A Salt-Tolerant SSR Molecular Marker, Reagent and Application of Fraxinus chinensis

Abstract

The present invention belongs to the technical field of forest tree breeding, and particularly relates to a salt-tolerant SSR molecular marker, reagent and application thereof for Fraxinus. The salt-tolerant SSR molecular marker for Fraxinus includes one or more combinations of 213, 167, 217, 92, 208, 203, 202 and 186. The molecular marker is significantly associated with the salt-tolerant trait of Fraxinus. Furthermore, the salt-tolerant SSR molecular marker combination and its primer set can be used to detect the tolerance of Fraxinus and assist in cultivating salt-tolerant Fraxinus varieties, effectively improving the efficiency of salt-tolerant breeding.

Description

Technical Field

[0001] The present invention belongs to the technical field of forest tree breeding, and specifically relates to a salt-tolerant SSR molecular marker, reagent and application thereof for Fraxinus chinensis Roxb.. Background Art

[0002] As an important product of the earth's environment and the history of life development, the asset value of soil natural resources determines that it provides multiple services for life and ecosystems. Good soil resources can benefit all mankind and support the sustainable development of the whole society. Soil salinization, as one of the top ten global soil changes, has become an ecological environment problem faced by all mankind.

[0003] The genus Fraxinus, also known as the ash genus, is collectively called ash trees, belonging to the subfamily Oleoideae of the family Oleaceae. There are about 70 species in the world and about 30 species in China, among which more than 10 species are introduced from abroad. The tree species of this genus are mainly distributed in temperate regions. Different species of ash are distributed in different geographical regions of China, including in the northeastern region, northwestern region, Yellow River Basin and Yangtze River Basin, including provinces such as Sichuan, Guangdong, and Fujian. The vertical distribution altitude is 400 - 2000m. Most ash trees are light-loving or slightly shade-tolerant, and have the advantages of rapid growth, long tree age, fast growth and reproduction, strong adaptability, strong resistance, and good tree materials. They can grow in many environments, showing strong stress resistance and adaptability. They are excellent tree species for wind prevention, sand fixation and saline-alkali land improvement in northern China. Therefore, it is particularly important to comprehensively study the germplasm resources of ash trees, deeply analyze and explore their salt tolerance value, and select highly salt-tolerant ash tree species for forestry construction in saline-alkali areas.

[0004] In existing research, SSR molecular markers have been widely used in the breeding of crops, forest trees and herbaceous plants, such as cotton, soybean, wheat and poplar. Domestic and foreign research on the molecular level of ash germplasm resources mostly focuses on the construction of fingerprint maps and genetic diversity analysis, and rarely associates its salt tolerance traits with SSR molecular markers. Summary of the Invention

[0005] The purpose of the present invention is to provide a salt-tolerant SSR molecular marker, reagent and application thereof for Fraxinus chinensis Roxb.. The salt-tolerant SSR molecular marker combination of the present invention has a significant correlation with multiple salt tolerance traits and can be used for the detection and breeding of salt-tolerant ash trees.

[0006] The present invention provides a salt-tolerant SSR molecular marker for Fraxinus chinensis Roxb., and the salt-tolerant SSR molecular marker for Fraxinus chinensis Roxb. includes one or more combinations of 213, 167, 217, 92, 208, 203, 202 and 186;

[0007] The primer pairs designed for 213, 167, 217, 92, 208, 203, 202 and 186 are S213, S167, S217, S92, S208, S203, S202 and S186 in sequence;

[0008] The nucleotide sequence of the upstream primer of S213 is shown in SEQ ID NO.1, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.2;

[0009] The nucleotide sequence of the upstream primer of S167 is shown in SEQ ID NO.3, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.4;

[0010] The nucleotide sequence of the upstream primer of S217 is shown in SEQ ID NO.5, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.6;

[0011] The nucleotide sequence of the upstream primer of S92 is shown in SEQ ID NO.7, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.8;

[0012] The nucleotide sequence of the upstream primer of S208 is shown in SEQ ID NO.9, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.10;

[0013] The nucleotide sequence of the upstream primer of S203 is shown in SEQ ID NO.11, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.12;

[0014] The nucleotide sequence of the upstream primer of S202 is shown in SEQ ID NO.13, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.14;

[0015] The nucleotide sequence of the upstream primer of S186 is shown in SEQ ID NO.15, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.16.

[0016] Preferably, the salt-tolerant SSR molecular markers of Fraxinus include 213, 167, 217, 92, 208, 203 and 202.

[0017] Preferably, the salt-tolerant SSR molecular markers of Fraxinus include 213, 167, 92, 208 and 202.

[0018] Preferably, the salt-tolerant SSR molecular marker of Fraxinus is 202.

[0019] The present invention provides an application of the salt-tolerant SSR molecular marker of the white wax described in the above technical solution in detecting the salt tolerance of the white wax.

[0020] The present invention provides a reagent for detecting the salt tolerance of the white wax, comprising the salt-tolerant SSR molecular marker of the white wax described in the above technical solution.

[0021] Preferably, the reagent further comprises a PCR amplification reagent.

