Indel molecular marker primer combination for identifying different populations of sargassum and detection method and application of Indel molecular marker primer combination

Abstract

The invention provides an Indel molecular marker primer combination for identifying different populations of sargassum and a detection method and application of the Indel molecular marker primer combination, and belongs to the technical field of germplasm identification and genetic diversity analysis of macroalgae. The Indel molecular marker primer combination disclosed by the invention comprises an Indel-F1 / R1 and an Indel-F2 / R2, wherein the Indel molecular marker primer combination comprises an Indel molecular marker primer and an Indel molecular marker primer; the sequence of the Indel-F1 is as shown in SEQ ID NO. 1, and the sequence of the Indel-R1 is as shown in SEQ ID NO. 2; the sequence of the Indel-F2 is as shown in SEQ ID NO.3, and the sequence of the Indel-R2 is as shown in SEQ ID NO.4. The Indel molecular marker primer combination disclosed by the invention can be used for accurately identifying a creeping branch population and a guede population of a guede line and quickly distinguishing from other common sargassum, and has important significance in the aspects of resource protection, research and utilization of the sargassum.

Description

Technical Field

[0001] The present invention relates to the technical field of identification of germplasm and genetic diversity analysis of large seaweeds, and particularly relates to an Indel molecular marker primer combination for identifying different populations of Sargassum, a detection method and an application thereof. Background Art

[0002] Sargassum spp. belongs to Phaeophyta, Fucales, and is a type of large seaweed with important ecological and economic values. Sargassum is widely distributed in subtropical and tropical waters of the Pacific, Indian, and Atlantic Oceans. Different populations of Sargassum differ in growth habits, morphological characteristics, and genetic backgrounds. Understanding these differences is crucial for understanding the ecological adaptability, resource protection, research, and utilization of Sargassum.

[0003] Data on germplasm resource evaluation is of great significance for maintaining the genetic diversity of species. However, Sargassum has a wide variety and high morphological plasticity. Traditional morphological identification methods are affected by environmental conditions and developmental stages, making it difficult to accurately distinguish different populations, and the morphological identification is difficult. Coupled with limited genetic information of Sargassum and very few studies on molecular markers, there is currently no molecular marker in the genus Sargassum that can be used to distinguish different populations. Therefore, developing an efficient and accurate molecular marker method is of great significance for the protection, development, and utilization of Sargassum resources, as well as for better understanding the genetic diversity level, interspecific hybridization, and gene exchange among populations of Sargassum. Summary of the Invention

[0004] In view of this, the present invention provides an Indel molecular marker primer combination for identifying different populations of Sargassum, a detection method and an application thereof. The molecular marker primers provided by the present invention can be used to identify the creeping branch population and the Binder population of the Binder series of the genus Sargassum, and can be quickly distinguished from other common Sargassum.

[0005] To achieve the above invention purposes, the present invention provides the following technical solutions:

[0006] The present invention provides an Indel molecular marker primer combination for identifying different populations of Sargassum, including Indel-F1 / R1 and Indel-F2 / R2; the sequence of Indel-F1 is as shown in SEQ ID NO.1, and the sequence of Indel-R1 is as shown in SEQ ID NO.2; the sequence of Indel-F2 is as shown in SEQ ID NO.3, and the sequence of Indel-R2 is as shown in SEQ ID NO.4.

[0007] The present invention also provides a reagent or kit for identifying different populations of Sargassum, and the reagent or kit contains the above-mentioned Indel molecular marker primer combination.

[0008] The present invention also provides the use of the Indel molecular marker primer combination and / or the reagent or kit in at least one of the following:

[0009] (1) Application in identification of Sargassum populations;

[0010] (2) Application in the analysis of Sargassum diversity;

[0011] (3) Application in the protection of Sargassum germplasm resources.

[0012] The present invention also provides a method for identifying different populations of Sargassum, comprising the following steps:

[0013] S1. extracting genomic DNA of the Sargassum to be tested;

[0014] S2. Using the genomic DNA of the Sargassum to be tested as a template, PCR amplification is performed using the Indel-F1 / R1 to obtain an amplified product;

[0015] S3. Perform electrophoresis on the amplified product of S2. If a fragment of 188 bp is amplified, it is determined to be a creeping population; if a fragment of 287 bp is amplified, it is determined to be a Binde population of the Binde system; if a fragment of 311 bp is amplified, it is determined to be other populations;

[0016] S4. Using the genomic DNA of the Sargassum to be tested as a template, PCR amplification is performed using the Indel-F2 / R2 to obtain an amplified product;

[0017] S5. Perform electrophoresis on the amplified product described in S4. If a fragment of 188 bp is amplified, it is determined to be the Binde population of the Binde system; if a fragment larger than 300 bp is amplified, it is determined to be other populations.

