CN116064834A - Method for evaluating genetic diversity of Chinese prawn population by using environmental DNA - Google Patents
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Abstract
The invention provides a method for evaluating genetic diversity of a Chinese prawn population by using environmental DNA, which is characterized in that seawater is collected and filtered in a target sea area, the seawater environmental DNA is enriched through a filter membrane, and eDNA extraction is completed in a laboratory. The sequence determination of the Penaeus chinensis eDNA in the sea water is carried out by an amplicon high-throughput sequencing technology, and the amplified target fragment is the Penaeus chinensis mitochondrial COI gene. And (3) carrying out SNP locus haplotype and abundance analysis on the amplified target fragment, and calculating to obtain SNP haplotype composition and abundance of each haplotype, thereby obtaining the genetic diversity level of the target sea area Chinese prawn population in the mitochondrial COI gene. Compared with the current method, the genetic diversity evaluation of population level is more accurate, the method is environment-friendly, and can be easily expanded to other genes and even whole genome level.
Description
Technical Field
The invention belongs to the technical field of marine animal resource protection, and particularly relates to a method for evaluating genetic diversity of a Chinese prawn population by using environmental deoxyribonucleic acid (eDNA).
Background
The Chinese prawn (Fenneropenaeus chinensis) is a special original species of the yellow Bohai sea in China, and is also named as Chinese prawn, oriental prawn and the like. The plant species belongs to Arthropoda (Arthropoda) in animal taxonomy, has the phylum of gill subgenera (Branchiata), crustacean (Crustaceae), octopus (Decapoda), pacific (Penaeidae) and Pacific (Penaeus) and is a large-sized economic shrimp, is an important fishing and breeding object in yellow Bohai sea in China, and has extremely outstanding economic value.
Historically, the Chinese prawn resources are abundant, but in recent decades, due to the influence of a plurality of factors such as increased fishing strength, ecological transition, environmental pollution, disease frequency and the like, the Chinese prawn resources are rapidly shrunken, the annual autumn flood fishing amount is reduced to about 1/10 of the highest peak of the history, and the genetic diversity level is seriously threatened.
The adaptability of the population to environmental changes is directly determined by the level of genetic diversity, the genetic diversity reflects an important index of whether the population is healthy or not, and is also a key factor for maintaining continuation of the population, and meanwhile, the genetic diversity is the basis for developing artificial breeding and good variety cultivation. Therefore, the method has extremely important application and ecological significance in carrying out genetic diversity level monitoring on the existing Chinese prawn population.
In the past, genetic diversity monitoring of populations has relied on collection of living samples, which are either limited by specialized tools or due to the scarcity of population resources (such as migratory parent shrimps), often fail to obtain satisfactory and sufficient numbers of samples, which are too small to accurately assess the genetic diversity level of the population. Conversely, a sufficient number of samples either impact population resources or represent a significant amount of laboratory work and high laboratory costs at a later stage. Meanwhile, satisfactory living samples cannot be obtained in the forbidden period and the early stage of the development of the offspring, so that the monitoring of the genetic diversity level of the population in the life history stage is limited. Therefore, a technology for evaluating genetic diversity level of a penaeus vannamei boone population without living sample collection is urgently needed to meet the requirements of population resource investigation and establishment and modification of relevant fishery management measures.
Disclosure of Invention
The invention aims to provide a method for realizing the genetic diversity level detection of a Chinese prawn population by carrying out eDNA high-throughput sequencing analysis on a sea area where a target population lives and carrying out statistical analysis on SNP locus haplotype composition and proportion in the sea area under the premise of no living body acquisition.
