DNA fingerprint spectrum of penaeus monodon and construction method of DNA fingerprint spectrum

Abstract

The invention discloses a penaeus monodon DNA (deoxyribonucleic acid) fingerprint spectrum which comprises at least 209 specific SNP (single nucleotide polymorphism) sites and realizes efficient and accurate distinguishing of different culture populations, so that technical guarantee is provided for identification and traceability work of penaeus monodon germplasm, and meanwhile, an accurate molecular technology platform for germplasm resource development, utilization and protection is built. The invention further discloses a construction method of the DNA fingerprint spectrum of the penaeus monodon and application of the DNA fingerprint spectrum of the penaeus monodon in identification of germplasm resources of the penaeus monodon and assistant breeding.

Description

Technical Field

[0001] This invention belongs to the field of aquatic genetic breeding and molecular marker technology, specifically relating to a DNA fingerprint of Penaeus monodon and its construction method, and particularly to a Penaeus monodon DNA fingerprint constructed based on whole-genome single nucleotide polymorphism (SNP) markers. Penaeus monodon DNA fingerprinting and its construction method. Background Technology

[0002] The tiger prawn (Litopenaeus monodon) holds unique value in meeting human nutritional needs due to its large size, rapid growth, and stable market price. As the world's second-largest farmed shrimp species, the tiger prawn began to take shape at the end of the 20th century. In recent years, its production as a percentage of marine farmed shrimp has been steadily increasing, rising from 5.8% in 2019 to 7.9% in 2023. However, with the rapid development of the global shrimp farming industry, tiger prawn production is far from meeting contemporary demand. Furthermore, the use of inbreeding and other unhealthy breeding methods by farmers has significantly reduced the quality of tiger prawns on the market. Therefore, breeding tiger prawns with high yield, good quality, and strong environmental adaptability has become an urgent problem for the industry.

[0003] However, current technologies still lack molecular tools for the efficient and accurate differentiation and identification of different geographical and breeding populations of *Litopenaeus monodon*. Existing research indicates that after years of breeding, the main farmed *Litopenaeus monodon* populations in my country have begun to show low genetic diversity, and the genetic structure and degree of differentiation among different breeding populations remain unclear. Traditional morphological markers are easily affected by the farming environment and developmental stage, resulting in insufficient stability and reproducibility. With the maturity of high-throughput sequencing technology, molecular marker technology based on single nucleotide polymorphisms (SNPs) has been widely used in the identification of plant and animal species and population genetic analysis due to its wide distribution in the genome, high detection throughput, and stable results, highlighting its unique value. DNA fingerprinting is a modern technology method established based on such molecular markers, enabling precise classification and identification of germplasm resources. Currently, this technology has been widely used in the assessment of genetic diversity and identification of aquaculture species; however, it has not yet been applied to the germplasm resource research of *Litopenaeus monodon*.

[0004] Therefore, developing novel molecular marker systems (such as SNP chips) based on high-throughput sequencing technology and covering the entire genome level, and constructing a stable, specific, and digitizable DNA fingerprint map of Penaeus monodon, is of great theoretical and practical significance for filling the gap in Penaeus monodon population identification technology, realizing precise management of Penaeus monodon germplasm resources, scientific and efficient breeding planning, and even the sustainable development of the entire industry. Summary of the Invention

[0005] The purpose of this invention is to provide a DNA fingerprint of Penaeus monodon and its construction method, so as to achieve efficient and accurate differentiation between different farming populations, thereby providing technical support for the identification and traceability of Penaeus monodon germplasm.

[0006] To achieve the above objectives, the present invention adopts the following technical solution.

[0007] In a first aspect, the present invention provides a DNA fingerprint of Penaeus monodon, the fingerprint containing at least 209 specific SNP sites, the SNP sites and their specific nucleotides as follows:

