A combination of ssr molecular markers for evaluating genetic diversity of ziziphus jujuba mill and application thereof

Abstract

The application discloses a kind of genetic diversity evaluation of Ziziphus silvestris and its application of SSR molecular marker combination, it is related to molecular genetics technical field.The SSR molecular marker combination includes molecular marker NSZ-43385, NSZ-9141, NSZ-37910, NSZ-240, NSZ-22900, NSZ-39803, NSZ-42088, NSZ-165, NSZ-26522, NSZ-39039, NSZ-41051, NSZ-1007, NSZ-36575, NSZ-37045, NSZ-34772 and NSZ-45650.The SSR molecular marker combination can be used for genetic diversity evaluation of Ziziphus silvestris, carries out genetic diversity research of different population of Ziziphus silvestris, and has important guiding significance to aspects such as formulating Ziziphus silvestris germplasm resource effective protection and sustainable utilization strategy.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of molecular genetics, in particular to a combination of SSR molecular markers for evaluating genetic diversity of Choerospondias axillaris and application thereof. BACKGROUND

[0002] Choerospondias axillaris (Roxb.) Burtt et Hill. is a single species of Choerospondias, and its fruit has high nutritional value and rich components. It is an economic, ecological and social tree species with good benefits. The leaves of Choerospondias axillaris can be used as fertilizer, and the wood has a wide range of uses. The bark and leaves can be used to extract tannin. The fruit can be eaten raw or brewed into wine. The fruit kernel can be used as raw material for activated carbon. The stem bark fiber can be used as a rope. The bark and fruit have medicinal value, and can be used to treat inflammation, detoxification, hemostasis and pain relief, and can be used externally to treat large area of burns and scalds. Since Choerospondias axillaris has strong adaptability, Li Dong et al. found that it can mainly be distributed in areas with high temperature and precipitation sensitivity. Xue Shensheng et al. found that due to different regional climates, the phenotypic variation of Choerospondias axillaris in different habitats is large, and the nutritional composition of the fruit also varies significantly. There are also large differences between the phenological periods and disease resistance. In addition, in the investigation and research of Tian Hualin et al. on the natural forest resources of Choerospondias axillaris and the study of seedling traits of Choerospondias axillaris, it was found that the seedling traits of Choerospondias axillaris have large intra-specific variation, and there are significant differences between provenances, and have high general heritability, which is helpful for the selection of superior trees. All of these can indicate that Choerospondias axillaris has accumulated rich intra-specific genetic variation in the long-term evolution process in the subtropical and tropical regions of China. Genetic variation is an important material basis for genetic improvement of species. At present, there are few reports on genetic diversity of Choerospondias axillaris. The related researches of molecular genetics include the analysis of the transcriptome of Choerospondias axillaris based on high-throughput sequencing by Yang Chunxia et al., and the establishment and optimization of the ISSR-PCR reaction system of Choerospondias axillaris by Ye Jinshan et al.

[0003] Microsatellite markers, also known as short tandem repeats (STRs), simple sequence repeats (SSRs), are composed of 1-6 base tandem repeat units, and widely exist in the genomes of eukaryotes and prokaryotes. Hamada et al. found that there are many microsatellites in eukaryotes (from yeast to vertebrates), and later Delseny, Tautz and Renz et al. found and confirmed that there are rich microsatellites in plants and many other eukaryotes. Due to the difference in repeated parts, the plant genome is rich in AT repeats and the animal genome is rich in AC repeats, thereby revealing the difference between plant and animal genomes. SSRs are distributed in the coding and non-coding regions of the genome and in the entire nuclear genome. There are about 104-105 SSRs in the genome, and the polymorphism is high due to different repeats in the microsatellite region, so it can be easily detected by PCR. Therefore, the SSR molecular marker is a very important and practical tool. After a long period of evolution, unequal exchanges occur between nucleotides in the organism, and then different repeat frequencies of bases are gradually accumulated. In different organisms, even at the same microsatellite site, the mutation of microsatellites due to the difference in the number of repeat units makes the microsatellites show rich polymorphism, and this mutation does not harm the organism, so the rich microsatellite variation in the chromosome of the organism can exist for a long time. SSR markers have the advantages of fast mutation, high polymorphism and stability, rich information content, wide distribution, and co-dominance, and can be used as an excellent genetic marker type (Kuroda et al., 2006). At present, this technology is widely used in the field of population genetic diversity research. Developing a SSR molecular marker for evaluating the genetic diversity of Ziziphus mauritiana and applying it to the genetic diversity research of different populations of Ziziphus mauritiana will have important guiding significance for analyzing the genetic structure and genetic diversity of different populations of the species, formulating effective protection and sustainable utilization strategies for Ziziphus mauritiana germplasm resources, and the like. SUMMARY

