CN114934130B - Southern sclerotium bacterium specific SSR marker and application thereof - Google Patents
Southern sclerotium bacterium specific SSR marker and application thereof Download PDFInfo
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Abstract
The invention provides a sclerotium rolfsii specific SSR marker and application thereof, belonging to the technical field of molecular biology. On the basis of determining the sclerotium affinity group of the southern blight bacteria, SSR locus searching and SSR primer design are carried out based on the sequencing result of a transcriptome of the southern blight bacteria, 9 pairs of SSR primers suitable for the southern blight bacteria are screened out, the genetic diversity and the group structure of the southern blight bacteria are researched by utilizing SSR molecular markers, the propagation path of the plant southern blight bacteria can be determined on the molecular level, and the method has important significance for guiding the control of the southern blight of the plant.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to a southern blight bacterium specific SSR marker and application thereof.
Background
Southern blight is a devastating soil-borne disease, and its host is reported to exceed 500 plants, mainly damaging dicotyledonous plants, and also damaging some monocotyledonous plants including grain crops and cash crops, and southern blight mainly infects roots and stems of plants to cause root rot or stem rot. The sclerotium rolfsii is sclerotium rolfsii in sclerotium rolfsii, belonging to asexual spore class, hyphomyceae, anospora and sclerotium; the active form is Artai rosenbergii belonging to Basidiomycota, polyporales, plaster and Mycoleptomycetaceae. In the early stage of infection of sclerotium rolfsii, the infected part is dark brown, the temperature and the humidity are proper, the infected part is enlarged, and white hyphae can be seen. With the aggravation of infection, the cortex of the rhizome part is rotten, transmission of nutrient substances, water and the like is influenced, and finally, the plants die. And at the final stage of infection, a large amount of brown rapeseed-like sclerotium appears at the infected part and the soil surface layer.
Genetic diversity may reflect the fitness of a species. The genetic diversity research methods which are relatively common at present comprise morphological markers, cytological markers, biochemical markers, immunological markers, biochemical markers, molecular markers and the like. The molecular marker can reflect the genetic diversity of biological populations from the microcosmic level, wherein the SSR molecular marker technology has the characteristics of high polymorphism, accurate result, high single base resolution, high information content, co-dominance, good repeatability and low sample DNA use amount, is considered to be an accurate marking mode at present, and is widely applied to researches on genetic distance, evolution, diversity analysis and the like among phytopathogen populations.
The SSR molecular marker mainly comprises a genome SSR and an expression sequence tag SSR. The former develops SSR primers from genomes, has large analysis data volume, complex steps and high development cost, and needs stronger data analysis capability. The latter obtains SSR primer based on sequencing of transcriptome and the like for related research, and has the advantages of simple steps, low cost and the like. Therefore, the development of SSR markers by using transcriptome data of pathogenic bacteria is an economic and effective DNA molecular marker method. However, at present, there is no report related to the SSR molecular marker of the sclerotium rolfsii at home and abroad.
Disclosure of Invention
In order to solve the problems in the prior art, the invention aims to provide the SSR marker of the plant southern blight, specifically comprises 9 pairs of primer pairs, and can be used for detection of the plant southern blight, genetic diversity and population structure analysis, determination of a bacterial source center and a propagation path and prevention and treatment of the plant southern blight.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a plant sclerotium rolfsii SSR marker, which is obtained by amplifying any one or more of the following primer pairs, wherein the sequences of the primer pairs are shown as SEQ ID NO: 1-2, SEQ ID NO:3 to 4, SEQ ID NO:5 to 6, SEQ ID NO:7 to 8, SEQ ID NO:9 to 10, SEQ ID NO:11 to 12, SEQ ID NO:13 to 14, SEQ ID NO:15 to 16, SEQ ID NO:17 to 18.
The invention also provides an SSR labeled primer pair of the plant sclerotium rolfsii, which comprises any one or more of the following primer pairs: the sequence of the primer pair is shown as SEQ ID NO: 1-2, SEQ ID NO:3 to 4, SEQ ID NO:5 to 6, SEQ ID NO:7 to 8, SEQ ID NO:9 to 10, SEQ ID NO: 11-12, SEQ ID NO:13 to 14, SEQ ID NO:15 to 16, SEQ ID NO:17 to 18.
