Polymorphic microsatellite molecular marker of sticktight, primer and application of polymorphic microsatellite molecular marker

Abstract

The invention provides a spanishneedles polymorphic microsatellite molecular marker, a primer and application thereof, and belongs to the technical field of molecular markers. The core sequences of the molecular markers are TTC, GA, TACA, AGA, CAC, CAA, AG, CT, AT, GTT, CT, CTT, CA, TA, AT, GA, AG, CAC, TC, TGA, TGG, CA, TC, TA, AG, AG and TC in sequence. The SSR markers can directly reflect genetic information of a spanishneedles genome, so that the genetic diversity of spanishneedles groups is better analyzed, and a powerful tool is provided for genetic map construction, target gene calibration, fingerprint drawing, variety identification, pedigree analysis, inter-group genetic distance analysis, evolution, genetic diversity, variety breeding and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular markers, and in particular to a polymorphic microsatellite molecular marker, primer and application thereof for Phallus odoratus. Background Art

[0002] The genus Phallus Phallus is an important group of economic fungi, and many species of this genus are also well-known edible and medicinal fungi, such as Dictyophora echinovolvata Phallus echinovolvatus , Dictyophora rubrovolvata P. rubrovolvatus , Winter Phallus P.dongsun and so on. Most of the fungi in the genus Phallus have a foul smell, while Phallus odoratus has a faint lilac smell after budding and can be eaten. Because it is extremely similar in appearance and living habits to Winter Phallus, it is often cultivated or sold as Winter Phallus. It is extremely difficult to identify the fungal species only based on morphological characteristics. At present, there are few reports on the breeding, germplasm resource identification, genetic relationship analysis, etc. of Phallus odoratus. Therefore, developing a set of rapid and effective molecular fingerprint identification systems for Phallus odoratus will help to solve the problem of identifying Phallus odoratus fungal species.

[0003] Molecular marker technology is a genetic marker different from morphological markers, cytological markers and biochemical markers, and can directly reflect certain differences in the genomes among biological individuals or populations. SSR (Simple Sequence Repeats) markers, also known as microsatellite DNA (Microsatellite DNA), are a kind of molecular marker technology based on specific primer PCR, which consists of a core sequence and flanking sequences. The core sequence is a tandem repeat sequence dozens of nucleotides long with 1-6 nucleotides as the repeat unit. Research has found that there are highly variable numbers of repeat units in microsatellites. These variations are manifested as integral multiple variations in the number of microsatellites or incomplete identity in the degree of repetition, thus resulting in polymorphisms at multiple loci. Revealing these variations can discover the polymorphisms of different SSRs among different species or even among different individuals of the same species.

[0004] Since the flanking sequences at both ends of microsatellite markers are conserved in species with relatively close genetic relationships, therefore, microsatellite markers developed for a certain species can be applied to related research on related species. This characteristic greatly reduces the workload of developing microsatellite markers. At the same time, microsatellite markers have the advantages of wide distribution and co-dominant markers, and are considered by the International Union for the Protection of New Varieties of Plants (UPOV) to be one of the ideal markers for constructing DNA fingerprint maps at present, and are widely used in genetic relationship identification, population genetic structure research, genetic linkage map construction and other aspects. Summary of the Invention

[0005] The object of the present invention is to provide a polymorphic microsatellite molecular marker, primer and its application for Phallus odoratus, so as to provide technical support for the analysis of population genetic diversity, variety identification, genetic map construction or molecular assisted breeding of Phallus odoratus.

