CN111286544B - Saline-alkali-resistant molecular marker C72 of portunus trituberculatus and application thereof - Google Patents
Saline-alkali-resistant molecular marker C72 of portunus trituberculatus and application thereof Download PDFInfo
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Abstract
The invention provides a salt and alkali tolerant molecular marker C72 of portunus trituberculatus and application thereof. The nucleotide sequence of the molecular marker C72 is shown as SEQ ID No.1, and the nucleotide sequence of the primer pair for detecting the molecular marker C72 is shown as SEQ ID No.2 and SEQ ID No. 3. The molecular marker C72 is an SNP marker, and the saline-alkali tolerant genotype is a CT genotype. The molecular marker C72 provided by the invention can be free from the limitation of the growth stage of the portunus trituberculatus, obviously quickens the breeding process of the portunus trituberculatus and quickly breeds the crab species with excellent saline-alkali tolerance, and the molecular marker C72 is accurate, reliable and simple to operate, is beneficial to the healthy breeding and development of the portunus trituberculatus, so that the molecular marker C72 has wide application prospect.
Description
Technical Field
The invention belongs to the technical field of aquatic animal DNA molecular markers, and particularly relates to a salt and alkali tolerant molecular marker C72 of portunus trituberculatus and application thereof.
Background
Portunus trituberculatus (Portugulus trituberculatus) belongs to Crustacea, decapod, Paramicidae, commonly called Portunus, and is an important large-scale marine economic crab in China. The swimming crabs are delicious in meat quality and rich in nutrition, and are deeply loved by consumers. The saline-alkali water area is a worldwide low-yield water resource, 4587 ten thousand hm2 saline-alkali water area exists in China, and due to the characteristics of high salinity, high alkalinity, high pH value, complex ion composition and the like, common aquatic animals cannot normally live and breed in the saline-alkali water area, so that the development and utilization of the water resource are greatly hindered. The portunus trituberculatus can affect the physiology and biochemistry of the portunus trituberculatus under the stimulation of high salt and alkali, and the high salt and alkali can reduce the ingestion rate, the metamorphosis rate and the survival rate of young crabs. Therefore, the saline-alkali tolerant character is one of important breeding characters of the portunus trituberculatus, and the method has important significance for improving the culture rate of the portunus trituberculatus and promoting the cultivation and popularization of the portunus trituberculatus in saline-alkali soil. However, the saline-alkali tolerant character has obvious low heritability characteristics, the inheritance of the traditional breeding method is slowly progressed, an advanced molecular marker-assisted breeding technology is urgently needed to accelerate the breeding process, and the identification and innovative application of the saline-alkali tolerant molecular marker are necessary preconditions and ways for developing molecular marker-assisted breeding.
At present, the research on the development of the saline-alkali tolerant molecular marker of the blue crab is less, and the industry lacks a marker which can be applied to molecular marker assisted breeding. Therefore, the development of the molecular marker related to the saline-alkali tolerant character has important significance for the healthy breeding and selective breeding of the portunid.
Disclosure of Invention
The invention provides a saline-alkali tolerant molecular marker C72 of portunus trituberculatus and application thereof, the invention utilizes methods of polymorphic site filtration, comparison analysis and PCR sequencing of sequencing data to obtain SNP and InDel markers, and a new saline-alkali tolerant molecular marker C72 of portunus trituberculatus is finally obtained through gradual screening and verification of the markers, and the molecular marker is beneficial to the breeding of the saline-alkali tolerant 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 salt and alkali tolerant molecular marker C72 of blue crab, and the nucleotide sequence of the molecular marker C72 is shown in SEQ ID No. 1.
Further, the molecular marker C72 is an SNP marker.
Further, the salt-base tolerance genotype of the molecular marker C72 is CT genotype.
The invention also provides a primer pair for detecting the molecular marker C72 as claimed in claim 1, wherein the nucleotide sequence of the forward primer in the primers is shown as SEQ ID No.2, and the nucleotide sequence of the reverse primer is shown as SEQ ID No. 3.
The invention also provides application of the molecular marker C72 in screening salt and alkali tolerant varieties of the portunus trituberculatus.
Further: the application steps are as follows: extracting DNA of a sample to be tested of the blue crab, taking the DNA as a template, carrying out PCR amplification by using an amplification primer of a molecular marker C72, sequencing a PCR product, and selecting the sample to be tested as a parent for cultivating the saline-alkali tolerant variety of the blue crab if the genotype of the molecular marker C72 in a sequencing result is CT.
