CN114836417B - Development and application of SSR (simple sequence repeat) marker of malus asiatica - Google Patents

Development and application of SSR (simple sequence repeat) marker of malus asiatica Download PDF

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CN114836417B
CN114836417B CN202210654426.3A CN202210654426A CN114836417B CN 114836417 B CN114836417 B CN 114836417B CN 202210654426 A CN202210654426 A CN 202210654426A CN 114836417 B CN114836417 B CN 114836417B
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CN114836417A (en
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张得芳
夏涛
王占林
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Qinghai Academy of Agricultural and Forestry Sciences
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Abstract

The application discloses development and application of SSR markers of malus haliotidis. The application provides an SSR primer combination for detecting malus hupehensis, which consists of all or part of 18 primer pairs, wherein the part is more than 2 and less than 17. The 18 primer pairs were selected from 10730 set primers designed based on the malus hupehensis transcriptome sequencing data. Experiments prove that the SSR primer combination consisting of the 18 primer pairs can effectively analyze the genetic diversity of the malus hupehensis population. The application has important significance for constructing the malus hupehensis genetic map and identifying the germplasm.

Description

Development and application of SSR (simple sequence repeat) marker of malus asiatica
Technical Field
The application relates to the technical field of biology, in particular to development and application of SSR (simple sequence repeat) markers of malus hupehensis.
Background
SSR molecular markers, also known as microsatellites, are simple repeats uniformly distributed in the genome of eukaryotic organisms, usually a simple repeat consisting of 1-6 oligonucleotides, which are conserved single copy sequences at both ends, and a common approach is to design and ligate a pair of specific primers at both ends of the sequence. SSR markers have the characteristic of co-dominance, microsatellites are distributed in the genome of organisms under most conditions, and mutation sites contain a plurality of genetic genes, so that the marker method has stable polymorphism, high repetition rate, convenient operation and good sensitivity. Microsatellite markers have wide applicability because the number of repetitions of the repeating unit varies highly between individuals and is abundant. The SSR marking method can be used for evaluating the diversity between varieties and populations, and can also be applied to the aspects of genetic map construction, germplasm identification and the like.
Malus hupehensis (subject to the name Malus transitoria (Batalin) c.k.schneid.) is a plant of the genus malus of the family rosaceae. Meanwhile, the product has edible, medicinal and ornamental values. At present, no relevant primer information is published in the germplasm research and genetic polymorphism analysis of the malus hupehensis.
Disclosure of Invention
The application aims to provide development and application of SSR markers of malus hupehensis.
In a first aspect, the application claims an SSR primer combination for detecting malus haliotidis.
The SSR primer combination for detecting the malus hupehensis disclosed by the application consists of all or part of the following 18 primer pairs, wherein the part is more than 2 and less than 17:
primer pair 1: consists of two single-stranded DNA shown in SEQ ID No.1 and SEQ ID No. 2;
primer pair 2: consists of two single-stranded DNAs shown as SEQ ID No.3 and SEQ ID No. 4;
primer pair 3: consists of two single-stranded DNAs shown as SEQ ID No.5 and SEQ ID No. 6;
primer pair 4: consists of two single-stranded DNAs shown as SEQ ID No.7 and SEQ ID No. 8;
primer pair 5: consists of two single-stranded DNAs shown as SEQ ID No.9 and SEQ ID No. 10;
primer pair 6: consists of two single-stranded DNAs shown as SEQ ID No.11 and SEQ ID No. 12;
primer pair 7: consists of two single-stranded DNAs shown as SEQ ID No.13 and SEQ ID No. 14;
primer pair 8: consists of two single-stranded DNAs shown in SEQ ID No.15 and SEQ ID No. 16;
primer pair 9: consists of two single-stranded DNAs shown as SEQ ID No.17 and SEQ ID No. 18;
primer pair 10: consists of two single-stranded DNAs shown as SEQ ID No.19 and SEQ ID No. 20;
primer pair 11: consists of two single-stranded DNAs shown as SEQ ID No.21 and SEQ ID No. 22;
primer pair 12: consists of two single-stranded DNAs shown as SEQ ID No.23 and SEQ ID No. 24;
primer pair 13: consists of two single-stranded DNAs shown in SEQ ID No.25 and SEQ ID No. 26;
primer pair 14: consists of two single-stranded DNAs shown in SEQ ID No.27 and SEQ ID No. 28;
primer pair 15: consists of two single-stranded DNAs shown as SEQ ID No.29 and SEQ ID No. 30;
primer pair 16: consists of two single-stranded DNAs shown as SEQ ID No.31 and SEQ ID No. 32;
primer pair 17: consists of two single-stranded DNAs shown as SEQ ID No.33 and SEQ ID No. 34;
primer pair 18: consists of two single-stranded DNAs shown as SEQ ID No.35 and SEQ ID No. 36.
