Michelia alba SSR molecular marker primer group and application thereof

Abstract

The invention discloses a michelia alba SSR (Simple Sequence Repeat) molecular marker primer group and application thereof. Belongs to the technical field of molecular markers. According to the method, high-throughput transcriptome sequencing data of michelia alba leaves is taken as a material, SSR marker sites are explored by utilizing MISA, corresponding primers are designed by utilizing Primer3 software, 34 parts of michelia alba germplasm are subjected to genetic diversity analysis by utilizing 47 pairs of screened polymorphic SSR primers, and the 34 parts of michelia alba germplasm is divided into three subgroups by clustering analysis. At present, research on michelia alba is mainly in the aspects of chemical components and pharmacological action, and previous research fully proves the development value of michelia alba. 47 pairs of SSR highly-polymorphic primers developed by the invention are suitable for michelia alba, the number of SSR molecular markers of michelia alba is enriched, a basis is provided for michelia alba genetic diversity research and germplasm resource analysis, and a theoretical basis is provided for michelia alba resource protection and utilization and other works.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of molecular markers, and more particularly to a Michelia alba SSR molecular marker primer set and application thereof. BACKGROUND

[0002] Michelia alba is a evergreen tree of Magnoliaceae and Miechelia, also known as white orchid, white Myanmar Gui, white Myanmar flower, etc. Michelia alba is a hybrid of M.chanpaca and M.montana. Michelia alba has a high and elegant tree shape, gray bark, and evergreen green branches and leaves. Michelia alba is rich in volatile oil, which is refreshing and pleasant, and is an important raw material for jasmine tea processing, which enhances the freshness of tea. It can also be used as an ingredient to process white orchid tea. In addition, its flowers can also be extracted for medicinal use, which can promote blood circulation and resolve turbidity, and treat cough. Therefore, Michelia alba is a tree species with high comprehensive utilization value in southern China, which is widely used as a spice, ornamental, timber and medicine.

[0003] Simple Sequence Repeat (SSR) is a DNA sequence composed of single nucleotide to six nucleotide units in tandem repeats. SSR sequences have the advantages of co-dominance, good specificity, multiple alleles, easy detection, and good repeatability, and are widely used in genetic diversity evaluation, variety identification, genetic map construction and other aspects. Transcriptome can accurately reveal the genetic coding information of genes, therefore, the SSR marker developed based on transcriptome sequence is a new type of SSR functional marker. With the maturation of sequencing technology and the significant reduction in cost, transcriptome sequencing technology has been widely used in the research of various species, and the accumulation of transcriptome data has laid a good foundation for the development of SSR markers. The SSR marker developed by using transcriptome data has the advantage of being more likely to be associated with genes. The SSR marker developed based on transcriptome has revealed the distribution types and characteristics of different species in transcriptome sequences in multiple species such as Eriobotrya japonica (Thunb.) Lindl., Fagopyrum cymosum, Michelia macclurei Dandy, Cumumis melo L. and Solanum melongena L., which helps to deeply understand the genetic characteristics and evolutionary history of species, and also provides a powerful tool for the protection and utilization of germplasm resources, genetic breeding, etc. However, the SSR marker of Michelia alba has not been reported so far.

[0004] In summary, how to provide a SSR marker based on the transcriptome of Michelia alba is a problem that those skilled in the art need to solve. SUMMARY

[0005] In view of this, the present invention provides a primer set for SSR molecular markers of white orchid and its application.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A white orchid SSR molecular marker primer set, the nucleotide sequences of the SSR molecular marker primer set are shown as SEQ ID No. 1 to SEQ ID No. 94.

[0008] The above-mentioned SSR molecular marker primer set is used for genetic analysis, and the application is used for cluster analysis of white orchid.

[0009] The application of the above-mentioned SSR molecular marker primer set in genetic analysis is used for genetic diversity analysis of different white orchid varieties.

[0010] The application of the above-mentioned SSR molecular marker primer set in drawing DNA fingerprints of different white orchid varieties.

