SSR (Simple Sequence Repeat) molecular marker primer group for oryzias chinensis and application

Abstract

The invention discloses an oryzias chinensis SSR molecular marker primer group and application, and belongs to the technical field of molecular biology. The oryzias chinensis SSR molecular marker primer group provided by the invention comprises 10 pairs of primers, and the primer sequences of the primers are sequentially shown as SEQ ID NO.1-20. The oryzias chinensis SSR molecular marker primer group can be used for polymorphism analysis of an oryzias chinensis population in combination with a PCR amplification technology, and a basis is provided for construction of a genetic structure and a genetic map of the oryzias chinensis population.

Description

Technical Field

[0001] This invention relates to a molecular marker primer set for SSR of the Chinese medaka and its application, belonging to the field of molecular biology technology. Background Technology

[0002] Chinese Blue Medaka ( Oryzias sinensis The mosquitofish (Siniperca muscatus) is a typical, widespread fish species in my country, extending north to the Korean Peninsula. The type locality is near Kunming, Yunnan Province. This species possesses rich genetic diversity and is an important model organism and subject of genetic research. Studying its genetic diversity is crucial for understanding species evolution, ecological adaptation, and biodiversity conservation. However, in recent years, due to the invasive species *Siniperca muscatus* (…), it has become a significant problem. Gambusia affinis The invasion of [unspecified species] has severely damaged the natural habitat of the Chinese medaka, significantly shrinking its distribution range and threatening some populations with extinction. Therefore, developing efficient molecular marker tools is crucial for monitoring the genetic diversity of the Chinese medaka, assessing population health, and formulating conservation strategies. This invention aims to address the lack of efficient and specific molecular marker tools for the Chinese medaka in existing technologies. It provides an SSR molecular marker primer set that can be effectively used for genetic diversity analysis, population genetic structure research, and genetic mapping of the Chinese medaka. Based on this primer set, an application method is provided to rapidly and accurately assess the genetic structure of Chinese medaka populations, providing technical support for conservation biology research and population management of the Chinese medaka. Summary of the Invention

[0003] To address the lack of efficient and specific molecular marker tools for *Pygmy jalapa* in existing technologies, one objective of this invention is to provide a primer set for *Pygmy jalapa* SSR molecular markers. This primer set can be effectively used for genetic diversity analysis, population genetic structure research, and genetic map construction of *Pygmy jalapa*. The *Pygmy jalapa* SSR molecular marker primer set comprises the following 10 primer pairs, the nucleotide sequences of which are shown below: The forward primer for OsSSR001 is shown in SEQ ID NO.1, and the reverse primer is shown in SEQ ID NO.2.

[0004] The forward primer for OsSSR002 is shown in SEQ ID NO.3, and the reverse primer is shown in SEQ ID NO.4.

[0005] The forward primer for OsSSR005 is shown in SEQ ID NO.5, and the reverse primer is shown in SEQ ID NO.6.

[0006] The forward primer for OsSSR035 is shown in SEQ ID NO.7, and the reverse primer is shown in SEQ ID NO.8.

[0007] The forward primer of OsSSR038 is shown as SEQ ID NO. 9, and the reverse primer is shown as SEQ ID NO. 10.

[0008] The forward primer of OsSSR062 is shown as SEQ ID NO. 11, and the reverse primer is shown as SEQ ID NO. 12.

[0009] The forward primer of OsSSR066 is shown as SEQ ID NO. 13, and the reverse primer is shown as SEQ ID NO. 14.

[0010] The forward primer of OsSSR070 is shown as SEQ ID NO. 15, and the reverse primer is shown as SEQ ID NO. 16.

[0011] The forward primer of OsSSR087 is shown as SEQ ID NO. 17, and the reverse primer is shown as SEQ ID NO. 18.

[0012] The forward primer of OsSSR088 is shown as SEQ ID NO. 19, and the reverse primer is shown as SEQ ID NO. 20.

