CN110283931B - SSR fingerprint of 6 good varieties of pomegranate in Anhui, Huaihei and China, and construction method and application thereof - Google Patents
SSR fingerprint of 6 good varieties of pomegranate in Anhui, Huaihei and China, and construction method and application thereof Download PDFInfo
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
The invention discloses an SSR fingerprint of 6 good varieties of pomegranate in Anhui-Huai-Bei, wherein the 6 good varieties of pomegranate in Anhui-Huai-Bei are respectively as follows: huaibei soft seed No. 2, Huaibei soft seed No. 3, crystal sugar seed, Jinxihong, Nanzhuang super-red and Tashan red; the SSR fingerprint is constructed based on 10 pairs of SSR primers, and the 10 pairs of SSR primers are respectively as follows: PG080, PG130, PG139, PG152, PG098, PG077, PG090, PG093, PG070 and PG153, wherein the nucleotide sequences are sequentially shown as SEQ ID NO.1-20, and the fingerprint maps of 6 varieties are shown as Table 1. The invention also discloses a construction method and application thereof. The invention provides a theoretical basis for the authenticity identification, breeding utilization and new variety right protection of 6 pomegranate varieties.
Description
Technical Field
The invention relates to the technical field of molecular markers, in particular to an SSR fingerprint of 6 good varieties of pomegranate in Anhui, Huaihei and the construction method and the application thereof.
Background
Pomegranate is an ancient health-care fruit, fruits are rich in anthocyanin, flavone, punicalagin and other polyphenols, particularly punicalagin and other ellagitannin substances, have high clinical medical and health-care values, have potential functions of oxidation resistance, cancer resistance, bacteria resistance and the like, are known as super fruits, and are classified as one of national planning special economic forest trees. It is recorded that the pomegranate has more than 2000 years of cultivation history in China, and a plurality of excellent pomegranate varieties with local characteristics are formed in the long-term cultivation process. The method is characterized in that Anhui Huaibei is a production base of six pomegranate in China, Huaibei pomegranate is mainly distributed in the tower village and the Huang-Li village in the local mountain area, Huaibei tower mountain is a base of Chinese soft-seed pomegranate, Tashan pomegranate is also a national geographical marker product, and main cultivated varieties comprise good varieties such as Jinxihong, Nanzhuang super-red and Tashan red; the excellent variety of Huangli mainly comprises rock candy seeds and soft seed series. Huaibei pomegranate is popular with consumers because of thin peel and big seed and unique flavor.
The unique economic, ecological and social effects of the pomegranate promote the rapid development of the pomegranate industry. In the rapid development process of the pomegranate industry, frequent introduction and variety exchange between different areas often lead to variety mixing, the phenomenon of homonymy or homonymy foreign matter appears, the storage, identification and utilization of pomegranate resources are very unfavorable, and the matching and pedigree analysis of parents in cross breeding are also adversely affected. In production, pure variety is the condition for guaranteeing high-efficiency cultivation. Therefore, the fingerprint spectrum of the variety is determined, and the method has important practical significance for the storage, identification and utilization of pomegranate resources. The molecular marker can directly detect the difference of different varieties or individuals on the DNA molecular level, has higher polymorphism and individual specificity, is not influenced by environment and growth stage, and can identify the individuals which are difficult to identify by the morphological marker. Therefore, the development of reliable molecular markers and the construction of pomegranate fingerprint spectra have important significance for rapid identification of pomegranate.
The Huaibei soft seed No. 2, the Huaibei soft seed No. 3, the rock candy seed, the brocade red, the Nanzhuang super red and the Tashan red are 6 excellent main pomegranate varieties in the North of Anhui province, and because the pomegranate varieties have excellent properties and high commodity value, the pomegranate varieties are always well-graded and have false and spurious phenomena in the processes of seedling production, fruit sales and introduction, so that the fingerprint spectrum of the 6 excellent pomegranate varieties is established, and the pomegranate varieties have important significance for scientific and reasonable utilization of the varieties.
