Ammopiptanthus nanus germplasm resource identification method based on EST-SSR (expressed sequence tag-simple sequence repeat) marker

Abstract

The invention discloses an Ammopiptanthus nanus germplasm resource identification method based on an EST-SSR (expressed sequence tag-simple sequence repeat) marker, and belongs to the technical field of molecular genetics. A bioinformatics method is used, an EST-SSR molecular marker combination suitable for identifying the ammopiptanthus nanus germplasm resources is screened out, and the EST-SSR molecular marker combination comprises molecular markers F1, F2, F3, F4, F5, F6, F7, F8, F9, F10, F11, F12, F13, F14 and F15. The source of the ammopiptanthus nanus germplasm resources of different populations can be well identified by utilizing the EST-SSR molecular marker system, and powerful support is provided for efficient utilization and protection of the ammopiptanthus nanus.

Description

Technical Field

[0001] The invention relates to the technical field of molecular genetics, and in particular to a method for identifying Xinjiang Ammopiptanthus mongolicus germplasm resources based on EST-SSR markers. Background Art

[0002] Ex situ conservation is an effective means of protecting species. Through ex situ conservation, scientists and practitioners can better understand the morphological characteristics, systematic and evolutionary relationships, and growth and development of protected organisms, thereby laying the foundation for population reconstruction of wild species and providing a reference for formulating effective protection and sustainable utilization of Xinjiang Ammopiptanthus germplasm resources. However, we do not know whether the existing ex situ plant species contain the same adaptive evolutionary potential and genetic resources as wild species. Therefore, it is crucial to use molecular genetics technology to analyze the genetic diversity of Xinjiang Ammopiptanthus in different populations, in order to identify the source of Xinjiang Ammopiptanthus germplasm resources and effectively protect it.

[0003] Microsatellite (simple sequence repeat, SSR) refers to a DNA simple tandem repeat sequence, which consists of a core sequence and flanking sequences on both sides. The core sequence is a repeating unit of 1-6 nucleotides. The differences in the number of repeats and repeating units between different alleles make it length polymorphic. The core sequence is composed of conservative flanking sequences on both sides, which have high homology between species and can therefore be used for primer design. Compared with other molecular markers, microsatellites have the advantages of large number, good stability, rich polymorphism information, co-dominance and easy detection. At the same time, microsatellite analysis based on PCR technology has low requirements for template DNA, and nanogram amounts or partially broken DNA can be effectively analyzed. Therefore, microsatellite markers are increasingly used in genetic studies of endangered species. The characteristics and co-dominance of SSR markers make them the first choice for assessing plant genetic diversity. The high conservatism of DNA markers, especially EST-SSR, makes them more accurate in predicting genetic differences and more common in similar species. For example, explaining the evolutionary history of endangered species, analyzing population genetic structure, assisting population surveys, identifying kinship and identifying closely related species and hybrid individuals are key conservation biology tasks. However, although some studies have found that microsatellite DNA flanking sequences are somewhat conserved among closely related species, for some rare and endangered species, when there is a lack of reference microsatellite sequence information of related closely related species, it is necessary to screen microsatellite loci and develop specific primers. In addition, the genetic diversity of endangered species is generally low, so the development of polymorphic microsatellite loci is more difficult and more important.

[0004] Xinjiang Ammopiptanthus nanus is a member of the genus Ammopiptanthus of the Leguminosae family and a national second-level key protected wild plant. Xinjiang Ammopiptanthus nanus is mainly distributed in Xinjiang, and is only distributed in Wuqia County, Atushi City and Akto County of Kizilsu Kirgiz Prefecture, Xinjiang, in the low mountain belt with an altitude of about 2100-2700 meters, and the soil matrix is ​​a gravel soil with strong lithification. The huge root structure of Xinjiang Ammopiptanthus nanus enables it to survive on barren mountains and rocky Gobi with severe drought, and it has the characteristics of anti-premature aging, cold resistance, barrenness resistance and well-developed root system. This shrub has broad leaves, is evergreen in desert areas, has thick leaves, golden corolla, and short plants, and can also be used as an ornamental plant. In addition, Xinjiang Ammopiptanthus nanus is also of great value in the research and development of the biological industry. Its stems, branches, leaves and seeds contain various alkaloids and flavonoid compounds. Xinjiang Ammopiptanthus is a relatively primitive legume plant. It is a third-generation surviving relict plant on the coast of the ancient Mediterranean Sea and existed before the retreat of the ancient Mediterranean Sea. Therefore, Xinjiang Ammopiptanthus also provides an important scientific basis for studying the climate characteristics of Central Asia in the Tertiary, the geographical effects of environmental changes, plant migration, and even the origin and formation of desert vegetation. Xinjiang Ammopiptanthus is mainly distributed in arid and cold environments, with fragile ecology and generally harsher environmental conditions. Due to changes in climate and geographical location and other ecological characteristics, the ex situ conservation of Xinjiang Ammopiptanthus has not been successful, and its distribution area is showing a trend of continuous and rapid shrinkage. There are few studies on the detailed analysis and evaluation of its ex situ conservation effectiveness using molecular marker technology. Therefore, the development of efficient and powerful molecular markers is crucial for the identification and efficient protection of Xinjiang Ammopiptanthus germplasm resources. Summary of the invention