[0013] Another object of the present application is to provide an application method based on the above-mentioned SSR molecular marker primer set, which is used for quickly and accurately evaluating the genetic diversity, genetic structure, constructing genetic map or polymorphism analysis of Oryzias sinensis population, and providing technical support for the protection biology research and population management of Oryzias sinensis.

[0014] Advantages of the present application (1) The SSR molecular marker primer set provided by the present application has high amplification efficiency and specificity after strict screening and verification, and can effectively distinguish the genetic differences of different individuals and populations of Oryzias sinensis.

[0015] (2) The primer set can be used in various application scenarios, including but not limited to population genetic structure analysis, genetic map construction, kinship identification, and population genetic diversity evaluation.

[0016] (3) Combined with conventional PCR amplification technology, the operation process of the present application is simple, the experimental cost is low, and it is suitable for genetic analysis of large-scale samples. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 UPGMA clustering results of 39 samples based on genetic distance.

[0018] Figure 2 Gene function annotation of transcript data.

[0019] Figure 3 GO function classification diagram of Oryzias sinensis gene Figure 4 KEGG pathway of O. latipes.

[0020] Figure 5 Statistical chart of SSR type.

[0021] Figure 6 UPGMA clustering results of 39 samples based on genetic distance. DETAILED DESCRIPTION

[0022] The technical solutions of the present application will be further described below in combination with the drawings and through specific embodiments. However, the following embodiments are only simple examples of the present application, and do not represent or limit the protection scope of the present application, and the protection scope of the present application is subject to the claims.

[0023] Example 1 Development of O. latipes SSR molecular marker primer set The transcriptome data of O. latipes was obtained by using Illumina sequencing platform. The original image data file obtained by sequencing was converted into sequence raw data by CASAVA base recognition analysis, and the reads with low quality, adapter contamination and high content of unknown base N were removed. Clean reads were de novo assembled by using Trinity. The transcripts were annotated by using Blast and diamond software for NT, NR, COG, KEGG and SwissProt, and GO annotation was performed by using Blast2GO and NR annotation results.

[0024] After strict filtering and quality control, a total of 48,367,500 clean reads, 7,176,400,050 nucleotides and GC content of 51.62% were obtained, and the average sequence length was 149 bp. Q20 and Q30 were 97.84% and 94.09%, respectively (Table 1). De novo assembly was performed by using Trinity software, and a total of 80494 transcripts were obtained, with an average length of 1069 bp, and N50 and N90 values of 2436 bp and 361 bp, respectively. The smallest fragment was 152 bp, and the longest fragment was 9327 bp. There were 25620 unigenes with a length greater than 1000 bp, accounting for 31.8% of the total number of transcripts ( Figure 1 ).

[0025] Table 1 Related results of O. latipes transcriptome data The transcript data was annotated for gene function by using six databases (NR, NT, GO, COG, KEGG, Swissprot) Figure 2). A total of 80494 unigenes were annotated, with an annotation rate of 100%, and the number of genes annotated by each database was 46389 (Nr: 57.63%), 79669 (Nt: 98.98%), 34916 (Swissprot: 43.38%), 38192 (KEGG: 47.45%), 7649 (COG: 9.50%), and 18579 (GO: 23.08%).

[0026] By aligning and annotating with the Nr library, it was found that the O. latipes gene sequences had high homology with O. latipes Oryzias latipes . Specifically, 36463 (78.6%) genes were annotated for O. latipes, 1128 (2.43%) genes were annotated for A. pulchellus Stegastes partitus , 1107 (2.39%) genes were annotated for P. olivaceus Larimichthys crocea , and 603 (1.30%) and 520 (1.12%) genes were annotated for O. niloticus Oreochromis niloticus and A. lineatus Austrofundulus limnaeus , respectively.