Disclosure of Invention
The invention provides SSR finger prints of 6 good varieties of pomegranate in Anhui-Huai-Bei, wherein the 6 good varieties of pomegranate in Anhui-Huai-Bei are respectively as follows: huaibei soft seed No. 2, Huaibei soft seed No. 3, crystal sugar seed, Jinxihong, Nanzhuang super-red and Tashan red;
the SSR fingerprint is constructed based on 10 pairs of SSR primers, and the 10 pairs of SSR primers are respectively as follows: PG080, PG130, PG139, PG152, PG098, PG077, PG090, PG093, PG070 and PG153, wherein the nucleotide sequences of the PG080, PG130, PG139, PG152, PG098, PG077, PG090, PG093, PG070 and PG153 are shown in SEQ ID NO.1-20 in sequence;
the fingerprint spectrums of 6 good varieties of the pomegranate in Anhui Huaihe province constructed by utilizing the 10 pairs of primers are shown in the table 1:
TABLE 16 SSR finger prints of good varieties of pomegranate in Anhui Huaihe province
The second aspect of the invention provides a method for constructing the SSR fingerprints of the 6 good varieties of pomegranate in Anhui, Huaihei and Heihei, which comprises the following steps:
(1) extracting DNA by CTAB method, adding 50 μ L of 0.1M TE buffer solution, dissolving overnight, and storing at-20 deg.C;
(2) 10 pairs of primers are adopted, the extracted DNA is taken as a template for PCR amplification, and the PCR amplification adopts a 20 mu L reaction system containing 1.0ng of DNA, 0.4 mu M forward primer, 0.4 mu M reverse primer and 4mM MgCl2400 μ M dNTPs, 1.0U Taq-DNase, plus ddH20 to the total volume of 20 mu L, and adopting touchdown reaction in PCR under the following reaction conditions: 5min, 95 ℃; the next 11 cycles, each after which the annealing temperature dropped by 0.8 ℃ {30s, 95 ℃; 30s, 65 ℃; 50s, 72 ℃; 22 cycles {30s, 95 ℃; 30s, 55 ℃; 50s, 72 ℃; finally, extending for 8min at 72 ℃;
(3) determining the fragment size of the PCR product by using capillary electrophoresis, wherein the capillary electrophoresis is carried out according to the following steps of diluting the 6-FAM or HEX fluorescence labeled PCR product by 30 times by using ultrapure water, and sucking 1 mu L of product to add into a deep-hole plate hole special for a DNA analyzer; 0.1 μ L of LIZ500 molecular weight internal standard and 8.9 μ L of deionized formamide were added to each well of the plate; denaturation at 95 deg.C for 5min, and cooling at 4 deg.C for 10 min; after instantaneous centrifugation for 10s, placing the sample on a DNA analyzer for automatic fluorescence detection;
(4) analyzing SSR amplification data by using Genemapper software, and detecting amplified fragment peaks by using SSR capillary electrophoresis, wherein the abscissa is the size of a fragment, and the ordinate is a fluorescence intensity value; the data of the size of the allelic variation of the homozygous locus is recorded as X/X, wherein X is the size of the allelic variation of the locus; allelic variation data for heterozygous loci were recorded as X/Y, where X, Y is two different allelic variations at the locus, small before and large after.
The third aspect of the invention provides application of SSR fingerprints of 6 good varieties of pomegranate in the aspects of pomegranate variety identification.
The invention has the beneficial effects that:
1. according to the invention, 10 pairs of SSR core primers with good repeatability and rich polymorphism are utilized to construct DNA finger prints of 10 pairs of core primers of 6 good varieties of pomegranate in Anhui, Huaihei and North China. Compared with the conventional morphological detection, the fingerprint has the advantages of short detection time, high accuracy, reliability, convenience, good repeatability and the like. The invention provides a theoretical basis for the authenticity identification, breeding utilization and new variety right protection of 6 pomegranate varieties.
2. The fingerprint spectrum of the variety constructed by the invention is very stable and is not influenced by the environment. When the variety is identified, the seedlings or the adult plants can be used, tissues or organs such as leaves, flowers, fruits, buds and the like can be used for detection, and the material taking is not limited by seasons. The present invention can be used directly in production practice.
Detailed Description
The present invention will be further explained with reference to examples. The following examples are provided only for illustrating the present invention and are not intended to limit the scope of the present invention.