[0018] Preferably, the reaction system of the PCR amplification in steps S2 and S4 is 20 μL: 1 μL of genomic DNA of the Sargassum to be tested, 0.5 μL of upstream primers, 0.5 μL of downstream primers, 10 μL of 2×PCRMix, and 8 μL of ddH2O.

[0019] Preferably, the reaction procedure of the PCR amplification in step S2 is: pre-denaturation at 94°C for 4 min; denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec, extension at 72°C for 30 sec, 35 cycles; and finally extension at 72°C for 10 min.

[0020] Preferably, the reaction procedure of the PCR amplification in step S4 is: pre-denaturation at 94°C for 4 min; denaturation at 94°C for 30 sec, annealing at 48°C for 30 sec, extension at 72°C for 30 sec, 35 cycles; and finally extension at 72°C for 10 min.

[0021] Preferably, the concentration of the genomic DNA of the Sargassum to be tested is 20 ng / μL, and the concentrations of the upstream primer and the downstream primer are 10 μM respectively.

[0022] Preferably, the electrophoresis in S3 and S5 is agarose gel electrophoresis, and the electrophoresis conditions are: 2% agarose gel, 150V voltage, and electrophoresis for 30 minutes.

[0023] By adopting the above technical solution, the present invention has the following beneficial effects: Compared with the traditional molecular marker method, the InDel molecular marker provided by the present invention has the following advantages:

[0024] 1. Stability: Ability to stably amplify PCR products from DNA of different Sargassum species for subsequent analysis.

[0025] 2. Accuracy: It can accurately identify creeping populations, binder populations of the binder system, and quickly distinguish them from other common Sargassum, and is not affected by environmental conditions and the individual development stage of Sargassum.

[0026] 3. Easy to operate: Sargassum has many species and great morphological plasticity, making morphological identification difficult. Using the InDel molecular marker combination provided by the present invention, population identification can be performed with simple PCR amplification and gel electrophoresis, and even researchers who lack rich morphological classification experience can quickly complete the operation. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The agarose electrophoresis diagram is a result of PCR amplification using primers Indel-F1 / R1 with genomic DNA of 32 Sargassum accessions as templates; M is a DNA marker, and the numbers 1-32 in the figure correspond to the Sargassum samples 1-32 in Table 1.

[0028] Figure 2 The agarose electrophoresis diagram is a result of PCR amplification using primers Indel-F2 / R2 with genomic DNA of 32 Sargassum accessions as templates; M is a DNA marker, and the numbers 1-32 in the figure correspond to the Sargassum samples 1-32 in Table 1. DETAILED DESCRIPTION

[0029] The technical solutions provided by the present invention will be described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention. The experimental methods in the embodiments are all conventional methods unless otherwise specified. The test materials used in the following embodiments are all commercially available products unless otherwise specified.

[0030] Example 1. Development of Indel molecular markers for identifying different populations of Sargassum

[0031] 1. Collection and taxonomic identification of Sargassum samples

[0032] In this example, 32 Sargassum resources in the South China Sea were collected, morphologically identified, and molecularly identified based on conventional DNA barcodes. The results of the collection and taxonomic identification of Sargassum germplasms are shown in Table 1.

[0033] Table 1 Sources and taxonomic identification of Sargassum germplasms

[0034]

[0035]

[0036] Through morphological and molecular identification, among the 32 Sargassum samples, there are 8 species in total. Among them, S. polycystum and S. granularium belong to the S. polycystum population, S. siliquastrum and S. binderi are the S. binderi population of the S. binderi series, and other Sargassum are represented by S. ilicifolium of the S. ilicifolium series, S. henslowianum of the S. carpophyllum series, and S. morii and S. cystophyllum of the Cystoseira section.

[0037] 2. Development of Indel molecular markers for identifying different populations of Sargassum

[0038] The genomic DNA of 32 Sargassum germplasms collected in the South China Sea was extracted using the optimized CTAB method. Using the universal primers of the conventional DNA barcode markers of Sargassum, the DNA of 32 samples was PCR amplified and sequenced, and combined with the known Sargassum sequence information in the GenBank database, sequence alignment analysis was carried out among species. One InDel interval with rich diversity located within ITS was screened out. According to the DNA fragment lengths of different species in this interval, different Sargassum populations can be divided into 3 types. Among them, S. polycystum and S. granularium of the S. polycystum population have a base deletion of about 120 bp in this Indel interval, S. siliquastrum and S. binderi of the S. binderi population have a base deletion of about 20 bp, and other Sargassum do not have large fragment deletions.