Further: the PCR amplification system is as follows: template 1. mu.l, forward primer (10. mu.M) 0.2. mu.l, reverse primer (10. mu.M) 0.2. mu.l, Buffer 1. mu.l, dNTPs 0.8. mu.l, HiFi 0.2. mu.l, ddH2O 6.6μl。
Further: the PCR amplification procedure is as follows: pre-denaturation at 94 deg.C for 2-5 min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 1-2kb/min, repeating for 35 cycles; finally, the extension is carried out for 5-10min at 72 ℃.
The invention also provides application of the molecular marker C72 in genetic diversity analysis, germplasm identification and genetic map construction of the blue crab.
Compared with the prior art, the invention has the following advantages and beneficial effects:
1. the salt and alkali tolerant molecular marker C72 of portunus trituberculatus provided by the invention can not be limited by the growth stage of portunus trituberculatus, and can be used for breeding early crab seedlings of portunus trituberculatus, so that the breeding process of portunus trituberculatus is obviously accelerated, and crab species with excellent salt and alkali tolerant properties are rapidly bred.
2. The molecular marker C72 provided by the invention is used for detecting the saline-alkali tolerant character of the portunus trituberculatus, the method is accurate and reliable, the operation is simple, the character meeting the requirements can be effectively and quickly screened, the short-time and low-cost early-stage breeding of the saline-alkali tolerant portunus trituberculatus is assisted, the number of the portunus trituberculatus with excellent quality is increased, the ingestion rate, the metamorphosis rate and the breeding rate of the portunus trituberculatus are improved, the yield of the portunus trituberculatus is improved, the healthy breeding of the portunus trituberculatus is promoted, and the application prospect is wide.
Drawings
FIG. 1 shows the result of gel electrophoresis of mixed template PCR products according to the present invention.
FIG. 2 shows the difference between the two groups of corresponding positions of the sensitive population mixed template and the resistant population mixed template according to the present invention.
FIG. 3 is the result of gel electrophoresis band of the product of PCR amplification with two sets of primers with obvious position difference corresponding to the screened sensitive population mixed template and the screened resistant population mixed template.
Detailed Description
The technical solution of the present invention will be described in further detail with reference to specific examples.
The blue crabs used in the invention are all from the experimental base of Changyi Haifeng aquatic products Limited company of yellow sea aquatic product research institute of Chinese aquatic product science institute, healthy and active blue crabs are randomly obtained from a pond by a trawl fishing method, are temporarily arranged in a net basket, are paved with a layer of waterweeds to prevent the crabs from fighting, and finally 300 blue crabs with the weight of 35 +/-3 g are obtained and are placed in 4 culture ponds (500cm multiplied by 300cm multiplied by 150cm) for temporary culture for 7d, the water temperature is kept at 22 +/-1 ℃ during the temporary culture, water is added to 20cm, the carbonate alkalinity is 4mmol/L, the pH value is 8.2 +/-0.5, oxygen is continuously supplied, fresh seawater is replaced at 8 am every day, fresh trash fish is fed at 5 pm, and the feeding amount is about 1/3 of the total weight of the crabs. And 7d, selecting the portunids with better vitality and shape for subsequent experiments.
During formal experiments, crabs with vigorous activity and perfect body surface are placed in 4 cement pools, 50 crabs are placed in each pool, the salinity of seawater carbonate is firstly adjusted to 25mmol/L by using sodium bicarbonate, the pH value of a water body is 8.0 +/-0.5, the water temperature is kept at 22 +/-1 ℃, and the water depth is 20 cm. Feeding fresh trash fish at 5 pm with 1/4 of crab weight to ensure no bait residue on the bottom of the pond and influence water quality. Recording the death time, the weight, the shelling period and other data of the 20 crabs which die firstly, increasing the alkalinity of carbonate to 35mmol/L after 24 hours, wherein the pH value of the water body is 8.5 +/-0.5, and the water temperature is kept at 22 +/-1 ℃. And (4) continuously recording data such as death time, number and the like until the number of the surviving crabs in the 4 pools is 20, and stopping the experiment. The first 20 dead individuals were considered as saline-alkali sensitive group (denoted as Q), the last 20 surviving individuals were considered as saline-alkali tolerant group (denoted as H), and muscle tissue was dissected out and placed in a freezing tube and stored in liquid nitrogen.