[0008] Serial number Chromosome number Position Nucleotide Serial number Chromosome number Position Nucleotide 1 chr1 1640407 C / A 106 chr14 37477991 C / T 2 chr1 1640751 C / G 107 chr14 47202443 T / G 3 chr1 1642068 C / T 108 chr15 6965681 A / G 4 chr1 22368440 A / G 109 chr15 31049336 T / A 5 chr1 27093538 C / A 110 chr16 10032 C / G 6 chr1 30640885 A / T 111 chr16 1482961 A / G 7 chr1 33488180 G / T 112 chr16 27093767 T / C 8 chr1 48107496 C / T 113 chr16 27509745 T / C 9 chr1 61517839 T / C 114 chr16 32139531 G / A 10 chr2 162820 G / T 115 chr16 32140642 C / T 11 chr2 9223575 G / A 116 chr16 46058532 T / C 12 chr2 24186050 A / T 117 chr17 5475840 G / T 13 chr2 25463597 T / C 118 chr17 18566810 G / T 14 chr2 40762096 G / T 119 chr17 25692517 G / A 15 chr2 40763592 T / C 120 chr17 47969616 G / A 16 chr2 41669378 A / G 121 chr18 740902 A / G 17 chr2 41806137 T / C 122 chr18 27907254 A / G 18 chr3 446388 G / A 123 chr18 28060228 T / C 19 chr3 22464169 G / C 124 chr18 34651601 T / C 20 chr3 30922205 C / T 125 chr18 47498628 T / C 21 chr3 33685637 C / T 126 chr19 1284533 A / C 22 chr3 49037603 G / C 127 chr19 31402393 G / A 23 chr3 52957717 T / A 128 chr20 215196 C / T 24 chr3 54937945 A / T 129 chr20 1562631 A / C 25 chr4 2449589 G / A 130 chr20 4471882 G / A 26 chr4 8525184 A / T 131 chr20 13208439 C / T 27 chr4 14151342 C / T 132 chr21 7610958 G / A 28 chr4 14372879 G / A 133 chr21 14565008 C / T 29 chr4 34411408 C / T 134 chr21 31596482 T / C 30 chr4 34504645 C / G 135 chr22 3172145 A / G 31 chr4 40200871 A / G 136 chr22 24311555 T / A 32 chr4 42523678 T / C 137 chr23 12555922 A / T 33 chr4 54125338 C / T 138 chr23 42748406 A / G 34 chr4 54125761 T / C 139 chr24 4832291 C / A 35 chr4 54161739 G / T 140 chr24 26897931 C / T 36 chr5 40599 A / G 141 chr24 44060390 T / C 37 chr5 50357 C / T 142 chr25 10483221 T / C 38 chr5 19839469 T / C 143 chr25 30682913 C / T 39 chr5 20823225 T / G 144 chr26 6927222 T / C 40 chr5 42136262 T / C 145 chr26 12992378 C / A 41 chr5 42526364 C / A 146 chr26 28616804 T / G 42 chr5 46603559 T / C 147 chr26 32572609 C / T 43 chr5 55381992 T / G 148 chr27 4216988 T / C 44 chr6 247480 T / C 149 chr27 7575487 C / A 45 chr6 1029395 A / G 150 chr28 3254016 A / G 46 chr6 21370307 C / T 151 chr28 6396578 C / T 47 chr6 21794069 T / C 152 chr28 18443506 A / C 48 chr6 24759353 C / T 153 chr29 8866928 G / T 49 chr6 25963679 C / T 154 chr29 24613943 A / T 50 chr6 35411961 A / G 155 chr30 3650426 C / T 51 chr6 35636829 G / A 156 chr30 33311123 C / T 52 chr6 52524034 T / C 157 chr31 4857885 T / A 53 chr6 55136790 G / A 158 chr31 7322432 G / T 54 chr7 1982526 A / G 159 chr32 12964248 T / C 55 chr7 10688931 T / A 160 chr32 22837650 A / G 56 chr7 32362134 C / T 161 chr33 8730749 T / A 57 chr7 33424176 C / T 162 chr33 19474464 C / T 58 chr7 33435237 T / C 163 chr34 1090451 G / A 59 chr7 49816530 T / G 164 chr34 4830221 G / A 60 chr7 51999279 T / C 165 chr35 2060987 C / T 61 chr7 53064934 A / G 166 chr35 4142045 T / G 62 chr8 115371 C / T 167 chr35 26849015 T / C 63 chr8 8902438 C / T 168 chr35 27049683 T / G 64 chr8 22271366 C / T 169 chr36 1143813 G / A 65 chr8 33358038 A / T 170 chr36 9027855 A / G 66 chr8 37008166 C / A 171 chr36 24413984 G / A 67 chr9 427201 C / T 172 chr36 24884105 G / A 68 chr9 5342107 A / G 173 chr37 3486729 T / C 69 chr9 5455985 T / C 174 chr37 6986394 T / C 70 chr9 8332210 T / C 175 chr37 28679967 A / T 71 chr9 13046653 G / A 176 chr37 29219624 G / A 72 chr9 13437678 A / G 177 chr38 2445807 A / C 73 chr9 25047936 G / A 178 chr38 3107430 G / A 74 chr9 36256279 C / G 179 chr38 14104225 G / A 75 chr9 39323637 C / A 180 chr38 14394344 C / T 76 chr9 40676985 A / G 181 chr38 28592475 T / A 77 chr9 51051703 C / A 182 chr38 31218546 G / A 78 chr10 1253463 T / C 183 chr39 105919 T / G 79 chr10 2747898 C / T 184 chr39 6220088 C / T 80 chr10 6009557 T / C 185 chr39 7579915 G / T 81 chr10 20093240 G / A 186 chr39 22486215 G / T 82 chr10 22133357 T / C 187 chr39 32623236 G / C 83 chr10 43116189 C / G 188 chr40 15128 C / T 84 chr10 46751782 G / A 189 chr40 3326721 C / T 85 chr10 48076568 G / A 190 chr40 7680711 C / T 86 chr11 422081 T / C 191 chr40 11770495 G / A 87 chr11 15195077 G / A 192 chr40 20304802 C / T 88 chr11 29790804 A / C 193 20661905 chr40 89 C / G 40393119 chr11 194 G / A 27054531 chr40 90 A / G 44386573 chr11 195 G / A 43186 chr41 91 C / T 5660780 chr12 196 T / C 7467271 chr41 92 A / G 17307888 chr12 197 T / C 23476698 chr41 93 C / T 33489258 chr12 198 A / G 27942816 chr41 94 A / G 35400882 chr12 199 T / C 29967461 chr41 95 A / C 929553 chr13 200 T / C 3981845 chr42 96 A / T 3661365 chr13 201 T / A 16721790 chr42 97 G / A 5258265 chr13 202 A / G 21934967 chr42 98 A / T 26464808 chr13 203 C / T 7625221 chr43 99 C / G 29109934 chr13 204 T / C 18873670 chr43 100 C / T 30178951 chr13 205 G / A 20624536 chr43 101 C / A 1312644 chr14 206 T / C 30107268 chr43 102 C / T 6857788 chr14 207 G / A 2632329 chr44 103 G / T 15135117 chr14 208 T / C 2909769 chr44 104 C / T 20010293 chr14 209 G / A 3972122 chr44 105 G / T 21660167 chr14

