Poplar cladosporium cucumerinum SSR primer and application thereof in genetic diversity and genetic relationship analysis
By developing 11 pairs of SSR primers of highly polymorphic Poplar cystellae, the problem of no SSR molecular markers of this species in the prior art was solved, and the genetic diversity and kinship analysis of the Poplar cystellae was achieved.
Patent Information
- Application Number
- CN202510120851.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-26
- Publication Date
- 2025-05-23
AI Technical Summary
There is no SSR molecular marker of Poplar Black Star bacteria in the prior art, and it is difficult to analyze genetic diversity and kinship.
11 pairs of highly polymorphic SSR primers of Poplar Black Astrology were developed, and these primers were used for PCR amplification, combined with POPGENE32 and NTSYSpc2.10 software for data processing, and analyzed genetic diversity and kinship.
The genetic diversity and kinship analysis of poplar black astrology bacteria was achieved, filling the research gap in the existing technology that there is no SSR molecular marker.
Smart Images
Figure CN120026126A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biotechnology, and particularly relates to SSR primers of Venturia phaeosepta and their application in genetic diversity and phylogenetic relationship analysis. Background Art
[0002] Venturia phaeosepta is an important plant pathogenic fungus that mainly harms poplars and willows. Venturia leaf spot is distributed in many countries such as Japan, Europe, and North America, and is an important poplar disease in Italy and Poland. In China, it is distributed in provinces (autonomous regions) such as Heilongjiang, Jilin, Hebei, Inner Mongolia, Shandong, Henan, Shaanxi, Sichuan, Guizhou, and Xinjiang. Venturia phaeosepta infects the current-year new leaves with conidia, symptoms appear in June, the disease incidence peak is in July, and there is another peak in September. It overwinters with conidia or mycelia on the tree tips or fallen leaves. It seriously harms poplars, and both nurseries and young forests can be infected. In mild cases, it affects growth, the tree vigor weakens continuously for several years, and it is vulnerable to other pests and diseases. In severe cases, the whole plant will wither and die.
[0003] Simple Sequence Repeat (SSR), also known as Microsatellite DNA, is a class of DNA sequences composed of tandem repeats of motifs consisting of 2 - 6 bases. There are differences in the number of repeats between different alleles, and the sequences on both sides of the repeat sequence are mostly relatively conserved single-copy sequences. Specific primers can be designed to perform PCR amplification on genomic DNA, and molecular markers can be developed based on the length polymorphism of the amplified fragments. Microsatellite markers have the advantages of rich polymorphism, good repeatability, co-dominant markers, and wide and uniform distribution in the genome. They have now been widely used in the study of genetic diversity of various eukaryotes. Currently, in the study of fungal genetic diversity and genetic structure, SSR molecular markers of many species have been developed and applied, but for Venturia phaeosepta, there has been no official report on the SSR molecular markers of this species yet. Summary of the Invention
[0004] In order to solve the deficiencies in the prior art, the purpose of the present invention is to provide SSR primers of Venturia phaeosepta and their application in genetic diversity and phylogenetic relationship analysis. The present invention has developed, for the first time, highly polymorphic SSR molecular markers of Venturia phaeosepta, and these molecular markers can be used to analyze the genetic diversity and phylogenetic relationship of Venturia phaeosepta.