[0006] In order to achieve the above object of the invention, the present invention provides the following technical solutions: The present invention provides a polymorphic microsatellite molecular marker for Phallus odoratus, and the molecular marker includes CPSSR-1382, CPSSR-692, CPSSR-1090, CPSSR-188, CPSSR-516, CPSSR-499, CPSSR-174, CPSSR-638, CPSSR-217, CPSSR-950, CPSSR-644, CPSSR-686, CPSSR-450, CPSSR-1071, CPSSR-292, CPSSR-733, CPSSR-164, CPSSR-518, CPSSR-1139, CPSSR-1295, CPSSR-1331, CPSSR-462, CPSSR-1107, CPSSR-957, CPSSR-154, CPSSR-166 and CPSSR-1134; The core sequences of each molecular marker are TTC, GA, TACA, AGA, CAC, CAA, AG, CT, AT, GTT, CT, CTT, CA, TA, AT, GA, AG, CAC, TC, TGA, TGG, CA, TC, TA, AG, AG and TC in sequence, and are amplified by the following primer pairs: The nucleotide sequences of the primer pair of CPSSR-1382 are shown in SEQ ID NO.28 and SEQ ID NO.29; The nucleotide sequences of the primer pair of CPSSR-692 are shown in SEQ ID NO.30 and SEQ ID NO.31; The nucleotide sequences of the primer pair of CPSSR-1090 are shown in SEQ ID NO.32 and SEQ ID NO.33; The nucleotide sequences of the primer pair of CPSSR-188 are shown in SEQ ID NO.34 and SEQ ID NO.35; The nucleotide sequences of the primer pair of CPSSR-516 are shown in SEQ ID NO.36 and SEQ ID NO.37; The nucleotide sequences of the primer pair of CPSSR-499 are shown in SEQ ID NO.38 and SEQ ID NO.39; The nucleotide sequences of the primer pairs of CPSSR-174 are shown as SEQ ID NO.40 and SEQ ID NO.41; The nucleotide sequences of the primer pairs of CPSSR-638 are shown as SEQ ID NO.42 and SEQ ID NO.43; The nucleotide sequences of the primer pairs of CPSSR-217 are shown as SEQ ID NO.44 and SEQ ID NO.45; The nucleotide sequences of the primer pairs of CPSSR-950 are shown as SEQ ID NO.46 and SEQ ID NO.47; The nucleotide sequences of the primer pairs of CPSSR-644 are shown as SEQ ID NO.48 and SEQ ID NO.49; The nucleotide sequences of the primer pairs of CPSSR-686 are shown as SEQ ID NO.50 and SEQ ID NO.51; The nucleotide sequences of the primer pairs of CPSSR-450 are shown as SEQ ID NO.52 and SEQ ID NO.53; The nucleotide sequences of the primer pairs of CPSSR-1071 are shown as SEQ ID NO.54 and SEQ ID NO.55; The nucleotide sequences of the primer pairs of CPSSR-292 are shown as SEQ ID NO.56 and SEQ ID NO.57; The nucleotide sequences of the primer pairs of CPSSR-733 are shown as SEQ ID NO.58 and SEQ ID NO.59; The nucleotide sequences of the primer pairs of CPSSR-164 are shown as SEQ ID NO.60 and SEQ ID NO.61; The nucleotide sequences of the primer pairs of CPSSR-518 are shown as SEQ ID NO.62 and SEQ ID NO.63; The nucleotide sequences of the primer pairs of CPSSR-1139 are shown as SEQ ID NO.64 and SEQ ID NO.65; The nucleotide sequences of the primer pairs of CPSSR-1295 are shown as SEQ ID NO.66 and SEQ ID NO.67; The nucleotide sequences of the primer pairs of CPSSR-1331 are shown as SEQ ID NO.68 and SEQ ID NO.69; The nucleotide sequences of the primer pairs of CPSSR-462 are shown as SEQ ID NO.70 and SEQ ID NO.71; The nucleotide sequences of the primer pairs of CPSSR-1107 are shown as SEQ ID NO.72 and SEQ ID NO.73; The nucleotide sequences of the primer pairs of CPSSR-957 are shown as SEQ ID NO.74 and SEQ ID NO.75; The nucleotide sequences of the primer pairs of CPSSR-154 are shown as SEQ ID NO.76 and SEQ ID NO.77; The nucleotide sequences of the primer pairs of CPSSR-166 are shown as SEQ ID NO.78 and SEQ ID NO.79; The nucleotide sequences of the primer pairs of CPSSR-1134 are shown as SEQ ID NO.80 and SEQ ID NO.81.

[0007] The present invention provides a primer pair designed according to the said molecular marker.

