CN112522423B

CN112522423B - Molecular marker of lumbrolumbricus microsatellite and polymorphic primer and application thereof

Info

Publication number: CN112522423B
Application number: CN202011482287.8A
Authority: CN
Inventors: 刘宏毅; 方玉; 阮宏华
Original assignee: Nanjing Forestry University
Current assignee: Nanjing Forestry University
Priority date: 2020-12-15
Filing date: 2020-12-15
Publication date: 2023-07-18
Anticipated expiration: 2040-12-15
Also published as: CN112522423A

Abstract

The invention discloses a molecular marker of a popular lumbricus microsatellite, and a polymorphic primer and application thereof, and belongs to the field of biotechnology detection. The invention utilizes RAD-SEQ high-throughput sequencing technology to screen 12 microsatellite loci from the genome DNA of the Lepidium meyenii, wherein the nucleotide sequences corresponding to the microsatellite loci are shown in SEQ ID No.1-12, and the microsatellite loci are respectively shown in Mv01, mv02, mv03, mv04, mv05, mv06, mv07, mv08, mv09, mv10, mv11 and Mv 12; and a specific primer is designed according to flanking sequences at two ends of a microsatellite locus, the nucleotide sequence of the primer is shown as SEQ ID No.13-36, and an amplification result obtained by using the primer has polymorphism and stability, so that the method is applicable to the fields of popular lumbricus population genetic diversity detection, individual identification and molecular assisted breeding, and lays a solid foundation for popular lumbricus microsatellite polymorphism detection.

Description

Molecular marker of lumbrolumbricus microsatellite and polymorphic primer and application thereof

Technical Field

The invention belongs to the field of biotechnology detection, and particularly relates to a molecular marker of a popular lumbricus microsatellite, a polymorphic primer and application thereof.

Background

The lumbricus (Metaphire vulgaris) belongs to the class clitella (Clitellata), the rear Kong Guamao mesh (Opisthopora), the family Megascolecidae (Megascolecidae), the genus lumbricus (Metaphire), and is widely distributed in Jiangsu, zhejiang and other places. The lumbricus body contains succinic acid with higher concentration, and can be used for relieving asthma, promoting urination, relieving spasm, etc.; also contains oleic acid, linoleic acid and other unsaturated fatty acids, and can be used for preventing and treating arteriosclerosis, promoting blood circulation, dispelling blood stasis, and preventing and treating cardiovascular and cerebrovascular diseases. Traditional Chinese medicine is commonly called as earthworm. In addition, earthworms are known as large decomposers in soil, and their ecological status is self-evident and called "ecological engineers".

Microsatellite markers, also known as simple sequence repeats (Simple sequence repeats, SSRs) or simple sequence length polymorphisms (Simple sequence length polymorphisms, SSLPs), are simple repeat sequences with 1-6 bases as repeat units, wherein 2-3 bases of repeat units predominate, and are known as microsatellites because of their shorter repeat units than the sequence of a microsatellite. Microsatellite DNA is typically flanked by relatively conserved single copy sequences and differs in the number of repeats between different alleles. Compared with other genetic molecular markers, the microsatellite DNA markers have extremely high individual specificity and repeatability, and can provide abundant polymorphic sites due to relatively high mutation rate, and genetic information such as heterozygosity, homozygosity and the like can be obtained through polymorphism analysis of the sites. At present, microsatellite DNA markers are widely applied in aspects of population genetic structure research, genetic relationship identification, variety identification and the like.

Microsatellite locus detection methods include agarose gel electrophoresis, polyacrylamide gel electrophoresis (PAGE), capillary electrophoresis, and fluorescent labeling full-automatic genome scanning. However, as the length difference of partial microsatellite DNA fragments is smaller and can not be distinguished by agarose gel electrophoresis, at present, microsatellite fluorescent marking full-automatic genotyping technology has been commonly adopted, and 3 fluorescent dyes with different colors are used for marking microsatellite DNA primers, so that PCR products with different fluorescent marks and different amplified fragments can be electrophoresed in the same sample-adding hole, and the PCR products to be detected and the intrinsic molecular weight standard of DNA can be simultaneously loaded, and the size of microsatellite allele fragments can be accurately calculated by sequential gel electrophoresis and image analysis by using software. The method has the advantages of high yield, low cost, rapidness, simplicity, convenience and the like, and has wide application prospect.

