CN114836417A - Development and application of malus spectabilis SSR marker - Google Patents

Development and application of malus spectabilis SSR marker Download PDF

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CN114836417A
CN114836417A CN202210654426.3A CN202210654426A CN114836417A CN 114836417 A CN114836417 A CN 114836417A CN 202210654426 A CN202210654426 A CN 202210654426A CN 114836417 A CN114836417 A CN 114836417A
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primer
ssr
primer pair
malus spectabilis
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CN114836417B (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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    • 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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    • C12Q1/6844—Nucleic acid amplification reactions
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Abstract

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

Description

Development and application of malus spectabilis SSR marker
Technical Field
The invention relates to the technical field of biology, in particular to development and application of malus spectabilis SSR markers.
Background
SSR molecular markers, also called microsatellites, are simple repetitive sequences uniformly distributed in eukaryotic genomes, usually a simple repetitive sequence consisting of 1-6 oligonucleotides, the two ends of which are conserved single copy sequences, and a common means is to design and connect a pair of specific primers at the two ends of the sequence. The SSR marker has the characteristic of codominance, in most cases, the microsatellites are distributed in the genome of organisms, and the variation sites comprise a plurality of genetic genes. Since the number of repetitions of the repeating unit is highly variable among individuals and is abundant, the microsatellite marker has wide applicability. The SSR marker method can be used for research on evaluation of diversity between varieties and populations, and can also be applied to aspects such as construction of genetic maps and identification of germplasm.
Malus spectabilis (scientific name: Malus fransitioia (Batalin) C.K.Schneid.) is a plant of Malus genus of Rosaceae family. Meanwhile, the utility model has edible, medicinal and ornamental values. At present, no relevant primer information is published in germplasm research and genetic polymorphism analysis of malus spectabilis.
Disclosure of Invention
The invention aims to provide development and application of an SSR marker of malus spectabilis.
In a first aspect, the invention claims an SSR primer combination for detecting malus spectabilis.
The SSR primer combination for detecting malus spectabilis claimed by the invention consists of all or part of the following 18 primer pairs, wherein the part comprises more than 2 and less than 17 primers:
primer pair 1: consists of two single-stranded DNAs shown as SEQ ID No.1 and SEQ ID No. 2;
and (3) primer pair 2: consists of two single-stranded DNAs shown as SEQ ID No.3 and SEQ ID No. 4;
and (3) primer pair: consists of two single-stranded DNAs shown as SEQ ID No.5 and SEQ ID No. 6;
and (3) primer pair 4: consists of two single-stranded DNAs shown as SEQ ID No.7 and SEQ ID No. 8;
and (3) primer pair 5: consists of two single-stranded DNAs shown as SEQ ID No.9 and SEQ ID No. 10;
and (3) primer pair 6: consists of two single-stranded DNAs shown as SEQ ID No.11 and SEQ ID No. 12;
and (3) primer pair 7: consists of two single-stranded DNAs shown as SEQ ID No.13 and SEQ ID No. 14;
and (3) primer pair 8: consists of two single-stranded DNAs shown as SEQ ID No.15 and SEQ ID No. 16;
and (3) primer pair 9: consists of two single-stranded DNAs shown as SEQ ID No.17 and SEQ ID No. 18;
a primer pair 10: consists of two single-stranded DNAs shown as SEQ ID No.19 and SEQ ID No. 20;
a 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;
a primer pair 13: consists of two single-stranded DNAs shown as SEQ ID No.25 and SEQ ID No. 26;
primer pair 14: consists of two single-stranded DNAs shown as 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;
a primer pair 17: consists of two single-stranded DNAs shown as SEQ ID No.33 and SEQ ID No. 34;
a 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 malus spectabilis can be a primer combination for detecting polymorphism of malus spectabilis.
In a second aspect, the invention claims SSR primers for detecting malus spectabilis.
The SSR primer for detecting malus spectabilis claimed by the invention is any one of the 18 primer pairs.
In a third aspect, the invention claims a combination of SSR markers for detecting malus spectabilis.
The SSR marker combination for detecting malus spectabilis claimed by the invention consists of all or part of 18 SSR markers, wherein the number of the part is more than 2 and less than 17; the 18 SSR markers are 18 amplification product sequences obtained by respectively adopting the 18 primer pairs to carry out PCR amplification by taking Malus spectabilis genome DNA as a template.