[0008] Preferably, the nucleotide sequences of the primer pairs of CPSSR-1382 are shown as SEQ ID NO.28 and SEQ ID NO.29; The nucleotide sequences of the primer pairs of CPSSR-692 are shown as SEQ ID NO.30 and SEQ ID NO.31; The nucleotide sequences of the primer pairs of CPSSR-1090 are shown as SEQ ID NO.32 and SEQ ID NO.33; The nucleotide sequences of the primer pairs of CPSSR-188 are shown as SEQ ID NO.34 and SEQ ID NO.35; The nucleotide sequences of the primer pairs of CPSSR-516 are shown as SEQ ID NO.36 and SEQ ID NO.37; The nucleotide sequences of the primer pairs of CPSSR-499 are shown as SEQ ID NO.38 and SEQ ID NO.39; The nucleotide sequences of the primer pairs of CPSSR-174 are shown as SEQ ID NO.40 and SEQ ID NO.41; The nucleotide sequences of the primer pairs of CPSSR-638 are shown as SEQ ID NO.42 and SEQ ID NO.43; The nucleotide sequences of the primer pairs of CPSSR-217 are shown as SEQ ID NO.44 and SEQ ID NO.45; The nucleotide sequences of the primer pairs of CPSSR-950 are shown in SEQ ID NO.46 and SEQ ID NO.47; The nucleotide sequences of the primer pairs of CPSSR-644 are shown in SEQ ID NO.48 and SEQ ID NO.49; The nucleotide sequences of the primer pairs of CPSSR-686 are shown in SEQ ID NO.50 and SEQ ID NO.51; The nucleotide sequences of the primer pairs of CPSSR-450 are shown in SEQ ID NO.52 and SEQ ID NO.53; The nucleotide sequences of the primer pairs of CPSSR-1071 are shown in SEQ ID NO.54 and SEQ ID NO.55; The nucleotide sequences of the primer pairs of CPSSR-292 are shown in SEQ ID NO.56 and SEQ ID NO.57; The nucleotide sequences of the primer pairs of CPSSR-733 are shown in SEQ ID NO.58 and SEQ ID NO.59; The nucleotide sequences of the primer pairs of CPSSR-164 are shown in SEQ ID NO.60 and SEQ ID NO.61; The nucleotide sequences of the primer pairs of CPSSR-518 are shown in SEQ ID NO.62 and SEQ ID NO.63; The nucleotide sequences of the primer pairs of CPSSR-1139 are shown in SEQ ID NO.64 and SEQ ID NO.65; The nucleotide sequences of the primer pairs of CPSSR-1295 are shown in SEQ ID NO.66 and SEQ ID NO.67; The nucleotide sequences of the primer pairs of CPSSR-1331 are shown in SEQ ID NO.68 and SEQ ID NO.69; The nucleotide sequences of the primer pairs of CPSSR-462 are shown in SEQ ID NO.70 and SEQ ID NO.71; The nucleotide sequences of the primer pairs of CPSSR-1107 are shown in SEQ ID NO.72 and SEQ ID NO.73; The nucleotide sequences of the primer pairs of CPSSR-957 are shown in SEQ ID NO.74 and SEQ ID NO.75; The nucleotide sequences of the primer pairs of CPSSR-154 are shown in SEQ ID NO.76 and SEQ ID NO.77; The nucleotide sequences of the primer pairs of CPSSR-166 are shown as SEQ ID NO.78 and SEQ ID NO.79; The nucleotide sequences of the primer pairs of CPSSR-1134 are shown as SEQ ID NO.80 and SEQ ID NO.81.

[0009] Preferably, a fluorescent group is labeled at the 5'-end of the forward primer; the fluorescent group is selected from any one of FAM, HEX, ROX and TAMRA.

[0010] Preferably, FAM is labeled at the 5'-end of the forward primers of CPSSR-1382, CPSSR-692, CPSSR-1090, CPSSR-188, CPSSR-516, CPSSR-1107, CPSSR-957, CPSSR-154, CPSSR-166, CPSSR-1134; HEX is labeled at the 5'-end of the forward primers of CPSSR-499, CPSSR-174, CPSSR-638, CPSSR-217, CPSSR-950, CPSSR-644; ROX is labeled at the 5'-end of the forward primers of CPSSR-686, CPSSR-450, CPSSR-1071, CPSSR-292, CPSSR-733; TAMRA is labeled at the 5'-end of the forward primers of CPSSR-164, CPSSR-518, CPSSR-1139, CPSSR-1295, CPSSR-1331, CPSSR-462.

[0011] The present invention provides a kit, which contains the above-mentioned primer pairs.

[0012] The present invention provides an application of the molecular marker or the primer pair or the kit in the genetic analysis and / or germplasm resource identification and / or molecular assisted breeding of Phallus odoratus.

[0013] The present invention also provides a method for detecting the microsatellite polymorphism of Phallus odoratus. Using the genomic DNA of Phallus odoratus as a template, PCR amplification is carried out by using the above-mentioned primer pairs, and genetic analysis and / or germplasm resource identification and / or molecular assisted breeding of Phallus odoratus are carried out according to the amplification products.