[0004] The purpose of the present application is to provide a SSR molecular marker combination for evaluating the genetic diversity of Ziziphus mauritiana and its application, so as to solve the problems existing in the prior art. The SSR molecular marker combination can be used for evaluating the genetic diversity of Ziziphus mauritiana, and can be used for genetic diversity research of different populations of Ziziphus mauritiana. It has important guiding significance for analyzing the genetic structure and genetic diversity of different populations of the species, formulating effective protection and sustainable utilization strategies for Ziziphus mauritiana germplasm resources, and the like.

[0005] To achieve the above purpose, the present application provides the following solutions:

[0006] The application provides a SSR molecular marker combination for evaluating genetic diversity of Ziziphus jujuba, which comprises molecular markers NSZ-43385, NSZ-9141, NSZ-37910, NSZ-240, NSZ-22900, NSZ-39803, NSZ-42088, NSZ-165, NSZ-26522, NSZ-39039, NSZ-41051, NSZ-1007, NSZ-36575, NSZ-37045, NSZ-34772 and NSZ-45650.

[0007] The NSZ-43385 is obtained by amplification of a primer pair as shown in SEQ ID NO. 1-2;

[0008] The NSZ-9141 is obtained by amplification of a primer pair as shown in SEQ ID NO. 3-4;

[0009] The NSZ-37910 is obtained by amplification of a primer pair as shown in SEQ ID NO. 5-6;

[0010] The NSZ-240 is obtained by amplification of a primer pair as shown in SEQ ID NO. 7-8;

[0011] The NSZ-22900 is obtained by amplification of a primer pair as shown in SEQ ID NO. 9-10;

[0012] The NSZ-39803 is obtained by amplification of a primer pair as shown in SEQ ID NO. 11-12;

[0013] The NSZ-42088 is obtained by amplification of a primer pair as shown in SEQ ID NO. 13-14;

[0014] The NSZ-165 is obtained by amplification of a primer pair as shown in SEQ ID NO. 15-16;

[0015] The NSZ-26522 is obtained by amplification of a primer pair as shown in SEQ ID NO. 17-18;

[0016] The NSZ-39039 is obtained by amplification of a primer pair as shown in SEQ ID NO. 19-20;

[0017] The NSZ-41051 is obtained by amplification of a primer pair as shown in SEQ ID NO. 21-22;

[0018] The NSZ-1007 is obtained by amplification of a primer pair as shown in SEQ ID NO. 23-24;

[0019] The NSZ-36575 is obtained by amplification with a primer pair as shown in SEQ ID NO. 25-26;

[0020] The NSZ-37045 is obtained by amplification with a primer pair as shown in SEQ ID NO. 27-28;

[0021] The NSZ-34772 is obtained by amplification with a primer pair as shown in SEQ ID NO. 29-30;

[0022] The NSZ-45650 is obtained by amplification with a primer pair as shown in SEQ ID NO. 31-32.

[0023] The application further provides a primer combination for evaluating genetic diversity of Ziziphus nummularia, which comprises 16 primer pairs, and the nucleotide sequences of the primer pairs are shown in SEQ ID NO. 1-32.

[0024] The application further provides application of the primer combination in the preparation of a kit for evaluating genetic diversity of Ziziphus nummularia.

[0025] The application further provides a kit for evaluating genetic diversity of Ziziphus nummularia, which comprises the primer combination.

[0026] The application further provides application of the SSR molecular marker combination, the primer combination or the kit in the evaluation of genetic diversity of Ziziphus nummularia.

[0027] The application further provides a method for evaluating genetic diversity of Ziziphus nummularia, which comprises the following steps:

[0028] (1) extracting genomic DNA of Ziziphus nummularia;

[0029] (2) performing PCR amplification with the primer combination;

[0030] (3) detecting the amplification product to obtain a test result;

[0031] (4) performing genetic diversity analysis, genetic differentiation analysis or genetic structure analysis of a Ziziphus nummularia population by using the test result of step (3).