The invention also provides a plant sclerotium rolfsii detection kit, which comprises the SSR labeled primer pair.
Preferably, the kit further comprises dNTPs, taq DNA polymerase, PCR reaction buffer and genome extraction reagent.
Preferably, the kit further comprises reagents in capillary electrophoresis.
The invention also provides application of the primer pair or the kit in plant southern blight bacterium detection.
The invention also provides application of the primer pair or the kit in genetic diversity and colony structure analysis of the southern sclerotium rolfsii.
The invention also provides application of the primer pair or the kit in determination of the sclerotium rolfsii origin center of the plant.
The invention also provides application of the primer pair or the kit in determining the propagation path of the plant sclerotium rolfsii.
The invention also provides application of the primer pair or the kit in plant southern blight prevention and treatment.
Compared with the prior art, the technical scheme of the invention has the following beneficial effects:
on the basis of determining the sclerotium affinity group of the southern blight, SSR locus searching and SSR primer design are carried out on the basis of a sequencing result of a transcriptome of the southern blight, 9 pairs of SSR primers suitable for the southern blight are screened out, genetic diversity analysis is carried out from a molecular level by using SSR molecular markers, the genetic diversity and the group structure of the southern blight can be analyzed, the bacteria source center and the propagation path of the plant southern blight are determined, and the method has important significance for guiding the control of the southern blight and reducing the pesticide using amount.
Drawings
FIG. 1: the SSR primers are used for amplifying gel electrophoresis images of 8 southern blight strains, wherein A is a primer BJ071, B is a primer BJ082, and C is a primer BJ112;
FIG. 2 is a schematic diagram: amplifying a capillary electrophoresis chart of the southern blight strain by using a polymorphic SSR primer (BJ 112);
FIG. 3: amplifying a capillary electrophoresis chart of the southern blight strain by using SSR primers (BJ 020) with poor polymorphism;
FIG. 4 is a schematic view of: 9 pairs of SSR primers to detect the result of the sclerotium rolfsii strain of konjak;
FIG. 5 is a schematic view of: the magnitude of Delta K on the K value level;
FIG. 6: structure results of 120 strains with K = 3;
FIG. 7 is a schematic view of: UPGMA clustering results of 120 southern blight bacteria.
Detailed Description
The invention provides a plant sclerotium rolfsii SSR marker, which is obtained by amplifying any one or more of the following primer pairs, wherein the sequences of the primer pairs are shown as SEQ ID NO: 1-2, SEQ ID NO:3 to 4, SEQ ID NO:5 to 6, SEQ ID NO:7 to 8, SEQ ID NO:9 to 10, SEQ ID NO: 11-12, SEQ ID NO:13 to 14, SEQ ID NO:15 to 16, SEQ ID NO:17 to 18.
The invention also provides an SSR marker primer pair of the plant sclerotium rolfsii, which comprises any one or more of the following primer pairs: the sequence of the primer pair is shown as SEQ ID NO: 1-2, SEQ ID NO:3 to 4, SEQ ID NO:5 to 6, SEQ ID NO:7 to 8, SEQ ID NO:9 to 10, SEQ ID NO: 11-12, SEQ ID NO:13 to 14, SEQ ID NO:15 to 16, SEQ ID NO:17 to 18.
The primer pairs are respectively named as BJ015, BJ071, BJ082, BJ112, BJ130, BJ152, BJ156, BJ157 and BJ184.