[0005] In order to achieve the above purpose, the specific solution adopted by the present invention is as follows:
[0006] In a first aspect, the present invention provides a poplar venison SSR primer set, comprising 11 pairs of primers, namely VP-SSR1, VP-SSR4, VP-SSR8, VP-SSR10, VP-SSR11, VP-SSR18, VP-SSR20, VP-SSR24, VP-SSR25, VP-SSR26 and VP-SSR27; The nucleotide sequences of the VP-SSR1 primer pair are shown in SEQ ID NO: 1 and SEQ ID NO: 2; The nucleotide sequences of the VP-SSR4 primer pair are shown in SEQ ID NO: 3 and SEQ ID NO: 4; The nucleotide sequences of the VP-SSR8 primer pair are shown in SEQ ID NO: 5 and SEQ ID NO: 6; The nucleotide sequences of the VP-SSR10 primer pair are shown in SEQ ID NO: 7 and SEQ ID NO: 8; The nucleotide sequences of the VP-SSR11 primer pair are shown in SEQ ID NO: 9 and SEQ ID NO: 10; The nucleotide sequences of the VP-SSR18 primer pair are shown in SEQ ID NO: 11 and SEQ ID NO: 12; The nucleotide sequences of the VP-SSR20 primer pair are shown in SEQ ID NO: 13 and SEQ ID NO: 14; The nucleotide sequences of the VP-SSR24 primer pair are shown in SEQ ID NO: 15 and SEQ ID NO: 16; The nucleotide sequences of the VP-SSR25 primer pair are shown in SEQ ID NO: 17 and SEQ ID NO: 18; The nucleotide sequences of the VP-SSR26 primer pair are shown in SEQ ID NO: 19 and SEQ ID NO: 20; The nucleotide sequences of the VP-SSR27 primer pair are shown in SEQ ID NO: 21 and SEQ ID NO: 22.
[0007] In a second aspect, the present invention provides a kit for analyzing the genetic diversity and kinship of poplar black spot pathogen, comprising the above-mentioned SSR primer set.
[0008] In a third aspect, the present invention provides the use of the above-mentioned SSR primer set or kit in the analysis of genetic diversity and kinship of poplar black spot pathogen.
[0009] Beneficial effects: The present invention performs whole genome sequencing on poplar venus pathogen, and then analyzes the types and quantities of SSRs in the genome of poplar venus pathogen, and develops highly polymorphic SSR molecular markers, which can be used to analyze the genetic diversity and kinship of poplar venus pathogen. The present invention fills the research gap of SSR molecular markers for poplar venus pathogen in the prior art. BRIEF DESCRIPTION OF THE DRAWINGS
[0010] Figure 1 This is the phylogenetic tree of 24 poplar scab pathogens; Figure 2 is the genetic similarity coefficient between the strains. DETAILED DESCRIPTION
[0011] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention.
[0012] 1 Materials and methods
[0013] 1.1 Test strains and culture medium
[0014] The 24 strains of poplar venison used in this study were isolated and purified from diseased plants in Jiyuan City, Henan Province by the single spore separation method, and transferred to PDA slants and stored at 8°C. The culture medium used was potato dextrose agar (PDA): 200 g potato, 20 g glucose, 15 g agar, and 1 L distilled water;
[0015] Table 1 Information on the pathogen of black spot on 24 poplars
[0016] 1.2 Analysis of genomic SSR loci and primer design
[0017] The VP23-1 strain of Venturia spp. was sent to Shanghai Yuanxin Biopharmaceutical Technology Co., Ltd. for whole genome sequencing. The SSR sites of 2-6 nucleotides in the genome sequence were searched using Krait software. The search conditions were that the number of repetitions of 2 nucleotides must be ≥6, and the number of repetitions of 3-6 nucleotides must be ≥5. Then, the 200bp sequence upstream and downstream of the SSR site was extracted, and the SSR primers were designed using Primer5.0 software. The parameters were set as follows: primer length 20-24bp, annealing temperature 50-65℃, GC content 40%-60%, and PCR product length 150-350bp. The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.
[0018] 1.3 DNA extraction and PCR
[0019] After culturing the Venturia populina strain for 40 days, 100 mg of mycelium was collected, and genomic DNA was extracted using the modified CTAB method. The concentration of the extracted DNA was adjusted to 50 ng / μL using a NanoDrop 1000 ultra-micro spectrophotometer (Thermo Fisher Scientific, USA).
[0020] PCR reaction system: 2.5 μL of 10×PCR reaction buffer, 1 μL of 2.5 mM dNTPs, 0.2 μL of 5 U / μL Taq DNA polymerase, 0.5 μL of 10 μM upstream and downstream primers each, 1 μL of DNA template, and sterile ultrapure water was added to make up to 25 μL. PCR amplification conditions: denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 54 - 56°C for 30 s, extension at 72°C for 30 s, 35 cycles, and extension at 72°C for 5 min. The PCR products were detected by electrophoresis on a 3% agarose gel for 6.5 h, and the size of the amplified fragments was determined by measuring the migration distance of the SSR alleles relative to the 50 bp DNA ladder on the agarose gel picture.
