CN113604587A - Molecular marker T5198 for rapidly identifying low-temperature tolerant variety of penaeus japonicus and application thereof - Google Patents
Molecular marker T5198 for rapidly identifying low-temperature tolerant variety of penaeus japonicus and application thereof Download PDFInfo
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Abstract
The invention provides a molecular marker T5198 for rapidly identifying a low-temperature tolerant variety of penaeus japonicus and application thereof. The nucleotide sequence of the molecular marker T5198 is shown as SEQ ID No.1, and the nucleotide sequence of the primer for detecting the molecular marker T5198 is shown as SEQ ID No.2 and SEQ ID No. 3. The molecular marker T5198 is Indel marker, and the low-temperature tolerant genotype is deletion homozygous genotype. The molecular marker T5198 provided by the invention can be free from the limitation of the growth stage of the penaeus japonicus, obviously quickens the breeding process of the penaeus japonicus and quickly breeds a new penaeus japonicus species with excellent low temperature tolerance character, and the molecular marker T5198 is accurate and reliable in low temperature tolerance detection and simple in operation, is beneficial to the healthy culture of the penaeus japonicus and has wide application prospect.
Description
Technical Field
The invention belongs to the field of molecular marker assisted breeding of aquatic animals, and particularly relates to a molecular marker T5198 for rapidly identifying a low-temperature tolerant variety of penaeus japonicus and application thereof.
Background
Japanese prawn (Marsupenaeus japonicus) Originally called Japanese prawn, belonging to the order of ten-foot, the family of prawns, the genus of marsupenaeus, distributed in the sea areas of India-Western Pacific tropical zone, African east coast, Malaysia, Japan, Korea, China southeast coast and the like, is one of the main prawn breeding varieties in China, and the Japanese prawn grows rapidly, is resistant to dew, bright in color and high in economic value, is popular with water product markets and breeders, and is bred in provinces and cities from south China to northern coast, and forms a certain scale. The improved breeding has great significance for the breeding industry of the penaeus vannamei boone. Low temperature resistance is an important stress resistance character of the penaeus japonicus. On one hand, because the penaeus japonicus has higher market demand, the penaeus japonicus is mainly cultured in northern water areas in China, including northern provinces of seawater areas such as Shandong, Hebei, Jiangsu and the like, and low temperature resistance becomes an important stress resistance character pursued by the penaeus japonicus culture industry in northern China; on the other hand, in the spring festival of China, the market price of fresh and alive Japanese prawns is more than 3 times of that of ordinary seasons, farmers in northern areas and other places often risk to bring the time to market to winter, the cultured prawns are usually killed in a large scale due to sudden drop of water temperature caused by cold tide in winter, and the breeding of low-temperature-resistant varieties is also an urgent need of the Japanese prawn breeding industry.
The molecular marker is a genetic marker based on nucleotide sequence variation of genetic materials among individuals, and is a direct reflection of DNA level genetic polymorphism. The molecular marker has remarkable advantages: most molecular markers are co-dominant, and selection of recessive characters is very convenient; the genome variation is extremely abundant, and the number of molecular markers is almost unlimited; the DNA of different tissues at different stages of biological development can be used for marking analysis; the molecular marker detection method is simple and rapid. At present, the research on the development of low temperature resistant molecular markers of penaeus japonicus is less, and the industry lacks markers applicable to molecular marker assisted breeding. Therefore, the development of molecular markers related to low-temperature resistance is of great significance to the healthy culture and the accelerated breeding process of the penaeus japonicus.
Disclosure of Invention
The invention provides a molecular marker T5198 for rapidly identifying a low-temperature tolerant variety of penaeus japonicus and application thereof. The molecular marker T5198 can accurately and efficiently identify the low-temperature tolerant individuals of the penaeus japonicus, and is beneficial to the cultivation of the low-temperature tolerant penaeus japonicus and the development of the breeding industry.
In order to realize the purpose of the invention, the invention adopts the following technical scheme to realize:
the invention provides a molecular marker T5198 for rapidly identifying a low-temperature tolerant variety of Penaeus japonicus, wherein the nucleotide sequence of the molecular marker T5198 is shown as SEQ ID No. 1.