The repetitive motif of the BJ015 primer pair is TGG, and the specific sequence is as follows:
F：5’-AGAAGACGGGAATAAGCCGT-3’(SEQ ID NO：1)；
R：5’-AGTTGATTGGGCAAACTTGG-3’(SEQ ID NO：2)。
the repeated motif of the BJ071 primer pair is GAT, and the specific sequence is as follows:
F：5’-GTCACGCAATTGGAACACAC-3’(SEQ ID NO：3)；
R：5’-GAGAGCGGACACTCTCCAAG-3’(SEQ ID NO：4)。
the primer pair BJ082 has repeated motifs of CYG and has the specific sequence:
F：5’-ATCCAAATCAAGCATCTGGC-3’(SEQ ID NO：5)；
R：5’-GATGTACGGGTCGTATTCGG-3’(SEQ ID NO：6)。
the repeated motif of the BJ112 primer pair is GT, and the specific sequence is:
F：5’-GGGAGGATGCAAGAACTGAA-3’(SEQ ID NO：7)；
R：5’-ATTTATACCCGCTCACACCG-3’(SEQ ID NO：8)。
the repetitive motif of the BJ130 primer pair is CT, and the specific sequence is as follows:
F：5’-TGGCTCTCTACGGACGAACT-3’(SEQ ID NO：9)；
R：5’-TATCATCGCCTTCCTTGGAC-3’(SEQ ID NO：10)。
the repeated motif of the BJ152 primer pair is GGAG, and the specific sequence is as follows:
F：5’-CCGAGAGAGCAAGAGAATGG-3’(SEQ ID NO：11)；
R：5’-AAAGGCCTGCTCGACTTGTA-3’(SEQ ID NO：12)。
the repeated motif of the BJ156 primer pair is GAT, and the specific sequence is as follows:
F：5’-TGATCGGGTAGGTGAGGTTC-3’(SEQ ID NO：13)；
R：5’-CGTTACATACGGCCAGTCCT-3’(SEQ ID NO：14)。
the repetitive motif of the BJ157 primer pair is GT, and the specific sequence is as follows:
F：5’-TGCTCTTGTCCAGTGGAGTG-3’(SEQ ID NO：15)；
R：5’-GGTTGCCTTTCCCTTCTCTC-3’(SEQ ID NO：16)。
the repeated motif of the BJ184 primer pair is AAT, and the specific sequence is as follows:
F：5’-CACACAAGGGAGCACAGAGA-3’(SEQ ID NO：17)；
R：5’-GGCAACAACGCTCATCAGTA-3’(SEQ ID NO：18)。
the plant southern sclerotium rolfsii SSR marker provided by the invention is a marker based on a microsatellite DNA sequence repeat marker (Simple sequence repeat), a primer of a specific SSR marker is selected from one or more of BJ015, BJ071, BJ082, BJ112, BJ130, BJ152, BJ156, BJ157 and BJ184, and a fragment obtained by amplification is the SSR marker.
Preferably, the plants comprise macleaya cordata, konjak, cynanchum glaucescens, dendrobium, coptis chinensis, peanuts, pinellia ternate, plantain, rice, jerusalem artichoke, bighead atractylodes rhizome, figwort root and sealwort.
The invention also provides a kit for detecting the plant sclerotium rolfsii, which comprises one or more SSR labeled primer pairs of BJ015, BJ071, BJ082, BJ112, BJ130, BJ152, BJ156, BJ157 and BJ184.
Preferably, the kit further comprises dNTPs, taq DNA polymerase, PCR reaction buffer and genome extraction reagent.
More preferably, the kit further comprises reagents in capillary electrophoresis. The invention adopts a capillary electrophoresis apparatus to detect the products obtained by the amplification of the SSR labeled primers, and can analyze the genetic differentiation and diversity of the southern blight bacteria according to the detection result of the capillary electrophoresis.
The invention adopts the amplification primer pair to carry out PCR amplification and capillary electrophoresis detection on the genome DNA of the plant sclerotium rolfsii to be detected. As an alternative embodiment, the invention adopts a fluorescence modified joint primer and a corresponding reverse primer to carry out fluorescence primer PCR (the fluorescence modification adopts a modification mode commonly used for capillary electrophoresis detection). The amplification conditions were as follows: 5min at 94 ℃; 30 cycles at 94 ℃ 30s and TM 30s; 30s at 72 ℃; 5min at 72 ℃; at 4 ℃ C- -.The PCR reaction system is calculated by 20 mu L: 10. Mu.L of Mastermix, 1. Mu.L of primer F (10. Mu.M), 1. Mu.L of primer R (10. Mu.M), 1. Mu.L of template DNA, 7. Mu.L of ddH 2 And (O). The resulting fluorescent PCR product was then used for detection by capillary electrophoresis using a 3730xl sequencer.
The invention also provides application of the primer pair or the kit in plant southern blight, which comprises application in detection of plant southern blight, application in analysis of genetic diversity and population structure of plant southern blight, application in determination of plant southern blight source center, application in determination of plant southern blight transmission pathway and application in prevention and control of plant southern blight.
