CN115323060A - Fish nuclear gene molecular marker primer, molecular marker and molecular marker database - Google Patents

Fish nuclear gene molecular marker primer, molecular marker and molecular marker database Download PDF

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CN115323060A
CN115323060A CN202210345227.4A CN202210345227A CN115323060A CN 115323060 A CN115323060 A CN 115323060A CN 202210345227 A CN202210345227 A CN 202210345227A CN 115323060 A CN115323060 A CN 115323060A
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牟希东
杨叶欣
刘奕
宋红梅
徐猛
房苗
刘超
汪学杰
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Pearl River Fisheries Research Institute CAFS
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Abstract

The invention discloses a fish universal nuclear gene molecular marker primer, a molecular marker and a molecular marker database, wherein the molecular marker primer comprises one or more of primers shown as SEQ NO.1-SEQ NO. 328. According to the invention, 82 universal nuclear gene molecular markers are screened by comparing 132 kinds of fish complete genome data, and the overall PCR success rate of the designed molecular marker primer set is up to 80%. According to the invention, a large number of universal nuclear gene molecular markers are screened out, so that germplasm resource protection is facilitated, and on the other hand, a fish nuclear gene database can be constructed and phylogenetic trees and classifications can be constructed on the basis of the molecular markers, so that rapid germplasm identification of fish is realized, and the problems of time consumption, labor consumption and high cost in the prior art are reduced on the premise of ensuring the accuracy. The method can be used for analyzing the evolutionary origin of the fishes and the like, providing data support and promoting the genetic evolution research of the related fishes.

Description

Fish nuclear gene molecular marker primer, molecular marker and molecular marker database
Technical Field
The invention relates to acquisition of a fish nuclear gene marker and a molecular marker database, in particular to acquisition of a fish nuclear gene molecular marker and establishment of a gene database, and more particularly relates to a fish nuclear gene molecular marker primer, a molecular marker and a molecular marker database.
Background
With the technical development of genomics, more and more fish genomes and transcriptomes around the world are determined, and the exploitation of universal nuclear gene molecular markers by using a plurality of fish genomes becomes a very effective means. RAG1 is a widely used nuclear gene molecular marker so far, mainly because it can be easily amplified by PCR in vertebrates and also has a good phylogenetic resolution. However, the nuclear gene molecular marker data which are common like RAG1 are very few at present. In recent years, many scholars have done many similar tasks. For example, li et al (Li et al 2007) screened 10 molecular marker primers suitable for teleost based on the whole genome sequences of two fishes (zebrafish and puffer fish). Townsend et al (Townsend et al.2008) used comparative data of puffer fish, human genome, and chicken to obtain 26 molecular markers suitable for scaly species. Fong and Fujita (Fong and Fujita 2011) reported research efforts to develop 75 nuclear gene molecular markers for vertebrates. However, the above work is achieved by comparing 2-3 existing species genome data, and the success rate of PCR in the distant research group is low. Shen et al (2011) utilized multi-species genome alignment data to screen nuclear coding gene molecular markers and summarized a set of simple, efficient and automated nuclear gene molecular marker screening methods. But if large samples of multiple species are involved, then a great deal of money, time, and labor is required.
Although recent molecular phylogenetic studies have developed a plurality of independent nuclear gene molecular markers, they have group specificity and are not generally widely applicable to studies in the field of conservation of specific fish germplasm resources and the like. Therefore, the fish universal nuclear gene molecular markers still have more defects, and a new fish universal nuclear gene molecular marker with universality and a set thereof are needed to achieve the function of identifying the germplasm resources of fishes.
Disclosure of Invention
The invention aims to overcome at least one defect of the prior art, provides a fish nuclear gene molecular marker primer, a molecular marker and a molecular marker database, is beneficial to protection of germplasm resources, quickly identifies germplasm, is convenient to analyze evolutionary origin relationship of fish, and promotes research on heredity and evolutionary direction of fish.
The group records over 34,000 species of capelin (actinomycetes), the predominant species in vertebrates. Finfish have evolved remarkable morphological and ecological diversity over the past 4 million years of evolution history. Many attempts to solve the phylogenetic problem at different taxonomic levels range from traditional Sanger sequencing using 20 genetic markers to the genome-scale method. However, despite the use of hundreds or thousands of tokens, some high-level relationships remain difficult to resolve or achieve consensus. Transcriptome sequencing is rapid and simple, but requires fresh samples and is therefore not suitable for ethanol preservation or for dry samples. In addition, the identification of orthologs is difficult due to the problems of many homologous isomers, and the difference in expression of genes in different tissues or life stages. The sequence capture method requires much lower sample quality and is even suitable for highly degraded DNA. However, sequence capture efficiency is strongly affected by probe-to-target DNA sequence differences.
Given that finfish is a highly diverse and ancient population, it can be difficult to design universal capture probes and efficiently capture sequences in highly differentiated lineages. Large-scale studies typically require multiple probe sets to fit different lineages, which greatly increases the budget for probe synthesis. Furthermore, these genomic methods require expensive equipment and many fish scientists around the world may still be burdened with next generation sequencing. Furthermore, many studies do not require hundreds to thousands of markers; based on empirical and simulated data, 20 to 50 loci are generally sufficient to accurately restore well-resolved phylogeny. One of the solutions to these problems is to revert to the traditional PCR-based method, which can be performed in almost every molecular laboratory. Downstream data analysis is simple and can be handled by many researchers. The major drawbacks of using this traditional approach in fish phylogeny are the high labor required to perform PCR on many loci and many samples (since PCR success rates tend to be unpredictable), the need to design universal PCR primers, and the high cost of standard Sanger sequencing.
However, these deficiencies can be largely overcome by careful selection of the optimal number of phylogenetic information markers, design of universal PCR primers with high PCR success using many publicly available fish genomes and transcriptomes, and then sequencing a large number of PCR products. If necessary, by next generation sequencing. The fish universal nuclear gene molecular marker provided by the invention can achieve the effect and overcome the problems, and the invention provides a group of Nuclear Protein Coding Loci (NPCL) with rich information so as to close the difference between the traditional marker sequencing and a more advanced high-throughput method.
In the invention, above one embodiment, single copy NPCL is identified from 132 fish genomes, and PCR amplification rates of 203 species of 31 finfish are further screened according to PCR primer specificity and PCR amplification rate to finally obtain 82 markers. In combination with homologous genes identified from genomes and transcriptomes of other fish species, our phylogenetic analysis yielded resolution comparable to phylogenetic studies using hundreds of markers, and by introducing new taxonomic groups, it was possible to provide new clues to the phylogeny of certain lineages. Thus, our 82 NPCL markers provide an alternative to high-throughput methods to perform phylogenetic project studies of various fish branches, and a new approach to fish germplasm resource identification.
The invention adopts the technical scheme that a universal nuclear gene molecular marker primer for fish comprises one or more of primers shown as SEQ NO.1-SEQ NO. 328.
Further, one or more primer pairs in NP 1-NP 82 are included, wherein the primer pair NP1 is shown as SEQ NO.1-SEQ NO.4, the primer pair NP2 is shown as SEQ NO.5-SEQ NO.8, the primer pair NP3 is shown as SEQ NO.9-SEQ NO.12, the primer pair NP4 is shown as SEQ NO.13-SEQ NO.16, the primer pair NP5 is shown as SEQ NO.17-SEQ NO.20, the primer pair NP6 is shown as SEQ NO.21-SEQ NO.24, the primer pair NP7 is shown as SEQ NO.25-SEQ NO.28, the primer pair NP8 is shown as SEQ NO.29-SEQ NO.32, the primer pair NP9 is shown as SEQ NO.33-SEQ NO.36, the primer pair NP10 is shown as SEQ NO.37-SEQ NO.40, the primer pair NP11 is shown as SEQ NO.41-SEQ NO.44, the primer pair NP12 is shown as SEQ NO.45-SEQ NO.48, the primer pair NP 13-SEQ NO.52, the primer pair NP 52-SEQ NO.53, the primer pair NP15 is shown as SEQ NO.57-SEQ NO.60, the primer pair NP16 is shown as SEQ NO.61-SEQ NO.64, the primer pair NP17 is shown as SEQ NO.65-SEQ NO.68, the primer pair NP18 is shown as SEQ NO.69-SEQ NO.72, the primer pair NP19 is shown as SEQ NO.73-SEQ NO.76, the primer pair NP20 is shown as SEQ NO.77-SEQ NO.80, the primer pair NP21 is shown as SEQ NO.81-SEQ NO.84, the primer pair NP22 is shown as SEQ NO.85-SEQ NO.88, the primer pair NP23 is shown as SEQ NO.89-SEQ NO.92, the primer pair NP24 is shown as SEQ NO.93-SEQ NO.96, the primer pair NP25 is shown as SEQ NO.97-SEQ NO.100, the primer pair NP26 is shown as SEQ NO.101-SEQ NO.104, the primer pair NP27 is shown as SEQ NO.105-SEQ NO.108, the primer pair NP28 is shown as SEQ NO.109-SEQ NO.112, and the primer pair NP 113-SEQ NO.116, the primer pair NP30 is shown as SEQ NO.117-SEQ NO.120, the primer pair NP31 is shown as SEQ NO.121-SEQ NO.124, the primer pair NP32 is shown as SEQ NO.125-SEQ NO.128, the primer pair NP33 is shown as SEQ NO.129-SEQ NO.132, the primer pair NP34 is shown as SEQ NO.133-SEQ NO.136, the primer pair NP35 is shown as SEQ NO.137-SEQ NO.140, the primer pair NP36 is shown as SEQ NO.141-SEQ NO.144, the primer pair NP37 is shown as SEQ NO.145-SEQ NO.148, the primer pair NP38 is shown as SEQ NO.149-SEQ NO.152, the primer pair NP39 is shown as SEQ NO.153-SEQ NO.156, the primer pair NP40 is shown as SEQ NO.157-SEQ NO.160, the primer pair NP41 is shown as SEQ NO.161-SEQ NO.164, the primer pair NP42 is shown as SEQ NO.165-SEQ NO.168, the primer pair NP43 is shown as SEQ NO. 172-SEQ NO.169, and the primer pair NP 169 as SEQ NO. 176-SEQ NO.169, the primer pair NP45 is shown as SEQ NO.177-SEQ NO.180, the primer pair NP46 is shown as SEQ NO.179-SEQ NO.184, the primer pair NP47 is shown as SEQ NO.185-SEQ NO.188, the primer pair NP48 is shown as SEQ NO.189-SEQ NO.192, the primer pair NP49 is shown as SEQ NO.193-SEQ NO.196, the primer pair NP50 is shown as SEQ NO.197-SEQ NO.200, the primer pair NP51 is shown as SEQ NO.201-SEQ NO.204, the primer pair NP52 is shown as SEQ NO.233-SEQ NO.208, the primer pair NP53 is shown as SEQ NO.209-SEQ NO.212, the primer pair NP54 is shown as SEQ NO.213-SEQ NO.216, the primer pair NP55 is shown as SEQ NO.217-SEQ NO.220, the primer pair NP56 is shown as SEQ NO.233-SEQ NO.224, the primer pair NP57 is shown as SEQ NO.225-SEQ NO.228, the primer pair NP58 is shown as SEQ NO.229-SEQ NO.232, and the primer pair NP59, the primer pair NP60 is shown as SEQ NO.237-SEQ NO.240, the primer pair NP61 is shown as SEQ NO.241-SEQ NO.244, the primer pair NP62 is shown as SEQ NO.245-SEQ NO.248, the primer pair NP63 is shown as SEQ NO.249-SEQ NO.252, the primer pair NP64 is shown as SEQ NO.253-SEQ NO.256, the primer pair NP65 is shown as SEQ NO.257-SEQ NO.260, the primer pair NP66 is shown as SEQ NO.261-SEQ NO.264, the primer pair NP67 is shown as SEQ NO.265-SEQ NO.268, the primer pair NP68 is shown as SEQ NO.269-SEQ NO.272, the primer pair NP69 is shown as SEQ NO.273-SEQ NO.276, the primer pair NP70 is shown as SEQ NO.277-SEQ NO.280, the primer pair NP71 is shown as SEQ NO.281-SEQ NO.284, the primer pair NP72 is shown as SEQ NO.285-SEQ NO.288, the primer pair NP73 is shown as SEQ NO.289-SEQ NO.292, the primer pair NP74 is shown as SEQ NO.293-SEQ NO.296, the primer pair NP75 is shown as SEQ NO.297-SEQ NO.300, the primer pair NP76 is shown as SEQ NO.301-SEQ NO.304, the primer pair NP77 is shown as SEQ NO.305-SEQ NO.308, the primer pair NP78 is shown as SEQ NO.309-SEQ NO.312, the primer pair NP79 is shown as SEQ NO.313-SEQ NO.316, the primer pair NP80 is shown as SEQ NO.317-SEQ NO.320, the primer pair NP81 is shown as SEQ NO.321-SEQ NO.324, and the primer pair NP82 is shown as SEQ NO.325-SEQ NO. 328.