Further, the low-temperature tolerant genotype of the molecular marker T5198 is a deletion homozygous genotype.
Further, the molecular marker T5198 shows GTGTGAGTATGT sequence deletion at the 508 th base.
Further, the molecular marker T5198 is an Indel marker.
Furthermore, the 508 th base to the 519 th base in the molecular marker T5198 are deleted.
The invention also provides a primer for detecting the molecular marker T5198, which is characterized by comprising the following specific components:
T5198-F: CGAGGGAAACTCTAGTGATGG；
T5198-R: CACGCGCTTAAGTACACAAAT。
the invention also provides application of the molecular marker T5198 or the primer in identifying the low-temperature tolerant variety of the penaeus japonicus.
Further, the specific steps for identifying the low-temperature tolerant penaeus japonicus variety are as follows:
(1) extracting the genomic DNA of the individual penaeus japonicus;
(2) carrying out PCR amplification on the extracted genome DNA by using primers T5198-F and T5198-R to obtain an amplification product;
(3) sequencing the amplification product; if the homozygous gene deletion mutation appears in the sequencing result, the individual Penaeus japonicus is a low-temperature tolerant variety.
Further, if the molecular marker T5198 is subjected to GTGTGAGTATGT homozygous deletion in the sequencing result in the step (3), the individual Penaeus japonicus is a low-temperature tolerant variety; if the molecular marker T5198 does not have GTGTGAGTATGT homozygous deletion, the individual Penaeus japonicus is a low-temperature intolerant variety.
Further, the PCR amplification conditions are as follows: pre-denaturation at 94 deg.C for 5 min; denaturation at 94 deg.C for 30s, annealing at 55 deg.C for 30s, extension at 72 deg.C for 30s, and repeating for 32 cycles; the circulation extends for 72 ℃ for 7 min.
The invention also provides application of the molecular marker T5198 or the primer in genetic diversity analysis, germplasm identification and genetic map construction of the Japanese prawn.
Compared with the prior art, the invention has the following advantages and beneficial effects:
1. according to the invention, Indel mutation sites are screened by genome sequencing and bioinformatics analysis methods of a low-temperature tolerant group and a low-temperature intolerant group, and a low-temperature tolerant molecular marker T5198 of the penaeus japonicus is obtained by further identifying and screening, wherein the molecular marker T5198 can not be limited by the growth stage of the penaeus japonicus, can be used for breeding early-stage young penaeus japonicus and parents of the penaeus japonicus, and further obviously accelerates the breeding process of the penaeus japonicus and rapidly breeds a new penaeus japonicus variety with excellent low-temperature tolerant properties.
2. The molecular marker T5198 and the primers thereof provided by the invention are used for identifying the low-temperature-resistant property of the penaeus japonicus, the method is accurate and reliable, is simple to operate, can effectively and quickly screen out the property meeting the requirement, assists in early-stage short-time and low-cost breeding of the low-temperature-resistant penaeus japonicus, increases the number of excellent-quality penaeus japonicus, improves the breeding rate and breeding period of the penaeus japonicus, further improves the yield of the penaeus japonicus, and promotes the healthy propagation of the penaeus japonicus, so that the molecular marker T5198 has a wide application prospect.
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FIG. 1: according to the invention, two groups of corresponding position sequencing result difference peak maps of the low-temperature intolerant population and the low-temperature tolerant population are obtained.
FIG. 2: in the invention, 1 is the genotype of the low-temperature intolerant population, and 2 is the genotype of the low-temperature tolerant population.
Detailed Description
The technical solution of the present invention will be described in further detail with reference to specific examples.
The penaeus japonicus used in the invention is from the experimental base of Retzhei treasure Co., Ltd. 300 Japanese prawns with the weight of 10 +/-3 g are placed in 4 culture ponds (500 cm multiplied by 300 cm multiplied by 150 cm) for temporary culture for 7d, the water temperature is kept at 28 +/-1 ℃ during the temporary culture, water is added to the pond to reach 20 cm, the pH value is 8.2 +/-0.5, oxygen is continuously supplied, fresh seawater is replaced at 8 o 'clock in the morning, fresh clam meat is fed at 5 o' clock in the afternoon, and the feeding amount is about 10% of the weight of the prawns. And 7d, selecting the penaeus japonicus with good activity and complete body surface for subsequent experiments.
