A kind of microsatellite locus of Platygyra daedalea and its application

Abstract

The present invention belongs to the technical field of molecular biology, and specifically discloses microsatellite loci of Platygyra daedalea. The microsatellite loci are one or more of JQ1026, JQ1086, JQ1610 and JQ55, and the nucleotide sequences of JQ1026, JQ1086, JQ1610 and JQ55 are SEQ ID NO.1-SEQ ID NO.4 respectively. The present invention also discloses the application of the above-mentioned microsatellite loci of Platygyra daedalea in population genetic structure analysis, paternity testing and / or molecular marker-assisted breeding.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology, and particularly relates to a microsatellite locus of Platygyra daedalea and its application. Background Art

[0002] Microsatellite DNA, also known as short tandem repeats (STRs), simple sequence repeats (SSRs), and simple sequence length polymorphisms (SSLP), refers to nucleotide sequences in the genome that are tandemly composed of 1-6 nucleotides as units. Microsatellite markers have the advantages of wide distribution, high polymorphism information content, co-dominance, compliance with Mendelian segregation laws, easy PCR amplification, and good reproducibility, and have been widely used in the fields of population genetic structure analysis, paternity testing, genetic linkage map construction, functional gene mapping, and genetic breeding of marine organisms.

[0003] Coral reef ecosystems have always been regarded as one of the ecosystems with the highest biodiversity in the world, with extremely important ecological, economic, and cultural values, providing habitats for at least a quarter of known marine species, and having the function of preventing land loss. Platygyra daedalea (Ellis & Solander, 1786) belongs to the order Scleractinia, family Faviidae, and genus Platygyra, and is widely distributed in the Indo-Pacific region and the South China Sea of China. The coral skeleton is convex or sub-spherical, or columnar-block-shaped, with many long valleys and fewer short valleys. The long valleys reach 80 mm, and the short valleys are only 5 mm.

[0004] At present, coral reefs have seriously degraded globally, and the same serious degradation trend has also occurred in China's coral reefs. In the past 30 years, the inshore coral reefs have degraded by at least 80%. The connectivity between Platygyra daedalea populations is an important basis for implementing appropriate protection and restoration measures. In order to protect the wild germplasm resources of Platygyra daedalea and formulate corresponding protection strategies, it is very urgent to carry out relevant work such as population genetic diversity analysis and population genetic structure analysis on it. Summary of the Invention

[0005] The purpose of the present invention is to provide a microsatellite locus of Platygyra daedalea and its application to solve the above technical problems.

[0006] To achieve the above purpose, one of the purposes of the present invention is to provide a microsatellite locus of Platygyra daedalea, and the microsatellite locus is one or more of JQ1026, JQ1086, JQ1610, and JQ55. The nucleotide sequences of JQ1026, JQ1086, JQ1610, and JQ55 are SEQ ID NO.1-SEQ ID NO.4 respectively.

[0007] The second object of the present invention is to provide the application of the above-mentioned microsatellite loci of Turbinaria elegans in population genetic structure analysis, paternity testing, and / or molecular marker-assisted breeding.

[0008] Preferably, the nucleotide sequences of the primer pairs for amplifying the JQ1026 microsatellite locus are SEQ ID NO.5 and SEQ ID NO.6.

[0009] Preferably, the nucleotide sequences of the primer pairs for amplifying the JQ1086 microsatellite locus are SEQ ID NO.7 and SEQ ID NO.8.

[0010] Preferably, the nucleotide sequences of the primer pairs for amplifying the JQ1610 microsatellite locus are SEQ ID NO.9 and SEQ ID NO.10.

[0011] Preferably, the nucleotide sequences of the primer pairs for amplifying the JQ55 microsatellite locus are SEQ ID NO.11 and SEQ ID NO.12.

[0012] Advantages of the present invention:

[0013] The four novel microsatellite loci of Turbinaria elegans provided by the present invention are respectively named JQ1026, JQ1086, JQ1610, and JQ55, and the nucleotide sequences are SEQ ID NO.1 - SEQ ID NO.4 respectively.

