Development and application of molecular marker related to millet viscosity

Abstract

The invention protects application of a substance for detecting polymorphism or genotype at the upstream 1218bp of the ATG of the SiUGP1 gene in a millet genome by a technical method in distinguishing or assisting in distinguishing millet viscosity and other millet variety quality. The polymorphic site is an SNP site on a SiUGP1 gene promoter in a millet genome, the nucleotide type of the polymorphic site is A or G, and the polymorphic site is the 1218th nucleotide on the upstream of the ATG of the SiUGP1 gene; in order to detect the 1218th nucleotide variation on the upstream of the ATG of the SiUGP1 gene in the millet genome, a marker site is designed, and the viscosity of millet of different millet varieties can be effectively distinguished by utilizing the marker site, so that the efficiency and the achievement of improving the cultivation of high-viscosity millet varieties are achieved.

Description

Technical Field

[0001] The present invention Belongs to the field of biotechnology, Involved A sort of Molecular markers, specifically Millet Development and application of viscosity-related molecular markers. Background Art

[0002] Millet (Setaria italica Beauv.) belongs to the subfamily Millet of the Poaceae family. It was called "millet" in ancient times. After the shell is removed, it is called millet. It was first used in China about 10,000 years ago. Taihang and Yanshan regions Start domestication cultivation , with a long history 。 Millet is considered a nutritious food with high energy output because it is rich in nutrients such as protein, fat, dietary fiber and vitamins. With the improvement of millet production technology and people's quality of life, people's consumption concepts and consumption patterns have changed, and people have put forward higher requirements for the quality of millet.

[0003] The main way to eat millet is to make millet porridge. "Delicious" and "easy to cook" are the goals pursued by consumers and breeders. The eating and cooking quality (ECQ) reflects the color, smell, taste, mouthfeel and hardness of millet after steaming. .in, The viscosity characteristic is Steaming Rapid viscosity analyzer (RVA) is an important parameter indicator reflecting viscosity characteristics. It simulates the cooking process and records the gelatinization curve caused by the change of starch viscosity in real time, namely RVA spectrum. RVA spectrum mainly includes three primary indicators: peak viscosity (PKV), hot paste viscosity (HPV), cold paste viscosity (CPV) and three secondary indicators: break down (BD), setback (SB), consistency (CSV) as well as pasting temperature (PTemp) and gelatinization time (Peak time, PTime).

[0004] In the process of millet breeding, the selection of millet varieties with high millet viscosity is important for the consumption and processing of millet. significance. However, due to The viscosity and other cooking characteristics of millet evaluate Have a certain Operational complexity ,and A certain amount of sample volume is required, making quality identification difficult, time-consuming and labor-intensive during millet breeding 。 In this situation, molecular marker-assisted selection can It plays an important role in parent selection and low-generation single plant quality detection.Rapidly improve the work efficiency and level of millet cooking and taste quality breeding, greatly promote the quality breeding process of millet, and serve the industrial production of high-quality millet varieties. Summary of the invention

[0005] The purpose of the present invention is to solve how to distinguish or assist in distinguishing Different millet Viscosity Millet varieties and breeding lists strain .

[0006] To achieve the above objectives, the present invention provides the following applications and methods.

[0007] The present invention provides a method for detecting the upstream of ATG of SiUGP1 gene in millet genome. 1218 bp polymorphism or genotype In distinguishing or assisting in distinguishing Millet viscosity of different millet varieties Applications in The polymorphic site is A SNP site on the promoter of SiUGP1 gene, whose nucleotide type is A or G, is located at the 1218th position upstream of ATG of SiUGP1 gene. nucleotide.

[0008] The present invention provides Detection of polymorphism or genotype at 1218 bp upstream of the ATG of SiUGP1 gene in foxtail millet genome The use of a substance in preparing a millet viscosity test for distinguishing or assisting in distinguishing different millet varieties; the polymorphic site is a millet base A SNP site on the promoter of the SiUGP1 gene in the genomic DNA, whose nucleotide type is A or G, is located upstream of the ATG of the SiUGP1 gene. Nucleotide position 1218.