The low-temperature tolerance test is carried out on 300-tailed Japanese prawns under the stress condition of 10 +/-0.5 ℃, the individual tolerance time is counted, 20-tailed prawns which die first are taken as a low-temperature intolerant group (UT), and 20-tailed prawns which survive last are taken as a low-temperature tolerant group (T). Genomic DNAs of Penaeus japonicus in the low-temperature intolerant group and the low-temperature tolerant group were extracted using a Tiangen marine animal tissue genome extraction kit (TIANGEN, Tiangen Biochemical technology Co., Ltd.).
Example 1
Screening of low temperature-resistant related candidate molecular markers
1. Sequencing data filtering and alignment
Genomic DNA of Penaeus japonicus was extracted, and 20 individuals were used for each of the low-temperature-tolerant group and the low-temperature-intolerant group. The genomic DNA of the penaeus japonicus is extracted by using a centrifugal adsorption column which can be specifically combined with the DNA and a unique buffer solution system. First, approximately 30 mg of tissue sample was placed into a 1.5 mL sterile enzyme centrifuge tube, 200. mu.L of buffer GA and 20. mu.L of protease K solution were added, vortexed, and incubated at 56 ℃ until the tissue was completely lysed. Then 200. mu.L of buffer GB was added, mixed well by inversion, left at 70 ℃ for 10 min, the solution cleared and centrifuged briefly to remove inner wall water droplets. Adding 200 μ L of anhydrous ethanol, mixing thoroughly, adding flocculent precipitate, centrifuging briefly, adding the obtained solution and flocculent precipitate into adsorption column CB3 (adsorption column is placed in collecting tube), centrifuging at 12000 rpm for 30s, discarding waste liquid, and returning adsorption column CB3 to collecting tube. Add 500. mu.L of buffer GD to adsorption column CB3, centrifuge at 12000 rpm for 30s, discard waste, place adsorption column CB3 in the collection tube. 600. mu.L of the rinsing solution PW was added to the adsorption column CB3, and centrifuged at 12000 rpm for 30s, and the waste liquid was discarded, and the adsorption column CB3 was put into the collection tube. Discard the waste liquid (repeat once), add 700. mu.L of rinsing liquid PW, centrifuge at 12000 rpm for 30s, and discard the waste liquid. The reaction mixture was then air-separated at 12000 rpm for 2min to completely remove the residual reagents. The adsorption column CB3 was placed in a clean centrifuge tube, 40. mu.L of distilled water was added to the center of the column, and the column was allowed to stand at room temperature for 15min, centrifuged at 12000 rpm for 2min, and the DNA was eluted. The concentration and purity of the DNA was determined using a Nanodrop spectrophotometer.
And equivalently mixing the DNA samples qualified by the test into two mixing pools, and respectively naming the mixing pools as a low-temperature intolerant DNA mixing pool (UT) and a low-temperature tolerant DNA mixing pool (T). Randomly breaking a mixed DNA sample into fragments with the length of 350bp by a Covaris crusher, constructing a Library by adopting a TruSeq Library Construction Kit, and completing the preparation of the whole Library by the steps of end repair, ployA tail addition, sequencing joint addition, purification, PCR amplification and the like of the DNA fragments. The constructed library was sequenced by Illumina HiSeq PE 150.
And strictly filtering sequencing data to obtain high-quality clean data. And (4) counting the output data of the 40 samples, wherein the statistics comprise effective data after sequencing filtration, the number of the compared fragments and the like. The total sequencing data amount is 611.10 Gb, the high-quality clean data amount is 605.67 Gb, and the sequencing data results are shown in Table 1.
Table 1: summary of sequencing data quality
In the table: t stands for low temperature tolerance and UT stands for low temperature intolerance.
