CN114107538A

CN114107538A - Core primer group based on Chinese wolfberry variety SSR (simple sequence repeat) markers and application thereof

Info

Publication number: CN114107538A
Application number: CN202111117613.XA
Authority: CN
Inventors: 陈金焕; 秦英之; 饶书培
Original assignee: Beijing Forestry University
Current assignee: Beijing Forestry University
Priority date: 2021-09-23
Filing date: 2021-09-23
Publication date: 2022-03-01
Anticipated expiration: 2041-09-23
Also published as: CN114107538B

Abstract

The invention provides a core primer group based on an SSR (simple sequence repeat) marker of a Chinese wolfberry variety, which is characterized by comprising the following 23 primer pairs; the invention also provides a medlar variety SSR fingerprint established based on the core primer group and application thereof in medlar variety identification. The invention utilizes the full-length transcript information of the Lycium plants and the SSR molecular marker technology to develop the SSR primers of the Lycium plants, can well distinguish different varieties, has the advantages of simplicity, rapidness and low cost, and can be used for identification of the Lycium plants and construction of variety fingerprints.

Description

Core primer group based on Chinese wolfberry variety SSR (simple sequence repeat) markers and application thereof

Technical Field

The invention relates to the technical field of molecular biology, in particular to a plant variety identification technology based on molecular markers, and especially relates to an SSR primer group for identifying varieties of Lycium plants and application thereof in variety identification of Lycium plants.

Background

Lycium barbarum L is a shrub of Lycium genus (Solanaceae) of Solanaceae, and has more than 80 species worldwide, mainly distributed in south America, south North America, south Africa and continental Eurasia. Chinese matrimony vine has 7 kinds of 3 varieties, wherein Ningxia matrimony vine is distributed most widely and is widely distributed and cultivated in Ningxia, Xinjiang, Tibet, Qinghai, Gansu, inner Mongolia and other areas in China.

The medlar has more than 4000 years of cultivation history in China, and in recent years, along with diversification of breeding targets, the number of new medlar varieties is continuously increased along with the development of medlar industry, and the new medlar varieties are developed to dozens of current main cultivars such as the earliest hemp leaves, Ningqi No. 1 and the like. Along with the increase of medlar varieties, the phenotype difference is smaller and smaller, the distinguishing difficulty is larger and larger, and meanwhile, the characters of the varieties which are easy to distinguish are concentrated in the flowering phase and the fruit phase and can not be shown in the seedling phase of the medlar, so that a method for distinguishing different medlar varieties without being influenced by the phenotype characters is needed. The variety forms of the Chinese wolfberry at the seedling stage cannot be well distinguished, and the protection of the new Chinese wolfberry variety faces a plurality of difficulties and problems, so that the development of a comprehensive and accurate identification method for the existing variety is urgent.

Microsatellite DNA (SSR), also known as Simple Sequence Repeat (SSR), is a repetitive Sequence of up to several tens of nucleotides in length consisting of 1-6 nucleotides in tandem as repeating units, and is usually flanked by highly conserved single-copy sequences. Therefore, primers can be designed based on the flanking sequences for PCR amplification, and polymorphism of the microsatellite locus is shown due to the difference in the size or the number of repeats of the repetitive fragment. Compared with other molecular markers, the SSR marker has the advantages of high polymorphism, co-dominant inheritance, simple technology, good repeatability, strong specificity, convenient operation and the like, and is widely applied to variety identification.

In order to develop molecular biology SSR markers to assist in identifying germplasm resources of Lycium plants, promote improved variety breeding of the Lycium plants, accelerate development and utilization of germplasm resources and protect diversity of germplasm resources of the Lycium plants, development of SSR primers of the Lycium plants is urgently needed, and scientific basis is provided for researches on assessment, variety identification, protection and the like of Lycium germplasm resources.

Disclosure of Invention

On the basis of Pacbio full-length transcription group sequencing, verification and polymorphism analysis are carried out on a selected part of SSRs through PCR detection on the basis of a large amount of obtained full-length CDS sequences and SSR information, so that the universal polymorphic primer is obtained. The invention aims to make clear the overall characteristics of the SSR of the Lycium plants by using an SSR molecular marker technology, develop SSR primers of main varieties of the Lycium plants, provide a method for identifying the varieties of the Lycium plants by using the SSR molecular markers, and lay a foundation for genetic diversity, linkage map construction and genetic relationship research of Lycium germplasm resources to solve the technical problems.