[0009] The present invention provides Detection of polymorphism or genotype at 1218 bp upstream of the ATG of SiUGP1 gene in foxtail millet genome The polymorphic site is SiUGP1 in the millet genome. A SNP site on the gene promoter, whose nucleotide type is A or G, is the 1218th nucleotide upstream of the ATG of the SiUGP1 gene acid.

[0010] The present invention also provides a method for distinguishing or assisting in distinguishing the viscosity of millet of different millet varieties, characterized in that: The genotype of millet is detected, and the millet viscosity characteristics of different millet varieties are distinguished or assisted in distinguishing the millet genotype. The genotype is a SNP site on the promoter of the SiUGP1 gene in the millet genome, and the nucleotide type is A or G is the 1218th nucleotide upstream of ATG in SiUGP1 gene.

[0011] Another technical problem to be solved by the present invention is how to carry out millet variety of Viscosity inheritance Breeding.

[0012] In order to solve the above problems, the present invention provides the following applications and methods.

[0013] The present invention provides a method for millet breeding comprising the method described in claim 1 SiUGP1 gene in foxtail millet genome The SNP at 1218 bp upstream of ATG was used to screen the homozygous millet with the nucleotide of the SNP site being A in the millet genome. Breeding parents and selecting hybrid offspring plants or strains.

[0014] Contains the upstream ATG of SiUGP1 gene in the foxtail millet genome 1218 Products of the nucleotide category also belong to the protection scope of the present invention, and the product is any one of C1)-C3);

[0015] C1) Detection and Millet Products of viscosity-related single nucleotide polymorphisms or genotypes;

[0016] C2) Differentiation or auxiliary differentiation Different varieties of millet Viscosity of the product;

[0017] C3) used to cultivate high viscosity Millet Millet variety products.

[0018] In the above application, the above method or the above product, the millet breeding is to cultivate millet with higher viscosity after cooking. variety Or breeding millet with higher viscosity after cooking variety .

[0019] In the above application, the above method or the above product, the detection of the upstream ATG of the SiUGP1 gene 1218 bp Polymorphic or genotypic The substance is S1 / S2 or S3 as follows;

[0020] S1. Detection of the upstream of ATG of SiUGP1 gene 1218 bp Polymorphism or genotype of substances The amplicon contains the upstream ATG of the SiUGP1 gene 1218 KASP-labeled primers for foxtail millet genomic DNA fragments including bp;

[0021] S2, the detection of the upstream of SiUGP1 gene ATG 1218 bp Polymorphism or genotype of substances A PCR reagent containing the KASP labeled primer;

[0022] S3. A kit containing the KASP labeled primers described in S1 or the PCR reagents described in S2.

[0023] Preferably, the KASP labeled primer is a primer set consisting of the single-stranded DNA shown in sequence 1 in the sequence list, the single-stranded DNA shown in sequence 2 in the sequence list, and the single-stranded DNA shown in sequence 3 in the sequence list.

[0024] The beneficial effects of the present invention are as follows:

[0025] The invention discloses a method for regulating the viscosity of millet by regulating the SiUGP1 gene, a key gene of millet viscosity. Haplotype analyze And further related to different Viscosity characteristics of millet varieties , designed a method that can effectively distinguish High viscosity millet varieties and low viscosity millet varieties Marker site, which can be used to Select low-generation breeding and achieve selection through marker selection Viscosity Higher The purpose of individual plants, strains and varieties, improve Millet High viscosity Quality breeding efficiency and results. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Schematic diagram of haplotype analysis of the promoter region of the SiUGP1 gene of the present invention , where the negative signs in the table are The numbers represent the position of the variant site upstream of ATG

[0027] Figure 2 Schematic diagram of the association analysis between the SiUGP1 promoter region haplotype and quality phenotype of the present invention;

[0028] Figure 3 Schematic diagram of the development and identification of the millet SiUGP1 gene KASP marker of the present invention , where each dot has a pair A test material should be used. Red dots indicate that the test material is homozygous genotype G:G, and blue dots indicate homozygous genotype A:A. Green indicates heterozygous genotype G:A, pink indicates no signal or weak signal, and black indicates blank control .