[0032] Further, in step (2), the reaction system of the PCR amplification is 2x Taq PCR Master Mix 12.5 μL, upstream primer 1 μL, downstream primer 1 μL, DNA template 1 μL and ddH2O 9.5 μL.

[0033] Further, in step (2), the reaction procedure of the PCR amplification is: 94℃ 5min; 94℃ 30s, 63℃ 30s, 72℃ 45s, 10 cycles; 94℃ 30s, 55℃ 30s, 72℃ 45s, 20 cycles; 72℃ 7min.

[0034] The present application discloses the following technical effects:

[0035] The present application utilizes the results of the transcriptome sequencing of Ziziphus sabaea to screen a combination of SSR molecular markers suitable for evaluating the genetic diversity of Ziziphus sabaea, which can be used to analyze the genetic structure and genetic diversity of the species among different populations. The combination of SSR molecular markers provided by the present application helps to analyze the distribution pattern of genetic variation of Ziziphus sabaea, and provides a theoretical basis for efficient utilization and protection of Ziziphus sabaea germplasm resources. BRIEF DESCRIPTION OF DRAWINGS

[0036] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced as follows. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0037] Figure 1 The fluorescence labels of the three primer pairs are as follows: A is the FAM fluorescence label of primer pair NSZ-37910; B is the HEX fluorescence label of primer pair NSZ-240; and C is the TAMRA fluorescence label of primer pair NSZ-39039.

[0038] Figure 2 The amplification results of two Ziziphus sabaea samples under 24 pairs of screening primers are shown in A and B; the odd-numbered Ziziphus sabaea sample in A and B is YF01 (Yifeng 01), and the even-numbered Ziziphus sabaea sample is YF07 (Yifeng 07); in A and B, 13 is a marker, and 1-12 and 14-25 are 24 pairs of screening primers.

[0039] Figure 3 The proportion of different types of SSR in total SSR is shown in the table.

[0040] Figure 4 The results of cluster analysis of 28 Ziziphus sabaea germplasm resources in four populations using 16 pairs of polymorphic SSR primers are shown in the table. DETAILED DESCRIPTION

[0041] The various exemplary embodiments of the present application will now be described in detail, which should not be considered as limiting the present application, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the present application.

[0042] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. In addition, where particular ranges of values are given, understand that each intervening value, to the upper or lower limit of the ranges is also specifically included. The upper and lower limits of these intervening values are also specifically included within the scope of the present application. These smaller ranges are not insubstantial.

[0043] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the documents are cited. In case of conflict between the content of the specification and that of any document incorporated herein by reference, the content of the specification prevails.

[0044] Many modifications and variations of the present application described in the specification are possible without departing from the scope or spirit of the application. Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples are illustrative only.

[0045] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean including, but not limited to.

[0046] In the following examples, the Zizyphus sativa materials used for SSR molecular marker screening and availability evaluation were from Zizyphus sativa germplasm resource bank in Chongyi County, Ganzhou City, Jiangxi Province, including 28 germplasm resources of 4 populations in Jiangxi (Table 1).

[0047] Table 1 Details of Zizyphus sativa germplasm resources for SSR screening

[0048]

[0049] Example 1

[0050] 1. Mining of SSR loci based on Zizyphus sativa transcriptome spliced sequences

[0051] (1) RNA sample preparation, library preparation and quality inspection

[0052] a) Extraction of total RNA sample preparation: tissue samples were collected, and then the samples were determined. After the RNA quality detection met the requirements, the library preparation was carried out.

[0053] b) After the eukaryotic mRNA with polyA tail is enriched by magnetic beads with Oligo(dT), the mRNA is broken by ultrasonic wave.

[0054] c) The first strand of cDNA is synthesized in M-MuLV reverse transcriptase system with the fragmented mRNA as template and random oligonucleotide as primer, and then the RNA strand is degraded by RNase H.

[0055] d) The second strand of cDNA is synthesized in DNA polymerase I system with dNTPs as raw material.

[0056] e) After purification, the double-stranded cDNA is subjected to end repair, A tailing and sequencing adapter installation, and then screened by AMPure XP beads for cDNA of about 200 bp, PCR amplification and purification of PCR products by AMPure XP beads again, and finally the library is obtained by agarose gel electrophoresis screening of fragment size.

[0057] f) After the library is constructed, Illumina HiseqTM PE150s is used for sequencing to obtain raw data.