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It should be apparent that the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
1. Southern blight transcriptome analysis
Selecting a southern sclerotium rolfsii LC1 strain for transcriptome data analysis, respectively taking samples of 3 periods of a hyphal period (S1), a sclerotium initial period (S2) and a sclerotium later period (S3) of the southern sclerotium rolfsii LC1, collecting 500mg samples in each period, freezing the samples by liquid nitrogen, and storing the samples at-80 ℃. The method for extracting RNA by using Trizol comprises the following steps: taking equal amount of mycelium in each period, fully grinding in liquid nitrogen environment, transferring to a 1.5mL centrifuge tube, adding 1mL Trizol (production company: life technologies), and immediately and fully mixing; standing the mixed mycelia at room temperature for 10min for full cracking; adding 200 μ L chloroform, shaking thoroughly, mixing, centrifuging at 4 deg.C at 12000r/min for 10min; taking 400 μ L of the upper layer water phase, adding 400 μ L of isopropanol, mixing, and standing at room temperature for 10min. Centrifuging at 12000rpm at 4 deg.C for 10min to obtain white RNA precipitate at the bottom of the tube; discarding the supernatant, adding 1mL of 75% ethanol without RNase, mixing uniformly by vortex, centrifuging for 5min at 4 ℃ at 10000 r/min; repeating the previous step; discarding the supernatant, drying at room temperature, and dissolving the precipitate in 20 μ L DEPC water; storing at-80 deg.C.
And carrying out agarose gel electrophoresis on the extracted RNA sample to ensure the integrity of the sample, and checking whether degradation occurs or not and whether protein and other pollution exists or not. The OD value of the sample was measured using a microspectrophotometer (NanoDrop 2000) to verify the purity. The Guangzhou Yoto Diao Biotechnology Limited company was entrusted to complete the construction and sequencing of the transcriptome library of the LC1 strain of southern sclerotium.
Microsatellite mining is carried out on the whole transcriptome by adopting microsatellite (MISA, http:// pgrc. Ipk-gatersleen. De/MISA /) software, and if the distance between two SSRs is less than 100bp, the SSRs are regarded as one SSR. And analyzing the type, the quantity, the distribution, the occurrence frequency and the like of the SSR, and designing a Primer pair of SSRs flanking regions by using Primer3 (http:// www.Broadadintitute.org/genome _ software/other/Primer3. Html) according to the MISA result.
2. Screening of bacterial strains of southern blight
8 southern blight bacteria (HS 8, QB2, ZB6, LC5, CB2, XH1, NS5 and EM 1) with larger difference of biological characteristics in 120 southern blight bacteria are selected for SSR primer PCR amplification screening, and 16 southern blight bacteria (EM 1, HG2, LC5, QJ2, HS3, LB4, LY2, NB3, XH3, YH4, ZH5, LH1, NH5, NC4, NS6 and NY 1) with different geographical sources and different hosts are selected for capillary electrophoresis screening. Genomic DNA of southern sclerotium was extracted using TSINGKE DNA extraction kit (general purpose type). Information on southern blight is shown in table 1.
TABLE 1 numbering and sources of the strains of Scutellaria alba
3. SSR primer prescreening
The principle of SSR primer preliminary screening is as follows: (1) Selecting sites with repeat units of 2, 3, 4, 5 bases and only one repeat type; (2) selecting fragments with the length more than 150bp and less than 300bp; (3) The positions of the genes are not suitable for being concentrated, polymorphic sites are preferentially selected, and repeat units with different combinations are selected in a balanced manner; (4) Selecting a primer with the length of 20-23 bp and the TM value of about 60 ℃; (5) selecting a primer having a base repetition number of 4 or less; (6) selecting that the 5 'end and the 3' end do not contain 2 continuous A/T bases; (7) the primer cannot have a repetitive sequence therein. Based on the principle of primer preliminary screening, 200 pairs of primers are screened from the designed primer pairs for PCR amplification detection.