[0029] Figure 4 The figure is a schematic diagram of the promoter sequence of the SiUGP1 gene in the present invention. DETAILED DESCRIPTION

[0030] The specific embodiments of the present invention are further described below. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the protection scope of the present invention.

[0031] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0032] Example 1 Determination of viscosity characteristics of millet and discovery of associated SNP markers

[0033] The viscosity characteristics of different millet varieties were determined by rapid viscosity analyzer, including material collection, sample grinding, 653 millet varieties from different ecological regions across the country were selected and harvested after maturity. The laboratory ridger and laboratory rice mill were used for two hulling and two rice milling processes, and the millet grains were subsequently whirled using a cyclone. Grind to powder, and pass millet powder through a 60-mesh sieve for viscosity measurement. Accurately weigh 3.0 g of the millet powder (water content 12%) and 25 mL of distilled water were placed in an aluminum can dedicated to the instrument and placed on the preheated RVA instrument. Program: 50℃ for 1min, heat to 95℃ at a rate of 12℃ / min, and keep at 95℃ for 2.5min, then heat at the same rate Reduce to 50℃ and keep at this temperature for 2min. Record the peak viscosity (PKV), hot slurry viscosity (HPV), cold slurry viscosity (CPV), Disintegration value (BD = PKV-HPV), recovery value (CSV = CPV-HPV), reduction value (SB = CPV-PKV), peak time (PTime) and peak temperature (PTemp)

[0034] about Millet Viscosity key gene SiUGP1 Positioning. Viscosity indicators PKV, HPV and CPV of RVA characteristics are fully Genome association analysis revealed that the SiUGP1 gene is the major gene regulating the viscosity characteristics of millet. The SiUGP1 gene encodes UDP-glucose pyrophosphorylase (UGPase). UGPase is a carbohydrate Key enzymes of metabolism,Its main function is to catalyze the formation of UDPG in photosynthetic tissues for the synthesis of sucrose, while in non-photosynthetic tissues such as seeds, UGPase degrades UDPG to provide the carbon skeleton glucose-1-phosphate (Glc-1-P) for the metabolic process. 。

[0035] Sample of this study Including 801 cultivars (299 cultivated varieties, 502 farmer varieties) and 348 wild Setaria Genomic sequence variation analysis revealed that SiUGP1 There are 15 SNPs and 5 Indel / Insert mutation sites in the 1900bp promoter region upstream of the start codon ATG. There are 20 variant sites in total. The 1149 materials were divided into 8 haplotypes, among which Hap1 had the largest number of materials, with 425 materials; Hap2, Hap3 and Hap4 contained 175, 198 and 280 varieties respectively; Hap5, Hap6, Hap7 and Hap8 had fewer varieties. ， For rare variants 。 Further analysis of the proportion of each haplotype in cultivated varieties, farm varieties and wild species (Setaria viridis) revealed that, except for 15 materials in Hap4 that were farm varieties, the haplotypes of Hap4, Hap5, Hap6, Hap7 and Hap8 only existed in wild species and were haplotypes unique to wild species, while Hap1, Hap2 and Hap3 were mainly cultivated variety haplotypes and mainly existed in modern cultivated varieties and farm varieties. . The mutation site is Figure 1 .

[0036] Analysis of the differences in haplotype variation revealed that there was only one base difference between the Hap1 haplotype and the Hap2 haplotype (marked in red, Chromosome 2, position 39,476,236bp ), It is located 1218 bp upstream of the ATG of the SiUGP1 gene. Hap1 is located upstream of the ATG of the SiUGP1 gene 1218 bp is A base, while Hap2 is 1218 bp upstream of the ATG of SiUGP1 gene The other six haplotypes (Hap3- Hap 8) This site is consistent with Hap2.

[0037] Figure 1 middle:

[0038] A, Mutation information and haplotype analysis of 1900 bp upstream of ATG of SiUGP1 gene;

[0039] B , Analysis of the proportion of each haplotype in cultivated varieties, farm varieties and wild species;

[0040] The numbers with negative signs in the table represent the positions of the variant sites upstream of ATG, and the right side of the table shows the number of cultivated varieties, farm varieties and wild species in each haplotype.