[0058] (2) Data quality control

[0059] When performing quality assessment and splicing, data filtering is performed on the raw data before information analysis to reduce data analysis interference. First, fastp is used to control the quality of the raw reads, filter low-quality data, and obtain clean reads. The filtering steps are as follows: remove reads containing adapters, containing N more than 10%, and all A bases, low quality (the number of bases with quality value Q≤20 accounts for more than 50% of the whole read).

[0060] (3) Raw sequence quality assessment and splicing

[0061] The high-throughput sequencing raw data obtained by the above steps (1), (2) is assembled by using Trinity assembly software on high-quality sequences, and finally 40341 Unigenes are obtained as the basic data of this experiment.

[0062] (4) SSR site mining based on Ziziphus sabaea transcriptome

[0063] The 40341 Unigenes assembled are used as the basic data of this experiment. MISA software is used to identify SSR in Ziziphus sabaea transcriptome data.

[0064] (5) Characteristics of SSR motif repeat type, number and frequency in transcriptome

[0065] There are five types of SSR repeats, i.e. di-nucleotide to hexa-nucleotide repeats. The statistics of the proportion of different types of SSR repeats in total SSR are as follows Figure 3 The short tandem repeat units of SSR (horizontal axis meaning: such as AAC / GTT, which is composed of the two most abundant SSRs of AAC, ACA, CAA, GTT, TGT and TTG (identical except the beginning and end part through shifting and reverse complementation) with the highest frequency of di-nucleotide repeats.

[0066] The repeat number of SSR repeat units in the transcriptome of Ziziphus silvestris is between 4 and 26 times, among which 4476 SSRs with 4-8 repeats account for 78.33% of the total; followed by 952 SSRs with 9-14 repeats, accounting for 16.66% of the total; and 286 SSRs with 15 repeats or more, accounting for 5%. Di-nucleotide and tri-nucleotide repeats are dominant, accounting for 46.95% and 34.27% of the total SSRs, respectively; the number of tetra-nucleotide, penta-nucleotide, hexa-nucleotide and other repeat types is relatively small, accounting for 11.64%, 3.64% and 3.50% of the total, respectively (see Table 2).

[0067] Table 2 Types, number and distribution frequency of SSRs in Ziziphus silvestris

[0068]

[0069] 2. SSR primer design and screening

[0070] A total of 5251 SSR sites were found, and Primer3.0 software was used to design primers for the identified SSRs. The main technical parameters for primer design were GC content of 40%-60%, annealing temperature of 55-65°C (optimal 60°C), Tm value difference between upstream and downstream primers ≤2°C, primer length of 18-24 bp, expected amplification product length of 100-300 bp, and no secondary structure and dimers. Then, SSR primers were synthesized according to the obtained nucleotide sequence, a total of 15753 pairs of primers were designed and synthesized. Among the screened primers with polymorphic potential (SSR sequence length > 15 bp), 100 pairs were randomly selected.

[0071] The selected 100 pairs of primers were used for pre-experiment, two different populations of Ziziphus silvestris individuals were randomly selected, and the PCR reaction system in Table 3 was used to amplify the 100 pairs of SSR primers. The size of the amplification product was detected by 2% agarose gel electrophoresis, and the primers with clear and single bands, no specific amplification, and the expected fragment size were selected as the primary screening qualified primers. A total of 55 pairs of primers were primary screening qualified (part of which is shown in Table 4). Figure 2). Then 8 samples (YF01, 02; CY01, 02; LN01, 02; GF01, 02) were selected from Yifeng, Chongyi, Longnan, Guangfeng, two samples from each group, and the DNA was extracted for repeated screening and verification (see Table 4 for amplification procedure). The stable, high polymorphic and repeatable effective SSR marker primers were selected for 16 pairs (see Table 5). The selected amplification products were sent to the company for capillary electrophoresis detection. Figure 1 The fluorescence labels (TAMRA, FAM, HEX) of 3 primer pairs were displayed.