[0005] The purpose of the present invention is to provide a method for identifying the germplasm resources of Ammopiptanthus mongolicus in Xinjiang based on EST-SSR markers to solve the problems existing in the above-mentioned prior art. The EST-SSR molecular marker combination provided by the present invention is used to detect the PCR amplification product, which can be used for the evaluation of the genetic diversity of Ammopiptanthus mongolicus in Xinjiang and the study of the kinship of different populations. It can quickly, accurately and high-throughput identify the sources of the germplasm resources of Ammopiptanthus mongolicus in Xinjiang from different populations, and has important guiding significance in formulating strategies for the identification, effective protection and sustainable utilization of Ammopiptanthus mongolicus germplasm resources in Xinjiang.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides an EST-SS molecular marker combination of Xinjiang Ammopiptanthus mongolicus, wherein the molecular marker combination comprises molecular markers F1, F2, F3, F4, F5, F6, F7, F8, F9, F10, F11, F12, F13, F14 and F15;

[0008] The F1 is obtained by amplifying the primer pair shown in SEQ ID NO.1-2;

[0009] The F2 is obtained by amplification using the primer pair shown in SEQ ID NO.3-4;

[0010] The F3 is obtained by amplification using the primer pair shown in SEQ ID NO.5-6;

[0011] The F4 is obtained by amplification using the primer pair shown in SEQ ID NO.7-8;

[0012] The F5 is obtained by amplification using the primer pair shown in SEQ ID NO.9-10;

[0013] The F6 is obtained by amplification using the primer pair shown in SEQ ID NO.11-12;

[0014] The F7 is obtained by amplification using the primer pair shown in SEQ ID NO.13-14;

[0015] The F8 is obtained by amplification using the primer pair shown in SEQ ID NO.15-16;

[0016] The F9 is obtained by amplification using the primer pair shown in SEQ ID NO.17-18;

[0017] The F10 is obtained by amplification of the primer pair shown in SEQ ID NO.19-20;

[0018] The F11 is obtained by amplification using the primer pair shown in SEQ ID NO.21-22;

[0019] The F12 is obtained by amplification using the primer pair shown in SEQ ID NO.23-24;

[0020] The F13 is obtained by amplification using the primer pair shown in SEQ ID NO.25-26;

[0021] The F14 was obtained by amplification using the primer pair shown in SEQ ID NO.27-28;

[0022] The F15 was obtained by amplification using the primer pair shown in SEQ ID NO. 29-30.

[0023] The present invention also provides a primer combination for amplifying the EST-SS molecular marker combination, wherein the primer combination comprises 15 pairs of primer pairs, specifically as shown in SEQ ID NO.1-30.

[0024] The present invention also provides an application of the primer combination in preparing a kit for identifying Xinjiang Ammopiptanthus mongolicus germplasm resources.

[0025] The present invention also provides a kit for identifying Xinjiang Ammopiptanthus mongolicus germplasm resources, comprising the primer combination.

[0026] The present invention also provides a use of the primer combination or the kit in any of the following:

[0027] (1) Evaluation of genetic diversity of Ammopiptanthus mongolicus in Xinjiang;

[0028] (2) Analysis of genetic structure of Ammopiptanthus mongolicus population in Xinjiang;

[0029] (3) Research on the evolution and genetic relationship of Ammopiptanthus mongolicus in Xinjiang;

[0030] (4) Identification of Ammopiptanthus mongolicus germplasm resources in Xinjiang.

