CN107868839A - A kind of SNP marker, primer and the application of analyzing rice genetic diversity identification of species - Google Patents

Abstract

The invention belongs to the technical field of molecular biology, and specifically discloses a SNP marker for analyzing rice genetic diversity identification varieties, the site of the SNP marker in rice, primers for obtaining the SNP marker, and related applications. The SNP marker has good versatility and does not require large-scale equipment and instruments; the SNP marker of the present invention can accurately distinguish the genotype of the variety to be tested and the genetic difference between the varieties, and the difference at the very close locus of the genetic distance can also be distinguished; After the primers for a certain site are designed, multiple individuals can be studied for this site, and the cost can be well controlled.

Description

A kind of SNP marker, primer and application for analyzing rice genetic diversity identification varieties

technical field

The invention belongs to the technical field of molecular biology, and in particular relates to a SNP marker for analyzing rice genetic diversity and identifying varieties and its application.

Background technique

Rice is an important food crop in my country, and hybrid rice has made great contributions to food security in my country and the world with its remarkable yield advantage. The promotion and use of new varieties has played an extremely important role in ensuring my country's food supply and meeting people's diverse needs. From 2001 to 2015, 6,310 rice varieties passed provincial-level and above approvals across the country. Effectively distinguishing and identifying these variety materials has important practical significance for rice variety breeding, testing, quality and market management, and property rights protection.

Varieties of morphological differences and differences in characteristics, in essence, are caused by differences in genes. The use of DNA molecular markers to identify differences in the genotypes of different rice varieties is currently a more effective and faster method for distinguishing varieties. Among them, SSR markers have the characteristics of simple and quick operation, high polymorphism and good stability, and can accurately reveal the SSR polymorphism at the same site among different varieties, and have been widely used in the construction of genetic maps, genetic diversity analysis and variety Verification of purity and authenticity. However, due to the poor or lack of correlation between SSR markers and the agronomic traits of varieties, it is difficult to distinguish near-isogenic lines or functional substitution lines. For such varieties, SNP markers (functional markers) of related characteristic functional genes are needed to be distinguished from the original species.

Single nucleotide polymorphism (single nucleotide polymorphism, SNP) mainly refers to the DNA sequence polymorphism caused by the variation of a single nucleotide at the genome level. The polymorphism shown by SNP only involves the variation of a single base, which can be caused by the transition or transversion of a single base, or by the insertion or deletion of a base. SNPs are widely distributed in biological genomes. The SNPs that occur in the coding regions are called cSNPs (coding regions SNPs). In addition, they occur in the 5′, 3′ non-coding regions of genes, and introns. The number of SNP markers is large and the distribution is wide. For example, in the maize genome, the average density of SNP markers is about 1 SNP/57bp; in the soybean genome, the average density of SNP markers is about 1 SNP/272bp; in the rice genome In , the average density of SNP markers is about 1 SNP/170bp. Therefore, it is necessary to use the polymorphism and versatility of SNP markers within and between species to develop a functional SNP marker that is closely related to rice agronomic shape and can analyze rice genetic diversity and identify varieties.

Contents of the invention

The invention provides a SNP marker for analyzing rice genetic diversity and identifying varieties and its application, and develops a functional SNP marker that is closely related to rice agronomic shape and can analyze rice genetic diversity and identify varieties.

The first object of the present invention is to provide a kind of SNP mark that analyzes rice genetic diversity identification variety, and the position of described SNP mark in rice species is as shown in Table 1:

Table 1 Different SNP marker sites and corresponding primer sequences

The second object of the present invention is to provide a primer for obtaining the above-mentioned SNP markers for analyzing rice genetic diversity identification varieties, and the primer sequences corresponding to the SNP markers are shown in Table 1.

The third object of the present invention is to provide an application of the SNP markers for analyzing rice genetic diversity and identifying varieties in molecular marker-assisted breeding of rice.

The fourth object of the present invention is to provide an application of the rice genetic diversity and SNP marker primers for identifying varieties in molecular marker-assisted breeding of rice.

Compared with the prior art, the present invention provides a SNP marker for analyzing rice genetic diversity identification varieties and its application, which has the following beneficial effects:

(1) The SNP marker has good versatility and does not require large-scale equipment; the SNP marker of the present invention can accurately distinguish the genotype of the variety to be tested and the genetic difference between the varieties, and the difference of the locus with a very close genetic distance can also be distinguished . (2) After the primers for a certain site are designed, multiple individuals can be studied for this site, and the cost can be well controlled. When the SNP marker of the present invention is used for fingerprint identification of different germplasm materials, by screening the characteristic SNP markers identified for different germplasm materials, the purpose of using one SNP marker to identify specific germplasm materials can be achieved.