[0072] The DNA of Ziziphus sabinoides was extracted by CTAB method. The method steps are as follows:

[0073] ① The dried Ziziphus sabinoides leaves were placed in a 2 mL EP tube, a steel ball was added, and the leaves were ground into powder with a plant tissue grinder (12000 rpm, 3 min). After grinding, the steel ball was removed;

[0074] ② 800 μL of 2×CTAB extraction solution and 8 μL of mercaptoethanol were added to the EP tube containing the powder, mixed well, and placed in a 65℃ preheated water bath for 20 min. The EP tube was inverted every 6 min to mix well;

[0075] ③ The EP tube was taken out, an equal volume of chloroform and isoamyl alcohol was added, and it was slowly inverted up and down to mix well. It was placed in a centrifuge with a temperature setting of 4℃ and a speed of 12000 rpm for 10 min;

[0076] ④ The supernatant was carefully aspirated into a new 2 mL EP tube, an equal volume of chloroform and isoamyl alcohol was added, and it was slowly inverted up and down to mix well. It was placed in a centrifuge with a temperature setting of 4℃ and a speed of 12000 rpm for 10 min;

[0077] ⑤ 500 μL of the supernatant was carefully aspirated into a new 1.5 mL EP tube, an equal volume of chloroform and isoamyl alcohol was added, and it was gently shaken to mix the two phases. It was placed in a -20℃ refrigerator for more than 3 hours;

[0078] ⑥ When the DNA precipitated, a white flocculent precipitate was visible. It was placed in a centrifuge with a temperature setting of 4℃ and a speed of 12000 rpm for 10 min. At this time, the DNA precipitated at the bottom of the tube;

[0079] ⑦ The solution in the tube was carefully poured out, and the precipitate at the bottom was retained. 500 μL of 75% ethanol was added to wash the precipitate. It was placed in a centrifuge with a temperature setting of 4℃ and a speed of 12000 rpm for 2 min;

[0080] 7. Repeat step 6, wash the precipitate again, pour off the ethanol, and air dry the EP tube containing the DNA precipitate at room temperature. Add 100 μL of lx TE to dissolve the DNA;

[0081] The purity and concentration of the DNA were detected by ultraviolet spectrophotometer. The extracted DNA was diluted to 50 ng / μL with ddH2O and stored at -20 °C.

[0082] Table 3 SSR-PCR reaction system

[0083]

[0084] Table 4 SSR-PCR amplification program

[0085]

[0086]

[0087] Table 5 16 pairs of polymorphic SSR markers

[0088]

[0089]

[0090] The 28 Ziziphus jujuba Mill. materials of 4 populations were clustered according to different geographical locations and genetic distances of the populations by UPGMA method using 16 pairs of polymorphic SSR primers shown in Table 5. The test materials were divided into 2 categories at genetic distance 0.15. Figure 4 The first category contained 21 sample materials, and at the genetic distance threshold of 0.17, the category was divided into 2 subcategories, in which the two populations of Yichun Yifeng and Ganzhou Chongyi were clustered into one category, and the population of Ganzhou Longnan was clustered into another category. In addition, the second category contained 7 sample materials, and the population of Nanjiaozhuo in Shangrao was clustered into one category.

[0091] The above-described embodiments are only to describe the preferred modes of the present application, and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements of the technical solutions of the present application made by those skilled in the art shall fall within the protection scope determined by the claims of the present application.

Claims

1. A primer combination for evaluating genetic diversity of Ziziphus mauritiana, characterized by, The primer combination comprises 16 pairs of primer pairs, and the correspondence of each primer pair is shown in the following table: 。 2. Use of the primer combination of claim 1 in the preparation of a kit for evaluating the genetic diversity of Ziziphus jujuba.

3. A kit for the evaluation of genetic diversity in Ziziphus mauritiana, characterized in that, The kit comprises the primer combination of claim 1.

4. Use of the primer combination of claim 1 or the kit of claim 3 in the evaluation of the genetic diversity of Ziziphus jujuba.

5. A method for evaluating genetic diversity of Ziziphus mauritiana characterized in that, The method comprises the following steps: (1) extracting the genomic DNA of Ziziphus jujuba; (2) performing PCR amplification using the primer combination of claim 1; (3) detecting the amplification product to obtain a test result; (4) using the test result of step (3) to analyze the genetic diversity, genetic differentiation or genetic structure of the population of Ziziphus jujuba.

6. The method of claim 5, wherein, In step (2), the reaction system of the PCR amplification is 2×Taq PCR Master Mix 12.5 μL, upstream primer 1 μL, downstream primer 1 μL, DNA template 1 μL, and ddH2O 9.5 μL.

7. The method of claim 5, wherein, In step (2), the reaction program of the PCR amplification is 94℃ 5min; 94℃ 30s, 63℃ 30s, 72℃ 45s, 10 cycles; 94℃ 30s, 55℃ 30s, 72℃ 45s, 20 cycles; 72℃ 7min.

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