Microsatellite molecular marker and specific primer for fantasis arundinacea, and preparation method and application of microsatellite molecular marker and specific primer
Developing microsatellite molecular markers and specific primers of scattered scattered scattered scattered scattered scattered scattered scattered scattered scattered genetics in the existing technology, realizing the diversity and population structure of germplasm resources, and providing resources for its The rational development and protection of the simplified development and protection provide a theoretical basis.
Patent Information
- Application Number
- CN202411415380.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-11
- Publication Date
- 2024-12-13
AI Technical Summary
There is a lack of reporting and research on genetic research on sequential germplasm resources using simple sequence repeat (SSR) microsatellite molecular markers in the prior art, resulting in a lack of effective technical means for the study of the genetic diversity, population structure and historical dynamics of its germplasm resources.
Microsatellite molecular markers of Fansaihu were developed through transcriptome data, specific primers were designed, and the third-generation sequencing technology and SSR site detection software were used to prepare and apply microsatellite molecular markers for germplasm resource identification, genetic diversity analysis and Population research.
Provides microsatellite molecular markers and specific primers for the genitalia, fills the gap in the existing technology, lays the theoretical basis for the rational development and protection of its germplasm resources, and supports the genetic diversity, population structure and Research on historical dynamics.
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Figure CN119120760A_ABST
Abstract
Description
Technical Field
[0001] The present application belongs to the field of biological technology, and in particular relates to microsatellite molecular markers, specific primers, preparation methods and applications of Achyranthes bidentata. Background Art
[0002] Littledalea racemosa belongs to the Poaceae family, the Bromeae tribe, and the Littledalea genus. This genus is a sparse, short-rooted grass unique to the plateau. Its main growth environment is in the Qinghai-Tibet Plateau and its surrounding alpine areas, with an average altitude of more than 4,000 meters. Only a few populations of this genus grow in nearby areas with slightly lower altitudes. The environment in which Littledalea racemosa is located is usually relatively extreme climatic conditions, such as alpine grass slopes, river valley beaches, and shrub meadows. This genus is also a common species on alpine meadows, with strong resistance to salt and alkali, drought, barrenness, diseases, pests, and wind and sand. At the same time, its root system is very developed, and it is one of the most important plants in soil and sand conservation. This genus also contains rich nutritional value and can be used as an excellent forage feed in animal husbandry. At present, many scholars at home and abroad have conducted multi-faceted research on Littledalea racemosa, including trait characteristics, distribution, classification methods, characteristics of chloroplast genomes, and germplasm resources. There are few reports on the molecular biology of Arundophylla, especially the research on its intra-genus relationships is still controversial. The development of polymorphic microsatellite molecular markers through transcriptome data can provide effective technical means for conducting genetic research on the genetic diversity of germplasm resources, population structure, and population historical dynamics of Arundophylla, laying a theoretical foundation for the subsequent rational development and protection of Arundophylla resources.
[0003] Simple sequence repeats (SSR), also known as microsatellites, are an indispensable part of the genome of eukaryotic organisms. They are mainly composed of short tandemly repeated DNA sequences, which are generally collectively referred to as simple sequence repeats (SSR). These SSR sequences are characterized by repeating units containing 1 to 5 bases, and their tandem arrangement characteristics give them unique biological significance. The high variability of SSR mainly stems from the diversity of nucleotide composition and the difference in the number of repetitions within its repeating units, which in turn leads to significant changes in its length. Following Mendel's law of inheritance, SSR presents co-dominant characteristics, has high polymorphism and reliability, and has a fast detection speed. It can be used as an effective molecular marker and plays an important role in population genetics analysis. It has been widely used in the drawing of genetic maps, in-depth research on the genetic diversity of germplasm resources, analysis of genetic relationships and differentiation, and analysis of population structure.
[0004] Therefore, the development of SSR microsatellite molecular markers using transcriptome data provides an effective technical means for conducting genetic studies on the genetic diversity, population structure, and population historical dynamics of Arundophylla germplasm resources. However, there are no reports or studies on the genetic research of Arundophylla using SSR methods in the existing technology. Summary of the invention
[0005] The purpose of the embodiments of the present application is to provide microsatellite molecular markers, specific primers, preparation methods and applications of Arundo donax, aiming to fill the gap in relevant reports and research on genetics of Arundo donax using SSR molecular markers in the current prior art.
