CN118147344A - Primer group and kit for identifying sunflower varieties and application of primer group and kit - Google Patents
Primer group and kit for identifying sunflower varieties and application of primer group and kit Download PDFInfo
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- 235000003222 Helianthus annuus Nutrition 0.000 title claims abstract description 61
- 241000208818 Helianthus Species 0.000 title claims abstract description 60
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- 238000003199 nucleic acid amplification method Methods 0.000 abstract description 17
- 238000001514 detection method Methods 0.000 abstract description 10
- 238000007403 mPCR Methods 0.000 abstract description 10
- 238000012163 sequencing technique Methods 0.000 abstract description 8
- 230000004907 flux Effects 0.000 abstract description 2
- 239000003550 marker Substances 0.000 description 31
- 238000000034 method Methods 0.000 description 13
- 108020004414 DNA Proteins 0.000 description 12
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- 239000007795 chemical reaction product Substances 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 3
- 210000000349 chromosome Anatomy 0.000 description 3
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- 239000003147 molecular marker Substances 0.000 description 2
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- 241000208838 Asteraceae Species 0.000 description 1
- 241000336315 Cistanche salsa Species 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 244000020551 Helianthus annuus Species 0.000 description 1
- 108091092878 Microsatellite Proteins 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 108091081062 Repeated sequence (DNA) Proteins 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
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- 230000009261 transgenic effect Effects 0.000 description 1
- 238000012070 whole genome sequencing analysis Methods 0.000 description 1
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Abstract
The present disclosure provides a primer set for identifying sunflower varieties, a kit and applications thereof. The primer set includes: the 1 st primer pair to the 552 st primer pair, each of which comprises a forward primer and a reverse primer, and the forward primer of the 1 st primer pair, the reverse primer of the 1 st primer pair to the forward primer of the 552 st primer pair and the reverse primer of the 552 st primer pair are sequentially shown as SEQ ID NOs in the sequence table: 1 to SEQ ID NO: 1104. The primer pair is not interfered with each other, the identification accuracy is high, the result reproducibility is strong, meanwhile, the primer group can carry out multiplex PCR amplification, and the amplification product is sequenced by combining a second generation sequencing platform, so that the sunflower variety detection method has the characteristics of high flux, high discrimination, high accuracy and the like, can meet the requirements of sunflower variety authenticity identification and substantial derivative identification, and realizes the sharing and free comparison of variety data in a DNA fingerprint database.
Description
Technical Field
The disclosure relates to the field of biotechnology, and in particular relates to a primer group and a kit for identifying sunflower varieties and application thereof.
Background
Sunflower (Helianthus annuus l.) is an annual herb of the genus sunflower of the family asteraceae, which plays a very important role in the agricultural economy as an important oil crop.
At present, the number of sunflower varieties which are accumulated and registered in China is approximately 2000, and along with the large-scale development of sunflower industry, the sunflower industry has the problems of insufficient utilization of germplasm resources, undefined variety rights and the like. The method is formally implemented in the year 2022, 3 and 1, and takes strengthening of intellectual property protection of seed industry as an important point, establishes a substantial derived variety system, and expands the protection range and the protection link of new plant variety rights. As a main means for protecting new varieties of plants, the variety DUS (DISTINCTNESS UNIFORMITY STABILITY, specificity, consistency and stability) test is based on the judging result of plant phenotypic character, and the international new varieties of plants protection alliance takes SSR (Simple Sequence Repeat, simple repeated sequence) and SNP (Single Nucleotide Polymorphism ) molecular marking method as auxiliary technical means for testing the variety DUS, which plays a positive role in screening approximate varieties and judging the specificity. The SSR molecular marker method for identifying the authenticity of sunflower varieties is formally implemented in 2021, 4 and 1, but the locus of the SSR molecular marker is low in genome and has single allele type. However, the original variety derives a plurality of substantial derived varieties (ESSENTIAL DERIVED VARIETY, EDV), and the substantial derived varieties are identical to the original variety in terms of expressing basic characters generated by genotypes or genotype combinations of the original variety except for character differences caused by derivation, so that the current SSR molecular marking method is difficult to satisfy accurate identification of the substantial derived varieties.
BRIEF SUMMARY OF THE PRESENT DISCLOSURE
In order to solve the problems of the prior art, the embodiment of the disclosure provides a primer group and a kit for identifying sunflower varieties and application thereof. The technical scheme is as follows:
In one aspect, the present disclosure provides a primer set for identifying a sunflower variety, the primer set comprising: the 1 st primer pair to the 552 st primer pair, each of which comprises a forward primer and a reverse primer, and the forward primer of the 1 st primer pair, the reverse primer of the 1 st primer pair to the forward primer of the 552 st primer pair and the reverse primer of the 552 st primer pair are sequentially shown as SEQ ID NOs in the sequence table: 1 to SEQ ID NO: 1104.