The variety sampled by the invention relates to a plurality of regions and different fruit colors, each variety collects 6 different individuals from pure lines, the result of the SSR molecular marker is more accurate and reliable, compared with the existing patent, no repeated primer exists, the variety which can be distinguished is more comprehensive, and the polymorphism is better than that of the existing SSR primer of the medlar.

The invention firstly provides a core primer group based on the SSR marker of a Chinese wolfberry variety, which comprises the following 23 primer pairs: lbs sr1474, lbs sr21443, lbs sr1690, lbs sr1156, lbs sr1866, lbs sr29, lbs sr79148, lbs sr7735, lbs sr40, lbs sr20799, lbs sr1895, lbs sr501, lbs sr0076, lbs sr0297, lbs sr0338, lbs sr0277, lbs sr0543, lbs sr0010, lbs sr0421, lbs sr0426, lbs sr0270, lbs sr0512, and lbs sr 0363; wherein the content of the first and second substances,

LBSR 1474 consists of SEQ ID NO. 1 and SEQ ID NO. 2;

LBSR 21443 consists of SEQ ID NO 3 and SEQ ID NO 4;

LBSSR1690 consists of SEQ ID NO 5 and SEQ ID NO 6;

LBSSR1156 consists of SEQ ID NO 7 and SEQ ID NO 8;

LBSSR1866 consists of SEQ ID NO 9 and SEQ ID NO 10;

LBSSR29 consists of SEQ ID NO 11 and SEQ ID NO 12;

LBSSR79148 consists of SEQ ID NO 13 and SEQ ID NO 14;

LBSSR7735 consists of SEQ ID NO 15 and SEQ ID NO 16;

LBSSR40 consists of SEQ ID NO 17 and SEQ ID NO 18;

LBSSR20799 consists of SEQ ID NO 19 and SEQ ID NO 20;

LBSSR1895 consists of SEQ ID NO 21 and SEQ ID NO 22;

LBSSR501 consists of SEQ ID NO 23 and SEQ ID NO 24;

LBSSR0076 consists of SEQ ID NO 25 and SEQ ID NO 26;

LBSSR0297 consists of SEQ ID NO 27 and SEQ ID NO 28;

LBSR 0338 consists of SEQ ID NO:29 and SEQ ID NO: 30;

LBSSR0277 consists of SEQ ID NO 31 and SEQ ID NO 32;

LBSSR0543 consists of SEQ ID NO 33 and SEQ ID NO 34;

LBSSR0010 consists of SEQ ID NO 35 and SEQ ID NO 36;

LBSSR0421 consists of SEQ ID NO 37 and SEQ ID NO 38;

LBSSR0426 consists of SEQ ID NO 39 and SEQ ID NO 40;

LBSSR0270 consists of SEQ ID NO 41 and SEQ ID NO 42;

LBSSR0512 consists of SEQ ID NO 43 and SEQ ID NO 44;

LBS SR0363 consists of SEQ ID NO 45 and SEQ ID NO 46.

The invention further provides a Chinese wolfberry variety SSR fingerprint spectrum established based on the core primer group, which is specifically shown in Table 2.

The invention further provides application of the core primer group or the Chinese wolfberry variety SSR fingerprint spectrum in Chinese wolfberry variety identification, Chinese wolfberry germplasm resource genetic diversity determination, Chinese wolfberry variety linkage map construction or Chinese wolfberry variety genetic relationship determination.

The invention also provides a method for identifying the variety of the medlar, which comprises the following steps:

1) based on the genome DNA of a sample to be detected, respectively carrying out PCR amplification by using the core primer group to obtain PCR amplification products;

2) detecting the PCR amplification product through capillary electrophoresis to obtain capillary original data;

3) obtaining a fragment size of a PCR amplification product in a lane corresponding to each primer pair based on analyzing the capillary raw data;

4) and determining the Chinese wolfberry variety according to the corresponding locus and the size of the allele of the amplification product.