Disclosure of Invention

Aiming at the problems existing in the prior art, the first technical problem to be solved by the invention is to provide a group of popular lumbricus microsatellite molecular markers; the second technical problem to be solved by the invention is to provide polymorphic primers of the molecular markers of the earthworm microsatellite; the third technical problem to be solved by the invention is the application of the molecular markers and/or polymorphic primers of the molecular markers in genetic polymorphism, genetic relationship identification and variety identification of the lumbricus in popular province.

In order to solve the problems, the technical scheme adopted by the invention is as follows:

the invention utilizes RAD-seq high-throughput sequencing technology to screen out 12 microsatellite loci from the genomic DNA of the lumbricus, and the marker number is Mv01-12. The nucleotide sequence corresponding to the molecular marker of the lumbricus microsatellite is shown as SEQ ID No. 1-12.

The microsatellite locus primers of the invention are forward (F) and reverse (R) primers designed from flanking sequences at two ends of the Mv01-12 microsatellite. The nucleotide sequence of the polymorphic primer of the molecular marker of the lumbricus microsatellite is shown as SEQ ID No.13-36, wherein the nucleotide sequence SEQ ID No.13-14 is used for amplifying Mv01, and so on.

Application of molecular markers of Leptospira occidentalis and/or polymorphic primers of molecular markers in genetic polymorphism, genetic relationship identification and variety identification of Leptospira occidentalis.

Furthermore, the polymorphic primer of the microsatellite molecular marker is used for detecting the genetic diversity of the lumbricus in popular life, and comprises the following steps:

1) Extracting genome DNA;

2) Microsatellite PCR amplification: amplifying popular lumbricus genome DNA by adopting FAM, HEX, TAMRA fluorescent marked microsatellite primers to obtain an amplified product;

3) Analysis of genetic diversity: genotype was taken according to the molecular weight of each individual microsatellite amplification product and genetic diversity parameters were calculated using Cervus 3.0.7.

The beneficial effects are that: compared with the prior art, the invention has the advantages that:

the invention screens out 12 microsatellite loci from the molecular DNA of the earthworms by utilizing the RAD-seq high-throughput sequencing technology, designs specific primers according to flanking sequences at two ends of the microsatellite loci, has polymorphism and stability by using the amplification result of the obtained primers, and can be suitable for the fields of genetic diversity detection, individual identification and molecular assisted breeding of the earthworms population in the popular field, thereby laying a solid foundation for microsatellite polymorphism detection of the earthworms in the popular field.

Detailed Description

The invention is further described below in connection with specific embodiments.

Example 1

1. Extracting DNA of lumbricus

30 popular lumbrous specimens are collected and stored in absolute ethyl alcohol, and DNA extraction is carried out by adopting a DNA extraction kit of Nanjinouzan biotechnology limited company, and the specific steps are as follows:

a. 1-20mg of earthworm tissues are sheared and transferred into a 1.5mL centrifuge tube by using clean scissors, 230 mu L of Buffer GA and 20 mu L of PK working solution are added, and vortex mixing is carried out.

b, water bath at 55 ℃ until the tissue is completely hydrolyzed. If the digestate is cloudy or there are significant particles, the tube is placed in a centrifuge, centrifuged for 3min at 12,000Xg, and the supernatant is transferred to a new 1.5mL centrifuge tube.

c. 250 μl Buffer GB was added to the digest solution and vortexed at maximum speed for 20sec with a water bath at 70deg.C for 10min.

d. 250 μl of absolute ethanol was added to the digest and centrifuged manually for 15-20sec.