Respectively taking genome DNA of 8 southern blight strains as templates, and selecting different primers for PCR amplification. The PCR reaction (20. Mu.L) was as follows: 10. Mu.L Master mix, 1. Mu.L primer F (adapter "CAG", 10. Mu.M), 1. Mu.L primer R (10. Mu.M), 1. Mu.L template DNA, 7. Mu.L ddH 2 And O. The amplification conditions were: pre-denaturation at 94 ℃ for 5min; denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 30s, and 35 cycles; finally, extension is carried out for 5min at 72 ℃.
mu.L of the amplification product was electrophoretically detected on a 1% agarose gel, detected on a Bio-Red ultraviolet light gel imaging system and photographed. And screening out primers with clear amplified bands. And (3) displaying the amplification band of each primer pair by the electrophoresis result of the PCR product, and screening out primers which can amplify clear and bright bands by taking 8 test strain genomes as templates to obtain 36 pairs of SSR primer pairs.
The results of gel electrophoresis on amplified bands are shown in FIG. 1. In FIG. 1, A is a primer BJ071, B is a primer BJ082, C is a primer BJ112, and 1-8 are HS8, QB2, ZB6, LC5, CB2, XH1, NS5 and EM1 in sequence. (since the electropherograms are more in result, only a portion of the results are provided for corroboration)
4. Capillary electrophoresis screening
Selecting 16 southern blight bacteria of different hosts from different geographical sources, and screening 36 pairs of primers obtained by screening in the step 2 by taking 16 southern blight bacterium genome DNAs of Hubei province (konjak EM1, cynanchum glaucescens HG2, coptis chinensis LC5 and pinellia ternate QJ 2), anhui province (dendrobium nobile HS 3), hunan province (macleaya cordata LB4, LY2, NB3, polygonatum XH3, YH4 and ZH 5), henan province (peanuts LH1 and NH 5), sichuan province (pinellia ternate NC 4) and Guangxi Zhuang autonomous region (rice NS6 and Jerusalem artichoke NY 1) as templates.
36 pairs of primers were screened by capillary electrophoresis and fluorescence primer PCR was performed using fluorescently modified adapter primers and corresponding reverse primers. The amplification conditions were as follows: 5min at 94 ℃; 30 cycles of 94 ℃ 30s and TM 30s; 30s at 72 ℃; 5min at 72 ℃; at 4 deg.C- -.
And (3) carrying out capillary electrophoresis detection on the obtained fluorescent PCR product by using a 3730xl sequencer, and judging whether the primer has site polymorphism or not by combining the analyzed site peak image and data information. After capillary electrophoresis, primers with high site specificity and good polymorphism show multiple fragment sizes (as shown in FIG. 2); if all 16 test strains have a single fragment size (see FIG. 3), the polymorphism of the primer is not ideal. (because the results are more, only part of the primers are provided for demonstration, FIG. 2 shows the amplification results of the primer pair BJ112 on EM1, HG2 and HS3, respectively, and FIG. 3 shows the amplification results of the primer pair BJ020 on CB5, HS8 and ZB6, respectively), primers with high site specificity, good polymorphism and good repeatability are screened out for genetic diversity analysis.
Combining the primer amplification effect and whether polymorphism exists after detection, and screening out a primer 17 pair altogether, wherein the polymorphism of the primer pair BJ112, BJ152, BJ156, BJ157 is the best; the polymorphism of the primer pair BJ015, BJ071, BJ082, BJ130 and BJ184 is better; the primer pairs BJ066, BJ078, BJ080, BJ086, BJ106, BJ109, BJ138, BJ163 polymorphisms are common. Thus, 9 total primers BJ015, BJ071, BJ082, BJ112, BJ130, BJ152, BJ156, BJ157, BJ184 were selected for genetic diversity analysis. The information of the 9 pairs of primers obtained by screening is shown in Table 2.
