A set of primers for identifying the genetic relationship of Paris plants and their applications

Abstract

The present invention belongs to the field of identification of genetic germplasm resources, and particularly relates to a set of primers for identifying the genetic relationships of Paris plants and their applications. The present invention provides a set of primers consisting of a total of 3 primer pairs, which can simply, effectively and quickly identify the genetic relationships of Paris plants of different species and different populations from a molecular perspective. It is not limited by the growth and development period of Paris itself, and breaks through the limitation of relying on the weak differences in phenotypes at specific periods for the classification and identification of various Paris plants, providing a favorable tool for tracking and identifying Paris germplasm resources, and having broad application prospects in the fields of genetics, breeding, drug development, etc.

Description

Technical Field

[0001] The invention belongs to the field of genetic resource identification, and in particular relates to a group of primers for identifying the phylogenetic relationship of Paris polyphylla plants and applications thereof. Background Art

[0002] Paris is a general term for plants of the genus Paris in the family Melanthiaceae of the order Liliales, and is the main raw material for many traditional Chinese medicines. There are currently 36 species and 15 varieties of Paris plants reported worldwide.

[0003] As the medicinal value of Paris polyphylla is constantly being explored, the consumption of Paris polyphylla medicinal materials has continued to grow in recent years, and wild resources have been severely depleted due to over-exploitation. Therefore, the development and application of germplasm resources are becoming increasingly important. However, current research still focuses on the two Paris polyphylla sources specified in the Chinese Pharmacopoeia, namely Paris polyphylla Smith var.yun-nanensis (Franch.) Hand.-Mazz. and P. polyphylla Smith var.chinensis (Franch.) Hara. In fact, in addition to Paris polyphylla and Paris polyphylla, other Paris polyphylla plants are also likely to have medicinal value worthy of research and application, but previous studies have found that the representative medicinal components of different Paris polyphylla plants are different, and the types of active substances and their content levels are different.

[0004] At the same time, the intraspecific relationships of the genus Paris are complex. The phenotypic differences of the same species in different populations and regions are small, and the phenotypic characteristics used for systematic classification of different species may not appear until the flowering or fruiting period. Moreover, the growth and development of Paris itself is characterized by a long hibernation period, a low germination rate, and slow growth and development. Under natural conditions, it usually takes two winters and one summer to germinate. After germination, it usually takes another three years to start flowering and fruiting. The key period for phenotypic classification and identification of Paris plants is also during the reproductive growth period. It is usually necessary to rely on pedicel length, sepal width, petal color, style length, and pericarp characteristics to separate different Paris plants. Moreover, the flowering and fruiting periods of different Paris plants are inconsistent. The flowering period can span 3 to 6 months, and the fruiting period can span 7 to 11 months. They are mostly distributed in the shade and moist places under forests in high-altitude areas, making it difficult to collect and track resources in the wild. This has caused great difficulties in the early and seedling classification and identification of Paris plants, especially wild Paris plants, and is also very unfavorable for tracking and investigating specific germplasm resources in the wild, which can easily lead to delays or even incorrect introduction of resources due to the inability to identify them.

[0005] For example, the research group of the inventors found that the saponin components and composition ratios of Paris vietnamensis (Takht.) H. Li, Paris polyphylla Smith and Paris vietnamensis (Takht.) H. Li in different populations showed different characteristics. Among them, the content of polyphyllin VI in Paris vietnamensis (Takht.) H. Li of one population was significantly higher than that of Paris vietnamensis (Takht.) H. Li in other populations, and the contents of polyphyllin I and total saponins in Paris vietnamensis (Takht.) H. Li were relatively high. This indicates that the medicinal ranges of different Paris plants are not the same. Even for the same Paris plant, there are differences in the active ingredients among the Paris resources of different populations. It is necessary to accurately discriminate the genetic relationships of different Paris plants, and strengthen the molecular tracking of Paris plants in the same wild population or adjacent populations, so as to facilitate the carrying out of related work such as introduction and hybridization breeding, promote the expansion of Paris medicinal resources, expand the medicinal range of Paris, and improve the medicinal value of Paris plants.