e. The gDNA Columns were placed in Collection Tubes 2 mL. Transferring the mixed solution (including sediment) obtained in the last step into an adsorption column. Centrifuging at a speed of 12,000Xg for 1min, and centrifuging at a speed of 14,000Xg for 3-5min if column blocking occurs.

f. Waste liquid is discarded, and the adsorption column is placed in a collecting pipe. 500 mu L Washing Buffer A was added to the column. Centrifuge at 12,000Xg for 1min.

g. Waste liquid is discarded, and the adsorption column is placed in a collecting pipe. 650 mu L Washing Buffer B was added to the column. Centrifuge at 12,000Xg for 1min. The operation is repeated once.

h. Waste liquid is discarded, and the adsorption column is placed in a collecting pipe. The 12,000Xg hollow tube was centrifuged for 2min.

i. The column was placed in a new 1.5mL centrifuge tube. 30-100. Mu.L of an absorption Buffer preheated to 70 ℃ is added to the center of the membrane of the adsorption column, and the mixture is left standing at room temperature for 3min. The 12,000Xg hollow tube was centrifuged for 2min.

j. Discarding the adsorption column, and storing DNA at 2-8deg.C for long term storage at-20deg.C.

2. Popular lumbro polymorphism microsatellite primer screening

According to the RAD-SSR sequence of the popular lumbrous genome, the RAD-SSR design of two to four nucleotide different repetitive motifs is selected, 20 pairs of microsatellite primers are designed by utilizing software Premier 5, and the main parameters of the primer design are as follows: the primer length is about 22bp, and the product length is 120-200bp; complementarity of a single primer of less than 3 nucleotides; ensuring that the Tm values of the two primers are above and below 55-60 ℃; the pairing between two primers is less than 3 continuous nucleotides, and the content of (G+C)% is approximate (40% -60%). 12 pairs of microsatellite primers were screened by preliminary detection.

The microsatellite loci, corresponding primer information, fluorescent markers and optimal annealing temperatures are shown in Table 1.

TABLE 1 popular lumbrolumbricus microsatellite loci and primers, fluorescent markers and optimal annealing temperature thereof

3. Popular lumbrolumbricus fluorescent labeling microsatellite PCR amplification and genetic diversity analysis

Labeling 12 pairs of microsatellite primers obtained by screening, and the forward primers with different fluorescent substances, wherein fluorescent labels of Mv09, mv10, mv11 and Mv12 are FAM; fluorescent markers of Mv01, mv02, mv03 and Mv07 are HEX; fluorescent markers of Mv04, mv05, mv06, mv08 are TAMRA. PCR amplification is carried out on the obtained popular lumbrolumbricus DNA sample by using the fluorescent primer pairs. The PCR reaction system is as follows: 12.5. Mu.L of 2 XTaq Master Mix (Dye Plus), 1. Mu.L of template genomic DNA, 0.5. Mu.L of each of the upstream and downstream primers (concentration 10. Mu.M), 10.5. Mu.L of ddH ₂ O, PCR amplification procedure: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 30s, annealing at 55-60℃for 30s, extension at 72℃for 30s, and 35 cycles; finally, the extension is carried out at 72 ℃ for 5min. The allele factor (Na), expected heterozygosity (He) and Polymorphism Information Content (PIC) for each microsatellite marker were calculated using Allele frequency analysis in software Cervus v.3.0.7, and the results indicated that the number of alleles per microsatellite locus varied from 2 to 8, the expected heterozygosity (He) ranged from 0.183 to 0.792, and the Polymorphism Information Content (PIC) ranged from 0.164 to 0.745, with the detailed results shown in table 2.