[0039] To obtain the sequence information of Sargassum within and flanking the Indel region, genomic DNA of the above 32 Sargassum samples was extracted for resequencing and sequence analysis. To compare with the sequences of more Sargassum species, Sargassum sequences with voucher specimens were retrieved and downloaded from NCBI. After screening, a total of 158 Sargassum ITS sequences were used for alignment analysis to find relatively conserved sites on both sides of the Indel region for designing upstream and downstream primers.

[0040] The nucleotide sequence of the upstream primer for amplifying Indel marker 1 is shown in SEQ ID NO.1, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.2.

[0041] Indel-F1: 5’-CAAGCCTAGAGAGYTACCGATTG-3’ (SEQ ID NO.1);

[0042] Indel-R1: 5’-TACCTACCGTCCYRAGCCTC-3’ (SEQ ID NO.2).

[0043] Furthermore, using e-PCR electronic amplification analysis, it was found that there were three fragment sizes in the Indel region of the analyzed Sargassum samples. The first fragment size was 188 bp, corresponding to the amplification product of Sargassum in the stoloniferous population; the second fragment size was 287 bp, corresponding to the amplification product of Sargassum binderi in the Binder series; the third fragment size was 311 bp, corresponding to the amplification products of other Sargassum.

[0044] Considering that the resolution of agarose gel is not particularly obvious for distinguishing DNA fragments with a size difference of about 20 bp, the mitochondrial genomes of 32 Sargassum samples were assembled using the resequencing data, and combined with the mitochondrial genome sequences of other Sargassum downloaded from NCBI. Primers for amplifying Indel marker 2 were designed based on their polymorphic sites and flanking conserved sequences to assist in distinguishing the products of the second and third fragment sizes in the above Indel marker 1.

[0045] The nucleotide sequence of the upstream primer for amplifying Indel marker 2 is shown in SEQ ID NO.3, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.4.

[0046] Indel-F2: 5’-TGGTGGGGGTTTAATGTAATA-3’ (SEQ ID NO.3);

[0047] Indel-R2: 5’-GTATTTGAAATTGTTCTAAAGGGC-3’ (SEQ ID NO.4).

[0048] The amplified fragment size of Sargassum binderi in the Binderi population is 188 bp, while that of Sargassum in other populations is greater than 300 bp. Since there is a deletion of more than 100 bp in the primer pair amplification interval of this marker for the Binderi population of Sargassum binderi, this marker can effectively distinguish Sargassum binderi from other Sargassum species.

[0049] Example 2. Application of Indel molecular marker primers

[0050] Using the genomic DNA of 32 Sargassum germplasms in Example 1 as a template, and using Indel-F1 and Indel-R1, or Indel-F2 and Indel-R2 as the upstream and downstream primers respectively for PCR amplification, a 20 μL PCR reaction system was prepared (Table 2):

[0051] Table 2. PCR reaction system

[0052]

[0053]

[0054] When amplifying with the primer pair Indel-F1 / R1, the PCR amplification reaction program was: pre-denaturation at 94 °C for 4 min; denaturation at 94 °C for 30 sec, annealing at 55 °C for 30 sec, extension at 72 °C for 30 sec, for 35 cycles; finally, extension at 72 °C for 10 min, and the amplified product was stored at 4 °C.

[0055] When amplifying with the primer pair Indel-F2 / R2, the PCR amplification reaction program was: pre-denaturation at 94 °C for 4 min; denaturation at 94 °C for 30 sec, annealing at 48 °C for 30 sec, extension at 72 °C for 30 sec, for 35 cycles; finally, extension at 72 °C for 10 min, and the amplified product was stored at 4 °C.

[0056] The PCR amplification products were detected using 2.0% agarose gel. The genomic DNA samples of Sargassum in the stoloniferous population were added to lanes 1 - 11, the genomic DNA samples of Sargassum binderi in the Binderi population were added to lanes 12 - 21, the genomic DNA samples of Sargassum ilicifolium were added to lanes 22 and 23, the genomic DNA samples of Sargassum henslowianum in the Caribica population were added to lanes 24 - 26, and the genomic DNA samples of Sargassum in the Cystoseira group were added to lanes 27 - 32. Electrophoresis was carried out at 150 V for 30 min and observed under ultraviolet light.

[0057] The agarose gel electrophoresis results of the genomic DNA of 32 Sargassum are as Figure 1 and Figure 2 shown. Figure 1It shows that among the PCR amplification products of Indel marker 1, Sargassum thunbergii of the stoloniferous population amplified a fragment of 188 bp in size, Sargassum binderi of the Binder population amplified a fragment of 287 bp in size, and other Sargassum thunbergii amplified a fragment of 311 bp in size.

