Robinia pseudoacacia core germplasm characteristic fingerprint spectrum and construction method thereof

Abstract

The invention relates to the technical field of plant germplasm resources. The invention provides an SSR-PCR (Simple Sequence Repeat-Polymerase Chain Reaction) primer group for a robinia pseudoacacia genome SSR molecular marker, a robinia pseudoacacia core germplasm fingerprint spectrum obtained by utilizing the primer group and a method for constructing a robinia pseudoacacia core germplasm bank. The SSR-PCR primer group disclosed by the invention is relatively high in polymorphism, good in stability, relatively good in expression in the operation of the SSR molecular marker of the robinia pseudoacacia germplasm genome, and good in screening effect. By adopting the construction method disclosed by the invention, excellent germplasm with special characters can be screened out to construct the fingerprint spectrum of the robinia pseudoacacia core germplasm, a basis is better provided for scientific management, efficient utilization and preservation of robinia pseudoacacia germplasm resources, and meanwhile, the breeding efficiency can be effectively improved.

Description

Technical Field

[0001] The present invention relates to the technical field of plant germplasm resources, and in particular to a Robinia pseudoacacia core germplasm characteristic fingerprint map and a construction method thereof. Background Art

[0002] Each species in nature has its own unique gene pool, and genetic diversity is usually the sum of genetic variation among individuals within a species. Based on the influence of various factors such as chromosome variation, mating mode, gene flow, geographical environment, differences in selection mechanisms, and human intervention, changes in the level of genetic diversity of biological populations can occur. Studies have found that the higher the genetic diversity of a species, the richer the genetic variation, and the stronger its adaptability to the environment. Rich genetic diversity is the core and guarantee of stable reproduction and continuous evolution of species. Research on species genetic diversity is of great significance to the formulation of more scientific species protection and utilization strategies and to ensure the sustainable utilization of species and ecosystems. The collection, preservation and protection of germplasm resources is a means for humans to protect species diversity. Under the current land policy, the concept of core collection has found an optimization strategy for more efficient protection, evaluation and application of germplasm resources. Core collection can help researchers obtain target germplasm more efficiently with the minimum number of resources and genetic duplication. Core collection is a dynamic preservation process, which requires timely updating of rare and typical germplasm to ensure a high level of genetic diversity. Germplasm resources other than core collection are used as reserve collection.

[0003] At present, the detection methods of forest genetic diversity include morphological markers, cell markers, biochemical markers and molecular markers. Phenotypic trait markers and molecular markers are mainly used to construct plant core germplasm banks. In research and application, it is found that the genetic stability of phenotypic morphological markers is unstable, cell markers are complex, time-consuming and labor-intensive, and the number of biochemical marker detection sites is limited, which cannot represent the variation of the entire plant genome. In recent years, molecular markers have been well developed and have overcome the above shortcomings to a certain extent. They have been proven to be accurate and effective in the construction of many forest and grass core germplasms such as natural Korean pine (Pinus koraiensis), monk fruit (Siraitiagrosvenorii), perilla (Perilla frutescens), chestnut (Castaneamollissima), wild strawberry (Fragaria vesca), and Chinese fir (Cunninghamia lanceolata).

[0004] Although Robinia pseudoacacia L. is native to the United States, it has shown good adaptability in China, Europe and many other parts of the world. Robinia pseudoacacia is a pioneer tree species for afforestation in saline-alkali land and difficult site conditions, and its germplasm resources are preserved in Shandong, Shanxi, Henan and other places. Although some researchers have studied the germplasm resources of Robinia pseudoacacia, they mostly focus on the evaluation of phenotypic traits of Robinia pseudoacacia germplasm resources. There are fewer screening and research on core germplasm, and the research population is also small. In summary, there has been no comprehensive study on the genetic diversity and genetic structure of Robinia pseudoacacia populations and the construction of core seed banks. Existing studies believe that only direct introductions from the United States belong to the original source, and the rest of the introductions belong to secondary sources. Because there are mutual introductions and direct introductions from the United States between domestic Robinia pseudoacacia bases, there are unclear records and unknown sources in the long-term introduction process, and there may be phenomena such as homonyms and homonyms. Summary of the invention

[0005] The purpose of the present invention is to provide an SSR-PCR primer set for SSR molecular markers of Robinia pseudoacacia genome, a Robinia pseudoacacia core germplasm characteristic fingerprint obtained by using the primer set, and a method for obtaining the Robinia pseudoacacia core germplasm characteristic fingerprint. The SSR-PCR primer set has high polymorphism and good stability. The construction method of the primer set in the present invention can screen out excellent germplasms with special traits to construct a Robinia pseudoacacia core germplasm bank, better provide a basis for the scientific management, efficient utilization and preservation of Robinia pseudoacacia germplasm resources, and can also effectively improve breeding efficiency.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The invention provides an SSR-PCR primer set for SSR molecular markers of Robinia pseudoacacia genome, comprising one or more of primer set MQ7, primer set MQ12, primer set MQ16, primer set MQ32, primer set MQ37, primer set MQ39, primer set MQ47, primer set MQ49, primer set MQ54, primer set MQ59, primer set MQ64, primer set CC2-48, primer set CC2-54, primer set CC2-61, primer set Rply02, primer set Rply22 and primer set Rply109.

[0008] Preferably, the primer set MQ7 includes primer MQ7-F and primer MQ7-R, the sequence of the primer MQ7-F is shown as SEQ ID NO.1, and the sequence of the primer MQ7-R is shown as SEQ ID NO.2.