Example 1
Screening of candidate molecular markers related to saline-alkali tolerance character
1. Sequencing data filtering and alignment
DNA extraction is carried out by adopting a kit of the whole gold company and by utilizing the principle that silica gel membrane centrifugal columns specifically adsorb DNA. First, approximately 30mg of a tissue sample was put into a sterile 1.5ml centrifuge tube, 200. mu.l of lysine Buffer 8(LB8) and 20. mu.l of RNaseA (10mg/ml) were added, the mixture was incubated at room temperature for about 10 seconds with shaking for 2min, 20. mu.l of protease K (20mg/ml) was added, the mixture was thoroughly mixed with shaking, incubated at 55 ℃ until complete Lysis, 1.5-fold volume of Binding Buffer 8(BB8) was added, the mixture was mixed with a centrifugal column, centrifuged at 12000rpm in a high-speed low-temperature refrigerated centrifuge (model Eppendorf 5804R) for 30 seconds, and the waste solution was discarded. Then 500. mu.l of Clean Buffer8(CB8) was added, centrifuged at 12000rpm for 30s, the waste solution was discarded (repeated), 500. mu.l of Wash Buffer8(WB8) was added, centrifuged at 12000rpm for 30s, the waste solution was discarded (repeated), and the mixture was left to stand at 12000rpm for 2min to completely remove the remaining WB 8. The column was placed in a clean centrifuge tube, 50. mu.l of Elution Buffer (EB) was added to the center of the column, and the column was allowed to stand at room temperature for 2min, centrifuged at 12000rpm for 1min, and the DNA was eluted. DNA purity and integrity was analyzed by agarose gel electrophoresis; the purity of the DNA (OD260/280 ratio) was measured by Nanodrop, and the DNA concentration was precisely quantified by Qubit.
Equivalently mixing the DNA samples qualified by the test into two mixing pools which are respectively named as a saline-alkali sensitive DNA mixing pool (SG) and a saline-alkali tolerant DNA mixing pool (TG). 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 filtering Raw reads obtained by sequencing to obtain Clean reads for subsequent analysis, wherein the sequencing data result is shown in table 1.
TABLE 1 summary of sequencing data quality
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 rate of all samples is between 84.94% and 86.21%, the average coverage depth is between 44.65X and 48.31X, and the 1X coverage (coverage of at least one base) is more than 80.37%. The comparison result is normal, and can be used for subsequent variation detection and related analysis.
2. Marker detection and annotation
SNP and InDel are detected by a UnifiedGentyper module in Genome analysis toolkit 3.8(GATK) software, with SNP filtration parameters set as: MQ is less than 40, QD is less than 4, FS is more than 60, and the total number of the SNP markers obtained finally is 3,295,951.
3. SNP frequency difference analysis
The SNP-index of each site of two groups of individuals is analyzed and calculated, and the polymorphic sites are filtered, and the filtering criteria are as follows:
(1) the SNP-index frequencies in both groups of individuals were less than 0.3;
(2) the site of SNP deletion in one individual was filtered out.
And simultaneously calculating the frequency difference distribution of the SNPs, wherein the directions are as follows: and (2) filtering sites with the delta index smaller than 0.3 to obtain 855 SNP (saline-alkali tolerance related candidate molecular markers), wherein the results of the candidate SNP are shown in Table 2.
TABLE 2 SNPs detection and annotation statistics
4. Marker screening
And screening the candidate SNP 1 marker, and selecting a site with the All-index close to 0 in the individual or selecting a site with the All-index close to 1 in the individual as a preferential selection site for next verification. The screening criteria were as follows:
according to the annotation information of SNP loci, on the basis of sorting from high to low according to delta index, loci of synonymous, non-synonymous mutation or upstream and downstream regions are preferentially selected;
finally, screening out the markers with large frequency difference before and after salt and alkali stress, and screening out 24 SNP markers in total
II, salt and alkali tolerance related molecular marker verification
The method for sequencing the PCR product is adopted to verify the salt and alkali tolerance character related candidate molecular markers in SG and TG populations:
(1) firstly, designing primers on flanking sequences of marker sites, wherein at least one primer is more than 70bp away from the marker sites;
(2) carrying out PCR amplification by using the designed primers and SG and TG mixed DNA materials as templates respectively, sequencing the PCR products successfully amplified, and selecting the primer far away from the marking site as a sequencing primer;
(3) analyzing the sequencing peak map by using Vector NTI software, selecting two groups of SG and TG with larger difference marks at the corresponding position of the sequencing peak map, and continuing to perform PCR amplification and sequencing analysis of an individual DNA template;
(4) and (4) counting the genotype of each individual according to the sequencing result, and analyzing whether the marker is related to the saline-alkali tolerance character or not by SPSS software.