The filtered effective data are compared by Burrows-Wheeler alignment tool (BWA) software, and the comparison result is subjected to SAMTOOLS to remove duplication. The alignment result shows that the alignment rate of all samples is more than 85%, the average sequencing depth is more than 25 x, and the method can be used for subsequent analysis.
2. Marker detection and annotation
INDEL assays were performed using SAMTOOLS software. Detecting polymorphic sites in the population by using a Bayesian model, and obtaining high-quality INDELs through the following filtering and screening:
1) q20 quality control;
2) the INDEL loci are at least 5 bp apart from each other;
3) the support number (depth of coverage) of INDEL is between [ 1/3, 5] times the average depth.
INDEL detection results were then annotated with ANNOVAR. The results are shown in Table 2, and the INDEL results are statistics after filtering according to Depth (DP) greater than 4, deletion rate 0.2, and minimum allele frequency 0.05.
Table 2: INDEL test result statistics
| Grouping | Indel number |
| Upstream of | 27,922 |
| Exon(s) | 87 |
| Intron | 392,013 |
| Shear-connecting body | 224 |
| Downstream | 33,304 |
| Between genes | 1,132,068 |
| Total | 1,585,618 |
3. INDEL differential analysis
Analytical statistics were performed for INDEL-index at each locus in the low temperature tolerant and low temperature intolerant groups as follows:
var _ T = number of individuals with variation (heterozygous mutation 0/1, homozygous mutation 1/1)/number of individuals in the tolerant group;
var _ UT = number of individuals with variation (heterozygous mutation 0/1, homozygous mutation 1/1)/number of individuals in the non-tolerant group.
The INDEL frequency difference distribution is also calculated, with the following directions: Δ (index) = Var _ UT (low temperature tolerant group) -Var _ T (low temperature intolerant group), filtering out sites with Δ index absolute value less than 0.55, and finally obtaining 51 INDELs of difference between groups, and then verifying these markers.
Second, verification of low temperature resistance related molecular marker
Verifying the candidate molecular markers related to the low-temperature resistance in a low-temperature resistant population and an intolerant population by adopting a PCR product sequencing method:
(1) firstly, designing primers on flanking sequences of marker sites, wherein at least one primer is more than 70 bp away from the marker sites;
(2) carrying out PCR amplification by using the designed primers and respectively taking mixed DNA materials of a low-temperature tolerant group and an intolerant group as templates, sequencing the PCR products which are successfully amplified, and selecting the primer which is far away from the marking site by using a sequencing primer;
(3) analyzing the sequencing peak maps by using Bio-edit software, selecting a marker with a large difference between the sequencing peak maps of the low-temperature tolerant group and the non-tolerant group at the corresponding positions, and continuing to perform PCR amplification and sequencing analysis on the individual DNA template;
(4) the genotype of each individual was counted from the sequencing results and analyzed by SPSS software whether the markers correlated with the low temperature resistance profile.
The specific operation steps are as follows:
1. DNA mixing pool construction
Mixing 1 μ g of DNA of each individual, preparing DNA mixing pools of a low-temperature tolerant group and a non-tolerant group, diluting the DNA mixing pools to a final concentration of 200 ng/μ L, and containing 20 individuals in each mixing pool for subsequent experiments.
2. Primer design
The INDEL mutation sites are screened by comparing the genomic sequencing and bioinformatics analysis methods in the two populations, and then the low temperature resistant molecular markers are screened. Primers were designed at both ends of the candidate INDEL sites resistant to low temperature using Primer Premier 5.0 software.
The design criteria for the primers were:
(1) the annealing temperature of the primer is 55-60 ℃;
(2) the formation of stable dimer and hairpin structures between the primers and the primers is avoided as much as possible;
(3) the fragment size is greater than 200 bp.