[0009] Preferably, the primer set MQ12 includes primer MQ12-F and primer MQ12-R, the sequence of the primer MQ12-F is shown in SEQ ID NO.3, and the sequence of the primer MQ12-R is shown in SEQ ID NO.4.

[0010] Preferably, the primer set MQ16 includes primer MQ16-F and primer MQ16-R, the sequence of the primer MQ16-F is shown in SEQ ID NO.5, and the sequence of the primer MQ16-R is shown in SEQ ID NO.6.

[0011] Preferably, the primer set MQ32 includes primer MQ32-F and primer MQ32-R, the sequence of the primer MQ32-F is shown in SEQ ID NO.7, and the sequence of the primer MQ32-R is shown in SEQ ID NO.8.

[0012] Preferably, the primer set MQ37 includes primer MQ37-F and primer MQ37-R, the sequence of the primer MQ37-F is shown in SEQ ID NO.9, and the sequence of the primer MQ37-R is shown in SEQ ID NO.10.

[0013] Preferably, the primer set MQ39 includes primer MQ39-F and primer MQ39-R, the sequence of the primer MQ39-F is shown in SEQ ID NO.11, and the sequence of the primer MQ39-R is shown in SEQ ID NO.12.

[0014] Preferably, the primer set MQ47 includes primer MQ47-F and primer MQ47-R, the sequence of the primer MQ47-F is shown in SEQ ID NO.13, and the sequence of the primer MQ47-R is shown in SEQ ID NO.14.

[0015] Preferably, the primer set MQ49 includes primer MQ49-F and primer MQ49-R, the sequence of the primer MQ49-F is shown in SEQ ID NO.15, and the sequence of the primer MQ49-R is shown in SEQ ID NO.16.

[0016] Preferably, the primer set MQ54 includes primer MQ54-F and primer MQ54-R, the sequence of the primer MQ54-F is shown in SEQ ID NO.17, and the sequence of the primer MQ54-R is shown in SEQ ID NO.18.

[0017] Preferably, the primer set MQ59 includes primer MQ59-F and primer MQ59-R, the sequence of the primer MQ59-F is shown in SEQ ID NO.19, and the sequence of the primer MQ59-R is shown in SEQ ID NO.20.

[0018] Preferably, the primer set MQ64 includes primer MQ64-F and primer MQ64-R, the sequence of the primer MQ64-F is shown in SEQ ID NO.21, and the sequence of the primer MQ64-R is shown in SEQ ID NO.22.

[0019] Preferably, the primer set CC2-48 includes primer CC2-48-F and primer CC2-48-R, the sequence of primer CC2-48-F is shown in SEQ ID NO.23, and the sequence of primer CC2-48-R is shown in SEQ ID NO.24.

[0020] Preferably, the primer set CC2-54 includes primer CC2-54-F and primer CC2-54-R, the sequence of primer CC2-54-F is shown in SEQ ID NO.25, and the sequence of primer CC2-54-R is shown in SEQ ID NO.26.

[0021] Preferably, the primer set CC2-61 includes primer CC2-61-F and primer CC2-61-R, the sequence of the primer CC2-61-F is shown in SEQ ID NO.27, and the sequence of the primer CC2-61-R is shown in SEQ ID NO.28.

[0022] Preferably, the primer set Rply02 includes primer Rply02-F and primer Rply02-R, the sequence of the primer Rply02-F is shown in SEQ ID NO.29, and the sequence of the primer Rply02-R is shown in SEQ ID NO.30.

[0023] Preferably, the primer set Rply22 includes primer Rply22-F and primer Rply22-R, the sequence of the primer Rply22-F is shown in SEQ ID NO.31, and the sequence of the primer Rply22-R is shown in SEQ ID NO.32.

[0024] Preferably, the primer set Rply109 includes primer Rply109-F and primer Rply109-R, the sequence of the primer Rply109-F is shown in SEQ ID NO.33, and the sequence of the primer Rply109-R is shown in SEQ ID NO.34.

[0025] The present invention also provides a Robinia pseudoacacia core germplasm characteristic fingerprint map, using the SSR-PCR primer set to perform SSR-PCR amplification on the genomes of different varieties of Robinia pseudoacacia, and then establishing SSR genotype information data based on the length of the SSR-PCR amplification product, encoding according to the primer sequence, and the obtained fingerprint code is the Robinia pseudoacacia variety characteristic fingerprint map.

[0026] Preferably, the characteristic fingerprints of 75 Robinia pseudoacacia varieties are shown in Table 10.

[0027] The present invention also provides a method for constructing a Robinia pseudoacacia core germplasm bank using the above SSR-PCR primer set, comprising the following steps:

[0028] (1) Collect Robinia pseudoacacia germplasm resources and extract genomic DNA from various germplasm resources;

[0029] (2) SSR molecular markers are applied to the genome of the Robinia pseudoacacia germplasm sample described in step (1), and then SSR genotype information data is established based on the length of the SSR-PCR amplification product and genetic diversity analysis is performed;

[0030] (3) Performing genome cluster analysis on the Robinia pseudoacacia germplasm samples described in step (1), gradually selecting samples based on the minimum genetic distance, and constructing a core germplasm library.

[0031] Preferably, the SSR molecular labeling process in step (2) includes an SSR-PCR reaction, and the reaction system of the SSR-PCR reaction is 25 μL, including 12.5 μL of 2×GS Taq PCR Mix, 1.0 μL of forward SSR-PCR primer, 1.0 μL of reverse SSR-PCR primer, 1 μL of DNA template, and ddH2O is supplemented to 25 μL.

[0032] Preferably, the reaction procedure of the SSR-PCR reaction is: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 20 s, annealing at 55°C for 20 s, extension at 72°C for 1 min, 35 cycles; final extension at 72°C for 10 min.

