Polymorphic EST-SSR Molecular Marker Primers and Their Application in Pinus elliottii

Abstract

The present invention relates to the field of molecular biology, and in particular to polymorphic EST-SSR molecular marker primers for longitudinal pine and their applications. The nucleotide sequence of the primer set is shown in SEQ ID NO.1-SEQ ID NO.40. The primer set provided by the present invention can specifically amplify the EST-SSR molecular markers, and has a high degree of polymorphism, and can be applied to research fields such as longitudinal pine genetic diversity and population genetic structure. Moreover, the EST-SS molecular markers amplified by the EST-SSR primer set of the present invention have the advantages of abundant quantity, high polymorphism and good stability. At the same time, the amplification experiment of the EST-SSR primer set in the present invention is simple to operate, and the bands are clear and easy to distinguish, which is suitable for genetic diversity and genetic structure analysis of longitudinal pine.

Description

Technical Field

[0001] The invention relates to the field of molecular biology, in particular to elliottii pine polymorphic EST-SSR molecular marker primers and applications thereof. Background Art

[0002] Longleaf pine (Pinus lliottii Engelm.) is an evergreen tree belonging to the genus Pinus of the Pinaceae family, native to the southeastern United States. Longleaf pine has the characteristics of fast growth, good wood quality, strong adaptability, high resin yield and good resin quality, and has become one of the important afforestation, resin and timber tree species in low mountains and coastal areas in the south. The main research on longleaf pine includes genetic improvement of high resin yield and wood quality, exploration of asexual reproduction technology, sustainable management and the use of modern biotechnology to assist breeding. There are few studies on genetic diversity and population structure analysis of longleaf pine populations.

[0003] Genetic diversity is the foundation and core of biodiversity, and determines the ability of plants to adapt to the environment and maintain the stability of the ecosystem. Understanding the changing patterns and influencing factors of genetic diversity is of great significance to the rational use and protection of plant resources. Therefore, studying plant genetic diversity can not only understand the level of genetic variation and the stability of genetic structure of the population, but also establish more targeted protection measures and provide efficient strategies for improving the level of genetic diversity of the population. Molecular markers directly reflect the sequence changes of individual genetic material at the DNA level and are currently a common method for studying plant genetic diversity. Among them, SSR (Simple Sequence Repeat) is a simple repeat sequence. According to the primer development sequence, it can be divided into genomic SSR markers and EST-SSR markers. It is composed of 1-6 repeated base sequences on the plant genome. The number of markers is large, the stability is good, the repeatability is good, and the polymorphism is high. It also has the characteristics of simple operation and co-dominant inheritance. It can be used to genetically distinguish individuals or groups. It is widely used in the research of genetic diversity, population structure analysis, core germplasm construction and kinship identification of forest germplasm resources at home and abroad. At present, the insufficient number of molecular markers is an important factor hindering the study of genetic diversity of Elliptic pine. Therefore, it is necessary to develop sufficient, stable and efficient SSR molecular markers. Summary of the invention

[0004] The purpose of the present invention is to provide polymorphic EST-SSR molecular marker primers for longevity pine and their applications to solve the problems existing in the above-mentioned prior art. The amplification experiment of the EST-SSR molecular marker primers provided by the present invention is simple to operate, the bands are clear and easy to distinguish, and it is suitable for genetic diversity and genetic structure analysis of longevity pine; and the EST-SS molecular markers amplified by the EST-SSR primer set of the present invention have the advantages of abundant quantity, high polymorphism and good stability.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a EST-SSR primer set for Elliot pine, the nucleotide sequence of which is shown in the following table:

[0007]

[0008] The present invention provides the use of the above EST-SSR primer set in the genetic diversity analysis of Pinus elliottii. The present invention provides the use of the above EST-SSR primer set in the population genetic structure analysis of Pinus elliottii.