The SSR primer combination for detecting the malus haliotidis can be a primer combination for detecting the malus haliotidis polymorphism.
In a second aspect, the application claims an SSR primer for detecting malus haliotidis.
The SSR primer for detecting the malus hupehensis disclosed by the application is any one of the 18 primer pairs.
In a third aspect, the application claims an SSR marker combination for detecting malus haliotidis.
The SSR marker combination for detecting the malus hupehensis disclosed by the application consists of all or part of 18 SSR markers, wherein the part is more than 2 and less than 17; the 18 SSR markers are 18 amplified product sequences obtained by PCR (polymerase chain reaction) amplification by using the Malus hupehensis genome DNA as a template and respectively adopting the 18 primer pairs.
In a fourth aspect, the application claims an SSR marker for detecting malus haliotidis.
The SSR marker for detecting the malus hupehensis disclosed by the application is any one of 18 SSR markers.
In a fifth aspect, the application claims a kit for detecting malus hupehensis.
The kit for detecting the malus hupehensis disclosed by the application contains the SSR primer combination or the SSR primer.
In a sixth aspect, the application claims the use of a SSR primer combination as hereinbefore described or a SSR primer as hereinbefore described or a SSR marker combination as hereinbefore described or a SSR marker as hereinbefore described or a kit as hereinbefore described in any of the following:
(A1) Detecting genetic diversity of the malus hupehensis;
(A2) Constructing a genetic map of the malus asiatica;
(A3) And (5) carrying out germplasm identification on the malus asiatica.
In a seventh aspect, the application claims a method for genetic diversity detection of malus hupehensis.
The method for detecting genetic diversity of the malus hupehensis disclosed by the application can comprise the following steps:
(B1) Respectively taking different to-be-detected malus hupehensis genome DNA as templates, and carrying out PCR amplification by adopting the SSR primer combination in the first aspect or the SSR primer in the second aspect to obtain amplification products;
(B2) And (3) carrying out polyacrylamide gel electrophoresis on the amplification product, and carrying out genetic diversity analysis on the malus asiatica according to the stripe polymorphism among the different malus asiatica to be detected. In particular, the analysis is performed based on the number and position of polymorphic bands between different samples.
In the step (B1), the ratio of the two single-stranded DNA molecules constituting the primer pair to the genomic DNA of Malus hupehensis as a template was 5pmol when the PCR amplification was performed: 5 pore: 125ng.
In the step (B1), when the PCR amplification is performed, the final concentration of the two single-stranded DNA molecules constituting the primer pair in the reaction system is 0.5. Mu. Mol/L; the final concentration of the Malus hupehensis genomic DNA as a template in the reaction system is 12.5 ng/. Mu.L.
Experiments prove that the finally screened SSR primer combination can effectively analyze genetic diversity of malus hupehensis populations. The application has important significance for constructing the malus hupehensis genetic map and identifying the germplasm.
Drawings
FIG. 1 shows the result of detecting DNA by 0.8% agarose electrophoresis. Lanes 1-24 represent leaf samples of Malus hupehensis numbered 1-24.
