CN114908088B - Carpinus SSR primer developed based on whole genome data and identification method thereof - Google Patents
Carpinus SSR primer developed based on whole genome data and identification method thereof Download PDFInfo
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Abstract
The invention discloses a carpinus wilfordii SSR primer developed based on whole genome data and an identification method thereof, wherein the carpinus wilfordii SSR primer sequence comprises one or more than one of 8 pairs of primer sequences. The invention develops the carpinus SSR molecular marker primer based on the whole genome sequence through bioinformatics analysis software, and provides a convenient and effective method for developing carpinus SSR markers.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a carpinus wilfordii SSR primer group developed based on whole genome data and an identification method thereof.
Background
Carpinus (carpinus) belongs to carpinus genus (Betulaceae) belonging to Betulaceae. China is the distribution center of carpinus plants, wherein carpinus of carpinus is widely distributed. At present, carpinus is in a wild state, a small amount of the seed is introduced in a plant garden, a campus, a park, etc. The tree has straight branches, purple red new leaves, luxuriant branches and leaves and strong resistance, and is a potential excellent landscaping tree species.
At present, the research on carpinus at home and abroad is mostly surrounded by systematic development, genetic diversity and population genetic structure analysis are still in a blank state through SSR molecular markers in China, and SSR primer screening is a primary condition for molecular marker experiments.
SSR molecular markers refer to tandem repeat units of 1-6bp, are widely distributed in genomes of eukaryotic organisms and prokaryotic organisms, and are popular in the fields of population genetics, kindred species definition and the like. And the SSR marker is adopted to carry out genetic diversity analysis on the carpinus, so that introduction, domestication, popularization and application of the carpinus are facilitated.
Disclosure of Invention
This section is intended to outline some aspects of embodiments of the invention and to briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section as well as in the description summary and in the title of the application, to avoid obscuring the purpose of this section, the description summary and the title of the invention, which should not be used to limit the scope of the invention.
The invention provides a carpinus wilfordii SSR primer developed based on whole genome data, wherein: the carpinus wilfordii SSR primer sequence comprises one or more of the following 8 pairs:
CV4 upstream primer: tgtcaaactcatgtttgtcgttt; a downstream primer: ttggaggatatggataacgc;
CV24 upstream primer: ttattatttcttgcttggcagtt; a downstream primer: tgtaaaattgtattcatacccattgat;
CV27 upstream primer: tgtaatttttaatgagaaactcaatcg; a downstream primer:
atatgtcaacacaaagggaggg；
CV65 upstream primer: atctatagtggtggccggtg; a downstream primer: gatcctccaattatttggcact;
CV67 upstream primer: gcaatatttacaaagtgatggca; a downstream primer: tggtttgtttgtagttgttattcttga;
CV71 upstream primer: tctaccacaatgctttgcagat; a downstream primer: tgcgccttttaataaagcagt;
CV81 upstream primer: atgtgatccacgtgacgc; a downstream primer: gcctcaaaattaaaacagtgtcat;
CV99 upstream primer: taaaacatggattggggcat; a downstream primer: aagcacttatattccttgacacca.
As another aspect of the present invention, the present invention provides a method for SSR identification of carpinus developed based on whole genome data, which consists of the steps of:
(1) Extracting genome DNA of carpinus;
(2) Taking the carpinus genome DNA as a template and using the SEQ ID NO: 1-8 primer, and detecting PCR product.
As a preferable scheme of the carpinus wilfordii SSR identification method developed based on whole genome data, the invention has the following advantages that: the method for extracting the carpinus genome DNA is to extract the carpinus genome DNA by adopting a CTAB method.
As a preferable scheme of the carpinus wilfordii SSR identification method developed based on whole genome data, the invention has the following advantages that: the method comprises the steps of extracting the carpinus genome DNA by adopting a CTAB method, adding the carpinus powder into CTAB extracting solution preheated by a water bath at 65 ℃, then adding the decomposing enzyme, adding the mercaptoethanol, uniformly mixing, and then placing into a water bath at 65 ℃ for 0.5-1 h; centrifuging at normal temperature of 12000rpm for 10min, collecting supernatant, adding an equal volume of extract, shaking and mixing for 5-10min, centrifuging, collecting supernatant, adding pre-cooled isopropanol, and precipitating in a refrigerator at-20deg.C for 1 hr; putting into a centrifuge 12000rpm for centrifugation for 10min; pouring out the liquid, adding 75% alcohol for washing, centrifuging, air-drying the precipitate, and adding ultrapure water for dissolution.
