CN113981112B - InDel marker C3082 for identifying ammonia nitrogen tolerance character of portunus trituberculatus, primers and application thereof - Google Patents

InDel marker C3082 for identifying ammonia nitrogen tolerance character of portunus trituberculatus, primers and application thereof Download PDF

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CN113981112B
CN113981112B CN202111606841.3A CN202111606841A CN113981112B CN 113981112 B CN113981112 B CN 113981112B CN 202111606841 A CN202111606841 A CN 202111606841A CN 113981112 B CN113981112 B CN 113981112B
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portunus trituberculatus
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孟宪亮
王黛霞
刘晓晨
张景琰
吕建建
刘萍
李健
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Abstract

The invention relates to an InDel marker C3082 for identifying ammonia nitrogen tolerance character of portunus trituberculatus, a primer and application thereof. Belongs to the technical field of DNA molecular markers, and the molecular marker C3082 is an InDel marker, the nucleotide sequence of which is shown as SEQ ID No.1, and the deletion homozygosis is an ammonia nitrogen tolerant 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 using the molecular marker C3082. The molecular marker C3082 and the amplification primer thereof can identify and screen ammonia nitrogen-resistant portunus trituberculatus individuals from a molecular level, the method is simple, accurate and reliable, and the breeding process of new ammonia nitrogen-resistant portunus trituberculatus varieties is effectively accelerated.