In one embodiment according to the present invention, the reaction conditions for the PCR amplification in step 1) are:

pre-denaturation at 95 ℃ for5 min;

denaturation: the temperature of the mixture is 94 ℃ for30s,

the annealing is carried out for30s for the first time,

extension: 30s at 72 ℃ and 30 cycles;

denaturation: the temperature of the mixture is 94 ℃ for30s,

secondary annealing: the temperature of the mixture is 53 ℃ for30s,

extension: 30s at 72 ℃ and 10 cycles;

further extension for 10min at 72 ℃; the annealing temperature of the primary annealing is determined by the annealing temperature of the downstream primers in each core primer pair; preferably, the annealing temperature of the primary annealing is as shown in table 2.

In one embodiment according to the invention, the analysis of the capillary raw data in step 3) is carried out by a method comprising the following steps:

the capillary raw data was analyzed using fragment (plant) fragment analysis function in GeneMarke, and the fragment size of each amplification product was obtained by comparing the position of the internal molecular weight standard in each lane with the position of each sample peak.

In one embodiment of the present invention, the step of determining the variety of lycium barbarum in step 4) comprises:

the sample to be detected comprises a variety to be detected and a comparison variety, the data of the amplification products of the variety to be detected and the comparison variety in the sample to be detected are compared, and whether the variety to be detected and the comparison variety are the same variety is judged according to the difference of the sizes of the loci and the alleles thereof corresponding to the amplification products;

in an embodiment according to the present invention, step 4) further comprises comparing the size of the fragment with the SSR fingerprint described above to determine the variety of lycium barbarum.

The technical scheme of the invention has the following beneficial effects:

the invention utilizes the full-length transcript information of the Lycium plants and the SSR molecular marker technology to develop the SSR primers of the Lycium plants, can well distinguish different varieties (lines), has simple, quick and low cost, can be used for identifying the Lycium plants and constructing variety fingerprints, and lays a foundation for genetic diversity protection, linkage map construction, genetic relationship research and identification and protection of the existing varieties in the market of the Lycium plants by utilizing the SSR molecular markers.

The core primer group provided by the invention has no repeated primers, and the Ningxia wolfberry variety and wild resource of the wild lycium plant wild resource are improved in education. According to the invention, the experiment precision is improved by a capillary fluorescence electrophoresis method, the fragment size is more accurate, 6 different individuals are selected for each strain to extract DNA, and the accuracy of primer detection is greatly improved.

Drawings

FIG. 1 is a diagram showing the result of LBSR 29 Ningqi No. 10 capillary electrophoresis;

FIG. 2 is a diagram showing the result of LBS SR1474 capillary electrophoresis of Ningnong Qi No. 9;

FIG. 3 is the result chart of LBSR 120 capillary electrophoresis of No. 4 Jingqi;

FIG. 4 is the result chart of capillary electrophoresis of LBS SR2144 day sperm No. 3;

FIG. 5 is a diagram showing the result of LBSSR/1474 capillary electrophoresis of a sample to be tested;

FIG. 6 is a diagram showing the result of capillary electrophoresis of LBSSR/20799 of a sample to be tested;

FIG. 7 is a diagram showing the result of LBSSR/1895 capillary electrophoresis of a sample to be tested;

FIG. 8 is a diagram showing the result of LBSSR/29 capillary electrophoresis of a sample to be tested;

FIG. 9 is a diagram showing the result of LBSSR/1474 capillary electrophoresis of a sample to be tested;

FIG. 10 is a diagram showing the result of capillary electrophoresis of LBSSR/20799 of a sample to be tested;

FIG. 11 is a diagram showing the result of LBSSR/1895 capillary electrophoresis of a sample to be tested;

FIG. 12 is a diagram showing the result of LBSSR/29 capillary electrophoresis of a sample to be tested.

Detailed Description

In order to make the technical problems, technical solutions and advantages to be solved by the present invention clearer, the following detailed description is made with reference to the accompanying drawings and specific embodiments.