As a preferable scheme of the carpinus wilfordii SSR identification method developed based on whole genome data, the invention has the following advantages that: the extract is chloroform: isoamyl alcohol volume ratio 24:1.
as a preferable scheme of the carpinus wilfordii SSR identification method developed based on whole genome data, the invention has the following advantages that: the PCR reaction is carried out, and the PCR reaction system is as follows: 2X Taq master Mix 10uL,10 mu mo1/L upstream primer: 0.75ul, 10. Mu. Mo1/L of downstream primer 0.75ul, 50ng of DNA, and sterilized ddH 2 07.5ul。
As a preferable scheme of the carpinus wilfordii SSR identification method developed based on whole genome data, the invention has the following advantages that: the PCR reaction is carried out, and the PCR amplification program is as follows: 94 ℃ for 5min;94℃for 40s,52℃for 40s,72℃for 120s,35 cycles, and finally 72℃for 10min.
As a preferable scheme of the carpinus wilfordii SSR identification method developed based on whole genome data, the invention has the following advantages that: the PCR products were detected by 2% agarose gel electrophoresis.
As a preferable scheme of the carpinus wilfordii SSR identification method developed based on whole genome data, the invention has the following advantages that: the carpinus of the Tripterygium wilfordii comprises 6 carpinus of the Tripterygium wilfordii, wherein the germ plasm herd of the 6 carpinus of the Tripterygium wilfordii is Zhejiang, hubei Shennong frame, and Guizhou, and the carpinus of the Tripterygium wilfordii, the Guangdong milk source, the Chongqing, the Nanchuan, and the Tibet, the Nie, and the Tripterygium wilfordii.
The invention has the beneficial effects that:
(1) Based on the whole genome sequence, the carpinus wilfordii SSR molecular marker primer is developed through bioinformatics analysis software.
(2) The invention provides a convenient and effective method for developing the carpinus pterinus SSR marker, enriches the current carpinus pterinus SSR marker primer, and provides a certain reference effect for researches on related aspects of kindred species.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the description of the embodiments will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art. Wherein:
FIG. 1 shows the amplified bands of 6 collected carpinus samples for 8 pairs of SSR primers.
Detailed Description
In order that the above-recited objects, features and advantages of the present invention will become more apparent, a more particular description of the invention will be rendered by reference to specific embodiments thereof.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways other than those described herein, and persons skilled in the art will readily appreciate that the present invention is not limited to the specific embodiments disclosed below.
Further, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic can be included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.
Example 1:
effective carpinus wilfordii SSR primer screening:
(1) Whole gene sequence acquisition: using the complete carpinus genome sequence (https:// doi.org/10.6084/m9.Figshare. 14988777);
(2) SSR site screening: constructing fastfile sequence files according to the whole genome sequence, screening SSR sites in the whole genome range through MISA (MIcroSAtellite identification tool) tools written by perl, repeating 10 times and more than 10 times according to 1 base; repeating 2 bases 6 times or more; repeating 3 bases 5 times or more; repeating 4 bases 5 times or more; repeating 5 or more times with 5 bases; the 6 bases are repeated for 5 times and more than 5 times, and the threshold value requires that the base repeated sequence is screened and identified as a microsatellite sequence;
(3) Primer design: designing primers in 200bp at the upstream and downstream of the SSR locus by using Primer3.0, and selecting three pairs of primers at each locus to obtain 3306 loci of primers which are designed to be uniquely matched with a whole genome;
(4) Effective primer screening: DNA extraction is carried out on carpinus resources with 6 different communities, preferably SSR primers are synthesized and amplified by PCR, then polymorphism detection is carried out by polyacrylamide gel electrophoresis, and effective SSR primers are screened.
The method for identifying the carpinus wilfordii SSR comprises the following steps:
1. extraction of carpinus wilfordii genome DNA
(1) 6 portions of carpinus resources with different communities are selected.
6 carpinus herd sources are shown in Table 1.