[0009] Secondly, the present invention provides a method for constructing the above-mentioned DNA fingerprint of Penaeus monodon, comprising the following steps:

[0010] (1) Sample collection and genotyping: Collect individual samples from multiple representative populations of Penaeus monodon, extract genomic DNA, and obtain the original SNP dataset by whole-genome resequencing or SNP chip genotyping technology;

[0011] (2) SNP data quality control and filtering: The original SNP data is subjected to quality control to obtain a high-quality, low-redundancy set of SNP sites;

[0012] (3) Population genetic analysis: Using quality-controlled SNP data, principal component analysis (PCA), phylogenetic tree and community structure analysis were performed on multiple breeding populations to assess the genetic relationships and degree of differentiation among populations;

[0013] (4) Core marker screening: Multiple samples are normalized to screen out specific SNP sites with high marker quality, strong representativeness, high material discrimination of marker (combination) and uniform distribution on the genome;

[0014] (5) Fingerprint mapping: The specific SNP sites selected in step (4) and their allele frequency distribution information in different populations are integrated to establish a DNA fingerprint database for Penaeus monodon.

[0015] Preferably, the tiger prawn population in step (1) includes at least five populations; specifically, the tiger prawn population includes typical cultured populations and wild parent populations, as well as populations covering different regions. In constructing the tiger prawn DNA fingerprint, this invention selects at least five populations as basic sample populations, which not only meets the statistical requirements of population genetics analysis but also fully considers the ecological distribution patterns and industrial structure layout of tiger prawns both domestically and internationally, demonstrating scientific validity and rationality.

[0016] Preferably, the quality control of the original SNP data in step (2) includes: using the variant detection tool GATK to identify the original SNP sites and performing quality filtering on the original SNP sites, excluding sites that meet any of the following conditions: Quality Depth (QD) < 2.0; Root Mean Square Mapping Quality (MQ) < 40.0; Fisher Bias (FS) > 60.0; Alignment Quality Rank Sum (MQRankSum) < -12.5; Read Position Rank Sum (ReadPosRankSum) < -8.0; Alignment Chain Bias (SOR) > 3.0; retaining high-confidence SNP sites that pass the above filtering conditions (marked as PASS).

[0017] Preferably, in step (2), PLINK software is used to perform secondary quality control on the high-confidence SNP sites that have passed the filtering conditions: sites with a genotype deletion rate of more than 20% are removed, and sites with a minor allele frequency (MAF) of less than 0.05 are removed.

[0018] Preferably, in step (3), principal component analysis (PCA), phylogenetic tree analysis, and community structure analysis are performed on the five aquaculture populations using R language.

[0019] Preferably, in step (4), after normalizing the sample genotype data, sites without polymorphism, sites with a deletion rate higher than 22.0%, sites with a second allele ratio (MAF) lower than 0.1%, sites with a Hardy-Weinberg test p-value lower than 0.001, and sites with a polymorphism information content (PIC) lower than 0.35 are preferentially removed to screen out specific SNP sites with population discrimination ability.

[0020] Preferably, the number of specific SNP sites screened in step (4) is 209, and these sites are distributed on 44 chromosomes of Penaeus monodon.