[0022] The present invention also provides an application of the salt-tolerant SSR molecular marker or the reagent of the white wax described in the above technical solution in the molecular breeding of the white wax.

[0023] Preferably, the molecular breeding of the white wax includes the molecular breeding of salt-tolerant white wax.

[0024] The present invention also provides a method for SSR molecular marker-assisted breeding of salt-tolerant white wax, comprising the following steps: extracting the DNA of the sample population of the white wax to be tested, and performing PCR amplification on the primer pair or the reagent in the salt-tolerant SSR molecular marker of the white wax described in the above technical solution to obtain the PCR amplification product of the sample to be tested;

[0025] Performing genetic diversity detection on the PCR amplification product of the sample to be tested to obtain the polymorphic bands of the primer of the salt-tolerant SSR molecular marker of the white wax to be tested;

[0026] Calculating and obtaining the population structure data of the white wax population sample based on the polymorphic bands of the primer of the salt-tolerant SSR molecular marker of the white wax to be tested;

[0027] Performing correlation analysis on the population structure data of the white wax population sample to be tested and the salt-tolerance trait data, and the white wax population sample corresponding to the population structure data significantly associated with the salt-tolerance trait data is the salt-tolerant white wax.

[0028] Beneficial effects:

[0029] The present invention provides a salt-tolerant SSR molecular marker of the white wax, and the salt-tolerant SSR molecular marker of the white wax comprises one or a combination of more than one of 213, 167, 217, 92, 208, 203, 202 and 186. The molecular marker is significantly associated with the salt-tolerance trait of the white wax. Furthermore, the salt-tolerance of the white wax can be detected by using the salt-tolerant SSR molecular marker combination and its primer set, and the salt-tolerant white wax variety can be assisted in cultivation, effectively improving the efficiency of salt-tolerant breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments.

[0031] Figure 1 For the PIC frequency of the SSR marker

[0032] Figure 2-1 is the value of L’(K) estimated based on the analysis of Structure software;

[0033] Figure 2-2 is the value of ΔK estimated based on the analysis of Structure software;

[0034] Figure 3 is the population structure of 173 Fraxinus materials estimated based on 14 pairs of SSR molecular markers. Detailed implementation manners

[0035] The present invention provides a salt-tolerant SSR molecular marker for Fraxinus, and the salt-tolerant SSR molecular marker for Fraxinus includes one or more combinations of 213, 167, 217, 92, 208, 203, 202, and 186; the primer pairs of 213, 167, 217, 92, 208, 203, 202, and 186 are S213, S167, S217, S92, S208, S203, S202, and S186 in sequence; the nucleotide sequence of the upstream primer of S213 is shown in SEQ ID NO.1, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.2; the nucleotide sequence of the upstream primer of S167 is shown in SEQ ID NO.3, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.4; the nucleotide sequence of the upstream primer of S217 is shown in SEQ ID NO.5, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.6; the nucleotide sequence of the upstream primer of S92 is shown in SEQ ID NO.7, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.8; the nucleotide sequence of the upstream primer of S208 is shown in SEQ ID NO.9, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.10; the nucleotide sequence of the upstream primer of S203 is shown in SEQ ID NO.11, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.12; the nucleotide sequence of the upstream primer of S202 is shown in SEQ ID NO.13, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.14; the nucleotide sequence of the upstream primer of S186 is shown in SEQ ID NO.15, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.16.

[0036] The primer group sequence of the salt-tolerant SSR molecular marker for Fraxinus of the present invention is preferably shown in Table 1:

[0037] Table 1 Salt-tolerant SSR molecular markers for Fraxinus and their primers

[0038]

[0039] The salt-tolerant SSR molecular markers of the present invention preferably include 213, 167, 217, 92, 208, 203 and 202, or include 213, 167, 92, 208 and 202, or are 202. The primer sets corresponding to the molecular marker combinations are the same as above and will not be elaborated here.

[0040] The present invention preferably also provides a method for screening salt-tolerant SSR molecular markers of Fraxinus chinensis, comprising the following steps: extracting DNA of Fraxinus chinensis population samples, performing PCR amplification using primers of candidate Fraxinus chinensis SSR markers with genetic diversity to obtain PCR amplification products; detecting the genetic diversity of the PCR amplification products by capillary electrophoresis to obtain polymorphic bands of the candidate Fraxinus chinensis SSR marker primers. Calculating the population structure data of the Fraxinus chinensis population samples based on the polymorphic bands of the candidate Fraxinus chinensis SSR marker primers. Performing association analysis on the population structure data of the Fraxinus chinensis population samples and the salt-tolerance trait data to obtain the explanation rate of the Fraxinus chinensis SSR markers for the variation of tolerance traits, and identifying the Fraxinus chinensis SSR markers with a P value less than 0.01 as salt-tolerant SSR molecular markers of Fraxinus chinensis; the salt-tolerant SSR molecular markers of Fraxinus chinensis include one or more combinations of 213, 167, 217, 92, 208, 203, 202 and 186; the salt-tolerance trait data include conductivity level, chlorophyll a content, chlorophyll b content, carotenoid content, superoxide dismutase activity, peroxidase activity measured by guaiacol method, catalase activity, malondialdehyde content and soluble protein content.

