CN112522423A

CN112522423A - Fuworm microsatellite molecular marker and polymorphism primer and application thereof

Info

Publication number: CN112522423A
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: 2021-03-19
Anticipated expiration: 2040-12-15
Also published as: CN112522423B

Abstract

The invention discloses a microsatellite molecular marker of a common Lupulus lumbricus, a polymorphic primer and application thereof, belonging to the field of biotechnology detection. The method utilizes RAD-SEQ high-throughput sequencing technology to screen 12 microsatellite loci from DNA of a Laurella furcifera genome, wherein the microsatellite loci are Mv01, Mv02, Mv03, Mv04, Mv05, Mv06, Mv07, Mv08, Mv09, Mv10, Mv11 and Mv12, and corresponding nucleotide sequences are shown as SEQ ID Nos. 1-12; and designing specific primers according to flanking sequences at two ends of the microsatellite locus, wherein the nucleotide sequence of the primers is shown as SEQ ID No.13-36, and the amplification result of the obtained primers has polymorphism and stability, so that the primers can be applied to the fields of genetic diversity detection, individual identification and molecular assisted breeding of the population of the Laureria praecox and lay a solid foundation for the microsatellite polymorphism detection of the Laureria praecox.

Description

Fuworm microsatellite molecular marker and polymorphism primer and application thereof

Technical Field

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

Background

The common lumbricus (Metaphire vulgaris) belongs to the class of Clitella (Clitella), the order of the posterior foramina (Opisthopora), the family Megascolecidae (Megascolecidae), the genus Metaphire (Metaphire), and is widely distributed in Jiangsu, Zhejiang, etc. The earthworm contains high-concentration succinic acid, and can be used for relieving asthma, promoting urination, relieving spasm, etc.; it also contains various unsaturated fatty acids such as oleic acid and linoleic acid, and can be used for preventing and treating arteriosclerosis, promoting blood circulation for removing blood stasis, and preventing and treating cardiovascular and cerebrovascular diseases. The traditional Chinese medicine is commonly called earthworm. In addition, earthworms are used as medium-large soil decomposers, and their ecological status is self-evident, and they are called "ecological engineers".

Microsatellite markers, also known as Simple Sequence Repeats (SSRs) or Simple Sequence Length Polymorphisms (SSLPs), are Simple repetitive sequences of 1-6 bases in repeat units, with repeat units of 2-3 bases being the majority and are called microsatellites because they are shorter in repeat units than microsatellite sequences. Microsatellite DNA is generally flanked by relatively conserved single copy sequences and there is a difference in the number of repeats between different alleles. Compared with other genetic molecular markers, the microsatellite DNA marker not only has extremely high individual specificity and repeatability, but also can provide abundant polymorphic sites due to relatively high variation rate, and obtains genetic information such as heterozygosity, purity and the like through polymorphism analysis of the sites. At present, microsatellite DNA markers are widely applied in population genetic structure research, genetic relationship identification, variety identification and other aspects.

The microsatellite locus detection method comprises agarose gel electrophoresis, polyacrylamide gel electrophoresis (PAGE), capillary electrophoresis and fluorescence labeling full-automatic genome scanning. However, because the length difference of partial microsatellite DNA fragments is small and cannot be distinguished by an agarose gel electrophoresis method, at present, a microsatellite fluorescent marking full-automatic genotyping technology is generally adopted, FAM, TAMRA and HEX are utilized, 3 fluorescent dyes with different colors are used for marking microsatellite DNA primers, PCR products of various fluorescent markers and different amplified fragments can be electrophoresed in the same sample adding hole, the PCR product to be detected and the DNA internal molecular weight standard can be simultaneously loaded, and the size of the microsatellite allelic gene fragments can be accurately calculated by sequential gel electrophoresis and using software to perform image analysis. The method has the advantages of high yield, low cost, rapidness, simplicity and convenience and the like, and shows wide application prospect.