Description

InDel marker C3082 for identifying ammonia nitrogen tolerance character of portunus trituberculatus, primers and application thereof
Technical Field
The invention belongs to the technical field of DNA molecular markers, and particularly relates to an Indel molecular marker C3082 for identifying ammonia nitrogen tolerance character of portunus trituberculatus, a primer and application thereof.
Background
Portunus trituberculatus (Portugulus trituberculatus) belongs to Arthropoda, Crustacea, decapod order, Paralithodes, 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 very popular with domestic and foreign consumers. 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 continuously increased, and the bait investment is continuously increased, so that the nitrogen source in the culture water body 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 survival rate of the culture and the culture benefit.
Insertion-deletion (InDel) markers refer to insertions or deletions of nucleotide fragments of different sizes in sequences at the same site of the genome between closely related species or between different individuals of the same species. The InDel marker has the advantages of low development cost, abundant polymorphism, stable variation and the like. In addition, the method is essentially a length polymorphism molecular marker, can be used for rapid typing through PCR amplification and agarose electrophoresis, and is simple and high in accuracy. At present, InDel markers have been widely applied to molecular marker-assisted breeding. The development of molecular markers 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 C3082 for identifying ammonia nitrogen tolerance character of portunus trituberculatus, a primer and application thereof. The molecular marker C3082 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 an InDel mark C3082 for identifying the ammonia nitrogen tolerance character of blue crab, wherein the nucleotide sequence of the InDel mark C3082 is shown as SEQ ID No. 1. The ammonia nitrogen tolerant genotype of the molecular marker C3082 is deletion homozygote, namely 202 nd to 222 th base deletion of SEQ ID No. 1.
The invention also provides the identifying primer of the molecular marker C3082, and the nucleotide sequence of the primer is shown as SEQ ID No.2 and SEQ ID No. 3.
The invention also provides application of the identifying primer in screening of portunus trituberculatus varieties with ammonia nitrogen tolerance characters.
Further, the screening step is as follows: extracting DNA of a to-be-detected sample of the portunus trituberculatus to be used as a template, carrying out PCR amplification by using an identifying primer C3082-Forward and a C3082-Reverse of the molecular marker C3082, carrying out agarose gel electrophoresis on an obtained PCR product, and if the genotype of the molecular marker C3082 in an electrophoresis result is deletion homozygous type and has an amplification strip at a position of 99bp, the to-be-detected sample of the portunus trituberculatus has ammonia nitrogen tolerance character.
Further, the PCR amplification conditions are as follows: pre-denaturation at 94 ℃ for 30S; denaturation at 98 ℃ for 10S, annealing at 55 ℃ for 30S, and extension at 72 ℃ for 30S, and repeating 35 cycles.
The invention also provides application of the identifying primer in preparing a detection reagent for screening the portunus trituberculatus ammonia nitrogen tolerant variety.
The invention also provides a kit for detecting the ammonia nitrogen tolerance of the portunus trituberculatus, and the kit comprises the primer.
Compared with the prior art, the invention has the following advantages and beneficial effects:
1. the InDel molecular marker C3082 is closely related to the ammonia nitrogen resistance of the portunus trituberculatus, the molecular marker C3082 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 environment 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 agarose gel electrophoresis 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 C3082, the primer pair designed by the molecular marker and the identification method have wide application prospects.
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FIG. 1 is a gel electrophoresis result of PCR products after amplification of genomic DNAs of an ammonia nitrogen tolerant population and an ammonia nitrogen sensitive population by using two primers of SEQ ID No.2 and SEQ ID No. 3; wherein M is a standard molecular weight; 1-20 are ammonia nitrogen sensitive groups; 21-40 are ammonia nitrogen tolerant groups.
Detailed Description
The technical solution of the present invention will be described in further detail with reference to specific examples.
Example 1
The portunus trituberculatus used by the invention is taken from a downstream proliferation station of the Chinese aquatic science research institute, 200 portunus trituberculatus crabs with the weight of 50 +/-5 g, good vitality and no scars are selected and placed in 4 indoor culture ponds (500cm multiplied by 300cm multiplied by 150cm) for temporary culture for 7 d. During the temporary culture period, the water temperature is kept at 23 + -1 deg.C, the salinity is 30 + -1, the pH is 8.0 + -0.3, the ammonia nitrogen concentration is lower than 0.01mg/L, the water depth is 20cm, and oxygen is continuously supplied. The filtered seawater is changed at 8 am for about 1/2 a day, and fresh trash fish is fed in the afternoon, wherein the feeding amount is about 1/5 of the total weight of the swimming 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 tank4Cl) is adjusted to 80mg/L, and other water quality conditions are kept consistent with those during temporary culture. And (5) the ammonia nitrogen in the seawater is maintained at 80 mg/L48 h before the stress, and the death time of the swimming crabs is recorded every 2 h. After 48 hours, gradually increasing the ammonia nitrogen concentration of the seawater by 40mg/L every two days. The number of surviving individuals in the culture pond was recorded and the experiment was terminated until 20 portunids remained. In the ammonia nitrogen stress process, the 20 individuals which die at first are considered AS an ammonia nitrogen sensitive group (marked AS an AS group), the 20 individuals which survive at last are considered AS an ammonia nitrogen tolerant group (marked AS an AR group), muscle tissues are dissected and placed in a freezing storage tube, quick-frozen in liquid nitrogen, and stored in a refrigerator at the temperature of minus 80 ℃.
Example 2
Screening of ammonia nitrogen resistance character related molecular marker
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 about 25mg of Portunus trituberculatus individual, putting the muscle tissues into a centrifugal tube filled with 200 mu l of buffer GA, carrying out vortex oscillation for 15S, adding 20 mu l of protease K (20mg/ml), carrying out vortex mixing, standing 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 10min 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 30S, pouring off waste liquid, and putting the adsorption column CB3 back into the collecting pipe; to adsorption column CB3, 500. mu.l of buffer GD was added, centrifuged at 12,000rpm for 30S, the waste liquid was discarded, and adsorption column CB3 was put into the collection tube.