In another aspect, the present disclosure provides a kit for identifying sunflower varieties, the kit comprising the primer set of claim 1.
In yet another aspect, the present disclosure provides a use of a primer set for identifying a sunflower variety, the use comprising: the primer set is used for identifying sunflower varieties.
Specifically, the application includes: the primer set is used for identifying the authenticity of sunflower varieties.
Specifically, the application includes: the primer set is used for identifying a substantial derivative variety of sunflower.
Specifically, the application includes: the primer set is used for constructing a DNA fingerprint database of sunflower varieties.
The technical scheme provided by the embodiment of the disclosure has the beneficial effects that: the embodiment of the disclosure provides a primer group and a kit for identifying sunflower varieties and application thereof, wherein the primer pair is not interfered with each other, the identification accuracy is high, the result reproducibility is strong, meanwhile, the primer group can carry out multiplex PCR amplification, and is combined with a second generation sequencing platform to carry out sequencing of amplification products, so that the detection of sunflower varieties has the characteristics of high flux, high discrimination, high accuracy and the like, the requirements of sunflower variety authenticity identification and substantial derivative identification can be met, the sharing and free comparison of variety data in a DNA fingerprint database are realized, the requirement of DNA fingerprint database construction is met, and the primer group can be applied to large-scale sunflower variety identification and has great application value in the aspects of market supervision of crop products, intellectual property protection, new variety cultivation and the like.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present disclosure, the drawings required for the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present disclosure, and other drawings may be obtained according to these drawings without inventive effort for a person of ordinary skill in the art.
FIG. 1 is a graph showing the distribution of MNP markers on sunflower chromosomes according to an embodiment of the present disclosure;
FIG. 2 is a plot of the number of sequenced fragments and the detection rate of MNP marker loci provided by example III of the present disclosure;
fig. 3 is a graph showing the MNP marker proportion difference profile between sunflower varieties provided in example three of the present disclosure.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the present disclosure more apparent, the embodiments of the present disclosure will be described in further detail below.
Example 1
The present disclosure provides a primer set for identifying sunflower varieties, the primer set comprising: the 1 st primer pair to the 552 st primer pair, each of which comprises a forward primer and a reverse primer, and the forward primer of the 1 st primer pair, the reverse primer of the 1 st primer pair to the 552 st primer pair and the reverse primer of the 552 st primer pair are sequentially shown as SEQ ID NO:1 to SEQ ID NO: 1104.
Table 1 shows 552 pairs of detection primer sequences
The screening of the primer group comprises the following steps: 25 sunflower varieties such as SH363, JK601, SH361 and X3939 are collected, genome is carried out, sequencing data covering more than 20X are obtained for each variety, 290 sunflower whole genome sequencing data (PRJNA 353001) are combined, a public sunflower genome sequence GCF_002127325.2 is taken as a reference genome, sequence analysis is firstly carried out by adopting Samtools (Version 1.2) and BCFtools (Version: 1.2) to obtain SNP loci on the sunflower genome, the SNP loci are compared and analyzed with an NCBI NT library, and marker screening is carried out according to the following principle: (1) The marker sequence is unique only to sunflower and not present in other species; (2) the sequence is a single copy in the genome; (3) The difference of at least three discontinuous SNP on the marker sequence; (4) the length of the marker sequence is less than 250bp. The obtained candidate MNP markers were analyzed and screened using the measured genome resequencing data of 25 sunflower varieties, and finally 552 MNP marker loci shown in Table 1 were selected, each chromosome contained 16.2 marker loci on average, and the number distribution of marker loci on each chromosome was shown in FIG. 1.
Example two
The present disclosure provides a kit for identifying sunflower varieties, comprising the primer set provided in example one. 5 mu L of each primer in the primer group is taken and mixed in equal amount to prepare the primer mix.
The kit further comprises: multiplex PCR premix.
Example III
The present disclosure provides an application of a primer set for identifying sunflower varieties, the application comprising: the primer set provided in example one was used to identify sunflower varieties.
Specifically, the application includes: the primer set provided in example one was used to identify the authenticity of sunflower varieties.
Specifically, the application includes: the primer set provided in example one above was used to identify a substantial derivative of sunflower.
Specifically, the application includes: the primer set provided in example one was used to construct a DNA fingerprint database of sunflower varieties.
The example uses 31 sunflower varieties for identification, and the specific process is as follows:
The sample to be tested in this example was 31 sunflower varieties, and the whole genome DNA of 31 sunflowers was extracted respectively to obtain the whole genome DNA of 31 samples to be tested. In this example, a kit method is used to extract whole genome DNA, and the kit is a novel plant genome DNA extraction kit for Tiangen DP320 (column membrane method) DNAsecure.