[0006] The embodiment of the present application is implemented as follows: the microsatellite molecular markers of the Achyranthes bidentata are characterized in that the microsatellite molecular markers of the Achyranthes bidentata are SR57, SR78, SR87, SR91, SR106, SR108, SR112, SR119, SR120, SR121, SR125, SR127, SR129, SR130 and SR131, and their nucleotide sequences are shown in SEQ.ID.No.1-SEQ.ID.No.15.
[0007] Another purpose of the present invention is to provide specific primers for microsatellite markers of Arundophyllum spicatum.
[0008] Forward primer sequence of SR57 (SEQ.ID.No.16): 5′-TGATGATTGGAGCGAGCGAAGT-3′;
[0009] Reverse primer sequence of SR57 (SEQ.ID.No.17): 5′-GAGAAGCGAAAAAAAGGGAGGC-3′;
[0010] Forward primer sequence of SR78 (SEQ.ID.No.18): 5′-GGGTCCTTTACCTGACTCCTC-3′;
[0011] Reverse primer sequence of SR78 (SEQ.ID.No.19): 5′-CACCCAATCGCAAACAGC-3′;
[0012] The forward primer sequence of SR87 (SEQ.ID.No.20): 5′-TCCCGCAAGGAAAAAGAG-3′;
[0013] The reverse primer sequence of SR87 (SEQ.ID.No.21): 5′-CAGCACAAACGAAAAATAAAAATA-3′;
[0014] The forward primer sequence of SR91 (SEQ.ID.No.22): 5′-ATCGCCCGTGTCTCCTGTTTC-3′;
[0015] The reverse primer sequence of SR91 (SEQ.ID.No.23): 5′-CGCAAGAAAACATCTCCACACAAGT-3′;
[0016] Forward primer sequence of SR106 (SEQ.ID.No.24): 5′-CAAAGTCGTGGGTCAGGTGTC-3′;
[0017] Reverse primer sequence of SR106 (SEQ.ID.No.25): 5′-CCACAAGAACCAGCCAATACAAA-3′;
[0018] Forward primer sequence of SR108 (SEQ.ID.No.26): 5′-AGGCACAACTAATAGCAAGCA-3′;
[0019] Reverse primer sequence of SR108 (SEQ.ID.No.27): 5′-CAATCGCTAACGCTGACG-3′;
[0020] The forward primer sequence of SR112 (SEQ.ID.No.28): 5′-GCAGCCGCCGACTACCTACT-3′;
[0021] Reverse primer sequence of SR112 (SEQ.ID.No.29): 5′-AAACCACCGCGAAGACAACA-3′;
[0022] Forward primer sequence of SR119 (SEQ.ID.No.30): 5′-GGACCTGACCATCCATCTAAACT-3′;
[0023] Reverse primer sequence of SR119 (SEQ.ID.No.31): 5′-TCGTTCTCGGTCGCTTTGT-3′;
[0024] Forward primer sequence of SR120 (SEQ.ID.No.32): 5′-CGGCTGCTCTTGGTTGATG-3′;
[0025] The reverse primer sequence of SR120 (SEQ.ID.No.33): 5′-CGGTTAGGTGGGCTGGTTC-3′;
[0026] Forward primer sequence of SR121 (SEQ.ID.No.34): 5′-GACAACGGAAATGTGCCTGAG-3′;
[0027] The reverse primer sequence of SR121 (SEQ.ID.No.35): 5′-TGGGTGGAGTTCCCTTTAGTTT-3′;
[0028] Forward primer sequence of SR125 (SEQ.ID.No.36): 5′-GCCGTCGCCAGTTCATTC-3′;
[0029] Reverse primer sequence of SR125 (SEQ.ID.No.37): 5′-CTGCCTGACTCCAAGAGGAAATA-3′;
[0030] Forward primer sequence of SR127 (SEQ.ID.No.38): 5′-GGGAATGGACAGATTGGTTGA-3′;
[0031] Reverse primer sequence of SR127 (SEQ.ID.No.39): 5′-GCTGACTGGTAACACGAAAGAAAAAAA-3′;
[0032] Forward primer sequence of SR129 (SEQ.ID.No.40): 5′-GCCGTCGCCAGTTCATTC-3′;
[0033] Reverse primer sequence of SR129 (SEQ.ID.No.41): 5′-CTGCCTGACTCCAAGAGGAAATA-3′;
[0034] Forward primer sequence of SR130 (SEQ.ID.No.42): 5′-CAGGTCCAGATGCCAAGC-3′;
[0035] The reverse primer sequence of SR130 (SEQ.ID.No.43): 5′-GACAACAGAATACTACATTACACGG-3′;
[0036] Forward primer sequence of SR131 (SEQ.ID.No.44): 5′-CAGGTCCAGATGCCAAGC-3′;
[0037] The reverse primer sequence of SR131 (SEQ. ID. No. 45): 5'-GACAACAGAATACTACATTACACGG-3'.
