CN111269993A - Saline-alkali-resistant molecular marker C261 of portunus trituberculatus and application thereof - Google Patents
Saline-alkali-resistant molecular marker C261 of portunus trituberculatus and application thereof Download PDFInfo
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Abstract
The invention provides a salt and alkali tolerant molecular marker C261 of Portunus trituberculatus and application thereof. The nucleotide sequence of the molecular marker C261 is shown in SEQ ID No.1, and the nucleotide sequence of the primer pair for detecting the molecular marker C261 is shown in SEQ ID No.2 and SEQ ID No. 3. The molecular marker C261 is Indel marker, and the alkali-resistant genotype is insertion homozygous genotype. The molecular marker C261 provided by the invention can not be limited by 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.
Description
Technical Field
The invention belongs to the technical field of aquatic animal DNA molecular markers, and particularly relates to a saline-alkali-resistant molecular marker C261 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 C261 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 C261 of portunus trituberculatus is finally obtained through gradual screening and verification of the markers, and the molecular marker is favorable for breeding 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 C261 of Portunus trituberculatus, and the nucleotide sequence of the molecular marker C261 is shown in SEQ ID No. 1.
Further, the molecular marker C261 is an Indel marker.
Further, the saline-alkali tolerance genotype of the molecular marker C261 is an insertion homozygous genotype.
The invention also provides a primer pair for detecting the molecular marker C261 of claim 1, wherein the nucleotide sequence of a forward primer in the primers is shown as SEQ ID No.2, and the nucleotide sequence of a reverse primer is shown as SEQ ID No. 3.
The invention also provides application of the molecular marker C261 in screening salt and alkali tolerant varieties of 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 C261, 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 C261 in a sequencing result is an insertion homozygous genotype.
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 C261 in genetic diversity analysis, germplasm identification and genetic map construction of portunus trituberculatus.
Compared with the prior art, the invention has the following advantages and beneficial effects:
1. the saline-alkali tolerant molecular marker C261 of the portunus trituberculatus provided by the invention can not be limited by the growth stage of the portunus trituberculatus, and can be used for breeding early-stage crab seedlings of the portunus trituberculatus, so that the breeding process of the portunus trituberculatus is obviously accelerated, and crab species with excellent saline-alkali tolerant characters are rapidly bred.
2. The method for detecting the saline-alkali tolerant character of the portunus trituberculatus by using the molecular marker C261 provided by the invention is accurate, reliable and simple to operate, can effectively and quickly screen out characters meeting requirements, assists in early-stage short-time and low-cost breeding of the saline-alkali tolerant portunus trituberculatus, increases the number of the portunus trituberculatus of good quality, improves the ingestion rate, the metamorphosis rate and the breeding rate of the portunus trituberculatus juvenile crabs, further improves the yield of the portunus trituberculatus, and promotes the healthy breeding of the portunus trituberculatus, so that the method has a wide application prospect.
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FIG. 1 shows the result of gel electrophoresis bands of the products 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 Lysis 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 times the volume of Binding Buffer 8(BB8) was added, the mixture was added to a centrifugal column, centrifuged at 12000rpm in a high-speed low-temperature refrigerated centrifuge (model Eppendorf58 5804R) for 30 seconds, and the waste liquid was discarded. Then 500. mu.l of Clean Buffer 8(CB8) was added, centrifuged at 12000rpm for 30s, the waste liquid was discarded (repeated), 500. mu.l of Wash Buffer 8(WB8) was added, centrifuged at 12000rpm for 30s, the waste liquid 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 UnifiedGenottyper module in Genome analysis toolkit 3.8(GATK) software, the filtration parameters of the InDel are set to be QD < 4, FS > 200, and the total number of finally obtained InDel markers is 1,427,108.
3. Indel frequency difference analysis
The analysis calculates the Indel-index of each site of two groups of individuals, and filters the polymorphic sites, with the following filter criteria:
(1) the Indel-index frequencies in both groups were less than 0.3;
(2) the sites of Indel deletion in one individual were filtered out.
And meanwhile, calculating the frequency difference distribution of the indels in the following direction of △ (index) -index (saline and alkaline tolerance property), filtering out sites with △ index smaller than 0.3, and finally obtaining indels 1051 with difference among groups, wherein the indels are saline and alkaline tolerance related candidate molecular markers, and the statistics of the results of the candidate InDel are shown in Table 2.
TABLE 2 Indel test and annotation statistics
4. Marker screening
And screening candidate Indel markers, 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:
based on the annotation information of InDel sites, sites with more than 5 inserted or deleted bases are preferably selected based on the high-to-low ordering by △ index.