[0014] Preferably, the reaction system for PCR amplification includes: 0.9 - 1.1 μL of the genomic DNA template of Phallus odoratus, 9 - 11 μL of 2×TSINGKE Master Mix, 0.14 - 0.16 μL of Primer F, 1.1 - 1.3 μL of Primer R, 1.1 - 1.3 μL of fluorescent Tag, 0.9 - 1.1 μL of Template gDNA, and 6.3 - 6.6 μL of ddH 2 O; the concentration of the genomic DNA template of Phallus odoratus is 80 - 120 ng / μL, and the concentrations of Primer F, Primer R, and Tag are all 9 - 11 μmol / L; Preferably, the reaction conditions for PCR amplification are: 94 °C for 5 min; 94 °C for 30 s, 60 °C for 30 s, 72 °C for 30 s, for a total of 35 cycles; 72 °C for 8 min.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The present invention uses bioinformatics methods to detect SSR sequences in the genomic DNA of the Phallus odoratus population, selects some SSR sequences for primer development, and uses Phallus odoratus strains from different sources to screen SSR loci, and finally screens out 27 SSR molecular markers with relatively high polymorphism.

[0016] The present invention also designs primers according to the developed SSR molecular markers. These primers have the advantages of specific amplification, cross-species universality, high polymorphism, co-dominance, and easy detection. At the same time, these SSR markers can directly reflect the genetic information of the Phallus odoratus genome, thus better analyzing the genetic diversity of the Phallus odoratus population, and providing a powerful tool for the construction of its genetic map, the calibration of target genes, the drawing of fingerprint maps, variety identification, pedigree analysis, analysis of genetic distance between populations, evolution, genetic diversity, and variety breeding. Brief Description of the Drawings

[0017] 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 for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained according to the provided drawings without creative efforts.

[0018] Figure 1 It is a BUSCO evaluation result diagram of the genomic assembly quality of 12 Phallus odoratus population samples for genomic SSR locus and primer development in the embodiment; Figure 2Distribution maps of alleles and allele frequencies amplified by 27 SSR loci in 12 Phallus odoratus strains in the examples; Figure 3 Gel images obtained by capillary electrophoresis of molecular markers CPSSR-1382, CPSSR-692, CPSSR-188, CPSSR-516, CPSSR-499, CPSSR-174, CPSSR-638, CPSSR-217, CPSSR-950 and CPSSR-644 in the examples; Figure 4 Gel images obtained by capillary electrophoresis of molecular markers CPSSR-1107, CPSSR-957, CPSSR-154, CPSSR-166 and CPSSR-1134 in the examples; Figure 5 Gel images obtained by capillary electrophoresis of molecular markers CPSSR-450, CPSSR-1071, CPSSR-292, CPSSR-733, CPSSR-164, CPSSR-518, CPSSR-1139, CPSSR-1295, CPSSR-1331, CPSSR-462, CPSSR-686 and CPSSR-1090 in the examples; Figure 6 UPGMA tree constructed for 12 Phallus odoratus strains based on 27 SSR loci in the examples. Detailed implementation manners

[0019] The technical solutions provided by the present invention will be described in detail below in conjunction with the examples, but they should not be construed as limiting the protection scope of the present invention.

[0020] Examples

[0021] 1. Extraction of genomic DNA of Phallus odoratus The genomic DNA of Phallus odoratus was extracted using the TSINGKE Plant DNA Extraction Kit (universal type), and the specific steps are as follows: (1) Place Spin Colu2 in the Collection Tube, add 250 μL Buffer BL, and centrifuge at 12000 rpm for 1 min to activate the silica gel membrane; (2) Take the dry tissue of the sample (not more than 20 mg), add liquid nitrogen and grind thoroughly. After grinding, place it in a 1.5 mL centrifuge tube, add 400 μL Buffer gP1, vortex for 1 min, and incubate in a water bath at 65°C for 20 min. During this period, it can be taken out and inverted to mix evenly to fully lyse; (3) Add 150 μL Buffer gP2, vortex for 1 min, and incubate on ice for 5 min; (4) Centrifuge at 12,000 rpm for 5 min, and transfer the supernatant to a new centrifuge tube. (5) Add an equal volume of absolute ethanol to the supernatant, immediately mix well by vigorous shaking, transfer all the liquid into Spin Colu2, centrifuge at 12,000 rpm for 30 s, and discard the waste liquid. (6) Add 500 μL of Buffer Pw (anhydrous ethanol has been added before use) to Spin Colu2, centrifuge at 12,000 rpm for 30 s, and discard the waste liquid. (7) Add 500 μL of Wash Buffer (anhydrous ethanol has been added before use) to Spin Colu2, centrifuge at 12,000 rpm for 30 s, and discard the waste liquid. (8) Repeat operation step (7). (9) Place Spin Colu2 back into Collection Tube, centrifuge at 12,000 rpm for 2 min, open the lid and air dry for 1 min. (10) Take out Spin Colu2, put it into a clean centrifuge tube, add 100 μL of TE Buffer (TE Buffer preheated at 65°C) at the center of the adsorption membrane, place at 25°C for 2 min, and centrifuge at 12,000 rpm for 2 min.