The specific operation steps are as follows:
1. PCR amplification
The PCR amplification system of the invention is as follows: template 1. mu.l, forward primer (10. mu.M) 0.2. mu.l, reverse primer (10. mu.M) 0.2. mu.l, Buffer 1. mu.l, dNTPs 0.8. mu.l, HiFi 0.2. mu.l, ddH2O6.6μl。
After the sample is added according to the system, PCR amplification is carried out according to the following reaction conditions: pre-denaturation at 94 deg.C for 2-5 min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 1-2kb/min, repeating for 35 cycles; finally, extending the temperature of 72 ℃ for 5-10 min; storing at 4 ℃.
2. Electrophoretic detection
Preparing 1% electrophoresis gel by agarose, mixing agarose and TAE in a certain proportion, heating the mixture in a microwave oven until the mixture is dissolved into colorless transparent liquid, pouring the colorless transparent liquid into a gel-making mould, inserting a comb, standing for 20min, solidifying, then pulling out the comb, putting the agarose gel into a horizontal electrophoresis tank, arranging a sample application hole at a negative electrode, taking 0.5% TAE as a buffer solution, selecting Genegeen as a nucleic acid coloring agent, sucking 3ul of PCR products of a mixed template out of the sample hole by a liquid-transferring gun, adjusting the voltage and the current to 120V and 60mA respectively, setting the time to be 30min for carrying out gel electrophoresis, stopping when a dyeing zone reaches 2/3, observing and photographing and recording by a gel imaging system after the electrophoresis is finished, selecting a bright sample with a single strip, sending the sample to a processor for DNA sequencing, carrying out data image analysis on returned sequencing results, selecting two groups of marks with obvious sequencing difference at corresponding positions for carrying out individual PCR amplification, the amplification products were also subjected to gel electrophoresis and the PCR products were sequenced.
3. Statistical analysis
According to the position and brightness of the gel electrophoresis band of the mixed template PCR product, selecting a band (shown in figure 1) meeting the requirement, and selecting 12 pairs of SNP primers to continue sequencing analysis.
Analyzing the sequenced peak map by using Vector NTI software, selecting primers (shown in figure 2) with obvious position difference corresponding to the sequenced peak map of the mixed template of sensitive and tolerant groups, continuously using the primers to perform PCR amplification by taking individuals as templates, keeping the amplification conditions unchanged, selecting bright strips with consistent sizes, sending the strips to be sequenced, completing the sequencing result by amplifying the individual strips with poor amplification effect again, and selecting 5 SNP labeled primers for continuous sequencing analysis.
And observing the returned individual sequencing result by using contig software, introducing the genotype information into SPSS software, calculating a user value by using a chi-square test method, selecting primers with the P less than 0.05, and finally verifying 5 SNP markers in total.
As shown in table 3, it can be seen that genotype CT accounts for more than 55% of the live group in C72, and 55% of genotype CC accounts for early dead individuals, and that the group data P is 0.007, which has significant difference, and thus genotype CT is considered to be a salt and alkali tolerant genotype at this site. The nucleotide sequence of the molecular marker C72 is shown as SEQ ID No.1, wherein the amplification primers for developing the molecular marker are shown as SEQ ID No.2 and SEQ ID No.3 (Table 4).
TABLE 3 genotype results for the C72 molecular marker
TABLE 4 amplification primers for molecular markers
The molecular marker C72 obtained by the invention can be used for assisting in breeding the salt and alkali tolerant varieties of blue crabs, and the application steps are simply as follows: extracting DNA of a sample to be tested of the portunus trituberculatus, taking the DNA as a template, carrying out PCR amplification by using an amplification primer of a molecular marker, sequencing a PCR product, and selecting the test sample as a parent for cultivating the saline-alkali tolerant variety of the portunus trituberculatus if the genotype of the molecular marker in a sequencing result is CT. In addition, the molecular marker can be used for analyzing the genetic diversity of the blue crab, identifying the germplasm and constructing the genetic map of the blue crab.