Example 1 establishment of a Lycium germplasm identification technology System

1. Full length transcriptome sequencing

Because the wolfberry has no complete genome at present, three clones of Ningqi No. 1 without plant diseases and insect pests are used. After the roots, stems, leaves, flowers and fruits are mixed, the PacBio RS system is used for full-length transcriptome sequencing and analysis, and the transcript information is assembled. The average read length of the full-length transcription group can reach 2500-.

2. Test material

TABLE 1 SSR polymorphism analysis materials of 28 varieties (lines) of Lycium

2.1 sampling

Young leaves of 28 varieties (lines) free from plant diseases and insect pests were collected as test samples, and six clones were collected for each variety (line), one replicate for each clone.

2.2 sample preservation

Selecting a proper amount of sample, loading into an EP tube, quickly freezing by liquid nitrogen, and storing in a refrigerator at-80 ℃ to be tested. Or selecting a proper amount of samples and putting the samples into a self-sealing bag with silica gel, and removing air in the bag as much as possible to store the samples in dark and ventilated places.

DNA extraction

DNA of 28 different medlar planting materials (No. 1-28, shown in Table 1) was extracted using a high-efficiency plant genome DNA extraction kit (DP350) and a polysaccharide polyphenol plant genome DNA extraction kit (DP360) from Tiangen Biochemical technology (Beijing) Ltd.

PCR reaction

4.1 PCR primer Synthesis

Primer screening was performed using PREMIER 5.0. The primer sequences were sent to the company for synthesis. After synthesis, PCR reaction is carried out.

4.2 PCR reaction System

2 XTaqMix7.5. mu.l, DNA 1.5. mu.l, upstream primer 0.06. mu.l, downstream primer 0.24. mu.l, ddH₂O5.4. mu.l, M13 fluorescent marker 0.3. mu.l. Wherein the DNA concentration of the PCR reaction system is 25 ng/. mu.l.

4.3 PCR amplification procedure

Pre-denaturation 95 ℃ for5min, denaturation: 94 ℃ for30s, annealing temperature determined by downstream primer temperature (° c) for30s, extension: 72 ℃ for30s, 30 cycles. Denaturation: 94 ℃ for30s, annealing: 53 ℃ for30s, extension: 72 ℃ for30s, 10 cycles. Further extension was carried out at 72 ℃ for 10 min.

5. And (3) capillary electrophoresis detection:

detection of PCR amplification products by capillary electrophoresis apparatus (ABI3730 XLNAnalyzer) was performed by Biotech limited of Borneo, Beijing, to obtain capillary raw data in FASTA file format.

6. Data analysis and result determination

7. Using genemark: fragment (plant) fragment analysis software in (1) analyzes the original data obtained by the sequencer, compares the position of the molecular weight internal standard in each lane with the position of the peak value of each sample, and records the size of each fragment.

8. As shown in fig. 1-5, which are capillary electrophoresis result diagrams of exemplary control varieties, the results of the SSR fingerprint comparison between the sample to be tested and the exemplary control sample are analyzed, and if the sample to be tested and the control variety have different loci with different alleles, the sample to be tested and the control variety are determined to be different varieties according to the loci and the differences of the alleles; and if all locus alleles of the variety to be detected and the control variety are the same, judging that the variety to be detected and the control variety are the same varieties of SSR loci completely.

23 pairs of primers with rich polymorphism and stable amplification are screened out based on a wolfberry transcriptome sequencing result, the number of gene loci, the number of allelic fragments and the size of the allelic fragments amplified from 28 parts of wolfberry materials of the 23 pairs of SSR primers are determined through genetic analysis of collected and stored 28 parts of wolfberry germplasm, and the strain, the original strain of the variety and asexual propagation offspring of the wolfberry can be effectively identified through the allelic fragment combination obtained through different SSR markers. Where the relationship of/represents sum is the result of homologous staining of 2 alleles.

Therefore, the SSR fingerprint of the lycium barbarum strain and variety can be identified by using the 23 pairs of SSR primers and the corresponding size combinations of the amplified product fragments as the SSR marker fingerprints of the lycium barbarum strain and variety, and whether the original lycium barbarum strain to be detected or the asexual propagation progeny thereof is the lycium barbarum strain and variety or not.