TABLE 2 microsatellite loci measurements

Site(s)	Core repeat sequence	Allelic factor Na	Desired heterozygosity He	Polymorphic information content PIC
					Mv01	(CA) ₉	8	0.787	0.742
Mv02	(GT) ₇	5	0.531	0.457
					Mv03	(CTGT) ₇	5	0.749	0.690
Mv04	(CT) ₁₅	7	0.738	0.686
					Mv05	(CAG) ₄	4	0.572	0.465
Mv06	(GT) ₁₁	4	0.545	0.484
					Mv07	(AG) ₁₅	8	0.792	0.745
Mv08	(CTCA) ₉	2	0.183	0.164
					Mv09	(CTG) ₆	6	0.327	0.385
Mv10	(CCG) ₅	7	0.653	0.589
					Mv11	(GAGG) ₅	3	0.488	0.412
Mv12	(TG) ₁₆	6	0.666	0.593

Sequence listing

<110> university of Nanjing forestry

<120> popular lumbricus microsatellite molecular marker and polymorphic primer and application thereof

<130> 1

<160> 36

<170> SIPOSequenceListing 1.0

<210> 1

<211> 218

<212> DNA

<213> Mv01

<400> 1

cccccctcag ccccataaac cgctttcgcc tctgtttctc cgttttgaaa ttatctgtcg 60

tatttttaca ttaacattag atgctacaca cgcacgcacg cacacacaca cacacacatg 120

aacacgggaa ctcggtgtga taaaagtggc gagagaaggc aactgggtga ccatgtgcag 180

ggcgtaagag taagggtaaa agtgatggcg ttatgggt 218

<210> 2

<211> 218

<212> DNA

<213> Mv02

<400> 2

cagggcattt gggagccatg gctacgctgc tttaattctt ttagtaaacg ttgctggtcc 60

agttgctatg gcaacatttc tatggcaatg gaccgatgtc gtgtgtgtgt gtgtgtgtgc 120

atgtgtggaa tgattgacag ctactgtctc gctgttttga tttcttattc ctagcaattg 180

cccatctgcc agttaatcgc cagccacacc cactgtct 218

<210> 3

<211> 196

<212> DNA

<213> Mv03

<400> 3

tggctgacgt catgtcttcg ccatcctaat cggcgccttc aactggagct cagtctgtct 60

gtctcagact gtctgtctgt ctgtctgtct gtctgtctct ctctctgtct ctctgtctct 120

ctgtctctct ctctctctcg ggggtagaga gaagggttca gtccactgga gcgccacctc 180

ggggccggtc ctcgtc 196

<210> 4

<211> 198

<212> DNA

<213> Mv04

<400> 4

tcagcacatt ctggaaatgc gccaaatgaa tacatttgca tgaaaagcaa gtcacgacat 60

gattaacgct ctctctctct ctctctctct ctctctctca cacacacaca cgcacacgct 120

atgctagtag cctactagat aatccagata ctgtatatcc agcatatggc atgcaatatt 180

aatgctggaa atttcgac 198

<210> 5

<211> 180

<212> DNA

<213> Mv05

<400> 5

atctttgtgc aagtctttgt aatctttaaa cttcgaccca cactgaagca acgaacgcaa 60

cttcccatca gcagcagcag acgacgagtt cggccgtgaa ctgacgttgt caagcgcaga 120

agccgtcagc atcgcatcag tcaggcccag gatgggactt cttaggtcag acgtttgaca 180

<210> 6

<211> 182

<212> DNA

<213> Mv06

<400> 6

catggctgta agtccaaaca tgtgatctgc ggccaagtta aactgtcctt gtcaagtttg 60

tgtggcgcgt gtgtgtgtgt gtggctgctt tgttaggtat ttgttcgagc agtaattcta 120

aacaggaatg cacaacaatc actcctatca tgacgaggca cttaacaacg ggaggcacag 180

tc 182

<210> 7

<211> 198

<212> DNA

<213> Mv07

<400> 7

tcgccataat tagctccact cggaagaaga gccgtgccaa gaagaaccgg aaagaagaac 60

aacatgaaag agagagagag agagagagag agagagagaa tttgctaatc gttctgttcc 120

tctgattaac acgcacgttg gcaatacaac atcctcctac cgtcctccac catcctccgc 180

ttcctccaac agcaaccg 198