[0011] It can be seen from the above technical solutions that, compared with the prior art, the present invention has the following beneficial effects:

[0012] Based on transcriptome sequencing data, the present invention retrieved SSR loci of white orchid leaves, deeply analyzed their basic characteristics, developed corresponding SSR primers, and conducted a preliminary evaluation of their usability. These primers are used for genetic diversity research and molecular marker-assisted breeding of white orchid, so as to further improve the efficiency of white orchid breeding and provide a theoretical basis for more effective utilization of white orchid resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0014] Figure 1 The SSR type distribution of the transcriptome of white orchid in Example 1 of the present invention;

[0015] Figure 2 The SSR repeat number distribution in the transcriptome of white orchid in Example 1 of the present invention;

[0016] Figure 3 This is the clustering result of 34 white orchid samples in Example 1 of the present invention. DETAILED DESCRIPTION

[0017] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0018] The reagents required for the present invention are conventional experimental reagents, purchased from commercial channels; the experimental methods not mentioned are conventional experimental methods and will not be described in detail here.

[0019] Example 1

[0020] 1 Materials and Methods

[0021] 1.1 Materials

[0022] A. Transcriptome sequencing materials: The present invention uses an asexual line of Orchidaceae as the material. After spraying with methyl jasmonate, its leaves are collected, stored in liquid nitrogen and frozen, and sent to Beijing Novogene Technology Co., Ltd. for library construction and sequencing using the Illumina platform.

[0023] B. Genetic diversity materials: The genetic diversity materials of the present invention were collected from 30 samples of white orchid leaves in Qinzhou City, Guangxi Zhuang Autonomous Region, 3 samples in Suqian City, Jiangsu Province, and 1 sample in Foshan City, Guangdong Province. A total of 34 samples were collected.

[0024] Table 134 White Orchid Sampling Information

[0025]

[0026]

[0027] 1.2 Experimental methods

[0028] A. Use the Plant Genomic DNA Kit to extract genomic DNA from Orchidaceae according to the kit instructions. Detect the quality of the extracted DNA using an ultra-micro-volume full-spectrum spectrophotometer and 1% agarose gel. Store the DNA at -20°C until ready for use.

[0029] B. The SSR-PCR reaction system is as follows: DNA template 1 μL, 2× Taq PCR Master Mix 5 μL, ddH2O 2 μL, upstream primer 1 μL, downstream primer 1 μL, primer concentration is 10 μmol / L -1 .

[0030] The SSR-PCR reaction procedure was as follows: pre-denaturation at 94°C for 5 min, followed by 5 cycles of denaturation at 94°C for 1 min, annealing at 37°C for 1 min, and extension at 72°C for 10 s; 33 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 10 s; and extension at 72°C for another 7 min. The reaction was terminated at 10°C and stored at 4°C.

[0031] After the SSR-PCR reaction, the cells were stored in a 4°C refrigerator.

[0032] C. Silver staining and data recording: Use a pipette to spot 2.0 μL of the SSR-PCR product onto a polyacrylamide gel. Run electrophoresis at 200 V for 1.5 hours. After electrophoresis, silver stain the gel and add developer to visualize the bands. Based on the banding results, manually read the major band of the SSR locus and record its molecular weight for subsequent analysis.

[0033] 1.3 Analysis methods

[0034] A. Assembly of the P. serrata transcriptome: FastQC software was used to perform quality control on the sequencing data. Trimmomatic software was used to trim and remove low-quality sequences and adapter sequences to obtain clean data. Trinity software was then used to assemble the transcriptome without a reference genome.

[0035] B. Development of SSR markers for white orchid: On this basis, the MISA script was used to identify the SSR sites in the white orchid transcripts. The screening criteria for SSR sites were set as follows: the number of repetitions of two to six nucleotides was not less than 10, 7, 5, 5, and 5 times, respectively. Data retrieval was completed based on this screening criteria, and two SSR sites with an interval of less than or equal to 100nt were regarded as a composite SSR site. In addition, the frequency of occurrence, distribution density, and repetition type of SSR were statistically analyzed. Primer3 software was used for primer design, and its specific standards were as follows: primer length was 18-22bp, GC content was 30%-70%, annealing temperature Tm was 55-65℃, and the difference in Tm value between upstream and downstream primers did not exceed 5℃.

[0036] C. Genetic diversity parameters such as the number of alleles (Na), effective number of alleles (Ne), observed heterozygosity (Ho), expected heterozygosity (He), Shannon information index (I), unbiased expected heterozygosity (uHe), and polymorphism information index (PIC) were calculated using PowerMarker V3.25 and GeneAlEx V6.5 software. The genetic distance of shared alleles was calculated using DARwin software, and cluster analysis was performed using the neighbor-joining method to construct a phylogenetic tree, which was then beautified using ITOL software.