[0031] The present invention also provides a method for identifying the germplasm resources of Ammopiptanthus mongolicus in Xinjiang, comprising the following steps:

[0032] Extract genomic DNA from Ammopiptanthus mongolicus;

[0033] Performing PCR amplification using the primer combination;

[0034] The amplified products were tested by capillary electrophoresis, and genotyping was performed based on the test results;

[0035] According to the results of genotyping, genetic diversity analysis, genetic differentiation analysis and genetic structure analysis of Xinjiang Ammopiptanthus population were carried out. According to the analysis results, the variety source of the Xinjiang Ammopiptanthus samples to be tested was identified, and the group to which the Xinjiang Ammopiptanthus germplasm resources belonged was determined.

[0036] Furthermore, the reaction system of the PCR amplification is: 1 μL of template DNA, 1 μL of 10 μM forward and reverse primers, 12.5 μL of 2×EasyTaqRPCR SuperMix, and 9.5 μL of Nuclease-free Water.

[0037] Furthermore, the reaction procedure of the PCR amplification is: pre-denaturation at 93°C for 3 min, denaturation at 94°C for 30 s, annealing at 56°C for 30 s, extension at 65°C for 90 s, for a total of 30 cycles, and finally repair extension at 65°C for 5 min.

[0038] The present invention discloses the following technical effects:

[0039] The present invention uses a bioinformatics method to screen out an EST-SS molecular marker combination suitable for identifying the source of Xinjiang Ammopiptanthus germplasm resources, and the marker combination can also be used to analyze the genetic structure of the species among different populations and the genetic diversity of the population. The EST-SS molecular marker combination provided by the present invention is helpful to analyze and understand the genetic variation distribution pattern of Xinjiang Ammopiptanthus in different populations, so as to identify the source of Xinjiang Ammopiptanthus seed resources in different populations, and provide a theoretical basis for the efficient utilization and protection of Xinjiang Ammopiptanthus germplasm resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0041] Figure 1 This is the EST-SSR typing map of 12 Xinjiang Ammopiptanthus mongolicus samples DNA amplified by primers at the F3 locus;

[0042] Figure 2 This is the EST-SSR typing map of 12 Xinjiang Ammopiptanthus mongolicus samples DNA amplified by primers at the F5 locus;

[0043] Figure 3 This is the EST-SSR typing map of 12 Xinjiang Ammopiptanthus mongolicus samples DNA amplified by primers at F6 locus;

[0044] Figure 4 This is the EST-SSR typing map of 12 Xinjiang Ammopiptanthus mongolicus samples DNA amplified by primers at the F7 locus;

[0045] Figure 5 This is the EST-SSR typing map of 12 Xinjiang Ammopiptanthus mongolicus samples DNA amplified by primers at the F9 locus;

[0046] Figure 6 This is the EST-SSR typing map of 12 Xinjiang Ammopiptanthus mongolicus samples DNA amplified by primers at the F10 locus;

[0047] Figure 7 This is the EST-SSR typing map of 12 Xinjiang Ammopiptanthus mongolicus samples DNA amplified by primers at the F11 locus;

[0048] Figure 8 This is the EST-SSR typing map of 12 Xinjiang Ammopiptanthus mongolicus samples DNA amplified by primers at the F12 locus;

[0049] Figures 1 to 8 A1 to A12 in the figure represent 12 Ammopiptanthus mongolicus samples from Xinjiang;

[0050] Fig. 9 To analyze the genetic structure of Ammopiptanthus mongolicus in Xinjiang based on Bayesian model;

[0051] Fig.10 This is the principal coordinate analysis of three Xinjiang Ammopiptanthus populations based on EST-SSR genetic distance. DETAILED DESCRIPTION

[0052] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0053] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0054] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0055] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0056] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0057] Example 1 Extraction of polymorphic microsatellite markers of Ammopiptanthus mongolicus and primer design

[0058] 1. Extraction of microsatellite loci and primer design

[0059] Based on the transcriptome sequencing of Ammopiptanthus nanus, the microsatellite loci were detected using MISA software, and the microsatellite sequences on the transcriptome of Ammopiptanthus nanus were screened. Primer 3.0 software was used to design primers on both sides of the SSR loci.

