Development and Application of Molecular Markers Related to the Viscosity of Millet

Abstract

The present invention protects the application of a substance for detecting polymorphisms or genotypes at 1218 bp upstream of the ATG of the SiUGP1 gene in the foxtail millet genome in distinguishing or assisting in distinguishing the quality of foxtail millet varieties such as the viscosity of millet; the polymorphic locus is an SNP locus on the promoter of the SiUGP1 gene in the foxtail millet genome, and its nucleotide type is A or G, which is the 1218th nucleotide upstream of the ATG of the SiUGP1 gene; in order to detect the variation of the 1218th nucleotide upstream of the ATG of the SiUGP1 gene in the foxtail millet genome, the present invention designs a marker locus, and using this marker locus can effectively distinguish the viscosities of different foxtail millet varieties of millet, so as to improve the efficiency and results of cultivating foxtail millet varieties with high viscosity.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to a molecular marker, specifically the development and application of a molecular marker related to the viscosity of millet. Background Art

[0002] Foxtail millet (Setaria italica Beauv.) belongs to the Panicoideae subfamily of the Poaceae family. It was called "millet" in ancient times and is millet after hulling. It began to be domesticated and cultivated in the Taihang Mountains and Yanshan Mountains regions of China about 10,000 years ago and has 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 foxtail 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 foxtail millet.

[0003] The main way of eating foxtail millet is to make millet porridge. "Tasty" and "easy to cook" are the goals pursued by consumers and breeders. The eating and cooking quality (ECQ) reflects the color, smell, taste, texture, and hardness of millet after cooking. Among them, the viscosity characteristic is an important feature of the eating and cooking quality. The Rapid Viscosity Analyzer (RVA) is an important parameter index reflecting the viscosity characteristic. It simulates the cooking process and records in real-time the gelatinization curve presented due to the change in the viscosity of starch, that is, the RVA spectrum. The RVA spectrum mainly includes 3 primary indexes: Peak viscosity (PKV), Hot paste viscosity (HPV), Cool paste viscosity (CPV) and 3 secondary indexes: Break down (BD), Setback (SB), Consistence (CSV) as well as Pasting temperature (PTemp) and Peak time (PTime).

[0004] In the process of foxtail millet breeding, selecting foxtail millet varieties with high millet viscosity is of great significance for the eating and processing of foxtail millet. However, due to the certain operational complexity in the evaluation of cooking characteristics such as the viscosity of millet and the need for a certain amount of sample volume, it is difficult and time-consuming to identify the quality in the process of foxtail millet breeding. In the face of this situation, molecular marker-assisted selection will play an important role in parental selection and quality detection of low-generation individual plants, rapidly improving the work efficiency and level of foxtail millet eating and cooking quality breeding, greatly promoting the quality breeding process of foxtail millet, and serving the industrial production of high-quality foxtail millet varieties. Summary of the Invention

[0005] The object of the present invention is to solve the problem of how to distinguish or assist in distinguishing foxtail millet varieties and breeding single plants with different millet viscosities.

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

[0007] The present invention provides the application of detecting the polymorphism or genotype at 1218 bp upstream of the ATG of the Si UGP1 gene in the foxtail millet genome in distinguishing or assisting in distinguishing the millet viscosities of different foxtail millet varieties; the polymorphic locus is an SNP locus on the promoter of the SiUGP1 gene in the foxtail millet genome, and its nucleotide type is A or G, which is the 1218th nucleotide upstream of the ATG of the Si UGP1 gene.

[0008] The present invention provides the application of a substance for detecting the polymorphism or genotype at 1218 bp upstream of the ATG of the Si UGP1 gene in the foxtail millet genome in preparing a product for distinguishing or assisting in distinguishing the millet viscosities of different foxtail millet varieties; the polymorphic locus is an SNP locus on the promoter of the Si UGP1 gene in the foxtail millet genome, and its nucleotide type is A or G, which is the 1218th nucleotide upstream of the ATG of the Si UGP1 gene.