The method for evaluating the genetic diversity of the Chinese prawn population by using the environmental DNA (eDNA) provided by the invention comprises the following steps:
1) Water sample collection
Collecting water samples of natural sea areas of the population distribution of the Chinese prawns to be monitored;
the water sample is collected, as a specific record of an embodiment, is collected at a position 2-5 meters away from the sea bottom, the collected seawater is filtered by using a glass fiber membrane of 0.45 mu m under the assistance of a vacuum pump, and a filter membrane enriched with eDNA is stored at-20 ℃ to a laboratory for extracting the eDNA in a dark place;
2) eDNA extraction
Extracting a DNA sample on the filter membrane enriched with eDNA, and diluting for later use;
specific description of the examples was that the extraction of the eDNA was completed using a micro DNA/RNA extraction kit DNeasy Blood and Tissue kit, while the eDNA was quantified using a micro spectrophotometer and diluted with sterilized double distilled water;
3) Chinese prawn eDNA-PCR amplification
Amplifying a fragment of a mitochondrial COI gene (cytochrome oxidase subunit I gene) of the Chinese prawn;
wherein the primer pair used has a forward primer sequence of 5'-TTTTTGACCCTGCAGGAGGT-3' (SEQ ID NO: 1) and a reverse primer sequence of 5'-CCGTGGAGGGTTCCTATTCA-3' (SEQ ID NO: 2);
the amplification reaction system consisted of 12. Mu.L of Monamp2 XTaq Mix Pro, 1. Mu.L of 10mM forward and reverse primers each, 1. Mu.L of template DNA at a concentration of 50 ng/. Mu.L, 10. Mu.L of ddH 2 O；
The Chinese prawn eDNA-PCR amplification reaction procedure is as follows: pre-denaturation at 94℃for 3min; denaturation at 94℃for 20s, annealing at 56.8℃for 20s, elongation at 72℃for 40s,35 cycles; re-extending at 72 deg.c for 10min; preserving at 4 ℃;
4) High throughput sequencing of PCR products
OTU (Operational Taxonomic Units) clustering the sequences of the amplified products according to 97% similarity, and comparing the sequences with NCBI database to obtain the sequence of the mitochondrial COI gene of the Chinese prawn derived from the eDNA of the water sample; and (3) BLAST comparison is carried out on the obtained COI sequence, variant base sites are screened, variant SNP sites form SNP site haplotypes, and the proportion of different SNP sites is counted to be used as the SNP site haplotype result of the Chinese prawn mitochondrial COI gene, so that the genetic diversity level of the Chinese prawn population living in the water sample acquisition area is reflected.
According to the invention, the genetic diversity level of the Chinese prawn population living in the sea water sample is indirectly estimated by directly analyzing the eDNA of the sea water sample, so that the requirement of the collected sample on facility equipment is avoided, the damage of the sample collection on the environment and population resources is avoided, and the influence of sampling errors on an estimation result is avoided. The method is a brand new, efficient and accurate method for evaluating the genetic diversity level of the Chinese prawn population, and can be conveniently applied to other species. The concrete explanation is as follows:
firstly, because the water sample is directly collected instead of the living body of the Chinese prawn, a large amount of funds are not required to be invested for purchasing or special personnel, nets, fishing boats and the like are not required to be hired for collecting the sample, and only one 2L water sampler is required to meet the sample collection requirement, so that the cost and the threshold for developing the test are greatly reduced;
secondly, the collection of living samples of the Chinese shrimps requires a special trawl, which not only can damage the increasingly fragile submarine environment, but also can catch other species by mistake while the trawl obtains the Chinese shrimps, thereby causing associated injuries. In the individual life stages of Chinese prawns, such as larval development and larval prawns, the existing net fishing mode cannot collect living samples due to the technical limitation. In other life history stages of the Chinese prawns, such as reproduction migration period, the collection of samples can seriously damage population resources due to the small number of parent prawns. The water sample collection is not limited by the problems;
thirdly, analyzing the genetic diversity level of the population by using eDNA theoretically includes all the individuals of the population of the penaeus vannamei in the sea area to be analyzed, and avoids the errors of the sampling quantity of living bodies or the sampling errors on the genetic diversity level evaluation of the population.
Drawings
Fig. 1: a sequence comparison diagram of the COI gene of prawn,
fig. 2: SNP locus haplotype network map based on four groups;
fig. 3: population cluster map based on genetic consistency.
Detailed Description
The technical scheme of the present invention is further explained by the following specific examples, but the scope of the present invention is not limited by any form of the examples.
Example 1
1) Chinese prawn living body sample and sea water sample collection
And 4, in the middle and upper ten days of 2022, respectively performing living body collection and water sample collection of parent shrimps of the reproduction and migration Chinese shrimps along with the fishing boat in the sand mouth sea area of the peninsula in the Shandong province and the sea-yang sea area. Living body collection adopts a bottom trawl net for fishing. Meanwhile, a seawater sampler (2L) is adopted to collect seawater in the trawl process, and the seawater collection depth is 2-5 meters away from the sea floor. Collected living Chinese prawns are stored by using ice cubes until the living Chinese prawns are transported back to a laboratory. Seawater is synchronously collected by a seawater collector (2L), the collection depth is 2-5 meters away from the sea bottom, and the same position is parallel to 3 times of technology. The collected seawater is filtered and eDNA enriched by using a glass fiber membrane of 0.45 mu m with the aid of a vacuum pump, and three monitoring stations are arranged in parallel. The water extractor, filter, etc. are rinsed twice with deionized water prior to each seawater sample filtration. The filters enriched for eDNA were stored in the dark at-20℃until the laboratory was ready for eDNA extraction. The sampling is carried out at this time, and 160 and 75 living samples of the Chinese prawn are respectively collected at a sand port and in the sea area; and collecting 12 water samples at the sand mouth and the sea area.