Description of drawings

Fig. 1 is a cluster analysis diagram of the application of SNP markers in rice genetic diversity analysis;

Figure 2 is the results of CAPs marker amplification and enzyme digestion of SNP markers in rice genetic diversity analysis;

In Fig. 2, M swimming lane represents 50bp Marker; 1-4 swimming lane represents sample father; 5-8 swimming lane represents sample female parent; 9-28 swimming lane represents 20 hybrid offspring;

Fig. 3 is the electrophoresis diagram of target gene identification based on SNP markers in rice molecular marker-assisted selection breeding;

In Figure 3, lane M represents 50bpmarker; lane 1-4 represents the male parent of the sample; lane 5-8 represents the female parent of the sample; lane 9-28 represents the 20 hybrid offspring.

Detailed ways

The present invention will be described in detail below in conjunction with specific examples, but should not be construed as a limitation of the present invention. The experimental methods in the following examples, unless otherwise specified, are conventional methods, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

1 Test materials and methods

245 samples of the main hybrid rice and its parents in the middle and lower reaches of the Yangtze River were collected (Table 2), including 157 samples of conventional species/parents, 38 samples of two-line hybrids, and 50 samples of three-line hybrids. The DNA of individual plants (10 individual plants of each variety) was extracted by CTAB method, and stored at -20°C as experimental materials for the next step.

Table 2 245 rice samples

2. Rice functional gene screening

Starting from the traits and characteristics that determine the characteristics of rice varieties, the known important functional genes that are closely related to the main agronomic traits of rice were selected. The specific information is shown in Table 3.

Table 3 Functions of known important genes closely related to main agronomic traits of rice

3. Functional SNP marker screening

3.1. Seed germination: randomly select 96 conventional species/parental samples from the rice samples collected in Table 1, take a few seeds each, and germinate at 28°C for one week.

3.2. DNA extraction: DNA was extracted by CTAB method.

3.3. PCR amplification reaction system: 1×PCR buffer, 2.5mmol/L Mg ²⁺ , 0.25mmol/L dNTPs, 0.1μmol/L upstream primer 1, 0.1μmol/L upstream primer 2, 0.2μmol/L downstream primer , 1.0U Taq DNA polymerase, 20ng-40ng sample DNA, total volume 10μL.

3.4. PCR reaction program: 94°C for 15 minutes, 94°C for 20s, 76°C for 30s (0.6°C drop per cycle), cycle 10 times, 94°C for 20s, 55°C for 1min, cycle 26 times, 72°C for 5 minutes, and store at 30°C. Run the denaturing program on the PCR machine.

3.5. Electrophoresis detection of PCR amplification products (non-denaturing polyacrylamide gel): add 0.5% volume of ammonium persulfate solution and 0.1% volume of TEMED to a certain volume of 6% non-denaturing polyacrylamide solution, and mix well After that, pour glue. 100V constant voltage, pre-electrophoresis 10-30min. Add 1-3 μL of sample to each well. 200-250V constant voltage, electrophoresis 1-2h, xylene cyanide FF electrophoresis to the middle. Turn off the power, peel off the gel from the glass plate, and make a mark in time to distinguish the gel plate. The gel was immersed in the fixative solution and shaken on a shaker for 5 min. Perform a quick rinse with appropriate amount of ddH ₂ O. Shake and stain in the newly prepared staining solution for 10 min. Rinse quickly with an appropriate amount of ddH ₂ O (containing 0.2% volume of 0.1% sodium thiosulfate) for no more than 10s. Shake in fresh developer solution until distinct bands appear. Fix in fixative solution for 5 min. Record results or take pictures directly on the film viewing light.

3.6 Fluorescence detection by capillary electrophoresis

PCR amplification: Amplify 4 individual plants per sample, the amplification reaction system is: 1×PCR buffer, 2.5mmol/L Mg ²⁺ , 0.25mmol/L dNTPs, 0.1μmol/L upstream primer 1, 0.1μmol/L L upstream primer 2, 0.1 μmol/L anchor primer 1, 0.1 μmol/L anchor primer 2, 0.2 μmol/L downstream primer, 1.0 U Taq DNA polymerase, 20ng-40ng sample DNA, total volume 10 μL. The reaction procedure is the same as 3.4.

Fluorescence detection: Dilute the PCR product 40 times, add 1 μL diluted PCR product, 9.05 μL deionized formamide, 0.05 μL Genescan500-LIZ molecular weight internal standard (ABI company), 4000r/min to each well of the 96-well plate Centrifuge for 1 min, denature at 95°C for 5 min, place on ice for 10 min, and perform capillary electrophoresis analysis on an ABI3730 DNA analyzer. Pre-electrophoresis at 15kV for 3min; electrosampling at 2kV for 10s; electrophoresis at 15kV for 20min. At the same time, raw data were collected with Data collection software. After electrophoresis, use Genemapper4.0 software to analyze the raw data collected by Data Collection software. The software system will compare the position of the target peak with the internal standard Genescan500-LIZ in the same swimming lane to determine the accuracy of the SSR amplified fragments of different samples. Length (unit: bp). Four independent repeated experiments were carried out for each sample, and the average value of the four repeated experiments was taken and rounded up as the size of the amplified fragment of the sample at this site.