Disclosure of Invention

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

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

the method utilizes RAD-seq high-throughput sequencing technology to screen 12 microsatellite loci from DNA of the genome of the Laurella furcifera, and the marker number is Mv 01-12. The nucleotide sequence corresponding to the molecular marker of the Fulviax furiosus is shown in SEQ ID No. 1-12.

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

The application of the molecular marker of the microsatellite of the Lasioderma aspergillum and/or the polymorphic primer of the molecular marker in genetic polymorphism, genetic relationship identification and variety identification of the Lasioderma aspergillum.

Further, the polymorphic primer of the microsatellite molecular marker is used for detecting genetic diversity of the Laureria vulgata, and comprises the following steps:

1) extracting genome DNA;

2) micro-satellite PCR amplification: adopting a fluorescent labeled microsatellite primer of FAM, HEX and TAMRA to amplify DNA of a genome of the common earthworm to obtain an amplification product;

3) genetic diversity analysis: genotypes were determined based on the molecular weight of each individual microsatellite amplification product and genetic diversity parameters were calculated using Cervus 3.0.7.

Has the advantages that: compared with the prior art, the invention has the advantages that:

the invention utilizes RAD-seq high-throughput sequencing technology to screen 12 microsatellite loci from the genome DNA of the Laureria pratense, and designs specific primers according to flanking sequences at two ends of the microsatellite loci, and the amplification result of the obtained primers has polymorphism and stability, so that the method is applicable to the fields of genetic diversity detection, individual identification and molecular assisted breeding of the Laureria pratense population, and lays a solid foundation for the microsatellite polymorphism detection of the Laureria pratense.

Detailed Description

The invention is further described with reference to specific examples.

Example 1

Firstly, extracting DNA of the common lumbricus

Collecting 30 Suo Lumbrialus samples, storing in absolute ethyl alcohol, and extracting DNA by adopting a DNA extraction kit of Nanjing NuoWei Zan biotechnology limited company, wherein the method comprises the following steps:

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

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

c. Add 250. mu.L Buffer GB to the digest, vortex at maximum speed and mix well for 20sec, water bath at 70 ℃ for 10 min.

d. Add 250. mu.L of absolute ethanol to the digest and centrifuge manually for 15-20 sec.

e. The gDNA Columns were placed in a Collection tube of 2 mL. Transferring the mixed liquid (including precipitate) obtained in the last step to an adsorption column. Centrifuging at 12,000 Xg for 1min, and centrifuging at 14,000 Xg for 3-5min if column blockage occurs.

f. Discarding the waste liquid, and placing the adsorption column in a collection tube. Add 500. mu.L Washing Buffer A to the adsorption column. Centrifuge at 12,000 Xg for 1min.

g. Discarding the waste liquid, and placing the adsorption column in a collection tube. 650. mu.L of Washing Buffer B was added to the adsorption column. Centrifuge at 12,000 Xg for 1min. The operation was repeated once.

h. Discarding the waste liquid, and placing the adsorption column in a collection tube. Centrifuge in empty tube at 12,000 Xg for 2 min.

i. The column was placed in a new 1.5mL centrifuge tube. Adding 30-100 μ L of solution Buffer preheated to 70 deg.C to the center of the membrane of the adsorption column, and standing at room temperature for 3 min. Centrifuge in empty tube at 12,000 Xg for 2 min.

j. The adsorption column is discarded, and the DNA is stored at 2-8 ℃ and stored at-20 ℃ for a long time.

Second, screening of polymorphic microsatellite primers of common Lupulus lumbricus

According to the RAD-SSR sequence of the genome of the common lumbricus, RAD-SSR design of two to four nucleotide different repetitive motifs is selected, 20 pairs of microsatellite primers are designed by using software Premier 5, and the main parameters of the primer design are as follows: the length of the primer is about 22bp, and the length of the product is 120-200 bp; a single primer is complementary by less than 3 nucleotides; ensuring the Tm values of the two primers to be above or below 55-60 ℃; the two primers are paired with less than 3 consecutive nucleotides, (G + C)% content is approximately (40% -60%). And screening 12 pairs of microsatellite primers through primary detection.

