CN112080581B - SSR primer group and application thereof - Google Patents

SSR primer group and application thereof Download PDF

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CN112080581B
CN112080581B CN202011144623.8A CN202011144623A CN112080581B CN 112080581 B CN112080581 B CN 112080581B CN 202011144623 A CN202011144623 A CN 202011144623A CN 112080581 B CN112080581 B CN 112080581B
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徐德林
邓宽平
陈荣辉
张宏扬
杨丹
周超
史军华
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Zunyi Medical University
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Abstract

The invention relates to an SSR primer group and application thereof, belonging to the technical field of molecular markers. The SSR primer group comprises 22 pairs of SSR primers, and the nucleotide sequence is shown as SEQ ID NO. 1-44. The SSR primer group not only can accurately identify different raspberry strains, but also can read genetic diversity information of the raspberry strains and explain genetic diversity caused by regional differences, can realize the application of the raspberry primer group in raspberry strain identification, genetic diversity analysis and molecular marker breeding auxiliary selection, and has higher reliability.

Description

SSR primer group and application thereof
Technical Field
The invention relates to the technical field of molecular markers, in particular to an SSR primer group and application thereof.
Background
Raspberry (Rubus corchorifolius l.), an alias raspberry, tray, marlin, etc., is a perennial shrub berry type plant belonging to the genus raspberry (Rubus, also known as Rubus) of the family Rosaceae (Rosaceae). The fruit has the characteristics of bright appearance, fine and smooth taste, aromatic flavor and the like, and can be processed into jam, fruit wine, fruit juice, jelly, thick soup, preserved fruit and other foods besides raw fruits. In addition, raspberry is also a medicinal plant, and can be used as a medicine for treating hangover, quenching thirst, stopping bleeding, removing toxic substances, eliminating phlegm, stopping dysentery, and relieving pain. The 27 provinces and autonomous areas in China have scattered distribution of raspberry plants, wherein the distribution of raspberry plants is the most widely distributed in Guizhou, guangxi, yunnan, fujian, sichuan and Guangdong, and the raspberry plants are the important economic crops for mountain agriculture development in Shaanxi, gansu, tibet, hunan and Hubei.
Based on good resource foundation, china is dedicated to large-area planting of raspberries in the 80 s, but the breeding work is still in a starting stage, and meanwhile, the breeding of new varieties of raspberries is also influenced by factors such as pollen incompatibility, reproduction apomixis, chromosome ploidy, low seed germination rate and the like, so that the breeding is difficult to break through. Therefore, in combination with the actual economic development of the region, the selection of the excellent tree species with developed root systems and strong drought resistance has great significance for improving the structure of the raspberry planting industry, increasing the benefits of farmers, driving the economic development of the region and the like. In view of the economic and medicinal value of raspberries and the urgent need to be solved, the genetic analysis and germplasm evaluation of the genetic resources of raspberries are discussed from the molecular level, the genetic relationship and genetic difference of raspberries are further known, and theoretical basis is provided for the research of genetic diversity of raspberries, the formulation of conservation strategies and the work of crossbreeding. However, there is no efficient method for raspberry diversity research.
Disclosure of Invention
The invention aims to provide an SSR primer group and application thereof. The SSR primer group not only can accurately identify different raspberry strains, but also can read genetic diversity information of the raspberry strains and explain genetic diversity caused by regional differences, can realize the application of the raspberry primer group in raspberry strain identification, genetic diversity analysis and molecular marker breeding auxiliary selection, and has higher reliability.