[0038] Another object of the present invention is to prepare a method for preparing specific primers for microsatellite molecular markers of Achyranthes paniculata, comprising:
[0039] RNA extraction and quality testing: Trizol method was used to extract RNA from Achyranthes bidentata samples. After RNA was obtained, Qubit was used to accurately quantify RNA concentration and Agilent 2100 was used to accurately detect RNA integrity. After the RNA samples were tested, equal amounts of different tissues were mixed.
[0040] Construction of cDNA library: SMARTer PCR cDNA Synthesis Kit was used to synthesize double-stranded cDNA chains; then the automatic nucleic acid collection system BluePippin was used for fragment screening, small molecule fragments in the library were removed, damage and end repair of the full-length cDNA were performed, and the library was obtained after ligation of SMRT dumbbell-shaped adapters and exonuclease digestion;
[0041] The constructed library was accurately quantified using the Qubit 2.0 fluorometer, and then the library size was tested using the Agilent 2100 to ensure the quality of the sequencing library.
[0042] The libraries that passed the library inspection were sequenced using the Pacbio Sequel platform. The software SMRTlink v6.0 was used to filter and process the data. The parameter --minLength=50 was used, and the final subreads were valid data.
[0043] Detection and search of SSR loci: MISA software was used for SSR detection and search;
[0044] SSR primer design: PrimerPremier 5 software was used to batch design SSR primers, and their polymorphism in different individuals of Arundophyllum indica was identified to obtain specific primers for Arundophyllum indica microsatellite molecular markers.
[0045] Another object of the present application embodiment is to use a specific primer of a microsatellite molecular marker of Agropyron truncatum in detecting genetic diversity of Agropyron truncatum population, including:
[0046] DNA extraction was performed on samples of individuals of the species of Puccinia splendens;
[0047] According to the specific primers of the microsatellite molecular marker of the above-mentioned Agropyron fasciatum, the DNA is subjected to PCR amplification reaction treatment;
[0048] The selected primers with high polymorphism were fluorescently labeled, and the 5' end of the fluorescent modification was labeled with 6-FAM. The fluorescently labeled forward primer and the common reverse primer were used for capillary electrophoresis amplification on the ABI3730XL DNA analyzer;
[0049] Population genetic diversity parameters were calculated to characterize microsatellite DNA polymorphisms associated with Achyranthes bidentata.
[0050] Another purpose of the embodiments of the present application is to apply the above-mentioned microsatellite molecular markers of Arundo donax or the above-mentioned specific primers in genetic diversity analysis, germplasm resource evaluation, gene positioning, variety identification, germplasm conservation, quantitative trait gene analysis and / or evolution and kinship research of Arundo donax.