[0022] (11) Detect the DNA concentration with a nucleic acid concentration detector, adjust the concentration to 100 ng / µL, and store it in a -20°C refrigerator.

[0023] 2. Development of SSR loci and their primers in the genome of Phallus odoratus After liquid culture of 12 strains collected from the wild and the market, whole-genome resequencing based on the second-generation sequencing technology was carried out. Using Ds-1 as the reference sequence, SSR loci in their population genomes were detected by CandiSSR software, and primers were developed within the range of 200 bp upstream and downstream of them. The primer development conditions were set to the software default values. The designed primers were first arranged in ascending order according to the deletion rate, and the primers with a deletion rate of 0 were retained. Then, they were arranged in descending order according to the standard deviation, and the top 30 pairs of primers ranked by the standard deviation were detected on Phallus odoratus strains.

[0024] Figure 1 BUSCO evaluation results for the genome assembly quality of 12 population samples of Phallus odoratus used for the development of genomic SSR loci and primers.

[0025] 3. PCR amplification of SSR loci First, arrange the designed primers in ascending order according to the deletion rate, retain the primers with a deletion rate of 0, then arrange them in descending order according to the standard deviation, and select the top 30 pairs of primers with the highest standard deviation to preliminarily screen 12 Phallus odoratus strains from different sources and detect the presence or absence of amplified bands. The PCR reaction system is as follows: 1 μL of 100 ng / µL Phallus odoratus genomic DNA template, 10 μL of 2×TSINGKE Master Mix (blue), 0.15 μL of 10 μmol / L Primer F (with adapter), 1.2 μL of 10 μmol / L Primer R, 1.2 μL of 10 μmol / L Tag (fluorescent), 1 μL of Template (gDNA), 6.45 μL of ddH 2 O. The PCR reaction conditions are as follows: 94 °C for 5 min; 94 °C for 30 s, 60 °C for 30 s, 72 °C for 30 s, for a total of 35 cycles; 72 °C for 8 min.

[0026] 4. Detection of PCR amplification products of SSR loci (1) Perform agarose gel electrophoresis on the above-mentioned amplified PCR products (2 μL of sample + 6 μL of bromophenol blue) at 300 V for 12 minutes to obtain an identification gel image, determine the template concentration through the gel image, and dilute it with water to the concentration required for capillary electrophoresis.

[0027] (2) Mix HiDi and the internal standard of GS500 at a ratio of 130:1 to prepare a mix.

[0028] (3) Aliquot the mix into a 96-well reaction plate, and add 10 μL of the mix to each well.

[0029] (4) Corresponding to adding 0.5 μL of the sample template to the 96-well plate, and centrifuge until it stops at 4000 rpm.

[0030] (5) Use a metal bath heater to heat the mixed plate at 95 °C for pre-denaturation for 5 minutes, take it out and immediately place it in -20 °C.

[0031] (6) Take it out after cooling, centrifuge at 4000 rpm, thaw and mix well.

[0032] (7) Perform capillary electrophoresis on the 3730 sequencer.

[0033] (8) Obtain the results after running off the machine and analyze them to screen out loci with better polymorphism.

[0034] 5. Locus analysis and screening (1)Analyze the accurate loci of the data using the software Gene mapper 4.1. Determine the accurate size of the loci according to the core base repeat number (e.g., (AG)6) corresponding to the primer in the analyzed data and the fragment size of 263 (16 bases with tags added, so the actual size is 279).

[0035] (2)Judge whether the detection primers have locus polymorphism based on the analyzed locus information (peak graph.PDF and data.excel). Select loci with high specificity and good polymorphism as the focus of subsequent research.

[0036] (3)Statistically analyze the specific bands of each individual in terms of band size (bp). According to the peak graph analyzed by Gene mapper 4.1, if the signal value is above 400, there is no interference from other miscellaneous peaks, and the peak shapes run out at the same locus are similar. If the peak shapes are not similar, even if the peak value is higher than 400 bp and there is no interference from other miscellaneous peaks, the data will not be adopted. Use this to screen whether the data is available. Finally, establish the original data matrix.