TABLE 2 core primer sequences and allelic variation information for plants of the genus Lycium (23 pairs)

Example 2 application of identifying the original strain of Lycium barbarum to be tested or its asexual reproduction progeny as existing strain or variety of Lycium barbarum

1. Extracting genome DNA:

respectively extracting genome DNA of 6 parts of Chinese wolfberry leaves; wherein N6-13-1, N6-13-2, N6-14, N6-15, N6-16 and N6-20 are asexual propagation seedlings of Ningqi No. 6, and N6 is seedlings of Ningqi No. 6;

2. PCR amplification of SSR markers:

carrying out PCR amplification on the extracted DNA by using the screened SSR primers respectively, wherein the SSR primer system of the PCR amplification (total 20u 1): 2 XTaqMix7.5. mu.l, DNA 1.5. mu.l, upstream primer 0.06. mu.l, downstream primer 0.24. mu.l, ddH₂O5.4. mu.l, M13 fluorescent marker 0.3. mu.l.

The PCR amplification reaction adopts the following cycle parameters: pre-denaturation 95 ℃ for5min, denaturation: 94 ℃ for30s, annealing temperature determined by downstream primer temperature (° c) for30s, extension: 72 ℃ for30s, 30 cycles. Denaturation: 94 ℃ for30s, annealing: 53 ℃ for30s, extension: 72 ℃ for30s, 10 cycles. Further extension was carried out at 72 ℃ for 10 min.

3. And (3) capillary electrophoresis detection:

Using genemark: fragment (plant) fragment analysis software in (1) analyzes the raw data obtained by the sequencer, compares the position of the molecular weight internal standard in each lane with the position of the peak value of each sample, and records the size of each fragment.

If the amplification results of the 23 pairs of primers of the to-be-detected medlar meet the following conditions, the to-be-detected medlar is or is a candidate of a medlar strain or variety; if the amplification results of the 23 pairs of primers of the to-be-detected medlar do not meet the following conditions, the to-be-detected medlar is not or is not a medlar strain or variety as a candidate;

the capillary current results of the amplification products of lycium barbarum to be tested are shown in fig. 6-12:

the amplification product of the primer pair of Ningqi No. 6 LBS SR/1474 is 245 bp; and the amplification products of the LBS SR/29 primer pair are 244bp and 286 bp; and the amplification products of the LBS SR/20799 primer pair are 252bp and 268 bp; and the amplification products of the LBSSSR/1895 primer pair are 219bp and 226 bp.

Six strains of N6-13-1, N6-13-2, N6-14, N6-15, N6-16 and N6-20 which are clone No. 6 of Ningqi are amplified by using 4 pairs of primers of LBSSR/1474, LBSSR/20799, LBSSR/1895 and LBSSR/29. The results of N6-13-1, N6-14, N6-16 and N6-20 are shown in FIG. 5-FIG. 8, and it can be seen that the amplification product of the LBS SR/1474 primer pair is 245 bp; and the amplification product of the LBS SR/29 primer pair is 286 bp; and the amplification products of the LBS SR/20799 primer pair are 252bp and 268 bp; and the amplification products of the LBSSSR/1895 primer pair are 219bp and 226 bp. The amplification results of N6-13-2 and N6-14 are shown in FIG. 9-FIG. 12, and it can be seen that the amplification results of 4 pairs of primers do not meet the identification standards of strains and varieties of Lycium barbarum, which proves that the primers are not Ningqi No. 6.

The target product is obtained, and the method is proved to be correct.

The foregoing is a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should be considered as the protection scope of the present invention.