[0051] In view of the current situation that the current technology has not yet developed microsatellite markers for Corydalis chinensis, the embodiments of the present application propose microsatellite molecular markers for Corydalis chinensis, specific primers, and preparation methods and applications thereof. Corydalis chinensis microsatellite molecular markers are developed and corresponding primers are obtained by using the transcriptome scanning technology obtained by the third-generation sequencing technology. A method for preparing microsatellite molecular markers for Corydalis chinensis is established, which provides molecular markers for the identification of Corydalis chinensis germplasm resources, genetic diversity analysis, construction of plant genetic maps, population genetic structure analysis, gene positioning, analysis of quantitative trait genes, evolution and kinship research, etc., lays a foundation for conducting genetic research on the genetic diversity, population structure, and historical population dynamics of Corydalis chinensis germplasm resources, and is conducive to the rational development and protection of Corydalis chinensis resources. BRIEF DESCRIPTION OF THE DRAWINGS
[0052] Figure 1 This is an electrophoresis gel image of RNA of a sample of Achyranthes bidentata provided in the examples of the present application.
[0053] Figure 2 This is a schematic diagram of the synthesis principle of double-stranded cDNA of Achyranthes paniculata provided in the examples of this application.
[0054] Figure 3 A distribution diagram of high-quality sequencing reads produced by a single molecule during sequencing provided in an embodiment of the present application, wherein the horizontal axis represents the sequence length and the vertical axis on the left represents the corresponding sequence quantity.
[0055] Figure 4 The subreads length distribution diagram provided in the embodiment of the present application, the horizontal axis is the sequence length, and the vertical axis on the left is the corresponding sequence amount.
[0056] Figure 5 This is a flow chart of transcriptome Iso-Seq analysis provided in the examples of this application.
[0057] Figure 6 Statistics on the number of different types of nucleotide SSRs in the transcriptome of Arundophylla provided in the examples of the present application.
[0058] Figure 7 This is the electrophoresis diagram of some SSR primer amplification of the Herba Cynoglossi provided in the examples of the present application.
[0059] Figure 8 This is the capillary electrophoresis diagram of three individuals in population No. 88 after amplification using primer No. 78 provided in the examples of the present application. DETAILED DESCRIPTION
[0060] In order to make the purpose, technical solution and advantages of the present application more clearly understood, the present application is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.
[0061] In view of the current situation that the current technology has not yet developed microsatellite markers for Corydalis chinensis, the embodiments of the present application propose microsatellite molecular markers, specific primers and their preparation methods and applications, develop Corydalis chinensis microsatellite molecular markers and obtain corresponding primers by using the transcriptome scanning technology obtained by the third-generation sequencing technology, establish a method for preparing microsatellite molecular markers for Corydalis chinensis, provide molecular markers for the identification of Corydalis chinensis germplasm resources, genetic diversity analysis, plant genetic map construction, population genetic structure analysis, gene positioning, analysis of quantitative trait genes, evolution and kinship research, lay a foundation for carrying out genetic research on genetic diversity, population structure, population historical dynamics and other aspects of Corydalis chinensis germplasm resources, and are conducive to the rational development and protection of Corydalis chinensis resources.
[0062] Example 1: A method for preparing specific primers for microsatellite molecular markers of Achyranthes paniculata, comprising the following steps:
[0063] S1: Extract RNA from P. truncatum and perform quality test: RNA from P. truncatum samples was extracted using the Trizol method. After RNA was obtained, the total RNA concentration and quality were tested using Nanodrop 2000 ultra-micro spectrophotometer, Agilent 2100 bioanalyzer, and agarose gel electrophoresis ( Figure 1 );
[0064] S2: cDNA library construction: After the RNA (or polyA+RNA) passes the test, the first cDNA chain is synthesized using the SMARTer PCR cDNA Synthesis Kit. Once SMARTScribe Reverse Transcriptase is added to the 5' end of the mRNA, the terminal repair transferase will add several additional base sequences to the 3' end of the cDNA. These bases serve as binding points for oligonucleotide synthesis, extending the first cDNA chain to the end of the oligonucleotide, generating a universal primer sequence that is conducive to the synthesis of the second cDNA chain and PCR amplification, thereby performing PCR amplification. The schematic diagram of double-stranded cDNA synthesis is shown in the figure below. Figure 2 As shown;
[0065] S3: After the library is constructed, Qubit 2.0 is used for preliminary quantification and the library is diluted. Then, the insert size of the library is detected using Agilent 2100. When the insert size is in line with expectations, the effective concentration of the library is accurately quantified using the Q-PCR method to ensure the quality of the library.