(4) To adsorption column CB3, 600. mu.l of rinsing solution PW was added, centrifuged at 12,000rpm for 30S, the waste solution was discarded, and adsorption column CB3 was put into the collection tube, and the procedure was repeated.
(5) The adsorption column CB3 was returned to the collection tube, centrifuged at 12,000rpm (13,400 Xg) for 2min, 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. The adsorption column CB3 is transferred into a clean centrifuge tube, 100 mu l of elution buffer TE is suspended and dripped into the middle part of the adsorption membrane, the mixture is placed for 5min at room temperature and centrifuged for 2min at 12,000rpm, and the DNA solution is collected into the centrifuge tube.
(6) Analyzing the integrity of the DNA by agarose gel electrophoresis to determine whether RNA and protein are polluted; detecting the purity and concentration (OD260/280 ratio) of the DNA by using Nanodrop; the DNA concentration was accurately quantified using the 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). Randomly breaking the genome DNA into fragments with the length of 350bp by a Covaris breaker, repairing the tail ends, adding A tails, and then respectively connecting joints at two ends of the fragments to prepare a DNA library. After the library is constructed, firstly, Qubit 2.0 is used for preliminary quantification, then Agilent2100 is used for detecting the insetsize of the library, and after the insetsize meets the expectation, the qPCR method is used for accurately quantifying the effective concentration of the library so as 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
Figure GDA0003504695410000051
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. Detecting the InDel locus by adopting Genome Analysis Toolkit (GATK) software, annotating the detected InDel locus by utilizing Annovar software, and determining information such as a mutation locus and a corresponding gene. The information of the InDel sites obtained is shown in Table 2.
Table 2 InDel detection statistics and site information
Figure GDA0003504695410000061
InDel marker screening
Respectively calculating InDel-index of each site of the ammonia nitrogen tolerant group and the sensitive group, 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) sites with InDel-index less than 0.2 and depth less than 7;
(2) sites less than 7 deep and heterozygous for the genotype.
Meanwhile, calculating InDel frequency difference distribution, filtering the sites with the delta index less than 0.5, wherein the delta index is an index (ammonia nitrogen tolerance character) -index (ammonia nitrogen sensitivity character), and finally obtaining 173 InDel with difference among groups. The Δ indexes are arranged in order from high to low, and the first 20 InDel marks are selected for verification.
Second, verifying ammonia nitrogen resistance character related molecular marker
By PCR amplification and agarose gel electrophoresisThe method of (1) verifies the related molecular markers in the ammonia nitrogen tolerant population and the ammonia nitrogen sensitive population. Specific primers were designed on both sides of the labeled sites using Primer 3.0 on-line system. And carrying out PCR amplification by using the designed specific primers and using the genomic DNA of the ammonia nitrogen tolerant group and the ammonia nitrogen sensitive group as templates. The PCR reaction system was 25. mu.l, containing: template (concentration about 100 ng/. mu.l) 1. mu.l, forward primer (10. mu.M) 0.5. mu.l, reverse primer (10. mu.M) 0.5. mu.l, 2 XPRO Taq Master Mix 12.5. mu.l, RNase free H2O10.5. mu.l. The PCR amplification program is set as follows: pre-denaturation at 94 ℃ for 30S; denaturation at 98 ℃ for 10S, annealing at 55 ℃ for 30S, and extension at 72 ℃ for 30S, and repeating 35 cycles. The PCR product was detected by 4% agarose gel electrophoresis and the genotype was determined from the bands. Through SPSS 19.0 software, the genotype frequency of the InDel locus of the difference between the ammonia nitrogen tolerant group and the ammonia nitrogen sensitive group is subjected to difference detection by chi-square detection, and if P is less than 0.05, the difference is regarded as significant difference, otherwise, the difference is regarded as not.
As shown in Table 3 and FIG. 1, in the C3082_12048 (C3082 for short) locus, the proportion of deletion homozygous genotype (one band at 99 bp) in the ammonia nitrogen tolerant population was 70%, and the proportion of insertion homozygous genotype (one band at 120bp only) in the ammonia nitrogen sensitive population was 50%. Chi-square test analysis results show that the group of data loci have significant differences (P <0.05) in a tolerance group and a sensitive group, which indicates that the marker has significant correlation with ammonia nitrogen tolerance traits, so that the locus is considered to be deficient and homozygous into ammonia nitrogen tolerance genotypes. The ammonia nitrogen resistant molecular marker C3082 of the blue crab is obtained, the nucleotide sequence of the molecular marker C3082 is shown in SEQ ID No.1, the partial electrophoresis result is shown in figure 1, wherein the nucleotide sequences of the amplification primers C3082-Forward and C3082-Reverse for amplifying the molecular marker are shown in SEQ ID No.2 and SEQ ID No.3 (see Table 4 for details).
TABLE 3 genotype results for C3082 molecular markers
Figure GDA0003504695410000071
TABLE 4 amplification primers and product lengths for molecular markers
Figure GDA0003504695410000072
The molecular marker C3082 obtained by the invention can be used for assisting in breeding the portunus trituberculatus ammonia nitrogen tolerant variety, 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 the amplification primer nucleotide sequences of the molecular marker C3082 such as C3082-Forward and C3082-Reverse shown in SEQ ID No.2 and SEQ ID No.3, the PCR product is subjected to 4% agarose gel electrophoresis, and if the genotype of the molecular marker C3082 in the electrophoresis result is deletion homozygous (only one band is arranged at the position of 99 bp), the sample to be tested can be selected as a parent for breeding the portunus trituberculatus ammonia nitrogen tolerant variety.
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
<110> research institute for aquatic products in yellow sea of China institute for aquatic science
<120> InDel mark C3082 for identifying ammonia nitrogen tolerance character of portunus trituberculatus, primers and application thereof
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 401
<212> DNA
<213> Portunus trituberculatus (Portulus trituberculatus)
<400> 1
gaggcgtgga gttggagcct tgtggagtgg tcagcggtgg agcgaggtgg agcagggtgg 60
agtggggcgt ggggcggggc atcactgagc gggcgtgggt tgaagagcac tgttgtacga 120
gtcataaaac aaatcattaa gaattaattg accttcgagg gaagcgatca ccatcaattg 180
cccgtgctag cttgaagaga gggaggagga ggaggaggag gaggaggagg aggaggattt 240
taacgaacat agaagagcaa aaaaggaaga acaaacagca ctggatttgt tgctccttat 300
aatgttattt atgatgatat actctccaga ctaagataaa aggtgttgga tactaaagtt 360
aaatgttcct ccccacccac cgttgcacca tccaccatct g 401
<210> 2
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
gatcaccatc aattgcccgt g 21
<210> 3
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
tccagtgctg tttgttcttc ct 22