[0006] With the development of molecular markers, molecular markers such as SSR have become effective tools for discriminating plant germplasm resources and studying the genetic relationships of germplasm resources. However, Paris, as a plant with the largest known genome in the world at present, has an extremely complex genome, and the relationships between and within species are very complicated. The development of molecular markers for efficiently screening different germplasm resources is very difficult. Chen Zhongsu et al. (2017) developed 8 pairs of SSR primers to analyze the genetic diversity of 5 different populations of Paris polyphylla var. yunnanensis, and found that the 5 populations could be divided into 2 categories, and there was still a certain degree of genetic differentiation within and between populations, which was consistent with the applicant's previous inferences. Yang Weize et al. (2014) used 15 pairs of polymorphic SSR primers to detect the polymorphism of 35 Paris polyphylla var. yunnanensis materials, showing that Paris polyphylla var. yunnanensis has relatively rich genetic diversity. Wang Haiming et al. (2019) used 16 pairs of SSR markers to analyze the germplasm resource diversity of 14 wild Paris polyphylla Smith var. chinensis in Fanjingshan, showing that they could be mainly grouped into 2 different groups.

[0007] However, generally speaking, the application of SSR markers in the genetic relationships of the entire Paris plants is relatively few. Moreover, for the existing developed molecular markers, at least 8 pairs or even more than 10 pairs of primers need to be used simultaneously to achieve detection and identification, and often only Paris polyphylla var. yunnanensis and Paris polyphylla Smith var. chinensis can be identified, and it cannot be extended to the entire Paris plants for identification, which limits the overall expandable application of Paris plants and is not conducive to efficiently distinguishing target resources. Therefore, if SSR primers that can identify the genetic relationships of multiple Paris plants and use relatively few primer pairs can be developed, it will have important value and practical significance in the fields of genetics, breeding, and drug development of Paris plants. Summary of the Invention

[0008] The object of the present invention is to provide a set of primers for identifying the genetic relationships of Paris plants and their applications.

[0009] To achieve the above-mentioned invention object, the technical solution adopted by the present invention is: a primer pair, characterized in that: it includes primer pair 1 and / or primer pair 2 and / or primer pair 3;

[0010] The forward sequence of the primer pair 1 is as shown in SEQ ID NO:1, and the reverse sequence is as shown in SEQ ID NO:2; the forward sequence of the primer pair 2 is as shown in SEQ ID NO:3, and the reverse sequence is as shown in SEQ ID NO:4; the forward sequence of the primer pair 3 is as shown in SEQ ID NO:5, and the reverse sequence is as shown in SEQ ID NO:6.

[0011] Preferably, it includes primer pair 1, primer pair 2 and primer pair 3 at the same time.

[0012] Correspondingly, the application of the primer pair in the identification, discrimination, classification and breeding of Paris plants.

[0013] Correspondingly, a kit, reagent paper, and reagent strip containing the primer pair 1 and / or the primer pair 2 and / or the primer pair 3.

[0014] Correspondingly, a method for identifying, discriminating, classifying and breeding Paris plants using the primer pair includes the following steps:

[0015] (1) Use the primer pair 1 and / or the primer pair 2 and / or the primer pair 3 to amplify the DNA of the sample to be tested;

[0016] (2) Perform polymorphism analysis on the sequencing results;

[0017] (3) Perform cluster analysis on the samples to analyze the genetic relationship.

[0018] Preferably, the amplification system in step (1) is: the total system is 20 μL, including 10 μL of mix, 0.3 μL each of the 20 μM forward primer and reverse primer, 2 μL of the DNA template at 50 - 200 ng / μL, and make up to 20 μL with ddH 2 O; each pair of primers corresponds to one reaction system.

[0019] Preferably, the amplification reaction program in step (1) is: pre-denaturation at 94 °C for 5 min; denaturation at 94 °C for 30 s, annealing at 55 °C for 40 s, extension at 72 °C for 30 s, 30 cycles; extension at 72 °C for 10 min, and store at 4 °C.

[0020] The present invention has the following beneficial effects: The present invention provides a set of SSR primers, which altogether include 3 primer pairs. It can simply, effectively and quickly identify the genetic relationships of Paris plants of different species and different populations from a molecular perspective, can conduct discrimination of genuine medicinal materials and field discrimination and tracking of specific germplasm resources, is not restricted by the growth and development period of Paris itself, provides a favorable tool for tracking and identifying Paris germplasm resources, and has broad application prospects in the fields of genetics, breeding, drug development, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is a schematic diagram of the band reading result of the first group of primers;

[0022] Figure 2 It is a schematic diagram of the band reading result of the second group of primers;

[0023] Figure 3 It is a schematic diagram of the band reading result of the third group of primers;