[0009] The present invention provides the application of a substance for detecting the polymorphism or genotype at 1218 bp upstream of the ATG of the Si UGP1 gene in the foxtail millet genome in foxtail millet breeding or preparing foxtail millet breeding products. The polymorphic locus is an SNP locus on the promoter of the SiUGP1 gene in the foxtail millet genome, and its nucleotide type is A or G, which is the 1218th nucleotide upstream of the ATG of the SiUGP1 gene.

[0010] The present invention also provides a method for distinguishing or assisting in distinguishing the millet viscosities of different foxtail millet varieties, which is characterized in that: it includes detecting the genotype of the foxtail millet, and using the genotype of the foxtail millet to distinguish or assist in distinguishing the millet viscosity characteristics of different foxtail millet varieties, and the genotype is an SNP locus on the promoter of the Si UGP1 gene in the foxtail millet genome, and its nucleotide type is A or G, which is the 1218th nucleotide upstream of the ATG of the Si UGP1 gene.

[0011] Another technical problem to be solved by the present invention is how to carry out viscosity genetic breeding of foxtail millet varieties.

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

[0013] The present invention provides a method for foxtail millet breeding, which includes the SNP at 1218 bp upstream of the ATG of the SiUGP1 gene in the foxtail millet genome as claimed in claim 1. Select the homozygous foxtail millet with the nucleotide at the SNP locus in the foxtail millet genome being the A base as a parent for breeding, and select individual plants or lines of the hybrid offspring.

[0014] Products containing substances for detecting the nucleotide category at the 1218th position upstream of the ATG of the SiUGP1 gene in the foxtail millet genome also fall within the protection scope of the present invention, and the products are any one of C1)-C3);

[0015] C1) Products for detecting single nucleotide polymorphisms or genotypes related to the viscosity of millet;

[0016] C2) Products for differentiating or assisting in differentiating the viscosity of different varieties of millet;

[0017] C3) Products for cultivating foxtail millet varieties with high viscosity.

[0018] In the above applications, methods or products, the foxtail millet breeding is to cultivate foxtail millet varieties with higher viscosity after cooking or select foxtail millet varieties with higher viscosity after cooking.

[0019] In the above applications, methods or products, the substances for detecting the polymorphism or genotype of the 1218 bp upstream of the ATG of the SiUGP1 gene are as follows S1 / S2 or S3;

[0020] S1. The substance for detecting the polymorphism or genotype of the 1218 bp upstream of the ATG of the SiUGP1 gene contains KASP marker primers for amplifying the foxtail millet genomic DNA fragment including the 1218 bp upstream of the ATG of the SiUGP1 gene;

[0021] S2. The substance for detecting the polymorphism or genotype of the 1218 bp upstream of the ATG of the SiUGP1 gene is a PCR reagent containing the KASP marker primers;

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

[0023] Preferably, the KASP marker primers are a primer group composed of the single-stranded DNA shown in sequence 1 in the sequence listing, the single-stranded DNA shown in sequence 2 in the sequence listing, and the single-stranded DNA shown in sequence 3 in the sequence listing

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

[0025] The present invention designs a marker locus capable of effectively distinguishing high-viscosity millet varieties from low-viscosity millet varieties by performing haplotype analysis on the key gene SiUGP1 for millet viscosity and further correlating the viscosity characteristics of different millet varieties. By using this marker locus, selection can be carried out in the early generations of millet breeding, so as to achieve the purpose of selecting individual plants, lines and varieties with higher viscosity through marker selection, and improve the efficiency and results of high-viscosity quality breeding of millet. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is a schematic diagram of haplotype analysis of the promoter region of the SiUGP1 gene of the present invention. Among them, the numbers with negative signs in the table represent the positions of the mutation sites upstream of ATG.

[0027] Figure 2 It is a schematic diagram of the correlation analysis between the haplotype of the SiUGP1 promoter region of the present invention and the quality phenotype;

[0028] Figure 3 It is a schematic diagram of the development and identification of the KASP marker of the SiUGP1 gene of millet of the present invention. Among them, each dot corresponds to a test material. The red dots indicate that the test material is a homozygous genotype G:G, the blue dots indicate a homozygous genotype A:A, the green indicates a heterozygous genotype G:A, the pink indicates no signal or weak signal, and the black indicates a blank control.