[0014] The present invention provides four novel microsatellite loci of Turbinaria elegans and primer sequences (SEQ ID NO.5 - SEQ ID NO.12) for amplifying these four microsatellite loci, which can be applied to the research in the fields of population genetic structure analysis, paternity testing, and molecular marker-assisted breeding of Turbinaria elegans, with good repeatability and being a reliable and effective molecular marker. Description of the Drawings

[0015] Figure 1 and Figure 2 are capillary electrophoresis signal maps of four individuals of Turbinaria elegans amplified by specific primers for the JQ1026 locus;

[0016] Figure 3 and Figure 4 are capillary electrophoresis signal maps of four individuals of Turbinaria elegans amplified by specific primers for the JQ1086 locus;

[0017] Figure 5 and Figure 6 are capillary electrophoresis signal maps of four individuals of Turbinaria elegans amplified by specific primers for the JQ1610 locus;

[0018] Figure 7 and Figure 8Capillary electrophoresis signal maps of four individuals of *Platygyra daedalea* amplified with specific primers for the JQ55 locus. Detailed implementation manners

[0019] The following is a further detailed description through specific implementation manners:

[0020] Examples

[0021] S1. RAD sequencing analysis:

[0022] After the living *Platygyra daedalea* is induced to be albino by high temperature, it is sent to Guangzhou Gene Denovo Biotechnology Co., Ltd. for genomic DNA extraction, enzyme digestion, end repair, A-tailing and adapter addition, fragment selection, PCR amplification and sequencing on the machine.

[0023] The specific steps are as follows. (1) DNA extraction: Genomic DNA is extracted using the CTAB (cetyltrimethylammonium bromide) method. The quality of the DNA is detected using Qubit (Thermo Fisher Scientific, Waltham, MA) and Nanodrop (Thermo Fisher Scientific, Waltham, MA).

[0024] (2) The genomic DNA with qualified quality inspection is digested by a restriction endonuclease, physically broken into sequences of 300 - 700 bp, then end repaired, A-tailed, and ΜLtra TM the Illumina sequencing adapter is added using the DNA Library Prep Kit library (NEB, USA).

[0025] The DNA fragments of 300 - 400 bp are amplified and enriched by PCR. Finally, the PCR products are purified using the AMPure XP system (Beckman Coulter, Brea, CA, USA), the sequencing library is detected using an Agilent 2100 bioanalyzer (Agilent, Santa Clara, CA), and the library is quantified using real-time PCR. The sequencing is carried out on a Novaseq 6000 sequencer using the PE 150 sequencing strategy.

[0026] (3) The raw data of the Illumina platform is filtered using FASTP (version 0.18.0), and the filtering criteria are as follows:

[0027] ① Remove reads containing ≥ 10% unknown nucleotides (N);

[0028] ② Remove reads with ≥ 50% bases having a phred quality score ≤ 20;

[0029] ③Delete the reads containing adapters. The filtered clean reads are used for assembly analysis.

[0030] (4) Use stack (version 1.46) to first perform separate clustering on the read1 data of all individuals to obtain individual stacks, and then cluster the stacks among individuals to obtain a set of population stacks. Classify read2 according to the read1 clustering results, and then splice read2. Connect the stack sequences obtained by splicing read1 and the contigs obtained by splicing read2 to construct RAD-tags. The RAD-tags will be used as reference sequences for subsequent variant detection and advanced analysis.

[0031] (5) Use the software MISA (http: / / pgrc.ipk-gatersleben.de / misa / ) to search all RAD-tags to find SSRs in the RAD-tags. After obtaining the SSRs in each sample, obtain polymorphic microsatellite loci.

[0032] Polymorphic microsatellite loci, that is, polymorphic SSRs, refer to SSRs that are different among different individuals and can be used for molecular marker development. Take the intersection of the obtained SSR results and InDel results, and select the SSRs whose sequences within the SSR intervals have changed, which are the polymorphic microsatellite loci.

[0033] Configuration parameter information:

[0034] definition(unit_size,min_repeats):2-6 3-5 4-4 5-4 6-4;

[0035] interruptions(max_difference_between_2_SSRs):100.