[0033] Preferably, the 5' end of the SSR-PCR primer is connected with a fluorescent group, and the fluorescent group includes TARMRA, FAM, ROX or HEX.

[0034] By adopting the above technical solution, the present invention has the following beneficial effects:

[0035] The SSR-PCR primer set for SSR molecular markers of Robinia pseudoacacia genome described in the technical solution of the present invention has high polymorphism and good stability, performs relatively well in the operation of SSR molecular markers of Robinia pseudoacacia germplasm genome, and has good screening effect. In addition, the construction method of the present invention can screen out germplasms with special traits and excellent properties to construct a Robinia pseudoacacia core germplasm bank, better provide a basis for the scientific management, efficient utilization and preservation of Robinia pseudoacacia germplasm resources, and can also effectively improve breeding efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The results of molecular variance analysis of genetic differentiation and gene flow among populations;

[0037] Figure 2 This is the analysis of the genetic structure of the total population of Robinia pseudoacacia clone germplasm;

[0038] Figure 3 This is an analysis diagram based on the genetic origin of Robinia pseudoacacia;

[0039] Figure 4 The PCoA analysis results of the original germplasm, initial core library, and supplementary core germplasm;

[0040] Figure 5 To detect the genome of Robinia pseudoacacia of FW01 variety using CC2-61 primer set;

[0041] Figure 6 To detect the genome of Robinia pseudoacacia L. 226 using the MQ39 primer set;

[0042] Figure 7 To detect the genome of Robinia pseudoacacia of FW05 variety using MQ12 primer set;

[0043] Figure 8 To detect the genome of Robinia pseudoacacia of FW04 variety using MQ54 primer set;

[0044] Fig. 9 To detect the genome of Robinia pseudoacacia of Meici 87-12 variety using MQ64 primer set;

[0045] Fig.10 To detect the genome of Robinia pseudoacacia cultivar Qingza 9 using the MQ64 primer set;

[0046] Fig.11 To detect the genome of Robinia pseudoacacia cultivar Liaocixing 23 using the MQ12 primer set;

[0047] Fig.12 To detect the genome map of Robinia pseudoacacia cultivar Lu 13 Zichao 1501 using the CC2-54 primer set;

[0048] Fig.13 To detect the genome map of Robinia pseudoacacia cultivar Meici 11 using the CC2-54 primer set;

[0049] Fig.14 To detect the genome of Robinia pseudoacacia of the variety Luci 133-Robinia pseudoacacia (15-2) using the MQ32 primer set;

[0050] Fig.15 To detect the genome of Robinia pseudoacacia L. variety 131 using the MQ39 primer set;

[0051] Fig.16 This is the detection map of the early-leaf variety of Robinia pseudoacacia genome using the MQ49 primer set. DETAILED DESCRIPTION

[0052] The invention provides an SSR-PCR primer set for SSR molecular markers of Robinia pseudoacacia genome, comprising one or more of primer set MQ7, primer set MQ12, primer set MQ16, primer set MQ32, primer set MQ37, primer set MQ39, primer set MQ47, primer set MQ49, primer set MQ54, primer set MQ59, primer set MQ64, primer set CC2-48, primer set CC2-54, primer set CC2-61, primer set Rply02, primer set Rply22 and primer set Rply109.

[0053] In the present invention, the primer set MQ7 includes primer MQ7-F and primer MQ7-R, the sequence of primer MQ7-F is shown in SEQ ID NO.1, and the specific sequence is 5'-AACACGAGTCGAGGCTATCAAT-3'; the sequence of primer MQ7-R is shown in SEQ ID NO.2, and the specific sequence is 5'-ACGGCTAGAGTCATTGGTTCAA-3'.

[0054] In the present invention, the primer group MQ12 includes primer MQ12-F and primer MQ12-R, the sequence of primer MQ12-F is shown in SEQ ID NO.3, and the specific sequence is 5'-AGAAAAGCGTCCATTGTCTAAT-3'; the sequence of primer MQ12-R is shown in SEQ ID NO.4, and the specific sequence is 5'-AGAGGGACAAGACAGAAAATG-3'.

[0055] In the present invention, the primer set MQ16 includes primer MQ16-F and primer MQ16-R, the sequence of primer MQ16-F is shown in SEQ ID NO.5, and the specific sequence is 5'-ACCCTTACGCTTTGCAGATATA-3'; the sequence of primer MQ16-R is shown in SEQ ID NO.6, and the specific sequence is 5'-TCAACGTCCAATTTTCGGTAGA-3'.

[0056] In the present invention, the primer set MQ32 includes primer MQ32-F and primer MQ32-R, the sequence of primer MQ32-F is shown in SEQ ID NO.7, and the specific sequence is 5'-TCCCTCTCTTTCCGTTGATGAT-3'; the sequence of primer MQ32-R is shown in SEQ ID NO.8, and the specific sequence is 5'-AATCCTGGACGTGAAGAATAAA-3'.

[0057] In the present invention, the primer set MQ37 includes primer MQ37-F and primer MQ37-R, the sequence of primer MQ37-F is shown in SEQ ID NO.9, and the specific sequence is 5'-ACGAATTCTTTCTTCCGCAAAA-3'; the sequence of primer MQ37-R is shown in SEQ ID NO.10, and the specific sequence is 5'-TCGCCCTCTATTTCTTGTTCTT-3'.

[0058] In the present invention, the primer set MQ39 includes primer MQ39-F and primer MQ39-R, the sequence of primer MQ39-F is shown in SEQ ID NO.11, and the specific sequence is 5'-CTTCGTGGTGGTGTCCTTTATA-3'; the sequence of primer MQ39-R is shown in SEQ ID NO.12, and the specific sequence is 5'-CGAAAGAATCCACTGCCAATTT-3'.