[0041] The present invention preferably extracts the DNA of the Fraxinus chinensis population to obtain Fraxinus chinensis population DNA samples. The present invention has no special limitation on the DNA extraction method, and any conventional plant DNA extraction method or kit in the art can be used. For example, in the examples of the present invention, the TIANGEN plant genomic DNA extraction kit is used, and the specific steps can be referred to the kit instructions. The Fraxinus chinensis population samples for DNA extraction in the present invention are preferably taken from the clonal test forest in Xiaobotou Town, Wudi County, Binzhou City, Shandong Province. The plant spacing of the test forest is preferably 2m×3m, with 3 repetitions and 4 plants in each plot. In the present invention, the clonal samples for establishing the clonal test forest are preferably obtained by grafting and breeding the Fraxinus chinensis population samples at the Shouguang saline-alkali land afforestation test station of the Shandong Academy of Forestry Sciences from 2014 to 2015. The Fraxinus chinensis population in the present invention preferably includes 173 Fraxinus chinensis germplasm resources, which are preferably collected from Beijing, Gansu, Hebei, Liaoning, Inner Mongolia Autonomous Region, Ningxia Hui Autonomous Region, Shandong, Shanxi and Shaanxi from 2013 to 2014. The specific sources and variety names are shown in Table 2:

[0042]

[0043]

[0044]

[0045]

[0046]

[0047]

[0048] After obtaining the DNA samples of the ash tree population, the present invention preferably uses primers of candidate ash tree SSR markers for PCR amplification to obtain a PCR amplification product. The primers of the candidate ash tree SSR markers of the present invention are shown in Table 3:

[0049] Table 3 Primers of Candidate Ash Tree SSR Markers

[0050]

[0051]

[0052] The PCR amplification system of the present invention preferably includes 10 μL of 2×Mix, 0.15 μL of upstream primer at 10 μM and downstream primer at 10 μM, 1 μL of DNA sample, and ddH2O is added to make up 20 μL. The PCR amplification program of the present invention preferably includes pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 52°C for 30 s, extension at 72°C for 30 s, for a total of 35 cycles; extension at 72°C for 10 min.

[0053] After obtaining the PCR amplification product, the present invention preferably uses capillary electrophoresis to detect the genetic diversity of the PCR amplification product to obtain polymorphic bands of the candidate ash tree SSR marker primers. The system for the capillary electrophoresis of the present invention preferably includes 0.3 μL of PCR amplification product, 0.5 μL of molecular weight internal standard (LIZ500), and 9.5 μL of deionized formamide. The present invention preferably denatures the system for capillary electrophoresis at 95°C for 5 min, cools it at 4°C and then centrifuges it, and detects it on a machine with 1×Buffer buffer.

[0054] After completing the detection of the genetic diversity, the present invention preferably calculates the population structure data of the ash population sample based on the polymorphic bands of the candidate ash SSR marker primers. The population structure data of the present invention preferably includes the number of alleles (Na), the effective number of alleles (Ne), gene diversity (He), Shannon-Weaver information index (H′), and polymorphic information content (PIC). The number of alleles (Na), the effective number of alleles (Ne), gene diversity (He), and Shannon-Weaver information index (H′) of the present invention are preferably calculated using the EXCEL add-in GenAlEx 6.51, and the polymorphic information content (PIC) is preferably calculated using the Powermarker 3.25 software.

[0055] After obtaining the population structure data of the ash population sample, the present invention preferably performs an association analysis between the population structure data of the ash population sample and the salt tolerance trait data to obtain the explanation rate of the ash SSR marker for the variation of the salt tolerance trait, and confirms the ash salt tolerance SSR molecular marker combination for the ash SSR markers with a P value less than 0.01. Before performing the association analysis, the present invention preferably further includes performing a population genetic structure analysis on the ash population sample using the Structure software to confirm the genetic composition of the ash population sample. The present invention has no special limitation on the version of the Structure software, and any version can be used to implement the population structure analysis in the present invention. For example, Structure 2.3.4 is used in the examples of the present invention. The salt tolerance trait data of the present invention preferably includes the conductivity level (EC), chlorophyll a content (Chla), chlorophyll b content (Chlb), carotenoid content (CAR), superoxide dismutase activity (SOD), peroxidase activity (POD), catalase activity (CAT), malondialdehyde content (MAD), and soluble protein content (SP). The salt tolerance trait data of the present invention is preferably obtained by detecting the ash leaf sample. The present invention preferably uses GLM (general linear model) and MLM (mixed linear model) in TASSEL 3.0 software to perform the association analysis. The present invention has no special limitation on the specific process of the association analysis, and the conventional process in the art can be used.

[0056] The present invention also provides the application of the ash salt tolerance SSR molecular marker described in the above technical solution in detecting the salt tolerance of ash. Based on the significant association characteristics between the ash salt tolerance SSR molecular marker and the salt tolerance trait, the ash SSR molecular marker can be used to detect the salt tolerance sample of ash, laying a foundation for the cultivation of salt tolerance ash varieties.