Table 1 6 carpinus herd population information
| Sequence number | Population source |
| 1 | Zhejiang Linan (Linan' an medicine for treating Zhejiang) |
| 2 | Hubei Shennong frame |
| 3 | Guizhou Pichia |
| 4 | Guangdong milk source |
| 5 | Chongqing nan Chuan |
| 6 | Tibet niehira wood |
Extracting the genome DNA of carpinus by an improved CTAB method: weighing 0.1g-0.2g of fresh and healthy leaves, adding liquid nitrogen, rapidly grinding into powder, and transferring the powder into a 2.0mL centrifuge tube by using a medicine spoon; 1000uL of 2 XCTAB extract preheated in a water bath at 65℃was added, followed by 10uL of 10ng/ml RNaseA (RNA degrading enzyme) and then 10uL of mercaptoethanol; mixing or shaking up and down for several minutes by vortex oscillator until the powder at the bottom of the tube is completely dissolved in CTAB extract, and then placing into a water bath of 65 ℃ for 0.5-1h, shaking once every five to ten minutes; centrifuging at 12000rpm for 10min at normal temperature, collecting supernatant, adding the supernatant into a new 2ml centrifuge tube, adding an equal volume of extract CI (chloroform/isoamyl alcohol=24/1), and shaking up and down for 5-10min until layering occurs; centrifuging at 12000rpm for 10min at normal temperature, collecting supernatant in transparent state, and placing into a new 2ml centrifuge tube; adding the extraction solution CI (chloroform/isoamyl alcohol=24/1) with the same volume again, shaking up and down for 5-10min until layering phenomenon occurs, and centrifuging at normal temperature of 12000rpm for 10min; placing the supernatant in transparent state into a new 1.5ml centrifuge tube, adding 2/3 volume of isopropanol frozen at-20deg.C in advance, shaking up and down for 5min to thoroughly mix, and placing the centrifuge tube in a refrigerator at-20deg.C for precipitation for 1 hr; taking out the centrifuge tube, wiping off water mist on the tube wall, and putting the tube wall into a centrifuge at 12000rpm for centrifugation for 10min; pouring out the liquid, adding 700uL of the existing 75% alcohol, bouncing up the precipitate at the bottom of the tube, fully washing the impurity in the precipitate, and centrifuging at 12000rpm for 2min; pouring out the liquid, adding 700uL of 75% alcohol, bouncing the precipitate at the bottom of the tube, and centrifuging at 12000rpm for 3min after 1 min; pouring out the liquid, adding 700uL absolute ethyl alcohol, fully washing the sediment at the bottom of the tube for 1min, and centrifuging at 12000rpm for 3min; pouring out the liquid, putting the inverted centrifugal tube into a baking oven at 37 ℃ for baking or drying by sterile air on an ultra-clean workbench until the bottom of the tube can spring up white solid particles; adding 50-100ul of sterilized ultrapure water, dissolving in a refrigerator at 4deg.C for 1 hr, and storing in a refrigerator at 4deg.C.
SSR primer synthesis and screening:
the synthesized 96 pairs of primers (synthesized by Nanjing qing family biotechnology Co., ltd.) are utilized to amplify the genome DNA of 6 carpinus with different communities and obvious phenotype differences, and then SSR primers which can be stably amplified, have clear bands and rich polymorphism are screened, and 8 pairs are added (Table 2).
TABLE 28 primer sequences pairs
And 3, establishing a PCR reaction system and detecting a PCR product: the PCR amplification adopts a 20ul PCR reaction system, and the concentration and the use amount of each component are as follows: 2 XTaq master Mix (Vazyme Co.) 10uL, primer (10. Mu. Mo 1/L) PrimerF:0.75ul,PrimerR 0.75ul,DNA:50ng (1 ul), sterilized ddH 2 0:7.5ul,Taq master Mix from Vazyme Inc.
PCR amplification procedure: 94 ℃ (5 min); 94 ℃ (40 s), 52 ℃ (40 s), 72 ℃ (120 s) (35 cycles), and finally 72 ℃ (10 min), the amplified product was stored at 4 ℃ for use.
PCR product detection PCR products were detected by electrophoresis on a 2% agarose gel (100 mL 1XTBE buffer plus 2g agarose). And loading the 3uLPCR product, performing 150V electrophoresis for 20min, and photographing and storing the electrophoresis result in a gel imaging system.