The designed primer information is shown in Table 3:
table 3: primer information
3. Mixed template PCR amplification and electrophoresis
Mixed gDNA is used as a template, enzyme of Kangji century company is used for PCR amplification, and the reaction program is set as follows: pre-denaturation at 94 deg.C for 5 min; denaturation at 94 deg.C for 30s, annealing at 55 deg.C for 30s, extension at 72 deg.C for 30s, and repeating for 32 cycles; the circulation extends for 72 ℃ for 7 min. The PCR system was as follows:
| 2×Matermix | 10 μL |
| forward primer | 0.5 μL |
| Reverse primer | 0.5 |
| gDNA | |
| 2 μL | |
| ddH2O | 7 μL |
| Total volume | 20 μL |
The obtained PCR amplification product is subjected to agarose gel electrophoresis. Performing first-generation sequencing on the residual products; as a result, as shown in FIG. 1, the Japanese prawn was a low temperature tolerant individual if the sequencing peak pattern showed GTGTGAGTATGT homozygous deletion after 507 (or at the beginning of 508), and was a low temperature intolerant individual if the sequencing peak pattern showed no GTGTGAGTATGT homozygous deletion after 507 (or at the beginning of 508).
4. Low temperature tolerant and intolerant individuals PCR amplification and electrophoresis
Extracting tissue DNA of 40 Penaeus japonicus individuals as a template to carry out PCR, wherein PCR primers are as follows:
T5198-F: CGAGGGAAACTCTAGTGATGG；
T5198-R: CACGCGCTTAAGTACACAAAT。
the PCR system and amplification conditions were the same as described above. After amplification, the individual template PCR products obtained were subjected to 1% agarose gel electrophoresis. The remaining PCR products were then sequenced, the results of which are shown in figure 2.
The low-temperature tolerant genotype of the molecular marker T5198 is a deletion homozygous genotype, the nucleotide sequence of the low-temperature tolerant genotype is shown in SEQ ID No.1, and the amplification primers for developing the molecular marker are T5198-F and T5198-R shown in Table 3.
Research results show that the molecular marker T5198 of the invention can accurately identify individuals with low temperature resistance in Penaeus japonicus, and the application steps can be summarized as follows: extracting genomic DNA in a penaeus japonicus sample, and performing PCR amplification reaction by using the extracted genomic DNA as a template and using molecular-labeled amplification primers T5198-F and T5198-R; and (3) carrying out agarose gel electrophoresis on the PCR amplification product and sequencing, wherein if T5198 in the sequencing result has GTGTGAGTATGT homozygous deletion, the penaeus japonicus is a low-temperature resistant individual, and if the sequencing result does not have GTGTGAGTATGT homozygous deletion, the penaeus japonicus is a low-temperature intolerant individual.
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> low-temperature-resistant molecular marker C6101 of penaeus japonicus and application thereof
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 989
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
attcaggaga gagactaaat tacttgcgcc ctcgttgttg agttcatttt tccttacgaa 60
catgaaggaa aaaaaagact taaataaaac agaaagatga atcatcctcc ttacattaaa 120
aaattggacg aactgcattt gcataattgc atcgcgaggg aaactctagt gatggcagac 180
ttttgtgtct cgtctttgct tagactaaat gagttaaata attttaatga agtgtgatga 240
tttggttgtt attattgtaa ttagtgatag tattcttttg attctcatca tcataagaat 300
tgatgttatc ataaaactat cattactatc ataatcataa ttataataga ggtgatcatc 360
atcaacattg attttgttat tacaggtaaa tgatttgcaa catttattct cgctttgtga 420
tgcattgtga tgtggaaata tgatataata tttatagatg tcattttgtg tgtgtgtcat 480
tgtgtgtagt ggcttatgtg tgtatgtggg ttgttttaat gctaggtaca tgtgcatgtg 540
tacgtgtgta aaaacatcac acaggattta tgtgtgcatt tcatttggta tttgtgtact 600
taagcgcgtg tgtctgtgtc ttcgcgtaca tgtgtgtgta tccttgtgtt catgtgtatg 660
tgtctgtagt tacaaagaaa aagaaggaca tttcattgct cttcatggaa cgatactgca 720
tggaacgata gtcaccaagt ccttcgtaag aaatgttaat ctagatttcc agagaaaatg 780
caattgcact ttgcagtttg aagagatttt cttgttatat cctacacttt tcttaccccc 840
cccccccccc tccccatccc acctcccacc ttttacatga aaaaaaaaac gagtagccaa 900
gatatgaaat acaagaacat caatacaacc aaacaacatt gcaagaaatc tcccatgtgc 960
aacaagctcc agcggaaacg ccattgttc 989
<210> 2
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
cgagggaaac tctagtgatg g 21
<210> 3
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
cacgcgctta agtacacaaa t 21
Claims (9)
1. A molecular marker T5198 for rapidly identifying a low-temperature tolerant variety of Penaeus japonicus is characterized in that the nucleotide sequence of the molecular marker T5198 is shown as SEQ ID No. 1.