[0060] 2. Screening of polymorphic microsatellite primers

[0061] 1) Extraction of leaf DNA: Genomic DNA from leaves was extracted using a plant genomic DNA extraction kit. A total of 308 DNA samples were extracted, and the collected DNA solution was divided into two equal parts, labeled with number information, and the mother solution was stored at -80°C for future molecular experiments, and the sub-liquid was stored at -20°C for subsequent molecular experiments to construct a Xinjiang Ammopiptanthus mongolicus DNA template library.

[0062] 2) After preliminary detection by agarose gel electrophoresis, 15 pairs of EST-SSR primers with bright bands were selected for synthesis among the designed microsatellite primers, and the 5' end of the single primer was fluorescently labeled (HEX-red). Templates with better quality were selected from the Xinjiang Ammopiptanthus mongolicus DNA template library (containing 308 Xinjiang Ammopiptanthus mongolicus leaf DNAs, and DNA quality was detected by agarose gel electrophoresis) for primer amplification specificity detection and initial polymorphism screening.

[0063] PCR amplification used the 2×EasyTaq PCR SuperMix kit produced by Tiangen Biochemical Technology (Beijing) Co., Ltd. The amplification system and reaction procedure were as follows: 25 μL reaction system: 1 μL template DNA, 1 μL each of 10 μM forward and reverse primers, 12.5 μL 2×EasyTaqRPCR SuperMix, and 9.5 μL Nuclease-free Water.

[0064] The PCR amplification program was as follows: pre-denaturation at 93°C for 3 min, denaturation at 94°C for 30 s, annealing at 56°C for 30 s, extension at 65°C for 90 s, for a total of 30 cycles, and finally repair extension at 65°C for 5 min.

[0065] 3) The PCR reaction products were subjected to 2% agarose gel electrophoresis at 130 V for 30 min. After the electrophoresis, the agarose was exposed for detection, clear bands were screened out based on the exposure results, and the optimal annealing temperature for each pair of primers was determined based on the clarity of the bands.

[0066] 4) After PCR amplification, the target product was sent to the company (Shanghai Shenggong Biotechnology Co., Ltd.) for capillary electrophoresis typing of microsatellite marker alleles, and the original electrophoresis data results were manually verified and confirmed SSR amplification bands, and finally the bands were interpreted. 15 stable microsatellite loci were obtained, marked as F1, F2, F3, F4, F5, F6, F7, F8, F9, F10, F11, F12, F13, F14 and F15. The 15 microsatellite loci and the corresponding primer sequences are shown in Table 1. The EST-SSR typing diagram of the DNA of some Xinjiang Ammopiptanthus samples amplified by primers for some microsatellite loci is shown in Figures 1 to 8 shown.

[0067] Table 115 microsatellite loci and corresponding primer sequences

[0068]

[0069]

[0070]

[0071] Example 2 Identification of polymorphism of microsatellite markers in Ammopiptanthus mongolicus

[0072] 15 pairs of primers corresponding to the 15 microsatellite loci screened and obtained in Example 1 were synthesized with fluorescent primers, and the 5' end of the single-side primer was fluorescently labeled (HEX-red); 308 leaf DNAs of Xinjiang Ammopiptanthus mongolicus in the Xinjiang Ammopiptanthus mongolicus DNA template library constructed in Example 1 were used as templates for PCR amplification, and the amplification system and reaction conditions were the same as in Example 1. The amplified products were detected by capillary fluorescence electrophoresis on an ABI 3730XL sequencer, and data such as alleles were counted according to the electrophoresis results.

[0073] GeneMaker software was used to separate the collected data by fluorescence color, calculate the allele size, and then determine the genotyping data of each locus. The GenAlex plug-in and Powermarker were used to determine the genetic index to further evaluate the polymorphism of the microsatellite loci. The results of the polymorphism detection are shown in Table 2.