[0021] Traditional methods for constructing DNA fingerprints rely on assessments of shrimp morphology and molecular markers based on microsatellite (SSR) or limited local SNP loci. However, these methods typically suffer from limitations such as limited locus distribution and low reproducibility. This invention proposes for the first time a method for constructing a DNA fingerprint of *Litopenaeus monodon* based on highly polymorphic SNP loci. Compared to traditional methods, this invention's method mines SNP loci across the entire genome, identifying 209 highly polymorphic SNP loci covering all 44 chromosomes of *Litopenaeus monodon*, thus achieving a technological advancement from local markers to a genome-wide marker system.

[0022] Furthermore, this invention employs more stringent screening criteria for highly polymorphic SNP sites, and its data processing features higher throughput and automation. The samples used encompass populations from diverse geographical origins, including typical farmed populations and wild parent resources, thereby ensuring the constructed DNA fingerprint has broad applicability and population representativeness.

[0023] Thirdly, the present invention provides the application of the DNA fingerprint of *Litopenaeus monodon* in the identification of *Litopenaeus monodon* germplasm resources; the present invention also provides the application of the DNA fingerprint of *Litopenaeus monodon* in assisting *Litopenaeus monodon* breeding.

[0024] The DNA fingerprinting of *Litopenaeus monodon* of this invention, with its high-throughput and high-stability technical advantages, can widely empower *Litopenaeus monodon* germplasm resource research and aquaculture breeding industrialization practices, covering multi-dimensional technical needs. In the field of germplasm resource identification, it can accurately locate the population affiliation of *Litopenaeus monodon* individuals of unknown origin, clarify their genetic origin information, effectively distinguish different geographical populations, cultured strains, and hybrid offspring, and provide data support for the standardized management of germplasm resources. In population genetic research, it can systematically assess the genetic diversity level and genetic structure differentiation characteristics of *Litopenaeus monodon* cultured populations, providing a scientific basis for the protection, rejuvenation, and rational development of population resources, and avoiding genetic resource degradation. In addition, in the breeding strategy of superior *Litopenaeus monodon*, this fingerprinting can serve as a core molecular basis for parent selection, assisting in the screening of parental combinations with appropriate genetic distance and complementary superior traits, reducing the risk of inbreeding depression. It can also be used for the genetic background identification of offspring individuals, verifying the genetic stability of breeding target traits, and contributing to the efficient breeding of high-quality, stress-resistant *Litopenaeus monodon* strains.

[0025] The present invention has the following beneficial effects:

[0026] (1) High throughput and high resolution: Based on whole-genome SNP markers, the DNA fingerprint of Penaeus monodon in this invention has a large amount of information, which can finely characterize the genetic characteristics of different populations at the molecular level, with strong discrimination ability and a comprehensive discrimination rate of 90.11%;

[0027] (2) Stable and reliable: The SNP markers used in the DNA fingerprinting of Penaeus monodon in this invention are biale genotype markers. Their genetic characteristics are determined by the genome sequence, which have extremely high detection repeatability and accuracy. They can effectively avoid interference caused by experimental operation errors and are not affected by the environment and developmental stage.

[0028] (3) High practicality: The DNA fingerprint spectrum of the present invention can be directly used for germplasm identification, resource assessment and breeding practice. By establishing a standardized molecular identification system, the standardized traceability, authenticity identification and quality grading of Penaeus monodon germplasm have been realized, filling the gap in the industry where there is a lack of unified molecular identification standards.

[0029] (4) Breeding guidance: This invention can clarify the genetic distance and the correlation characteristics of excellent traits among different populations, providing accurate molecular references for the selection of hybrid parents. By screening parental combinations with strong genetic complementarity and rich in excellent genes, the probability of inbreeding depression can be effectively reduced, and the breeding process of high-quality and stress-resistant tiger prawn strains can be accelerated. Attached Figure Description

[0030] A / G This is a three-dimensional scatter plot of principal component analysis (PCA) of five cultured populations of tiger prawns in Example 1 of this invention;

[0031] Figure 1 This is a phylogenetic tree of 349 samples from 5 cultured populations of Penaeus monodon in Example 1 of this invention;

[0032] Figure 2 This is a population structure diagram of 349 samples from 5 cultured populations of tiger prawns in Example 1 of the present invention;

[0033] Figure 3 This is a heatmap showing the fingerprint patterns of the 209 specific SNP sites selected in Example 1 of this invention on 44 chromosomes. Detailed Implementation

[0034] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention. Unless otherwise specified, the reagents or materials used in the embodiments are all from commercial sources.