The invention provides an SSR primer group, which comprises 22 pairs of SSR primers: rubus166b, rubus1b, rubus105b, rubus153a, rubus25a, rubus223a, rubus22a, rubus167a, rubus253a, rubus24a, rubus110a, rubus260a, ZA005, ZA004, rubus263f, rubus279a, rubus285a, rubus 56a, rubus45c, rubus 35a, rubus 19a and Rubus243a; the nucleotide sequences of the primer pair of the Rubus166b are respectively shown in SEQ ID NO.1 and SEQ ID NO. 2; the nucleotide sequences of the primer pair of the Rubus1b are respectively shown in SEQ ID NO.3 and SEQ ID NO. 4; the nucleotide sequences of the primer pair of the Rubus105b are respectively shown in SEQ ID NO.5 and SEQ ID NO. 6; the nucleotide sequences of the primer pair of the Rubus153a are respectively shown in SEQ ID NO.7 and 8; the nucleotide sequences of the primer pair of the Rubus25a are respectively shown in SEQ ID NO.9 and 10; the nucleotide sequences of the primer pair of the Rubus223a are respectively shown in SEQ ID NO.11 and 12; the nucleotide sequences of the primer pair of the Rubus22a are respectively shown in SEQ ID NO.13 and 14; the nucleotide sequences of the primer pair of the Rubus167a are respectively shown in SEQ ID NO.15 and 16; the nucleotide sequences of the primer pair of the Rubus253a are respectively shown in SEQ ID NO.17 and 18; the nucleotide sequences of the primer pair of the Rubus24a are respectively shown in SEQ ID NO.19 and SEQ ID NO. 20; the nucleotide sequences of the primer pair of the Rubus110a are respectively shown in SEQ ID NO.21 and SEQ ID NO. 22; the nucleotide sequences of the primer pair of the Rubus260a are respectively shown in SEQ ID NO.23 and SEQ ID NO. 24; the nucleotide sequences of the primer pair of ZA005 are respectively shown in SEQ ID NO.25 and SEQ ID NO. 26; the nucleotide sequences of the primer pair of ZA004 are respectively shown as SEQ ID NO.27 and 28; the nucleotide sequences of the primer pair of the Rubus263f are shown in SEQ ID NO.29 and SEQ ID NO. 30 respectively; the nucleotide sequences of the primer pair of the Rubus279a are respectively shown in SEQ ID NO.31 and 32; the nucleotide sequences of the primer pair of the Rubus285a are respectively shown in SEQ ID NO.33 and 34; the nucleotide sequences of the primer pair of the rubus 56a are respectively shown in SEQ ID NO.35 and SEQ ID NO. 36; the nucleotide sequences of the primer pair of the Rubus45c are respectively shown in SEQ ID NO.37 and 38; the nucleotide sequences of the primer pair of the rubus 35a are respectively shown as SEQ ID NO.39 and SEQ ID NO. 40; the nucleotide sequences of the primer pair of the rubus 19a are respectively shown in SEQ ID NO.41 and SEQ ID NO. 42; the nucleotide sequences of the primer pair of the Rubus243a are respectively shown in SEQ ID NO.43 and SEQ ID NO. 44. SSRFinder is applied in the early stage of the invention to scan SSR of transcriptome databases of a plurality of species such as raspberry and the like, markers with the consistency rate higher than 90% are screened out from SSR markers obtained by scanning, 75 pairs of markers are randomly selected for subsequent verification experiments, and finally 22 pairs of SSR primers with stable amplification and good polymorphism are selected.
The invention also provides application of the SSR primer group in identification of raspberry germplasm resources.
The invention also provides application of the SSR primer group in identification of raspberry strains.
The invention also provides application of the SSR primer group in raspberry genetic diversity analysis.
The invention also provides application of the SSR primer group in raspberry molecule assisted breeding.
Preferably, the application comprises the steps of:
1) Extracting raspberry genome DNA;
2) Taking the raspberry genome DNA obtained in the step 1) as a template, and respectively carrying out PCR amplification by using the primer pairs in the SSR primer group in the technical scheme to obtain an amplification product;
3) And (3) respectively carrying out electrophoresis separation on the amplified products obtained in the step (2), and carrying out marking, processing and analysis according to the polymorphism of the amplified bands.
Preferably, the electrophoresis comprises non-denaturing polyacrylamide gel electrophoresis.
Preferably, the electrophoresis is performed by using 8% non-denaturing polyacrylamide gel for electrophoresis separation, 150V 90min.
Preferably, the method of processing comprises: after the polymorphism of the amplified band is marked, the original (0, 1) matrix is established by manually reading the band with the occurrence frequency and the size of the amplified band as references and marking the band as '1' and the band as '0' with unclear or missing bands.
Preferably, the analysis comprises: according to the result of the original (0, 1) matrix, the NTsys2.10e software is utilized to obtain the genetic similarity coefficient and the upgma cluster map between samples, so that the genetic relationship between the samples is obtained, and the germplasm resource identification diversity analysis and molecular breeding are realized.
The invention provides an SSR primer group. The SSR primer group not only can accurately identify different raspberry strains, but also can read genetic diversity information of the raspberry strains and explain genetic diversity caused by regional differences, can realize the application of the raspberry primer group in raspberry strain identification, genetic diversity analysis and molecular marker breeding auxiliary selection, and has higher reliability.
