CN113789394B - Molecular marker C13 for identifying ammonia nitrogen tolerance character of portunus trituberculatus and application thereof - Google Patents
Molecular marker C13 for identifying ammonia nitrogen tolerance character of portunus trituberculatus and application thereof Download PDFInfo
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Abstract
The invention provides a molecular marker C13 for identifying ammonia nitrogen tolerance character of portunus trituberculatus and application thereof. The molecular marker C13 is an SNP marker, the nucleotide sequence of the SNP marker is shown as SEQ ID No.1, the 590 th base is A or G, and the ammonia nitrogen tolerance genotype is AA genotype. The invention also designs a pair of effective amplification primers with nucleotide sequences shown as SEQ ID No.2 and SEQ ID No.3 by utilizing the molecular marker C13. The molecular marker C13 and the amplification primer thereof can identify and screen individual portunus trituberculatus resistant to ammonia nitrogen from molecular level, the method is simple, accurate and reliable, and the breeding process of new species of portunus trituberculatus resistant to ammonia nitrogen is effectively accelerated.
Description
Technical Field
The invention belongs to the technical field of DNA molecular markers, and particularly relates to a molecular marker C13 for identifying ammonia nitrogen tolerance traits of portunus trituberculatus and application thereof.
Background
Portunus trituberculatus (blue crab)Portunus trituberculatus) Belongs to Arthropoda, crustacea, decapod order, halocyanidae, and genus Thieladaceae, and is an important marine economic crab mainly distributed in Bohai sea, yellow sea, and east sea. The meat is delicious and rich in nutrition, contains protein, vitamin A, B1, B12, nicotinic acid, calcium, phosphorus, iron and other elements, and is popular with consumers at home and abroad. Since the last 90 s, the artificial culture of portunus trituberculatus has rapidly developed in China, and the yield reaches 11.38 ten thousand tons in 2019. However, with the increase of the culture intensification degree, the culture density is increased, and the bait investment is increasedSo that the nitrogen source in the aquaculture water is excessive. Excess organic nitrogen can be converted to inorganic nitrogen. Ammonia nitrogen is considered to be the most toxic inorganic nitrogen in the aquaculture water. The ammonia nitrogen accumulation can obviously inhibit the growth of portunus trituberculatus, cause tissue damage and even lead to mass death of individuals. Therefore, the ammonia nitrogen tolerance character is one of the important breeding characters of the portunus trituberculatus, and is very important for improving the breeding survival rate and the breeding benefit.
Molecular markers are genetic markers that reflect genetic polymorphisms at the molecular level of DNA between individuals. As a third generation molecular marker, single Nucleotide Polymorphism (SNP) has the characteristics of high abundance, high density, strong stability, co-dominance and the like, and has been widely applied to molecular marker assisted breeding. At present, molecular markers of ammonia nitrogen tolerance of the blue crab are not reported. Therefore, the development of the molecular marker related to the ammonia nitrogen resistance character has important significance for the genetic breeding of the portunus trituberculatus.
Disclosure of Invention
The invention provides a molecular marker C13 for identifying ammonia nitrogen tolerance character of portunus trituberculatus and application thereof. The molecular marker C13 can accurately and efficiently identify the ammonia nitrogen tolerance character of the portunus trituberculatus.
In order to realize the purpose of the invention, the invention adopts the following technical scheme to realize:
the invention provides a molecular marker C13 for identifying ammonia nitrogen tolerance character of portunus trituberculatus, and the nucleotide sequence of the molecular marker C13 is shown in SEQ ID No. 1.
Further, the 590 th base in the nucleotide sequence of the molecular marker C13 is A or G.
Further, the ammonia nitrogen tolerance genotype of the molecular marker C13 is an AA genotype.
Further, the molecular marker C13 is an SNP marker.
The invention also provides an amplification primer of the molecular marker C13, and the nucleotide sequence of the amplification primer is shown as SEQ ID No.2 and SEQ ID No. 3.
The invention also provides application of the molecular marker C13 in screening of portunus trituberculatus varieties with ammonia nitrogen tolerance characters.
Further, the screening step is: extracting DNA of a portunus trituberculatus sample to be detected as a template, performing PCR amplification by using amplification primers C13-Forward and C13-Reverse of the molecular marker C13, sequencing the obtained PCR product, and if the genotype of the molecular marker C13 in the sequencing result is AA, determining that the portunus trituberculatus sample to be detected has ammonia nitrogen tolerance character.
Further, the PCR amplification conditions are as follows: 94. pre-denaturation at 30 sec; 98. denaturation at 55 ℃ for 10 sec, annealing at 55 ℃ for 15 sec, and extension at 72 ℃ for 10 sec, and 35 cycles were repeated.