[0009] The present invention discloses the following technical effects:

[0010] The present invention obtains a DNA sequence containing microsatellites by performing SSR sites on the unigenes obtained from the transcriptome of slash pine, and at the same time designs specific primers to perform polymorphism detection on these microsatellite sites, develops a group of slash pine EST-SSR molecular marker combinations with rich polymorphism, and provides a group of primer sets for amplifying the slash pine EST-SSR molecular marker combinations. The primer set provided by the present invention can specifically amplify the EST-SSR molecular markers, and has a high degree of polymorphism, and can be applied to research fields such as genetic diversity and population genetic structure of slash pine. Moreover, the EST-SSR molecular markers amplified by the primer set provided by the present invention are abundant in number, highly polymorphic, and have good stability. At the same time, the amplification experiment of the EST-SSR molecular marker primers in the present invention is simple to operate, and the bands are clear and easy to distinguish; it is suitable for genetic diversity and genetic structure analysis of slash pine. The materials used in the present invention are selected from slash pine germplasm resources collected from many places in Jiangxi Province, and can more comprehensively reveal the genetic diversity level of slash pine germplasm resources in Jiangxi Province. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] 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.

[0012] Figure 1 This is the capillary electrophoresis diagram of EST-SSR primer Pe145169 of Pinus elliottii in some individuals; a, b, c and d are 4 of the 417 materials;

[0013] Figure 2 To determine the number of subpopulations (K) of a population based on the LnP(D) value and ΔK;

[0014] Figure 3 This is a population structure analysis diagram of 417 individuals of Pinus elliottii (K=2);

[0015] Figure 4 This is the principal coordinate analysis (PCoA) diagram based on Nei's genetic distance of the longitudinal pine population. DETAILED DESCRIPTION

[0016] 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.

[0017] 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.

[0018] 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.

[0019] 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.

[0020] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0021] Unless otherwise specified, the experimental methods used in the present invention are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial channels.

[0022] The present invention uses 417 slash pine germplasm resources currently collected and preserved in Jiangxi Province as experimental materials, which are mainly divided into three groups:

[0023] 1. Introduced family population (IP): The slash pine provenance / family test forest built by Baiyunshan Forest Farm in Qingyuan District, Ji'an City in 1990 has a total of 113 families, which were introduced from seed gardens in Georgia, Mississippi and Florida, USA, and eventually 112 samples were effectively expanded; the slash pine family test forest built by Fengshushan Forest Farm in Jingdezhen in 2011 and 2018 included 30 families and 10 families respectively, which were introduced from Florida, USA, and eventually 18 samples were effectively expanded; the slash pine excellent family testing forest built by Ganzhou Forestry Institute in 2018 has a total of 40 families, which were introduced from Arkansas, USA, and eventually 40 samples were effectively expanded;

[0024] 2. First cycle superior trees breeding population (FP): mainly from the early introduced slash pine plantations in Jiangxi, including 44 samples from the first cycle slash pine seed garden built by Baiyunshan Forest Farm in Qingyuan District, Ji'an City in May 1992; and 77 samples from the first cycle slash pine seed garden built by Xijiang County Forest Seed Farm in 1980.

[0025] 3. Improved generation superior trees breeding population (IGP): mainly comes from 126 samples of experimental forests selected and constructed throughout the province, including 79 samples from Baiyunshan Forest Farm in Qingyuan District, Ji'an City, and 47 samples from Xijiang County Forest Seed Farm.

[0026] Example 1: Development of EST-SSR Molecular Marker Primers for Pinus elliottii Transcriptome

[0027] 1 Materials and methods

[0028] 1.1 Materials

[0029] A total of 417 slash pine germplasm materials were collected in Jiangxi Province, and the young shoots or leaves of the current year were taken, quickly frozen with liquid nitrogen and stored in a -80℃ refrigerator for later use. Eleven samples were randomly selected for primer polymorphism verification.

[0030] 1.2 Methods

[0031] 1.2.1 Screening of EST-SSR loci and EST-SSR molecular markers in Pinus elliottii transcriptome;

[0032] The MISA software was used to search for SSR loci in the unigenes obtained from the transcriptome of Pinus elliottii 10 times, with dinucleotide repeats >6 times and tri-, tetra-, penta-, and hexa-nucleotide repeats >5 times.

[0033] 1.2.2EST-SSR primer design;

[0034] Primer 3.0 software was used to design and randomly select 103 pairs of EST-SSR primers, and 24 pairs of EST-SSR primers of Pinus elliottii were selected from published literature. A total of 127 pairs of primers were synthesized by Shanghai Shenggong Bioengineering Technology Service Co., Ltd.

[0035] 1.2.3 Extraction of genomic DNA

[0036] DNA was extracted using the DNAsecure Plant Kit (DP320) from Tiangen Biochemical Company. The concentration and quality of the extracted DNA were tested using 1% agarose gel, and the sample DNA stock solution was then uniformly diluted to 50 ng.μL. -1 , and store in a -20℃ refrigerator for later use.