Drawings
Fig. 1 is a clustering result of 45 raspberry materials by the SSR primer set provided by the invention.
Detailed Description
The invention provides an SSR primer group, which comprises 22 pairs of SSR primers: rubus166b, rubus1b, rubus105b, rubus153a, rubus25a, rubus223a, rubus22a, rubus167a, rubus253a, rubus24a, rubus110a, rubus260a, ZA005, ZA004, rubus263f, rubus279a, rubus285a, rubus 56a, rubus45c, rubus 35a, rubus 19a and Rubus243a; the nucleotide sequences of the primer pair of the Rubus166b are respectively shown in SEQ ID NO.1 and SEQ ID NO. 2; the nucleotide sequences of the primer pair of the Rubus1b are respectively shown in SEQ ID NO.3 and SEQ ID NO. 4; the nucleotide sequences of the primer pair of the Rubus105b are respectively shown in SEQ ID NO.5 and SEQ ID NO. 6; the nucleotide sequences of the primer pair of the Rubus153a are respectively shown in SEQ ID NO.7 and 8; the nucleotide sequences of the primer pair of the Rubus25a are respectively shown in SEQ ID NO.9 and 10; the nucleotide sequences of the primer pair of the Rubus223a are respectively shown in SEQ ID NO.11 and 12; the nucleotide sequences of the primer pair of the Rubus22a are respectively shown in SEQ ID NO.13 and 14; the nucleotide sequences of the primer pair of the Rubus167a are respectively shown in SEQ ID NO.15 and 16; the nucleotide sequences of the primer pair of the Rubus253a are respectively shown in SEQ ID NO.17 and 18; the nucleotide sequences of the primer pair of the Rubus24a are respectively shown in SEQ ID NO.19 and SEQ ID NO. 20; the nucleotide sequences of the primer pair of the Rubus110a are respectively shown in SEQ ID NO.21 and SEQ ID NO. 22; the nucleotide sequences of the primer pair of the Rubus260a are respectively shown in SEQ ID NO.23 and SEQ ID NO. 24; the nucleotide sequences of the primer pair of ZA005 are respectively shown in SEQ ID NO.25 and SEQ ID NO. 26; the nucleotide sequences of the primer pair of ZA004 are respectively shown as SEQ ID NO.27 and 28; the nucleotide sequences of the primer pair of the Rubus263f are shown in SEQ ID NO.29 and SEQ ID NO. 30 respectively; the nucleotide sequences of the primer pair of the Rubus279a are respectively shown in SEQ ID NO.31 and 32; the nucleotide sequences of the primer pair of the Rubus285a are respectively shown in SEQ ID NO.33 and 34; the nucleotide sequences of the primer pair of the rubus 56a are respectively shown in SEQ ID NO.35 and SEQ ID NO. 36; the nucleotide sequences of the primer pair of the Rubus45c are respectively shown in SEQ ID NO.37 and 38; the nucleotide sequences of the primer pair of the rubus 35a are respectively shown as SEQ ID NO.39 and SEQ ID NO. 40; the nucleotide sequences of the primer pair of the rubus 19a are respectively shown in SEQ ID NO.41 and SEQ ID NO. 42; the nucleotide sequences of the primer pair of the Rubus243a are respectively shown in SEQ ID NO.43 and SEQ ID NO. 44.
In the present invention, specific nucleotide sequences of the SSR primer set are shown in table 1.
TABLE 1 nucleotide sequences of SSR primer sets
The invention also provides application of the SSR primer group in identification of raspberry germplasm resources.
The invention also provides application of the SSR primer group in identification of raspberry strains.
The invention also provides application of the SSR primer group in raspberry genetic diversity analysis.
The invention also provides application of the SSR primer group in raspberry molecule assisted breeding.
In the present invention, the application includes the steps of:
1) Extracting raspberry genome DNA;
2) Taking the raspberry genome DNA obtained in the step 1) as a template, and respectively carrying out PCR amplification by using the primer pairs in the SSR primer group in the technical scheme to obtain an amplification product;
3) And (3) respectively carrying out electrophoresis separation on the amplified products obtained in the step (2), and carrying out marking, processing and analysis according to the polymorphism of the amplified bands.
The invention extracts raspberry genomic DNA. The method for extracting the genome of the present invention is not particularly limited, and a conventional genome extraction method known to those skilled in the art may be used.