Example 1
Obtaining primer pairs for constructing SSR fingerprints of 6 good varieties of pomegranate in Anhui, Huaihei and Heihei:
(1) pomegranate genome data was downloaded from the DDBJ/ENA/GenBank database, serial number MTKT 00000000. And (3) mining SSR sites of different repeat units in a whole genome range by using MISA (MicrosAtellites identification tool, http:// pgrc. ipk-gatersleen. de/MISA) software, wherein the search criteria of the SSR are that the repeat times of dinucleotides, trinucleotides, tetranucleotides, pentanucleotides and hexanucleotides are respectively 11, 8, 6, 5 and 4.
(2) SSR primer design
5 pairs of SSR loci obtained above are randomly selected on each chromosome, conserved flanking sequences at two ends of a repetitive sequence are utilized, primers are designed by using Primer 3.0, and the conditions for Primer design are as follows: the length of the PCR product is 100-350 bp; the annealing temperature (Tm) is 50-70 ℃, and the Tm difference between two primers is not more than 4 ℃; the GC% content is 40-65%; the length of the primer is 18-28 bp; the 5 'end of the primer is preferably G/C, and the 3' end avoids A. In order to ensure the specificity of the primer, the conservative flanking sequence for designing the primer is at least separated from the microsatellite locus by 20-23 bases. 45 pairs of primers were successfully designed and synthesized by Biotechnology engineering (Shanghai) GmbH.
(3) Primer screening
By adopting 45 pairs of synthesized primers, the 6 Anhui Huaibei pomegranate varieties (Huaihei soft seed No. 2, Huaihei soft seed No. 3, crystal sugar seed, brocade embroidered red, Nanzhuang super red and Tashan red) are used as materials, 10 pairs of primers (shown in Table 2) which can be stably amplified, have clear banding patterns and rich polymorphism are screened according to the amplification result, and are used for constructing SSR fingerprint spectrums of the 6 Anhui Huaihei pomegranate excellent varieties.
TABLE 210 primer sequences
Example 2
Constructing SSR fingerprints of 6 good varieties of pomegranate in Anhui and Huaibei:
(1) extracting the DNA of the test variety genome by adopting a CTAB method: weighing 0.2-0.3 g of fresh leaves, adding liquid nitrogen, quickly grinding into powder, and transferring the powder into a 2.0mL centrifuge tube; adding 1.0mL of a preheated 3 xCTAB extracting solution at 65 ℃ into a water bath at 65 ℃ for 1 h; centrifuging at 12000rpm for 10min at room temperature; sucking the supernatant into a 2.0mL centrifuge tube; adding phenol/chloroform/isoamyl alcohol (25:24:1, V/V/V) with the same volume as the supernatant, and slightly reversing and mixing to obtain emulsion; centrifuging at 12000r/min for 8min, collecting supernatant, adding equal volume of chloroform/isoamyl alcohol (24:1, V/V), mixing, and centrifuging at 12000r/min for 8 min; adding isovoluminal frozen isopropanol and 10 μ L of 3M sodium acetate into the supernatant, and standing at-20 deg.C for precipitation for half an hour; centrifuging at 12000r/min for 8min, carefully discarding the supernatant, and keeping the DNA in a centrifuge tube; adding 75% ethanol, washing for 2 times, washing with anhydrous ethanol, centrifuging, and removing anhydrous ethanol; air-dried at room temperature, added with 50. mu.L of TE buffer (0.1M), and dissolved overnight, and stored at-20 ℃ for further use.
(2) PCR amplification was carried out using 10 pairs of primers of example 1 and the extracted DNA as a template, using a 20. mu.L reaction system containing 1.0ng of DNA and 0.4. mu.M of forward primer,0.4. mu.M reverse primer, 4mM MgCl2400 μ M dNTPs, 1.0U Taq-DNase, plus ddH20 to the total volume of 20 mu L, and adopting touchdown reaction in PCR under the following reaction conditions: 5min, 95 ℃; the next 11 cycles, each after which the annealing temperature dropped by 0.8 ℃ {30s, 95 ℃; 30s, 65 ℃; 50s, 72 ℃; 22 cycles {30s, 95 ℃; 30s, 55 ℃; 50s, 72 ℃; finally, extension is carried out for 8min at 72 ℃.