The invention screens the SSR primer of carpinus based on the whole genome sequence, and 8 pairs of SSR molecular marker primers which can be stably amplified, have clear bands and rich polymorphism are screened out of 96 pairs of SSR marker primers by agarose gel electrophoresis by adopting 6 pairs of carpinus germplasm resources (shown in figure 1). Wherein, the primer CV4 can distinguish the carpinus of Guangdong milk source from other 5 collection areas; primer CV24 distinguishes 6 populations; primer CV27 distinguishes the population located in the Shennong's frame in Hubei and the Tibet nigella wood from the other 4 populations; primer CV65 distinguishes 2 populations (Pichia and Guangdong milk sources) from 4 populations (Zhejiang Linan, hubei Shennong's frame, chongqing nan Chuan and Tibet Kela wood); primer CV67 distinguishes 2 clusters of Chongqing Nanchuan and Tibet Nula from the other 4 clusters; primer CV71 distinguishes Guangdong milk source population from other 5 populations; primer CV81 distinguishes the Guangzhou Pichia of the herd from the other 4 herds of Zhejiang Linan; primer CV99 distinguishes the Hubei Shennong frame, pichia 2 communities from the other 4 communities. In conclusion, it is proved that the development of the carpinus pterinus SSR primer based on the whole genome sequence is feasible.
FIG. 1 is an 8 pairs of SSR primer pairs for amplifying bands of 6 collected carpinus, wherein the numbers 1-6 in the figure represent 6 collected sites of carpinus, wherein 1 is Zhejiang Linan, 2 is Hubei Shennong's frame, 3 is Pijie in Guizhou, 4 is Guangdong milk source, 5 is Chongqing nan Chua, and 6 is Tibet nieshi.
In conclusion, the invention develops a set of SSR primers belonging to carpinus based on the whole genome sequence through bioinformatics analysis software and PCR amplification detection, and provides a certain technical support for carrying out genetic diversity analysis, population structure research and kindred species definition of carpinus in the future.
It should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted without departing from the spirit and scope of the technical solution of the present invention, which is intended to be covered in the scope of the claims of the present invention.
Sequence listing
<110> university of Zhejiang industry college of Zhejiang
University of Nanjing forestry
<120> carpinus SSR primer developed based on whole genome data and identification method thereof
<141> 2022-05-31
<160> 16
<170> SIPOSequenceListing 1.0
<210> 1
<211> 23
<212> DNA
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<400> 1
tgtcaaactc atgtttgtcg ttt 23
<210> 2
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
ttggaggata tggataacgc 20
<210> 3
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 3
ttattatttc ttgcttggca gtt 23
<210> 4
<211> 27
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 4
tgtaaaattg tattcatacc cattgat 27
<210> 5
<211> 27
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 5
tgtaattttt aatgagaaac tcaatcg 27
<210> 6
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 6
atatgtcaac acaaagggag gg 22
<210> 7
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 7
atctatagtg gtggccggtg 20
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<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 8
gatcctccaa ttatttggca ct 22
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gcaatattta caaagtgatg gca 23
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<211> 27
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 10
tggtttgttt gtagttgtta ttcttga 27
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<213> Artificial sequence (Artificial Sequence)
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tctaccacaa tgctttgcag at 22
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tgcgcctttt aataaagcag t 21
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atgtgatcca cgtgacgc 18
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gcctcaaaat taaaacagtg tcat 24
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taaaacatgg attggggcat 20
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<213> Artificial sequence (Artificial Sequence)
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aagcacttat attccttgac acca 24
Claims (8)
1. The carpinus SSR primer developed based on whole genome data is characterized in that: the sequence of the carpinus pterygium SSR primer comprises one or more of the following 8 pairs:
CV4 upstream primer: tgtcaaactcatgtttgtcgttt; a downstream primer: ttggaggatatggataacgc;
CV24 upstream primer: ttattatttcttgcttggcagtt, downstream primer: tgtaaaattgtattcatacccattgat;
CV27 upstream primer: tgtaatttttaatgagaaactcaatcg; a downstream primer: atatgtcaacacaaagggaggg;
CV65 upstream primer: atctatagtggtggccggtg; a downstream primer: gatcctccaattatttggcact;
CV67 upstream primer: gcaatatttacaaagtgatggca; a downstream primer: tggtttgtttgtagttgttattcttga;
CV71 upstream primer: tctaccacaatgctttgcagat; a downstream primer: tgcgccttttaataaagcagt;
CV81 upstream primer: atgtgatccacgtgacgc; a downstream primer: gcctcaaaattaaaacagtgtcat;
CV99 upstream primer: taaaacatggattggggcat; a downstream primer: aagcacttatattccttgacacca.