3.7, SNP marker screening

Through the relevant website (http://ricevarmap.ncpgr.cn/), 2687 SNP sites of the above functional genes were downloaded and sorted. Combined with relevant literature, the deletion effect is non-functional sites such as nonsense mutation (Synonymous), intron (INNTRON), 5-terminal untranslated region (UTR-5-PRIME), 3-terminal untranslated region (UTR-3-PRIME) Points, retain non-sense mutations (Non-Synonymous), introduce initiation codons, disrupt initiation codons, introduce stop codons, disrupt splicing sites (disrupt splice sites), etc., and the sites with frequency ≤ 0.95 finally obtained 1319 functional SNP sites. Different SNP marker sites, corresponding primer sequences, and agronomic traits are shown in Table 1 of the Summary of the Invention. In Table 1, we designed two upstream primers and one downstream primer respectively. The main function is to use the specific matching of the end bases of the primers to realize the detection of SNP typing, specifically including: 1. Two upstream primers with different end bases The allele forms a primer mix with a downstream primer, and the 5' ends of the two upstream primers are different. 2. The template is combined with the matching primers in the primer mix. 3. The two upstream primers can be used for the synthesis of the complementary chain of the allele-specific end sequence respectively. 4. Realize the classification difference, so as to achieve the purpose of identification. The 2 upstream primers and 1 downstream primer we designed are actually combined and amplified, which is a matching upstream primer and downstream primer. In addition, the unmatched upstream primer has no function, and it is actually 2 primers. Since the matching between different species and the two upstream primers is different, it is to use the characteristics and innovations of our SNP primer design. Each SNP marker site in Table 1 corresponds to two primers, wherein the numbering of the marker with the sequence number 1 corresponds to SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 in sequence, and the sequence number is 2 The numbering of the primers in the sequence table corresponds to SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, and so on, and the corresponding primers are numbered according to the sequence of the SNP marker sites, and the final sequence number is 1 The numbers of the primers in the sequence listing correspond to SEQ ID NO.370, SEQ ID NO.371 and SEQ ID NO.372 in turn. Each sequence in the sequence listing is a DNA artificial sequence. The SNP marker design of the present invention: design amplification primers within the range of 60 bp upstream and downstream of the SNP site, and design requirements for 2 upstream primers: length 14-35 bp, 3' ends are complementary to two allelic types, of which 1 The penultimate base of each primer is mutated from G/C to A/T, or from A/T to G/C; downstream primer design requirements: length 14-25bp, annealing temperature optimum 63°C, minimum 58°C, 68°C max. Finally, the two upstream primers are respectively anchored to two primers that have no homology with rice and have a length difference of at least 4bp. The anchor primers used in the present invention are: M13-1-gtaaaacgacggccagt, M13-2-cgccagggttttcccagtcacgac, the anchor primers make the length difference of the two gene amplification products at least 4bp, which is beneficial to polyacrylamide gel electrophoresis detection, and is convenient for labeling fluorescent markers , which is conducive to capillary electrophoresis detection.

4. Application of functional SNP markers in rice genetic diversity analysis

4.1 Germination of seeds: From the 245 rice samples collected, several seeds were taken from each, and germinated at 28°C for one week.

4.2 DNA extraction: the operation steps are the same as 3.2; amplification reaction system: the operation steps are the same as 3.3; PCR amplification reaction procedure: the operation steps are the same as 3.4; PCR amplification product electrophoresis detection: the operation steps are the same as 3.5.

4.6 SNP marker electrophoresis detection band data record.

4.7 Cluster analysis of rice varieties: According to all 125 SNP markers, polymorphisms were detected among 245 materials, and the detection efficiency reached 100%. Figure 1 is a cluster analysis diagram of the application of functional SNP markers in the analysis of rice genetic diversity; as can be seen from Figure 1, the cluster analysis can clearly divide these 245 materials into different groups according to the distance of genetic distance. Structural analysis showed that these 245 materials had obvious population structures, which could accurately distinguish the genotypes of the tested varieties and the genetic differences among the varieties.

5. Application of functional SNP markers in molecular marker-assisted selection breeding of rice

Rice male parent: carrying the GG allele type of the main gene (waxy gene Wx) that determines the amylose content of rice; female parent: carrying the TT allele type of Wx. Use CAPs markers and functional SNP markers to perform DNA amplification analysis on parents and hybrid offspring, and compare whether the genotypes of the two comparatively screened offspring are consistent, so as to verify the effectiveness of the application of functional SNP markers in rice molecular marker-assisted selection breeding sex.