[0058] Since the sizes of the PCR amplification products of the Binder series and other Sargassum thunbergii only differ by about 20 bp, it is difficult to clearly distinguish them during 2.0% agarose gel electrophoresis. Therefore, the resolution effect for Sargassum binderi and other Sargassum thunbergii is limited. To obtain more accurate identification results, the PCR amplification results of Indel marker 2 can be combined for judgment. Figure 2 It shows that for the PCR amplification products of Indel marker 2, the amplified fragment size of Sargassum binderi of the Binder population is 188 bp, which is more than 100 bp smaller than the amplification products of Sargassum thunbergii of the stoloniferous type and other Sargassum thunbergii.

[0059] Since Indel marker 1 and Indel marker 2 are respectively located in the ITS and the hypervariable region of the mitochondrial genome of the genus Sargassum, in addition to the size polymorphism of the inserted / deleted fragments in these two regions, there is also abundant nucleotide polymorphism at the sequence level (Table 3). Using the two pairs of specific primers of the present invention for PCR amplification of Sargassum samples and sequencing and analyzing their products, preliminary identification of the genetic relationship and genetic diversity analysis of Sargassum germplasm can be carried out.

[0060] Table 3 Comparison of polymorphisms of different DNA barcode markers in the genus Sargassum

[0061]

[0062]

[0063] As can be seen from the above embodiments, the present invention provides an Indel molecular marker primer combination for identifying different populations of Sargassum, as well as its detection method and application. The InDel molecular marker primer combination of the present invention for identifying or distinguishing Sargassum populations has high specificity and stability, and can accurately identify the stoloniferous population and the Binder population of the Binder series, and can be quickly distinguished from other common Sargassum thunbergii.

[0064] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. An Indel molecular marker primer combination for identifying different populations of Sargassum, characterized in that, Including Indel-F1 / R1 and Indel-F2 / R2; the sequence of Indel-F1 is shown as SEQ ID NO.1, the sequence of Indel-F1 is shown as SEQ ID NO.2; the sequence of Indel-F2 is shown as SEQ ID NO.3, and the sequence of Indel-R2 is shown as SEQ ID NO.

4.

2. A reagent or kit for identifying different populations of Sargassum, characterized in that, The reagent or kit comprises the Indel molecular marker primer combination according to claim 1.

3. Use of the Indel molecular marker primer combination according to claim 1 and / or the reagent or kit according to claim 2 in at least one of the following: (1) Application in identification of Sargassum populations; (2) Application in the analysis of Sargassum diversity; (3) Application in the protection of Sargassum germplasm resources.

4. A method for identifying different populations of Sargassum, characterized in that, The following steps are involved: S1. extracting genomic DNA of the Sargassum to be tested; S2. Using the genomic DNA of the Sargassum to be tested as a template, PCR amplification is performed using the Indel-F1 / R1 to obtain an amplified product; S3. Perform electrophoresis on the amplified product of S2. If a fragment of 188 bp is amplified, it is determined to be a creeping population; if a fragment of 287 bp is amplified, it is determined to be a Binde population of the Binde system; if a fragment of 311 bp is amplified, it is determined to be other populations; S4. Using the genomic DNA of the Sargassum to be tested as a template, PCR amplification is performed using the Indel-F2 / R2 to obtain an amplified product; S5. Perform electrophoresis on the amplified product described in S4. If a fragment of 188 bp is amplified, it is determined to be the Binde population of the Binde system; if a fragment larger than 300 bp is amplified, it is determined to be other populations.

5. The method according to claim 4, wherein The reaction system of the PCR amplification in steps S2 and S4 is 20 μL: 1 μL of genomic DNA of the Sargassum to be tested, 0.5 μL of upstream primers, 0.5 μL of downstream primers, 10 μL of 2×PCR Mix, and 8 μL of ddH2O.

6. The method according to claim 4, characterized in that The reaction procedure of the PCR amplification in step S2 is: pre-denaturation at 94°C for 4 min; denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec, extension at 72°C for 30 sec, 35 cycles; and finally extension at 72°C for 10 min.

7. The method according to claim 4, characterized in that, The reaction procedure of the PCR amplification in step S4 is: pre-denaturation at 94°C for 4 min; denaturation at 94°C for 30 sec, annealing at 48°C for 30 sec, extension at 72°C for 30 sec, 35 cycles; and finally extension at 72°C for 10 min.

8. The method according to claim 4, characterized in that, The concentration of the genomic DNA of the Sargassum to be tested is 10-20 ng / μL, and the concentrations of the upstream primer and the downstream primer are 10 μM respectively.

9. The method according to claim 8, characterized in that, The electrophoresis described in S3 and S5 is agarose gel electrophoresis, and the electrophoresis conditions are: 2.0% to 2.5% agarose gel, 150V voltage, and electrophoresis for 30 minutes.

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