[0059] In the present invention, the primer set MQ47 includes primer MQ47-F and primer MQ47-R, the sequence of primer MQ47-F is shown in SEQ ID NO.13, and the specific sequence is 5'-TGGTTCCGTTGCTTTCTTATTC-3'; the sequence of primer MQ47-R is shown in SEQ ID NO.14, and the specific sequence is 5'-AGACTATGCCTACGCCATATAA-3'.

[0060] In the present invention, the primer set MQ49 includes primer MQ49-F and primer MQ49-R, the sequence of primer MQ49-F is shown in SEQ ID NO.15, and the specific sequence is 5'-ACTGCCGCCACTTATCTTTATT-3'; the sequence of primer MQ49-R is shown in SEQ ID NO.16, and the specific sequence is 5'-GAACCGCTGGATCATCACTTAT-3'.

[0061] In the present invention, the primer set MQ54 includes primer MQ54-F and primer MQ54-R, the sequence of primer MQ54-F is shown in SEQ ID NO.17, and the specific sequence is 5'-CAGTCCAAGGGTTGAAGTAAAA-3'; the sequence of primer MQ54-R is shown in SEQ ID NO.18, and the specific sequence is 5'-GTCGTTAAACCCGGCAAAATA-3'.

[0062] In the present invention, the primer set MQ59 includes primer MQ59-F and primer MQ59-R, the sequence of primer MQ59-F is shown in SEQ ID NO.19, and the specific sequence is 5'-AAATCGTCGAGAACCCTTTAAA-3'; the sequence of primer MQ59-R is shown in SEQ ID NO.20, and the specific sequence is 5'-TTCTGGGCCGAGAGAATTATTT-3'.

[0063] In the present invention, the primer set MQ64 includes primer MQ64-F and primer MQ64-R, the sequence of primer MQ64-F is shown in SEQ ID NO.21, and the specific sequence is 5'-GGATAGAGATATCGCCCGTTAT-3'; the sequence of primer MQ64-R is shown in SEQ ID NO.22, and the specific sequence is 5'-TCGTGTGGATCAAAAGAAAGAT-3'.

[0064] In the present invention, the primer set CC2-48 includes primer CC2-48-F and primer CC2-48-R, the sequence of primer CC2-48-F is shown in SEQ ID NO.23, and the specific sequence is 5'-GACACTGAAATAGGCTCCTGAT-3'; the sequence of primer CC2-48-R is shown in SEQ ID NO.24, and the specific sequence is 5'-ATTTTGGCAGGAGGGGAAATTA-3'.

[0065] In the present invention, the primer set CC2-54 includes primer CC2-54-F and primer CC2-54-R, the sequence of primer CC2-54-F is shown in SEQ ID NO.25, and the specific sequence is 5'-TCAGTCCAAGGGTTGAAGTAAA-3'; the sequence of primer CC2-54-R is shown in SEQ ID NO.26, and the specific sequence is 5'-GTCGTTAAACCCGGCAAAATA-3'.

[0066] In the present invention, the primer set CC2-61 includes primer CC2-61-F and primer CC2-61-R, the sequence of primer CC2-61-F is shown in SEQ ID NO.27, and the specific sequence is 5'-TGAGGCCCCATATTGACTATCA-3'; the sequence of primer CC2-61-R is shown in SEQ ID NO.28, and the specific sequence is 5'-TCGGGAGTCACAAATTCGTTAA-3'.

[0067] In the present invention, the primer set Rply02 includes primer Rply02-F and primer Rply02-R, the sequence of primer Rply02-F is shown in SEQ ID NO.29, and the specific sequence is 5'-TGTGAATGGTTGGTGGACAT-3'; the sequence of primer Rply02-R is shown in SEQ ID NO.30, and the specific sequence is 5'-CGTTGCTTGGAGGAGAATAA-3'.

[0068] In the present invention, the primer set Rply22 includes primer Rply22-F and primer Rply22-R, the sequence of primer Rply22-F is shown in SEQ ID NO.31, and the specific sequence is 5'-ATCACCATCTGTTCCTCCAC-3'; the sequence of primer Rply22-R is shown in SEQ ID NO.32, and the specific sequence is 5'-TTCTCCTCAGCCACTTCTTT-3'.

[0069] In the present invention, the primer set Rply109 includes primer Rply109-F and primer Rply109-R, the sequence of primer Rply109-F is shown in SEQ ID NO.33, and the specific sequence is 5'-GAGGAATCACAAAACCGTTTGG-3'; the sequence of primer Rply109-R is shown in SEQ ID NO.34, and the specific sequence is 5'-TGGGATTTGAGAGAGTGGTGGTG-3'.

[0070] The present invention also provides a characteristic fingerprint map of Robinia pseudoacacia varieties. The SSR-PCR primer set is used to perform SSR-PCR amplification on the genomes of different varieties of Robinia pseudoacacia. Then, SSR genotype information data is established according to the length of the SSR-PCR amplification product, and the data is encoded according to the primer sequence. The obtained fingerprint code is the characteristic fingerprint map of Robinia pseudoacacia varieties.

[0071] In the present invention, the characteristic fingerprints of 75 Robinia pseudoacacia varieties are shown in Table 11, wherein the detection result of the SSR-PCR forward primer is before the “ / ”, and the detection result of the SSR-PCR reverse primer is after the “ / ”.