[0041] Specifically, we will Viscosity characteristics of millet from different millet varieties Haplotype Classification Make an association, Related Specifically, SPSS software was used to analyze the viscosity phenotype characteristics of the haplotype grouped materials according to each variation site. The t-test was used for significant analysis and the differences in viscosity phenotypes between haplotypes were detected to analyze molecular variation. Correlation analysis between site and viscosity phenotype.

[0042] According to the statistical results, the peak viscosity PKV, hot pulp viscosity HPV and cold pulp viscosity of Hap1 millet varieties are The average CPV levels of Hap2 millet varieties were 1019.3 cP, 836.6 cP and 1823.9 cP, respectively. The average levels of PKV, hot slurry viscosity HPV and cold slurry viscosity CPV were 938.7cP, 755.6cP and 1710.8cP respectively. The peak viscosity PKV, hot slurry viscosity HPV and cold slurry viscosity CPV of millet in type Hap2 were significantly higher than those in type Hap2 ( Figure 2 AC) ; this outside, Hap1 Types of Grains The gelatinization time PTime of Hap2 and Hap3 types was significantly higher than that of Hap2 and Hap3 types ( Figure 2 D); Hap1 type The apparent amylose content of Hap3 was also significantly lower than that of Hap3 ( Figure 2 E); Hap1 Type Xiaomi The adhesion and cohesion of the Hap3 type were also significantly higher than those of the Hap3 type ( Figure 2 FG); The protein content of PC showed significant differences between Hap1 and Hap2. Type Millet Variety The protein content of Figure 2 H) 。 above result illustrate, Compared with Hap2 type, According to the phenotypic association, Hap1 is the superior haplotype of SiUGP1 in terms of quality phenotype, showing the characteristics of high viscosity, low AAC and low protein content. , these characteristics It is the characteristic of the taste quality of high-quality millet .

[0043] Figure 2 The significance was analyzed by t test, *P<0.05, **P<0.01, ***P<0.001.

[0044] Combined with the above Results Hap1 and Hap2 type between Viscosity characteristic Differences in mutation sites , it is believed that the ATG upstream of the SiUGP1 gene promoter region 1218 A / G single base variation at bp position Highly correlated with the viscosity characteristics of millet, this base variation can able Is the impact Millet viscosity of millet varieties key functional sites.

[0045] Example 2 Millet Development of the Viscosity KASP Marker

[0046] 1. Primer Design

[0047] According to the haplotype analysis results of the SiUGP1 gene promoter region, 1218 bp upstream of the SiUGP1 gene ATG The A / G mutation site at the position is of great significance for distinguishing the viscosity characteristics of millet. When the base here is A, the viscosity of millet is relatively High, when the base here is G, the viscosity of millet is low. The polymorphism of this functional site was used to design KASP markers for distinguishing Millet of Viscosity Characteristics, three primers were designed, including a universal primer COM and two competitive primers FAM and VIC, of ​​which the competitive primers carried fluorescent probes of different colors. The fluorescent signal can be detected by an enzyme-labeled PCR instrument or a fluorescent quantitative PCR instrument. The 3' end of the FAM primer carries an A base, and the 3' end of the VIC primer carries a G base. If the detection site is A, the FAM primer will bind there and amplify a fluorescent product. If the detection site is G, the VIC primer will bind there and amplify a product with another fluorescent color. Primer information is shown in Table 1.

[0048] surface 1 : KASP marker primer sequence

[0049]

[0050]

[0051] 2. KASP marker detection SNP site

[0052] After the primers were synthesized, 139 millet varieties were randomly selected for KASP typing to test the practicality of the primers.