2. The molecular marker T5198 according to claim 1, wherein GTGTGAGTATGT sequence deletion occurs at base 508 of said molecular marker T5198.
3. The molecular marker T5198 according to claim 1, wherein said molecular marker T5198 is an Indel marker.
4. A primer for detecting the molecular marker T5198 of claim 1, which is specifically as follows:
T5198-F: CGAGGGAAACTCTAGTGATGG；
T5198-R: CACGCGCTTAAGTACACAAAT。
5. the use of the molecular marker T5198 of claim 1 or the primer of claim 4 for identifying low temperature tolerant species of Penaeus japonicus.
6. The use according to claim 5, wherein the specific steps for identifying the low temperature tolerant Penaeus japonicus variety are as follows:
(1) extracting the genomic DNA of the individual penaeus japonicus;
(2) carrying out PCR amplification on the extracted genome DNA by using primers T5198-F and T5198-R to obtain an amplification product;
(3) sequencing the amplification product; if the homozygous gene deletion mutation appears in the sequencing result, the individual Penaeus japonicus is a low-temperature tolerant variety.
7. The use of claim 6, wherein if the molecular marker T5198 is homozygous GTGTGAGTATGT deletion in the sequencing result in step (3), the Penaeus japonicus individual is a low temperature tolerant variety; if the molecular marker T5198 does not have GTGTGAGTATGT homozygous deletion, the individual Penaeus japonicus is a low-temperature intolerant variety.
8. The use of claim 6, wherein the PCR amplification conditions are: pre-denaturation at 94 deg.C for 5 min; denaturation at 94 deg.C for 30s, annealing at 55 deg.C for 30s, extension at 72 deg.C for 30s, and repeating for 32 cycles; the circulation extends for 72 ℃ for 7 min.
9. Use of the molecular marker T5198 of claim 1 or the primer of claim 4 for genetic diversity analysis, germplasm identification and genetic map construction in penaeus japonicus.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116732199A (en) * | 2023-08-07 | 2023-09-12 | 中国水产科学研究院黄海水产研究所 | Molecular marker T3368 for identifying low temperature resistance of penaeus japonicus and application thereof |
Citations (2)
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|---|---|---|---|---|
| CN105002267A (en) * | 2015-06-15 | 2015-10-28 | 浙江省海洋开发研究院 | Penaeus japonicus molecule marking method and application |
| CN105567865A (en) * | 2016-03-23 | 2016-05-11 | 厦门大学 | SNP (single-nucleotide polymorphism) marker related to Marsupenaeus japonicus heat resistance and detection method thereof |
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2021
- 2021-09-10 CN CN202111060191.7A patent/CN113604587B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105002267A (en) * | 2015-06-15 | 2015-10-28 | 浙江省海洋开发研究院 | Penaeus japonicus molecule marking method and application |
| CN105567865A (en) * | 2016-03-23 | 2016-05-11 | 厦门大学 | SNP (single-nucleotide polymorphism) marker related to Marsupenaeus japonicus heat resistance and detection method thereof |
Non-Patent Citations (1)
| Title |
|---|
| ANONYMITY: "Penaeus japonicus isolate Guangxi Mjap_648616_991_991, whole genome shotgun sequence", 《GENBANK数据库》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116732199A (en) * | 2023-08-07 | 2023-09-12 | 中国水产科学研究院黄海水产研究所 | Molecular marker T3368 for identifying low temperature resistance of penaeus japonicus and application thereof |
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