[0037] 1.2.4SSR-PCR amplification and capillary electrophoresis;

[0038] SSR-PCR reaction system of Pinus elliottii: 20 μL system contains 0.1 μL Taq enzyme, 0.8 μL upstream and downstream SSR primers, 1.6 μL 10 mol / L dNTPs, and 1 μL 50 ng / μL DNA template, 10× PCR Buffer (containing Mg 2+ )2 μL, and the rest was made up with ddH2O;

[0039] The SSR-PCR reaction procedure of Pinus elliottii was as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 55-60°C for 30 s, extension at 72°C for 60 s, 30 cycles; extension at 72°C for 7 min, and storage at 4°C.

[0040] The PCR products were then placed on an ABI 3730XL DNA sequencer for capillary electrophoresis. The capillary electrophoresis graphs of the EST-SSR primers used to amplify SSR molecular markers in some individuals are shown in Figure 2. Figure 1 As shown, these primers can amplify stable and clear bands.

[0041] 1.2.5 EST-SSR polymorphism primer data analysis.

[0042] GeneMarker V1.91 software was used to analyze the readings of the original peak results of capillary electrophoresis of each slash pine sample, and the genotype data were sorted and entered into Excel to establish the original data information matrix.

[0043] The software Popgene32 was used to calculate genetic diversity parameters: the number of alleles (Na), the effective number of alleles (Ne), Shannon's information index (I), observed heterozygosity (Ho), and expected heterozygosity (He). The Powermarker software was used to calculate the polymorphic information content (PIC) of primers.

[0044] 2 Experimental results

[0045] 2.1 Polymorphism analysis of EST-SSR markers

[0046] The detection results of EST-SSR molecular markers by capillary electrophoresis showed that among 127 pairs of primers, 112 pairs amplified bands, and the amplification rate was 88.19%. Among them, 20 pairs were highly polymorphic SSR primers, and the ratio of polymorphic loci was 15.75%. The final 20 pairs of primers screened are shown in Table 1. Among the 20 pairs of EST-SSR primers, 7 pairs had PIC > 0.5, 10 pairs had 0.25 < PIC ≤ 0.5, and 3 pairs had PIC ≤ 0.25. The proportion of highly and moderately polymorphic primers was 85%.

[0047] Table 1 Information of 20 pairs of EST-SSR primer pairs

[0048]

[0049]

[0050] Note: PIC: Polymorphic information content, the same below.

[0051] 3 Conclusions

[0052] This invention shows that the development of EST-SSR molecular markers from the transcriptome of slash pine has good effects. The number of SSR loci is large and the types are rich, with great polymorphism potential and availability. Through the screening of EST-SSR primer polymorphism, 20 pairs of EST-SSR molecular marker primers with good stability and high polymorphism were successfully developed, providing stable and reliable molecular marker materials for the analysis of genetic diversity and genetic structure of slash pine.

[0053] Example 2 Analysis of genetic diversity of slash pine germplasm resources

[0054] 1 Materials and methods

[0055] 1.1 Materials

[0056] The test materials were the same as those in Example 1, which were 417 slash pine germplasm resources.

[0057] 1.2 Methods

[0058] The molecular marker data obtained by the 20 pairs of EST-SSR primers in Table 1 of Example 1 were input into the software Popgene32 to calculate the genetic diversity parameters: the number of alleles (Na), the effective number of alleles (Ne), the Shannon information index (I), the observed heterozygosity (Ho), the expected heterozygosity (He), the inbreeding coefficients (Fis) and (Fit) within and between populations, and the genetic differentiation coefficient (Fst). The polymorphism information content PIC was calculated using the PowerMarker3.25 software; and the molecular variance analysis was performed using the software Arlequin.

[0059] The principal coordinates PCoA analysis was performed based on genetic distance using the software GenAlex, and the genetic structure analysis of 417 individuals of Pinus elliottii was performed using the software Structure 2.3.4 based on the Bayesian cluster analysis method.

[0060] 2 Test results

[0061] 2.1 Analysis of genetic diversity of Pinus elliottii germplasm resources

[0062] The results of genetic diversity analysis are shown in Table 2.