FIG. 2 is a diagram of polyacrylamide gel electrophoresis of different factor level orthogonal designs of SSR-PCR reaction. 1-11 represents the electrophoresis result of primer 11 under the 1 st reaction system; 1-70 shows the electrophoresis result of the primer number 70 under the 1 st reaction system, and 2-11 shows the electrophoresis result of the primer number 11 under the 2 nd reaction system; 2-70 represents the electrophoresis result of the primer number 70 under the 2 nd reaction system; 3-11 represents the electrophoresis result of primer 11 under the 1 st reaction system; 3-70 shows the result of electrophoresis of the primer number 70 in the 1 st reaction system. Wherein, the 1 st reaction system corresponds to factor 1 in table 2; the 2 nd reaction system corresponds to factor 2 in table 2; the 3 rd reaction system corresponds to factor 3 in table 2.
FIG. 3 shows a partial primer screening result. 1-19 are 19 primer pairs in Table 1.
FIG. 4 shows a partial primer screening result. 1-18 are the 18 primer pairs in Table 1.
FIG. 5 is an electrophoretogram of PCR products of primer number 11 (SEQ ID No.7 and SEQ ID No. 8) of Table 1 on 42 individual samples.
FIG. 6 is a graph showing the effect of PCR products of the primers No.29 (SEQ ID No.21 and SEQ ID No. 22) on 35 individual samples in Table 1.
FIG. 7 is a graph showing the effect of PCR products of primers No. 50 (SEQ ID No.27 and SEQ ID No. 28) on 38 individual samples in Table 1.
Detailed Description
The following detailed description of the application is provided in connection with the accompanying drawings that are presented to illustrate the application and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the application in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Example 1 development and application of SSR markers for Malus hupehensis
1. Acquisition of SSR data and primer design
A malus hupehensis transcriptome library was constructed with Roche GS Rapid Library Preparation Kit and end sequencing was performed by the IlluminaHiseq4000 sequencing platform. After sequencing, filtering out data with low sequencing quality, performing SSR site detection by adopting MISA (version 1.0, default parameters: the minimum repetition times of corresponding unit size are 1-10,2-6,3-5,4-5,5-5, 6-5) Unigene, performing SSR Primer design by adopting Primer3 (version 2.3.5, default parameters), and respectively designing 10730 groups of primers.
81 pairs of primers were selected for synthesis by comparative analysis of primer length, product length and Tm.
The primers selected are shown in Table 1.
TABLE 1 information Table of synthesized primers
2. Verification of SSR
1. Leaf DNA extraction
The genomic DNA of the leaf blade of Malus hupehensis was extracted and purified by the modified CTAB method, the integrity of the genomic DNA was checked by 0.8% agarose gel electrophoresis, and the concentration and purity (OD value) of the DNA were checked by an ultraviolet spectrophotometer. The detection results are shown in FIG. 1. The DNA band is complete and clear from the figure.
2. Amplification System construction
For 2X Taq PCR MasterMix, primer, diluted DNA template and ddH 2 The concentration of O in the PCR system and the annealing temperature are designed and optimized. Wherein the final concentration of the DNA template is 50 ng/. Mu.L, and the concentration of the upstream primer and the downstream primer is 10. Mu. Mol/L. The PCR reaction system design is shown in Table 2.
TABLE 2 SSR-PCR reaction different factor level design Table
Factors of 2×Taq PCR MasterMix(μL) Primer (mu L) Template DNA (mu L) ddH 2 O(μL) Total system (μL)
1 5 2 2 1 10
2 5 0.5 3.5 1.5 10
3 5 1 2.5 1.5 10
4 5 1.5 1.5 2 10
5 5 2 1.5 1.5 10
6 5 0.5 2.5 2 10
The SSR-PCR amplification procedure was: pre-denaturation at 94℃for 5min, denaturation at 94℃for 30s, annealing at 58℃for 30s, extension at 72℃for 45s, 25 cycles of the whole, 10min at 72℃and preservation at 4 ℃.