<210> 8

<211> 218

<212> DNA

<213> Mv08

<400> 8

agcatcatca tcttcttctc ctcctcctcc tagttccttc atgaaatgaa gctgagatga 60

cagcgggaat ttcccttgtc tatagcctgt cttttcctga ctgctgctgc tgctgctgac 120

atttgcagcc ctgtcctcct gagtcctcca cgtcacccgc ccaacgtcct ttggcagccc 180

tctatgtttc gttccagaga ccgtttcctt gtgttgac 218

<210> 9

<211> 182

<212> DNA

<213> Mv09

<400> 9

taataattag attgttagaa attatttctt aaaataactg atcaccaagt gaacgggcac 60

gcacacaaac acacacacac acataaatca cgcatgcaaa aaatcactaa tataggagag 120

caaacggaac tggttatagt cgatgacaat cggctaatct gtttgcaccg tttgaagcca 180

ga 182

<210> 10

<211> 183

<212> DNA

<213> Mv10

<400> 10

cgccggaggg ggaagagtcg gtcgacgaca gcaacacggc cgctggggtg acggggcgcc 60

ggtgtgcacc gccgccgccg ccggatccgg cacgacggtg catctcgatc gcgtcttcgt 120

cgtcggaccg tgacgtccaa accggcgacg gggtctggtc cgacgatagg gtggtggaaa 180

ggc 183

<210> 11

<211> 188

<212> DNA

<213> Mv11

<400> 11

atcgtgaggt ggccagggtg tgccaaaagg atcgggcaac cgcacaaagg acgagcgccg 60

agggaaacga gggagggagg gagggaggga gagagggacg gagggaggaa aggccgacgg 120

tcctccgcgc cacagccggc tggcttccgg cggcgctctg ccttccatct catctttggt 180

gctaattg 188

<210> 12

<211> 200

<212> DNA

<213> Mv12

<400> 12

gaagctgtgt gcattttaaa tagattctat agactcaaga cttacaatag tattttacgg 60

tacctctctg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg cgcgcgcgcg cgagcgcagt 120

ggcggttcca cgtgggggat gagtggggat gcatccccca ccggactctt cttgcttgtc 180

aaactatttt tgggtaaaag 200

<210> 13

<211> 19

<212> DNA

<213> Mv01F primer sequence (artificial)

<400> 13

gttttgaaat tatctgtcg 19

<210> 14

<211> 20

<212> DNA

<213> Mv01R primer sequence (artificial)

<400> 14

tctcgccact tttatcacac 20

<210> 15

<211> 21

<212> DNA

<213> Mv02F primer sequence (artificial)

<400> 15

attattttga cgcttccata c 21

<210> 16

<211> 20

<212> DNA

<213> Mv02R primer sequence (artificial)

<400> 16

gttcctttga tctctcgtaa 20

<210> 17

<211> 20

<212> DNA

<213> Mv03F primer sequence (artificial)

<400> 17

tggagctcag tctgtctgtc 20

<210> 18

<211> 20

<212> DNA

<213> Mv03R primer sequence (artificial)

<400> 18

tgaacccttc tctctacccc 20

<210> 19

<211> 21

<212> DNA

<213> Mv04F primer sequence (artificial)

<400> 19

tcccaagagt attgaggatt t 21

<210> 20

<211> 20

<212> DNA

<213> Mv04R primer sequence (artificial)

<400> 20

actagcatag cgtgtgcgtg 20

<210> 21

<211> 20

<212> DNA

<213> Mv05F primer sequence (artificial)

<400> 21

taaacttcga cccacactga 20

<210> 22

<211> 20

<212> DNA

<213> Mv05R primer sequence (artificial)

<400> 22

cgtctgacct aagaagtccc 20

<210> 23

<211> 21

<212> DNA

<213> Mv06F primer sequence (artificial)

<400> 23

atatggttgc aaaaacaatc a 21

<210> 24

<211> 21

<212> DNA

<213> Mv06R primer sequence (artificial)