[0035] Example 1: Establishment of DNA fingerprinting for Penaeus monodon

[0036] (1) Sample collection and genotyping:

[0037] Sample Source: A total of 349 healthy individuals were collected from major tiger prawn farming companies / bases along the Chinese coast (see Table 1). The five tiger prawn populations selected in this embodiment are from commonly farmed varieties currently on the market: Nanhai No. 1, Nanhai No. 2, African strain, Thai strain, and Hainan population. Among them, Nanhai No. 1 and Nanhai No. 2 are tiger prawn varieties independently developed in my country, mainly derived from hybridization of individuals with superior traits selected from the Hainan, African, and Thai strains. They possess significant farming advantages such as rapid growth and high survival rates. Furthermore, these five populations cover the main farming zones and core breeding areas of tiger prawns currently on the market, exhibiting clear representativeness in geographical, ecological, and genetic diversity, and comprehensively reflecting the genetic characteristics and differentiation patterns of tiger prawn germplasm resources both domestically and internationally. This sample design ensures the universality, accuracy, and long-term application value of the constructed DNA fingerprint, fully supporting the needs of genetic identification, strain differentiation, and superior breed selection of tiger prawns in my country.

[0038] Table 1 Sample Information Table

[0039]

[0040] DNA extraction and sequencing: Muscle tissue was collected from each tiger prawn, and genomic DNA was extracted using the conventional phenol-chloroform method or a commercial kit. After passing quality inspection, the DNA samples were randomly fragmented into 350bp-500bp fragments using a Covaris ultrasonic disruptor. Then, end repair, A-tailing, adapter ligation, and PCR amplification were performed to construct a sequencing library. Whole genome resequencing (average depth ≥10×) was performed using a sequencing platform. Sequence alignment and SNP calling of tiger prawns were performed using standard procedures such as BWA and GATK to obtain raw VCF format SNP data.

[0041] (2) SNP data quality control and screening:

[0042] The GATK mutation detection tool was used to identify raw SNP sites in the sequencing data of Penaeus monodon, and the identified raw SNP sites were subjected to strict quality filtering. The screening criteria are as follows:

[0043] Quality Depth (QD) < 2.0; Root Mean Square Mapping Quality (MQ) < 40.0; Fisher Bias (FS) > 60.0; Alignment Quality Rank Sum Test (MQRankSum) < -12.5; Read Position Rank Sum Test (ReadPosRankSum) < -8.0; Alignment Chain Bias (SOR) > 3.0.

[0044] The raw SNP data after GATK quality filtering was further rigorously quality controlled using PLINK software (v1.9) to remove low-reliability variants and retain core SNPs suitable for subsequent genetic analysis. The commands and results are as follows:

[0045] Remove sites with a genotype deletion rate >20%: plink --vcf snpfilter2.vcf --geno 0.2 –autosome-num 24 --make-bed --out snpfilter41.

[0046] Sites with minor allele frequency (MAF) < 0.05 were removed using the following command: `plink --bfile snpfilter41 --maf0.05 --make-bed --out snpfilter42`. This resulted in 6,790,260 unique SNP sites remaining for subsequent analysis.

[0047] (3) Population genetic analysis: Using 6,790,260 SNPs after quality control, PCA, phylogenetic tree analysis and community structure analysis were performed using tools such as R language (results are shown in the figure). Figure 4 (As shown in the figure); the analysis shows that there is obvious genetic differentiation among the 349 samples in the 5 groups. Based on the population structure analysis, the samples can be divided into 5 genetic subgroups.

[0048] (4) Core marker screening: The 6,790,260 SNPs after quality control were normalized, and core markers were screened using VCFtools or similar software; sites with a deletion rate higher than 22.0% were removed; sites with a second allele ratio (MAF) lower than 0.1% were removed; sites with a Hardy-Weinberg test p-value lower than 0.001 were removed; sites with a polymorphism information content (PIC) lower than 0.35 were removed, and their distribution on each chromosome was considered (e.g., Figures 1-3 As shown in the figure, 209 specific SNP loci with high population discrimination ability were finally screened out. These loci are distributed on 44 chromosomes of Penaeus monodon.

[0049] (5) Fingerprint pattern construction: The specific SNP sites selected in step (4) and their allele frequency distribution information in different populations are integrated to establish a DNA fingerprint database of Penaeus monodon. The list of these sites is shown in Table 2 below.