[0072] The present invention also provides a method for constructing a Robinia pseudoacacia core germplasm bank using the SSR-PCR primer set, comprising the following steps:

[0073] (1) Collect Robinia pseudoacacia germplasm resources and extract genomic DNA from various germplasm resources;

[0074] (2) SSR molecular markers are applied to the genome of the Robinia pseudoacacia germplasm sample described in step (1), and then SSR genotype information data is established based on the length of the SSR-PCR amplification product and genetic diversity analysis is performed;

[0075] (3) Performing genome cluster analysis on the Robinia pseudoacacia germplasm samples described in step (1), gradually selecting samples based on the minimum genetic distance, and constructing a core germplasm library.

[0076] In the present invention, the SSR molecular marking process in step (2) includes an SSR-PCR reaction, and the reaction system of the SSR-PCR reaction is 25 μL, including 12.5 μL of 2×GS Taq PCR Mix, 1.0 μL of forward SSR-PCR primer, 1.0 μL of reverse SSR-PCR primer, 1 μL of DNA template, and ddH2O is supplemented to 25 μL.

[0077] In the present invention, the reaction procedure of the SSR-PCR reaction is: 94°C pre-denaturation for 3 min, 94°C denaturation for 20 s, 55°C annealing for 20 s, 72°C extension for 1 min, 35 cycles; 72°C final extension for 10 min.

[0078] In the present invention, the 5' end of the SSR-PCR primer is connected with a fluorescent group, and the fluorescent group includes TARMRA, FAM, ROX or HEX.

[0079] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0080] Example 1

[0081] (I) Test materials

[0082] 323 Robinia pseudoacacia clones were collected by the research team in the early stage through introduction, hybrid breeding, seedling selection, etc., including 41 from the United States (original source), 16 from Beijing, 187 from Shandong, 24 from Shanxi, 26 from Henan, and 29 from Liaoning, a total of 6 geographical origins. They are preserved off-site at the Daqingshan Robinia pseudoacacia germplasm resource base in Shandong Province (35°387′N, 118°189′E). At least 6 strains of each acacia are preserved. In April 2023, the outer tender leaves of 323 Robinia pseudoacacia clones (free of diseases and pests) were collected, quickly dried with silica gel, and brought back to the laboratory for use. (two)

[0084] 1. Extraction and PCR Amplification of Robinia pseudoacacia Genome

[0085] The genomic DNA of the samples was extracted by the modified CTAB method (Doyle JJ, Doyle JL (1987). Arapid DNA isolation procedure for small quantities of fresh leaf tissue Phytochem Bull 19, 11-15.), and the DNA concentration and quality were detected by NanoDrop 2000 and 1% agarose gel electrophoresis, respectively, and stored at -20°C for future use. Based on the previous data of Robinia pseudoacacia chloroplast genome and transcriptome of the research group, MISA software was used to search for SSR sites. A total of 100 pairs of SSR primers were designed and synthesized by Shandong Huabo Gene Engineering Co., Ltd. 8 sample DNA templates were randomly selected for SSR primer screening, and 17 pairs of SSR primers with high polymorphism and good stability were screened out for subsequent research (see Table 1).

[0086] The reaction system of the SSR-PCR reaction is 25 μL, including 12.5 μL of 2×GS Taq PCR Mix, 1.0 μL of forward SSR-PCR primer, 1.0 μL of reverse SSR-PCR primer, 1 μL of DNA template, and ddH2O is supplemented to 25 μL.

[0087] The reaction procedure of the SSR-PCR reaction is: pre-denaturation at 94°C for 3 min, denaturation at 94°C for 20 s, annealing at 55°C for 20 s, extension at 72°C for 1 min, 35 cycles; final extension at 72°C for 10 min.

[0088] Table 1 SSR primer information

[0089]

[0090]

[0091] 2. Data Analysis

[0092] After SSR-PCR amplification, capillary electrophoresis was performed using an ABI3730XL sequencer. 0.3 μL of PCR product, 0.5 μL of molecular weight internal standard (LIZ500) and 9.5 μL of deionized formamide were mixed and added to the PCR plate, denatured at 95°C for 5 min, cooled at 4°C and centrifuged, and tested on the machine with 1× Buffer. The original file of the capillary electrophoresis result was imported into Genemarker 2.2.0, and the peak graph and Excel site information table were exported by site.

[0093] The data of capillary electrophoresis were analyzed by Gene Marker 2.2.0 to obtain the fragment length of each SSR locus of different samples. The observed allele number Na, effective allele number Ne, observed heterozygosity Ho, expected heterozygosity He, Shannon diversity index I, Nei's gene diversity index H, F value and gene flow Nm value between populations were calculated by POPGENE32 software. The polymorphism information content PIC value was obtained by PowerMaker software. The population structure of Robinia pseudoacacia was analyzed by Structure software, with ploidy set to 2 and K value set to 1-10. Each K value was calculated 20 times, using the correlated alleles frequencies model and admixture model, supplemented by 10,000 Markov chain Monte Carlo (MCMC) repetitions and 100,000 burn-ins. The optimal K value was inferred and calculated by the Evanno2005 method in the python 3 algorithm. The optimal number of groups was determined based on the optimal K value.