[0024] Figure 4 It is a molecular phylogenetic tree for analyzing the genetic relationships of 43 Paris genetic resources using the 3 groups of primers of the present invention;

[0025] Figure 5 It is a schematic diagram of the phenotypic appearance during the flowering period of the second group of genetic resources, from left to right: MS2017-1, MS2018-5, MS2018-6;

[0026] Figure 6 It is a schematic diagram of the phenotypic appearance during the flowering period of the fourth group of genetic resources, from left to right: GY2018-4, MS2018-3, MS2018-4. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0027] The present invention provides 3 pairs of SSR primer sets, and the sequences of each primer set are shown in Table 1 (F is forward and R is reverse).

[0028] Table 1 Primer set sequence information

[0029]

[0030] The present invention also provides a method for identifying the genetic relationships of Paris plants using the said primer sets, which specifically includes the following steps:

[0031] 1. Respectively use different markers to label the forward primer and the reverse primer. Use the primers to amplify the DNA of the sample to be tested.

[0032] An optional amplification system is: The total system is 20 μL, including 10 μL of mix, 0.3 μL each of 20 μM forward primer and reverse primer, 2 μL of 50 - 200 ng / μL DNA template, and the rest is filled with ddH 2Make up to 20 μL with O (7.4 μL). Each pair of SSR primers corresponds to a reaction system.

[0033] An optional amplification reaction program is as follows: pre-denaturation at 94 °C for 5 min; denaturation at 94 °C for 30 s, annealing at 55 °C for 40 s, extension at 72 °C for 30 s, for 30 cycles; extension at 72 °C for 10 min, and store at 4 °C.

[0034] 2. Perform capillary electrophoresis, and conduct polymorphism analysis on the sequencing results. Determine the size of the core site fragments according to the peak map and perform band reading.

[0035] 3. According to the band reading results, conduct cluster analysis on the Paris resources, construct its molecular evolutionary phylogenetic tree, and analyze its genetic relationship.

[0036] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. If not specifically specified, the technical means used in the embodiments are conventional means well-known to those skilled in the art. The obtained data are all the averages obtained after at least 3 repetitions, and all the repetitions obtain valid data.

[0037] Example: Display of SSR primer effects

[0038] 1. From 2016 to 2019, wild Paris resources were collected in Hongya County, Meishan City, Sichuan Province (in 2017 and 2018), Danling County, Meishan City, Sichuan Province (in 2019), Beichuan County, Mianyang City (in 2017 and 2019), Kangding County, Ganzi Prefecture (in 2018), and Qingchuan County, Guangyuan City (in 2016 and 2018). A total of 43 Paris plant resources were collected, as shown in Table 2 specifically.

[0039] Table 2 Comparison table of information of collected Paris samples

[0040]

[0041]

[0042] Conduct preliminary registration on the biological traits of the wild resources of different regions and different Paris. Collect fresh and tender leaves of each resource, place them in an ice bag and bring them back to the laboratory, and store them at -20 °C for later use.

[0043] 2. Extract total DNA using the CTAB method, and detect the quality of the DNA samples using 1.2% agarose.

[0044] For the 3 primer pairs shown in Table 1, the forward primers were labeled with FAM fluorescein and the reverse primers were labeled with HEX fluorescein. The total DNA of the above-mentioned Paris vietnamensis resources was amplified using the 3 primer pairs respectively.

[0045] The amplification system was as follows: the total system was 20 μL, including 10 μL of mix, 0.3 μL each of 20 μM forward primer and reverse primer, 2 μL of DNA template at 50 - 200 ng / μL, and the rest was made up to 20 μL with ddH 2 O (7.4 μL). Each pair of SSR primers corresponded to a reaction system.

[0046] The amplification reaction procedure was: pre-denaturation at 94 °C for 5 min; denaturation at 94 °C for 30 s, annealing at 55 °C for 40 s, extension at 72 °C for 30 s, for 30 cycles; extension at 72 °C for 10 min, and storage at 4 °C.

[0047] 3. Genetic diversity analysis. Capillary electrophoresis was performed using an ABI 3730XL automatic DNA sequencer, and polymorphism analysis of the sequencing results was carried out using GeneMapper 4.1. The size of the core locus fragments was determined according to the peak map and band reading was performed. Taking the amplification results of the 3 primer pairs in the GY2016 - 1 sample as an example, the results are as Figures 1 to 3 shown. Figures 1 to 3 They are respectively the schematic diagrams of the amplification results of the 1st, 2nd, and 3rd primer pairs. According to the peak sites for band reading, the "presence" and "absence" of the bands were recorded as a "1" and "0" matrix.