[0027] After GO annotation of the genes, the successfully annotated genes were classified according to the next layer of the three categories, and the results are shown in Figure 3 . Among biological processes, the largest group consisted of genes of cellular processes (9957; 53.59%), followed by metabolic processes (8088; 43.53%). Among cell components, the largest group consisted of genes of cellular structures (10655; 57.34%), followed by genes related to intracellular components (3401; 18.3%). Among molecular functions, the largest group consisted of genes related to binding (9483; 51%), followed by genes related to catalytic activity (6444; 34.68%).

[0028] For the KEGG database, 38192 genes were classified into six categories, including 43 pathways. The largest category was metabolism (19489; 51.02%), followed by human diseases (9986; 26.15%), biological systems (15934, 41.72%), environmental information processing (9353, 24.49%), cellular processes (8230, 21.54%), and genetic information processing (3253; 8.51%) ( Figure 4 ).

[0029] A total of 11494 SSRs were identified in 8979 transcripts, with a frequency of 11.15% and an occurrence frequency of 14.28%. Among them, 1865 genes had more than one SSR site, and 663 were identified as compound microsatellites (Table 1). The most common type was single nucleotide repeat (4210, 36.63%), followed by trinucleotide (3933, 34.22%) and dinucleotide (2840, 24.71%); the proportions of tetranucleotide, pentanucleotide and hexanucleotide were smaller, accounting for 2.71%, 1.26% and 0.5%, respectively Figure 5 ).

[0030] Among the single nucleotide repeat types, A / T had the highest occurrence frequency, with a total of 3301 occurrences, accounting for 78.41% of the total number of single nucleotides; the most common type in dinucleotide was AC / GT, accounting for 70.32% of the total; the most common type in trinucleotide was AGG / CCT, accounting for 35.98%. In addition, the repeat number of SSR in O. latipes mainly concentrated in 5-39 times, among which the repeat number of single nucleotide mainly concentrated in 12-25 times, the repeat number of dinucleotide mainly concentrated in 6-12 times, and the repeat number of trinucleotide mainly concentrated in 5-8 times.

[0031] MISA was used to detect SSR in Unigenes, and sites with repeat units of 2, 3, 4, and 5 bases and repeat numbers of 4 or more were selected. Primer3 software was used to design and synthesize 100 pairs of initial screening primers according to the SSR primer selection principles. The primers were subjected to fluorescence PCR amplification, and the amplification products were detected and analyzed using Genemapper software to analyze the polymorphism of each primer, and finally 10 pairs of primers with high polymorphism were selected for subsequent analysis.

[0032] Table 2 SSR primer information of O. latipes Example 2 Polymorphism of SSR primers of O. latipes Liquid nitrogen storage. The 39 samples of O. latipes used for genetic diversity analysis were collected from Fuxian Lake (24°21' N, 102°49' E) in August 2022 and fixed with absolute ethanol.

[0033] Genomic DNA was extracted from 39 individuals of the Chinese medaka using the TSINGKE Animal DNA Extraction Kit. PCR amplification was performed using 10 pairs of SSR primers selected from the above screening. The total PCR reaction volume was 20 µL, containing 50 ng of template genomic DNA, with other components added according to the Mixtaq enzyme instructions. PCR conditions were: 98℃ for 2 min; 98℃ for 10 s, TM value 10 s, 72℃ for 10 s, 35 cycles; 72℃ for 2 min; stored at 4℃. PCR products were validated by capillary electrophoresis, and the electrophoretic patterns were analyzed using Genemapper 4.1 software. Popgene32 and GenAlEx version 6.501 software were used to calculate various genetic diversity indicators for SSR loci and populations, including observed alleles (Na), average effective allele count (Ne), Shannon information index (I), polymorphism information content (PIC), observed heterozygosity (Ho), and expected heterozygosity (He). The results are shown in Table 3. Table 3 shows that the minimum number of alleles is 2 and the maximum number of alleles is 7. Os SSR035, with an average of 3.0000 alleles per locus. The total number of valid alleles was 23.5904, with a range of 1.7717. Os SSR066) -3.6962 ( Os SSR088), with an average of 2.3590 effective alleles per locus. The Shannon index (I) ranged from 0.6273 ( Os SSR066)-1.4353 ( Os SSR035), with an average value of 0.8848. The polymorphic information content (PIC) ranges from 0.3407 ( Os SSR066) -0.6797 ( Os SSR088), with an average value of 0.4582, and all 10 primer pairs showed high polymorphism information (PIC>0.25). In summary, the 10 EST-SSR primer pairs developed in this experiment exhibited high polymorphism and can be applied to related studies on *Meretrix sinensis*. The observed heterozygosity (Ho) and expected heterozygosity (He) ranged from 0.1538 (…). Os SSR062) -0.7692 Os SSR005) and 0.4412 ( Os SSR066) -0.7389 ( Os SSR088), with means of 0.4256 and 0.5555 respectively. The Hardy-Weinberg equilibrium test results show that, except for... Os SSR001 Os SSR005 OsSSR070 and Os SSR087, all of which deviated from Hardy-Weinberg equilibrium P <0.05).