[0066] S4: Transcriptome sequencing: After the library is qualified, the SMRT (single molecule Real-time) single molecule real-time sequencing technology (third-generation sequencing) of the PacBio Sequel sequencing platform is used for full-length transcriptome sequencing analysis. The original sequencing data will contain data measured with 0, 1 or more than 2 molecules as templates, but the information of 0 or more than 2 molecules will cause great interference to the subsequent information analysis. The high-quality sequencing reads produced by a single molecule during the sequencing process are called polymerase reads ( Figure 3 ), this application uses the software SMRTlink v6.0 to filter and process the offline data, using the parameter --minLength=50, and the final Subreads obtained are valid data. The subread length distribution diagram is shown in Figure 4 , the original data of Achyranthes paniculata off-line are shown in Table 1 and Table 2;
[0067] Table 1: Statistics of raw sequence data
[0068]
[0069]
[0070] Table 2: Subreads statistics
[0071]
[0072] S5: Sequencing data quality assessment: The analysis process of obtaining the full-length transcriptome using the SMRTLink software Iso-Seq process mainly includes three stages: obtaining CCS sequences (Circular Consensus Sequences), full-length sequence identification (classify), and isoform level clustering (cluster). The analysis process is shown in Figure 5 .
[0073] S5.1: CCS: CCS sequences were extracted from the bam files of the subreads. The statistics of CCS sequences are shown in Table 3.
[0074] Table 3: Statistics of raw sequence data
[0075] Sequencing sample name Total number of CCS sequences Total base number of CCS sequence Average CCS sequence length hun 592753 671506184bp 1132bp
[0076] S5.2: Classify: According to whether there are 3' primers, 5' primers and PolyA in the sequence, the CCS sequence is divided into full-length sequence (FL) and non-full-length sequence (NFL). The primers and polyA / T tail sequences at both ends of the CCS sequence are removed, and the artificial chimeric sequences (artificial concatemers) of the full-length sequence are filtered out to obtain full-length non-chimeric sequences (FLNC, Full-Length, Non-Chimeric) and non-full-length sequences (NFL, Non-Full-Length). At the same time, the sequence strand information can also be identified based on the primer information and polyA / T tail.
[0077] The classification criteria include the following four parts:
[0078] (1) Contains 5' and 3' primers
[0079] (2) Sequence length greater than 300 bp
[0080] (3) Contains Poly A tail
[0081] (4) Does not contain chimeric sequences
[0082] The statistics of the full-length non-chimeric sequences obtained from the transcriptome of Arundophyllum spicatum are shown in Table 4 below;
[0083] Table 4: Statistics of classification results of Achyranthes paniculata CCS sequences
[0084]
[0085] S5.3: Cluster: Use the ICE (Iterative isoform-clustering) algorithm to cluster similar FLNC sequences into clusters through alignment, and obtain a consensus sequence (consensus isoforms) for each cluster. Combined with non-full-length sequences, the Quiver (or Arrow) algorithm is used to polish the consensus isoforms obtained by clustering, and high-quality (HQ, polished High Quality) sequences are selected for subsequent analysis.
[0086] The screening criteria for high-quality transcripts are:
[0087] (1) Full-length sequence (including both the cDNA primer and the polyA signal tail);
[0088] (2) High quality (predicted accuracy >= 0.99);
[0089] (3) Supported by more than 2 full-length sequences.