2) Extraction of genome DNA of Chinese prawn sample and mitochondrial COI sequence analysis
The extraction tissue of the genome of the Chinese prawn is muscle tissue of swimming appendage, the extraction method is a traditional phenol/chloroform extraction method, and the obtained genome DNA is subjected to constant volume to 50 ng/. Mu.L concentration by using sterile double distilled water.
The PCR amplification primer sequence of the Chinese prawn mitochondrial COI gene is as follows: forward primer (F, 5'-3' end orientation): TTTTTGACCCTGCAGGAGGT, reverse primer (R) (5 '-3' end orientation): CCGTGGAGGGTTCCTATTCA.
The Chinese prawn eDNA-PCR reaction system comprises the following components: 25 μl of the reaction system specifically comprises 12 μl of Monamp2×Taq Mix Pro, 1 μl of each forward and reverse primer (10 mM), 1 μl of template DNA (50 ng/. Mu.L), and ddH 2 O10. Mu.L; the Chinese prawn eDNA-PCR amplification reaction procedure is as follows: pre-denaturation at 94℃for 3min; denaturation at 94℃for 20s, annealing at 56.8℃for 20s, elongation at 72℃for 40s,35 cycles; re-extending at 72 deg.c for 10min; preserving at 4 ℃.
The mitochondrial COI-PCR product was sent to the biological engineering Co.Ltd for the determination of the COI sequence. All 235 samples of mitochondrial COI sequences were aligned by DNAman (fig. 1) and subjected to SNP locus haplotype composition and proportion statistics using DnaSP v 6. The data is the evaluation result of genetic diversity level of mitochondrial SNP loci of the parent shrimp population of the reproduction of the Chinese shrimps in the Shake mouth and the sea yang sea area, and is used as a comparison reference for judging whether the eDNA analysis result is accurate.
3) Seawater eDNA extraction and high throughput sequencing of China prawn eDNA-COI gene
Extracting sea water eDNA. The extraction of the eDNA was accomplished using a micro DNA/RNA extraction kit DNeasy Blood and Tissue kit. Simultaneously, a micro-spectrophotometer was used to quantify the eDNA and the eDNA was diluted to a fixed concentration with sterilized double distilled water. The PCR amplification of seawater eDNA-COI adopts the same primer and the PCR amplification of living tissues, and the PCR flow and the PCR reaction system are the same as those of the PCR amplification of living tissues (the same content as the part 2). High throughput sequencing of PCR product of seawater eDNA (deoxyribonucleic acid) Chinese prawn COI gene by utilizing Illumina Miseq TM /Hiseq TM The second generation high throughput sequencing platform is completed. The obtained original data is subjected to primer joint sequence removal, splicing, identification and distinctionAnd obtaining effective data of the sample after a series of processing such as quality control and the like. Carrying out OUT (Operational Taxonomic Units) clustering on the sample effective data, comparing all the gene sequences belonging to the COI of the Chinese prawns with SNP locus haplotypes, and carrying out proportion statistics to obtain a COI gene SNP locus haplotype polymorphism statistical result of the eDNA of the Chinese prawns.
4) And (3) comparing the genetic polymorphism of the mitochondrial COI of the living body sample with the genetic diversity of the mitochondrial COI of the eDNA high-throughput sequencing.
9 SNP locus haplotypes were detected from 160 Litopenaeus vannamei living mitochondrial COI genes in the Qingdao sea area, and 4 SNP locus haplotypes were detected from 75 Litopenaeus vannamei living mitochondrial COI genes in the Qingdao sea area (Table 1). It is apparent that the number of SNP locus haplotypes detected in the mitochondrial COI gene increases with the number of samples.
The number of SNP locus haplotypes of the mitochondrial COI genes detected by high-throughput from the Qingdao and sea-yang sea area water sample eDNA is between 1500 and 2200, and the number of SNP locus haplotypes of the mitochondrial COI genes detected by the eDNA is far more than that of a living body detection result, because the living body is a 'sampling' detection, and the water sample eDNA is a full-coverage detection of all samples. The SNP locus haplotype of the mitochondrial COI gene of the Chinese prawn is characterized in that the haplotype is an absolute majority (the haplotype accounts for more than 86 percent), and the rest haplotypes are scattered and small, so that if more haplotype individuals are detected by living bodies, the number of samples is definitely greatly increased, and the disadvantage of the living bodies in the aspect of genetic diversity detection of the water sample eDNA is just reflected. Haplotypes detected by Qingdao and sea yang living body samples are detected in water samples eDNA corresponding to two places, the results of the water samples and living bodies are consistent no matter how the haplotype proportion and the sequencing of the proportion are, and the statistical detection result shows that no significant difference exists (Table 2).