Claims (6)

1. An InDel mark C3082 for identifying the ammonia nitrogen tolerance character of portunus trituberculatus is characterized in that the nucleotide sequence of the InDel mark C3082 is shown in SEQ ID No. 1; the ammonia nitrogen tolerant genotype of InDel marker C3082 is deletion homozygous, i.e. base 202 to 222 of SEQ ID No.1 is deleted.
2. The InDel marker C3082 identifying primer as claimed in claim 1, wherein the nucleotide sequence of the identifying primer is shown as SEQ ID No.2 and SEQ ID No. 3.
3. The application of the identifying primer according to claim 2 in screening of portunus trituberculatus varieties with ammonia nitrogen tolerance traits.
4. Use according to claim 3, characterized in that the screening step is: extracting DNA of a portunus trituberculatus sample to be detected as a template, performing PCR amplification by using identifying primers shown in SEQ ID No.2 and SEQ ID No.3, performing agarose gel electrophoresis on an obtained PCR product, and if the genotype of the marker C3082 in the electrophoresis result is deletion homozygous type and has an amplification strip at 99bp, the portunus trituberculatus sample to be detected has ammonia nitrogen tolerance.
5. The use according to claim 4, characterized in that the PCR amplification conditions are: pre-denaturation at 94 ℃ for 30S; denaturation at 98 ℃ for 10S, annealing at 55 ℃ for 30S, and extension at 72 ℃ for 30S, and repeating 35 cycles.
6. The application of the identifying primer according to claim 2 in preparing a detection reagent for portunus trituberculatus ammonia nitrogen tolerant varieties.
CN202111606841.3A 2021-12-27 2021-12-27 InDel marker C3082 for identifying ammonia nitrogen tolerance character of portunus trituberculatus, primers and application thereof Expired - Fee Related CN113981112B (en)

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Denomination of invention: InDel marker C3082, primer and its application in identification of ammonia nitrogen tolerance of Portunus trituberculatus

Granted publication date: 20220322

License type: Common License

Record date: 20221123

CF01 Termination of patent right due to non-payment of annual fee

Granted publication date: 20220322