[0090] The statistical results of HQ and LQ transcripts obtained from different library constructions are shown in Table 5 below:
[0091] Table 5: Statistics of high-quality full-length non-chimera sequences
[0092]
[0093] S6: De-redundancy analysis: Due to the possible degradation of the 5' end of the RNA sequence during the experimental library construction process, and the high sensitivity and specificity standards of the cluster algorithm (ICE), redundant transcripts may exist in the polished sequence obtained by the Iso-Seq process. The polished high-quality transcript sequences were screened and de-redundancy was performed using the cd-hit software. The number of transcripts after de-redundancy was 30624, the total number of transcript bases after de-redundancy was 94880976, and the average length of the transcript sequence after de-redundancy was 3098bp;
[0094] S7: Detection and search of SSR sites: MISA (MIcroSAtellite: http: / / pgrc.ipk-gatersleben.de / misa) software was used to detect SSRs for mononucleotide, dinucleotide, trinucleotide, tetranucleotide, pentanucleotide and hexanucleotide transcripts with a minimum number of repeats of 10, 6, 5, 5, 5 and 5, respectively. A total of 14,089 SSR sites were searched, as shown in Table 6. The number of different types of nucleotide SSRs is shown in Figure 6 ;
[0095] Table 6: Statistics of the number of SSRs of different types
[0096] Repeat Type number Proportion frequency Average distribution distance (kb) Total length (bp) Average length (bp) Single Nucleotide 5196 36.88％ 16.97％ 18.26 67040 12.90 Dinucleotides 2977 21.13％ 9.72％ 31.87 25448 8.55 Trinucleotides 5543 39.34％ 18.10％ 17.12 31536 5.69 Tetranucleotide 269 1.91％ 0.88％ 352.73 1515 5.63 Pentanucleotide 53 0.38％ 0.17％ 1790.21 243 4.58 Hexanucleotide 51 0.36％ 0.17％ 1860.41 312 6.12 total 14089 100.00％ 46.00％ 6.73 192100 13.63
[0097] S8: SSR primer design: Based on the SSR loci screened out from the transcriptome data of Arundo donax, a total of 160 pairs of primers were designed, including 12 pairs of primers of 2-nucleotide repeat type, 10 pairs of primers of trinucleotide repeat type, and 80, 24, and 34 pairs of primers of tetranucleotide repeat type, five-nucleotide repeat type, and six-nucleotide repeat type. Six Arundo donax samples from different populations were randomly selected for primer screening. By judging the presence and size of the amplified bands, a total of 45 pairs of SSR primers with the required size range were screened out, and the remaining primers had no bands or multiple bands, with an amplification efficiency of 28.13%. In order to verify the universality and amplification efficiency of the primers, the 45 pairs of SSR primers screened out were selected for rescreening of 12 individuals from different populations, and finally 15 pairs of SSR primers with stable amplification, specificity, and no mixed bands were obtained ( Figure 7 ), including 13 pairs of tetranucleotide repeat type primers and 2 pairs of pentanucleotide repeat type primers.
[0098] Table 7: Information of 15 pairs of polymorphic SSR primers of Achyranthes bidentata
[0099]
[0100] The primer numbers of the 15 pairs of polymorphic primers for microsatellite molecular markers of the genus Paniculatus shown in Table 7 are SR57, SR78, SR87, SR91, SR106, SR108, SR112, SR119, SR120, SR121, SR125, SR127, SR129, SR130, and SR131. The nucleotide sequences are shown in SEQ.ID.No.1 to SEQ.ID.No.15, respectively.
[0101] Example 2: A method for analyzing the genetic diversity of Achyranthes paniculata using microsatellite molecular markers, comprising the following steps:
[0102] S1: 27 naturally distributed Achyranthes paniculata population samples were selected, 3 individuals were selected for each sample, and a total of 81 individuals were used for primer polymorphism analysis. The sample information is detailed in Table 8.
[0103] Table 8: Information of 27 population samples
[0104]
[0105]
[0106] S2: Using the above 15 pairs of primers, PCR amplification was performed on the extracted DNA of different individuals of P. sphenanthera. The PCR amplification reaction procedure was as follows: the first step, pre-denaturation at 94°C for 5 min; the second step (36 cycles), denaturation at 94°C for 30 s, annealing for 30 s (annealing temperature was set according to different primers in Table 6), and extension at 72°C for 30 s; the third step, extension at 72°C for 5 min; and storage at 4°C.
[0107] S3: Capillary electrophoresis detection: The selected primers with high polymorphism are fluorescently labeled, and the fluorescently modified 5' end is labeled with 6-FAM. The fluorescently labeled forward primer and the common reverse primer are used for capillary electrophoresis amplification on the ABI3730XL DNA analyzer. Figure 8 This is the capillary electrophoresis diagram of three individuals in population No. 88 after amplification with primer No. 78.
[0108] S4: Popgene v1.32 software was used to calculate population genetic diversity parameters to describe the characteristics of microsatellite DNA polymorphism associated with P. truncatula.