[0053] Specifically, the detection reaction system was prepared by mixing 12 μL of each specific primer, 30 μL of universal primer and 46 μL of ddH2O. Then, 2.5 μL DNA and 2.5 μL HiGeno2×ProbeMixA (Beijing Jiacheng Biotechnology Co., Ltd.) were added to each reaction system. Technology Co., Ltd.), 0.07 μL of mixed primers were used for PCR. The PCR program was set as follows: 95°C for 10 min; 95°C for 30 s, 65-55°C for 25 s, 10 cycles (Touchdown reaction program decreases 1.0℃ per cycle); 95℃30s, 55℃30s, 35 cycles. Fluorescence scanning should be performed in an ELISA reader, and the genotyping results should be displayed using SNPviewer software. If the fluorescence signal is not shown, the genotype of the test material at 1218 bp upstream of the ATG of the SiUGP1 gene is A:A; if only VIC The fluorescence signal of the gene, the genotype of the detection material at 1218bp upstream of the ATG of the SiUGP1 gene is G:G; if If the fluorescent signal of the FAM gene and the VIC gene are displayed, the detection material is upstream of the ATG of the SiUGP1 gene. The genotype at 1218 bp was A:G.

[0054] The classification results are as follows Figure 3 As shown, the KASP marker can be effectively typed, and 73 of the 139 materials were detected as A : Genotype A (blue dots), 60 copies Material testing is G :G genotype (red dots), 2 copies Material testing is G:A heterozygous base Cause type (green dots), 3 copies Material The signal is weak (pink dot), and 1 material cannot be judged (purple dot) ( Figure 3 A). Further the above The test results were compared with the resequencing results, and all 73 A genotype materials and 60 G genotype materials were correct. The accuracy of KASP labeling reached 95.6%.

[0055] 3. KASP classification and phenotype association

[0056] right AboveThe material was classified into two types: homozygous A:A and homozygous G:G Viscosity Features Relationship analyze, Related Specifically, the EXECL software was used to compare the viscosity phenotype characteristics of the materials separated by KASP labeling with double t test. Are the viscosity characteristics of the two types of homozygous A:A and homozygous G:G significant? The results show that: : The amylose content of type A materials was significantly lower than that of type G : G type, A : The peak viscosity, hot slurry viscosity and cold slurry viscosity of type A material are significantly higher than those of type G. : G Type( Figure 3 BE) The test results show that the success Developed a startup for SiUGP1 Nucleotide 1218 of subregion of Can distinguish the viscosity characteristics of millet KASP mark, When the test result of the tested variety is base A, it has a higher viscosity. When the test result of the tested variety is G, it has a lower viscosity. This marker is used as a molecular assisted selection Parent selection in breeding, hybrid It is very practical in the selection of single plants in the segregation generation of hybrid offspring and the identification of stable lines, and can be used in Identification of millet varieties at the seedling stage The viscosity of millet produced by each plant or row Features, select the target type according to your needs, Significant Accelerate the quality breeding process of millet.

[0057] Figure 3 It should be noted that:

[0058] A: KASP marker typing diagram of 139 materials, each dot corresponds to one test material;

[0059] Red dots indicate that the test material is homozygous genotype G:G, blue dots indicate homozygous genotype A:A, green indicates heterozygous genotype G:A, pink indicates no signal or weak signal, and black indicates blank control;

[0060] B- E, represents AAC, PKV, HPV and CPV of two types of materials identified by KASP labeling; n=73 (A:A), n=60 (G:G).

[0061] It should be noted that if Figure 4 As shown, the bold position in the last line of the sequence is the start codon;

[0062] At the same time, the bold position in the middle line of the sequence is the 1218bp upstream of the ATG of the SiUGP1 gene.

[0063] Finally, it should be noted that in the description of the present invention, it should be noted that the terms "vertical", "up", "down", "horizontal", etc. indicate orientations or positional relationships based on the orientations or positional relationships shown in the accompanying drawings. They are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation. Therefore, they cannot be understood as limitations on the present invention.

[0064] In the description of the present invention, it is also necessary to explain that, unless otherwise clearly stipulated and limited, the terms "set", "installed", "connected" and "connected" should be understood in a broad sense. For example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium, or it can be the internal connection of two elements. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to the specific circumstances.

[0065] The above content is a further detailed description of the present invention in combination with specific implementation methods. It cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, several simple deductions or substitutions can be made without departing from the concept of the present invention, which should be regarded as belonging to the protection scope determined by the claims submitted for the present invention.