[0063] Table 2 Genetic diversity parameters of 20 microsatellite loci of Pinus elliottii

[0064]

[0065]

[0066] Note: Na: observed allele number; Ne: effective allele number; Ho: observed heterozygosity; He: expected heterozygosity; I: Shannon information index, same as the following table.

[0067] As shown in Table 2, the 417 Ginkgo germplasm resources had a high level of genetic diversity. A total of 105 alleles were detected in 417 elliottii samples by 20 pairs of EST-SSR primers, with an average of 5.250 alleles per locus. The Shannon information index (I) ranged from 0.305 (Pe144142) to 1.638 (Pe145169), with an average of 0.835. The observed heterozygosity (Ho) and expected heterozygosity (He) ranged from 0.138 (Pe114582) to 0.795 (Pe123077) and 0.149 (Pe114582) to 0.777 (Pe145169), with means of 0.390 and 0.442, respectively. The PIC value ranged from 0.150 (Pe114582) to 0.744 (Pe145169), with a mean of 0.402. Among them, there were 4 sites with PIC < 0.25, which were low-level polymorphic sites; 10 sites with 0.25 < PIC < 0.5, which were medium-level polymorphic sites; and 6 sites with PIC > 0.5, which were high-level polymorphic sites.

[0068] 2.2 Analysis of diversity of different elliottii populations

[0069] The results of diversity analysis of 20 pairs of EST-SSR primers in three populations are shown in Table 3.

[0070] Table 320 Polymorphism analysis of EST-SSR primers in three populations

[0071]

[0072]

[0073]

[0074] Note: IP: Introduced family population; FP: First cycle superior trees breeding population; IGP: Improved generation superior trees breeding population, the same below.

[0075] As recorded in Table 3, 85 and 97 alleles were detected in the first-generation superior tree breeding population and the improved-generation superior tree breeding population of Longleaf pine, respectively, and only 84 alleles were detected in the introduced family population. In addition, the ratios of Ne to Na in the first-generation superior tree breeding population and the improved-generation superior tree breeding population were higher than that in the introduced family population. The Shannon information index (I) of the introduced family population, the first-generation and improved-generation superior tree breeding populations were 0.713, 0.811 and 0.930, respectively, and the polymorphism information content (PIC) values ​​were 0.338, 0.394 and 0.443, respectively; the average observed heterozygosity (Ho) was 0.361, 0.409 and 0.410, respectively, and the average expected heterozygosity (He) was 0.387, 0.442 and 0.494, respectively; overall, the genetic diversity of the first-generation and improved-generation superior tree breeding populations of Pinus elliottii was higher than that of the introduced family population. SPSS software was used to perform variance analysis on the genetic diversity parameters of 20 SSR loci of the three populations, and the results showed that there were no significant differences among the populations (P>0.05).

[0076] 2.3 Genetic differentiation among populations

[0077] The genetic differentiation coefficients of Pinus elliottii populations are shown in Table 4.

[0078] Table 4 Genetic differentiation coefficients of Pinus elliottii populations

[0079] Location Fis Fit F Location Fis Fit F 145169 0.175 0.183 0.009 106732 -0.061 -0.019 0.039 144426 0.373 0.376 0.006 145380 0.144 0.148 0.005 138370 0.124 0.131 0.008 119033 -0.078 -0.060 0.017 130187 0.131 0.145 0.017 135178 0.083 0.090 0.008 132622 0.101 0.106 0.006 140688 0.310 0.335 0.037 146453 0.397 0.406 0.015 110222 0.100 0.129 0.032 134815 0.036 0.042 0.006 113019 0.244 0.252 0.010 131259 0.094 0.106 0.014 139538 0.401 0.451 0.084 103802 0.055 0.067 0.012 114582 0.079 0.086 0.007 123077 -0.655 -0.634 0.013 144142 0.010 0.010 0.000 Mean 0.103 0.118 0.017

[0080] Note: Fis: inbreeding coefficient within a population, Fit: inbreeding coefficient between populations, Fst: genetic differentiation coefficient, the same below.

[0081] As shown in Table 4, the Fis variation range of the 20 SSR loci was -0.655 (123077)-0.401 (139538), with an average of 0.103>0; the Fit variation range among populations was -0.634 (123077)-0.451 (139538), with an average of 0.118; the overall Fst variation range was 0.000 (144142)-0.084 (139538), with an average of 0.017, indicating that there was low genetic differentiation among the slash pine populations.

[0082] The results of AMOVA analysis based on 20 loci are shown in Table 5.