[0036] S2. Screen primers

[0037] Select one piece of Acropora tenuis from Sanya and Wenchang respectively, which are far apart geographically, and two pieces of corals from the Xisha Islands, and extract the genomic DNA of 4 individuals of Acropora tenuis, and then perform RCR amplification. The amplification reaction system is 25 μL.

[0038] The PCR system is: PCR Master Mix (2×) 12.5 μL, forward primer 10 μmol / L 0.2 μL, reverse primer 10 μmol / L 0.6 μL, FAM-labeled M13 primer 10 μmol / L 0.4 μL, template DNA < 100 ng, and finally make up to 25 μL with ddH2O.

[0039] The reaction procedure was as follows: 95°C for 2 min; 30 cycles: denaturation at 95°C for 20 s, annealing at 55°C for 20 s, extension at 72°C for 20 s; 8 cycles: denaturation at 95°C for 20 s, annealing at 53°C for 20 s, extension at 72°C for 30 s; final extension at 72°C for 5 min.

[0040] Allele typing was performed using an ABI 3130 genetic analyzer and Gene Mapper software 3.7 (Applied Biosystems). A total of four polymorphic microsatellite loci were obtained. The specific experimental results are as Figures 1 - 8 shown.

[0041] Figure 1 and Figure 2 are the capillary electrophoresis signal maps of JQ1026.

[0042] Figure 3 and Figure 4 are the capillary electrophoresis signal maps of JQ1086.

[0043] Figure 5 and Figure 6 are the capillary electrophoresis signal maps of JQ1610.

[0044] Figure 7 and Figure 8 are the capillary electrophoresis signal maps of JQ55.

[0045] It can be seen from Figures 1 - 8 that the lengths of the four microsatellite loci obtained in this example showed polymorphisms in all four tested Acropora tenuis. They have the advantages of high amplification stability and strong specificity. Thus, these four microsatellite loci can be used for the evaluation of germplasm resources and the construction of genetic linkage maps of Acropora tenuis in the future.

[0046] The polymorphic microsatellite loci and their primers obtained are shown in the following table:

[0047]

[0048]