The genomic DNA of 31 samples to be tested was subjected to multiplex PCR (Polymerase Chain Reaction ) amplification using the primer set provided in example I of the present invention, and the amplification system in this example is shown in Table 2.
Table 2 shows the amplification system
| Component (A) | Volume of |
| MNPPrimerPanelMix | 4μL |
| DNA | 50～200ng |
| GenoPlexs3×TMasterMix | 10μL |
| H2O | The amplification system was supplemented to 30. Mu.L with H 2 O |
In this example, the amplification procedure is shown in Table 3.
Table 3 shows the amplification procedure
After the multiplex PCR amplification reaction is finished, a multiplex PCR amplification reaction product is obtained, and the multiplex PCR amplification reaction product is purified by a magnetic bead method. Purifying the multiplex PCR amplification reaction product by using DNA purification magnetic beads of Norflu biological technology Co., ltd, and removing redundant primers to obtain the purified multiplex PCR amplification reaction product. See the description for specific operations.
The kit provided in the second embodiment is used for multiplex PCR amplification and sequencing library construction, and the 31 sunflower DNA samples to be tested are subjected to multiplex amplification, second-generation high-throughput sequencing and data analysis, so that the detection of 552×31= 17112 marker loci in one experiment is realized, the average coverage multiple of the sequencing of each sample to be tested is more than 20×times, and the high efficiency of MNP marker locus detection is shown.
The distribution diagram of the number of the detected MNP marker loci of sunflower is shown in FIG. 2, and the number of the detected MNP marker loci in the sequencing data of the 31 samples to be tested is counted, specifically shown in Table 4.
Table 4 shows the results of MNP molecular markers of 31 sunflower varieties
As shown in Table 4, 531 MNP marker loci can be detected on average for each variety, the variety with the lowest marker locus detection rate is 93.48%, and the average marker locus detection rate is 96.23%, which indicates that the sunflower MNP marker locus detection rate provided by the embodiment is high and stable. The detection rate of the marker loci is stable, and the problem that the variation is large due to the fact that the variation of the detected marker loci is large in different experiments, so that the genetic similarity coefficients are large in different experiments, and finally, the problem that the variation is large due to the fact that the loci are sampled in the variety identification conclusion is solved can be prevented.
(2) Accuracy analysis
The number of marker loci with differences and the same genotype among varieties is the basis for decision of the conclusion of variety identification, and therefore, the accuracy of variety identification ultimately depends on the accuracy of genotyping of the marker loci. Since the true value is unknown, the absolute accuracy of any method is not computable. In practice, either the accuracy is calculated assuming the reference value as a true value or the accuracy is estimated with accuracy. Since the reference value of the marker locus genotype of the variety is also unknown, accuracy is calculated by using three reproducibility experiments, and thus the accuracy of typing is calculated with accuracy=1- (1-accuracy)/2. The accuracy refers to the proportion of marking sites with consistent typing results of two experiments to all marking sites, and the repeatability refers to two independent experiments performed by different personnel, different batches of reagents and different laboratories. The reproducibility experiments simulate the identification of different batches, and the high reproducibility means that the identification results of different laboratories can be accurately compared with each other.
In order to test the accuracy of the primer set, a reproducibility experiment is carried out on 8 varieties, wherein the reproducibility experiment is respectively completed in Jiang Handa and 2 units of Barbary institute of agriculture and animal husbandry, each variety is respectively constructed into 2 libraries, and the serial numbers are library 1 and library 2; library sequencing typing results from different laboratories were compared pairwise. The comparison results are shown in Table 5.
Table 5 shows the accuracy analysis of sunflower MNP marker loci
As shown in Table 5, the total of 4261 MNP marker loci in the reproducibility test, the number of reproducible marker loci was 4254 MNP marker loci, and the accuracy of typing the marker loci was 99.92%. In one identification, the marking sites of the typing errors are not more than 552× (1-99.92%) =0.44, and the deviation is not more than 0.01% compared with the true value of the genetic similarity coefficient. Therefore, the primer group provided by the embodiment of the disclosure has high consistency in variety identification conclusion among different laboratories or different batches of the same laboratory, so that parallel experiments are not needed to reduce experimental errors, which means that accurate comparison can be performed among DNA fingerprint data of different sources, and great convenience is provided for variety identification.