Claims

1. The substance detecting the polymorphism or genotype at 1218 bp upstream of ATG of SiUGP1 gene in millet genome is used to distinguish or The polymorphic site is the promoter of the SiUGP1 gene in the millet genome. A SNP site on the promoter, whose nucleotide type is A or G, is the 1218th nucleotide upstream of the ATG of the SiUGP1 gene 。 2. The material for detecting the polymorphism or genotype at 1218 bp upstream of the ATG of the SiUGP1 gene in the millet genome is prepared in the The polymorphic site is the SiUGP1 gene in the millet genome. A SNP site on the promoter, whose nucleotide type is A or G, is the 1218th nucleotide upstream of the ATG of the SiUGP1 gene. 。 3. Detection of polymorphism or genotype of the 1218 bp upstream of the ATG of SiUGP1 gene in millet genome The invention relates to an application in planting or preparing millet breeding products; the polymorphic site is a polymorphic site on the promoter of the SiUGP1 gene in the millet genome. A SNP site, whose nucleotide type is A or G, is the 1218th nucleotide upstream of ATG of SiUGP1 gene.

4. A method for distinguishing or assisting in distinguishing the viscosity of millet of different millet varieties, characterized in that: Including testing the genes of millet The millet viscosity characteristics of different millet varieties are distinguished or assisted in distinguishing the millet viscosity characteristics of different millet varieties according to the millet genotype, wherein the genotype is A SNP site on the promoter of SiUGP1 gene in foxtail millet genome, whose nucleotide type is A or G, is The 1218th nucleotide upstream of ATG.

5. Claims 4 The invention relates to an application of the method in millet breeding.

6. A method for millet breeding, characterized in that: The method comprises the upstream of ATG of SiUGP1 gene in the millet genome according to claim 1 1218 bp SNPs, screening the SNP sites in the millet genome Homozygous foxtail millet with nucleotide A ， As Millet breeding Parent Selection and hybrid progeny selection markers Carry out breeding.

7. Contains Detection ATG upstream of SiUGP1 gene in foxtail millet genome 1218 bp Polymorphism or genotype The product is characterized by: The product is any one of products C1)-C3): C1) Detection and Millet Products of viscosity-related single nucleotide polymorphisms or genotypes; C2) Differentiation or auxiliary differentiation Different varieties of millet Viscosity of the product; C3) used to cultivate high viscosity Millet Millet variety products.

8. The use according to any one of claims 1 to 3 and 5, the method according to claim 4 or 6 or the product according to claim 7, characterized in that: The millet breeding is to cultivate millet with higher viscosity after cooking variety Or breeding millet with higher viscosity after cooking variety .

9. The use according to any one of claims 1 to 3 and 5, the method according to claim 4 or 6 or the product according to claim 7, characterized in that: Detection of the detection of the SiUGP1 gene ATG upstream 1218 bp Polymorphic or genotypic The substance is S1 / S2 or S3 as follows; S1. Detection of the upstream of ATG of SiUGP1 gene 1218 bp Polymorphism or genotype of substances The amplicon contained the ATG upstream of the SiUGP1 gene 1218 KASP-labeled primers for foxtail millet genomic DNA fragments including bp; S2, the detection of the upstream of SiUGP1 gene ATG 1218 bp Polymorphism or genotype of substances A PCR reagent containing the KASP labeled primer; S3. A kit containing the KASP labeled primers described in S1 or the PCR reagents described in S2.

10. The use, method or product according to claim 9, characterized in that: The KASP labeled primer is a primer set consisting of the single-stranded DNA shown in sequence 1 in the sequence list, the single-stranded DNA shown in sequence 2 in the sequence list, and the single-stranded DNA shown in sequence 3 in the sequence list; they are as follows: The sequence corresponding to the KASP marker primer is as follows: Primer: FAM, sequence: GGCGAGCGCATGGAGCTGA; Primer: VIC, sequence: GCGAGCGCATGGAGCTGG Primer: COM, sequence: TGAGAAAGCCCTTCAGCCAAGCAAA.

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