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

TABLE 1 Gentle Lumbriae microsatellite loci and primers, fluorescent markers and optimal annealing temperatures thereof

Fluorescence labeling microsatellite PCR amplification and genetic diversity analysis of Laureria praecox

Labeling 12 pairs of microsatellite primers obtained by screening, wherein forward primers are labeled by different fluorescent substances, and fluorescence labels of Mv09, Mv10, Mv11 and Mv12 are FAM; the fluorescent labels of Mv01, Mv02, Mv03 and Mv07 are HEX; the fluorescent markers Mv04, Mv05, Mv06, Mv08 are TAMRA. The DNA sample of the lumbricus pratense obtained by the fluorescent primer pairs is subjected to PCR amplification. 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, and upstream and downstream primers0.5. mu.L (concentration 10. mu.M), 10.5. mu.L each of ddH₂O, the PCR amplification program is as follows: pre-denaturation at 95 ℃ for 5 min; 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 for 5min at 72 ℃. The Allele number (Na), the desired heterozygosity (He) and the Polymorphic Information Content (PIC) of each microsatellite marker were calculated using the allee frequency analysis in the software Cervus v.3.0.7, and the results showed that the number of alleles at each microsatellite locus varied from 2 to 8, the desired heterozygosity (He) ranged from 0.183 to 0.792, and the Polymorphic Information Content (PIC) ranged from 0.164 to 0.745, with the detailed results shown in table 2.

TABLE 2 detection of microsatellite loci

Site of the body	Core repeat sequences	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> Nanjing university of forestry

<120> popular Lumbricidae microsatellite molecular markers, and polymorphism 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. A group of popular Lumbria punctata microsatellite molecular markers are characterized by being Mv01, Mv02, Mv03, Mv04, Mv05, Mv06, Mv07, My08, Mv09, Mv10, Mv11 and Mv12, and the nucleotide sequences corresponding to the popular Lumbria punctata microsatellite molecular markers are shown in SEQ ID Nos. 1-12.

2. The polymorphic primer of the lumbricus vulvosa microsatellite molecular marker as claimed in claim 1, wherein the nucleotide sequence of the polymorphic primer of the lumbricus vulvosa microsatellite molecular marker is shown as SEQ ID No.13-36, wherein the nucleotide sequence SEQ ID No.13-14 is used for amplifying Mv01, and the like.

3. The polymorphic primers for the microsatellite molecular markers of the lumbricus solvolensis as claimed in claim 2, wherein the polymorphic primers for the microsatellite molecular markers can be used in combination.

4. The polymorphic primer of the microsatellite molecular marker of the lumbricus solvolensis as claimed in claim 2, wherein the nucleotide sequence of the polymorphic primer of the microsatellite molecular marker is fluorescently labeled.

5. The polymorphic primer of a marks of the Calla variegata microsatellite molecule according to claim 3, wherein the fluorescent mark is FAM, HEX or TAMRA.

6. The use of the lumbricus solvolvata microsatellite molecular marker of claim 1 in genetic polymorphism, genetic relationship identification, and breed identification of lumbricus solvolvata.

7. The application of the polymorphic primer of the molecular marker of the lumbricus vulgare in the genetic polymorphism, genetic relationship identification and variety identification of the lumbricus vulgare in the claim 2.

8. The use as claimed in claim 6 or 7, wherein the detection of genetic polymorphism of Lupulus obliquus comprises the following steps:

1) extracting DNA of the earthworm;

2) carrying out PCR amplification by using the polymorphic primers shown as SEQ ID No.13-36 in claim 2 and DNA of the Lupulus communis as a template to obtain an amplification product;

3) determining the genotype according to the molecular weight of the amplified product, and calculating the genetic diversity parameter.