[0057] The present invention also provides a reagent for detecting salt tolerance of white wax, comprising the white wax salt tolerance SSR molecular marker described in the above technical solution. The reagent of the present invention preferably also includes a PCR amplification reagent. The present invention does not specifically limit the source and type of the PCR amplification reagent, and a conventional commercially available PCR amplification reagent in the art can be used.

[0058] The present invention also provides the use of the salt-tolerant SSR molecular markers or reagents of fraxinus chinensis described in the above technical solution in molecular breeding of fraxinus chinensis. The molecular breeding of fraxinus chinensis described in the present invention preferably includes molecular breeding of salt-tolerant fraxinus chinensis. The salt-tolerant SSR molecular markers of fraxinus chinensis described in the present invention provide reliable molecular marker site support for molecular breeding of fraxinus chinensis, especially obtaining SSR molecular markers that can be used to guide the practice of salt-tolerant breeding of fraxinus chinensis, laying the foundation for selecting related genes that control excellent salt-tolerant traits, and effectively improving the efficiency of salt-tolerant breeding.

[0059] The present invention also provides a method for SSR molecular marker-assisted breeding of salt-tolerant wax trees, comprising the following steps: extracting DNA of a sample of a wax tree population to be tested, performing PCR amplification using the wax tree salt-tolerant SSR molecular marker or the reagent described in the above technical scheme to obtain a PCR amplification product of the sample to be tested; performing genetic diversity detection on the PCR amplification product of the sample to be tested to obtain a polymorphic band of the SSR molecular marker primers for salt tolerance of the wax tree to be tested; calculating population structure data of the sample of the wax tree population to be tested based on the polymorphic bands of the SSR molecular marker primers for salt tolerance of the wax tree to be tested; performing correlation analysis on the population structure data of the sample of the wax tree population to be tested and salt tolerance trait data, and the sample of the wax tree population to be tested corresponding to the population structure data that is significantly correlated with the salt tolerance trait data is the salt-tolerant wax tree.

[0060] The present invention preferably extracts DNA from the tested Fraxinus chinensis population to obtain a DNA sample of the tested Fraxinus chinensis population. The present invention has no particular limitation on the DNA extraction method, and conventional plant DNA extraction methods or kits in the art can be used for extraction.

[0061] After obtaining the DNA sample of the fraxinus population, the present invention uses the fraxinus salt-tolerant SSR molecular marker or the reagent described in the above technical solution to perform PCR amplification to obtain the PCR amplification product of the sample to be tested. The fraxinus salt-tolerant SSR molecular marker and its primers described in the present invention are preferably as shown in Table 1. The PCR amplification system described in the present invention preferably includes 2×Mix 10μL, 10μM upstream primer, 0.15μL of 10μM downstream primer, 1μL of DNA sample, and ddH2O to make up to 20μL. The PCR amplification procedure described in the present invention preferably includes 95°C pre-denaturation for 5min; 95°C denaturation for 30s, 52°C annealing for 30s, 72°C extension for 30s, a total of 35 cycles; 72°C extension for 10min.

[0062] After obtaining the PCR amplification product of the sample to be tested, the present invention conducts genetic diversity detection on the PCR amplification product of the sample to be tested to obtain polymorphic bands of the salt-tolerant SSR molecular marker primers for the ash tree to be tested. The present invention has no special limitation on the method of the genetic diversity detection, and conventional methods in the art can be used, such as electrophoresis and sequencing, etc.

[0063] After completing the genetic diversity detection, the present invention preferably calculates and obtains the population structure data of the ash tree population sample to be tested based on the polymorphic bands of the salt-tolerant SSR molecular marker primers for the ash tree. The population structure data of the present invention preferably includes the number of alleles (Na), the effective number of alleles (Ne), gene diversity (He), Shannon-Weaver information index (H′), and polymorphic information content (PIC). The number of alleles (Na), the effective number of alleles (Ne), gene diversity (He), and Shannon-Weaver information index (H′) of the present invention are preferably calculated using the EXCEL add-in GenAlEx 6.51, and the polymorphic information content (PIC) is preferably calculated using the Powermarker3.25 software.

[0064] After completing the genetic diversity detection, the present invention conducts association analysis on the population structure data of the ash tree population sample to be tested and the salt-tolerance trait data. The ash tree population sample corresponding to the population structure data significantly associated with the salt-tolerance trait data is the salt-tolerant ash tree. The present invention preferably uses GLM (general linear model) and MLM (mixed linear model) in the TASSEL3.0 software to conduct association analysis. The present invention has no special limitation on the specific process of the association analysis, and a conventional process in the art can be used. The present invention has no special limitation on the determination standard for significant association, and a conventional determination standard in the art can be used. The salt-tolerance trait data of the present invention preferably includes conductivity level (EC), chlorophyll a content (Chla), chlorophyll b content (Chlb), carotenoid content (CAR), superoxide dismutase activity (SOD), peroxidase activity (POD), catalase activity (CAT), malondialdehyde content (MAD), and soluble protein content (SP).