[0037] 2 Results and Analysis

[0038] 2.1 Analysis of SSR distribution characteristics

[0039] The 145,297 Unigenes obtained by sequencing the transcriptome of white orchid were retrieved using the software MISA. From the perspective of SSR distribution (Table 2), 32,863 SSR sites were retrieved from 27,971 Unigene sequences. The SSR occurrence frequency (the proportion of SSR sites to the total number of Unigenes) was 22.62%, and the SSR occurrence frequency (the proportion of Unigenes containing SSR sites to the total number of Unigenes) was 19.25%. On average, one SSR site appeared every 8.83 kb. Among them, the number of Unigenes containing more than one SSR site was 4,084, and the number of Unigenes containing complex SSRs was 1,750, accounting for 5.33% of the total number of SSR sites. The above results indicate that the SSR sites contained in the white orchid transcriptome are relatively rich.

[0040] Table 2 Information on SSR sites in the transcriptome of white orchid

[0041]

[0042] There are many types of repeat sequences in the transcriptome SSR data of P. serrata, ranging from dinucleotide to hexanucleotide repeat types. The number of different nucleotide repeat types varies greatly ( Figure 1 ), among which dinucleotide repeat types were the most numerous, with 20,989 types, accounting for 63.87% of the total SSR loci; followed by trinucleotide repeat types and tetranucleotide repeat types, with 9,009 and 2,281 types, accounting for 27.41% and 6.94% of the total SSR loci, respectively; pentanucleotide repeat types and hexanucleotide repeat types were the least numerous, with only 474 and 110 types, accounting for 1.44% and 0.33% of the total SSR loci.

[0043] There are different SSR repeat numbers in the transcriptome of white orchid ( Figure 2 ), the number of SSR loci ranged from 5 to 41 repetitions. As the number of repetitions increased, the number of SSR loci decreased significantly. Among them, the number of SSR loci with a repetition of 5 to 12 times accounted for 66.76% of the total; the number of SSR loci with a repetition of 13 to 28 times accounted for 31.94% of the total; and the number of SSR loci with a repetition of 29 to 41 times accounted for only 1.30% of the total. In terms of the number of SSR repetitions, the loci with a repetition of 6, 11, and 12 times were the most numerous, with the number of SSR loci being 4520, 4247, and 3712, respectively, accounting for 13.75%, 12.92%, and 11.30% of all loci.

[0044] 2.2 Polymorphic primer screening

[0045] Based on transcriptome data, 50 pairs of SSR primers were synthesized and screened for polymorphism using DNA from 6 randomly selected DNA samples from 34 accessions of white orchid. Based on the SSR-PCR amplification results, the amplification performance of the primers was statistically analyzed, ultimately selecting 47 primer pairs with distinct main bands, good polymorphism, and accurate readability (Table 3).

[0046] Table 3 Information of 47 pairs of SSR primers in the transcriptome of white orchid

[0047]

[0048] 2.3 Genetic diversity analysis

[0049] The genetic diversity of 34 white orchid samples was analyzed using the results of 47 polymorphic SSR primer pairs screened (Table 4). A total of 369 alleles were detected using the 47 primer pairs. The Na ranged from 4 to 15, with an average of 7.851; the Ne ranged from 2.438 to 10.371, with an average of 4.842; the I ranged from 1.088 to 2.493, and the Shannon information index of primer MASSR-9 was the largest, with an average of 1.690; the Ho ranged from 0 to 1, with an average of 0.835; the He ranged from 0.59 to 0.904, with an average of 0.768 (He>0.5), indicating that the genetic diversity of the tested materials was high; the uHe ranged from 0.599 to 0.917, with an average of 0.780, which was higher than the He (0.769). The uHe of each primer was higher than the He, indicating that the white orchid population had high genetic diversity. The mean Ho value (0.835) was greater than the mean He value (0.769), indicating an excess of heterozygotes within the population. The F ranged from -0.653 to 1, with an average of -0.089. The PIC ranged from 0.525 to 0.896, with an average of 0.735. The PIC values ​​for all primers were greater than 0.500, indicating that the developed primers were highly polymorphic. These results indicate that the developed SSR primers are highly polymorphic and suitable for analyzing the genetic diversity of white orchid.