[0021] 1.4 Data processing
[0022] The bands in the electrophoresis pattern were converted into a digital matrix. The polymorphism information index of the SSR primers was calculated using Cervus 3.0.7 software; the apparent allele number, effective allele number, gene diversity index, and Shannon information index of the population were calculated using POPGENE 32 software; the genetic similarity coefficients between individual strains were calculated using NTSYSpc 2.10 software, and a phylogenetic tree was constructed using the UPGMA method.
[0023] 2 Results and analysis
[0024] 2.1 Distribution of genomic SSRs
[0025] The total length of the whole genome sequence of Venturia populina VP23-1 is 87.50 Mb, with a total of 59 Contigs. The Krait software was used to search for SSR loci with 2 - 6 nucleotides in the genome, and a total of 7503 SSR loci were found. On average, there are 85.75 SSR loci per Mb of the genome sequence (Table 2). Among them, the number of trinucleotide SSR loci is the largest, with a total of 4069, accounting for 54.23% of the total number of SSR loci. Followed by hexanucleotide and dinucleotide SSR loci, with 1442 and 1000 respectively, accounting for 19.22% and 13.33% of the total number of SSR loci. The number of tetra- and pentanucleotide repeat loci is relatively small, with 705 and 287 respectively, accounting for 9.40% and 3.83% of the total number of SSR loci.
[0026] Table 2 Distribution of genomic SSRs in Venturia populina VP23-1
[0027] 2.2 Screening of polymorphic SSR primers
[0028] Thirty-six dinucleotide and trinucleotide SSR loci with a repeat number ≥10 were randomly selected, and the 200bp upstream and downstream sequences of the SSR loci were extracted. Thirty-six pairs of SSR primers were designed using Primer 5.0 software. Then, PCR was performed using six strains from different geographical origins (VP23-1, VP23-8, VP23-12, VP23-25, VP23-36 and VP23-42) to detect the effectiveness and polymorphism of the 36 pairs of SSR primers. The results showed that there were 11 pairs of SSR primers, namely VP-SSR1 (sequences such as SEQ ID NO: 1 and SEQ ID NO: 2), VP-SSR4 (sequences such as SEQ ID NO: 3 and SEQ ID NO: 4), VP-SSR8 (sequences such as SEQ ID NO: 5 and SEQ ID NO: 6), VP-SSR10 (sequences such as SEQ ID NO: 7 and SEQ ID NO: 8), VP-SSR11 (sequences such as SEQ ID NO: 9 and SEQ ID NO: 10), VP-SSR18 (sequences such as SEQ ID NO: 11 and SEQ ID NO: 12), VP-SSR20 (sequences such as SEQ ID NO: 13 and SEQ ID NO: 14), VP-SSR24 (sequences such as SEQ ID NO: 15 and SEQ ID NO: 16), VP-SSR25 (sequences such as SEQ ID NO: 17 and SEQ ID NO: 18), VP-SSR26 (sequences such as SEQ ID NO: 19 and SEQ ID NO: 20) and VP-SSR27 (sequences such as SEQ ID NO: 21 and SEQ ID NO: 22). NO: 21 and SEQ ID NO: 22), clear bands could be amplified from all 6 strains and all had obvious polymorphism, so 11 SSR molecular markers were successfully developed (Table 3). These 11 pairs of SSR primers include 6 pairs of dinucleotide SSR primers and 5 pairs of trinucleotide SSR primers, with a repetition number of 10-26 times, a number of alleles of 5 to 10, and a polymorphism information index between 0.627 and 0.850. These SSR primers can be used for genetic diversity and phylogenetic analysis of black spot pathogen on poplar.