The invention also provides application of the amplification primer in preparation of a reagent for screening the portunus trituberculatus ammonia nitrogen tolerant variety.
Compared with the prior art, the invention has the following advantages and beneficial effects:
1. the SNP marker C13 is closely related to the ammonia nitrogen resistance of the portunus trituberculatus, the molecular marker C13 can identify and screen individual portunus trituberculatus with ammonia nitrogen resistance from a molecular level, the method is simple, accurate and reliable, is not limited by external environmental conditions and the growth and development stage of the individual, and can be used for selecting in an early stage, so that the breeding process of new species of portunus trituberculatus with ammonia nitrogen resistance is effectively accelerated.
2. The identification method disclosed by the invention is strong in practicability, has no specific requirements on the extraction method of the genomic DNA of the portunus trituberculatus and the DNA sequencing method, is simple and efficient, has low detection cost and wide applicability, can be used for screening individuals with ammonia nitrogen resistance in a large range, and effectively reduces the breeding cost. Therefore, the molecular marker C13, the primer pair designed by the molecular marker and the identification method have wide application prospects.
Drawings
FIG. 1 shows the partial sequencing result of the molecular marker C13.
Detailed Description
The technical solution of the present invention is further described in detail with reference to the specific embodiments.
The portunus trituberculatus used by the invention is all taken from a downstream proliferation station of the Chinese academy of aquatic science, 200 portunus trituberculatus crabs with the weight of 50 +/-5 g, good vitality and no scar are selected and put in 4 indoor culture ponds (500 cm multiplied by 300cm multiplied by 150 cm) for temporary culture for 7 days. During the temporary culture period, the water temperature is kept at 23 +/-1 ℃, the salinity is 30 +/-1, the pH is 8.0 +/-0.3, the ammonia nitrogen concentration is lower than 0.01 mg/L, the water depth is 20 cm, and oxygen is continuously supplied. Replacing filtered seawater at 8 am every day by about 1/2, and feeding fresh trash fish in the afternoon, wherein the feeding amount is about 1/5 of the total weight of the blue crabs.
After the temporary rearing is finished, 200 swimming crabs are randomly distributed into 4 indoor rearing ponds, and 50 swimming crabs are placed in each rearing pond. Ammonium chloride (NH) is used for seawater ammonia nitrogen concentration in the tank 4 Cl) is adjusted to 80 mg/L, and other water quality conditions are kept consistent with those in the temporary culture period. And (5) the ammonia nitrogen of the seawater is maintained at 80 mg/L in the first 48h of the ammonia nitrogen stress, and the death time of the portunus trituberculatus is recorded every 2 h. After 48h, gradually increasing the ammonia nitrogen concentration of the seawater by 40 mg/L every two days, recording the number of the living individuals of the blue crabs in the culture pond, and terminating the experiment until 20 blue crabs remain. In the ammonia nitrogen stress process, the first dead 20 blue crab individuals are considered AS an ammonia nitrogen sensitive group (marked AS an AS group), and the last alive 20 individuals are considered AS an ammonia nitrogen tolerant group (marked AS an AR group). Dissecting the portunus trituberculatus individual, taking muscle tissue, placing in a freezing tube, quickly freezing in liquid nitrogen, and storing in a refrigerator at-80 deg.C for use.
Example 1
1. Screening of molecular markers related to ammonia nitrogen resistance
1. Library construction, sequencing and data analysis
The method adopts a marine animal tissue genome DNA extraction kit of Tiangen Biotechnology limited company to extract genome DNA through a silicon substrate material adsorption column. The method comprises the following specific steps:
(1) Taking muscle tissues of Portunus trituberculatus of about 25 mg, putting the muscle tissues into a centrifuge tube filled with 200 ul of buffer GA, carrying out vortex oscillation for 15 sec, adding 20 ul of Proteinase K (20 mg/ml) solution, carrying out vortex mixing, placing at 56 ℃ until the tissues are completely dissolved, and carrying out brief centrifugation to remove water drops on the inner wall of a tube cover.
(2) Adding 200 μ l buffer GB, mixing thoroughly, standing at 70 deg.C for 10 min until the solution becomes clear, adding 200 μ l anhydrous ethanol, and mixing thoroughly.
(3) Adding the solution and flocculent precipitate obtained in the previous step into an adsorption column CB3, centrifuging at 12,000rpm for 30 sec, pouring off waste liquid, and putting the adsorption column CB3 back into the collecting pipe; to the adsorption column CB3, 500. Mu.l of buffer GD was added, centrifuged at 12,000rpm for 30 sec, the waste liquid was discarded, and the adsorption column CB3 was put into the collection tube.