Another objective of the invention is to provide a fish universal nuclear gene molecular marker, which is formed by amplification of the molecular marker primer set.
The invention also aims to provide a fish universal nuclear gene molecular marker database, which comprises the fish universal nuclear gene molecular marker.
The invention also aims to provide the application of the fish universal nuclear gene molecular marker primer, the fish universal nuclear gene molecular marker and the fish universal nuclear gene molecular marker database in germplasm resource protection. Further provides application of the fish germ plasm resource and category identification reagent.
Another object of the present invention is to provide a fish species identification method, comprising the steps of:
a1, obtaining DNA of a fish to be identified;
a2, carrying out PCR amplification through the primer of claim 1;
and A3, performing high-throughput sequencing on the PCR amplification product obtained in the step A2, comparing the PCR amplification product with the fish universal nuclear gene molecular marker database of claim 4, and acquiring species information of the fish to be identified according to molecular markers matched with the fish to be identified in the molecular marker database and a phylogenetic classification tree established on the basis of the molecular marker database.
Another object of the present invention is to provide a kit comprising the above-mentioned fish-universal nuclear gene molecular marker and/or the above-mentioned fish-universal nuclear gene molecular marker primer. Further comprises reagents required by a PCR amplification system of the fish universal nuclear gene molecular marker primer.
The invention further aims to provide a phylogenetic model, which is constructed by the fish universal nuclear gene molecular marker primer, the fish universal nuclear gene molecular marker and/or the fish universal nuclear gene molecular marker database. Further, a phylogenetic tree is included
The screening method of the fish universal type nuclear gene molecular marker database comprises the following steps:
(1) Similarity alignment is carried out on all gene sequences of 132 fishes with published genome sequences, the alignment method is blastn (version 2.10.1 +), the alignment strategy is blastn, the maximum expectation value is 1e -10 If non-unique alignment occurs, selecting an optimal alignment sequence; according to the comparison result, the candidate genes are screened according to the following standards: more than 80% of species can detect the same similar sequence, and the alignment length of more than 60% of the species in the related species is required to be more than or equal to 1000bp;
(2) Performing multi-sequence comparison on the homologous gene set established in the step (1) by adopting a multi-sequence comparison strategy, wherein the comparison software is mafft (v 7.475), and screening conserved segments among genes according to the multi-sequence comparison result; according to the multiple sequence comparison result, counting the position of the first non-blank comparison base and the position of the last non-blank comparison base of each gene sequence, converting the positions into 100bp position units, and respectively taking the position with the largest number of genes as a candidate conservative segment; carrying out multi-sequence comparison on the selected conserved segments by reusing mafft (v 7.475), screening the conserved segments, and counting the lengths of the conserved segments of various species to obtain the length range of the conserved segments of each gene;
(3) According to the result of multi-sequence comparison, selecting a conserved region by combining primer3 software to design a nested PCR primer, selecting the conserved region by primer design, particularly controlling the 3 'end conservation, wherein the 3' end does not contain degenerate bases, the amplification length is controlled within the range of 1.0-1.5 Kb, the GC proportion is controlled within the range of 40% -70%, the primer length is controlled within the range of 20 bp-50 bp, and the designed primer is compared back to a gene sequence again to evaluate the amplification success rate of the primer and the correct rate of site combination;
(4) Respectively comparing the similarity of the designed nested PCR primers with other genes of a gene family, if the similarity of the PCR primers and other non-target sequences is higher than 95%, redesigning the primers, and if a proper region cannot be found for redesigning, not considering the gene sequence;
(5) Randomly selecting a plurality of species in different orders by taking the order as a unit to perform a PCR experiment, performing high-throughput sequencing on a PCR product, and assembling sequencing fragments.
Compared with the prior art, the invention has the following beneficial effects: the invention utilizes the comparison of 132 kinds of fish complete genome data to screen 82 general nuclear gene molecular markers, and the integral PCR success rate of the correspondingly designed primer is up to 80 percent. According to the invention, a large number of universal nuclear gene molecular markers are screened out, so that germplasm resource protection is facilitated, and on the other hand, a fish nuclear gene database can be constructed and phylogenetic trees can be constructed and classified on the basis of the molecular markers, so that rapid germplasm identification of fish is realized, and the problems of time consumption, labor consumption and high cost in the prior art are reduced on the premise of ensuring accuracy. The method can be used for analyzing the evolutionary origin of the fishes, providing data support and promoting the genetic evolution research of the related fishes.
Drawings
FIG. 1 shows the PCR amplification success rate (a) and FishPIE marker search rate (b) between targets.
FIG. 2 shows the genetic distance (percent distance) of 82 FishPIE markers at the order (blue), family (green) and genus (yellow) levels; genes have been ordered by intergeneric level genetic distance.
Fig. 3 shows a graph of tree distance (normalized RF index) of the ML tree, constructed by concatenating the fisherpe labels one-to-one with the reference tree, showing that the dip at 40 labels tends to plateau.
FIG. 4 shows a mesh stem tree of a radial finfish based on a peptide sequence ML analysis; the numbers in parentheses after the item names indicate the number of families and species analyzed.
FIG. 5 shows the constructed identification classification map and an example enlargement.
FIG. 6 shows the results of gel electrophoresis of the PCR amplification products of example 3 for the kcnf and mab genes.
FIG. 7 shows the results of gel electrophoresis of the PCR amplification products labeled with NP11 and NP12 in example 3.
FIG. 8 shows the results of gel electrophoresis of the PCR amplification products labeled with NP13 and NP14 in example 3.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
The present invention will now be further described with reference to specific examples, which are intended to be illustrative only and not to be limiting. The test samples and test procedures used in the following examples include the following (generally, according to the conventional conditions or according to the conditions recommended by the reagent company if the specific conditions of the experiment are not specified in the examples; and reagents, consumables and the like used in the following examples are commercially available unless otherwise specified).
Example 1
1. Orthologous identification
To identify homologous genes, 132 were first iterated over a 5kb sliding windowThe genome of fish (shown in Table 1) was fragmented into 10kb sequences. The sequences of each species were then aligned to the zebrafish genome (GCF — 000002035.6) using BLASTN with a maximum e value of 1e -10 The minimum alignment length is 1000bp.
Using a maximum e value of 1e -10 BLASTN (R), detects one-to-one orthologs by the mutual best hits. Then, the following are reserved: (1) Orthologs located within annotated protein coding regions in the zebrafish genome, (2) exon length>1000bp, and (3) a match can be found in 80% of the tested fish. A total of 2093 genes met the above criteria.
2. Primer design
The sequences in each homologue were then aligned using MAFFT (ver 7.455). The conserved regions with the least number of gaps and the most conserved sites were calculated in 25bp sliding steps within a 50bp window using custom scripts.
According to the nested PCR strategy, target PCR amplicons can be obtained efficiently, and the inventors designed two pairs of primers for these homologous sequences, respectively.
The primers were selected based on the following criteria: (1) the primer should be located within a conserved region; (2) The 3' end of the primer must be particularly conserved and must not contain ambiguous sites; (3) The target region should contain 40% -70% of GC content, and the size is larger than 800bp; (4) the length of the primer should be between 20-40 bp. Primers were then mapped onto the genome of 132 fish and the specificity was assessed using BLASTN. Primers matching greater than 95% similarity to non-target regions, primers whose length is greater than 80% inconsistent with the target region by >90%, and primers that show mismatches in the last three bases of the 3' end are considered to be non-specific.
The nested PCR primer sets for each ortholog were ranked according to the number of species specific for the primers, and then 102 loci of the top-ranked primer set were selected as the fisherpe test primer set. The inner primers are indexed for PCR amplification.
3. Primer testing
To examine the general applicability of primers to ray-fined fish, we performed primer tests on each of 102 sites from 203 species of 31-purpose fish. Wherein the fresh or ethanol preserved sample is from the national fresh water genetic resource center, which is the preservation center of the national aquatic organism samples and life specimens.