2. A carpinus wilfordii SSR identification method based on whole genome data development is characterized by comprising the following steps of: the method comprises the following steps:
(1) Extracting genome DNA of carpinus;
(2) Carrying out PCR reaction by using the carpinus genome DNA as a template and using the 8 pairs of primers as set forth in claim 1, and detecting PCR products;
the 8 pairs of primers are used for distinguishing Tripterygium wilfordii of Zhejiang Linan, hubei Shennong's frame, pijie in Guizhou, guangdong milk source, chongqing nan Chuan and Tibet draws carpinus of the group source, wherein the primer CV4 distinguishes Tripterygium wilfordii of Guangdong milk source from other 5 groups; primer CV24 distinguishes 6 populations; primer CV27 distinguishes the Hubei Shennong frame, tibet nielamu from the other 4 clusters; primer CV65 distinguishes 2 clusters of Pichia and Guangdong milk sources from 4 clusters of Linan Zhejiang, shennong's frame, chongqing nan Chuan and Tibet Keyanmu; primer CV67 distinguishes 2 clusters of Chongqing Nanchuan and Tibet Nula wood from other 4 clusters; primer CV71 distinguishes Guangdong milk source population from other 5 populations; primer CV81 distinguishes the Guangzhou Pichia of the herd from the other 4 herds of Zhejiang Linan; primer CV99 distinguishes the Hubei Shennong frame, guizhou Pichia 2 herd from the other 4 herds.
3. The method for identifying the carpinus wilfordii SSR developed based on whole genome data according to claim 2, wherein the method is characterized by comprising the following steps of: the method for extracting the carpinus genome DNA is to extract the carpinus genome DNA by adopting a CTAB method.
4. A method for SSR identification of carpinus developed based on whole genome data as claimed in claim 3, wherein: the CTAB method is adopted to extract the genome DNA of the carpinus, and 65 th is added with carpinus powder o C water bath preheatingCTAB extract of (C), then adding the decomposing enzyme, then adding the mercaptoethanol, mixing uniformly, and then adding 65 o C, water bath of the water bath pot is carried out for 0.5-1 h; centrifuging at normal temperature of 12000rpm for 10min, collecting supernatant, adding an equal volume of extract, shaking and mixing for 5-10min, centrifuging, collecting supernatant, adding precooled isopropanol, and precipitating in a refrigerator at-20deg.C for 1h; putting into a centrifuge 12000rpm for centrifugation for 10min; pouring out the liquid, adding 75% alcohol for washing, centrifuging, air-drying the precipitate, and adding ultrapure water for dissolution.
5. The method for identifying the carpinus wilfordii SSR developed based on whole genome data according to claim 4, wherein the method comprises the following steps: the extract is chloroform: isoamyl alcohol volume ratio 24:1.
6. the method for identifying the carpinus wilfordii SSR developed based on whole genome data according to claim 2, wherein the method is characterized by comprising the following steps of: the PCR reaction is carried out, and the PCR reaction system is as follows: 2X Taq master Mix 10uL,10 mu mo1/L upstream primer: 0.75ul, 10. Mu. Mo1/L of downstream primer 0.75ul, 50ng of DNA, sterilized ddH 2 0 7.5ul。
7. The method for identifying the carpinus wilfordii SSR developed based on whole genome data according to claim 2, wherein the method is characterized by comprising the following steps of: the PCR reaction is carried out, and the PCR amplification program is as follows: 94 ℃ for 5min;94℃40s,52℃40s,72℃120s,35 cycles, and finally 72℃10min.
8. The method for identifying the carpinus wilfordii SSR developed based on whole genome data according to claim 2, wherein the method is characterized by comprising the following steps of: the PCR products were detected by 2% agarose gel electrophoresis.
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| CN109207620A (en) * | 2018-09-30 | 2019-01-15 | 江苏省中国科学院植物研究所 | A kind of precious magnificent carpinus turczaninowii ISSR-PCR molecular labeling system |
| WO2019223245A1 (en) * | 2018-05-23 | 2019-11-28 | 南京林业大学 | Method for identifying genetic relationship of liriodendron hybrids |
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| WO2019223245A1 (en) * | 2018-05-23 | 2019-11-28 | 南京林业大学 | Method for identifying genetic relationship of liriodendron hybrids |
| CN109207620A (en) * | 2018-09-30 | 2019-01-15 | 江苏省中国科学院植物研究所 | A kind of precious magnificent carpinus turczaninowii ISSR-PCR molecular labeling system |
Non-Patent Citations (2)
| Title |
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| DEVELOPMENT AND CHARACTERIZATION OF EST-SSR MARKERS IN O STRYOPSIS (BETULACEAE);BING-BING LIU et al.;《Applications in Plant Sciences》;第2卷(第2期);第1-3页 * |
| 多脉铁木叶绿体基因组的序列特征和系统发育;蒋明等;《林业科学》;第56卷(第5期);第60-68页 * |
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