Sequence listing

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Claims

1. A core primer group based on an SSR marker of a Chinese wolfberry variety is characterized by comprising the following 23 primer pairs: lbs sr1474, lbs sr21443, lbs sr1690, lbs sr1156, lbs sr1866, lbs sr29, lbs sr79148, lbs sr7735, lbs sr40, lbs sr20799, lbs sr1895, lbs sr501, lbs sr0076, lbs sr0297, lbs sr0338, lbs sr0277, lbs sr0543, lbs sr0010, lbs sr0421, lbs sr0426, lbs sr0270, lbs sr0512, and lbs sr 0363; wherein the content of the first and second substances,

LBSR 1474 consists of SEQ ID NO. 1 and SEQ ID NO. 2;

LBSR 21443 consists of SEQ ID NO 3 and SEQ ID NO 4;

LBSSR1690 consists of SEQ ID NO 5 and SEQ ID NO 6;

LBSSR1156 consists of SEQ ID NO 7 and SEQ ID NO 8;

LBSSR1866 consists of SEQ ID NO 9 and SEQ ID NO 10;

LBSSR29 consists of SEQ ID NO 11 and SEQ ID NO 12;

LBSSR79148 consists of SEQ ID NO 13 and SEQ ID NO 14;

LBSSR7735 consists of SEQ ID NO 15 and SEQ ID NO 16;

LBSSR40 consists of SEQ ID NO 17 and SEQ ID NO 18;

LBSSR20799 consists of SEQ ID NO 19 and SEQ ID NO 20;

LBSSR1895 consists of SEQ ID NO 21 and SEQ ID NO 22;

LBSSR501 consists of SEQ ID NO 23 and SEQ ID NO 24;

LBSSR0076 consists of SEQ ID NO 25 and SEQ ID NO 26;

LBSSR0297 consists of SEQ ID NO 27 and SEQ ID NO 28;

LBSR 0338 consists of SEQ ID NO:29 and SEQ ID NO: 30;

LBSSR0277 consists of SEQ ID NO 31 and SEQ ID NO 32;

LBSSR0543 consists of SEQ ID NO 33 and SEQ ID NO 34;

LBSSR0010 consists of SEQ ID NO 35 and SEQ ID NO 36;

LBSSR0421 consists of SEQ ID NO 37 and SEQ ID NO 38;

LBSSR0426 consists of SEQ ID NO 39 and SEQ ID NO 40;

LBSSR0270 consists of SEQ ID NO 41 and SEQ ID NO 42;

LBSSR0512 consists of SEQ ID NO 43 and SEQ ID NO 44;

LBS SR0363 consists of SEQ ID NO 45 and SEQ ID NO 46.

2. The SSR fingerprint of the Chinese wolfberry variety established based on the core primer group of claim 1 is characterized by being shown in the following table:

3. the use of the core primer set according to claim 1 or the SSR fingerprint of the variety of Lycium barbarum according to claim 2 for variety identification of Lycium barbarum, determination of genetic diversity of Lycium barbarum germplasm resources, construction of linkage maps of the variety of Lycium barbarum, or determination of genetic relationship between varieties of Lycium barbarum.

4. A method for identifying a variety of Lycium barbarum comprising:

1) respectively carrying out PCR amplification by using the core primer group according to claim 1 based on the genome DNA of the variety to be detected to obtain PCR amplification products;

5. The method of claim 4, wherein the reaction conditions for PCR amplification in step 1) are:

pre-denaturation at 95 ℃ for5 min;

denaturation: the temperature of the mixture is 94 ℃ for30s,

the annealing is carried out for30s for the first time,

extension: 30s at 72 ℃ and 30 cycles;

denaturation: the temperature of the mixture is 94 ℃ for30s,

secondary annealing: the temperature of the mixture is 53 ℃ for30s,

extension: 30s at 72 ℃ and 10 cycles;

further extension for 10min at 72 ℃;

the annealing temperature of the primary annealing is determined by the annealing temperature of the downstream primer in each primer pair; preferably, the annealing temperature of the primary annealing is as shown in the following table:

6. the method of claim 4, wherein the analysis of the capillary raw data in step 3) is achieved by a method comprising the steps of:

7. The method of claim 4, wherein the step of determining the variety of Lycium barbarum of step 4) comprises:

and comparing the amplification product data of the variety to be detected and the comparison variety in the sample to be detected, and judging whether the variety to be detected and the comparison variety are the same variety or not according to the difference of the sizes of the loci and the alleles thereof corresponding to the amplification products.

8. The method according to claim 4, wherein step 4) further comprises comparing the fragment size to the SSR fingerprint of claim 2 to determine the variety of Lycium barbarum.