[0048] The format was converted into the corresponding format of Popgene32 software using Data Formater, and genetic diversity parameters such as the number of alleles (Na), effective number of alleles (Ne), Shannon information index (I), expected heterozygosity (He), observed heterozygosity (Ho), and polymorphism information content (PIC) were statistically analyzed using Popgene32. The results are shown in Tables 3 - 5.

[0049] Table 3 Allele frequencies of SSR primer groups

[0050]

[0051] According to Table 3, 3 primer pairs amplified 8 - 24 allele loci in 43 Paris resources respectively, with an average of 14 allele loci per locus. The allele frequency ranges were 0.0233 - 0.5134, 0.0116 - 0.2326, and 0.0116 - 0.1512 respectively. The results showed that: the 3 primer pairs detected more allele loci. Especially, the band pattern of Marker3 (the 3rd primer pair) was relatively rich, with a total of 24 loci, and the least was Marker1 (the 1st primer pair), but there were also 8 loci. It was proved that the 3 primer pairs provided by the present invention could be jointly used for the detection of genetic diversity of Paris resources.

[0052] Table 4 Genetic diversity analysis of primer pairs in Paris resources

[0053]

[0054] According to Table 4, the number of effective alleles of the 3 primer pairs was 2.7072 - 12.7079, and the average number of effective alleles per locus was about 7. The observed heterozygosity was 0.0163 - 0.8837, with an average of 0.5736. While the expected heterozygosity was 0.638 - 0.9321, and the average was only 0.7886. There was a large difference between the expected heterozygosity and the observed heterozygosity, indicating an imbalance in mating. Considering that there were various different Paris resources in the resources and the reproductive growth periods were different, this phenomenon was in line with expectations. Nei's diversity index was 0.6306 - 0.9213, with an average of 0.7794, indicating that Paris resources had high polymorphism and genetic diversity.

[0055] Table 5 Genetic diversity analysis of different sampling areas based on primer pairs

[0056]

[0057] As can be seen from Table 5, taking different geographical locations as the classification criteria, the Paris resources collected at the 3 sampling points of Qingchuan, Beichuan and Meishan by the primer pairs showed rich polymorphism (since there were only 1 - 2 resources in Kangding and Danling, the population diversity data was not reflected). The percentage of polymorphic loci reached 100%. The average number of alleles per sampling site was 7.8889, and the average number of effective alleles was about 5.20. The observed heterozygosity and expected heterozygosity of each sampling site were inconsistent and different, indicating deviation. As an important index of genetic diversity, the Shannon information index ranged from 0.7467 to 0.7887, with an average of 0.763833. The value was relatively high, indicating that whether in different sampling sites or overall, the genetic diversity of Paris resources was relatively high, and the genetic diversity levels in different sampling areas were similar, which was in line with the characteristics of Paris often self - pollinating, further indicating the effectiveness and reliability of the primer pairs in the genetic diversity analysis of Paris.

[0058] 4. Kinship analysis. According to the read tape results, the resources of Paris genus were clustered and analyzed based on UPGMA using NTSYS2.1 software to construct a molecular phylogenetic tree, analyze the kinship, and the results of the molecular phylogenetic tree are as Figure 4 shown.

[0059] From Figure 4 it can be seen that at the level of genetic similarity coefficient of 0.80, the resources can be divided into 5 major categories. Among them, the first category contains 33 materials, including the resources from each sampling point; the second category contains 4 Paris genus resources, including 3 Beichuan resources and 1 Hongya resource; the third, fourth, and fifth categories contain 2, 3, and 1 resources respectively, all from Qingchuan. And at the level of 0.93, most of the genetic resources can be divided into different branches of the phylogenetic tree, indicating that each resource has a certain kinship, which is in line with the preliminary phenotypic discrimination results during sampling, that is, it is recognized that all resources are Paris genus resources; but at the same time, it shows that there is rich genetic diversity among the resources, which is consistent with the previous genetic diversity analysis results. It is worth noting that: even at the genetic similarity level of 0.93, there are still 7 groups of branches containing 2 or more resources, indicating that the kinship between these corresponding resources is relatively close. Specifically: GY2016-4 and GY2016-6 (Group 1), MS2017-1, MS2018-5 and MS2018-6 (Group 2), MS2017-2, MS2018-8 and MS2018-9 (Group 3), GY2018-4, MS2018-3 and MS2018-4 (Group 4), BC2019-1 and BC2019-2 (Group 5), GY2016-2 and GY2016-3 (Group 6), BC2017-1 and BC2017-3 (Group 7). Except for Group 4, the resources in other groups are all from the same sampling point and are clustered together, indicating that the resources in the similar sampling range have relatively close kinship, which is in line with expectations and further proves the effectiveness and reliability of the primer set.