[0034] Table 3 Genetic diversity analysis of O. sinensis population Note: Na: observed alleles, Ne: effective alleles, I: Shannon index, PIC: polymorphic information content, Ho: observed heterozygosity, He: expected heterozygosity, P HWE : Hardy-Weinberg equilibrium. P <0.05, **: P <0.01, ***: P <0.001, ns: P>0.05.

[0035] Based on genetic distance, the clustering tree of individuals was established by unweighted group method with arithmetic means (UPGMA). The specific method was as follows: the UPGMA tree was constructed in the populations-1_2_30 software, and the numerical setting was 1000. The beautification and editing of the clustering tree were performed in the software FigTree version 1.4.2. According to Nei's genetic distance, 39 individuals were subjected to UPGMA clustering analysis, and the dendrogram showed that all individuals were divided into two branches at a genetic distance of about 0.28 Figure 6 Through clustering analysis, the genetic similarity and difference between different individuals could be clearly observed, which provided direct evidence for the study of the population genetic structure of O. sinensis.

Claims

1. A set of SSR molecular marker primers for Oryzias latipes (Japanese rice fish) (Oryzias latipes) characterized by: Oryzias sinensis ) SSR molecular marker primer set for Oryzias latipes (Japanese rice fish) (Oryzias latipes) characterized by: The O. sinensis SSR molecular marker primer set consists of 10 pairs of primers, and the nucleotide sequences of the primers are as follows: The forward primer of OsSSR001 is shown as SEQ ID NO. 1, and the reverse primer is shown as SEQ ID NO. 2; The forward primer of OsSSR002 is shown as SEQ ID NO. 3, and the reverse primer is shown as SEQ ID NO. 4; The forward primer of OsSSR005 is shown as SEQ ID NO. 5, and the reverse primer is shown as SEQ ID NO. 6; The forward primer of OsSSR035 is shown as SEQ ID NO. 7, and the reverse primer is shown as SEQ ID NO. 8; The forward primer of OsSSR038 is shown as SEQ ID NO. 9, and the reverse primer is shown as SEQ ID NO. 10; The forward primer of OsSSR062 is shown as SEQ ID NO. 11, and the reverse primer is shown as SEQ ID NO. 12; The forward primer of OsSSR066 is shown as SEQ ID NO. 13, and the reverse primer is shown as SEQ ID NO. 14; The forward primer of OsSSR070 is shown as SEQ ID NO. 15, and the reverse primer is shown as SEQ ID NO. 16; The forward primer of OsSSR087 is shown as SEQ ID NO. 17, and the reverse primer is shown as SEQ ID NO. 18; The forward primer of OsSSR088 is shown as SEQ ID NO. 19, and the reverse primer is shown as SEQ ID NO.

20.

2. The O. sinensis SSR molecular marker primer set of claim 1 is applied in O. sinensis population polymorphism analysis or genetic diversity analysis.

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