(3) The PCR product was fragment sized by capillary electrophoresis. The capillary electrophoresis was performed by diluting 6-FAM or HEX fluorescently labeled PCR product 30 times with ultrapure water, and pipetting 1. mu.L of the product to add to the well of a deep well plate dedicated to a DNA analyzer. 0.1. mu.L of LIZ500 molecular weight internal standard and 8.9. mu.L of deionized formamide were added to each well of the plate. Denaturation at 95 deg.C for 5min, and cooling at 4 deg.C for 10 min. After being subjected to instantaneous centrifugation for 10s, the sample was placed on a DNA analyzer (ABI3730XL) for automatic fluorescence detection.
(4) And analyzing SSR amplification data by using Genemapper software, and detecting amplified fragment peaks by using SSR capillary electrophoresis, wherein the abscissa is the size of the fragment and the ordinate is the fluorescence intensity value. The data of the size of the allelic variation of the homozygous locus is recorded as X/X, wherein X is the size of the allelic variation of the locus; allelic variation data for heterozygous loci were recorded as X/Y, where X, Y is two different allelic variations at the locus, small before and large after. The data of 10 sites are integrated together to form SSR fingerprints of 6 pomegranate varieties. The number of the different points of the variety difference between the varieties is more than or equal to 3, and the varieties are different; the number of the ectopic points of the variety difference between the varieties is less than 3, which is an approximate variety. The SSR fingerprints of 6 constructed pomegranate varieties are shown in table 1, and the number of ectopic points of difference between 6 varieties is more than or equal to 3.
Sequence listing
<110> horticultural research institute of academy of agricultural sciences of Anhui province
SSR fingerprint of <120> 6 good varieties of pomegranate in Anhui, Huaihei and North China, and construction method and application thereof
<160> 20
<170> SIPOSequenceListing 1.0
<210> 1
<211> 24
<212> DNA
<213> PG080 Forward primer (Artificial sequence)
<400> 1
ctgactgttg cagagagtag gctg 24
<210> 2
<211> 24
<212> DNA
<213> PG080 reverse primer (Artificial sequence)
<400> 2
aggaggtgaa acaacgaata gctg 24
<210> 3
<211> 24
<212> DNA
<213> PG130 Forward primer (Artificial sequence)
<400> 3
ctcatatggc gattctctgt cctt 24
<210> 4
<211> 24
<212> DNA
<213> PG130 reverse primer (Artificial sequence)
<400> 4
aagttcgata aattgcactg gtgg 24
<210> 5
<211> 22
<212> DNA
<213> PG139 Forward primer (Artificial sequence)
<400> 5
gtttccttcc ctcaacccaa aa 22
<210> 6
<211> 24
<212> DNA
<213> PG139 reverse primer (Artificial sequence)
<400> 6
agtgggattt taccaagtcg aaca 24
<210> 7
<211> 21
<212> DNA
<213> PG152 Forward primer (Artificial sequence)
<400> 7
catcagaatc gtccccttgt g 21
<210> 8
<211> 24
<212> DNA
<213> PG152 reverse primer (Artificial sequence)
<400> 8
cagagagaag aagagagacc gagc 24
<210> 9
<211> 24
<212> DNA
<213> PG098 Forward primer (Artificial sequence)
<400> 9
tgccttctta aggacttcac caac 24
<210> 10
<211> 24
<212> DNA
<213> PG098 reverse primer (Artificial sequence)
<400> 10
ctaacctcat gcacttgtca tcca 24
<210> 11
<211> 24
<212> DNA
<213> PG077 forward primer (Artificial sequence)
<400> 11
gtcagtctcc tccttcttca atgg 24
<210> 12
<211> 23
<212> DNA
<213> PG077 reverse primer (Artificial sequence)
<400> 12
agacgaagca cctgagaagg aat 23
<210> 13
<211> 28
<212> DNA
<213> PG090 Forward primer (Artificial sequence)
<400> 13
attcttttat actaaccaaa atttgcga 28
<210> 14
<211> 22
<212> DNA
<213> PG090 reverse primer (Artificial sequence)
<400> 14
atgtcatgag aggacccaca aa 22
<210> 15
<211> 24
<212> DNA
<213> PG093 Forward primer (Artificial sequence)
<400> 15
cgtcaatagg acgtccctga gata 24
<210> 16
<211> 24
<212> DNA
<213> PG093 reverse primer (Artificial sequence)
<400> 16
gatgacgtgg cagagtaaga gagc 24
<210> 17
<211> 24
<212> DNA
<213> PG070 forward primer (Artificial sequence)
<400> 17
cacctctgct tcagcaaaca aata 24
<210> 18