[0094] (1) Analysis of the diversity of the total population of Robinia pseudoacacia clones

[0095] The results of the diversity analysis of 323 Robinia pseudoacacia clones by SSR fluorescent labeling are shown in Table 2. A total of 135 alleles were amplified by 17 pairs of SSR-PCR primers. The observed allele number Na ranged from 3 (CC2-61) to 23 (Rply109), with an average of 7.941 alleles amplified per primer pair. The effective allele number Ne ranged from 1.372 (MQ16) to 6.675 (RPly109), with an average of 2.808. The Shann information index I ranged from 0.556 (MQ37) to 2.334 (RPly10 9), with a mean of 1.2; the observed heterozygosity Ho varied from 0.012 (MQ420) to 0.690 (RPly109), with a mean of 0.191; the expected heterozygosity He varied from 0.271 (MQ16) to 0.850 (RPly109), with a mean of 0.588; the Nei's diversity index H varied from 0.272 (MQ16) to 0.852 (RPly109), with a mean of 0.589; the polymorphic information content PIC varied from 0.2567 (MQ37) to 0.8382 (RPly109), with a mean of 0.5443.

[0096] Table 2 Diversity of the total population of Robinia pseudoacacia clones

[0097] Locus Na Ne I Ho He H PIC MQ49 6.000 4.261 1.588 0.016 0.765 0.767 0.7296 MQ54 5.000 2.576 1.158 0.356 0.612 0.613 0.5661 MQ7 7.000 2.832 1.263 0.133 0.647 0.648 0.5922 CC2-61 3.000 2.093 0.855 0.015 0.522 0.523 0.4414 CC2-54 5.000 2.762 1.209 0.378 0.638 0.639 0.5914 CC2-48 7.000 3.348 1.301 0.526 0.701 0.702 0.6458 MQ59 7.000 2.752 1.294 0.016 0.637 0.638 0.5898 MQ32 9.000 2.753 1.286 0.019 0.637 0.638 0.5883 MQ37 7.000 1.391 0.556 0.099 0.281 0.282 0.2567 MQ39 9.000 2.856 1.359 0.019 0.650 0.651 0.6089 MQ47 7.000 2.107 0.953 0.012 0.525 0.526 0.4508 MQ12 8.000 2.977 1.361 0.028 0.664 0.665 0.6211 MQ64 6.000 2.379 1.016 0.016 0.580 0.581 0.5001 MQ16 7.000 1.372 0.606 0.015 0.271 0.272 0.2578 Rply02 10.000 3.062 1.468 0.557 0.673 0.674 0.6418 Rply22 9.000 1.538 0.797 0.347 0.350 0.350 0.3339 Rply109 23.000 6.675 2.334 0.690 0.850 0.852 0.8382 Mean 7.941 2.808 1.200 0.191 0.588 0.589 0.5443

[0098] Note: Na——Observed number of different alleles; Ne——Number of effective alleles; Ho——Observed heterozygosity; He——Expected heterozygosity; I——Shannon diversity index; H——Nei's diversity index; PIC——Polymorphic information content.

[0099] (2) Analysis of genetic diversity of Robinia pseudoacacia germplasm population

[0100] The results of genetic differentiation and gene flow analysis among populations are shown in Table 3. The genetic differentiation coefficient (Fst) among populations ranged from 0.018 to 0.055, with an average of 0.038. Fst < 0.05 indicates that the degree of population differentiation is small, indicating that the degree of genetic differentiation among Robinia acacia clone populations is low. Gene flow (Nm) is the amount of net inflow or outflow of genes between populations. In the analysis, the Nm among the six populations ranged from 4.285 to 13.445, with an average of 6.964, indicating that there was a certain amount of gene exchange among populations. The results of variance analysis showed that ( Figure 1 ), the variation between groups is very small, accounting for 2%, the variation between individuals accounts for 66%, and the variation based on individual gene mutations accounts for 32%.

[0101] Table 3 Analysis of genetic differentiation and gene flow among populations

[0102]

[0103]

[0104] (3) Analysis of genetic structure of Robinia pseudoacacia population

[0105] As geographical factors have little influence, genetic structure analysis was conducted on 323 clone germplasm populations. Figure 2 ), when △k is the maximum, K = 2, 323 clones can be divided into 2 groups; Figure 3 Based on the gene sources of individuals and populations, the 323 asexual lines had two main gene sources, and the germplasm from six geographical sources was evenly distributed in the two groups.

[0106] (III) Construction of core germplasm bank

[0107] According to the existing research on the relationship between the core germplasm and the number of samples, 12 sampling ratios were set, including 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, and 60%. After clustering analysis of germplasm resources using the UPGMA method using NTSYSpc2.11 software, the least distance stepwise sampling method (LDSS) was used to construct the core germplasm. The specific method is: according to the clustering results of germplasm resources, find a group with the smallest genetic distance, randomly delete one of the two samples in the group, and keep the other sample; if the group contains more than two samples, select one sample to enter the next round of clustering until the set ratio value is reached.

[0108] 1. Initial core germplasm library construction

[0109] The Na, Ne, Ho, He, I, H values ​​and the total number of alleles in the germplasm pool under each sampling ratio were calculated, and the optimal sampling ratio was screened out to determine the initial core germplasm pool. The genetic diversity parameters of the initial core germplasm pool were tested using SPSS22.0 software.

[0110] The minimum distance was used to stepwise cluster the above 12 proportions and calculate the genetic diversity indices Na, Ne, Ho, He, I, and H of the Robinia pseudoacacia germplasm bank. According to the results comparison and analysis (Table 4), except for the 30% proportion, Na generally decreased with the decrease of the sampling proportion. The values ​​of Ne, He, I, and H first increased with the decrease of the sampling proportion and decreased after the proportion reached 20%. Ho had the largest value at the 10% proportion. Considering the genetic diversity index comprehensively, 20% was selected as the initial core germplasm bank construction proportion.