After the raspberry genome DNA is extracted, the invention uses the raspberry genome DNA as a template, and uses the primer pairs in the SSR primer group in the technical scheme to carry out PCR amplification to obtain an amplification product. The method of amplification in the present invention is not particularly limited, and conventional amplification methods well known to those skilled in the art may be employed.
After the amplified products are obtained, the obtained amplified products are respectively subjected to electrophoresis separation, and are marked, processed and analyzed according to the polymorphism of the amplified bands. In the present invention, the electrophoresis includes non-denaturing polyacrylamide gel electrophoresis. In the invention, the electrophoresis is carried out by utilizing 8% non-denaturing polyacrylamide gel for electrophoresis separation, and 150V 90min. In the invention, the electrophoresis further comprises a color development operation. In the present invention, the coloring preferably includes a silver staining method, and the staining agent is preferably: 1gAgNO 3 +500mL H 2 O, the color developer is preferably: 2mL of formaldehyde 7.5g of NaOH, ddH 2 O. In the present invention, the method of processing includes: after marking polymorphism of the amplified bands, manually reading the bands with the occurrence frequency and the size of the amplified bands as references, marking the bands as '1', marking the bands as '0' with unclear or missing bands, establishing an original (0, 1) matrix, obtaining genetic similarity coefficients and upgma cluster diagrams of samples by using NTsys2.10e software, obtaining genetic relationships and genetic differences among the samples, and realizing germplasm resource identification diversity analysis. Meanwhile, when in breeding, materials with relatively far genetic relationship are selected as parents to hybridize according to the clustering result, so that the hybridization advantage is maximized, and more excellent raspberry types are created.
The following describes in further detail an SSR primer set and application thereof in combination with specific examples, and the technical scheme of the invention includes but is not limited to the following examples.
Example 1
SSR primer set information for plant line identification, comprising 22 pairs of SSR primers, namely, rubus166b, rubus1b, rubus105b, rubus153a, rubus25a, rubus223a, rubus22a, rubus167a, rubus253a, rubus24a, rubus110a, rubus260a, ZA005, ZA004, rubus263f, rubus279a, rubus285a, rubusr56a, rubus45c, rubusr35a, rubusr19a and Rubus243a; the primer sequences of each pair of SSR primers are shown in Table 1.
Example 2
45 parts of raspberry strain materials popularized and planted in Guizhou are taken for amplification and polymorphism data collection, and the 22 pairs of primers are used for distinguishing and identifying the 45 parts of raspberries. 45 parts of the test material information are shown in Table 2.
Table 245 parts of test material information
The application of SSR primer set information in 45 parts of raspberry test materials comprises the following specific steps:
step 1): extracting genome DNA of raspberry test materials by using a plant genome DNA extraction kit (model: DP 350-02), and preserving for later use;
step 2): taking the genomic DNA extracted in the step 1) as a template, and carrying out PCR amplification by utilizing the SSR primer information;
step 3): the PCR amplified product of step 2) was separated by electrophoresis using 8% non-denaturing polyacrylamide gel at 150V for 90min. Silver staining developed after electrophoresis (stain: 1g AgNO) 3 +500mL H 2 O, color developer: 2mL of formaldehyde 7.5g of NaOH, ddH 2 O), record the developed picture. Finally, manually reading the polymorphism marker of the amplified band by taking the occurrence frequency and the size of the amplified band as a reference, marking the band as '1', marking the condition that the band is unclear or missing as '0', and thus establishing an original (0, 1) matrix.
Step 4): and (3) according to the statistical result of the step (3), obtaining a genetic similarity coefficient and an upgma cluster map of the samples by using NTsys2.10e software, and further obtaining the genetic relationship between the samples.