Genomic DNA of these species was extracted using fish DNA extraction kit (cwbiotech, china). PCR was performed using the standard protocol of the kit, and the annealing temperatures are shown in Table 2. The success rate of PCR was assessed by gel electrophoresis on a 1% agarose gel. Successful PCR must produce products that match the predicted size, and in the case of non-specific priming, the band for non-specific products must be less than 4, and the band is easily distinguishable, and the concentration of non-specific products must be low.
Equal amounts of PCR products of the same species were then pooled, fragmented and treated with Taq DNA polymerase for end repair, 5' phosphorylation and dA tailing, followed by T-a ligation to add adaptors at both ends. And (3) sequencing on an Illumina Hiseq 4000 platform by adopting a 150bp opposite-end sequencing method. The generated readings were assembled from a number of Kmer sizes, SOAPdenovo (ver 2.04) at the default setting. Then, we use a maximum e value of 1e -10 The resulting sequences were matched to the reference genome of the most closely related species (as shown in table 1). Among the orthologous sequences that best hit these choices are considered valid sequences. And finally screening 82 NPCL as molecular markers (the screened NPCL marker set forms a FishPIE set in the text before and after), wherein the corresponding primer sequences are shown as SEQ ID NO.1-SEQ ID NO. 328.
4. Testing NPCL markers for phylogenetic reconstruction
To examine the phylogenetic utility of selected sites to resolve phylogenetic relationships at different taxonomic levels, we obtained homologous sequences of these sites from genomic and transcriptomic data of NCBI SRA, thereby improving the taxonomic coverage of the dataset.
Genome sequencing reads were assembled using soaptovo 2, transcriptomes were assembled using Trinity ver 2.8.5 (Grabherr et al, 2011), using default parameters. Using a maximum e value of 1e -10 BLASTN ofThe resulting sequence is mapped to the reference genome of the most closely related species. The mutually best hits of the selected loci are considered as valid sequences.
Potential paralogous sequences were then filtered by examining each gene tree. A gene tree was constructed by aligning the sequences of each selected locus using MAFFT. Phylogenetic analyses were then performed using IQtree2 ver 2.0.5 (min et al, 2020) and edge-associated scale partition models, ultra-fast boot replication, and 1000-fold SH-approximation likelihood ratio tests.
The phylogenetic resolution of all orthologous markers was examined jointly. Selected homologous sequences were aligned again using MAFFT and trimmed using the trimAL ver 1.4.Rev15 ("gappyout" setting of Capella-guillerez, silla-Martinez and Gabaldon, 2009). On the nucleotide and polypeptide data sets, the optimal surrogate model for each marker was selected using IQtree2, respectively, followed by gene partitioning (same branch length but different evolution rate) and 1000 ultrafast bootstrap reconstruction using the same software. We used ExaBayes ver 1.5.1 to perform bayesian inference (run at least 10000 generations using four chains and ASDSF < 5.00%).
5. Evolution rate of FishPIE
To examine the evolution rate, we used the original pairwise distance computed by the dist.dna function of R package ape as a proxy. We examined the average pairwise distances between genera within a family, between families within an order, and between orders within a cohort (taxonomic classification follows Rabosky et al).
6. Phylogenetic information content of FishPIE marker set
To test whether the number of markers in the fisher phiie set is sufficient to produce a credible phylogeny, we examined whether the phylogeny tree converged to a similar topology as the number of markers increased. Since this analysis is very time consuming, we narrow the dataset by selecting only individuals with >50% sequenced markers and at most two representatives from one genus.
Then, we obtained the identity of phylogenetic signals between the fisherpe markers by calculating the tree distance (normalized RF index) between each nucleotide gene tree and the reference species tree using the rf.dist function of R package phangorn (Schliep, 2010). The IQtree is calculated using the default settings and the reference species is reconstructed using the joined data sets based on the same method. Then, we constructed multiple phylogenetic trees by linking FishPIE tags together in ascending order of normalized RF according to the respective gene tree. The tree distance (normalized RF index) between the individual reconstruction and the reference species tree is calculated as described above.
7. Annotation of FishPIE tags
BLASTP-based 1e -10 Cutoff value of E-value a representative sequence for each orthologue was searched in the Uniport and nr databases. Genes were initially identified based on best hits to known sequences. And Gene function was inferred using the KEGG, gene Ontology, PANTHER, and KOG databases.
8. Results
1. Obtaining FishPIE database
From the 132 species genome, 2093 single copy orthologs were identified, of which 102 were selected for primer design. 82 NPCL markers showed high PCR success (> 75%) in 203 species from 67 families and 31 orders. After trimming the primer sequences and ambiguous region, the average PCR product size for each marker ranged from 544 to 1143 bp, with an average of 1051bp. PCR products obtained by PCR of 82 NPCL marker primer sets form a molecular marker database which can be used for sequence alignment and identification of fish germplasm resources.
We obtained orthologous sequences from the RNA-seq data (268 species), genome sequencing data (151 species) and assembled genomes (132 species) of public databases. The trimmed alignment length of the 82 NPCL marker sequences in the combined dataset varied from 420 to 1244bp (mean =712 bp), amounting to 58,386bp, with gc contents ranging from 37.58% to 58.58% (mean 47.66%) (table 3)). The number of abbreviated information sites ranged from 113 to 602bp (Table 3).
The labels are functionally diverse. For example, in the PANTIER database, 80 markers can be annotated to 74 gene families, where "teneurin and n-acetylglucosamine-1-phosphodiester α -n-acetylglucosaminidase", "voltage-gated potassium channel", "chondroitin synthase", and "serine/threonine-protein kinase" each contain two markers, and three of these are annotated as "PTHR24028: unnamed family ". Gene ontology analysis showed that "biological processes", "molecular functions" and "cellular components" were associated with 65, 64 and 57 markers, respectively.
2. Broad application of FishPIE in classification
Using the nested PCR strategy, the PCR success rate of the fisherpe primers averaged 74.68% across 203 species. Species from the "crown" group Otomorpha (81.1%) and euteleosteomorph (71.4%) had higher PCR success rates than species from elocomorpha (46.1%), ostelossomorpha (47.9%) and outliers (47.0%) (fig. 1). This is probably because most of the sequences used for primer design are from Otomorpha and Euteleosteomorpha. Nevertheless, these two lines cover more than 96% of the fish (Fricke et al, 2021). Therefore, the FishPIE primers can be widely applied to all taxonomic groups.
3. Evolution rates of FishPIE at different classification levels
In selecting markers for phylogenetic analysis at different classification levels, the evolutionary rate (using genetic distance as a proxy) is one of the most important attributes, since it is the main determinant of the resolution of the phylogenetic reconstruction. The genetic distance of the FishPIE markers at the interocular, family and genus levels is shown in FIG. 2. At the interocular level, the genetic distance of 82 markers ranged from 2.52% to 14.47%, with an average of 6.70%; RAG1 is a NPCL marker widely used in fish phylogenetic analysis with a genetic distance of 6.81%. The genetic distance between each family is 1.48% -9.45%, the average is 4.20%, while the genetic distance of RAG1 is 4.58%. At the intergeneric level, genetic distance averages 1.88% (0.58% to 5.19%), whereas that of RAG1 is 1.69%.
Therefore, our FishPIE tags provide evolution rates comparable to widely used tags, and thus can provide a large amount of information for resolving phylogeny at different evolutionary time scales.
4. Information content of FishPIE for phylogenetic reconstruction
One of the most common problems in phylogenetic studies is: whether enough information has been collected to resolve the target phylogeny. To solve this problem, we constructed phylogenetic trees by linking FishPIE tags one by one in ascending order of normalized RF based on individual gene trees. The graph shows that at 40 markers, the drop in RF levels off, with a normalized RF of 0.224 (fig. 3). The number of markers (or genetic data) is consistent with the view of Capella-Gutierrez, kauff and Gabald6n, i.e., not all relationships require genome-scale datasets to resolve, rather, judicious selection of markers may be sufficient to produce fully resolved target phylogeny. Since the dataset of the present invention covers a wide range of taxonomic levels and shows high phylogenetic accuracy at the three taxonomic levels of analysis (see below), convergence of the tree topology at around 40 markers means that 82 well-chosen fisherpie markers are sufficient to resolve a significant part of the fish phylogeny.
5. Overall phylogenetic Properties of FishPIE
Linked genetic and species trees are usually well supported. The mean ultrafast navigation (BP) support rates for the nucleotide and peptide ML trees were 96.26% and 97.00%, respectively, the mean Posterior Probability (PP) for the two BI trees was 0.96 and the mean PP for the ASTRAL tree was 0.87.
To further quantify the phylogenetic performance of FishPIE, we calculated a phylogenetic accuracy index for each gene tree, defined as the average of ML BP support for four widely accepted clades of genus, family and order. If no uniline of branches is recovered in the gene tree, bootstrap support will be considered negative.
All these clades were found to be well supported in the linked gene tree and ASTRAL species tree (ML tree BP =100%, BI tree PP >0.9, ASTRAL PP =1, except Euteleosteomorpha was 0.74).
In general, the phylogenetic properties of fisherpe are satisfactory at three levels. The average accuracy index was highest at the intergeneric level (74.71, ranging from-7.25 to 100), followed by the internodal (69.51, ranging from-50 to 100), and lowest at the interocular level (55.68, ranging from-100 to 100). Among these markers, ashll, prdmll and LOC100333177 showed complete support for all clades available for analysis, making them excellent markers for phylogenetic analysis at different taxonomic levels. The overall average accuracy index of the other 19 markers, including the commonly used RAG2 marker, exceeded 90. Thus, these markers can also be used in broad-spectrum phylogenetic analysis.
Our fisherpe primer set costs less per base sequencing compared to other systematic genomics approaches. Therefore, we believe that they constitute an important tool package for facilitating fish phylogenetic studies. The FishPIE can be used in combination with other sequencing methods to increase taxon coverage for further phylogenetic studies.