[0083] Table 5 AMOVA analysis of Pinus elliottii

[0084] Sources of variation Degrees of Freedom sum of squares Variance components Percent Variation Intergroup 2 54.608 0.085 2.06％ In-group 831 3352.779 4.035 97.94％ sum 833 3407.387 4.120 100％

[0085] As shown in Table 5, the genetic variation between populations accounted for 2.06% of the total population variation, and the variation between individuals within a population and within individuals accounted for 97.94%, indicating that the genetic variation of longitudinal pine mainly occurred between individuals and within individuals, and the genetic variation between the introduced family population, the first-generation superior tree breeding population and the improved-generation superior tree breeding population was relatively small.

[0086] Nei's method was used to calculate the genetic similarity coefficient and genetic distance among the three populations. The results are shown in Table 6.

[0087] Table 6 Genetic distance (lower left corner) and genetic similarity (upper right corner) between 3 populations

[0088] POPID IP FP IGP IP **** 0.988 0.983 FP 0.012 **** 0.984 IGP 0.017 0.016 ****

[0089] As recorded in Table 6, the genetic distance between the introduced family group and the first-generation superior tree breeding group is 0.012, and the genetic similarity is 0.988. The genetic distance between the introduced family group and the improved-generation superior tree breeding group is 0.017, and the genetic similarity is 0.983. The genetic distance between the first-generation and improved-generation superior tree breeding groups is 0.016, and the genetic similarity coefficient is 0.984, indicating that the introduced family group is more closely related to the first-generation superior tree breeding group than to the improved-generation superior tree breeding group.

[0090] 2.4 Population genetic structure

[0091] In order to further clarify the population genetic structure and the relationship between individuals of 417 slash pine samples, 20 pairs of EST-SSR primers were used to analyze them based on Structure software. Assuming that the population number K = 1-10, each K value was repeated 10 times. The results were processed by Structure Haevester to obtain the mean lnP(D) corresponding to each K value (the log-likelihood value obtained after the Markov chain is not iterated) and the ΔK value (the rate of change between two consecutive LnP(D)) that can determine the optimal grouping. The double Y-axis line graph was drawn in Origin software with it as the ordinate and the corresponding K value as the abscissa. Figure 2 ); As can be seen from the figure, with the increase of K value, lnP(D) continues to increase. When K value changes from 1 to 2, the LnP(D) value changes most significantly. When k = 2, ΔK is at the maximum peak, indicating that the 417 slash pine samples tested are divided into two subgroups ( Figure 3 )

[0092] 2.5 Principal coordinate analysis

[0093] PCoA analysis of Pinus elliottii based on Nei's genetic distance was performed. Figure 4As shown in the figure, all the slash pine germplasm can be roughly divided into two groups, and 11.20% and 17.23% of the genetic variation can be explained by the principal coordinates 1 and 2 respectively; the introduced family population, the first generation and the improved generation of superior tree breeding populations are distributed in both groups, and the germplasm resources of Group I are more than those of Group II. It can be seen from the figure that most of the samples have more overlapping distribution areas, and the improved generation of superior tree breeding populations deviate more significantly from the introduced family population than the first generation of superior tree breeding populations. The results show that the genetic relationship between the existing introduced family populations, the first generation and the improved generation of superior tree breeding populations is close, and there is genetic differentiation but the degree is low.

[0094] 3 Conclusion

[0095] The 20 pairs of EST-SSR primers developed based on the transcriptome of Pinus elliottii in the present invention have a moderately high polymorphism level (PIC=0.402<0.500), and are suitable for genetic diversity analysis and genetic structure analysis of Pinus elliottii.

[0096] Furthermore, the present invention conducted genetic diversity analysis on 417 elliotrigine germplasm resources using the above-mentioned EST-SSR primers, indicating that elliotrigine has rich genetic diversity (I=0.835, He=0.442), the genetic diversity levels of each population are different, and the degree of genetic differentiation between populations is low (Fst=0.017).

[0097] 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. A Pinus elliottii EST-SSR primer set, characterized in that: The nucleotide sequence of the EST-SSR primer set is shown in the following table:

2. Use of the EST-SSR primer set described in claim 1 in the genetic diversity analysis of Pinus elliottii.

3. Use of the EST-SSR primer set of Pinus elliottii according to claim 1 in the population genetic structure analysis of Pinus elliottii.

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