[0065] In order to further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below in conjunction with the drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0066] Example 1

[0067] Extraction, PCR amplification, and detection of the amplification product of the ash tree population sample DNA:

[0068] 1.1 General situation of the test site:

[0069] The experimental site is located in Xiaobotou Town, Wudi County, Binzhou City, Shandong Province. It is located on the south bank of the lower reaches of the Yellow River in the northern plain of Shandong, on the southwest coast of the Bohai Sea, and in the hinterland of the Yellow River Delta. It is between 117°58′ and 117°66′ east longitude and between 37°98′ and 38°07′ north latitude. It belongs to the continental climate in the East Asian monsoon region of the north temperate zone, with distinct seasons and pleasant climate. It has the characteristics of hot and rainy summers, long and cold winters, windy and dry springs, and mild and cool autumns. It has sufficient sunlight, abundant water sources, an average annual temperature of about 12°C, 2350.76 hours of annual sunshine hours, an annual precipitation of 570.1 mm, an annual evaporation of 1285.5 mm, and a frost-free period of 180 days. The soil type at the experimental site is salinized fluvo-aquic soil, with a salinity content of 0.2% - 0.3%, belonging to moderately saline soil.

[0070] 1.2 Experimental materials:

[0071] From 2013 to 2014, the research group collected Fraxinus germplasm resources in nine provinces including Beijing, Gansu, Hebei, Liaoning, Inner Mongolia Autonomous Region, Ningxia Hui Autonomous Region, Shandong, Shanxi, and Shaanxi. From 2014 to 2015, they were grafted and propagated at the Shouguang saline-alkali land afforestation experimental station of the Shandong Academy of Forestry Sciences to establish a germplasm resource nursery. In March 2019, excellent clones were selected and planted in Xiaobotou Town, Wudi County, Binzhou City, Shandong Province to establish a clone measurement forest. The plant spacing was 2m×3m, with 3 replicates, and 4 plants in each replicate. This experiment used 173 Fraxinus germplasm resource clones as experimental materials, as shown in Table 2 for details.

[0072] 1.3 DNA extraction

[0073] The genomic DNA of the leaves of 173 Fraxinus germplasm was extracted using the TIANGEN plant genomic DNA extraction kit, specifically referring to the steps of the kit. The purity and concentration of DNA were measured by ultraviolet spectrophotometry, diluted and stored in a -20°C refrigerator for standby.

[0074] 14 pairs of SSR primers are shown in Table 2. For SSR analysis, the assembled Unigene was used as the reference sequence. The SSR software MicroSAtellite (MISA, http: / / pgrc.ipk-gatersleben.de / misa / ) was used to find all SSRs, and the software primer3 - 2.2.2 was used to design primers, which were synthesized by Shandong Vaughan Technology Co., Ltd.

[0075] 1.5 PCR amplification

[0076] Using the primer pairs in 1.4, PCR amplification was performed on the genomic DNA of the Fraxinus germplasm extracted in 1.3 to obtain PCR amplification products. The PCR amplification system was as follows: 2×Mix 10 μL, 0.15 μL each of the 10 μM upstream primer / downstream primer, 1 μL of the DNA stock solution, and ddH2O was added to make up to 20 μL. The PCR amplification program was: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 52°C for 30 s, extension at 72°C for 30 s, for a total of 35 cycles; extension at 72°C for 10 min.

[0077] 1.6 Detection of amplification products:

[0078] Using capillary electrophoresis for on-machine detection, 0.3 μL of the PCR product, 0.5 μL of the molecular weight internal standard (LIZ500), and 9.5 μL of deionized formamide were mixed and added to the PCR plate. After denaturation at 95°C for 5 min and cooling to 4°C followed by centrifugation, it was detected on the machine with 1×Buffer buffer to obtain primer polymorphism bands.

[0079] Example 2

[0080] Genetic diversity detection of Fraxinus primers

[0081] Using the EXCEL add-in GenAlEx 6.51, the number of alleles (Na), effective number of alleles (Ne), gene diversity (He), Shannon-Weaver information index (H′) were calculated based on the polymorphism bands of the primers, and the polymorphism information content (PIC) was calculated using the Powermarker 3.25 software. The results are shown in Table 4:

[0082] Table 4 Polymorphism analysis of 14 SSR markers in 173 Fraxinus samples

[0083]

[0084] As can be seen from Table 4, 14 pairs of SSR primers screened were used for genetic diversity analysis with 173 Fraxinus samples. A total of 91 Na were generated by the 14 pairs of primers, with each pair of primers mainly generating from 4 - 12 Na, and the average frequency being 6.5. Ne was between 1.310 - 5.071, with an average frequency of 2.672; H′ was between 0.505 - 1.867, with an average frequency of 1.138; PIC was between 0.221 - 0.777, with an average frequency of 0.527. Sorted in ascending order of PIC value, they were Marker 112, 213, 167, 93, 123, 95, 217, 92, 82, 208, 187, 203, 186, 202. Among them, 3 markers (accounting for 21.4% of the 14 SSRs) had relatively high PIC values (i.e., >0.7), which were Marker 203, 186, and 202; 7 markers had PIC values greater than 0.5, accounting for 50% of the total number of SSR molecular markers; Marker 112 had the lowest PIC value (0.221), while Marker 202 had the highest PIC value (0.777)( Figure 1 ). Thus, it can be concluded that the polymorphism and stability of the 14 pairs of SSR primers are good and can be amplified within the 173 Fraxinus resources.