[0037] The detected SSR molecular markers are: CPSSR-1382, CPSSR-692, CPSSR-1090, CPSSR-188, CPSSR-516, CPSSR-499, CPSSR-174, CPSSR-638, CPSSR-217, CPSSR-950, CPSSR-644, CPSSR-686, CPSSR-450, CPSSR-1071, CPSSR-292, CPSSR-733, CPSSR-164, CPSSR-518, CPSSR-1139, CPSSR-1295, CPSSR-1331, CPSSR-462, CPSSR-1107, CPSSR-957, CPSSR-154, CPSSR-166, and CPSSR-1134. The core sequences of each molecular marker are TTC, GA, TACA, AGA, CAC, CAA, AG, CT, AT, GTT, CT, CTT, CA, TA, AT, GA, AG, CAC, TC, TGA, TGG, CA, TC, TA, AG, AG, and TC respectively. The gel diagrams obtained by capillary electrophoresis detection of each molecular marker are as Figures 3 to 5 shown. The nucleotide sequences of the primer pairs designed according to each molecular marker and the types of fluorescent groups labeled at the 5' end of the forward primers are shown in Table 1. The nucleotide sequences of each molecular marker used when designing the primer pairs are shown as SEQ ID NO.1~SEQ ID NO.27 in sequence.

[0038] Table 1 Primer pair sequences and types of fluorescent groups labeled for each molecular marker

[0039]

[0040] 6. Assessment of population genetic diversity Distribution maps of alleles and allele frequencies amplified by 27 SSR loci in 12 Phallus odorabilis strains are shown as Figure 2 follows. The obtained SSR data were used to calculate various genetic diversity indices of SSR loci and populations in GenAlEx version 6.501 and PowerMarker V3.25 software respectively. The results are shown in Table 2. The UPGMA tree constructed based on 27 SSR loci for 12 Phallus odorabilis strains is shown as Figure 6 follows.

[0041] Table 2 Results of genetic diversity assessment of 27 SSR loci

[0042] As can be seen from Table 2, the observed alleles (Na) of 27 SSR loci in 12 Phallus odorabilis samples were 2 - 8, with an average of 4. The average value of the Shannon diversity index (I) was 1.189, the average value of the observed heterozygosity (Ho) was 0.342, the average value of the expected heterozygosity (He) was 0.632, and the average value of the polymorphism information content (PIC) was 0.577. The 27 SSR loci had high polymorphism in Phallus odorabilis samples and could distinguish and classify all 12 strains. As Figure 3 can be seen, Ds - 1, Ds - 2, and Ds - 3 all originated from the germplasm market, the Dscb strain originated from the artificially cultivated fruiting bodies sold in the vegetable market, and the other strains were collected from the wild. Ds - 1, Ds - 2, and Ds - 3 clustered into one branch, indicating that these strains had the same genetic background and their production sources might be the same. The other strains from the wild environment and the strains collected from the vegetable market formed different branches and were distinguished from each other.

[0043] In summary, the 27 SSR loci developed in the present invention can not only distinguish strains with inconsistent genetic backgrounds, but also accurately identify strains with the same genetic background in the germplasm market. The microsatellite molecular markers and primers of the present invention can be used for the construction of genetic maps of Phallus odorabilis, the calibration of target genes, the drawing of fingerprint maps, variety identification, pedigree analysis, analysis of genetic distance between populations, evolution and genetic diversity, and variety breeding, etc.