(3) Differentiation degree
The core task of variety identification is to distinguish the variety to be detected from the control variety by using the detected marker loci. Two important factors influence the core task of variety identification, and firstly, the stronger the distinguishing capability of a single marker locus is, the stronger the capability of the primer group to distinguish varieties is; second, the larger the number of labels used, the more discriminating the variety of the primer set. In recent years, the problems of sunflower variety homogenization and imitated varieties become serious day by day, and in the embodiment, 700 sunflower variety samples are subjected to DUS test and subjected to clustering analysis according to phenotype characteristics, and representative varieties with large phenotype differences and varieties with very similar phenotypes which are difficult to distinguish are selected from the sunflower varieties (see table 4) and MNP molecular marking is carried out.
The primer set provided in this example was used to identify the authenticity of 31 sunflower varieties, and 531 MNP markers were detected in each variety on average, and the proportion of the MNP markers of the differences between each pair of sunflower varieties was analyzed to obtain 465 comparison results. The ratio of MNP markers of each pair of differences between varieties is called the distance between varieties, and the distance between varieties directly shows the distinguishing capability of the primer group to the varieties. The results show that the average distance between varieties reaches 70.39%, and particularly as shown in fig. 3, the primer group has strong variety distinguishing capability and can distinguish any variety combination of 99.99%.
In the pairwise alignment of 31 sunflower varieties MNP markers, 2 pairs of varieties are similar in phenotype, namely a known main-cultivated variety XRK0009 and variety XRK0017, and 40 varieties of the pairs of varieties have character code differences of 0 in 43 DUS test basic characters, and belong to similar varieties. Through MNP molecular marking, 5 different sites exist on 552 MNP sites of the variety XRK0009 and phenotype and the variety XRK0017, the genetic similarity reaches 99.06%, and the method belongs to similar varieties according to the judgment conclusion of GB/T38551-2020 MNP marking method for plant variety identification. The other pair is that the main cultivated variety XRK0001 and the variety XRK0018 have 40 character code differences of 0 on 43 DUS test basic characters, 32 difference sites exist on 552 MNP marking sites, and the genetic similarity is 93.9 percent. The MNP-labeling results of these two varieties were consistent with the DUS-test-similar variety screening results. 7 pairs of modified varieties were collected in 2023, and MNP molecular markers were performed on the varieties, and specific results are shown in Table 6.
Table 6 shows the MNP results of the substantial derivative variety
The variety 6 to be detected and the variety 7 to be detected in the table 6 are sister lines bred by a conventional breeding means, and the genetic similarity is between 80% and 85%. The varieties 1 to 3 to be tested are transgenic resistant broomrape varieties bred in a laboratory of a certain company, and the obvious difference in phenotype among the 3 pairs of varieties except the resistance is observed through field test. Meets the definition of the substantive derivative variety. The to-be-detected variety 5 (s 23-4) is obtained by mutating a comparison variety (s 23-5), the to-be-detected variety 4 (23 SJ 18) is identical to a female parent of the comparison variety (23 SJ 19), and a male parent of the to-be-detected variety 4 (23 SJ 18) is obtained by mutating a male parent of the comparison variety (23 SJ 19). Therefore, the primer group can be used for rapid screening of similar varieties among plant varieties and identification of substantial derivative varieties.
Identification of the substantial derivative requires calculation of the genetic similarity coefficient, and therefore, the number of marker loci used is sufficient to accurately calculate the genetic similarity coefficient. The international seed union (ISF) requires the use of more than 300 SSR markers for substantial derivative variety decision. MNP marker polymorphisms have been demonstrated to be higher than SSR markers in rice varieties and the standard number used in the present invention amounts to 552, and therefore, determination of substantial derivative varieties is also sufficient.
The foregoing description of the preferred embodiments of the present disclosure is provided for the purpose of illustration only, and is not intended to limit the disclosure to the particular embodiments disclosed, but on the contrary, the intention is to cover all modifications, equivalents, alternatives, and alternatives falling within the spirit and principles of the disclosure.
Claims (6)
1. A primer set for identifying a sunflower variety, said primer set comprising: the 1 st primer pair to the 552 st primer pair, each of which comprises a forward primer and a reverse primer, and the forward primer of the 1 st primer pair, the reverse primer of the 1 st primer pair to the forward primer of the 552 st primer pair and the reverse primer of the 552 st primer pair are sequentially shown as SEQ ID NOs in the sequence table: 1 to SEQ ID NO: 1104.
2. A kit for identifying sunflower varieties, comprising the primer set of claim 1.
3. Use of a primer set for identifying sunflower varieties, comprising: use of the primer set of claim 1 for identifying sunflower varieties.
4. The application according to claim 3, characterized in that it comprises: use of the primer set of claim 1 for identifying the authenticity of sunflower varieties.
5. The application according to claim 3, characterized in that it comprises: use of the primer set of claim 1 for identifying a substantial derivative of sunflower.
6. The application according to claim 3, characterized in that it comprises: use of the primer set of claim 1 for constructing a DNA fingerprint database of sunflower varieties.
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