[0085] Example 3

[0086] Population genetic structure analysis of 173 Fraxinus samples

[0087] The software Structure2.3.4 was used to analyze the population genetic structure of this natural population. According to the principle of the maximum likelihood value and in combination with the ΔK value, a suitable K value was determined (Evanno G, Regnaut S, Goudet J. Detecting the number of clusters of individuals using the software structure: a simulation study[J]. Molecular Ecology, 2005, 14: 2611 - 2620.), the number of sub - populations K of this population was determined, the probability (i.e., Q value) of each material belonging to each sub - population was calculated, and a population result analysis graph was drawn.

[0088] The results showed that the log - likelihood function value L'(K) had an obvious peak when K = 2( Figure 2-1 ), and further calculation found that the ΔK value was the largest when K = 2( Figure 2-2 ), indicating that the suitable number of sub - populations of this natural population was divided into 2 categories, and the number of materials in the two sub - populations was 154 and 19 respectively( Figure 3)。The Q values of all materials in Subpopulation 1 are higher than 0.85, and the Q values of all materials in Subpopulation 2 are higher than 0.95. It is speculated that the genetic components of the two parts of Fraxinus chinensis materials are relatively simple, accounting for 89% and 11% respectively. At the same time, the Q matrix generated when K = 2 is used for subsequent trait-marker association analysis.

[0089] Example 4

[0090] Physiological determination indexes of salt tolerance

[0091] Randomly select 3 - 5 functional leaves from the same part of Fraxinus chinensis plants. Put the samples into self - sealing bags and place them in an insulated box with ice, then bring them back to the laboratory. Cut the samples into pieces for the determination of relevant physiological indexes, with 3 replicates for each index. Determination of 9 relevant physiological indexes: The determination of the conductivity (EC) level uses a DDS - 12A conductivity meter; the contents of chlorophyll (Chla, Chlb) and carotenoid (CAR) are determined by ethanol extraction method (Zhao Shijie, Liu Huashan, Dong Xinchun. Plant Physiology Experiment Guide [M]. Beijing: China Agriculture Press, 1997: 152 - 154, 161 - 163.); the determination of the content of malondialdehyde (MDA) uses the thiobarbituric acid (TBA) method (Gao Junfeng. Plant Physiology Experiment Technology [M]. Beijing: World Book Publishing Company, 2000: 921.); the activity of peroxidase (POD) is determined by the guaiacol method, and the activity of superoxide dismutase (SOD) is determined by the nitroblue tetrazolium (NBT) reduction method (Lin Xuefeng, Xie Hongtao, Yu Mukui, Chen Shunwei. Morphological and Physiological Response Characteristics and Salt Tolerance Differences of Three Seashore Plants under Salt Stress [J]. Forest Research, 2018, 31(03): 95 - 103); the activity of catalase (CAT) is determined by ultraviolet spectrophotometry (Wu Mingjian, Sun Xianjun, Lei Qifu, Li Hui, Leng Hailan. Determination of Sulfur in Tobacco by Barium Sulfate Sol Colloidal Turbidity Method [J]. Tobacco Science & Technology, 2005(01): 24 - 26 + 37); the content of soluble protein (SP) is determined with reference to the ultraviolet absorption method (Li Xi, Wang Lihua, Liu Wei, Sun Lingxia. Comparison of the Resistance of Three Warm - Season Turfgrasses to Sulfur Dioxide [J]. Acta Ecologica Sinica, 2014, 34(05): 1189 - 1197).

[0092] Determine the EC level, Chla content, Chla content, CAR content, SOD activity, POD activity, CAT activity, MDA content and SP content of 173 Fraxinus chinensis materials under salt stress. Organize and calculate the measured experimental data and draw charts using Excel, and use SPSS 25.0 software to statistically analyze each salt - tolerance physiological index, calculate its coefficient of variation, mean square, F - value and significance. The results are shown in Table 5:

[0093] Table 5 Analysis of Salt - Tolerance Trait Indexes of Fraxinus chinensis

[0094]

[0095] It can be concluded from Table 5 that the coefficient of variation of 9 salt tolerance traits shows that the variation degrees of various physiological indexes are relatively large. The average coefficient of variation of each salt tolerance trait is 114.25%. The variation degree of CAT activity is the largest (261.19%), followed by SP content (200.74%) and POD activity (167.83%). The variation degree of SOD activity is the smallest, but its coefficient of variation is also as high as 50.11%. This result indicates that the salt tolerance traits of Fraxinus chinensis show high differences among different tested materials and can be used for association analysis.

[0096] Example 5

[0097] Association Analysis between SSR Molecular Markers and Salt Tolerance

[0098] The association between 14 polymorphic SSR molecular marker loci and MP, Chla content, Chlb content, CAR content, SOD activity, POD activity, CAT activity, MDA content, and SP content of 173 Fraxinus chinensis materials was calculated using GLM (general linear model) and MLM (mixed linear model) in the TASSEL 3.0 software package.