The selection of molecular markers is probably the most critical decision in any molecular phylogenetic study. The selection must be balanced between DNA quality, compatibility with existing equipment and data sets, phylogenetic resolution and budget. The fisherpe label has advantages in all of these areas. Since PCR is now a standard procedure in molecular laboratories, the fisherpe labeling provides a smooth transition from traditional protocols to the latest high-throughput methods. We also demonstrated high rates of retrieval of fisherpe orthologs from NGS datasets, allowing ready access to existing data. Although the total number of features of fisherpe is less than that used in phylogenetic studies (typically more than ten thousand sites), our markers exhibit comparable phylogenetic resolution at different taxonomic levels. Taking PCR as a routine approach, the initial investment in fish genomics using FishPEE was only the cost of primers, and less than $ 1000 for all 102 tested primers in our study was sufficient to test >200 samples. For other fish phylogenetic toolkits, such as UCE, AHE and exon trapping, the initial investment is at least three to four times higher. For approximately 200 samples, we estimated the cost of sequencing all the fisherpe labels per sample to be approximately $ 25. Therefore, the FishPIE has good cost performance.
TABLE 1 reference genomic fish species and genomic information Table
Figure BDA0003576281250000111
Figure BDA0003576281250000121
Figure BDA0003576281250000131
Figure BDA0003576281250000141
Figure BDA0003576281250000151
TABLE 2 primer sequence information and PCR conditions
Figure BDA0003576281250000152
Figure BDA0003576281250000161
Figure BDA0003576281250000171
Figure BDA0003576281250000181
Figure BDA0003576281250000191
Figure BDA0003576281250000201
Figure BDA0003576281250000211
Figure BDA0003576281250000221
Fishpie marker details show alignment length, GC content, reduced information sites and the proportion of reduced information sites in the complete data set (sequences generated in connection with this study and obtained from publicly available transcriptome and genomic data).
Figure BDA0003576281250000222
Figure BDA0003576281250000231
Figure BDA0003576281250000241
Example 2
Fish nuclear gene molecular marker library building process
The NPCL marker obtained by screening can be used for constructing a molecular marker database for sequence comparison, identification of germplasm resources of family species and the like. The partial library building process represented by random 10 markers in 82 molecular markers and random 10 fishes in the previous embodiment is exemplified.
Step 1, sample pretreatment, randomly selecting 10 fishes, shearing fin rays, and extracting DNA. From the 82 molecular markers screened, 10 were randomly selected for PCR amplification verification.
Step 2, nested PCR:
out PCR using the extracted DNA as a template and an outer primer (out PCR Prim)er) amplifying it. Reaction system: 2xPCR Mix 5. Mu.l, primer F1.5. Mu.l, primer R1.5. Mu.l, RNA Free H 2 O3. Mu.l, template 1. Mu.l, 10. Mu.l in total. Reaction conditions are as follows: performing pre-denaturation at 94 deg.C for 3min; denaturation at 94 ℃ for 30s, and annealing at 52 ℃ for 40s for 30 cycles; extension at 72 deg.C for 2min; finally, extension was carried out at 72 ℃ for 7min.
inner PCR: using the out PCR product as a template, it was amplified using inner primers (inner PCR Primer). Reaction system: 2xPCR Mix 10. Mu.l, primer F2. Mu.l, primer R2. Mu.l, RNA Free H 2 O7. Mu.l, template 1. Mu.l, 20. Mu.l in total. The reaction conditions are as follows: pre-denaturation at 94 ℃ for 2min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 40s, for 35 cycles; extension at 72 ℃ for 1min for 40 s; finally, extension was carried out at 72 ℃ for 7min. ( Note that: if no band is seen after electrophoresis detection of inner PCR products of partial reaction, the number of cycles of out PCR reaction can be adjusted to 35 or the annealing temperature of inner PCR reaction can be adjusted to 50 DEG C )
And 3, detecting by agarose gel electrophoresis. Glue concentration: gel concentration 1%, voltage 120V, time 30min. Marker: marker is DM2000. The DM2000 DNA Marker consists of 6 DNA fragments which are respectively 2,000bp, 1,000bp, 750bp, 500bp, 250bp and 100bp. 4. Mu.l of the sample was subjected to direct electrophoresis, and the amount of the 750bp DNA fragment was about 120ng, showing a bright band, and the amount of the DNA in the other bands was about 40ng. Taking 4ul of inner PCR product for electrophoresis detection.
And 4, mixing the PCR product. And preliminarily judging the concentration of the PCR products according to the agarose gel electrophoresis detection result of each PCR product, and mixing the inner PCR products of the same sample to make different inner PCR products have similar total amount in the mixture as much as possible.
And 5, purifying and recovering magnetic beads of the PCR product mixture. And (3) carrying out magnetic bead purification and recovery on the mixed PCR product, and comprising the following steps:
and 6, preparing 1.5ml of EP tubes, adding 100 mul of N411 magnetic beads into each tube (the mixture is balanced at room temperature for more than 30min and fully shaken and uniformly mixed before use), and marking. The inner PCR product mixture (about 100. Mu.l) was added to the corresponding EP tube, pipetted well and allowed to stand at room temperature for 10min.
Step 7, placing the incubated solution on a magnetic frame until the solution is clear (about 1 min), and absorbing the supernatant by using a pipette; step 8, keep the EP tube on the magnetic frame, add 200. Mu.l 80% ethanol (ready for use) per tube, incubate for 1min, remove the supernatant. And 9, repeating the step 3. And step 10, completely absorbing and removing the liquid by using a small-range liquid-transferring gun. And 11, uncovering and drying at room temperature for 3-5min, wherein the specific time is based on the reflection of the magnetic beads seen by naked eyes. Step 12, after drying, adding 100. Mu.l ddH into each tube 2 And O, sucking and beating the gun head and uniformly mixing. After incubation for 10min at room temperature, the magnetic frame was mounted. Step 13, when the solution is clear (about 1 min), the supernatant is transferred to a new 0.5ml EP tube using a pipette and labeled. And 14, carrying out Illumina on the PCR product to construct a high-throughput sequencing library, wherein the library construction process is carried out according to the instruction of an Illumina library construction kit.
Example 3
Species classification and identification by DNA molecular marker
The molecular marker database established by the invention is used for classifying and identifying the sequencing species, and classification and identification of the detected species at the level of the order, family, genus and species can be realized. The following description will be made by taking, as an example, a procedure for identifying a target gene by kcnf, mab gene, and primers NP11, NP12, NP13, and NP 14.
1. DNA extraction:
adopting an animal tissue genome DNA rapid extraction kit (CW 2089S, beijing kang is century science and technology limited company) to extract DNA of a fish tissue sample, wherein the operation method is consistent with the specification of the kit; DNA quality detection is carried out by 1% agarose gel electrophoresis at 150V for 25 min.
2. And (3) molecular marker amplification:
(1) The reagents used were:
(1) PCR Mix cat P2011 Dongsheng Biotech limited, guangzhou; (2) agarose sigma; (3) DM2000 cat: CW0632M well is a century; (4) N411 magnetic bead cat: n411-02 Nanjing Novozan Biotech, inc.;
(2) Sample dilution:
since the volume of the DNA sample received in this project is about 10ul, the sample needs to be diluted before the experiment is formally started. Each sample was taken 8ul, and 152ul RNA free H2O (diluted 20-fold) was added.
(3) Nested PCR
out PCR: the diluted template was amplified with an external Primer (out PCR Primer)
a) Reaction system:
TABLE 1 out PCR System
Figure BDA0003576281250000261
b) Reaction conditions are as follows:
TABLE 2 out PCR reaction conditions
Figure BDA0003576281250000262
inner PCR: using out PCR product as template, it was amplified with inner Primer (inner PCR Primer)
a) Reaction system:
TABLE 3 inner PCR System
Figure BDA0003576281250000271
b) The reaction conditions are as follows:
TABLE 4 inner PCR reaction conditions
Figure BDA0003576281250000272
Description of the invention: if no band is visible after electrophoretic detection of the inner PCR product of a partial reaction, the number of cycles of the out PCR reaction can be adjusted to 30 or the annealing temperature of the inner PCR reaction can be adjusted to 50 ℃.
(4) Agarose gel electrophoresis detection
Glue concentration: gel concentration 1%, voltage 120V, time 30min.
Marker: marker is DM2000. The DM2000 DNA Marker consists of 6 DNA fragments which are respectively 2,000bp, 1,000bp, 750bp, 500bp, 250bp and 100bp. 4. Mu.l of the sample was subjected to direct electrophoresis, and the amount of the 750bp DNA fragment was about 120ng, showing a bright band, and the amount of the DNA in the other bands was about 40ng.
Taking 4ul of inner PCR product for electrophoresis detection. Representative results are shown in FIGS. 6, 7 and 8.
3. Library construction:
(1) PCR product mix
And preliminarily judging the concentration of the PCR products according to the agarose gel electrophoresis detection result of each PCR product, and mixing the inner PCR products of the same sample to make different inner PCR products have similar total amount in the mixture as much as possible.
(2) Purification and recovery of magnetic beads from PCR product mixture
And (3) carrying out magnetic bead purification and recovery on the mixed PCR product, and comprising the following steps: 1.5ml EP tubes were prepared, and 100. Mu.l of N411 magnetic beads (equilibrated at room temperature for more than 30min, mixed well by shaking thoroughly before use) were added to each tube and labeled. The inner PCR product mixture (about 100. Mu.l) was added to the corresponding EP tube, pipetted well and allowed to stand at room temperature for 10min. (2) The incubated solution was placed on a magnetic rack until the solution cleared (about 1 min) and the supernatant was aspirated off with a 100. Mu.l pipette. (3) The EP tubes were kept on a magnetic rack, 200. Mu.l of 80% ethanol (ready for use) was added to each tube, and the supernatant was removed after 1min incubation. (4) The EP tubes were kept on the magnetic rack, 200. Mu.l of 80% ethanol (now ready for use) was added to each tube again, and the supernatant was removed after 1min incubation. (5) use 10. Mu.l pipette to aspirate the liquid thoroughly. (6) And (4) uncapping and drying at room temperature for 3-5min, wherein the specific time is based on the condition that the naked eyes see the light reflection of the magnetic beads. (7) After drying, add 100. Mu.l ddH per tube 2 And O, sucking and beating the gun head and mixing uniformly. Incubate for 10min at room temperature and then put on a magnetic rack. (8) When the solution cleared (about 1 min), the supernatant was transferred to a new 0.5ml EP tube using a pipette gun and labeled. Long-term storage at (9) -20 deg.C.
4. High-throughput sequencing:
and (3) sequencing by adopting an Illumina series sequencing platform, reading the sequence with a double end of 150bp, and measuring the sequence amount to be 4 Gb/library.