In a fourth aspect, the invention claims an SSR marker for detecting malus spectabilis.
The claimed SSR marker for detecting malus spectabilis of the present invention is any one of the 18 SSR markers described above.
In a fifth aspect, the invention claims a kit for detecting malus spectabilis.
The kit for detecting malus spectabilis claimed by the invention contains the SSR primer combination or the SSR primer.
In a sixth aspect, the present invention claims the use of the SSR primer combination described hereinbefore or the SSR marker combination described hereinbefore or the kit described hereinbefore in any one of:
(A1) detecting genetic diversity of the malus spectabilis;
(A2) constructing a genetic map of the malus spectabilis;
(A3) and (4) performing germplasm identification on malus spectabilis.
In a seventh aspect, the invention claims a method for detecting genetic diversity in malus spectabilis.
The method for detecting the genetic diversity of the malus spectabilis, which is claimed by the invention, can comprise the following steps:
(B1) respectively taking different genome DNAs of the Malus spectabilis to be detected as templates, and performing PCR amplification by adopting the SSR primer combination in the first aspect or the SSR primer in the second aspect to obtain an amplification product;
(B2) and performing polyacrylamide gel electrophoresis on the amplification product, and performing genetic diversity analysis on the malus mosaic according to the polymorphism of the bands among the different malus mosaic to be detected. Specifically, the analysis is carried out according to the number and the position of polymorphic bands among different samples.
In the step (B1), when the PCR amplification is performed, the ratio of the two single-stranded DNA molecules constituting the primer pair to the malus mosaic genomic DNA as a template is 5 pmol: 5 pom: 125 ng.
In the step (B1), when the PCR amplification is carried out, the final concentration of the two single-stranded DNA molecules which form the primer pair in the reaction system is 0.5 mu mol/L; the final concentration of the Malus spectabilis genomic DNA as a template in the reaction system was 12.5 ng/. mu.L.
Experiments prove that the SSR primer combination screened finally by the invention can effectively perform genetic diversity analysis on malus spectabilis groups. The invention has important significance for constructing the genetic map of the malus spectabilis and identifying the germplasm.
Drawings
FIG. 1 shows the result of DNA detection by 0.8% agarose electrophoresis. Lanes 1-24 represent the Malus spectabilis leaf samples numbered 1-24.
FIG. 2 is an electrophoresis diagram of polyacrylamide gel orthogonally designed according to different factor levels in SSR-PCR reaction. 1-11 represents the electrophoresis result of primer No.11 in the reaction system No. 1; 1 to 70 represent the electrophoresis results of the primer No. 70 in the reaction system No.1, and 2 to 11 represent the electrophoresis results of the primer No.11 in the reaction system No. 2; 2-70 represents the electrophoresis result of primer No. 70 in the reaction system No. 2; 3-11 represent the electrophoresis results of primer No.11 in the reaction system No. 1; 3-70 show the results of electrophoresis of primer No. 70 in reaction system No. 1. Wherein, the 1 st reaction system corresponds to factor 1 in table 2; reaction system 2 corresponds to factor 2 in table 2; the 3 rd reaction system corresponds to factor 3 in table 2.
FIG. 3 shows the partial primer screening results. 1-19 are 19 primer pairs in Table 1.
FIG. 4 shows the partial primer screening results. 1-18 are 18 primer pairs in Table 1.
FIG. 5 is an electrophoresis chart of PCR products of primers No.11 (SEQ ID No.7 and SEQ ID No.8) in Table 1 against 42 individual samples.
FIG. 6 is a graph showing the effect of primers No.29 (SEQ ID Nos. 21 and 22) on PCR products of 35 individuals in Table 1.
FIG. 7 is a graph showing the effect of primers No. 50 (SEQ ID No.27 and SEQ ID No.28) on PCR products of 38 individual samples in Table 1.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 development and application of Malus spectabilis SSR marker
First, SSR data acquisition and primer design
The crab malus floribunda transcriptome Library was constructed using the Roche GS Rapid Library prediction Kit and subjected to end sequencing by the IlluminaHiseq4000 sequencing platform. And filtering out data with low sequencing quality after sequencing, adopting MISA (version 1.0, default parameters: the minimum repetition times of corresponding unit sizes are respectively 1-10, 2-6, 3-5, 4-5, 5-5, 6-5) Unigene to carry out SSR locus detection, adopting Primer3 (version 2.3.5, default parameters) to carry out SSR Primer design, and respectively designing 10730 groups of primers.