[0099] GLM used the Q value of each material as a covariate, and the MLM model used the Q value and the kinship coefficient K (Kinship) between individuals as covariates to perform a regression analysis on the markers one by one using the variation data of salt tolerance physiological indexes. The explained rate (R2) of the markers for the variation of salt tolerance physiological indexes was calculated, and data such as F value, P value, and associated loci were obtained. The results are shown in Table 6 and Table 7.

[0100] Table 6 Correlation between SSR Markers and Physiological Indexes of Fraxinus chinensis (GLM)

[0101]

[0102]

[0103] Note: *P < 0.05; **P < 0.01.

[0104] It can be concluded from Table 6 that the analysis results of the GLM model show that a total of 8 marker loci, namely markers 92, 167, 186, 202, 203, 208, 213, and 217, are associated with the contents of CAR, CAT activity, Chla content, Chlb content, EC level, MDA content, POD activity, and SP content, and a total of 25 marker-trait pairs show a highly significant correlation level (P<0.01). The average contribution rate of each marker showing a highly significant association to the salt tolerance traits is 22.34%. Among them, the contribution rate of marker 202 to CAT activity is the highest, reaching 45.77%. Followed by the contribution rates of markers 202 and 203 to SP content, which are 42.45% and 33.92% respectively. This study found that CAT activity, Chlb content, EC level, POD activity, and SP content are respectively highly significantly associated with two or more SSR marker loci, while markers 92, 167, 202, 203, 208, 213, and 217 are highly significantly correlated with two or more salt tolerance traits simultaneously, indicating that the alleles controlling the salt tolerance traits of Fraxinus chinensis may be pleiotropic or polygenic.

[0105] Table 7 Correlation between SSR Markers and Physiological Indexes of Fraxinus chinensis (MLM)

[0106]

[0107] Note: *P < 0.05; **P < 0.01.

[0108] The analysis results of the MLM model show that a total of 8 marker loci, namely markers 92, 167, 186, 202, 203, 208, 213, and 217, are associated with the contents of CAR, CAT activity, Chlb content, EC level, POD activity, and SP content, and a total of 15 marker-trait pairs show a highly significant correlation level (P<0.01) (Table 6). The average contribution rate of each marker showing a highly significant association to the salt tolerance traits is 21.68%. Among them, the contribution rate of marker 202 to CAT activity is the highest, reaching 45.82%. Followed by the contribution rates of markers 202 and 208 to SP content and CAT activity, which are 44.12% and 23.94% respectively.

[0109] Through association analysis, it was found that CAT activity, POD activity, and SP content are respectively highly significantly associated with two or more SSR marker loci, while markers 92, 167, 202, 208, and 213 are highly significantly correlated with two or more salt tolerance traits simultaneously, indicating that the alleles controlling the salt tolerance traits of Fraxinus chinensis may be pleiotropic or polygenic.

[0110] From the above results, it can be concluded that: by associating the 14 pairs of SSR molecular markers screened with 9 salt tolerance-related traits, a total of 5 loci were detected to be extremely significantly associated with CRA content, CAT activity, EC level, POD activity, and SP content. These loci are markers 92, 167, 202, 208, and 213. Among them, except for CRA content and EC level, other traits are associated with more than two loci at the same time. Marker 202 has the largest contribution rate to CAT activity and is extremely significantly associated with it (P<0.01).