5. Data analysis
(1) The sequencing molecular markers are assembled and spliced, and second-generation sequencing assembly software such as Spads (v3.15.2), SOAPDennovo (Version 2.04) and the like can be used for assembling.
(2) And comparing the assembled fragments to a molecular marker database by adopting blastn (version 2.10.1 +) comparison software, distinguishing gene sequences corresponding to the molecular markers, and selecting the optimal comparison result as a gene corresponding to the sequencing marker.
(3) And (3) combining the classified molecular markers with the database molecular marker sequences, and performing multi-sequence comparison by adopting mafft (v 7.475) multi-sequence comparison software.
(4) And (4) carrying out optimal model substitution selection by adopting IQTree (version 2.0.5) software, and constructing a system classification tree by iterating for not less than 100 ten thousand times.
(5) And (3) checking the classification tree by adopting analysis software such as FigTree (v1.4.4) and the like in combination with the phylogenetic tree constructed in the embodiment, identifying species, acquiring the classification status corresponding to the detected species, and completing the identification process.
It should be understood that the above-mentioned embodiments of the present invention are only examples for clearly illustrating the technical solutions of the present invention, and are not intended to limit the specific embodiments of the present invention. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention claims should be included in the protection scope of the present invention claims.
Sequence information is shown below
SEQ NO.1CTyTAyGGmGAyGAyAGyTGGGA
SEQ NO.2TAAGGrTCwGCyTChArrGGCAT
SEQ NO.3 GAyAGyTGGGAyrCTCCTGTvAArGArTAC
SEQ NO.4 CAAArGGyArrCTyTCyCTGCGrCyrTAGG
SEQ NO.5 AArGACCAGGAryTGCTGTGGGA
SEQ NO.6 CArCAkGGrGTTTTrCTbCGACT
SEQ NO.7 GCmTCwGAyTCyGGyTGyTTTGATGATGA
SEQ NO.8 CAATdGArGGAGAyTCAAArCTrCCmAGTG
SEQ NO.9 CAGyTGGGnyTGGAGCArGACTT
SEQ NO.10 AGCCrTGyACyArTCCkrCTCTT
SEQ NO.11 GTvAGyCGbGGbATGAAyCGhAACTTTGA
SEQ NO.12 TAGGdrTCyCCrCACATyArrCACATGAC
SEQ NO.13 AArGArGArCAGGAyCCmCCTGT
SEQ NO.14 CTGAArATkGTrGGCArsAGGTC
SEQ NO.15 GCCAwAGrAArCGwGGrCAGhCCCAAGACG
SEQ NO.16 CTGnCGdGAsCGyAGTCTGyArGTACTTCT
SEQ NO.17 CATGCAGTGGmGwCGGCGrCACT
SEQ NO.18 TGvGAkGTGATsAGGTGrCTGTA
SEQ NO.19 ACCArAGCArCTChCTGmGrrGCCTbTGGA
SEQ NO.20 CCCyGAGAACACrTAsCCyGTsCCbGAGCA
SEQ NO.21 CGCATGCACAACCTbGAGAGACT
SEQ NO.22 AAyTCrAAyTGsGGCAGrCTGCA
SEQ NO.23 CGCTTyCTGGArGCyCArCTvCGCAGhGAGAGA
SEQ NO.24 CGyGGGCAyACyTTrATTGGCACTGGGCAGCTGG
SEQ NO.25 CCCCAyCGvCTrGAACGCCTTAG
SEQ NO.26 TGCACyGTCAGrTTGCTrCAGTT
SEQ NO.27 TGGGyyTmATCTCmCAGCAyCCCAGTGCCT
SEQ NO.28 GAkGArGTGGCrAAkGGmGATTGTCTGGA
SEQ NO.29 GAyrTsCCwGArGAmAsCTGGCT
SEQ NO.30 TTGCGrTTnGGsCGCCkrCAkCG
SEQ NO.31 GArGAmAsCTGGCTbCAGnTGTG
SEQ NO.32 CAkCGyTCrTCrATGCTyGGCACrCATTC
SEQ NO.33 CGCAChhTrGCyAChTTyAACTC
SEQ NO.34 TyTCCAGyTTbCkCTCrATGGAC
SEQ NO.35 CCACTCyCyhCAGACnAyrCArAArGTGGA
SEQ NO.36 CnGCyykkCkdGCyTTCTGrATCTGyTTCT
SEQ NO.37 GArGCyyTsTTTCGAyAGyCGCT
SEQ NO.38 CAGyTGrAAvACCTCkGGGATGT
SEQ NO.39 AAyGCbCCbCAGTTCAGyGArAArCGCTA
SEQ NO.40 TCrAACACrGGvGGGTTGTCrTTCAC
SEQ NO.41 AAyGGvTACmGrmGmTTyGACCC
SEQ NO.42 TAyTkCTGryGyArnGCyGGCTC
SEQ NO.43 GGbATGGAGTACACsyTrGACyTnCAGsT
SEQ NO.44 GCyGGCTCnAynGCyCkGAAhACrTG
SEQ NO.45 CAACAAGAACCCnCCyTACCAGT
SEQ NO.46 GArCCCAGrCACATyTTrTAyTG
SEQ NO.47 ACCAGTAyCGCCTdCACAGCTACmTGyTsAGCCGvAAGAT
SEQ NO.48 TAnAChACyTTdGGGTCrTACTGrCTGAAGATGAT
SEQ NO.49 ATGGTrGTGACnvArTGyGGCTG
SEQ NO.50 CCrTCnGArGTrCCrTCrAAGCA
SEQ NO.51 ACdGGmTACAArGGhACmTTCTChrTCCA
SEQ NO.52 GTCnAGdGGbGChArCATdGTGAATCTC
SEQ NO.53 GAGAACTTyATGGACATGGAGTG
SEQ NO.54 TkyCTGTTCTGrCAGTCbGACTC
SEQ NO.55 ACATGGAGTGyTTCATGATmCTGACyCCbAGCCAGCAGCT
SEQ NO.56 CTCCTCArrGCrTAGATGATbGGrTTGACrGTGGAGTTGA
SEQ NO.57 CmCGyAArCGyAGCCAyAGyAGC
SEQ NO.58 GTGGyyGTGATvAGbCCdATCCA
SEQ NO.59 AAyTGGTACAGyTGyGGkGArGGkGAGGT
SEQ NO.60 AhGTCATdGGyTGyTGwGGrTTsACrTGCT
SEQ NO.61 GAyGArGTbCCCAydGCvCAyTC
SEQ NO.62 TArTGdGCrTCCCACACrTCCAT
SEQ NO.63 CTbTChAAryACAGrGsvTGGGGvAArCAG
SEQ NO.64 CGyArCCACTTdGmrCAyTCyCCyArCT
SEQ NO.65 GArGCCATCCChCAGCATATCAA
SEQ NO.66 GCrTGbCCnGCnGCCATGTACTC
SEQ NO.67 TrGGvAAGCTGCAGTACCTCTACCTCCAG
SEQ NO.68 ATrTACTTsGAbTCTGACTTGTAGGACG
SEQ NO.69 GGCAGCCGyTCCTTyCCsTGCCA
SEQ NO.70 GCyTGrCAGCTCATbCkyTTGGC
SEQ NO.71 TGmGrGArAGyCAGGCCAGyGAGGTbGACT
SEQ NO.72 TCrTAGCACTGmACyTTGGGsAGyTTGTC
SEQ NO.73 CTsACvTGGCTCAGyTTCATGCT
SEQ NO.74 CCTCyCkyTCrTAhGTmAGCATG
SEQ NO.75 CTTCCCdCTyATyGChCAyTACTACCTCAC
SEQ NO.76 TCvCCrTTGTCrGCCAkGTTrGCrTC
SEQ NO.77 TCyCGvTACGGACAyyTrGAGGT
SEQ NO.78 TCbGCrCCrCACTCyAGsAGCAG
SEQ NO.79 GCTsTGGGCGGCbTCkGCvGCsGGTCACCT
SEQ NO.80 TTGTCyACrGCCATGTGsAGvGGvGTGAA
SEQ NO.81 ATmTChGCvCTsACkGCCTCCAA
SEQ NO.82 TGCTGyGAGAGmAryTGrCTGAT
SEQ NO.83 CGmAArTCrGAGAryGArkCmTGGCAGGAG
SEQ NO.84 GAGGTCCrAAbArdAGsAkrCTsCGAGGTA
SEQ NO.85 CArGAdCArGAvrCCATGACTGC
SEQ NO.86 GGdsCyCCrCTTGTsGGkATGAC
SEQ NO.87 CTCAGCmGrCAGyTnGATGAGCT
SEQ NO.88 AGnGTCTCCATyTCTCkGGCCAG
SEQ NO.89 GArmGsAGCCTGACvTCwGTGCC
SEQ NO.90 CCrTTrGGhGTGTArATGGGCT
SEQ NO.91 CTyTTCCTyACCAArAACCACyTrAGCAG
SEQ NO.92 AGCTGyTCATTGTTyAGhGmCACTATCTG
SEQ NO.93 TCnTCyCCmACwGATGAyGAGCG
SEQ NO.94 GGrCTyTTyTCmACrCTyGCyAC
SEQ NO.95 rGArACAGATGArCGhmGdGChCAryTkCA
SEQ NO.96 TGkGTbTGTGGyTCCTCyTCyTCywCCTC
SEQ NO.97 GCmGGrCTbGAGGTsCACCAGTT
SEQ NO.98 GTGATbCCsACkATCATGGTCAT
SEQ NO.99 GGvTGyGArGCsCTsATGAAyAArTTyGGyTTCCAGTGGC
SEQ NO.100 TCATGAACACbGTGAAGTCbGGvGwCATsGGGGCrAAGTT
SEQ NO.101 GyTCwCGTTCdCGCwsmyACAGC
SEQ NO.102 GGdGTGTGrTGrAGbGCAGArGC
SEQ NO.103 AGrCGbAGCAGCTAyAGyCGnAG
SEQ NO.104 AGrTGrTGyTGrGGwATrTGrTGyACCTG