[0109] Based on the 15 pairs of SSR primers screened for Arundinaceae, PCR amplification products of 81 samples from 27 naturally distributed Arundinaceae populations were detected by capillary electrophoresis, and all 81 individuals were successfully amplified. Figure 8 The capillary electrophoresis diagram of primer 78 after amplification of 3 individuals in population 88. The polymorphism of 15 primer pairs based on 27 populations of Agropyron lanceolatum was analyzed, as shown in Table 9: 15 primer pairs amplified a total of 132 alleles in 81 individuals, with an average of 8.8 alleles per primer pair; the number of effective alleles ranged from 1.9719 to 7.9152, with an average of 4.7799; Shannon's information index (I) ranged from 0.9952 to 2.2747, with an average of 1.6959; the polymorphism information content (PIC) ranged from 1.9719 to 2.9915 ... The PIC value of primer S78 was the largest, indicating that the SSR locus showed high polymorphism; the observed heterozygosity (Ho) ranged from 0.3793 to 1, with an average observed heterozygosity of 0.8575; the expected heterozygosity (He) ranged from 0.6341 to 0.8814, with an average expected heterozygosity of 0.7648; the average expected heterozygosity of all SSR markers was lower than the average observed heterozygosity, indicating that the heterozygosity of the tested Achyranthes bidentata sample population was relatively high.
[0110] Table 9: Genetic diversity index information of 15 pairs of SSR primers based on 27 populations of Agropyron sphenanthera
[0111]
[0112]
[0113] The above shows and describes the basic principles, main features and advantages of the present application. Those skilled in the art should understand that the present application is not limited to the above embodiments. The above embodiments and descriptions are only for explaining the principles of the present application. Without departing from the spirit and scope of the present application, the present application may have various changes and improvements, which fall within the scope of the present application to be protected. The scope of protection claimed in the present application is defined by the attached claims and their equivalents.
Claims
1. Microsatellite molecular markers of Arundophyllum spicatum, characterized in that: The microsatellite molecular markers of the Achyranthes bidentata are SR57, SR78, SR87, SR91, SR106, SR108, SR112, SR119, SR120, SR121, SR125, SR127, SR129, SR130 and SR131, and their nucleotide sequences are shown in SEQ.ID.No.1-SEQ.ID.No.
15.
2. The microsatellite molecular marker of Achyranthes paniculata according to claim 1, characterized in that: The repeating sequence of SR57 is (GTTGG)5; the repeating sequence of SR78 is (GTAG)5; The repeat sequence of SR87 is (CAAT)5; the repeat sequence of SR91 is (GAGCCT)5; The repeating sequence of SR106 is (GGTT)7; the repeating sequence of SR108 is (TGTA) 10 ; The repeating sequence of SR112 is (GCCT)6; the repeating sequence of SR119 is (ATTT)5; The repeating sequence of SR120 is (CTAT) 10 ; The repeating sequence of SR121 is (TGAC)6; The repeating sequence of SR125 is (ACGC)9; the repeating sequence of SR127 is (GTAT)5; The repeating sequence of SR129 is (ACGC)9; the repeating sequence of SR130 is (AGAT)5; The repeating sequence of SR131 is (AGAT)5.