[0111] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, not all embodiments. People can also obtain other embodiments based on this embodiment without creative efforts, and these embodiments all fall within the protection scope of the present invention. Sequence Listing <110> Shandong Academy of Forestry Sciences Shandong Huabo Genetic Engineering Co., Ltd. <120> A Salt-Tolerant SSR Molecular Marker, Reagent and Application of Fraxinus chinensis <160> 28 <170> SIPOSequenceListing 1.0 <210> 1 <211> 23 <212> DNA <213> Artificial Sequence <400> 1 gacaacatgc ctaaattgga ctc 23 <210> 2 <211> 23 <212> DNA <213> Artificial Sequence <400> 2 aattctgaac ttcaaggtgg gat 23 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <400> 3 tgagcaaatg tgaagaccgt ag 22 <210> 4 <211> 23 <212> DNA <213> Artificial Sequence <400> 4 taatttcatc caccagtttc cac 23 <210> 5 <211> 23 <212> DNA <213> Artificial Sequence <400> 5 ttgttttggc atttcttttt gtt 23 <210> 6 <211> 23 <212> DNA <213> Artificial Sequence <400> 6 ttatgataga atggtcctcc cct 23 <210> 7 <211> 23 <212> DNA <213> Artificial Sequence <400> 7 gattctccat acatgtgctc tcc 23 <210> 8 <211> 23 <212> DNA <213> Artificial Sequence <400> 8 taaaaagtga aaccctttcg ttg 23 <210> 9 <211> 23 <212> DNA <213> Artificial Sequence <400> 9 cctcctattg aatcattcgc tta 23 <210> 10 <211> 23 <212> DNA <213> Artificial Sequence <400> 10 attttgattt ccctcctctg aag 23 <210> 11 <211> 23 <212> DNA <213> Artificial Sequence <400> 11 gttatcagta gatgcaaccg cac 23 <210> 12 <211> 22 <212> DNA <213> Artificial Sequence <400> 12 aacaccggtt ttcaacattt ct 22 <210> 13 <211> 23 <212> DNA <213> Artificial Sequence <400> 13 agttttcacc gctttcagtg tta 23 <210> 14 <211> 23 <212> DNA <213> Artificial Sequence <400> 14 gggaatgaac atgagtttca gta 23 <210> 15 <211> 23 <212> DNA <213> Artificial Sequence <400> 15 tcttcacgtc ttctgtttgt tca 23 <210> 16 <211> 23 <212> DNA <213> Artificial Sequence <400> 16 gaaaacgtgt gaatgagttt ggt 23 <210> 17 <211> 23 <212> DNA <213> Artificial Sequence <400> 17 ttgactcgtg tttagggatg aat 23 <210> 18 <211> 23 <212> DNA <213> Artificial Sequence <400> 18 agctcttgaa gggaaaattt gaa 23 <210> 19 <211> 23 <212> DNA <213> Artificial Sequence <400> 19 agaatagatg aggatgaagg gga 23 <210> 20 <211> 22 <212> DNA <213> Artificial Sequence <400> 20 ctaactcatc cctctgcgaa ac 22 <210> 21 <211> 23 <212> DNA <213> Artificial Sequence <400> 21 gaaaaggagg agagtgggaa tac 23 <210> 22 <211> 23 <212> DNA <213> Artificial Sequence <400> 22 gctccatttc acttcaactc ttc 23 <210> 23 <211> 23 <212> DNA <213> Artificial Sequence <400> 23 ggacaaaatg gttcagaatt tca 23 <210> 24 <211> 24 <212> DNA <213> Artificial Sequence <400> 24 aaagaaagaa tcaaattcgt cgtc 24 <210> 25 <211> 23 <212> DNA <213> Artificial Sequence <400> 25 tcgatctttc catctaaaca agc 23 <210> 26 <211> 23 <212> DNA <213> Artificial Sequence <400> 26 aacgtgtgaa tgagtttggt ttt 23 <210> 27 <211> 23 <212> DNA <213> Artificial Sequence <400> 27 ccattgtcaa tttgcagatt ctt 23 <210> 28 <211> 23 <212> DNA <213> Artificial Sequence <400> 28 gtctggaaat gttgatcctg aaa 23

Claims

1. Application of salt-tolerant SSR molecular markers of Fraxinus chinensis in detecting salt tolerance of Fraxinus chinensis; the salt-tolerant SSR molecular markers of Fraxinus chinensis are 213, 167, 217, 92, 208, 203 and 202; the primer pairs designed for 213, 167, 217, 92, 208, 203 and 202 are S213, S167, S217, S92, S208, S203 and S202 in sequence; The nucleotide sequence of the upstream primer of S213 is shown in SEQ ID NO.1, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.2 The nucleotide sequence of the upstream primer of S167 is shown in SEQ ID NO.3, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.4; The nucleotide sequence of the upstream primer of S217 is shown in SEQ ID NO.5, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.6; The nucleotide sequence of the upstream primer of S92 is shown in SEQ ID NO.7, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.8; The nucleotide sequence of the upstream primer of S208 is shown in SEQ ID NO.9, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.10; The nucleotide sequence of the upstream primer of S203 is shown in SEQ ID NO.11, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.12; The nucleotide sequence of the upstream primer of S202 is shown in SEQ ID NO.13, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.

14.

2. Application of Fraxinus velutina salt-tolerant SSR molecular marker in molecular breeding of Fraxinus velutina, characterized in that, The salt-tolerant SSR molecular markers of Fraxinus chinensis are 213, 167, 217, 92, 208, 203 and 202; the nucleotide sequences of the primer pairs designed for 213, 167, 217, 92, 208, 203 and 202 are shown in SEQ ID NO.1-14.

3. A method for SSR molecular marker-assisted breeding of salt-tolerant Fraxinus chinensis, characterized in that, Comprising the following steps: extracting DNA of a sample of a population of Fraxinus chinensis to be tested, performing PCR amplification using the primer pairs of the salt-tolerant SSR molecular markers of Fraxinus chinensis to obtain a PCR amplification product of the sample to be tested; the salt-tolerant SSR molecular markers of Fraxinus chinensis are 213, 167, 217, 92, 208, 203 and 202; the nucleotide sequences of the primer pairs designed for 213, 167, 217, 92, 208, 203 and 202 are shown in SEQ ID NO.1-14; Performing genetic diversity detection on the PCR amplification product of the sample to be tested to obtain polymorphic bands of the salt-tolerant SSR molecular marker primers of Fraxinus chinensis to be tested; Calculating population structure data of a Fraxinus chinensis population sample based on the polymorphic bands of the salt-tolerant SSR molecular marker primers of Fraxinus chinensis to be tested; Performing correlation analysis on the population structure data of the Fraxinus chinensis population sample to be tested and the salt-tolerance trait data, and the Fraxinus chinensis population sample corresponding to the population structure data significantly correlated with the salt-tolerance trait data is the salt-tolerant Fraxinus chinensis.

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