[0029] Figure 4 It is a schematic diagram of the promoter sequence of the SiUGP1 gene in the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0030] The following further describes the specific embodiments of the present invention. It should be noted here that the description of these embodiments is for helping to understand the present invention, but does not constitute a limitation to 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. Those skilled in the art should understand that the details and forms of the technical solutions 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 embodiments are all conventional methods unless otherwise specified. The test materials used in the following embodiments can be obtained through conventional commercial channels unless otherwise specified.

[0032] Example 1

[0033] Determination of the Viscosity Characteristics of Millet and Discovery of Associated SNP Markers

[0034] The viscosity characteristics of millet from different foxtail millet varieties were determined by a rapid visco analyzer, including material collection, sample grinding, and measurement. 653 foxtail millet varieties from different ecological regions across the country were selected. After each variety matured, it was harvested and naturally air-dried. Then, it underwent two processes of hulling and two processes of milling using a laboratory hulling machine and a laboratory rice milling machine. Subsequently, the millet grains were ground into powder using a cyclone mill. The millet flour passed through a 60-mesh sieve and was used to measure the viscosity characteristics. Exactly 3.0 g of the above millet flour (with a moisture content of 12%) and 25 mL of distilled water were placed in a special aluminum can of the instrument and placed on a preheated RVA instrument. A heating and cooling program was adopted: 50 °C for 1 min, heated to 95 °C at a rate of 12 °C / min, and maintained at 95 °C for 2.5 min, then reduced to 50 °C at the same rate and maintained at this temperature for 2 min. Record the peak viscosity (PKV), hot paste viscosity (HPV), cold paste viscosity (CPV), breakdown value (BD = PKV - HPV), setback value (CSV = CPV - HPV), breakdown reduction value (SB = CPV - PKV), peak time (PTime), and peak temperature (PTemp).

[0035] Mapping of the key gene SiUGP1 for foxtail millet viscosity. Through genome-wide association analysis of the viscosity indexes PKV, HPV, and CPV of the RVA characteristics, it was found that the SiUGP1 gene is the major gene regulating the viscosity characteristics of foxtail millet. The SiUGP1 gene encodes uridine diphosphate glucose pyrophosphorylase (UDP-glucosepyrophosphorylase, UGPase). UGPase is a key enzyme in carbohydrate metabolism. Its main function is to catalyze the formation of UDPG in photosynthetic tissues for sucrose synthesis, while in non-photosynthetic tissues such as seeds, UGPase degrades UDPG to provide the carbon skeleton glucose-1-phosphate (Glc-1-P) for metabolic processes.

[0036] The samples in this study included 801 cultivated species (299 improved varieties and 502 landraces) and 348 wild green foxtails. Genome sequence variation analysis found 20 variation sites, including 15 SNPs and 5 Indel / Insert variation sites, in the 1900 bp promoter region upstream of the start codon ATG of SiUGP1. These sites divided 1149 materials into 8 haplotypes. Among them, the number of materials in Hap1 was the largest, with 425 materials; Hap2, Hap3, and Hap4 contained 175, 198, and 280 varieties respectively; the number of varieties in Hap5, Hap6, Hap7, and Hap8 was small, being rare variations. Further analysis of the proportions of each haplotype in improved varieties, landraces, and wild species (green foxtails) found that except for 15 materials in Hap4 being landraces, Hap4, Hap5, Hap6, Hap7, and Hap8 haplotypes 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 improved varieties and landraces. Variation sites such as Figure 1 。

[0037] Analyzing the differences in variation among haplotypes, it was found that there was only a one-base difference between Hap1 haplotype and Hap2 (the marked red position, at 39,476,236 bp on chromosome 2), which was located 1218 bp upstream of the ATG of the SiUGP1 gene. Hap1 was A base at 1218 bp upstream of the ATG of the SiUGP1 gene, while Hap2 was G base at 1218 bp upstream of the ATG of the SiUGP1 gene, and the other 6 haplotypes (Hap3 - Hap8) were consistent with Hap2 at this site.