By comparing and analyzing the length of the primer, the length of the product and the Tm value, 81 pairs of primers are selected for synthesis.
The primers selected are shown in table 1.
TABLE 1 primer information Table for Synthesis
Figure BDA0003688761930000041
Figure BDA0003688761930000051
Figure BDA0003688761930000061
Second, verification of SSR
1. Leaf DNA extraction
Extracting and purifying the genome DNA of the leaf of malus spectabilis by a modified CTAB method, detecting the integrity of the genome DNA by 0.8 percent agarose gel electrophoresis, and detecting the concentration and the purity (OD value) of the DNA by an ultraviolet spectrophotometer. The results of the detection are shown in FIG. 1. The complete and clear DNA bands can be seen from the figure.
2. Amplification System construction
For 2 XTaq PCR MasterMix, primers, 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 concentrations of the upstream primer and the downstream primer are both 10. mu. mol/L. The PCR reaction system design is shown in Table 2.
TABLE 2 design table of different factor levels of SSR-PCR reaction
Factors of the fact 2×Taq PCR MasterMix(μL) Primer (mu L) Template DNA (μ L) ddH 2 O(μL) Total amount of System (μ L)
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 deg.C for 5min, denaturation at 94 deg.C for 30s, annealing at 58 deg.C for 30s, and extension at 72 deg.C for 45s, and performing 25 cycles, preservation at 72 deg.C for 10min, and preservation at 4 deg.C.
According to the test design of different factor levels, 2 pairs of synthesized primers and 6 DNA templates are randomly selected for system screening, the selected primers are respectively No.11 primers and No. 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 leaf sample numbers of malus spectabilis of different population sources). And (3) performing polyacrylamide gel electrophoresis on the amplification product. The results are shown in FIG. 2.
FIG. 2 shows the results of polyacrylamide gel electrophoresis, and although PCR products can be obtained by various system designs, the 3 rd combination shows good product quality and high definition of the obtained band, so that the 3 rd combination is selected for the subsequent PCR amplification process. The method specifically comprises the following steps: SSR-PCR reaction system with total amount of 10 μ L, wherein 2 XTaq PCR MasterMix 5 μ L, positive and negative primers total 1 μ L (each 0.5 μ L), DNA template 2.5 μ L, ddH 2 O 1.5μL。
3. Primer screening
The PCR system selected in step 2 was used to screen 81 pairs of primers (Table 1) for biosynthesis, and 142 individual samples were amplified. All primers are screened by selecting 2 template DNAs, and 18 pairs of primers with good specificity are finally obtained. FIGS. 3 and 4 show the results of partial primer screening.
In the process of primer screening, primers without any band and without specific band were knocked out. In total, 18 pairs of primers with good repeatability, strong polymorphism and clear bands are screened out.
Primer sequence information that allows population genetic analysis was obtained by primer screening, see table 3.
TABLE 3 information table of screened primer sequences
Figure BDA0003688761930000081
Note: "primer numbers" correspond to those in Table 1. Polymorphism ratio (PPL) is the ratio of the number of polymorphic bands to the total number of amplified bands.
In total, 18 pairs of specific primers are screened out, and in order to clarify the genetic characteristics of the leaves of the malus spectabilis, the genetic information of the test material is reflected by a method of performing polyacrylamide gel electrophoresis on the population.
Thirdly, detecting genetic diversity of malus spectabilis based on SSR
And (3) respectively detecting 142 samples of malus spectabilis in different distribution areas of Qinghai province by using 18 pairs of screened primers (table 3) according to the optimal PCR system screened in the step two, wherein different strips appear among different single plants when the same primer is amplified, and the genetic diversity is detected by analyzing according to the polymorphism of the strips. These 142 samples were derived from 8 different regions of the Qinghai province (Table 4).
TABLE 4 sample sources
Group of people Number of samples Degree 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 of farmland and river bank
WD
16 35°46′49″101°23′30.55″ 2180 River bank and 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, beside the farmland
TR
12 35°20′54″101°55′25″ 2810 Reservoir inundation area
MKH 27 32°29′50″101°00′25″ 3205 Forest edge
Total up to 142
The results of polyacrylamide gel electrophoresis after partial primer amplification are shown in FIGS. 5, 6 and 7.