SEQ NO.105 AAyATGwCyAACCTsrGyGGCTC
SEQ NO.106 TCrGArGAsCkvAGATTGTTCGC
SEQ NO.107 ACAACTAyATyTTyCArGGnGGbCAGCATC
SEQ NO.108 CCArGAAGCGrTCCTTCTCyTTbAGGCTGA
SEQ NO.109 GCdTGTGCmAArCCmAGyGTGCT
SEQ NO.110 GwvTGwCCnGGwGTdGTdGGCAT
SEQ NO.111 GCTkCTmAAACACATmmGrTCyCACACAGG
SEQ NO.112 GrGrdGGwACrGArAGvAGrAAyACTGG
SEQ NO.113 AArAACCAGAGTkGCyhTGAACC
SEQ NO.114 CCAGCTGGTTGTTCTCyAGCCAG
SEQ NO.115 GTbCTGCAGrATCATCAAyGAGACCsACrGCdGCmGCCAT
SEQ NO.116 TTGCTsAGbCkGCCyTTrTCrTTGGTGATkGTGATyTTGT
SEQ NO.117 CCbTTyGTGGArGArTCwGACCT
SEQ NO.118 AAAdGCCATGTGGGAdyGrTAGC
SEQ NO.119 AGrCCywTbGAAGGyATGACyATCATGGA
SEQ NO.120 GrTAGCTGGGrCTTGyyCTyCkGCTGACC
SEQ NO.121 GAGCGyCTbmGrAGyTCTATGTC
SEQ NO.122 ATGCTGTCrTTrTCvCCrTCnGA
SEQ NO.123 GyTTTCbTTyGArGGACCTGArAAAGTCCA
SEQ NO.124 GAArTCvGACTCnGAyTTGCTyTGbCkGTT
SEQ NO.125 GArmGrGTGGTCATyAACATCTC
SEQ NO.126 GCyTCyTGrATyTCCATrTAGTC
SEQ NO.127 AGrATGCGsTAyTTyGAyCCdCTrAGGAAyGArTACTT
SEQ NO.128 TCdCGrTGrTAGAAGTAGTTrAArTTkGAsACdATGACAG
SEQ NO.129 GAGmGvGACTTyTTyTTCCACGA
SEQ NO.130 TArAACATrACsGTkGCrAAGAT
SEQ NO.131 TTCCACGAGGAGAsvAryGArTwCTTyTTyGACCGyGACC
SEQ NO.132 TGATGGCCATGGTGAGvGArAAkAGbAGGAAGCCsAGyTC
SEQ NO.133 ATGATyCGnCGCAAyTTCCACAA
SEQ NO.134 TTyTGbGGCATkGGGTAvCGCTG
SEQ NO.135 GCAAyTTCCACAArGTyATCCAGGAyGAsGArTTyTACAC
SEQ NO.136 TTGCGrTACTGCTTrTChACrTCrTCrCTrTTCTTCCA
SEQ NO.137 GshCCmCCyGTbATGAACTACAT
SEQ NO.138 CvGGbGAbGTyTGrATCTGCTTC
SEQ NO.139 ATGTACmChCCdGGTCCvGCmCmGCA
SEQ NO.140 GGrTAvGGTGGhGGnGGrCAGC
SEQ NO.141 TTyGArmGrGAGCGvATGGCnTA
SEQ NO.142 CAGyTCnGCCArdGTCGATGCAC
SEQ NO.143 GGrGArGCnGGdGAyTTyGCyvkyATGGC
SEQ NO.144 GATvACnGAGTTGCrbGGyTCdATGT
SEQ NO.145 TyCCvTACCTbGTbGTbATCCAC
SEQ NO.146 GTTbTCyyTACAGGTCTCArCTC
SEQ NO.147 CTbGvsAAGATGyTTyGAyTCnCCbTGGAC
SEQ NO.148 AGGCTrAArATCrGCrCTbGGGATnCGyTC
SEQ NO.149 CCCAGyTTyCCrTGGAsyGAGTT
SEQ NO.150 TCnCCrAArTCkGACAGsGACTC
SEQ NO.151 ATyCAGACGCGyAAyGAyGGvAGCAAGTT
SEQ NO.152 GCvGArAGCGGhGGCTGnCCrTTGTCCT
SEQ NO.153 GChAAyAThTCnGArGACATGCC
SEQ NO.154 CCbGTyCChACrAAGGTGTTGG
SEQ NO.155 GAyTCyGGnAAyCCdGCnCTvTChAGCAC
SEQ NO.156 GTCCTGvACnGCyTGGTGyTTCTkdGAGGC
SEQ NO.157 GTsGCbGArAAyGAGmmrGAGCA
SEQ NO.158 CrChAmrTCTGACsCCTCvTCCA
SEQ NO.159 CTCTGArACnCCbTGyGTkAACyTnCAGCT
SEQ NO.160 AAvACvTCmCkrTTCCAvCCCATrGCCATG
SEQ NO.161 ATGGAGGGnrTCAshGAGTTyAC
SEQ NO.162 TTGbGTkGChAChGGrTAbCkCA
SEQ NO.163 TTyGACCTbCTkGAGCCnCCmACnTCyGGA
SEQ NO.164 GTTyTGyGGyGCrTTyCCrGGrAACArmAC
SEQ NO.165 CGhCCbAAyCArCTbGTsGGyTC
SEQ NO.166 GTrCyrTCryTGTACTGyTCCCA
SEQ NO.167 GCdGCdyTmAGCAACATGyTdGGbGGmATG
SEQ NO.168 AGnGCyCTvACwGCCArrTAGTTGATG
SEQ NO.169 CACdGsCAArCTGmGGwCTGACT
SEQ NO.170 AGrTTyCCyCTkGGrCGhACCTC
SEQ NO.171 CAyTCvCCyAAyCACAACAChyTdCArGC
SEQ NO.172 GCrCTGGAbCCyGGrCTbGGrAArATCAT
SEQ NO.173 AyGCmTGyGAryTGAGrTGyCAG
SEQ NO.174 CdbCCCTGTCCrTAvGAyTTGCT
SEQ NO.175 GArrCCrGTbGArGTbCGyrTbGTGCArCG
SEQ NO.176 TCCAkrCCvArhCCyTCyCTGGwrAACTG
SEQ NO.177 GACACvGTkCTdGyrTCyGGrGA
SEQ NO.178 CyACdGGdCCrATrTTGACAGTA
SEQ NO.179 GGvAArAGCACmykrCTyCAGmGrCTrCAC
SEQ NO.180 TGCATGCTyTTCTTyTChCCyksyACAGG
SEQ NO.181 GGhCCrCTbATyGACmGrCAGAT
SEQ NO.182 TCyCkbyknCCCATGTGsACCAG
SEQ NO.183 GATyTTymGnTTCAGyGArGArGGCATGGT
SEQ NO.184 CAsGCmACrTArAACTCrTCsCCrCTGCT
SEQ NO.185 TCyAGCAChGCCATyCTsCArGT
SEQ NO.186 TTGATyTGrAAbGTdGGCTGAGG
SEQ NO.187 GACAAyGTyCChTCyATAGACAC
SEQ NO.188 GTrGGGTCdTCAGGCTTGGATTC
SEQ NO.189 CAsACGACCAsvGCyTCmAmCAC
SEQ NO.190 TGCTTCATrAACTCvCkGCkyTG
SEQ NO.191 GACTAyrCnGACrChGACTTyyTGGGTGAC
SEQ NO.192 TCyTCyTTGCTCCAGTAsCGdCCCATCTTC
SEQ NO.193 CACTAyAACACmAAGCTGGGmTA
SEQ NO.194 TGCTCCTGAGGTTGhGGTTGCAA
SEQ NO.195 CGvAArGACTTyCTrTGCCAGTACTGyGCC
SEQ NO.196 CCCAGCAGrTGrGArAArTCCATGTTAGCA
SEQ NO.197 ATGGTsTGyACyGGsAAGATGCA
SEQ NO.198 ATGTTyTCwGArAAvGTrTAGTT
SEQ NO.199 CyGGsAAGATGCACAChGAyCGCATyTGCCGCTTyGACTA
SEQ NO.200 TrTAAGGCATrTAbGTrTTyTCTCCyTGCTCyTGkATCCA
SEQ NO.201 GACTACyCrCTGCAGAGCAACAG
SEQ NO.202 TTyTCyTTCTGTCTTCCTGTTAC
SEQ NO.203 CTGCAGAGCAACAGyCACmCGCTCAGCCACGCrCACCAGT
SEQ NO.204 TTCCTGTTACAAAACCAAACyCksACyACyTCTTTyTCCA
SEQ NO.205 GTkCCwGAyCChCAryTdGATGA
SEQ NO.206 TCCCArTCrCAyTCyTGrCTCTG
SEQ NO.207 GArACvGArvTsAAAGArGGrGCwCAGCA
SEQ NO.208 GArTGyTCyTTyTCyTGyTGrGGrTCTCTG
SEQ NO.209 ACvCTbAAyGArATGTGGTGCCA
SEQ NO.210 AGCAGrTCrTTsATCTGbTCCAG
SEQ NO.211 GGyTCCAAGCArTTyAArATCCACACCAT
SEQ NO.212 AGvGGrAArCGyTGCyTGTryCTGvAGATG
SEQ NO.213 TCTAyATyATCAACsTTyGGCAT
SEQ NO.214 GTGAGvAGGGAGCAyTTGAACAG
SEQ NO.215 ATCAACsTTyGGCATGGGwGCyAGCCCyTTyACyAACAT
SEQ NO.216 AGTGTAvAGrTGkGGkGCACAGTGATCCAT
SEQ NO.217 TGGGTsCGsCACAsCTACArCCA
SEQ NO.218 CATCCCTTTCTTyTTsCGGTTGA
SEQ NO.219 AAyAGyGAyGArGACAGCAAAGAyGGCA
SEQ NO.220 CGnGCnGGCCACCArGGGAAkCCrTGrAT
SEQ NO.221 TTyCAysmvTTTGArTGGCAGCC
SEQ NO.222 CTGAAsAGyTTGTTbsChGACTC
SEQ NO.223 AArGAyCTbGAGAGAGGACAThATGGArGGdCTGA
SEQ NO.224 GTCATyAryykbCGrGCrTAGTTbCCATTCATCCTCAT
SEQ NO.225 TTWGGNCARAARGGNTGGCCNAA
SEQ NO.226 CATRCAYTGNGCRTGNACCCARTG
SEQ NO.227 AGGGTTTTCCCAGTCACGACGGWGGKAARACNCCNAAYAAYGA
SEQ NO.228 AGATAACAATTTCACACAGGCARCAYTTDATCCARTANCC
SEQ NO.229 ATGCTsTCvGGvTCyCCdCAGTC
SEQ NO.230 AGmCCyCTrGCyTkCTCCACCCA
SEQ NO.231 CAGTCyTACTGCrTbGTrGArAArTGGAG
SEQ NO.232 ChAGyTGsGTTyTkATCCTCTCyTCyTCCA
SEQ NO.233 TCCAGAyCChACnTAyGArCTCA
SEQ NO.234 ACrGTdCGnCCdGTCTCCATGCA
SEQ NO.235 GGyAAAGTyCCyhTrmGrCAGCTkGTsTAC
SEQ NO.236 GTTCATyTTkGCyGGrTCvAGrGCCCAGT
SEQ NO.237 GArCAGACyCArGTGGTGGCyAT
SEQ NO.238 TyTGwkGbkGGTGGTGsAkCTGC
SEQ NO.239 GTrrTyACyAArGAvCAGCCmATGAGCATG
SEQ NO.240 ATsGGkATyTGCATGGGrTArAAGTTCTGC
SEQ NO.241 AAYATHACNAAYGCNTGYTAYAA
SEQ NO.242 GCRAARTGNCCRTTNACRTGRAA
SEQ NO.243 AGGGTTTTCCCAGTCACGACTGYTAYAAYGAYTGYCCNTGGAT
SEQ NO.244 AGATAACAATTTCACACAGGCKGTGRGGYTTYTTRTARTTRTG
SEQ NO.245 GGArCAGyTrGTswGyCTyCAGC