[0038] Figure 1 In:

[0039] A, Variation information of 1900 bp upstream of the ATG of the SiUGP1 gene and haplotype analysis;

[0040] B, Analysis of the proportions of each haplotype in improved varieties, landraces, and wild species;

[0041] The numbers with negative signs in the table represent the positions of the variation sites upstream of the ATG, and on the right side of the table are the numbers of improved varieties, landraces, and wild species in each haplotype.

[0042] Specifically, we correlated the viscosity characteristics of the millet of different foxtail millet varieties with the haplotype typing. The specific process of the correlation was to use SPSS software to perform multiple comparisons on the viscosity phenotypic characteristics of the materials grouped by haplotypes according to each variation site, and the significance analysis used the t - test to detect the differences in the corresponding viscosity phenotypes among each haplotype, and to conduct a correlation analysis between the molecular variation sites and the viscosity phenotypes.

[0043] According to the statistical results, the average levels of peak viscosity (PKV), hot paste viscosity (HPV), and cold paste viscosity (CPV) of millet from Hap1-type foxtail millet varieties were 1019.3 cP, 836.6 cP, and 1823.9 cP, respectively, while those of Hap2-type foxtail millet varieties were 938.7 cP, 755.6 cP, and 1710.8 cP, respectively. The PKV, HPV, and CPV of millet in the Hap1 type were all significantly higher than those in the Hap2 type ( Figure 2 A-C); in addition, the pasting time (PTime) of Hap1-type foxtail millet was significantly higher than that of Hap2 and Hap3 types ( Figure 2 D); the apparent amylose content of the Hap1 type was also significantly lower than that of Hap3 ( Figure 2 E); the adhesiveness and cohesiveness of Hap1-type millet were also significantly higher than those of Hap3 type ( Figure 2 F-G); there was a significant phenotypic difference in protein content (PC) between Hap1 and Hap2, and the protein content of Hap1-type foxtail millet varieties was significantly lower than that of Hap2 ( Figure 2 H). The above results indicate that, compared with the Hap2 type, Hap1 is an excellent haplotype of SiUGP1 in terms of quality phenotypes, showing characteristics of high viscosity, low AAC, and low protein content, which are exactly the characteristics of high-quality millet taste quality.

[0044] Figure 2 In the analysis, t-test was used for significance analysis, *P<0.05, **P<0.01, ***P<0.001.

[0045] Combined with the differences in viscosity characteristics and variation sites between Hap1 and Hap2 types in the above results, it is considered that the A / G single-base variation at the position 1218 bp upstream of ATG in the promoter region of the SiUGP1 gene is highly correlated with the viscosity characteristics of millet, and this base variation may be the key functional site affecting the millet viscosity of foxtail millet varieties.

[0046] Example 2 Development of KASP markers for millet viscosity

[0047] 1. Primer design

[0048] According to the haplotype analysis results of the SiUGP1 gene promoter region, the A / G variant site 1218 bp upstream of the ATG of the SiUGP1 gene is of great significance for distinguishing the viscosity characteristics of millet. When the base here is A, the viscosity of millet is higher, and when the base here is G, the viscosity of millet is lower. Using the polymorphism of this functional site, KASP markers were designed to distinguish the viscosity characteristics of millet. Three primers were designed, including a common primer COM and two competitive primers FAM and VIC. The competitive primers carried fluorescent probes of different colors, and the fluorescence signals could be detected by a microplate reader or a real-time fluorescence quantitative PCR instrument. The 3' end of the FAM primer carried the A base, and the 3' end of the VIC primer carried the G base. If the detected site is A, the FAM primer will bind here and amplify a fluorescent product. If the detected site is G, the VIC primer will bind here and amplify a product with another fluorescent color. The primer information is shown in Table 1.