[0050] Table 2. Database of DNA fingerprints of Penaeus monodon

[0051] Figure 4 Serial number Chromosome number Position Nucleotide Serial number Chromosome number Position 1 Nucleotide 1640407 chr1 106 C / A 37477991 chr14 2 C / T 1640751 chr1 107 C / G 47202443 chr14 3 T / G 1642068 chr1 108 C / T 6965681 chr15 4 A / G 22368440 chr1 109 A / G 31049336 chr15 5 T / A 27093538 chr1 110 C / A 10032 chr16 6 C / G 30640885 chr1 111 A / T 1482961 chr 7 ​ 33488180 ​ 112 ​ 27093767 ​ 8 ​ 48107496 ​ 113 ​ 27509745 ​ 9 ​ 61517839 ​ 114 ​ 32139531 ​ 10 ​ 162820 ​ 115 ​ 32140642 ​ 11 ​ 9223575 ​ 116 ​ 46058532 ​ 12 ​ 24186050 ​ 117 ​ 5475840 ​ 13 ​ 25463597 ​ 118 ​ 18566810 ​ 14 ​ 40762096 ​ 119 ​ 25692517 ​ 15 ​ 40763592 ​ 120 ​ 47969616 ​ 16 ​ 41669378 ​ 121 ​ 740902 ​ 17 ​ 41806137 ​ 122 ​ 27907254 ​ 18 ​ 446388 ​ 123 ​ 28060228 ​ 19 ​ 22464169 ​ 124 ​ 34651601 ​ 20 ​ 30922205 ​ 125 ​ 47498628 ​ 21 ​ 33685637 ​ 126 ​ 1284533 ​ 22 ​ 49037603 ​ 127 ​ 31402393 G / A 23 chr3 52957717 T / A 128 chr20 215196 C / T 24 chr3 54937945 A / T 129 chr20 1562631 A / C 25 chr4 2449589 G / A 130 chr20 4471882 G / A 26 chr4 8525184 A / T 131 chr20 13208439 C / T 27 chr4 14151342 C / T 132 chr21 7610958 G / A 28 chr4 14372879 G / A 133 chr21 14565008 C / T 29 chr4 34411408 C / T 134 chr21 31596482 T / C 30 chr4 34504645 C / G 135 chr22 3172145 A / G 31 chr4 40200871 A / G 136 chr22 24311555 T / A 32 chr4 42523678 T / C 137 chr23 12555922 A / T 33 chr4 54125338 C / T 138 chr23 42748406 A / G 34 chr4 54125761 T / C 139 chr24 4832291 C / A 35 chr4 54161739 G / T 140 chr24 26897931 C / T 36 chr5 40599 A / G 141 chr24 44060390 T / C 37 chr5 50357 C / T 142 chr25 10483221 T / C 38 chr5 19839469 T / C 143 chr25 30682913 C / T 39 chr5 20823225 T / G 144 chr26 6927222 T / C 40 chr5 42136262 T / C 145 chr26 12992378 C / A 41 chr5 42526364 C / A 146 chr26 28616804 T / G 42 chr5 46603559 T / C 147 chr26 32572609 C / T 43 chr5 55381992 T / G 148 chr27 4216988 T / C 44 chr6 247480 T / C 149 chr27 7575487 C / A 45 chr6 1029395 A / G 150 chr28 3254016 A / G 46 chr6 21370307 C / T 151 chr28 6396578 C / T 47 chr6 21794069 T / C 152 chr28 18443506 A / C 48 chr6 24759353 C / T 153 chr29 8866928 G / T 49 chr6 25963679 C / T 154 chr29 24613943 A / T 50 chr6 35411961 A / G 155 chr30 3650426 C / T 51 chr6 35636829 G / A 156 chr30 33311123 C / T 52 chr6 52524034 T / C 157 chr31 4857885 T / A 53 chr6 55136790 G / A 158 chr31 7322432 G / T 54 chr7 1982526 A / G 159 chr32 12964248 T / C 55 chr7 10688931 T / A 160 chr32 22837650 A / G 56 chr7 32362134 C / T 161 chr33 8730749 T / A 57 chr7 33424176 C / T 162 chr33 19474464 C / T 58 chr7 33435237 T / C 163 chr34 1090451 G / A 59 chr7 49816530 T / G 164 chr34 4830221 G / A 60 chr7 51999279 T / C 165 chr35 2060987 C / T 61 chr7 53064934 A / G 166 chr35 4142045 T / G 62 chr8 115371 C / T 167 chr35 26849015 T / C 63 chr8 8902438 C / T 168 chr35 27049683 T / G 64 chr8 22271366 C / T 169 chr36 1143813 G / A 65 chr8 33358038 A / T 170 chr36 9027855 A / G 66 chr8 37008166 C / A 171 chr36 24413984 G / A 67 chr9 427201 C / T 172 chr36 24884105 G / A 68 chr9 5342107 A / G 173 chr37 3486729 T / C 69 chr9 5455985 T / C 174 chr37 6986394 T / C 70 chr9 8332210 T / C 175 chr37 28679967 A / T 71 chr9 13046653 G / A 176 chr37 29219624 G / A 72 chr9 13437678 A / G 177 chr38 2445807 A / C 73 chr9 25047936 G / A 178 chr38 3107430 G / A 74 chr9 36256279 C / G 179 chr38 14104225 G / A 75 chr9 39323637 C / A 180 chr38 14394344 C / T 76 chr9 40676985 A / G 181 chr38 28592475 T / A 77 chr9 51051703 C / A 182 chr38 31218546 G / A 78 chr10 1253463 T / C 183 chr39 105919 T / G 79 chr10 2747898 C / T 184 chr39 6220088 C / T 80 chr10 6009557 T / C 185 chr39 7579915 G / T 81 chr10 20093240 G / A 186 chr39 22486215 G / T 82 chr10 22133357 T / C 187 chr39 32623236 G / C 83 chr10 43116189 C / G 188 chr40 15128 C / T 84 chr10 46751782 G / A 189 chr40 3326721 C / T 85 chr10 48076568 G / A 190 chr40 7680711 C / T 86 chr11 422081 T / C 191 chr40 11770495 G / A 87 chr11 15195077 G / A 192 chr40 20304802 C / T 88 chr11 29790804 A / C 193 chr40 20661905 C / G 89 chr11 40393119 G / A 194 chr40 27054531 A / G 90 chr11 44386573 G / A 195 chr41 43186 C / T 91 chr12 5660780 T / C 196 chr41 7467271 A / G 92 chr12 17307888 T / C 197 chr41 23476698 C / T 93 chr12 33489258 A / G 198 chr41 27942816 A / G 94 chr12 35400882 T / C 199 chr41 29967461 A / C 95 chr13 929553 T / C 200 chr42 3981845 A / T 96 chr13 3661365 T / A 201 chr42 16721790 G / A 97 chr13 5258265 A / G 202 chr42 21934967 A / T 98 chr13 26464808 C / T 203 chr43 7625221 C / G 99 chr13 29109934 T / C 204 chr43 18873670 C / T 100 chr13 30178951 G / A 205 chr43 20624536 C / A 101 chr14 1312644 T / C 206 chr43 30107268 C / T 102 chr14 6857788 G / A 207 chr44 2632329 G / T 103 chr14 15135117 T / C 208 chr44 2909769 C / T 104 chr14 20010293 G / A 209 chr44 3972122 G / T 105 chr14 21660167 A / G