(4) To the adsorption column CB3, 600. Mu.l of the rinsing liquid PW was added, centrifuged at 12,000rpm for 30 sec, the waste liquid was discarded, and the adsorption column CB3 was put into a collection tube, and this step was repeated.
(5) The adsorption column CB3 was returned to the collection tube, centrifuged at 12,000rpm (-13,400 Xg) for 2 min, and the waste liquid was discarded. The adsorption column CB3 was left at room temperature for several minutes to completely dry the residual rinse solution in the adsorption material. Transferring the adsorption column CB3 into a clean centrifuge tube, suspending and dripping 100 mu l of elution buffer TE into the middle part of the adsorption membrane, standing at room temperature for 5 min, centrifuging at 12,000rpm for 2 min, and collecting the DNA solution into the centrifuge tube.
(6) Analyzing the integrity of the DNA by agarose gel electrophoresis to determine whether the DNA is polluted by RNA and protein; detecting the purity and concentration (OD 260/280 ratio) of the DNA by using Nanodrop; the DNA concentration was accurately quantified using Qubit.
And equivalently mixing the qualified genomic DNA samples into two mixing tanks, namely an ammonia nitrogen tolerant group DNA mixing tank (AR) and an ammonia nitrogen sensitive group DNA mixing tank (AS). The genomic DNA was randomly fragmented into fragments of length 350 bp using a Covaris disruptor, and after end repair and A-tailing, linkers were attached to both ends of the fragments to prepare DNA libraries. After the library is constructed, firstly, qubit 2.0 is used for preliminary quantification, then Agilent2100 is used for detecting the insertsize of the library, and after the insertsize meets the expectation, the qPCR method is used for accurately quantifying the effective concentration of the library to ensure the quality of the library. And performing Illumina HiSeq2500 platform PE150 sequencing after the library inspection is qualified. The original image data file obtained by high-throughput sequencing is converted into raw reads through CASAVA Base recognition (Base Calling) analysis. And (3) filtering raw reads, removing reads containing the joint and the reads with the proportion of N being more than 10% to obtain clean reads for subsequent data analysis, and detailed sequencing data results are shown in table 1.
TABLE 1 summary of sequencing data quality
| Group of | Raw data (bp) | Filtered data (bp) | Effective rate (%) | Error Rate (%) | Q30 (%) |
| AS | 29,420,319,900 | 28,274,686,500 | 99.65 | 0.032 | 86.77 |
| AR | 24,673,666,800 | 23,884,505,700 | 99.72 | 0.032 | 87.00 |
2. Marker detection and annotation
Clean reads were aligned to the reference genome using Burrows-Wheeler alignment tool (BWA) software, format converted and sorted using Samtools software. Adopting Genome Analysis Toolkit (GATK) software Call SNP, and utilizing Annovar software to annotate SNP sites and determine information such as variant sites and corresponding genes. The obtained SNP site information is shown in Table 2.
TABLE 2 SNP detection statistics and site information
| Species of | Number of SNPs |
| Upstream of | 6,458 |
| Downstream | 5,168 |
| Exon(s) | 5,227 |
| Intron | 80,316 |
| Intergenic region | 274,726 |
| Total up to | 371,895 |
3. SNP frequency difference analysis and marker screening
Calculating SNP-index of each site of the ammonia nitrogen tolerant group and the sensitive group respectively, and filtering polymorphic sites to reduce the influence caused by sequencing errors and comparison errors as far as possible, wherein the filtering standard is as follows:
(1) A site with SNP-index less than 0.2 and SNP depth less than 7;
(2) Sites less than 7 deep and heterozygous for the genotype.
And calculating SNP frequency difference distribution, filtering the sites with the delta index smaller than 0.4, and obtaining 362 SNP of the difference among groups by the delta index = index (ammonia nitrogen tolerance character) -index (ammonia nitrogen sensitivity character). The Δ indexes are arranged in order from high to low, and the first 50 SNPs are selected for validation.
2. Ammonia nitrogen resistance character related molecular marker verification
And verifying the related molecular markers in the ammonia nitrogen tolerant group and the ammonia nitrogen sensitive group by adopting a PCR product DNA sequencing method. Specific primers are designed on two sides of the marked site by using a Primer 3.0 online system, and at least one Primer is ensured to be not less than 80 bp away from the marked site. And performing PCR amplification by using the designed specific primers and using mixed genome DNA of the ammonia nitrogen tolerant group and the ammonia nitrogen sensitive group as templates. The PCR reaction system was 25. Mu.l, containing: mu.l template (concentration of about 100 ng/. Mu.l), 0.5. Mu.l forward primer (10. Mu.M), 0.5. Mu.l reverse primer (10. Mu.M), 12.5. Mu.l 2X ApexHF FS PCR Master Mix, RNase free H 2 O10.5. Mu.l. The PCR amplification program is set as follows: 94. pre-denaturation at 30 sec; 98. denaturation at 55 ℃ for 10 sec, annealing at 55 ℃ for 15 sec, and extension at 72 ℃ for 10 sec, 35 cycles were repeated. The PCR product was detected by 1% agarose gel electrophoresis, and single and clear samples were selected and sent to Biotechnology engineering, inc. for DNA sequencing.