3. The specific primers for the microsatellite molecular marker of Achyranthes paniculata according to any one of claims 1 or 2, characterized in that: The forward and reverse sequences of the primers of SR57 are shown in SEQ.ID.No.16-SEQ.ID.No.17; The forward and reverse sequences of the primers of SR78 are shown in SEQ.ID.No.18-SEQ.ID.No.19; The forward and reverse sequences of the primers of SR87 are shown as SEQ.ID.No.20-SEQ.ID.No.21; The forward and reverse sequences of the primers of SR91 are shown as SEQ.ID.No.22-SEQ.ID.No.23; The forward and reverse sequences of the primers of SR106 are shown in SEQ.ID.No.24-SEQ.ID.No.25; The forward and reverse sequences of the primers of SR108 are shown in SEQ.ID.No.26-SEQ.ID.No.27; The forward and reverse sequences of the primers of SR112 are shown in SEQ.ID.No.28-SEQ.ID.No.29; The forward and reverse sequences of the primers of SR119 are shown in SEQ.ID.No.30-SEQ.ID.No.31; The forward and reverse sequences of the primers of SR120 are shown in SEQ.ID.No.32-SEQ.ID.No.33; The forward and reverse sequences of the primers of SR121 are shown in SEQ.ID.No.34-SEQ.ID.No.35; The forward and reverse sequences of the primers of SR125 are shown in SEQ.ID.No.36-SEQ.ID.No.37; The forward and reverse sequences of the primers of SR127 are shown in SEQ.ID.No.38-SEQ.ID.No.39; The forward and reverse sequences of the primers of SR129 are shown in SEQ.ID.No.40-SEQ.ID.No.41; The forward and reverse sequences of the primers of SR130 are shown in SEQ.ID.No.42-SEQ.ID.No.43; The forward and reverse sequences of the primers of SR131 are shown as SEQ.ID.No.44-SEQ.ID.No.
45.
4. The method for preparing specific primers for microsatellite molecular markers of Achyranthes paniculata according to claim 3, characterized in that: include: RNA extraction and quality testing: Trizol method was used to extract RNA from Achyranthes bidentata samples. After RNA was obtained, Qubit was used to accurately quantify RNA concentration and Agilent 2100 was used to accurately detect RNA integrity. After the RNA samples were tested, equal amounts of different tissues were mixed. Construction of cDNA library: SMARTerPCR cDNA Synthesis Kit was used to synthesize double-stranded cDNA chains; then the automatic nucleic acid collection system BluePippin was used for fragment screening, small molecule fragments in the library were removed, damage and end repair of the full-length cDNA were performed, and the library was obtained after ligation of SMRT dumbbell-shaped adapters and exonuclease digestion; The constructed library was accurately quantified using the Qubit 2.0 fluorometer, and then the library size was tested using the Agilent 2100 to ensure the quality of the sequencing library. The libraries that passed the library inspection were sequenced using the Pacbio Sequel platform. The software SMRTlink v6.0 was used to filter and process the data. The parameter --minLength=50 was used, and the final subreads were valid data. Detection and search of SSR loci: MISA software was used for SSR detection and search; SSR primer design: Primer 5 software was used to batch design SSR primers, and their polymorphism in different individuals of Arundophyllum indica was identified to obtain specific primers for Arundophyllum indica microsatellite molecular markers.
5. The method for using the specific primers of the microsatellite molecular markers of the Agropyron truncatum according to claim 3 in detecting the genetic diversity of Agropyron truncatum populations, characterized in that: include: DNA extraction was performed on samples of individuals of the species of Puccinia splendens; According to the specific primers of the microsatellite molecular marker of Achyranthes paniculate according to claim 3, the DNA is subjected to PCR amplification reaction treatment; The selected primers with high polymorphism were fluorescently labeled, and the 5' end of the fluorescently modified primer was labeled with 6-FAM. The fluorescently labeled forward primer and the common reverse primer were used for capillary electrophoresis amplification on the ABI3730XL DNA analyzer. Population genetic diversity parameters were calculated to characterize microsatellite DNA polymorphisms associated with Achyranthes bidentata.
6. The application method according to claim 5, characterized in that: The PCR amplification reaction procedure is: (1) Pre-denaturation at 94°C for 5 min; (2) denaturation at 94°C for 30 s, annealing at different annealing temperatures for 30 s, and extension at 72°C for 30 s, for 36 cycles; The annealing temperatures of the 15 primer pairs are as follows: SR87: 51°C; SR108: 55°C; SR78: 56.5°C; SR57, SR91, SR106, SR119, SR121, SR130 are 57.5°C; SR112, SR125, SR129, SR131 are 59.5°C; SR120, SR127 are 60.5°C; (3) 72°C extension for 5 min; (4) Store at 4℃.
7. Application of the microsatellite molecular marker of Agropyron truncatum according to claim 1 or the specific primer of claim 3 in genetic diversity analysis, germplasm resource evaluation, gene positioning, variety identification, germplasm conservation, quantitative trait gene analysis and / or evolution and kinship research of Agropyron truncatum.