According to the experimental design of different factor levels, 2 pairs of synthesized primers and 6 DNA templates are randomly selected for systematic screening, wherein the selected primers are respectively 11 # primers and 70 # primers in the table 1, and the selected DNA templates are respectively BS-2, XQ-2, QJ-17, MH-28, WD-2 and WD-10 (randomly selected malus asiatica leaf sample numbers of different group sources). And (5) performing polyacrylamide gel electrophoresis on the amplified product. The results are shown in FIG. 2.
In FIG. 2, the result of polyacrylamide gel electrophoresis is shown, and although PCR products can be obtained by various system designs, the 3 rd combination is shown to have good product quality, and the obtained band has high definition, so that the 3 rd combination is selected for the subsequent PCR amplification process. The method comprises the following steps: 10. Mu.L total of SSR-PCR reaction system, wherein 2X Taq PCR MasterMix. Mu.L total of 1. Mu.L of forward and reverse primer (0.5. Mu.L each), 2.5. Mu.L of DNA template and ddH 2 O 1.5μL。
3. Primer screening
The 81 pairs of primers (Table 1) were screened for biosynthesis using the PCR system selected in step 2, and 142 individual samples were amplified. And selecting 2 template DNAs to screen all primers, and finally obtaining 18 pairs of primers with good specificity. FIGS. 3 and 4 show partial primer screening results.
During primer screening, primers without any bands and without specific bands are rejected. And 18 pairs of primers with good repeatability, strong polymorphism and clear bands are screened out in total.
Primer sequence information, which can be used for population genetic analysis, is obtained by primer screening, and is shown in Table 3.
TABLE 3 information table of primer sequences selected
Note that: the "primer number" corresponds to Table 1. The polymorphism ratio (PPL) is the ratio of the number of polymorphic bands to the total number of amplified bands.
And 18 pairs of specific primers are screened out, and in order to clarify the genetic characteristics of the malus halliana leaves, the genetic information of the tested materials is reflected by a method of performing polyacrylamide gel electrophoresis on the group.
3. SSR-based detection of malus hupehensis genetic diversity
According to the optimal PCR system screened in the second step, 142 samples of Malus asiatica in different distribution areas of Qinghai province are detected by 18 pairs of primers (table 3) respectively, different bands appear among different single plants when the same primer is amplified, and genetic diversity is detected by analysis according to the polymorphism of the bands. These 142 samples were derived from 8 different regions of Qinghai province (Table 4).
TABLE 4 sample Source
Group of people Number of samples Number of north latitude and east longitude Altitude (m) Plant living environment
MH 29 36°14′2″102°32′11″ 1903 Side and sunny slope of farmland
XQ 8 36°20′44″101°55′21″ 2117 Side and river bank of farmland
WD 16 35°46′49″101°23′30.55″ 2180 River bank, sunny slope
HY 19 36°40′52.3″101°22'7.6 2510 River bank, mountain foot, forest edge
BS 14 37°03′10″102°24′55″ 2535 Forest edge
QJ 17 36°15′3.3″101°42′6.0″ 2648 Roadside and farmland side
TR 12 35°20′54″101°55′25″ 2810 Reservoir inundation area
MKH 27 32°29′50″101°00′25″ 3205 Forest edge
Totalizing 142
The results of the polyacrylamide gel electrophoresis after the amplification of a part of the primers are shown in FIG. 5, FIG. 6 and FIG. 7.
After completing SSR labeling of 142 individual samples for all primer pairs, statistical results as shown in table 4 were obtained. Carrying out SSR (simple sequence repeat) marking on 142 single malus hupehensis samples, wherein 105 bands are total, 56 polymorphic bands are provided, the average polymorphism ratio is 54.05%, products amplified by different SSR primers have differences in the aspects of the number of the bands, the size of the bands and the like, and the polymorphism of the bands amplified by other primers except for the number 9, the number 17, the number 23, the number 27 and the number 70 is more than 50%, which indicates that the malus hupehensis in Qinghai province has higher polymorphism.
