 191 ​ ​ 116 192 ​ ​ 116 193 ​ ​ 117 194 ​ ​ 117 195 ​ ​ 118 196 ​ ​ 118 197 ​ ​ 119 198 ​ ​ 119 199 ​ ​ 120 200 ​ ​ 120 Table 2 Genotypes and gene frequencies of SNP loci in the two groups

[0023]

[0024] Note: The values ​​in the table are expressed as mean ± standard deviation. P <0.05 indicates a significant difference.

[0025] The results indicate that at locus 1850, individuals with the AA genotype exhibited significantly stronger hypoxia tolerance than those with the CC genotype; and at locus 2779, individuals with the GG genotype exhibited significantly stronger hypoxia tolerance than those with the AA genotype. Therefore, using the dominant genotype (AA or GG) at either locus alone can effectively screen for hypoxia-tolerant individuals; furthermore, using both loci (i.e., simultaneously satisfying AA and GG) can further improve the accuracy of screening.

[0026] Example 5: Genotyping of the dominant SNP locus of the SMO gene in the Chinese mitten crab As mentioned above, at locus 1850, individuals with the AA genotype showed significantly stronger hypoxia tolerance than those with the CC genotype; at locus 2779, individuals with the GG genotype showed significantly stronger hypoxia tolerance than those with the AA genotype. P <0.05). This demonstrates that the AA genotype at position 1850 and the GG genotype at position 2779 of the SNP locus are the dominant genotypes of the SMO gene in the Chinese mitten crab, and are also significantly associated with the hypoxia tolerance trait of the Chinese mitten crab.

[0027] The above embodiments demonstrate that the SNP molecular marker and detection method provided by the present invention can efficiently and accurately identify the low dissolved oxygen tolerance of individual Chinese mitten crabs, providing a reliable tool for marker-assisted selection (MAS) breeding of Chinese mitten crabs.

Claims

1. A SNP molecular marker for identifying the low dissolved oxygen tolerance of the Chinese mitten crab, characterized in that, The SNP molecular marker is at least one of the following SNP sites: (1) The base located at position 1850 from the 5' end of the nucleic acid sequence of SEQ ID NO: 1 is A or C; (2) The base at position 2779 from the 5' end of the nucleic acid sequence of SEQ ID NO: 1 is G or A.

2. The molecular marker according to claim 1, characterized in that, The dominant genotypes for the SNP molecular markers are: the AA genotype at position 1850 and / or the GG genotype at position 2779.

3. A primer set for detecting the SNP molecular marker as described in claim 1 or 2, characterized in that, This includes a first primer set for detecting site 1850 and / or a second primer set for detecting site 2779.

4. The primer set according to claim 3, characterized in that, The first primer set is used to detect the 1850th site and consists of two allele-specific upstream primers and one universal downstream primer, the nucleotide sequences of which are shown in SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4, respectively. The second primer set is used to detect the 2779th site and consists of two allele-specific upstream primers and one universal downstream primer, the nucleotide sequences of which are shown in SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7, respectively.

5. A reagent kit, characterized in that, Includes the primer set as described in claim 3.

6. A method for identifying the low dissolved oxygen tolerance of the Chinese mitten crab, characterized in that, Genotyping was performed using the SNP molecular markers described in claim 1. If the genotype of the Chinese mitten crab at the 1850 locus is AA and / or the genotype at the 2779 locus is GG, it is determined to be a low dissolved oxygen tolerant individual.

7. The method according to claim 6, characterized in that, Specifically, the following steps are included: (1) Take tissue from Chinese mitten crab and extract its genomic DNA; (2) The primer set described in claim 3 was used to perform PCR amplification of the genomic DNA of the Chinese mitten crab; (3) Obtain the genotype of the SNP site described in claim 1 based on the amplification products; (4) Determine whether the Chinese mitten crab to be tested is a low dissolved oxygen tolerant individual based on its genotype: If the genotype at the 1850th locus is AA and / or the genotype at the 2779th locus is GG, it is determined to be a low dissolved oxygen tolerant individual.

8. The method according to claim 7, characterized in that, The PCR amplification was performed using KASP real-time PCR.

9. The method according to claim 8, characterized in that, The KASP real-time PCR reaction system includes 2×KASP Master Mix, primer mixture and DNA template; the reaction procedure includes: pre-denaturation; Touchdown amplification, with decreasing annealing temperature per cycle; routine amplification; finally, reading the fluorescence signal and determining the genotypes at loci 1850 and 2779 based on the fluorescence signal results.

10. The application of the SNP molecular marker of claim 1 or 2, the primer set of claim 3 or 4, or the kit of claim 5 in screening hypoxia-tolerant parents in molecular marker-assisted breeding of Chinese mitten crab.

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