TABLE 2 SSR primer information
Example 2
Polymorphism analysis of SSR primers:
the SSR primers 9 in example 1 were used for analysis of genome DNA of 120 southern sclerotium, and 66 allele sites were detected in total (Na, allele with highest frequency is prototype). The number of alleles is totally 6, the primer BJ184 with the highest number of alleles is used, and the number of the primers BJ184 reaches 11; secondly, primer BJ112, the allele number is 10; the least allele factors were BJ015 and BJ071, both allele factors were 4, and the average number of alleles per locus was 7.3333. The total number of valid alleles (Ne, the more evenly distributed the alleles are in the population, the closer Ne is to the number of alleles actually detected) was 30.3318, the range of values varied from 1.9625 (BJ 184) to 5.9925 (BJ 112), and the average number of valid alleles per locus was 3.3702. The shannon index (I) ranged from 1.0435 (BJ 071) to 1.9814 (BJ 112) with an average value of 1.4139. The Polymorphic Information Content (PIC) ranged from 0.4767 (BJ 184) to 0.8140 (BJ 112), with an average value of 0.6303, with 9 pairs of primers having higher polymorphic information (PIC > 0.25), and the results are shown in Table 3.
TABLE 3 polymorphisms for SSR primers
Na: observing an allele; ne: a useful allele; i: a shannon index; PIC: a polymorphic information index; ho: observing the heterozygosity; he: heterozygosity is desired.
As can be seen from Table 3, the obtained 9 pairs of SSR primers have high polymorphism and can be used for genetic diversity analysis of southern blight flora.
Example 3
Taking genome DNA of plant southern blight bacteria as a template, and respectively carrying out capillary electrophoresis detection on 9 pairs of SSR primers by the following steps: and carrying out fluorescent primer PCR on the fluorescent modified joint primer and the corresponding reverse primer. The amplification conditions were as follows: 5min at 94 ℃; 30 cycles of 94 ℃ 30s and TM 30s; 30s at 72 ℃; 5min at 72 ℃; at 4 deg.C- -. The resulting fluorescent PCR product was used for detection by capillary electrophoresis using a 3730xl sequencer. (because the results are more, only part of the primers are provided for demonstration, and FIG. 4 is the detection result of 9 pairs of SSR primers on the sclerotium rolfsii) of konjak.
Example 4
The genome DNA of 120 southern sclerotium rolfsii strains were used as templates, 9 pairs of SSR primers were used for capillary electrophoresis detection, and the detection results were analyzed by the method described in example 3.
1. Genetic diversity analysis
Genetic diversity of various populations of southern sclerotium were analyzed using the software GenAlEx version 6.501.
Effective alleles (Ne) for 7 geographic populations ranged between 1.231 (Henan) -3.281 (Hunan), with an average of 2.277; the Shannon index (I) is between 0.174 (Henan) and 1.290 (Hunan), and the average value is 0.813. The results are shown in Table 4.
TABLE 4 genetic diversity among different geographical origins of southern blight flora
Na: observing the allele; ne: a potent allele; i: a shannon index; ho: observing the heterozygosity; he: a desired heterozygosity; f: and (4) fixing the index.
As can be seen from table 4, the genetic diversity index was the lowest for the group in the south of hewn province and the highest for the group in the south of hunnan province.
The effective allele (Ne) of 13 host populations was between 1.222 (MY) -2.989 (SH), with an average of 1.949; the Shannon index (I) was between 0.154 (MY) and 1.115 (SH), with an average value of 0.651, and the results are shown in Table 5.
TABLE 5 genetic diversity among different host-derived southern blight flora
Na: observing an allele; ne: a potent allele; i: a shannon index; ho: observing the heterozygosity; he: a desired heterozygosity; f: and (4) fixing the index.
As can be seen from Table 5, the genetic diversity index of the konjak population (MY) was the lowest, and the genetic diversity index of the dendrobe population (SH) was the highest. The fixed indexes (F) of the geographical population and the host population are both obviously greater than 0 or less than 0, which indicates that the phenomenon of heterozygote deletion or homozygote deletion exists.
2. Molecular analysis of variance
Molecular analysis of variance (AMOVA) was performed on the different geographic source populations and the results are shown in table 6.
Table 6 different geographical source population AMOVA analysis
As can be seen from Table 6, the ratio of genetic variation among geographical populations is 28%, and the ratio of genetic variation within the populations is 72%, indicating that variation exists between geographical populations and within populations of southern sclerotium, and the variation within the populations is 2.5 times that between distant populations, and the genetic variation mainly comes from within the populations.
Further analysis of the gene flow and genetic differentiation results between two populations revealed a gene flow value between 0.055 (Sichuan/Henan) -10.254 (Hunan/Anhui) and a genetic differentiation value between 0.024 (Anhui/Hunan) -0.819 (Henan/Sichuan), with the results shown in Table 7.