[0029] Table 3 Information of 11 pairs of polymorphic SSR primers
[0030] 2.3 Analysis of genetic diversity of two poplar venison pathogen populations
[0031] Based on the results of PCR amplification of 24 poplar venison pathogens using 11 pairs of SSR primers, the apparent allele number, effective allele number, gene diversity index and Shannon information index of the population were calculated using POPGENE32 software (Table 4). These four parameters are important indicators for measuring the level of genetic diversity. Among the populations from two towns in Jiyuan City, Henan Province, the genetic diversity level of the Potou Town population was higher, with the apparent allele number, effective allele number, Nei's gene diversity index and Shannon information index of 6.1818, 4.5431, 0.7579 and 1.6086, respectively, while the genetic diversity level of the Chengliu Town population was lower, with the apparent allele number, effective allele number, Nei's gene diversity index and Shannon information index of 4.9091, 3.8311, 0.6982 and 1.3945, respectively.
[0032] Table 4 Genetic diversity analysis of two poplar venison pathogen populations
[0033] Note: Na represents the apparent allele number, Ne represents the effective allele number, H represents Nei's gene diversity index, and I represents Shannon information index.
[0034] 2.4 Analysis of the relationship between poplar black spot pathogen strains
[0035] Based on the PCR amplification results of 11 pairs of SSR primers for 24 strains of poplar venison, the genetic similarity coefficients ( Figure 2 ), and the UPGMA method was used to construct a phylogenetic tree ( Figure 1 ). Based on the genetic similarity coefficient between strains or the constructed phylogenetic tree, the relationship between each strain can be determined. It can be seen that the genetic similarity coefficient between the 24 strains is between 0 and 0.5455. The genetic similarity coefficient between strains VP23-34 and VP23-45 is the largest, with a value of 0.5455, indicating that the relationship between these two strains is the closest; the genetic similarity coefficient between multiple strains is 0, which is the minimum value, such as between strains VP23-1 and VP23-20, and between strains VP23-2 and VP23-25, indicating that the relationship between them is the farthest in this group.
[0036] It should be noted that the above-described embodiments should be understood as illustrative rather than limiting the scope of protection of the present invention, and the scope of protection of the present invention shall be subject to the claims. For those skilled in the art, some non-essential improvements and adjustments made to the present invention still fall within the scope of protection of the present invention without departing from the essence and scope of the present invention.
Claims
1. A SSR primer set for Venturia spp., characterized in that: It contains 11 pairs of primers, namely VP-SSR1, VP-SSR4, VP-SSR8, VP-SSR10, VP-SSR11, VP-SSR18, VP-SSR20, VP-SSR24, VP-SSR25, VP-SSR26 and VP-SSR27; The nucleotide sequences of the VP-SSR1 primer pair are shown in SEQ ID NO: 1 and SEQ ID NO: 2; The nucleotide sequences of the VP-SSR4 primer pair are shown in SEQ ID NO: 3 and SEQ ID NO: 4; The nucleotide sequences of the VP-SSR8 primer pair are shown in SEQ ID NO: 5 and SEQ ID NO: 6; The nucleotide sequences of the VP-SSR10 primer pair are shown in SEQ ID NO: 7 and SEQ ID NO: 8; The nucleotide sequences of the VP-SSR11 primer pair are shown in SEQ ID NO: 9 and SEQ ID NO: 10; The nucleotide sequences of the VP-SSR18 primer pair are shown in SEQ ID NO: 11 and SEQ ID NO: 12; The nucleotide sequences of the VP-SSR20 primer pair are shown in SEQ ID NO: 13 and SEQ ID NO: 14; The nucleotide sequences of the VP-SSR24 primer pair are shown in SEQ ID NO: 15 and SEQ ID NO: 16; The nucleotide sequences of the VP-SSR25 primer pair are shown in SEQ ID NO: 17 and SEQ ID NO: 18; The nucleotide sequences of the VP-SSR26 primer pair are shown in SEQ ID NO: 19 and SEQ ID NO: 20; The nucleotide sequences of the VP-SSR27 primer pair are shown in SEQ ID NO: 21 and SEQ ID NO:
22.
2. A kit for analyzing the genetic diversity and kinship of poplar venison pathogen, characterized in that: Comprising the SSR primer set as described in claim 1.
3. Use of the SSR primer set according to claim 1 or the kit according to claim 2 in the analysis of genetic diversity and kinship of poplar black spot pathogen.