Analyzing the sequencing peak map by using contigeexpress software, selecting two groups of primers with obvious difference in corresponding positions of the sensitive population mixed template and the tolerant population mixed template, continuously using the primers, carrying out PCR amplification by using individual DNA as a template, keeping the amplification condition unchanged, selecting a bright band with a consistent size, and sending the band to a userAnd (5) sequencing. Through SPSS 19.0 software, the genotype frequency of the SNP locus of the difference between the ammonia nitrogen tolerant group and the ammonia nitrogen sensitive group is subjected to difference detection by using chi-square detection,P<0.05 was considered to be a significant difference, whereas no significant difference was considered.
According to the table 3 and fig. 1, there are 3 genotypes at the C13 locus, wherein the genotype is that AA accounts for 60.0% in the ammonia nitrogen tolerant population, and the genotype is that GG accounts for 55.0% in the ammonia nitrogen sensitive group (table 3). The ammonia nitrogen tolerant group and the sensitive group have significant difference at the site (P<0.05 Showing that the marker has significant correlation with the ammonia nitrogen tolerance character, therefore, the genotype AA at the site can be considered as the ammonia nitrogen tolerance genotype. Thus obtaining the ammonia nitrogen resistant molecular marker C13 of the blue crab, wherein the nucleotide sequence of the molecular marker C13 is shown in SEQ ID No.1, the partial sequencing result is shown in figure 1, and the nucleotide sequences of the primers C13-Forward and C13-Reverse of the amplified molecular marker C13 are shown in SEQ ID No.2 and SEQ ID No.3 (see the table 4 for details).
TABLE 3 genotype results for the molecular marker C13
TABLE 4 amplification primers for molecular markers
The molecular marker C13 obtained by the invention can be used for assisting in breeding portunus trituberculatus ammonia nitrogen tolerant varieties, the application steps are simple, only DNA of a sample to be tested of the portunus trituberculatus needs to be extracted and used as a template, PCR amplification is carried out by using C13-Forward and C13-Reverse with the nucleotide sequences of amplification primers of the molecular marker C13 shown in SEQ ID No.2 and SEQ ID No.3, a PCR product is sequenced, and if the genotype of the molecular marker C13 in the sequencing result is AA, the sample to be tested can be selected as a parent for cultivating the portunus trituberculatus ammonia nitrogen tolerant varieties.
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions.
Sequence listing
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Claims (4)
1. A molecular marker C13 for identifying the ammonia nitrogen tolerance character of Portunus trituberculatus is characterized in that the nucleotide sequence of the molecular marker C13 is shown in SEQ ID No. 1; the 590 th base in the nucleotide sequence of the molecular marker C13 is A or G, and the ammonia nitrogen tolerance genotype of the molecular marker C13 is AA genotype.
2. The application of the molecular marker C13 in screening of blue crab varieties with ammonia nitrogen tolerance traits as claimed in claim 1, wherein the screening comprises the following steps: extracting DNA of a portunus trituberculatus sample to be detected as a template, performing PCR amplification by using the amplification primer of the molecular marker C13, sequencing the obtained PCR product, and if the genotype of the molecular marker C13 in the sequencing result is AA, determining that the portunus trituberculatus sample to be detected has the ammonia nitrogen tolerance character.
3. The use according to claim 2, wherein the PCR amplification conditions are: 94. pre-denaturation at 30 sec; 98. denaturation at 55 ℃ for 10 sec, annealing at 55 ℃ for 15 sec, and extension at 72 ℃ for 10 sec, 35 cycles were repeated.
4. The application of the amplification primer of the molecular marker C13 in the preparation of the reagent for screening the portunus trituberculatus ammonia nitrogen tolerant varieties is characterized in that the nucleotide sequence of the amplification primer is shown in SEQ ID No.2 and SEQ ID No. 3.
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| Portunus trituberculatus isolate SZX2019 chromosome 45, whole genome shotgun sequence;anonymity;《Genbank数据库》;20210326;第1页 * |
| 三疣梭子蟹生长相关SNP位点的鉴定;张德宁等;《中国水产科学》;20150515(第03期);第393-401页 * |
| 氨氮胁迫对三疣梭子蟹酚氧化酶原系统和免疫指标的影响;岳峰等;《中国水产科学》;20100715(第04期);第761-770页 * |
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