[0049] Table 1: Primer sequences of KASP markers

[0050]

[0051] 1. Detection of SNP sites by KASP markers

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

[0053] Specifically, the detection reaction system was prepared by mixing 12 μL of each specific primer, 30 μL of the common primer, and 46 μL of ddH2O to form a primer system. Then, 2.5 μL of DNA, 2.5 μL of HiGeno2×ProbeMixA (Beijing Jiacheng Biotechnology Co., Ltd.), and 0.07 μL of the mixed primer were added to each reaction system 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 (the touchdown reaction program decreases by 1.0 °C per cycle); 95 °C for 30 s, 55 °C for 30 s, 35 cycles. The above PCR reaction was subjected to fluorescence scanning in a microplate reader, and the genotyping results were displayed by SNPviewer software. If only the fluorescence signal of the FAM gene is shown, the genotype of the test material at 1218 bp upstream of the ATG of the SiUGP1 gene is A:A; if only the fluorescence signal of the VIC gene is shown, the genotype of the test material at 1218 bp upstream of the ATG of the SiUGP1 gene is G:G; if both the fluorescence signal of the FAM gene and the fluorescence signal of the VIC gene are shown, the genotype of the test material at 1218 bp upstream of the ATG of the SiUGP1 gene is A:G.

[0054] The genotyping results are as Figure 3As shown in the figure, the KASP marker can be effectively genotyped. Among the 139 materials, 73 materials were detected as A:A genotype (blue dots), 60 materials were detected as G:G genotype (red dots), 2 materials were detected as G:A heterozygous genotype (green dots), 3 materials had weak signals (pink dots), and 1 material could not be judged (purple dots). Figure 3 A). Further, the above test results were compared with the resequencing results. All 73 A-genotype materials and 60 G-genotype materials were correct, and the accuracy rate of this KASP marker reached 95.6%.

[0055] 3. KASP Genotyping Associated with Phenotype

[0056] An association analysis was performed on the viscosity characteristics of the materials genotyped as homozygous A:A and homozygous G:G. Specifically, during the association process, the EXCEL software was used to conduct a two-sample t-test on the viscosity phenotypic characteristics of the materials separated by the KASP marker to test whether the viscosity characteristics of the two types of homozygous A:A and homozygous G:G were significant. The results showed that: the amylose content of the A:A type materials was extremely significantly lower than that of the G:G type, and the peak viscosity, hot paste viscosity, and cold paste viscosity of the A:A type materials were extremely significantly higher than those of the G:G type( Figure 3 B-E). The test results indicated that a KASP marker targeting the 1218th nucleotide in the promoter region of SiUGP1 for distinguishing the viscosity characteristics of millet was successfully developed. When the test result of the variety to be tested is the A base, it has a higher viscosity, and when the test result is G, it has a lower viscosity. This marker has strong practicability in parental selection in breeding, individual plant selection in the segregating generations of hybrid offspring, and line identification of stable lines as a molecular assistant selection. Moreover, it can identify the viscosity characteristics of the millet produced by millet varieties (individual plants, lines) at the seedling stage, select the target type according to requirements, and significantly accelerate the quality breeding process of millet.

[0057] Figure 3 It should be noted that

[0058] A: KASP marker genotyping map of 139 materials, each dot corresponding to a test material; the red dot indicates that the test material is homozygous genotype G:G, the blue dot indicates homozygous genotype A:A, the green indicates heterozygous genotype G:A, the pink indicates no signal or weak signal, and the black indicates the blank control; B-E indicate the AAC, PKV, HPV, and CPV of the two types of materials identified by the KASP marker respectively;

[0059] n = 73 (A:A), n = 60 (G:G).

[0060] It should be noted that, such as Figure 4As shown, the bold position in the last line of the sequence is the start codon; at the same time, the bold position in the middle line of the sequence is the position of the 1218th bp upstream of ATG of the SiUGP1 gene.

[0061] The above content further elaborates on the present invention in combination with specific embodiments. It cannot be determined that the specific implementation of the present invention is only limited to these descriptions. For those of ordinary skill in the technical field to which the present invention pertains, without departing from the concept of the present invention, several simple deductions or substitutions can still be made, which should all be regarded as falling within the protection scope determined by the claims submitted for the present invention.