[0060] However, the inventor also observed that although the resources in Group 2 and Group 3 are all from the same sampling point, they cover resources of different sampling years. In particular, it is found that the genetic similarity between MS2017-1 and MS2018-5 in Group 2 is almost 1, indicating that they are basically germplasm resources within the same genus, with extremely close genetic kinship and more likely to have the same or similar medicinal values. Based on this result, the inventor rechecked the resource phenotypes during the flowering period (when the phenotypic indicators for Paris classification and identification are relatively rich), and the results are as Figure 5As shown. The inventor found that in Group 2, the phenotypic characteristics of MS2017-1 and MS2018-5 were similar, which was consistent with the results of molecular research on genetic relationships. This result further proved that the primer set provided by the present invention could indeed be used to track the genetic relationships of specific Paris vietnamensis germplasm resources in the wild, facilitating the collection of target resources. Another resource in this group (Group 2), namely MS2018-6, clustered with MS2017-1 and MS2018-5 at the 0.93 level. Its leaf phenotype was consistent with that of the other two resources. Although the floral and fruit classification characteristics have not been observed yet, it can be inferred from the molecular marker results that its genetic relationship with MS2017-1 and MS2018-5 is relatively close, and it is more likely to have similar active substance characteristics and medicinal values. It can be further tracked and observed, greatly reducing the workload of resource identification.

[0061] Surprisingly, in Group 4, resources from Qingchuan (GY2018-4) and Hongya (MS2018-3 and MS2018-4) were clustered together at a relatively high genetic similarity coefficient level. The inventor's back-check results of the resource phenotypes are as Figure 6 shown. The back-check found that the phenotypic results of the resources were relatively consistent, which was also in line with the genetic similarity results analyzed by the primer set. This result proved that even for Paris vietnamensis resources from different regions, there may still be relatively close genetic relationships, and the habitat is not the only indicator for judging the genetic relationship. It provided an identification method for the expansion of backup resources of genuine medicinal materials in the future.

[0062] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations, variations, modifications, and substitutions made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. Primer pair Characterized in that: It includes primer pair 1, primer pair 2 and primer pair 3; The forward sequence of the primer pair 1 is shown in SEQ ID NO:1, and the reverse sequence is shown in SEQ ID NO:2; the forward sequence of the primer pair 2 is shown in SEQ ID NO:3, and the reverse sequence is shown in SEQ ID NO:4; the forward sequence of the primer pair 3 is shown in SEQ ID NO:5, and the reverse sequence is shown in SEQ ID NO:

6.

2. Use of the primer pair according to claim 1 in the identification of genetic relationships in Paris plants.

3. Kit containing the primer pair 1, primer pair 2 and primer pair 3 according to claim 1.

4. Test strip containing the primer pair 1, primer pair 2 and primer pair 3 according to claim 1.

5. Method for identifying the genetic relationship of Paris plants using the primer pair according to claim 1, comprising the following steps: (1) Amplify the DNA of the sample to be tested using the primer pair 1, primer pair 2 and primer pair 3; (2) Conduct polymorphism analysis on the sequencing results; (3) Conduct cluster analysis on the samples to analyze the genetic relationship.

6. According to the method described in claim 5, Characterized in that: Step (1) The amplification system in the amplification is as follows: the total system is 20 μL, including 10 μL of mix, 0.3 μL each of 20 μM forward primer and reverse primer, 2 μL of 50 - 200 ng / μL DNA template, and supplemented with ddH 2 O to 20 μL; each pair of primers corresponds to one reaction system.

7. According to the method described in claim 5, Characterized in that: The amplification reaction program in step (1) is: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 55°C for 40 s, extension at 72°C for 30 s, 30 cycles; extension at 72°C for 10 min, and preservation at 4°C.

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