[0044] The above are only the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A polymorphic microsatellite molecular marker for Psoralea corylifolia, characterized in that: The molecular markers include CPSSR-1382, CPSSR-692, CPSSR-1090, CPSSR-188, CPSSR-516, CPSSR-499, CPSSR-174, CPSSR-638, CPSSR-217, CPSSR-950, CPSSR-644, CPSSR-686, CPSSR-450, CPSSR-1071, CPSSR-292, CPSSR-733, CPSSR-164, CPSSR-518, CPSSR-1139, CPSSR-1295, CPSSR-1331, CPSSR-462, CPSSR-1107, CPSSR-957, CPSSR-154, CPSSR-166 and CPSSR-1134; The core sequences of each molecular marker are TTC, GA, TACA, AGA, CAC, CAA, AG, CT, AT, GTT, CT, CTT, CA, TA, AT, GA, AG, CAC, TC, TGA, TGG, CA, TC, TA, AG, AG and TC, which are amplified by the following primer pairs: The nucleotide sequences of the primer pair of CPSSR-1382 are shown in SEQ ID NO.28 and SEQ ID NO.29; The nucleotide sequences of the primer pair of CPSSR-692 are shown in SEQ ID NO.30 and SEQ ID NO.31; The nucleotide sequences of the primer pair of CPSSR-1090 are shown in SEQ ID NO.32 and SEQ ID NO.33; The nucleotide sequences of the primer pair of CPSSR-188 are shown in SEQ ID NO.34 and SEQ ID NO.35; The nucleotide sequences of the primer pair of CPSSR-516 are shown in SEQ ID NO.36 and SEQ ID NO.37; The nucleotide sequences of the primer pair of CPSSR-499 are shown in SEQ ID NO.38 and SEQ ID NO.39; The nucleotide sequences of the primer pair of CPSSR-174 are shown in SEQ ID NO.40 and SEQ ID NO.41; The nucleotide sequences of the primer pair of CPSSR-638 are shown in SEQ ID NO.42 and SEQ ID NO.43; The nucleotide sequences of the primer pair of CPSSR-217 are shown in SEQ ID NO.44 and SEQ ID NO.45; The nucleotide sequences of the primer pair of CPSSR-950 are shown in SEQ ID NO.46 and SEQ ID NO.47; The nucleotide sequences of the primer pair of CPSSR-644 are shown in SEQ ID NO.48 and SEQ ID NO.49; The nucleotide sequences of the primer pair of CPSSR-686 are shown in SEQ ID NO.50 and SEQ ID NO.51; The nucleotide sequences of the primer pair of CPSSR-450 are shown in SEQ ID NO.52 and SEQ ID NO.53; The nucleotide sequences of the primer pair of CPSSR-1071 are shown in SEQ ID NO.54 and SEQ ID NO.55; The nucleotide sequences of the primer pair of CPSSR-292 are shown in SEQ ID NO.56 and SEQ ID NO.57; The nucleotide sequences of the primer pair of CPSSR-733 are shown in SEQ ID NO.58 and SEQ ID NO.59; The nucleotide sequences of the primer pair of CPSSR-164 are shown in SEQ ID NO.60 and SEQ ID NO.61; The nucleotide sequences of the primer pair of CPSSR-518 are shown in SEQ ID NO.62 and SEQ ID NO.63; The nucleotide sequences of the primer pair of CPSSR-1139 are shown in SEQ ID NO.64 and SEQ ID NO.65; The nucleotide sequences of the primer pair of CPSSR-1295 are shown in SEQ ID NO.66 and SEQ ID NO.67; The nucleotide sequences of the primer pair of CPSSR-1331 are shown in SEQ ID NO.68 and SEQ ID NO.69; The nucleotide sequences of the primer pair of CPSSR-462 are shown in SEQ ID NO.70 and SEQ ID NO.71; The nucleotide sequences of the primer pair of CPSSR-1107 are shown in SEQ ID NO.72 and SEQ ID NO.73; The nucleotide sequences of the primer pair of CPSSR-957 are shown in SEQ ID NO.74 and SEQ ID NO.75; The nucleotide sequences of the primer pair of CPSSR-154 are shown in SEQ ID NO.76 and SEQ ID NO.77; The nucleotide sequences of the primer pair of CPSSR-166 are shown in SEQ ID NO.78 and SEQ ID NO.79; The nucleotide sequences of the primer pair of CPSSR-1134 are shown in SEQ ID NO.80 and SEQ ID NO.

81.