[0050] Table 4 Polymorphism analysis of SSR primers

[0051]

[0052]

[0053] 2.4 Cluster Analysis

[0054] The present invention conducted population structure analysis based on the corresponding genetic distances among 34 samples of white orchid ( Figure 3), the results showed that 34 samples of white orchids were divided into 3 groups. It is worth noting that the division of these 3 groups was not based on geographical origin, which indicates that the 34 white orchid varieties were subject to more human intervention. Group I contained 7 germplasms, namely ZC-02, SY-03, JZ-01, SY-02, HL-01, NK-01 and HS-01, which were respectively from Zicai East Street Auxiliary Road, Qinbei District, Qinzhou City, Guangxi Zhuang Autonomous Region, Shuyang County, Suqian City, Jiangsu Province, No. 1 Collective Housing of Forestry Research Institute, Qinnan District, Qinzhou City, Guangxi Zhuang Autonomous Region, Sucheng District, Suqian City, Jiangsu Province, Auxiliary Road of Huancheng West Road, Qinnan District, Qinzhou City, Guangxi Zhuang Autonomous Region, Nongken Homeland, Qinnan District, Qinzhou City, Guangxi Zhuang Autonomous Region and Heshun Market, Qinbei District, Qinzhou City, Guangxi Zhuang Autonomous Region; Group II contained PM The 14 germplasms of X-01, PXW-01, PXJ-01, PXW-02, NL-02, PBW-02, PLM-01, NL-03, PBW-01, NL-01, SL-01, PS-02, CG-01, and SY-01 were respectively obtained from Minxing Road, Pubei County, Qinzhou City, Guangxi Zhuang Autonomous Region; the compound of the Pubei County Party Committee, Qinzhou City, Guangxi Zhuang Autonomous Region; Xiaojiang Primary School, Pubei County, Qinzhou City, Guangxi Zhuang Autonomous Region; Nali Town, Qinnan District, Qinzhou City, Guangxi Zhuang Autonomous Region; and the Baiwanzhu Nursery, Pubei County, Qinzhou City, Guangxi Zhuang Autonomous Region. Breeding base, Longmen Town Street, Pubei County, Qinzhou City, Guangxi Zhuang Autonomous Region, Auxiliary Road of West Huancheng Road, Qinnan District, Qinzhou City, Guangxi Zhuang Autonomous Region, Penshan Village, Lingshan County, Qinzhou City, Guangxi Zhuang Autonomous Region, Changgangling of Forestry Research Institute, Qinnan District, Qinzhou City, Guangxi Zhuang Autonomous Region, Jianling Middle Road, Siyang County, Suqian City, Jiangsu Province; Group III includes 13 germplasms: WD-02, WD-01, Mm-01, HL-01, PS-04, MP-01, PS-03, SQ-01, ZC-01, PS-01, BJ-01, CG-02, SQ-02, which come from The samples originated from Beibu Gulf University in Qinnan District, Qinzhou City, Guangxi Zhuang Autonomous Region; X323 Muqian Village in Lingshan County, Qinzhou City, Guangxi Zhuang Autonomous Region; the auxiliary road of Huancheng West Road in Qinnan District, Qinzhou City, Guangxi Zhuang Autonomous Region; Penshan Village in Lingshan County, Qinzhou City, Guangxi Zhuang Autonomous Region; the central nursery of the Institute of Forestry in Qinnan District, Qinzhou City, Guangxi Zhuang Autonomous Region; Siqiaojiangbian in Qinbei District, Qinzhou City, Guangxi Zhuang Autonomous Region; the auxiliary road of Zicai East Street in Qinbei District, Qinzhou City, Guangxi Zhuang Autonomous Region; Tianning Road in Shunde District, Foshan City, Guangdong Province; Changgangling of the Institute of Forestry in Qinnan District, Qinzhou City, Guangxi Zhuang Autonomous Region; and Jianling Middle Road in Siyang County, Suqian City, Jiangsu Province. Cluster analysis results showed that geographically close samples did not preferentially cluster together, indicating that the relationship between genetic diversity of each sample may not be related to geographical distance, but is more dependent on gene flow, or more directly, on artificial introduction.

[0055] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0056] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A primer set for SSR molecular markers of P. glabra, characterized in that: The nucleotide sequences of the SSR molecular marker primer set are shown in SEQ ID No. 1 to SEQ ID No.

94.

2. An application of the SSR molecular marker primer set according to claim 1 for genetic analysis, characterized in that: The application is used for cluster analysis of P. fulvum.

3. An application of the SSR molecular marker primer set according to claim 1 for genetic analysis, characterized in that: The application is used for genetic diversity analysis of different white orchid varieties.

4. Use of the SSR molecular marker primer set as claimed in claim 1 in drawing DNA fingerprints of different white orchid varieties.

Images