[0111] Table 4 Comparison of genetic diversity under different sampling ratios

[0112]

[0113]

[0114] 2. Optimize the construction of core germplasm library

[0115] The core germplasm pool construction strategy was optimized by Chen Cun et al. (Chen Cun, Ding Changjun, Zhang Jing, et al. Population structure analysis and core germplasm pool construction of American Poplar [J]. Forestry Science, 2020, 56(9):10.), specifically, the distribution and loss of alleles in the initial core germplasm pool were analyzed, and the minimum number of germplasms in the reserved germplasm pool were selected to complete the alleles (100%) to form the final core germplasm pool. The representativeness of the optimized core germplasm pool was evaluated by t-test, and the distribution map of the reserved germplasm, initial core germplasm, and supplementary germplasm was drawn using the principal coordinate (PcoA).

[0116] Through comparative analysis (Table 5), it was found that the 17 pairs of SSR primers in the initial core germplasm library under the sampling ratio of 20% had a total of 123 alleles, and 12 alleles were lost compared with the original germplasm. Based on the excellent and specific comprehensive representativeness of the core germplasm, the original allele situation was analyzed, and 12 Robinia pseudoacacia clones were selected from the retained germplasm as supplementary germplasm and added to the initial core germplasm library to obtain an optimized core germplasm library with 75 clone germplasms and a screening ratio of 23.22%. The comparison of the diversity indexes of the original germplasm, initial core library and optimized core germplasm library showed that the genetic diversity index of the initial core library, optimized core germplasm library and core germplasm t test was not significant at the 0.05 level, indicating that the two libraries can fully represent the genetic diversity of the original germplasm. The I, Ho, He and H of the core germplasm library were higher than those of the original germplasm, indicating that the core germplasm effectively eliminated the genetic redundancy of the original germplasm. The number of observed alleles Na in the optimized core germplasm library was 100% compared with the initial optimized core germplasm library, and the diversity index was not significantly different at the 0.05 level. PCoA analysis was performed on 248 reserved germplasms, 63 initial core germplasms and 12 supplementary germplasms based on genetic distance ( Figure 4 ), 63 initial core germplasms and 12 supplementary germplasms were evenly distributed, indicating that the selected core germplasms were well representative. In summary, the construction of the optimized core germplasm bank was scientific. The geographical origins of the 75 core germplasms (Table 6) were 6 from Beijing, 8 from Henan, 17 from the United States, 8 from Shanxi, 29 from Shandong, and 7 from Liaoning.

[0117] Table 5 Comparison of genetic diversity indicators between original germplasm and initial core library

[0118]

[0119] Table 6 Comparison of geographical origins of original germplasm and core germplasm bank

[0120] group Beijing Shanxi Shandong USA Liaoning Henan Number of germplasms Original germplasm 16 24 187 41 26 29 323 Initial core collection 5 5 23 15 7 8 63 Optimizing core germplasm bank 6 8 29 17 7 8 75

[0121] 3. Construction of Robinia pseudoacacia core germplasm bank

[0122] Based on the genetic differentiation and genetic structure of 323 Robinia pseudoacacia populations from geographical origins, 75 core accessions were screened out, as shown in Tables 7, 8 and 9.

[0123] Table 7 Genetic diversity analysis of 75 core germplasm resources

[0124] Locus Na Ne I Ho He u MQ49 6.000 5.067 1.700 0.053 0.803 0.808 MQ54 5.000 3.650 1.370 0.539 0.726 0.731 MQ7 7.000 3.828 1.548 0.184 0.739 0.744 CC2-61 3.000 2.792 1.059 0.066 0.642 0.646 CC2-54 5.000 3.928 1.424 0.592 0.745 0.750 CC2-48 7.000 2.888 1.299 0.270 0.654 0.658 MQ59 7.000 4.994 1.729 0.068 0.800 0.805 MQ32 9.000 5.023 1.754 0.079 0.801 0.806 MQ37 7.000 1.970 0.965 0.237 0.492 0.496 MQ39 9.000 5.776 1.872 0.079 0.827 0.832 MQ47 7.000 3.451 1.442 0.053 0.710 0.715 MQ12 8.000 4.762 1.706 0.079 0.790 0.795 MQ64 6.000 2.645 1.192 0.056 0.622 0.626 MQ16 7.000 1.900 1.044 0.066 0.474 0.477 Rply02 10.000 4.195 1.759 0.658 0.762 0.767 Rply22 9.000 1.765 1.020 0.395 0.433 0.436 Rply109 23.000 9.294 2.536 0.645 0.892 0.898

[0125] Table 8 Sources of 75 Robinia pseudoacacia core germplasms

[0126]

[0127]

[0128]

[0129] Table 975 core germplasm fingerprints details

[0130]

[0131]

[0132]

[0133]

[0134] In summary, it can be seen that the SSR-PCR primer set for SSR molecular markers of Robinia pseudoacacia genome described in the present invention has high polymorphism and good stability. The construction method of the primer set can screen out germplasm with special traits and excellence to construct a Robinia pseudoacacia core germplasm bank, which can better provide a basis for the scientific management, efficient utilization and preservation of Robinia pseudoacacia germplasm resources in production, and can also effectively improve breeding efficiency.