[0074] Table 2 Polymorphism of microsatellite loci in Ammopiptanthus mongolicus

[0075]

[0076] The data in Table 2 show that the total genetic diversity (Ht) of the 15 primer pairs detected in 308 samples ranged from 0.691 to 0.904, with an average of 0.83. The observed mean heterozygosity (Mean Ho) ranged from 0 to 0.55, with an average of 0.25. The mean expected heterozygosity (Mean He) ranged from 0.218 to 0.716, with an average of 0.47. The inbreeding coefficient within the population (Fis), the total inbreeding coefficient of the population (Fit), and the genetic differentiation coefficient (Fst) ranged from -0.021 to 1, 0.326 to 1, and 0.208 to 0.733, respectively. The gene flow (Nm) ranged from 0.091 to 0.951, with an average of 0.43. Nm values ​​less than 1 indicate that the differentiation between populations may be caused by genetic drift, and the level of gene flow between most populations is low. As shown in Table 2, F6 is the most effective primer, followed by F3 and F1. F2 produced fewer effective results. It can be seen that the 15 microsatellite molecular markers of the present invention can be used for germplasm resource identification, genetic diversity, population genetic structure, evolution and kinship research, etc., and have good application prospects.

[0077] Example 3 Application of Microsatellite Molecular Markers in Identifying the Germplasm Resources of Ammopiptanthus mongolicus in Xinjiang

[0078] Based on the known distribution points investigated in the past, we queried and collected literature and consulted the specimens of A. mongolica collected and preserved in botanical gardens, herbariums, natural museums and herbariums of various scientific research institutions. After visiting relevant plant experts and scholars, we determined the population distribution points of A. mongolica in Xinjiang, and conducted a detailed field survey of the A. mongolica population in Xinjiang. We also collected plant samples for identifying the efficacy of ex situ conservation based on ETS-SSR markers from Pop1 Wuqia (50 samples) and Pop2 ex situ conservation areas (24 samples). In addition, we collected 13 A. mongolica plant samples from Pop3 Kyrgyzstan (the specific geographical locations of the three populations are shown in Table 3) to analyze the genetic diversity of different populations and provide strong support for the protection of A. mongolica germplasm resources in Xinjiang.

[0079] Table 3 Geographical location of samples

[0080] social quantity latitude longitude altitude Pop1UQIA 50 servings 39.7°N 75.01°E 2128m Pop2 Ex Situ Conservation Area 24 servings 39.72°N 75.27°E 2178m Pop3 Kyrgyzstan 13 servings 41.71°N 74.34°E 1698m

[0081] The leaf DNA of 87 Ammopiptanthus mongolicus from different regions was used as a template, and PCR amplification was performed after fluorescence synthesis of 15 pairs of primers screened in Example 1. The amplification system and reaction conditions were the same as in Example 1. The amplified product was detected by capillary fluorescence electrophoresis on an ABI 3730XL sequencer, and data such as alleles were statistically analyzed based on the electrophoresis results. GeneMaker software was used to perform fluorescence color separation on the collected data, calculate the allele size, and then determine the genotyping data of each locus. Genetic index determination was performed using the GenAlex plug-in and Powermarker. The results of the genetic diversity index determination of the Ammopiptanthus mongolicus population in Xinjiang are shown in Table 3, the genetic distance and genetic differentiation coefficient are shown in Table 4, and the molecular variance analysis of genetic variation within and between Ammopiptanthus mongolicus populations in Xinjiang is shown in Table 5.

[0082] Table 3 Genetic diversity index of Ammopiptanthus mongolicus population in Xinjiang

[0083] Pop N Na Ne I Ho He u F PPB Uchia 31.867 6.333 2.598 1.121 0.29 0.535 0.552 0.514 0.9333 Ex situ conservation areas 16.667 5 2.714 0.957 0.231 0.453 0.476 0.437 0.8 Kyrgyzstan 7.533 3.6 2.429 0.827 0.242 0.409 0.449 0.456 0.7333 average value 18.689 4.978 2.58 0.969 0.254 0.466 0.492 0.472 0.8222 total 56.067 14.933 7.741 2.905 0.763 1.397 1.477 1.407 2.4666

[0084] Table 4 Genetic distance and genetic differentiation coefficient of Ammopiptanthus mongolicus population in Xinjiang

[0085]

[0086] Table 5 Molecular variance analysis of genetic variation within and between populations of Ammopiptanthus mongolicus in Xinjiang

[0087] Source DF SS MS Est.Var. Proportion AmongPops 2 211.259 105.629 1.956 29％ AmongIndiv 84 707.678 8.425 3.583 53％ WithinIndiv 87 109.5 1.259 1.259 18％ Total 173 1028.437 6.797 1