[0052] Example 2: Germplasm Identification

[0053] (1) Sample source and testing purpose

[0054] A total of 91 individuals of tiger prawns of unknown origin were collected from aquaculture farms in Fujian, Guangdong, Hainan, Guangxi, and Zhanjiang as test samples to verify the accuracy of population identification based on fingerprint patterns.

[0055] (2) DNA extraction and genotyping

[0056] Approximately 50 mg of abdominal muscle tissue was collected from each sample, and genomic DNA was extracted using the TIANamp Marine Animal DNA Kit. After the DNA concentration and purity were determined by Nanodrop, the sample was diluted to 50 ng / μL for later use.

[0057] For the 209 loci constructed in this invention, SNP genotyping primers were designed and high-throughput genotyping was performed using KASP (Kompetitive Allele Specific PCR) technology. The PCR amplification products were read by the fluorescence detection system of QuantStudio 7 Flex (quantitative PCR instrument) to obtain the allele information of each sample at each locus.

[0058] (3) Data analysis and group identification

[0059] Genotypic data for each sample were standardized using PLINK and R, and allele frequencies, heterozygosity (He), polymorphism information content (PIC), and genetic distance matrices between samples were calculated. Phylogenetic trees were then constructed based on Euclidean distance and the Neighbor-Joining (NJ) method, and principal component analysis (PCA) was used for visualization validation.

[0060] The analysis results show that the 209 SNP loci screened in this invention can accurately distinguish five known populations into five genetic subpopulations. Furthermore, using core marker SNP loci for germplasm identification of 91 samples from unknown sources, 82 samples were accurately identified, achieving an accuracy rate of 90.11%. This effectively verifies that the DNA fingerprinting method of this invention has high accuracy and stability in the tracing of Penaeus monodon populations and strain identification.

[0061] (4) Technical effects and application value

[0062] The results of this embodiment demonstrate that the DNA fingerprinting method of the present invention has the following significant effects in the germplasm identification of Penaeus monodon:

[0063] High-precision population identification: By using 209 specific SNP markers to perform multi-dimensional quantitative analysis of genetic differences among individuals, different geographical populations and breeding strains can be accurately distinguished at the molecular level, with an accuracy of up to 90.11%.

[0064] High reproducibility and objectivity: All SNP loci in this fingerprint are biallelic genotype markers, determined by the genome sequence. The results are not affected by sample tissue, developmental stage, or detection experimental environment, and have good reproducibility.

[0065] Wide range of applications: Applicable to various scenarios such as germplasm resource tracing, variety authenticity identification, kinship analysis and hybridization breeding verification, and can serve as the core technical support for the standardized molecular detection system of the tiger prawn industry.