After completing SSR labeling of 142 individual samples of all primer pairs, statistical results as shown in Table 4 were obtained. The SSR labeling is carried out on 142 single-plant malus spectabilis samples, 105 bands are shared, 56 polymorphism bands are carried, the average polymorphism ratio is 54.05%, products amplified by different SSR primers are different in the aspects of band number, band size and the like, and the polymorphism of the bands amplified by other primers except No.9, No.17, No.23, No.27 and No. 70 is more than 50%, which indicates that the polymorphism of the malus spectabilis is higher.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
<110> academy of agriculture and forestry of Qinghai province
<120> development and application of malus spectabilis SSR marker
<130> GNCLN201523
<160> 36
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Claims (9)

1. The SSR primer combination for detecting malus spectabilis consists of all or part of the following 18 primer pairs, wherein the part comprises more than 2 and less than 17 primers:
primer pair 1: consists of two single-stranded DNAs shown as SEQ ID No.1 and SEQ ID No. 2;
and (3) primer pair 2: consists of two single-stranded DNAs shown as SEQ ID No.3 and SEQ ID No. 4;
and (3) primer pair: consists of two single-stranded DNAs shown as SEQ ID No.5 and SEQ ID No. 6;
and (3) primer pair 4: consists of two single-stranded DNAs shown as SEQ ID No.7 and SEQ ID No. 8;
and (3) primer pair 5: consists of two single-stranded DNAs shown as SEQ ID No.9 and SEQ ID No. 10;
and (3) primer pair 6: consists of two single-stranded DNAs shown as SEQ ID No.11 and SEQ ID No. 12;
and (3) primer pair 7: consists of two single-stranded DNAs shown as SEQ ID No.13 and SEQ ID No. 14;
and (3) primer pair 8: consists of two single-stranded DNAs shown as SEQ ID No.15 and SEQ ID No. 16;
and (3) primer pair 9: consists of two single-stranded DNAs shown as SEQ ID No.17 and SEQ ID No. 18;
and (3) primer pair 10: consists of two single-stranded DNAs shown as SEQ ID No.19 and SEQ ID No. 20;
a 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;
a primer pair 13: consists of two single-stranded DNAs shown as SEQ ID No.25 and SEQ ID No. 26;
primer pair 14: consists of two single-stranded DNAs shown as 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;
a primer pair 17: consists of two single-stranded DNAs shown as SEQ ID No.33 and SEQ ID No. 34;
and (3) primer pair 18: consists of two single-stranded DNAs shown as SEQ ID No.35 and SEQ ID No. 36.
2. The SSR primer for detecting malus spectabilis is any one of 18 primer pairs in claim 1.
3. The SSR marker combination for detecting malus spectabilis consists of all or part of 18 SSR markers, wherein the number of the part is more than 2 and less than 17; the 18 SSR markers are 18 amplification product sequences obtained by respectively adopting 18 primer pairs in claim 1 to perform PCR amplification by taking Malus spectabilis genome DNA as a template.
4. The SSR marker for detecting malus spectabilis, which is any one of the 18 SSR markers described in claim 3.
5. A kit for detecting malus spectabilis comprising the SSR primer combination of claim 1 or the SSR primer of claim 2.
6. Use of a SSR primer combination of claim 1 or a SSR primer of claim 2 or a SSR marker combination of claim 3 or a SSR marker of claim 4 or a kit of claim 5 in any of:
(A1) detecting genetic diversity of the malus spectabilis;
(A2) constructing a genetic map of the malus spectabilis;
(A3) and (4) performing germplasm identification on malus spectabilis.
7. A method for detecting genetic diversity of malus spectabilis comprises the following steps:
(B1) respectively taking different genome DNAs of Malus spectabilis to be detected as templates, and carrying out PCR amplification by adopting the SSR primer combination of claim 1 or the SSR primer of claim 2 to obtain amplification products;
(B2) and performing polyacrylamide gel electrophoresis on the amplification product, and performing genetic diversity analysis on the malus mosaic according to the polymorphism of the bands among the different malus mosaic to be detected.
8. The method of claim 7, wherein: in the step (B1), when the PCR amplification is performed, the ratio of the two single-stranded DNA molecules constituting the primer pair to the genomic DNA of malus spectabilis serving as a template is 5 pmol: 5 pom: 125 ng.
9. The method according to claim 7 or 8, 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 the reaction system is 0.5 mu mol/L; the final concentration of the Malus spectabilis genomic DNA as a template in the reaction system was 12.5 ng/. mu.L.
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