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> saline-alkali-tolerant molecular marker C72 of portunus trituberculatus and application thereof
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1001
<212> DNA
<213> Portunus trituberculatus (Portulus trituberculatus)
<400> 1
cttaatactc ctctagcggg acttgggcgc gtgttaagct cccttcttcc tcctccctgg 60
cctctccctg ccttgcttta tgttgtgtgt gcacatggaa cggccaacga aattaattca 120
taccatcttg tcggatttcc aaggcttatc ataatggata tatattcgag ttttgagcct 180
aaattggatt catgcatgca aaagaccatt cccttgaacc tcttcttcca caatctcctg 240
ccttcttttc cctccctacc tcttcctctt cttctttttc ttcctcctcc tcctcctcaa 300
acagctcctc ctcttcctct tctttcagac aacacccgga tatggcgcta atgtctccta 360
ccctaatgaa agtgatgagt gacgttatta aaaacgcgag gtgacagctg accacacctg 420
cctaatctac ctgttttcct ccggccaccc acacttgttc gcctctcccc gcacctccag 480
ttttctacct gtatctccat ttcacccacg ccagcctcac cacgccagcc tcaccacgcc 540
agcttcacaa tgccagcttt accgcgccag ttacacaacg ccagctacac cactagctac 600
acagtctagc ttcacctgtt gcatcagtaa atatctcaca gaaacactgg aatcttgcac 660
tcttaaacag taataatagt aatgatgata ataatgataa taatccagtt aaatagtaca 720
gtgggagtaa agatggtaat gataacaaca acgacaatga taataacgat aataataata 780
ataatagtaa taataataat aataataata ataataataa taataatcca ttatagttat 840
ttttatggga caacttaccc atattaacct tactaaaata aacctgacct taccatacgc 900
aaagtaaaaa atatcataat aataataata ataataataa taataataat aataataatt 960
ataataacat tgtttaaact aatccccttg ttgttgatgt t 1001
<210> 2
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
accacacctg cctaatctac c 21
<210> 3
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
caagattcca gtgtttctgt gag 23
Claims (1)
1. A saline-alkali-resistant molecular marker C72 of blue crab is characterized in that the nucleotide sequence of the molecular marker C72 is shown in SEQ ID No.1, wherein the 501 th base is T or C.
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| CN102154500A (en) * | 2011-03-25 | 2011-08-17 | 中国水产科学研究院黄海水产研究所 | Sextuple PCR (polymerase chain reaction) detection method of portunus trituberculatus miers microsatellite marker |
| CN103387980A (en) * | 2013-08-22 | 2013-11-13 | 宁波大学 | Microsatellite loci of portunus trituberculatus and primer |
| CN103789301A (en) * | 2013-06-05 | 2014-05-14 | 浙江海洋学院 | Specific primers of portunus trituberculatus microsatellite marker and screening method |
| CN108208318A (en) * | 2018-01-05 | 2018-06-29 | 浙江新篁生物技术有限公司 | A kind of sea crab feed addictive and its manufacturing method |
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2020
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102154500A (en) * | 2011-03-25 | 2011-08-17 | 中国水产科学研究院黄海水产研究所 | Sextuple PCR (polymerase chain reaction) detection method of portunus trituberculatus miers microsatellite marker |
| CN103789301A (en) * | 2013-06-05 | 2014-05-14 | 浙江海洋学院 | Specific primers of portunus trituberculatus microsatellite marker and screening method |
| CN103387980A (en) * | 2013-08-22 | 2013-11-13 | 宁波大学 | Microsatellite loci of portunus trituberculatus and primer |
| CN108208318A (en) * | 2018-01-05 | 2018-06-29 | 浙江新篁生物技术有限公司 | A kind of sea crab feed addictive and its manufacturing method |
Non-Patent Citations (2)
| Title |
|---|
| Portunus trituberculatus scaffold72832,whole genome shotgun sequence;Jeong,J.-H.等;《GenBank》;20190912;第1页 * |
| 逆环境因素对三疣梭子蟹幼蟹致死效应分析;高焕等;《海洋科学》;20101231;第34卷(第2期);第57-61页 * |
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