TABLE 7 Gene flow between geographic populations (upper triangles) and genetic differentiation results (lower triangles)
As can be seen from table 7, the sclerotium rolfsii genes in anhui and hunan provinces flow frequently and have insignificant variation, and the sclerotium rolfsii genes in sichuan province and hennan provinces have small flow values and large genetic variation, which may be related to geographical location differences.
Molecular analysis of variance (AMOVA) was performed on different host populations. The results are shown in Table 8.
TABLE 8 different geographic Source population AMOVA analysis
As can be seen from Table 8, the percentage of genetic variation among host populations is 39%, and the percentage of genetic variation within the population is 61%, indicating that variation exists between geographical populations and within populations of southern sclerotium, and the variation within the populations is much larger than that between populations, and the genetic variation mainly comes from within the populations.
Further analysis of gene flow and genetic differentiation results between two populations, except for the two populations MY/HS, BX/BQ, resulted in gene flow values between 0.003 (MY/BZ) -17.531 (XS/BX) and genetic differentiation values between 0.014 (XS/BX) -0.990 (MY/BZ), as shown in Table 9.
TABLE 9 Gene flow between host populations (upper triangle) and genetic differentiation results (lower triangle)
TABLE 9 Gene streams between host populations (upper triangle) and genetic differentiation results (lower triangle)
As can be seen from table 9, the genes of konjac and peanut, pinellia tuber and cynanchum glaucescens have no barrier and no obvious genetic differentiation, the genetic differentiation of scrophularia ningpoensis and pinellia tuber is low, and the genetic differentiation of southern blight of konjac and atractylodes macrocephala is obvious.
3. The genetic structure of 120 southern blight strain groups is analyzed, and the genetic identity and the genetic distance among the groups are analyzed by using PowerMark version 3.25 software.
In geographic populations, genetic identity is between 0.2084 and 0.9896, and genetic distance is between 0.0105 and 1.5681, see table 10.
TABLE 10 reason genetic identity (upper triangle) and genetic distance (lower triangle) between populations
As can be seen from table 10, the closest genetic distance between Shandong (Shandong) and Henan (Henan) is the highest, and the farthest genetic distance between Shandong (Shandong) and Sichuan (Sichuan) is the lowest, which corresponds to the actual difference in geographic location.
In the host population, the genetic identity was between 0.0547 and 0.9991 and the genetic distance was between 0.0009 and 2.9060, and the results are shown in Table 11.
TABLE 11 genetic identity (upper triangle) and genetic distance (lower triangle) between host populations
TABLE 11 genetic identity (upper triangle) and distance between host populations (lower triangle)
As can be seen from table 11, the genetic distance between peanut (HS) and konjac (MY) is the closest, the genetic identity is the highest, and the genetic distance between atractylodes macrocephala (BZ) and rice (SD) is the farthest, and the genetic identity is the lowest.
4. 120 southern blight genomic DNAs were analyzed, and the optimal delta K value calculated by the method of Evano (Evano et al 2005) in the Structure Harvester was 3, indicating that 3 gene pools existed in 120 southern blight groups of 22 colonies, and the results are shown in FIG. 5.
As can be seen from fig. 5, when K =3, the gene composition of the white atractylodes rhizome (QB) in hunan province, the sealwort (XH, ZH), the dendrobium (HS) in anhui province, and the plantain (NN) southern blight flora in guangxi autonomous region mainly originated from the gene bank 1; the genetic components of macleaya cordata (LB) in Hunan province, rhizoma polygonati (YH), coptis chinensis (LC) in Hubei province, pinellia ternata (NC) in Sichuan province, rice (NS) in Guangxi Zhuang autonomous region and sclerotium rolfsii (NY) sclerotium rolfsii flora are mainly derived from a gene bank 2; the genetic components of Macleaya cordata (CB, LY, NB) in Hunan province, radix scrophulariae (QX), konjak (EM) in Hubei province, cynanchum glaucescens (HG), pinellia ternate (QJ, ZB), peanuts (LH, NH) in Henan province, and peanuts (RH) in Shandong province are mainly derived from Genbank 3. Indicating that these populations are heterogeneous with distinct genetic structures.