5.1 DNA extraction: Take 4 young leaves of the male and female parents of rice and 20 young leaves of the hybrid offspring. The CTAB method was used to extract DNA, and the specific operation steps were the same as 3.2.

5.2 (1) CAPs marker amplification and enzyme digestion: first use the (GG/TT) site specificity of Wx to detect CAPS-AccI markers: upstream primer: F: 5'-gcttcacttctctgcttgtg-3', downstream primer: R: 5 '-atgatttaacgagagttgaa-3'. PCR reaction system (25 μL): 2.5 μL 10×PCR buffer, 2.0 μL MgCl2 (25 mM), 2.0 μL dNTPs (2.0 mM), 2 μL Primer-F (10 μM), 2 μL Primer-R (10 μM), 0.2 μL Taq enzyme (5u/ μL), 4 μL template DNA, 10.3 μL ddH ₂ O. (2) Amplification reaction program: 95°C for 5 min, 94°C for 40 s, 55°C for 40 s, 72°C for 60 s, 35 cycles; 72°C for 7 min. AccⅠdigestion: 10μL PCR amplification product, 1.5μl 10×digestion buffer, 5U AccI enzyme, make up the total volume to 15μL with sterile ddH ₂ O, mix well and incubate at 37.0℃ for 1-4h. (3) Electrophoresis detection: using 2% agarose gel electrophoresis detection, the GG genotype produces 403bp and 57bp fragments; the TT genotype cannot be digested, only a 460bp band; the hybrid GT genotype can be partially digested , 460bp and 403bp bands can appear at the same time. The result is shown in Figure 2. In Figure 2, lanes 1-4, 10, 17, and 24 represent samples that produce fragments of 403bp and 57bp for the GG genotype; lanes 5-8, 11, 14, 15, 26, and 27 represent samples for the TT genotype that cannot be digested by the enzyme Cut, only the 460bp band; Lanes 9, 12, 13, 16, 18, 19, 21, 22, 23, 25, 28 represent samples that are heterozygous GT genotypes and can be partially digested, and 460bp and 403bp can appear at the same time of strips. The restriction endonuclease digestion method is suitable for laboratory detection of relatively small amounts of SNPs, but all conventional experimental reagents are required, and the detection work can be completed more conveniently and quickly. According to the information of 124 polymorphic SNP markers, the genetic distance among 245 tested varieties was calculated by using NTSYS software, and the genetic relationship clustering diagram was drawn according to the UPGMA method (see Figure 1). The results show that the developed SNP markers are helpful to analyze the genetic differences of rice varieties, and the use of these molecular markers in molecular assisted breeding is a current research hotspot.

5.3 SNP marker amplification analysis: Then use the (GG/TT) site-specific marker SNP-sf0601764762 of Wx screened out to perform amplification analysis on the above individual plants. The reaction system is the same as 3.3, and the reaction procedure is the same as 3.4. Upstream primer 1: F: 5'-GTAAAACGACGGCCAGTcaggaagaacatctgcaCgt-3', Upstream primer 2: F: 5'-CGCCAGGGTTTTTCCCAGTCACGACcaggaagaacatctgcaagg-3', Downstream primer: R: 5'acgagcaatgaaagatgcatgtga3'. Electrophoresis detection: non-denaturing polyacrylamide gel electrophoresis detection, the GG genotype amplified a 114bp fragment; the TT genotype amplified a 121bp fragment; the heterozygous GT type could amplify two fragments of 114 and 121bp, the results As shown in Figure 3.

5.4 Selection and comparison of hybrid offspring: Use Caps-AccI markers to analyze the genotype of the hybrid offspring Wx, and keep the individual plants that produce fragments of 403bp and 57bp GG genotype; use SNP-sf0601764762 to analyze the above individual plants, and keep the single plants that amplify large fragments According to the amplification results of the two markers, the Caps-AccI marker is completely consistent with the Wx genotypes of the SNP-sf0601764762 amplified parent and offspring, indicating that the rice functional SNP marker can be used for authenticity identification of rice varieties.

Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention also intends to include these modifications and variations.

Claims (4)

1. a kind of SNP marker of analyzing rice genetic diversity identification of species, it is characterised in that the SNP marker is in rice Site it is as shown in table 1：

The different SNP marker sites of table 1, corresponding primer sequence

2. a kind of rice genetic diversity obtained described in claim 1, the primer of the SNP marker of identification of species, its feature exist In obtaining shown in the primer sequence table 1 corresponding to the SNP marker.

3. a kind of SNP marker of analyzing rice genetic diversity identification of species as claimed in claim 1 marks in rice molecular Application in assistant breeding.

4. a kind of rice genetic diversity obtained described in claim 1, the primer of the SNP marker of identification of species are in rice molecular Application in marker-assisted breeding.