[0088] Since plant populations with higher genetic diversity are more likely to contain rare or unique genes, these genes are of great significance for the protection and utilization of germplasm resources. By protecting these genetic diversities, rich genetic resources can be provided for future breeding work. As shown in Table 3, 15 pairs of EST-SSR primers measured the population genetic diversity of Xinjiang Ammopiptanthus. The number of alleles (Na) in different populations was different, which indicated that there were differences in the number of alleles in different populations, and the number of alleles in Pop1 Wuqia population was relatively large; the effective number of alleles (Ne) ranged from 2.429 to 2.714, with an average of 2.58; the Shannon information index (I) is an indicator to measure the genetic diversity of a population. The higher the value, the higher the genetic diversity. Here it is shown that there are differences in this index between different populations, among which the genetic diversity of Pop1 Wuqia population is relatively high; the observed heterozygosity The expected heterozygosity (Ho) ranges from 0.231 to 0.29, with an average of 0.254. The expected heterozygosity (He) ranges from 0.409 to 0.535, with an average of 0.466. The population genetic diversity parameter (PPB) ranges from 0.7333 to 0.9333, with an average of 82.22%, the highest of which is 93.33% in Wuqia area. Therefore, in terms of protecting the germplasm resources of A. nanus, Pop1 Wuqia area should be the priority area.

[0089] Genetic differentiation leads to genetic differences within and between plant populations. This difference increases the diversity of germplasm resources and helps plants adapt to different environmental conditions. Understanding the laws of plant genetic differentiation is helpful to scientifically protect and utilize germplasm resources and avoid the loss and loss of germplasm resources. In Table 4, the Nei genetic distance between Xinjiang Ammopiptanthus populations is 2.659-4.9, among which the genetic distance between the Wuqia population and the Kyrgyzstan population is the largest, and the genetic distance between the ex situ population and the Kyrgyzstan population is the smallest. The genetic differentiation coefficient (FST) between populations is between 0.352 and 0.431, the genetic differentiation index between the Wuqia and Kyrgyzstan populations is the largest, and the genetic differentiation index between the Wuqia and Kyrgyzstan populations is the smallest.

[0090] In the molecular variance analysis of genetic variation, the sources of variation can be decomposed into different levels, such as variation between populations, variation between individuals within a population, etc. Through this analysis, we can understand the impact of different environmental factors or geographical isolation on the genetic diversity of germplasm resources, so as to formulate effective germplasm resource protection strategies and management measures. Table 5 shows the molecular variance analysis of genetic variation within and between populations of Ammopiptanthus mongolicus in Xinjiang. The data show that among the 87 Ammopiptanthus mongolicus germplasms collected from three populations, 29% of the genetic variation originated from between populations, while nearly 71% of the genetic variation originated from within populations. StructureHarvester can help identify different population structures in germplasm resources, which is of great significance for understanding the evolutionary history and geographical distribution of species. Structure Harvester was used to analyze the population genetic structure and calculate the average value corresponding to each K value. The maximum inflection point appeared when K=3. This result also revealed that the 87 Ammopiptanthus mongolicus samples in Xinjiang can be divided into three genetic populations in theory. It shows that there is obvious genetic differentiation in the Ammopiptanthus mongolicus population in Xinjiang, which may be related to the different natural conditions in which they are located. This genetic structure analysis helps to understand the genetic differences between germplasm resource populations and provides a scientific basis for formulating effective germplasm resource protection strategies ( Fig. 9 ).

[0091] Fig.10 This is the principal coordinate analysis of three Xinjiang Ammopiptanthus populations based on EST-SSR genetic distance. The figure shows the distribution of the three Xinjiang Ammopiptanthus populations in terms of genetic distance, and the principal coordinate analysis is used to reveal the differences and similarities in their genetic relationships. Different colors and shapes represent different populations: blue diamonds for the Wuqia population, red squares for the ex situ population, and green triangles for the Kyrgyzstan population. The axis labels refer to the percentage of explained variance for each principal coordinate. The results showed that although the Xinjiang Ammopiptanthus samples from the three populations still belong to the same population, the degree of differentiation is extremely significant due to different natural conditions. There are a large number of genetic differences both within and between populations, and the variation within populations is greater. The high degree of genetic differentiation between populations may be the result of genetic drift, or it may be the combined effect of natural selection and genetic differentiation caused by gene flow between places. Principal component analysis ( Fig.10 ) further verified the group structure ( Fig. 9 ) and also revealed the closeness of relationship and degree of genetic differentiation between germplasm resource groups, and then classified and identified the Xinjiang Ammopiptanthus germplasm resources of different populations, which is of great value for the collection, preservation and utilization of species germplasm resources.