[0049] The preferred specific embodiments of the present invention have been described in detail above. It should be understood that those of ordinary skill in the art can make many modifications and variations based on the concept of the present invention without creative work. Therefore, all technical solutions that can be obtained by those skilled in the art in the technical field of the present invention through logical analysis, reasoning, or limited experiments based on the concept of the present invention on the basis of the prior art should be within the protection scope determined by the claims. Sequence Listing <110>Hainan Academy of Ocean and Fisheries Sciences <120>A kind of microsatellite locus of Platygyra daedalea and its application <160>12 <170>Patentln version 3.5 <210>1 <211> 563 <212>DNA <213>Platygyra daedalea <400>1 tacacgactt tggaaaacaa aacacctgcg gaaacgagat ttaagcgagt tttgatatat 60 ttgccctacg gtgtcataat aaattattat acagcccaga atataccttg caacaggtcg 120 ttttgtcgct gcttagatgc atgtaagagc ggtgcaaagg atgaccatgt ttcgtccctc 180 gattttggca gacgtttttg cttttttaaa gcaacgagcc agaacgctat atatctatta 240 ccaccgtttt cggagtgtag gttttgtcga gggtcaaggg tcgagggtga catatcgagg 300 gtcgagggtc gatggtcgag ggtcgagggt cgagggtggt aaaaaagtta aaaagttaat 360 atcctatcct atattaaatt atcaaaatat ataacgctaa tcgcgcggtt tgtctcgcat 420 tgcgcttgtt attatgtaag cgtttgaatt tattcttctc ttttcttttc ttttcttttc 480 tttgtttcta accatacgga aaagagagta gagcgtttag gatacgacgt tgccttcaat 540 ccgcgacaaa aatataaggg acg 563 <210>2 <211>468 <212>DNA <213>Platygyra daedalea <400>2 aattcaaagt actgttgaat aaaagaaggg actacggact ggattgctga gggaaagaac 60 tcttaaaata ggtctgtttt ttaaattcat tattggtaac ctttttgttt tgttttgttt 120 tgttttggtg ctttcaattg caaatacgag ggggaatgcg aaccgtattt tttatttcaa 180 aaatcgaatc ttaaatgcat aagttacacc tgcaaaaatg cttggtaatt tcattaaatg 240 taagaggtct tagagatgcg gttaagagaa ggagcatttt cacgtatctg aaagatcagg 300 aggccaattt ttattttcta caagaaacgt tttccaaagt aagtgacgaa gccgtttgga 360 gaaatgaatg gggaggtgaa atttactttt ctcacgggac ttctcatagc aaaggtgttt 420 gtattttgat taatcgagcg ataaaagaaa aagtgacctt cacatcca 468 <210>3 <211>142 <212>DNA <213>Platygyra daedalea <400>3 aattcaactt ttggcactta agaaatgtag gaggtcttta aaagggccaa tgtcaatgtg 60 agagtagatt ttgaaaacta actacagaat atacaccaac caaccaacca acaaatcagg 120 tttgcatcat atcttttaat at 142 <210>4 <211>511 <212>DNA <213>Platygyra daedalea <400>4 tttaatacag aatcaccctt cactgagtga aatatacaga gatcctccca tcatctcgta 60 caaaaggggc aaatcgctca aggacatact cgttagatct aaactctaga ggcaaagaat 120 ccgcacttgc gaacagcggg agacttgcga acagcgggag tcgtgtagtc ctgtcacaac 180 tttttattaa tgagtaaatg acgtcatttt ctctagatcc agccctctga ggtctaatca 240 aatattgtaa catgagatat gtacttttta tcttgtgaag aaataaagac catcatcatc 300 atcatcataa tcggtctgtt ttgaacgtga gtaatcgcgg aacttgaaat ccaaaactta 360 cactcaaaat aaacagtgta ggcctgtcac aactttttac tattgcttat gccacgacga 420 gttgtgaaat atcacggggt ctagaactga aaaacccatc tatgtggcag gaagagagcg 480 caatggtacc aactttttat atcatctttt g 511 <210>5 <211>20 <212>DNA <213>Platygyra daedalea <400>5 atttttgtcg cggattgaag 20 <210>6 <211>20 <212>DNA <213>Platygyra daedalea <400>6 tcgcattgcg cttgttatta 20 <210>7 <211>20 <212>DNA <213>Platygyra daedalea <400>7 attggcctcc tgatctttca 20 <210>8 <211>20 <212>DNA <213>Platygyra daedalea <400>8 gactggattg ctgagggaaa 20 <210>9 <211>24 <212>DNA <213>Platygyra daedalea <400>9 ttaaaagata tgatgcaaac ctga 24 <210>10 <211>20 <212>DNA <213>Platygyra daedalea <400>10 ggaggtcttt aaaagggcca 20 <210>11 <211>20 <212>DNA <213>Platygyra daedalea <400>11 ttttggattt caagttccgc 20 <210>12 <211>20 <212>DNA <213>Platygyra daedalea <400>12 cgggagtcgt gtagtcctgt 20

Claims

1. A primer pair for detecting microsatellite loci of Platygyra daedalea, characterized in that, The microsatellite loci are JQ1026, JQ1086, JQ1610 and JQ55, and the nucleotide sequences of JQ1026, JQ1086, JQ1610 and JQ55 are SEQ ID NO.1 - SEQ ID NO.4 respectively; the nucleotide sequences of the primer pairs for amplifying the JQ1026 microsatellite locus are SEQ ID NO.5 and SEQ ID NO.6; the nucleotide sequences of the primer pairs for amplifying the JQ1086 microsatellite locus are SEQ ID NO.7 and SEQ ID NO.8; the nucleotide sequences of the primer pairs for amplifying the JQ1610 microsatellite locus are SEQ ID NO.9 and SEQ ID NO.10; the nucleotide sequences of the primer pairs for amplifying the JQ55 microsatellite locus are SEQ ID NO.11 and SEQ ID NO.

12.

2. To detect the application of the primer pairs described in claim 1 in the analysis of the population genetic structure or paternity testing of Platygyra daedalea.

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