SEQ NO.246 CArATkGGrCAnGAGTGyTGCAT
SEQ NO.247 CArCArCTbTCTGCwGCAGCwGChCTvATw
SEQ NO.248 GTkGwGAAnGCvCkGCCrCAGATyTTGCA
SEQ NO.249 TCATyTTyTCyATmTTyGGCATG
SEQ NO.250 TTbTCrGGyTTGGTyACrCTGTC
SEQ NO.251 GAChTGTAyAACTTyGArACnTTyGGmAACAGyATGATC
SEQ NO.252 GTGCyCTCyTTyTTyTCTTGyTCyTGrTTG
SEQ NO.253 CCdCAyGCyCTrTChCTsCGsAG
SEQ NO.254 ACCTCrCAvsCrsAACTGCTGCC
SEQ NO.255 AGhTGGAGGTrTChTCwCCmAArmTCTACA
SEQ NO.256 TCCTCCTmCTmCTsCrCCTsCryyGGsCAT
SEQ NO.257 GAyAGyTCwGGhGGdGChGTbCA
SEQ NO.258 GCmACyyTyTCnGGyTCrCTGTC
SEQ NO.259 GAymGmTGyACyACbGTvAGyCCnGTGGT
SEQ NO.260 AGGmAywTGGCArGGyTTyAGdAkyTGCTC
SEQ NO.261 GTvATAGAyGGyCArGAGAGACT
SEQ NO.262 GGyCTGAAnGCvGAbAyGTAGCT
SEQ NO.263 GTbGCbCTkGGvATCACyTGyGTsCAGATG
SEQ NO.264 GyrGGCCArAACATsGGrAArGArkGrTAG
SEQ NO.265 ATGCTbTACATGGTvATyGTGCC
SEQ NO.266 AGCAGmAGCArvGCyGGyGCGTA
SEQ NO.267 CTbTTyGCnTChAArGCCATybTsCAGCT
SEQ NO.268 TChGCdATdGCrTAmATCrCTrCCrTACAC
SEQ NO.269 TACATGTTCyTGGGyATGTCCAT
SEQ NO.270 TrGCCTCrTGrCAGCTCACCA
SEQ NO.271 TGTTCyTGGGyATGTCCATCAThGCvGAyCGbTTCATGTC
SEQ NO.272 GCrTGCTTyTTCArrATGTTyCCnGCyCCGATCATCAT
SEQ NO.273 GArGArATmGAyGAGCCyTGCTT
SEQ NO.274 ATvAGvAChGAsAGhGGvACAGC
SEQ NO.275 CArGAyAThCAyGybGGnGCdTTyAATCGG
SEQ NO.276 ATrTCrCAnGAGCAvTCCCAdGGrTTCTG
SEQ NO.277 ACyGTsTAymGrCCbACrCAGCT
SEQ NO.278 TCvTCvAGyTCrTCrATGGTGCT
SEQ NO.279 GArAACyTGGAATACCTyCAGGCyGACTAC
SEQ NO.280 AGGCArAArGCACACrAAvACwGTnAGGAT
SEQ NO.281 GAyTTyGTCATGCTyCAGCAGCC
SEQ NO.282 TAyTGyAGvGArCGCACTTGCAT
SEQ NO.283 GArGTvAAAGCyCAGmGrCCCyTnAGAT
SEQ NO.284 CCyTCbACrTCdGGCTGCTCrTArAAGCT
SEQ NO.285 ATyCChCGnGAGGTGGAGATGGA
SEQ NO.286 CGyTChAGnArCCACTGCATGGT
SEQ NO.287 ACvCCnGAGTGGGAyGTkTCnGGGGAGACT
SEQ NO.288 TThCGCAGAwGTGCCdGACTTkGwGTAGC
SEQ NO.289 GCyCGTyTGArrGArGCbCGdGA
SEQ NO.290 CTvAGAGCnGCCACdATyCTvAG
SEQ NO.291 AGCArCArCCyGCyCTrTCyGArGCCATGA
SEQ NO.292 CmACyTGCATrGCCATbGCyTkrTGGTCCT
SEQ NO.293 GACAGCACvmGrGTyAGCTTCAC
SEQ NO.294 TArTCTCykCTCbCCrAAGTGCA
SEQ NO.295 GACmGGCAGAThTTyCAGsTTCAGyGArGAyGGCATGGT
SEQ NO.296 TArAACTCyTCnCCrCTrCTdATTCTCCAT
SEQ NO.297 AGyCCsTGyTCCAAyGGyTACAT
SEQ NO.298 CCTGrTAGkCyGGmGGrGAAGGT
SEQ NO.299 CCTsGTCATGCTCTAyGTGGTyTACCTkGT
SEQ NO.300 TGGhvCGrTGGCAGCGyTCrCArTTCTGCC
SEQ NO.301 GAGCArbTGGCrkCnGAyCACAG
SEQ NO.302 CCCAryCTyyTyTCCTTCTGCTC
SEQ NO.303 GGnATGAArGArGArAACAGyCAyyTGAAAG
SEQ NO.304 TTvACyGTbGTGATsACCTGCwkCTTCCACT
SEQ NO.305 AACTCyCCACTyAGCAATGGCAC
SEQ NO.306 GGAGyTGrTArGCrGCrTGAATG
SEQ NO.307 TGCAAAyyTGATyTCdGAyGCyTCyTGGTC
SEQ NO.308 TGrGCyTTrCTGATwGCACTkGArACwGT
SEQ NO.309 GAGCTGGTGAArGTrATGGGdCT
SEQ NO.310 TACTGGAACCArTAmACnGTCTC
SEQ NO.311 GGvAGCCTbATmTACCTsCAyGACACyyTGGAGGAG
SEQ NO.312 CChTCCCAGwArTGyTTGAAsCCrCAbCCrCACTT
SEQ NO.313 CATATCdCCACGdGAyAGTACCA
SEQ NO.314 ATGGCTTCryThACAATGAGGTC
SEQ NO.315 TGAGrCAyGTrCGAAGGGCTCAyCCCACTG
SEQ NO.316 TGGCTGAAGATCCAGTGCrAGCATTTCTGC
SEQ NO.317 ACbAACACyGCCATywCCCAGCT
SEQ NO.318 GCTGGCyTGCAysGGGGGCATGA
SEQ NO.319 GACTTCAAyGACTAyAAGCTsATGATGGC
SEQ NO.320 TTGGACAGrTArGCCArGTTsArmGGCTC
SEQ NO.321 GGbGAGsysAAdTTyGGnGACAC
SEQ NO.322 TCTkryGCTCkvTCnGAGAAGTC
SEQ NO.323 GGrCTCTTyTGGGGnCCnyTsTGCTGCTC
SEQ NO.324 GGrAGGAArGCvGGrTCrkTrGwCTTCTG
SEQ NO.325 GyCArCCwATyAGCAGyCAGATG
SEQ NO.326 GTsGTGGAdCGGCTGATGGACTG
SEQ NO.327 TGyGGCAArCGyTTCCGyTTyAACAGCATC
SEQ NO.328 GCkGvTGCTGsTGCTGTGmTCyTTnGGCAT。
SEQUENCE LISTING
<110> Zhujiang aquatic research institute of Chinese aquatic science research institute
<120> molecular marker primer, molecular marker and molecular marker database for fish nuclear genes
<130>
<160> 328
<170> PatentIn version 3.3
<210> 1
<211> 23
<212> DNA
<213> unknown
<400> 1
ctytayggmg aygayagytg gga 23
<210> 2
<211> 23
<212> DNA
<213> unknown
<400> 2
taaggrtcwg cytcharrgg cat 23
<210> 3
<211> 30
<212> DNA
<213> unknown
<400> 3
gayagytggg ayrctcctgt vaargartac 30
<210> 4
<211> 30
<212> DNA
<213> unknown
<400> 4
caaarggyar rctytcyctg cgrcyrtagg 30
<210> 5
<211> 23
<212> DNA
<213> unknown
<400> 5
aargaccagg arytgctgtg gga 23
<210> 6
<211> 23
<212> DNA
<213> unknown
<400> 6
carcakggrg ttttrctbcg act 23
<210> 7
<211> 29
<212> DNA
<213> unknown
<400> 7
gcmtcwgayt cyggytgytt tgatgatga 29
<210> 8
<211> 30
<212> DNA
<213> unknown
<400> 8
caatdgargg agaytcaaar ctrccmagtg 30
<210> 9
<211> 23
<212> DNA
<213> unknown
<400> 9
cagytgggny tggagcarga ctt 23
<210> 10
<211> 23
<212> DNA
<213> unknown
<400> 10
agccrtgyac yartcckrct ctt 23
<210> 11
<211> 29
<212> DNA
<213> unknown
<400> 11
gtvagycgbg gbatgaaycg haactttga 29
<210> 12
<211> 29
<212> DNA
<213> unknown
<400> 12
taggdrtcyc crcacatyar rcacatgac 29
<210> 13
<211> 23
<212> DNA
<213> unknown
<400> 13
aargargarc aggayccmcc tgt 23
<210> 14
<211> 23
<212> DNA
<213> unknown
<400> 14
ctgaaratkg trggcarsag gtc 23
<210> 15
<211> 30
<212> DNA
<213> unknown
<400> 15
gccawagraa rcgwggrcag hcccaagacg 30
<210> 16
<211> 30
<212> DNA
<213> unknown
<400> 16
ctgncgdgas cgyagtctgy argtacttct 30
<210> 17
<211> 23
<212> DNA
<213> unknown
<400> 17
catgcagtgg mgwcggcgrc act 23
<210> 18
<211> 23
<212> DNA
<213> unknown
<400> 18
tgvgakgtga tsaggtgrct gta 23
<210> 19
<211> 30
<212> DNA
<213> unknown
<400> 19
accaragcar ctchctgmgr rgcctbtgga 30
<210> 20
<211> 30
<212> DNA
<213> unknown
<400> 20
cccygagaac acrtasccyg tsccbgagca 30
<210> 21
<211> 23
<212> DNA
<213> unknown
<400> 21
cgcatgcaca acctbgagag act 23
<210> 22
<211> 23
<212> DNA
<213> unknown
<400> 22
aaytcraayt gsggcagrct gca 23
<210> 23
<211> 33
<212> DNA
<213> unknown
<400> 23
cgcttyctgg argcycarct vcgcaghgag aga 33
<210> 24
<211> 34
<212> DNA
<213> unknown
<400> 24
cgygggcaya cyttrattgg cactgggcag ctgg 34
<210> 25
<211> 23
<212> DNA
<213> unknown
<400> 25
ccccaycgvc trgaacgcct tag 23
<210> 26
<211> 23
<212> DNA