Clustering analysis is carried out according to the similarity coefficient obtained by the SSR molecular marking result of 45 raspberry varieties, and the clustering result (shown in figure 1) shows that when the threshold value is 0.71, the 45 varieties can be divided into 2 large groups. The first major class consisted of 21 raspberry samples numbered 1-17, 19, 21-34, etc., with the exception of 19, 21, 34, which were introduced from the national academy of agricultural liaison, all from Guizhou, which can be further divided into six subclasses at a threshold of 0.7455. The first class consists of samples numbered 1-4, 8-10, 13, 15-17, 19; the second class consists only of samples numbered 14; the third class consisted of samples numbered 9, 17, 28, 29; the fourth class consisted of samples numbered 5 only; the fifth category consists of samples numbered 21, 22; the sixth category consisted of only sample number 34. The second major class consists of 24 raspberry samples numbered 18, 20, 23-33, 35-45, etc. Wherein, except for 18, 20, 23 and 24, the other raspberry samples are all derived from the Liaoning national academy of sciences. They can be further divided into five subclasses at the threshold 0.7348 standard. The first class consisted of samples numbered 18, 32, 35, 36, 39; the second class consists of samples numbered 20, 24, 26, 27, 41-45; the third class consisted of samples numbered 23, 30, 33, 37, 40; the fourth class consisted of samples numbered 25, 28, 29, 31; the fifth class consisted of samples numbered 38 only. As shown by the clustering result, the genetic similarity among the raspberry varieties of different types is smaller, and the genetic relationship among the raspberry varieties of different sources is also far. Proved by the SSR primer information provided by the invention, the relatedness of partial raspberry varieties in Guizhou province is clarified, and 22 pairs of SSR primers can be applied to distinguish the 45 varieties of raspberries popularized and planted in Guizhou. Meanwhile, further analysis shows that 285 bands are obtained by amplifying 22 pairs of primers, the clear bands amplified by different primers are 7-25, 12.9 bands are generated by each primer on average, wherein the polymorphism rate is 96.8% and the polymorphism percentage of each primer is 85.71% -100%. The genetic similarity coefficient of the introduction sample 44 from the national academy of sciences of Liaoning and the introduction sample 45 from the national academy of sciences of Liaoning is the closest, and is 0.9176. Sample 5 from Cauzhou Hong Guan has the smallest genetic similarity coefficient of 0.6380 to sample 12 introduced from the national academy of sciences of Liaoning, and the largest genetic difference between them, and can be selected as hybrid parent material for heterosis utilization in breeding practice.
In conclusion, the SSR primer information provided by the invention can be successfully applied to identification of raspberry germplasm lines, analysis of genetic diversity and molecular marker assisted breeding selection, can lay a solid foundation for technical requirements in aspects of germplasm resource identification, analysis of genetic diversity, molecular assisted selection, genetic map construction, functional gene positioning and the like, and can provide abundant marker resources for related applications of raspberries and related species based on SSR molecular markers, so that the application prospect is very broad.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
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<213> Artificial sequence (Artificial Sequence)
<400> 28
ggattcttgc caaagtcgaa 20
<210> 29
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 29
attccgccct gcataaatc 19
<210> 30
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 30
ggaaattgga aaccattgga 20
<210> 31
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 31
tcgacatggc tagttctaca cag 23
<210> 32
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 32
ccccaactta aaccattctc a 21
<210> 33
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 33
tcgagaagct tgctatgctg 20
<210> 34
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 34
ggatacctca atggctttct tg 22
<210> 35
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 35
tggagattcc aaataaacaa atacc 25
<210> 36
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 36
tgtgtaaacc gttggatgaa 20
<210> 37
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 37
gaggggcaat taaagggttt 20
<210> 38
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 38
tgttgtaatt tggtttatcc ttgg 24
<210> 39
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 39
ttggaagcac aaaagcgata 20
<210> 40
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 40
gcgacagcca aaacaaaag 19
<210> 41
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 41
gcagatcaat gaaagcccat t 21
<210> 42
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 42
cggatcctcc aaccttcat 19
<210> 43
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 43
tgagcgagat gattggagtg 20
<210> 44
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 44
tatgtggtga tcatgcaagc 20

Claims (10)