<211> 24
<212> DNA
<213> PG070 reverse primer (Artificial sequence)
<400> 18
caactcaaca caatatccaa ccca 24
<210> 19
<211> 24
<212> DNA
<213> PG153 Forward primer (Artificial sequence)
<400> 19
gtgtttgatg ctcccatttc attt 24
<210> 20
<211> 24
<212> DNA
<213> PG153 reverse primer (Artificial sequence)
<400> 20
gccttcaacg gtctttcttc ttct 24
Claims (3)
1. An SSR primer group for constructing SSR fingerprints of 6 good varieties of pomegranate in Anhui, Huaihei and Heihei is characterized in that,
the 6 good varieties of the pomegranate in Anhui Huaihe are respectively as follows: huaibei soft seed No. 2, Huaibei soft seed No. 3, crystal sugar seed, Jinxihong, Nanzhuang super-red and Tashan red;
the SSR primer group comprises 10 pairs of SSR primers, and the 10 pairs of SSR primers respectively comprise: PG080, PG130, PG139, PG152, PG098, PG077, PG090, PG093, PG070 and PG153, wherein the nucleotide sequences are shown in SEQ ID NO.1-20 in sequence.
2. The method for constructing the SSR fingerprint of 6 good varieties of pomegranate in Anhui Huaihei by using the SSR primer group in claim 1 is characterized by comprising the following steps of:
(1) extracting DNA by CTAB method, adding TE buffer solution of 50 μ L0.1M, dissolving overnight, and storing at-20 deg.C;
(2) 10 pairs of primers are adopted, the extracted DNA is taken as a template for PCR amplification, and the PCR amplification adopts a 20 mu L reaction system containing 1.0ng of DNA, 0.4 mu M forward primer, 0.4 mu M reverse primer and 4mM MgCl2400 μ M dNTPs, 1.0U Taq-DNase, plus ddH2And O, until the total volume is 20 mu L, performing a touchdown reaction on the PCR by adopting the following reaction conditions: 5min, 95 ℃; following 11 cycles, the annealing temperature decreased by 0.8 ℃ after each cycle, and the procedure for each cycle was as follows: 30s, 95 ℃; 30s, 65 ℃; 50s, 72 ℃; 22 cycles, each cycle as follows: 30s, 95 ℃; 30s, 55 ℃; 50s, 72 ℃; finally, extending for 8min at 72 ℃;
(3) determining the size of the fragment of the PCR product by using capillary electrophoresis, wherein the capillary electrophoresis is carried out according to the following steps of diluting the 6-FAM or HEX fluorescence labeled PCR product by 30 times by using ultrapure water, and sucking 1 mu L of product to add into a deep-hole plate hole special for a DNA analyzer; 0.1 μ L of LIZ500 molecular weight internal standard and 8.9 μ L of deionized formamide were added to each well of the plate; denaturation at 95 deg.C for 5min, and cooling at 4 deg.C for 10 min; placing the mixture on a DNA analyzer for automatic fluorescence detection after instantaneous centrifugation for 10 s;
(4) analyzing SSR amplification data by using Genemapper software, and detecting amplified fragment peaks by using SSR capillary electrophoresis, wherein the abscissa is the size of a fragment, and the ordinate is a fluorescence intensity value; the data of the size of the allelic variation of the homozygous locus is recorded as X/X, wherein X is the size of the allelic variation of the locus; allelic variation data for the heterozygous locus is recorded as X/Y, wherein X, Y is two different allelic variations at the locus, small before and large after;
the data of 10 sites are integrated together to form SSR fingerprints of 6 pomegranate varieties, and the number of different spots between varieties is more than or equal to 3; the number of the ectopic points of the difference between the varieties is less than 3, and the varieties are similar; the SSR fingerprints of the 6 constructed pomegranate varieties are shown in the table 1:
TABLE 16 SSR finger prints of good varieties of pomegranate in Anhui Huaihe province
The number of the ectopic points of the 6 varieties is more than or equal to 3.
3. The application of the SSR fingerprints of the 6 good varieties of pomegranate in Anhui Huaihei province constructed by the method in claim 2 in the identification of pomegranate varieties.
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