[0092] The above-mentioned 15 A. mongolica microsatellite loci can be used to identify the germplasm resource sources, genetic diversity and kinship of different populations of A. mongolica. In addition, the A. mongolica microsatellite system can also evaluate the ex situ conservation effectiveness of A. mongolica, and has very important application value for the evaluation, identification and utilization of its germplasm resources.

[0093] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. An EST-SSR molecular marker combination of Ammopiptanthus mongolicus, characterized in that: The EST-SSR molecular marker combination includes molecular markers F1, F2, F3, F4, F5, F6, F7, F8, F9, F10, F11, F12, F13, F14 and F15; The F1 is obtained by amplifying the primer pair shown in SEQ ID NO.1-2; The F2 is obtained by amplification using the primer pair shown in SEQ ID NO.3-4; The F3 is obtained by amplification using the primer pair shown in SEQ ID NO.5-6; The F4 is obtained by amplification using the primer pair shown in SEQ ID NO.7-8; The F5 is obtained by amplification using the primer pair shown in SEQ ID NO.9-10; The F6 is obtained by amplification using the primer pair shown in SEQ ID NO.11-12; The F7 is obtained by amplification using the primer pair shown in SEQ ID NO.13-14; The F8 is obtained by amplification using the primer pair shown in SEQ ID NO.15-16; The F9 is obtained by amplification using the primer pair shown in SEQ ID NO.17-18; The F10 is obtained by amplification of the primer pair shown in SEQ ID NO.19-20; The F11 is obtained by amplification using the primer pair shown in SEQ ID NO.21-22; The F12 is obtained by amplification using the primer pair shown in SEQ ID NO.23-24; The F13 is obtained by amplification using the primer pair shown in SEQ ID NO.25-26; The F14 was obtained by amplification using the primer pair shown in SEQ ID NO.27-28; The F15 was obtained by amplification using the primer pair shown in SEQ ID NO. 29-30.

2. A primer combination for amplifying the EST-SSR molecular marker combination according to claim 1, characterized in that: The primer combination includes 15 pairs of primers, as shown in SEQ ID NO.1-30.

3. Use of the primer combination as claimed in claim 2 in preparing a kit for identifying Xinjiang Ammopiptanthus germplasm resources.

4. A kit for identifying the germplasm resources of Ammopiptanthus mongolicus in Xinjiang, characterized in that: Comprising the primer combination of claim 2.

5. Use of the primer combination according to claim 2 or the kit according to claim 4 in any of the following: (1) Evaluation of genetic diversity of Ammopiptanthus mongolicus in Xinjiang; (2) Analysis of genetic structure of Ammopiptanthus mongolicus population in Xinjiang; (3) Research on the evolution and genetic relationship of Ammopiptanthus mongolicus in Xinjiang; (4) Identification of Ammopiptanthus mongolicus germplasm resources in Xinjiang.

6. A method for identifying the germplasm resources of Ammopiptanthus mongolicus in Xinjiang, characterized in that: The following steps are involved: Extract genomic DNA from Ammopiptanthus mongolicus; Performing PCR amplification using the primer combination described in claim 2; The amplified products were tested by capillary electrophoresis, and genotyping was performed based on the test results; According to the results of genotyping, genetic diversity analysis, genetic differentiation analysis and genetic structure analysis of Xinjiang Ammopiptanthus population were carried out. According to the analysis results, the variety source of the Xinjiang Ammopiptanthus samples to be tested was identified, and the group to which the Xinjiang Ammopiptanthus germplasm resources belonged was determined.

7. The method according to claim 6, characterized in that The reaction system of the PCR amplification is: 1 μL of template DNA, 1 μL of 10 μM forward and reverse primers, 12.5 μL of 2×EasyTaq PCR SuperMix, and 9.5 μL of Nuclease-free Water.

8. The method according to claim 6, characterized in that The reaction procedure of the PCR amplification was as follows: pre-denaturation at 93°C for 3 min, denaturation at 94°C for 30 s, annealing at 56°C for 30 s, extension at 65°C for 90 s, for a total of 30 cycles, and finally repair extension at 65°C for 5 min.

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