Claims

1. Use of a substance for detecting the polymorphism or genotype at 1218 bp upstream of the ATG of the SiUGP1 gene in the foxtail millet genome in differentiating or assisting in differentiating the viscosity of millet of different foxtail millet varieties; the position at 1218 bp upstream of the ATG of the SiUGP1 gene is a SNP site on the promoter of the SiUGP1 gene in the foxtail millet genome, the nucleotide type thereof is A or G, and it is the 1218th nucleotide upstream of the ATG of the SiUGP1 gene; the promoter sequence of the SiUGP1 gene is as shown in Sequence 4 in the sequence listing.

2. Use of a substance for detecting the polymorphism or genotype at 1218 bp upstream of the ATG of the SiUGP1 gene in the foxtail millet genome in preparing a product for differentiating or assisting in differentiating the viscosity of millet of different foxtail millet varieties; the position at 1218 bp upstream of the ATG of the SiUGP1 gene is a SNP site on the promoter of the SiUGP1 gene in the foxtail millet genome, the nucleotide type thereof is A or G, and it is the 1218th nucleotide upstream of the ATG of the SiUGP1 gene; the promoter sequence of the SiUGP1 gene is as shown in Sequence 4 in the sequence listing.

3. Use of a substance for detecting the polymorphism or genotype at 1218 bp upstream of the ATG of the SiUGP1 gene in the foxtail millet genome in foxtail millet breeding or in preparing foxtail millet breeding products; the position at 1218 bp upstream of the ATG of the SiUGP1 gene is a SNP site on the promoter of the SiUGP1 gene in the foxtail millet genome, the nucleotide type thereof is A or G, and it is the 1218th nucleotide upstream of the ATG of the SiUGP1 gene; the promoter sequence of the SiUGP1 gene is as shown in Sequence 4 in the sequence listing, and the foxtail millet breeding is to cultivate or select foxtail millet varieties with higher viscosity after cooking.

4. Method for differentiating or assisting in differentiating millet viscosities of different millet varieties, characterized in that: It includes detecting the genotype of foxtail millet, and differentiating or assisting in differentiating the viscosity characteristics of millet of different foxtail millet varieties according to the genotype of the foxtail millet. The genotype is the genotype of a SNP site on the promoter of the SiUGP1 gene in the foxtail millet genome, the nucleotide type thereof is A or G, and it is the 1218th nucleotide upstream of the ATG of the SiUGP1 gene; the promoter sequence of the SiUGP1 gene is as shown in Sequence 4 in the sequence listing.

5. Use of the method according to claim 4 in foxtail millet breeding.

6. A method for foxtail millet breeding, characterized in that: It includes detecting the SNP site at 1218 bp upstream of the ATG of the SiUGP1 gene in the foxtail millet genome as described in claim 1, screening the homozygous foxtail millet with the nucleotide of the SNP site in the foxtail millet genome being A base, and using it as a marker for foxtail millet breeding parent selection and hybrid offspring selection for breeding. The foxtail millet breeding is to cultivate or select foxtail millet varieties with higher viscosity after cooking.

7. The application according to any one of claims 1 to 3, characterized in that: The substance for detecting the polymorphism or genotype at 1218 bp upstream of the ATG of the SiUGP1 gene is as follows S1, S2 or S3; S1. The substance for detecting the polymorphism or genotype at 1218 bp upstream of the ATG of the SiUGP1 gene is a KASP marker primer for amplifying a foxtail millet genome DNA fragment including the position at 1218 bp upstream of the ATG of the SiUGP1 gene. S2. The substance for detecting the polymorphism or genotype at 1218 bp upstream of the ATG of the SiUGP1 gene is a PCR reagent containing the KASP marker primer described in S1; S3. A kit containing the KASP marker primer described in S1 or the PCR reagent described in S2.

8. The application according to claim 7, characterized in that: The KASP marker primer is a primer set composed of the single-stranded DNA shown in Sequence 1 in the Sequence Listing, the single-stranded DNA shown in Sequence 2 in the Sequence Listing, and the single-stranded DNA shown in Sequence 3 in the Sequence Listing.

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