Table 1: living sample of Chinese prawn and SNP locus haplotype statistical table of water sample eDNA mitochondria COI gene
* Only SNP locus haplotypes of mitochondrial COI genes detected by both living organisms and water sample eDNA are listed here
Table 2: chi-square test chart with obvious SNP locus haplotype difference of mitochondrial COI genes detected by sea-yang and Qingdao sea-area living bodies and eDNA
The Qingdao eDNA and the sea yang eDNA are simulated into two groups A and B with the quantity of 1000 according to the haplotype data, and the Qingdao living bodies (150) and the sea yang living bodies (75) are two groups C and D. For group A, haplotypes with a frequency higher than 0.1% were selected for group B, the haplotype gene frequency lower than 0.1% was calculated as 0, and false positive data in only one parallel sample was deleted.
Population genetic analysis was performed on four populations a, B, C, and D, first using DNAman for sequence comparison, dnaSP v6 software for defining haplotypes, arlequin3.5 software for calculating genetic differentiation index, molecular variance variation (AMOVA) analysis, and genetic distance calculation, popart-1.7 software for plotting a haplotype network map (fig. 2), and ntsyspc2.1 software for constructing a cluster map from genetic identity (fig. 3).
The four populations altogether define 25 haplotypes, with 18, 21,9,4 for a, B, C, D, respectively. The genetic differentiation index was calculated to be 0.015 (< 0.05), and the molecular variance variation (AMOVA) analysis was shown in table 3, and the results indicated that the variation was mainly from within the population, with a 98.46% ratio. The results of the genetic distances are shown in Table 4, with a maximum of 0.0351 (< 0.05) for populations B and C, indicating minimal genetic differentiation, and no consideration. The above results further demonstrate that there is no significant difference between population genetic analysis and living organisms based on the eDNA data, and that eDNA can obtain more data information.
TABLE 3 analysis of variance of four populations of molecules (AMOVA)
TABLE 4 genetic distance
The results show that the evaluation of the genetic diversity of the Chinese prawn population by a water sample eDNA high-throughput sequencing method instead of in-vivo sampling is feasible and the results are reliable. Moreover, the high-throughput sequencing method for the water sample eDNA is far superior to the existing in-vivo sampling method in aspects of sample collection, injury to population, cost, result reliability and the like.
Claims (6)
1. A method for evaluating genetic diversity of a population of chinese prawns, said method comprising the steps of:
1) Water sample collection
Collecting water samples of natural sea areas of the population distribution of the Chinese prawns to be monitored;
2) eDNA extraction
Extracting a DNA sample on the filter membrane enriched with eDNA, and diluting for later use;
3) Chinese prawn eDNA-PCR amplification
Amplifying a fragment of a mitochondrial COI gene of the Chinese prawn;
4) High throughput sequencing of PCR products
OTU clustering is carried out on the sequences of the amplified products according to 97% similarity, and the sequences are compared with NCBI database to obtain the sequences of the mitochondrial COI genes of the Chinese prawns derived from the eDNA of the water sample; and (3) BLAST comparison is carried out on the obtained COI sequence, variant SNP loci are screened, variant SNP loci form SNP locus haplotypes, and the proportion of different SNP loci is counted to be used as the SNP locus haplotype result of the Chinese prawn mitochondrial COI gene, so that the genetic diversity level of the Chinese prawn population living in the water sample acquisition area is reflected.
2. The method according to claim 1, wherein the water sample is collected at a position 2-5 m away from the sea floor, the collected seawater is filtered by using a glass fiber membrane of 0.45 μm with the aid of a vacuum pump, and the eDNA is extracted from the filter membrane enriched with eDNA.
3. The method of claim 1, wherein 2) is performed using a micro DNA/RNA extraction kit DNeasy Blood and Tissue kit for the extraction of the eDNA, while using a micro spectrophotometer for the quantification of the eDNA and dilution with sterilized double distilled water.
4. The method according to claim 1, wherein the primer set used in 3) has a forward primer sequence of SEQ ID NO. 1 and a reverse primer sequence of SEQ ID NO. 2.
5. The method according to claim 1, wherein the amplification reaction system composition in 3) comprises 12. Mu.L of Monamp2 XTaq Mix Pro, 1. Mu.L of 10mM forward and reverse primers each, 1. Mu.L of 50 ng/. Mu.L of template DNA, 10. Mu.L of ddH 2 O。
6. The method of claim 1, wherein the amplification reaction procedure in 3) is as follows: pre-denaturation at 94℃for 3min; denaturation at 94℃for 20s, annealing at 56.8℃for 20s, elongation at 72℃for 40s,35 cycles; re-extending at 72 deg.c for 10min; preserving at 4 ℃.
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