[0135] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An SSR-PCR primer set for SSR molecular markers of Robinia pseudoacacia genome, characterized in that: Including one or more of primer set MQ7, primer set MQ12, primer set MQ16, primer set MQ32, primer set MQ37, primer set MQ39, primer set MQ47, primer set MQ49, primer set MQ54, primer set MQ59, primer set MQ64, primer set CC2-48, primer set CC2-54, primer set CC2-61, primer set Rply02, primer set Rply22, and primer set Rply109; The primer set MQ7 includes primer MQ7-F and primer MQ7-R, the sequence of the primer MQ7-F is shown in SEQ ID NO.1, and the sequence of the primer MQ7-R is shown in SEQ ID NO.2; The primer set MQ12 includes primer MQ12-F and primer MQ12-R, the sequence of the primer MQ12-F is shown in SEQ ID NO.3, and the sequence of the primer MQ12-R is shown in SEQ ID NO.4; The primer set MQ16 includes primer MQ16-F and primer MQ16-R, the sequence of the primer MQ16-F is shown in SEQ ID NO.5, and the sequence of the primer MQ16-R is shown in SEQ ID NO.6; The primer set MQ32 includes primer MQ32-F and primer MQ32-R, the sequence of the primer MQ32-F is shown in SEQ ID NO.7, and the sequence of the primer MQ32-R is shown in SEQ ID NO.8; The primer set MQ37 includes primer MQ37-F and primer MQ37-R, the sequence of the primer MQ37-F is shown in SEQ ID NO.9, and the sequence of the primer MQ37-R is shown in SEQ ID NO.10; The primer set MQ39 includes primer MQ39-F and primer MQ39-R, the sequence of the primer MQ39-F is shown in SEQ ID NO.11, and the sequence of the primer MQ39-R is shown in SEQ ID NO.12; The primer set MQ47 includes primer MQ47-F and primer MQ47-R, the sequence of the primer MQ47-F is shown in SEQ ID NO.13, and the sequence of the primer MQ47-R is shown in SEQ ID NO.14; The primer set MQ49 includes primer MQ49-F and primer MQ49-R, the sequence of the primer MQ49-F is shown in SEQ ID NO.15, and the sequence of the primer MQ49-R is shown in SEQ ID NO.16; The primer set MQ54 includes primer MQ54-F and primer MQ54-R, the sequence of the primer MQ54-F is shown in SEQ ID NO.17, and the sequence of the primer MQ54-R is shown in SEQ ID NO.18; The primer set MQ59 includes primer MQ59-F and primer MQ59-R, the sequence of the primer MQ59-F is shown in SEQ ID NO.19, and the sequence of the primer MQ59-R is shown in SEQ ID NO.20; The primer set MQ64 includes primer MQ64-F and primer MQ64-R, the sequence of the primer MQ64-F is shown in SEQ ID NO.21, and the sequence of the primer MQ64-R is shown in SEQ ID NO.22; The primer set CC2-48 includes primer CC2-48-F and primer CC2-48-R, the sequence of primer CC2-48-F is shown in SEQ ID NO.23, and the sequence of primer CC2-48-R is shown in SEQ ID NO.24; The primer set CC2-54 includes primer CC2-54-F and primer CC2-54-R, the sequence of primer CC2-54-F is shown in SEQ ID NO.25, and the sequence of primer CC2-54-R is shown in SEQ ID NO.26; The primer set CC2-61 includes primer CC2-61-F and primer CC2-61-R, the sequence of primer CC2-61-F is shown in SEQ ID NO.27, and the sequence of primer CC2-61-R is shown in SEQ ID NO.28; The primer set Rply02 includes primer Rply02-F and primer Rply02-R, the sequence of the primer Rply02-F is shown in SEQ ID NO.29, and the sequence of the primer Rply02-R is shown in SEQ ID NO.30; The primer set Rply22 includes primer Rply22-F and primer Rply22-R, the sequence of the primer Rply22-F is shown in SEQ ID NO.31, and the sequence of the primer Rply22-R is shown in SEQ ID NO.32; The primer set Rply109 includes primer Rply109-F and primer Rply109-R, the sequence of the primer Rply109-F is shown in SEQ ID NO.33, and the sequence of the primer Rply109-R is shown in SEQ ID NO.

34.

2. A Robinia pseudoacacia core germplasm characteristic fingerprint, characterized in that: The SSR-PCR primer set described in claim 1 is used to perform SSR-PCR amplification on the genomes of different varieties of Robinia pseudoacacia, and then SSR genotype information data is established according to the length of the SSR-PCR amplification product, and encoded according to the primer sequence. The obtained fingerprint code is the Robinia pseudoacacia core germplasm characteristic fingerprint map.

3. The Robinia pseudoacacia core germplasm characteristic fingerprint according to claim 2, characterized in that: The characteristic fingerprints of 75 Robinia species are as follows:

4. A method for constructing a Robinia pseudoacacia core germplasm bank using the SSR-PCR primer set of claim 1, characterized in that: The steps include: (1) Collect Robinia pseudoacacia germplasm resources and extract genomic DNA from various germplasm resources; (2) SSR molecular markers are applied to the genome of the Robinia pseudoacacia germplasm sample described in step (1), and then SSR genotype information data is established based on the length of the SSR-PCR amplification product and genetic diversity analysis is performed; (3) Stepwise screening samples of the Robinia pseudoacacia germplasm sample genomes described in step (1) based on the minimum genetic distance to construct a core germplasm library.

5. The method according to claim 4, characterized in that The SSR molecular labeling process in step (2) includes an SSR-PCR reaction. The reaction system of the SSR-PCR reaction is 25 μL, including 12.5 μL of 2×GS TaqPCRMix, 1.0 μL of forward SSR-PCR primer, 1.0 μL of reverse SSR-PCR primer, 1 μL of DNA template, and ddH2O is supplemented to 25 μL.

6. The method according to claim 4, characterized in that The reaction procedure of the SSR-PCR reaction is: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 20 s, annealing at 55°C for 20 s, extension at 72°C for 1 min, 35 cycles; final extension at 72°C for 10 min.

7. The method according to claim 4, characterized in that The 5' end of the SSR-PCR primer is connected with a fluorescent group, and the fluorescent group includes TARMRA, FAM, ROX or HEX.

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