[0066] Assisted breeding decision-making: By analyzing the genetic distance and differential loci between different populations, a scientific basis can be provided for the selection of parental pairs, and the pairing populations with strong genetic complementarity can be selected to improve the growth, stress resistance and reproductive performance of offspring.

[0067] The DNA fingerprinting of *Litopenaeus monodon* constructed in this invention can also be used in the following aspects, for example: a) Using 209 specific SNP loci as core markers, or combining all quality-controlled SNP loci data, to calculate key genetic parameters such as observed heterozygosity (Ho), expected heterozygosity (He), polymorphism information content (PIC), and allele richness of target culture population samples. Through systematic analysis of these parameters, the genetic diversity level, genetic homozygosity, and gene flow status of the population can be quantitatively assessed, and populations with a high risk of genetic decline can be accurately identified. b) Breeding assistance: In hybridization breeding, by using fingerprinting to analyze the genetic distance and degree of differentiation between different populations, populations with significant fingerprint differences and strong genetic complementarity can be preferentially selected as parental combinations to maximize the potential of hybrid vigor and improve the growth performance, stress resistance, and other superior traits of offspring.

[0068] In other words, this invention successfully constructed the first high-resolution DNA fingerprint map of Penaeus monodon based on whole-genome SNPs; this map is highly systematic and accurate, providing an indispensable molecular tool for the germplasm resource management, breeding of superior varieties, and industrial standardization of Penaeus monodon.

[0069] The above embodiments are only used to illustrate the present invention, and the scope of protection of the present invention is not limited to the above embodiments. Those skilled in the art can achieve the purpose of the present invention based on the above disclosure. Any improvements and modifications made based on the concept of the present invention fall within the scope of protection of the present invention, the specific scope of protection being as described in the claims.

Claims

1. A DNA fingerprint of Penaeus monodon, characterized in that, The map contains at least 209 specific SNP sites, the information of which is shown below: 。 2. A method for constructing a DNA fingerprint of Penaeus monodon, characterized in that, Includes the following steps: (1) Sample collection and genotyping: Collect individual samples from multiple representative populations of Penaeus monodon, extract genomic DNA, and obtain the original SNP dataset by whole-genome resequencing or SNP chip genotyping technology; (2) SNP data quality control and filtering: The original SNP data is subjected to quality control to obtain a high-quality, low-redundancy set of SNP sites; (3) Population genetic analysis: Using quality-controlled SNP data, principal component analysis (PCA), phylogenetic tree analysis and community structure analysis were performed on multiple aquaculture populations to assess the genetic relationships and degree of differentiation among populations; (4) Core marker screening: Multiple samples are normalized to screen out specific SNP sites with high marker quality, strong representativeness, high material discrimination of the markers, and uniform distribution on the genome; (5) Fingerprint mapping: The specific SNP sites selected in step (4) and their allele frequency distribution information in different populations are integrated to establish a DNA fingerprint database for Penaeus monodon.

3. The method for constructing a DNA fingerprint of Penaeus monodon according to claim 2, characterized in that, The tiger prawn population mentioned in step (1) includes at least 5 populations, including typical cultured populations and wild parent populations.

4. The method for constructing a DNA fingerprint of Penaeus monodon according to claim 3, characterized in that, The quality control of the original SNP data in step (2) includes: using the variant detection tool GATK to identify the original SNP sites and filtering the original SNP sites to exclude sites that meet any of the following conditions: quality depth < 2.0; root mean square mapping quality < 40.0; Fisher bias > 60.0; alignment quality rank-sum test < -12.5; read position rank-sum test < -8.0; alignment chain bias > 3.0; and retaining high-confidence SNP sites that pass the above filtering conditions.

5. The method for constructing a DNA fingerprint of Penaeus monodon according to claim 4, characterized in that, In step (2), PLINK software is used to perform secondary quality control on the high-confidence SNP sites that have passed the filtering conditions: sites with a genotype deletion rate of more than 20% are removed, and sites with a minor allele frequency (MAF) of less than 0.05 are removed.

6. The method for constructing a DNA fingerprint of Penaeus monodon according to claim 5, characterized in that, In step (3), R language was used to perform principal component analysis, phylogenetic tree analysis and community structure analysis on the five aquaculture groups.

7. The method for constructing a DNA fingerprint of Penaeus monodon according to claim 6, characterized in that, In step (4), after normalizing the sample genotype data, sites with no polymorphism, missing rates higher than 22.0%, second allele proportions lower than 0.1%, Hardy-Weinberg test p-values ​​lower than 0.001, and polymorphism information content lower than 0.35 are selected to identify specific SNP sites with population discrimination ability.

8. The application of the DNA fingerprint of Penaeus monodon as described in claim 1 in the identification of Penaeus monodon germplasm resources.

9. The application of the DNA fingerprint of Penaeus monodon as described in claim 1 in assisting Penaeus monodon breeding.

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