Finally, screening out the marks with large frequency difference before and after salt and alkali stress, and screening out 11 Indel marks 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 designed primers and SG and TG group individual DNA materials as templates respectively, and carrying out 2% agarose gel electrophoresis analysis on PCR products successfully amplified;
(3) and (4) counting the genotype of each individual according to the electrophoresis 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, ddH2O 6.6μl。
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 2% of electrophoresis gel by using agarose, mixing the agarose and TAE in a certain ratio, heating the mixture in a microwave oven until the mixture is dissolved into colorless transparent liquid, pouring the colorless transparent liquid into a gel preparation mould, inserting a comb, standing for 20min, solidifying, then pulling out the comb, putting the prepared 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 using a liquid transfer 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 by using a gel imaging system after the electrophoresis is finished, and taking a picture and recording.
3. Statistical analysis
Marking and typing individuals according to the electrophoresis bands, counting the number of each genotype, introducing the genotype information of the individuals into SPSS software, calculating a P value by using a chi-square test method, selecting primers with the P value less than 0.05, and finally verifying 7 Indel marks in total.
As shown in table 3, it can be seen that the insertion of the homozygous genotype into C261 accounts for 90% of the survival group, while the insertion of the heterozygous genotype accounts for 65% of the early death individuals, and the group data has a P value of 0.000, which is significantly different, and thus it can be considered that the insertion of the homozygous genotype into the site is a saline-alkali tolerant genotype. The nucleotide sequence of the C261 molecular marker is shown in SEQ ID No.1, wherein the amplification primers for developing the molecular marker are shown in SEQ ID No.2 and SEQ ID No.3 (Table 4).
TABLE 3 genotype results for C261 molecular markers
TABLE 4 amplification primers for molecular markers
The molecular marker C261 obtained by the invention can be used for assisting in breeding the salt and alkali tolerant varieties of portunus trituberculatus, and the application steps are simply 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, 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 C261 in a sequencing result is an insertion homozygous genotype. 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.
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gcccccttcc cccccaccgc tctgataagg ctcccaaact gtgatccctg gatccgccac 300
tgaatgagag agatcgagga ggtgggaggt gtaagagaag cgaaaatcca actacgacaa 360
tcttatgaga tacgatagtg tttgatacct tgattttgcc tctcttgggt gttgtcaatc 420
tgtgatggct taattgatat tcaactttat atataaactt tcaatgattg agtacataaa 480
tgaaataaat aatgtaccat agattctttg tcattgtaac actgtcatga ctcctcagtc 540
cagtgaccca gtgggactga gcctgggcca gtcgtctcgt cagtcagtca gtcagtgttc 600
cgtgtgctgc ggtgctggta aggcgtgaga caagaggtga tttattggag tatttccatc 660
gtagtgaggt gccaatacct caatagtgtg tacagtttag cgatgtatcc cacattctct 720
gctatgaaga aaagtttagt gggaatgaac aaaacacagc caagatgaaa gaaaatatgg 780
tttattgaga acatttacca cagtgaagat gaaaactatt tgtgcagtag tgtgtggctt 840
ctcagtaaag taagcttcac ttttgtatgt ggctgaaggc gtaaggcagg tgtgagaggg 900
tgggacgggc tgtgttggtg ccctgctggt ccacggaccg tttgggggca tagtggcggg 960
gcggggcagg cttcacaagt aaacatggat tcctttcata taattcaaca tttcacggtc 1020
atgagacg 1028
<210>2
<211>25
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>2
tgggtgttgt caatctgtga tggct 25
<210>3
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>3
ctgactgacg agacgactgg 20
Claims (9)
1. A saline-alkali-resistant molecular marker C261 of Portunus trituberculatus is characterized in that the nucleotide sequence of the molecular marker C261 is shown in SEQ ID No. 1.
2. The portunus trituberculatus saline-alkali tolerant molecular marker C261 of claim 1, wherein the molecular marker C261 is an Indel marker.
3. The portunus trituberculatus saline-alkali tolerant molecular marker C261 of claim 1 or 2, wherein the saline-alkali tolerant genotype of the molecular marker C261 is an insertion homozygous genotype.
4. The primer pair for detecting the molecular marker C261 of 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.
5. The application of the molecular marker C261 of claim 1 in screening of salt and alkali tolerant varieties of Portunus trituberculatus.
6. The application of the molecular marker C261 in screening of salt and alkali tolerant varieties of Portunus trituberculatus according to claim 5, which is characterized in that: 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 C261, 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 C261 in a sequencing result is an insertion homozygous genotype.
7. The application of the molecular marker C261 in screening of salt and alkali tolerant varieties of Portunus trituberculatus according to claim 6, which is characterized in that: 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。
8. The application of the molecular marker C261 in screening of salt and alkali tolerant varieties of Portunus trituberculatus according to claim 6, which is characterized in that: 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 ℃.
9. The use of the molecular marker C261 of claim 1 in the genetic diversity analysis, germplasm identification and genetic map construction of portunus trituberculatus.
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Cited By (1)
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| CN113981112A (en) * | 2021-12-27 | 2022-01-28 | 中国水产科学研究院黄海水产研究所 | InDel marker C3082 for identifying ammonia nitrogen tolerance character of portunus trituberculatus, primers and application thereof |
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