2. A primer pair designed according to the molecular marker of claim 1.

3. The primer pair according to claim 2, characterized in that The nucleotide sequences of the primer pair of CPSSR-1382 are shown in SEQ ID NO.28 and SEQ ID NO.29; The nucleotide sequences of the primer pair of CPSSR-692 are shown in SEQ ID NO.30 and SEQ ID NO.31; The nucleotide sequences of the primer pair of CPSSR-1090 are shown in SEQ ID NO.32 and SEQ ID NO.33; The nucleotide sequences of the primer pair of CPSSR-188 are shown in SEQ ID NO.34 and SEQ ID NO.35; The nucleotide sequences of the primer pair of CPSSR-516 are shown in SEQ ID NO.36 and SEQ ID NO.37; The nucleotide sequences of the primer pair of CPSSR-499 are shown in SEQ ID NO.38 and SEQ ID NO.39; The nucleotide sequences of the primer pair of CPSSR-174 are shown in SEQ ID NO.40 and SEQ ID NO.41; The nucleotide sequences of the primer pair of CPSSR-638 are shown in SEQ ID NO.42 and SEQ ID NO.43; The nucleotide sequences of the primer pair of CPSSR-217 are shown in SEQ ID NO.44 and SEQ ID NO.45; The nucleotide sequences of the primer pair of CPSSR-950 are shown in SEQ ID NO.46 and SEQ ID NO.47; The nucleotide sequences of the primer pair of CPSSR-644 are shown in SEQ ID NO.48 and SEQ ID NO.49; The nucleotide sequences of the primer pair of CPSSR-686 are shown in SEQ ID NO.50 and SEQ ID NO.51; The nucleotide sequences of the primer pair of CPSSR-450 are shown in SEQ ID NO.52 and SEQ ID NO.53; The nucleotide sequences of the primer pair of CPSSR-1071 are shown in SEQ ID NO.54 and SEQ ID NO.55; The nucleotide sequences of the primer pair of CPSSR-292 are shown in SEQ ID NO.56 and SEQ ID NO.57; The nucleotide sequences of the primer pair of CPSSR-733 are shown in SEQ ID NO.58 and SEQ ID NO.59; The nucleotide sequences of the primer pair of CPSSR-164 are shown in SEQ ID NO.60 and SEQ ID NO.61; The nucleotide sequences of the primer pair of CPSSR-518 are shown in SEQ ID NO.62 and SEQ ID NO.63; The nucleotide sequences of the primer pair of CPSSR-1139 are shown in SEQ ID NO.64 and SEQ ID NO.65; The nucleotide sequences of the primer pair of CPSSR-1295 are shown in SEQ ID NO.66 and SEQ ID NO.67; The nucleotide sequences of the primer pair of CPSSR-1331 are shown in SEQ ID NO.68 and SEQ ID NO.69; The nucleotide sequences of the primer pair of CPSSR-462 are shown in SEQ ID NO.70 and SEQ ID NO.71; The nucleotide sequences of the primer pair of CPSSR-1107 are shown in SEQ ID NO.72 and SEQ ID NO.73; The nucleotide sequences of the primer pair of CPSSR-957 are shown in SEQ ID NO.74 and SEQ ID NO.75; The nucleotide sequences of the primer pair of CPSSR-154 are shown in SEQ ID NO.76 and SEQ ID NO.77; The nucleotide sequences of the primer pair of CPSSR-166 are shown in SEQ ID NO.78 and SEQ ID NO.79; The nucleotide sequences of the primer pair of CPSSR-1134 are shown in SEQ ID NO.80 and SEQ ID NO.

81.

4. The primer pair according to claim 3, characterized in that The 5' end of the forward primer is labeled with a fluorescent group; the fluorescent group is selected from any one of FAM, HEX, ROX and TAMRA.

5. The primer pair according to claim 4, characterized in that The 5′ end of the forward primers of CPSSR-1382, CPSSR-692, CPSSR-1090, CPSSR-188, CPSSR-516, CPSSR-1107, CPSSR-957, CPSSR-154, CPSSR-166, and CPSSR-1134 were labeled with FAM; The 5′ end of the forward primers of CPSSR-499, CPSSR-174, CPSSR-638, CPSSR-217, CPSSR-950, and CPSSR-644 were labeled with HEX; The 5′ end of the forward primers of CPSSR-686, CPSSR-450, CPSSR-1071, CPSSR-292, and CPSSR-733 were labeled with ROX; The 5' end of the forward primers of CPSSR-164, CPSSR-518, CPSSR-1139, CPSSR-1295, CPSSR-1331, and CPSSR-462 were labeled with TAMRA.

6. A kit, characterized in that: The kit contains the primer pair according to any one of claims 2 to 5.

7. Use of the molecular marker according to claim 1, the primer pair according to any one of claims 2 to 5, or the kit according to claim 6 in genetic analysis of P. fulvicinense and / or identification of germplasm resources and / or molecular-assisted breeding.

8. A method for detecting microsatellite polymorphism of Psoralea corylifolia, characterized in that: Using the genomic DNA of P. fulgens as a template, PCR amplification is performed using the primer pair described in any one of claims 2 to 5, and genetic analysis and / or germplasm resource identification and / or molecular assisted breeding of P. fulgens are performed based on the amplified products.

9. The method according to claim 8, characterized in that The PCR amplification reaction system includes: 0.9~1.1 μL of P. crassifolia genomic DNA template, 9~11 μL of 2×TSINGKE Master Mix, 0.14~0.16 μL of Primer F, 1.1~1.3 μL of Primer R, 1.1~1.3 μL of fluorescent Tag, 0.9~1.1 μL of Template gDNA, and 6.3~6.6 μL of ddH2O; the concentration of the P. crassifolia genomic DNA template is 80~120 ng / μL, and the concentrations of Primer F, Primer R and Tag are all 9~11 μmol / L.

10. The method according to claim 8, characterized in that The reaction conditions for the PCR amplification were: 94°C for 5 min; 94°C for 30 s, 60°C for 30 s, 72°C for 30 s, for a total of 35 cycles; 72°C for 8 min.

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