Based on the genetic distance of Nei, UPGMA trees of 120 strains are constructed and subjected to cluster analysis, and the results are shown in FIG. 6. As can be seen from fig. 6, there was more cross-mixing of strains in each population, indicating that there was more genetic variation in the population, mainly divided into 3 branches, consistent with Structure analysis.
Through analyzing the relationship between UPGMA cluster and hosts, geographical sources and hypha affinity groups of 120 southern blight strains, the same cluster is related to the geographical sources, and the strains from the same geographical sources are mostly classified into one type. The same hyphal affinity groups were distributed approximately in uniform clusters, and the results are shown in FIG. 7.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and amendments can be made without departing from the principle of the present invention, and these modifications and amendments should also be considered as the protection scope of the present invention.
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Claims (9)
1. An SSR marker primer group for detecting plant sclerotium rolfsii is characterized by consisting of the following primer pairs: the sequence of the primer pair is shown as SEQ ID NO:1 to 2, SEQ ID NO:3 to 4, SEQ ID NO:5 to 6, SEQ ID NO:7 to 8, SEQ ID NO:9 to 10, SEQ ID NO:11 to 12, SEQ ID NO:13 to 14, SEQ ID NO:15 to 16, SEQ ID NO:17 to 18.
2. A plant southern sclerotium rolfsii detection kit, which is characterized by comprising the SSR marker primer group according to claim 1.
3. The kit of claim 2, further comprising dNTPs, taq DNA polymerase, PCR reaction buffer, and genome extraction reagents.
4. The kit of claim 3, further comprising reagents in capillary electrophoresis.
5. Use of the primer set of claim 1 or the kit of any one of claims 2 to 4 in the detection of southern blight.
6. Use of the primer set of claim 1 or the kit of any one of claims 2 to 4 for genetic diversity and population structure analysis of southern blight.
7. Use of the primer set according to claim 1 or the kit according to any one of claims 2 to 4 for determination of the origin of a plant sclerotium rolfsii.
8. Use of the primer set of claim 1 or the kit of any one of claims 2 to 4 for determining the propagation pathway of southern blight.
9. Use of the primer set according to claim 1 or the kit according to any one of claims 2 to 4 for the control of southern blight of a plant.
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| CN101801992A (en) * | 2007-05-31 | 2010-08-11 | 孟山都技术公司 | soybean polymorphisms and methods of genotyping |
| CN113755632A (en) * | 2021-10-08 | 2021-12-07 | 中国热带农业科学院环境与植物保护研究所 | SSR primer group for genetic structure analysis of rubber tree powdery mildew flora and application thereof |
| CN114426987A (en) * | 2022-01-14 | 2022-05-03 | 湖南省蔬菜研究所 | Genetic transformation method of capsicum sclerotium rolfsii |
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|---|---|---|---|---|
| CN101801992A (en) * | 2007-05-31 | 2010-08-11 | 孟山都技术公司 | soybean polymorphisms and methods of genotyping |
| CN113755632A (en) * | 2021-10-08 | 2021-12-07 | 中国热带农业科学院环境与植物保护研究所 | SSR primer group for genetic structure analysis of rubber tree powdery mildew flora and application thereof |
| CN114426987A (en) * | 2022-01-14 | 2022-05-03 | 湖南省蔬菜研究所 | Genetic transformation method of capsicum sclerotium rolfsii |
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| Analysis of Gene Expression Profiles in Leaf Tissues of Cultivated Peanuts and Development of EST-SSR Markers and Gene Discovery;Baozhu Guo等;《International Journal of Plant Genomics》;20090624;第2009卷;第1-14页 * |
| 华重楼灰霉病菌灰葡萄孢ITS分型及SSR遗传多样性分析;王帆帆等;《菌物学报》;20201127;第40卷(第03期);第566-579页 * |
| 博落回白绢病的病原鉴定及防治;游景茂等;《中国现代中药》;20171016;第19卷(第10期);第1429-1433页 * |
| 基于SSR标记分析蓝莓品种‘蓝美1号’自由授粉子代遗传多样性及群体遗传结构;王亮等;《植物资源与环境学报》;20220331;第31卷(第3期);第35-43页 * |
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