1. An SSR primer set, wherein the SSR primer set comprises 22 pairs of SSR primers: rubus166b, rubus1b, rubus105b, rubus153a, rubus25a, rubus223a, rubus22a, rubus167a, rubus253a, rubus24a, rubus110a, rubus260a, ZA005, ZA004, rubus263f, rubus279a, rubus285a, rubus 56a, rubus45c, rubus 35a, rubus 19a and Rubus243a; the nucleotide sequences of the primer pair of the Rubus166b are respectively shown in SEQ ID NO.1 and SEQ ID NO. 2; the nucleotide sequences of the primer pair of the Rubus1b are respectively shown in SEQ ID NO.3 and SEQ ID NO. 4; the nucleotide sequences of the primer pair of the Rubus105b are respectively shown in SEQ ID NO.5 and SEQ ID NO. 6; the nucleotide sequences of the primer pair of the Rubus153a are respectively shown in SEQ ID NO.7 and 8; the nucleotide sequences of the primer pair of the Rubus25a are respectively shown in SEQ ID NO.9 and 10; the nucleotide sequences of the primer pair of the Rubus223a are respectively shown in SEQ ID NO.11 and 12; the nucleotide sequences of the primer pair of the Rubus22a are respectively shown in SEQ ID NO.13 and 14; the nucleotide sequences of the primer pair of the Rubus167a are respectively shown in SEQ ID NO.15 and 16; the nucleotide sequences of the primer pair of the Rubus253a are respectively shown in SEQ ID NO.17 and 18; the nucleotide sequences of the primer pair of the Rubus24a are respectively shown in SEQ ID NO.19 and SEQ ID NO. 20; the nucleotide sequences of the primer pair of the Rubus110a are respectively shown in SEQ ID NO.21 and SEQ ID NO. 22; the nucleotide sequences of the primer pair of the Rubus260a are respectively shown in SEQ ID NO.23 and SEQ ID NO. 24; the nucleotide sequences of the primer pair of ZA005 are respectively shown in SEQ ID NO.25 and SEQ ID NO. 26; the nucleotide sequences of the primer pair of ZA004 are respectively shown as SEQ ID NO.27 and 28; the nucleotide sequences of the primer pair of the Rubus263f are shown in SEQ ID NO.29 and SEQ ID NO. 30 respectively; the nucleotide sequences of the primer pair of the Rubus279a are respectively shown in SEQ ID NO.31 and 32; the nucleotide sequences of the primer pair of the Rubus285a are respectively shown in SEQ ID NO.33 and 34; the nucleotide sequences of the primer pair of the rubus 56a are respectively shown in SEQ ID NO.35 and SEQ ID NO. 36; the nucleotide sequences of the primer pair of the Rubus45c are respectively shown in SEQ ID NO.37 and 38; the nucleotide sequences of the primer pair of the rubus 35a are respectively shown as SEQ ID NO.39 and SEQ ID NO. 40; the nucleotide sequences of the primer pair of the rubus 19a are respectively shown in SEQ ID NO.41 and SEQ ID NO. 42; the nucleotide sequences of the primer pair of the Rubus243a are respectively shown in SEQ ID NO.43 and SEQ ID NO. 44.
2. The use of the SSR primer set of claim 1 in identification of raspberry germplasm resources.
3. Use of the SSR primer set of claim 1 in identification of raspberry lines.
4. Use of the SSR primer set of claim 1 in raspberry genetic diversity analysis.
5. The use of the SSR primer set of claim 1 in raspberry molecule assisted breeding.
6. The use according to any one of claims 2 to 5, characterized in that it comprises the following steps:
1) Extracting raspberry genome DNA;
2) Taking the raspberry genome DNA obtained in the step 1) as a template, and respectively carrying out PCR amplification by using the primer pair in the SSR primer group of the claim 1 to obtain an amplification product;
3) And (3) respectively carrying out electrophoresis separation on the amplified products obtained in the step (2), and carrying out marking, processing and analysis according to the polymorphism of the amplified bands.
7. The use of claim 6, wherein the electrophoresis comprises non-denaturing polyacrylamide gel electrophoresis.
8. The use according to claim 7, wherein the electrophoresis is an electrophoresis separation using an 8% non-denaturing polyacrylamide gel, 150v 90min.
9. The use of claim 6, wherein the method of processing comprises: after the polymorphism of the amplified band is marked, the original (0, 1) matrix is established by manually reading the band with the occurrence frequency and the size of the amplified band as references and marking the band as '1' and the band as '0' with unclear or missing bands.
10. The use according to claim 9, wherein the analysis comprises: according to the result of the original (0, 1) matrix, the NTsys2.10e software is utilized to obtain the genetic similarity coefficient and the upgma cluster map between samples, so that the genetic relationship between the samples is obtained, and the germplasm resource identification diversity analysis and molecular breeding are realized.
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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110079628A (en) * 2019-04-30 2019-08-02 遵义医科大学 One group of EST-SSR primer information and its application in the identification of bletilla strain

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110079628A (en) * 2019-04-30 2019-08-02 遵义医科大学 One group of EST-SSR primer information and its application in the identification of bletilla strain

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