The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.
<110> national academy of sciences of agriculture and forestry in Qinghai province
<120> development and application of SSR marker of Malus hupehensis
<130> GNCLN201523
<160> 36
<170> PatentIn version 3.5
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Claims (6)

1. The SSR primer combination for detecting the malus hupehensis consists of all of the following 18 primer pairs:
primer pair 1: consists of two single-stranded DNA shown in SEQ ID No.1 and SEQ ID No. 2;
primer pair 2: consists of two single-stranded DNAs shown as SEQ ID No.3 and SEQ ID No. 4;
primer pair 3: consists of two single-stranded DNAs shown as SEQ ID No.5 and SEQ ID No. 6;
primer pair 4: consists of two single-stranded DNAs shown as SEQ ID No.7 and SEQ ID No. 8;
primer pair 5: consists of two single-stranded DNAs shown as SEQ ID No.9 and SEQ ID No. 10;
primer pair 6: consists of two single-stranded DNAs shown as SEQ ID No.11 and SEQ ID No. 12;
primer pair 7: consists of two single-stranded DNAs shown as SEQ ID No.13 and SEQ ID No. 14;
primer pair 8: consists of two single-stranded DNAs shown in SEQ ID No.15 and SEQ ID No. 16;
primer pair 9: consists of two single-stranded DNAs shown as SEQ ID No.17 and SEQ ID No. 18;
primer pair 10: consists of two single-stranded DNAs shown as SEQ ID No.19 and SEQ ID No. 20;
primer pair 11: consists of two single-stranded DNAs shown as SEQ ID No.21 and SEQ ID No. 22;
primer pair 12: consists of two single-stranded DNAs shown as SEQ ID No.23 and SEQ ID No. 24;
primer pair 13: consists of two single-stranded DNAs shown in SEQ ID No.25 and SEQ ID No. 26;
primer pair 14: consists of two single-stranded DNAs shown in SEQ ID No.27 and SEQ ID No. 28;
primer pair 15: consists of two single-stranded DNAs shown as SEQ ID No.29 and SEQ ID No. 30;
primer pair 16: consists of two single-stranded DNAs shown as SEQ ID No.31 and SEQ ID No. 32;
primer pair 17: consists of two single-stranded DNAs shown as SEQ ID No.33 and SEQ ID No. 34;
primer pair 18: consists of two single-stranded DNAs shown as SEQ ID No.35 and SEQ ID No. 36.
2. A kit for detecting malus hupehensis comprising the SSR primer combination of claim 1.
3. Use of the SSR primer combination of claim 1 or the kit of claim 2 in any of the following:
(A1) Detecting genetic diversity of the malus hupehensis;
(A2) Constructing a genetic map of the malus asiatica;
(A3) And (5) carrying out germplasm identification on the malus asiatica.
4. A method for detecting genetic diversity of malus hupehensis comprises the following steps:
(B1) Respectively taking the genomic DNAs of different to-be-detected malus hupehensis as templates, and carrying out PCR amplification by adopting the SSR primer combination of claim 1 to obtain amplification products;
(B2) And (3) carrying out polyacrylamide gel electrophoresis on the amplification products, and carrying out genetic diversity analysis on the malus asiatica according to the stripe polymorphism among the different malus asiatica to be detected.
5. The method according to claim 4, wherein: in the step (B1), the ratio of the two single-stranded DNA molecules constituting the primer pair to the genomic DNA of Malus hupehensis as a template was 5pmol when the PCR amplification was performed: 5pmol:125ng.
6. The method according to claim 4 or 5, characterized in that: in the step (B1), when the PCR amplification is carried out, the final concentration of the two single-stranded DNA molecules forming the primer pair in a reaction system is 0.5 mu mol/L; the final concentration of the Malus hupehensis genomic DNA as a template in the reaction system is 12.5 ng/. Mu.L.
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