<400> 24

gttgtgcatt cctgtttaga a 21

<210> 25

<211> 19

<212> DNA

<213> Mv07F primer sequence (artificial)

<400> 25

cataattagc tccactcgg 19

<210> 26

<211> 21

<212> DNA

<213> Mv07R primer sequence (artificial)

<400> 26

gttgtgcatt cctgtttaga a 21

<210> 27

<211> 20

<212> DNA

<213> Mv08F primer sequence (artificial)

<400> 27

gaaatgaagc tgagatgaca 20

<210> 28

<211> 19

<212> DNA

<213> Mv08R primer sequence (artificial)

<400> 28

tggaacgaaa catagaggg 19

<210> 29

<211> 20

<212> DNA

<213> Mv09F primer sequence (artificial)

<400> 29

tgaggactgg tttgacactt 20

<210> 30

<211> 21

<212> DNA

<213> Mv09R primer sequence (artificial)

<400> 30

taaccagttc cgtttgctct c 21

<210> 31

<211> 23

<212> DNA

<213> Mv10F primer sequence (artificial)

<400> 31

aggtcagcat cgacgacgac aac 23

<210> 32

<211> 20

<212> DNA

<213> Mv10R primer sequence (artificial)

<400> 32

cctttccacc accctatcgt 20

<210> 33

<211> 20

<212> DNA

<213> Mv11F primer sequence (artificial)

<400> 33

aggaggagat gaaaatatcg 20

<210> 34

<211> 20

<212> DNA

<213> Mv11R primer sequence (artificial)

<400> 34

agcaccaaag atgagatgga 20

<210> 35

<211> 20

<212> DNA

<213> Mv12F primer sequence (artificial)

<400> 35

cgacgtccat ctactttgaa 20

<210> 36

<211> 20

<212> DNA

<213> Mv12R primer sequence (artificial)

<400> 36

caaaaatagt ttgacaagca 20

Claims

1. The molecular marker fragments of the lumbricus in popular form are characterized by comprising Mv01, mv02, mv03, mv04, mv05, mv06, mv07, mv08, mv09, mv10, mv11 and Mv12, and the nucleotide sequences corresponding to the molecular marker fragments of the lumbricus in popular form are shown as SEQ ID No. 1-12.

2. The polymorphic primer of the molecular marker fragment of the lumbricus in the popular sense as set forth in claim 1, wherein the nucleotide sequence of the polymorphic primer of the molecular marker fragment of the lumbricus in the popular sense is shown as SEQ ID No.13-36, wherein the nucleotide sequence of SEQ ID No.13-14 is used for amplifying Mv01, and so on.

3. The polymorphic primer of the molecular marker fragment of lumbricus in the popular sense of claim 2, wherein the polymorphic primer of the molecular marker fragment of lumbricus is used in combination.

4. The polymorphic primer of the molecular marker fragment of lumbricus in the popular sense of claim 2, wherein the nucleotide sequence of the polymorphic primer of the molecular marker fragment of lumbricus is fluorescently labeled.

5. The polymorphic primer of the molecular marker fragment of lumbricus microsatellite according to claim 3 wherein said fluorescent marker is FAM, HEX or TAMRA.

6. The application of the molecular marker fragment of the lumbricus in the genetic polymorphism, genetic relationship identification and variety identification of the lumbricus in the popular field.

7. The application of polymorphic primers of molecular marker fragments of Leptospira collocaliae in genetic polymorphism, genetic relationship identification and variety identification of Leptospira collocaliae in claim 2.

8. The use according to claim 6 or 7, wherein the detection of genetic polymorphisms of the lumbricus colloquialis comprises the steps of:

1) Extracting DNA of the popular lumbricus;

2) PCR amplification is carried out by adopting polymorphic primers shown in SEQ ID No.13-36 in claim 2 and using DNA of common lumbricus as a template to obtain an amplified product;

3) Genotype is determined based on the molecular weight of the amplified product and genetic diversity parameters are calculated.