<213> unknown
<400> 26
tgcacygtca grttgctrca gtt 23
<210> 27
<211> 30
<212> DNA
<213> unknown
<400> 27
tgggyytmat ctcmcagcay cccagtgcct 30
<210> 28
<211> 29
<212> DNA
<213> unknown
<400> 28
gakgargtgg craakggmga ttgtctgga 29
<210> 29
<211> 23
<212> DNA
<213> unknown
<400> 29
gayrtsccwg argamasctg gct 23
<210> 30
<211> 23
<212> DNA
<213> unknown
<400> 30
ttgcgrttng gscgcckrca kcg 23
<210> 31
<211> 23
<212> DNA
<213> unknown
<400> 31
gargamasct ggctbcagnt gtg 23
<210> 32
<211> 29
<212> DNA
<213> unknown
<400> 32
cakcgytcrt cratgctygg cacrcattc 29
<210> 33
<211> 23
<212> DNA
<213> unknown
<400> 33
cgcachhtrg cyachttyaa ctc 23
<210> 34
<211> 23
<212> DNA
<213> unknown
<400> 34
tytccagytt bckctcratg gac 23
<210> 35
<211> 30
<212> DNA
<213> unknown
<400> 35
ccactcycyh cagacnayrc araargtgga 30
<210> 36
<211> 30
<212> DNA
<213> unknown
<400> 36
cngcyykkck dgcyttctgr atctgyttct 30
<210> 37
<211> 23
<212> DNA
<213> unknown
<400> 37
gargcyytst ttcgayagyc gct 23
<210> 38
<211> 23
<212> DNA
<213> unknown
<400> 38
cagytgraav acctckggga tgt 23
<210> 39
<211> 29
<212> DNA
<213> unknown
<400> 39
aaygcbccbc agttcagyga raarcgcta 29
<210> 40
<211> 26
<212> DNA
<213> unknown
<400> 40
tcraacacrg gvgggttgtc rttcac 26
<210> 41
<211> 23
<212> DNA
<213> unknown
<400> 41
aayggvtacm grmgmttyga ccc 23
<210> 42
<211> 23
<212> DNA
<213> unknown
<400> 42
taytkctgry gyarngcygg ctc 23
<210> 43
<211> 29
<212> DNA
<213> unknown
<400> 43
ggbatggagt acacsytrga cytncagst 29
<210> 44
<211> 26
<212> DNA
<213> unknown
<400> 44
gcyggctcna yngcyckgaa hacrtg 26
<210> 45
<211> 23
<212> DNA
<213> unknown
<400> 45
caacaagaac ccnccytacc agt 23
<210> 46
<211> 23
<212> DNA
<213> unknown
<400> 46
garcccagrc acatyttrta ytg 23
<210> 47
<211> 40
<212> DNA
<213> unknown
<400> 47
accagtaycg cctdcacagc tacmtgytsa gccgvaagat 40
<210> 48
<211> 35
<212> DNA
<213> unknown
<400> 48
tanachacyt tdgggtcrta ctgrctgaag atgat 35
<210> 49
<211> 23
<212> DNA
<213> unknown
<400> 49
atggtrgtga cnvartgygg ctg 23
<210> 50
<211> 23
<212> DNA
<213> unknown
<400> 50
ccrtcngarg trccrtcraa gca 23
<210> 51
<211> 29
<212> DNA
<213> unknown
<400> 51
acdggmtaca argghacmtt ctchrtcca 29
<210> 52
<211> 28
<212> DNA
<213> unknown
<400> 52
gtcnagdggb gcharcatdg tgaatctc 28
<210> 53
<211> 23
<212> DNA
<213> unknown
<400> 53
gagaacttya tggacatgga gtg 23
<210> 54
<211> 23
<212> DNA
<213> unknown
<400> 54
tkyctgttct grcagtcbga ctc 23
<210> 55
<211> 40
<212> DNA
<213> unknown
<400> 55
acatggagtg yttcatgatm ctgacyccba gccagcagct 40
<210> 56
<211> 40
<212> DNA
<213> unknown
<400> 56
ctcctcarrg crtagatgat bggrttgacr gtggagttga 40
<210> 57
<211> 23
<212> DNA
<213> unknown
<400> 57
cmcgyaarcg yagccayagy agc 23
<210> 58
<211> 23
<212> DNA
<213> unknown
<400> 58
gtggyygtga tvagbccdat cca 23
<210> 59
<211> 29
<212> DNA
<213> unknown
<400> 59
aaytggtaca gytgyggkga rggkgaggt 29
<210> 60
<211> 30
<212> DNA
<213> unknown
<400> 60
ahgtcatdgg ytgytgwggr ttsacrtgct 30
<210> 61
<211> 23
<212> DNA
<213> unknown
<400> 61
gaygargtbc ccaydgcvca ytc 23
<210> 62
<211> 23
<212> DNA
<213> unknown
<400> 62
tartgdgcrt cccacacrtc cat 23
<210> 63
<211> 30
<212> DNA
<213> unknown
<400> 63
ctbtchaary acagrgsvtg gggvaarcag 30
<210> 64
<211> 28
<212> DNA
<213> unknown
<400> 64
cgyarccact tdgmrcaytc yccyarct 28
<210> 65
<211> 23
<212> DNA
<213> unknown
<400> 65
gargccatcc chcagcatat caa 23
<210> 66
<211> 23
<212> DNA
<213> unknown
<400> 66
gcrtgbccng cngccatgta ctc 23
<210> 67
<211> 29
<212> DNA
<213> unknown
<400> 67
trggvaagct gcagtacctc tacctccag 29
<210> 68
<211> 28
<212> DNA
<213> unknown
<400> 68
atrtacttsg abtctgactt gtaggacg 28
<210> 69
<211> 23
<212> DNA
<213> unknown
<400> 69
ggcagccgyt ccttyccstg cca 23
<210> 70
<211> 23
<212> DNA
<213> unknown
<400> 70
gcytgrcagc tcatbckytt ggc 23
<210> 71
<211> 30
<212> DNA
<213> unknown
<400> 71
tgmgrgarag ycaggccagy gaggtbgact 30
<210> 72
<211> 29
<212> DNA
<213> unknown
<400> 72
tcrtagcact gmacyttggg sagyttgtc 29
<210> 73
<211> 23
<212> DNA
<213> unknown
<400> 73
ctsacvtggc tcagyttcat gct 23
<210> 74
<211> 23
<212> DNA
<213> unknown
<400> 74
cctcyckytc rtahgtmagc atg 23
<210> 75
<211> 30
<212> DNA
<213> unknown
<400> 75
cttcccdcty atygchcayt actacctcac 30
<210> 76
<211> 26
<212> DNA
<213> unknown
<400> 76
tcvccrttgt crgccakgtt rgcrtc 26
<210> 77
<211> 23
<212> DNA
<213> unknown
<400> 77
tcycgvtacg gacayytrga ggt 23
<210> 78
<211> 23
<212> DNA
<213> unknown
<400> 78
tcbgcrccrc actcyagsag cag 23
<210> 79
<211> 30
<212> DNA
<213> unknown
<400> 79
gctstgggcg gcbtckgcvg csggtcacct 30
<210> 80
<211> 29
<212> DNA
<213> unknown
<400> 80
ttgtcyacrg ccatgtgsag vggvgtgaa 29
<210> 81
<211> 23
<212> DNA
<213> unknown
<400> 81
atmtchgcvc tsackgcctc caa 23
<210> 82
<211> 23
<212> DNA
<213> unknown
<400> 82
tgctgygaga gmarytgrct gat 23
<210> 83
<211> 30
<212> DNA
<213> unknown
<400> 83
cgmaartcrg agarygarkc mtggcaggag 30
<210> 84
<211> 30
<212> DNA
<213> unknown
<400> 84
gaggtccraa bardagsakr ctscgaggta 30
<210> 85
<211> 23
<212> DNA
<213> unknown
<400> 85
cargadcarg avrccatgac tgc 23
<210> 86
<211> 23
<212> DNA
<213> unknown
<400> 86
ggdscyccrc ttgtsggkat gac 23
<210> 87
<211> 23
<212> DNA
<213> unknown
<400> 87
ctcagcmgrc agytngatga gct 23
<210> 88
<211> 23
<212> DNA
<213> unknown
<400> 88
agngtctcca tytctckggc cag 23
<210> 89
<211> 23
<212> DNA
<213> unknown
<400> 89
garmgsagcc tgacvtcwgt gcc 23
<210> 90
<211> 22
<212> DNA
<213> unknown
<400> 90
ccrttrgghg tgtaratggg ct 22
<210> 91
<211> 29
<212> DNA
<213> unknown
<400> 91
ctyttcctya ccaaraacca cytragcag 29
<210> 92
<211> 29
<212> DNA
<213> unknown
<400> 92
agctgytcat tgttyaghgm cactatctg 29
<210> 93
<211> 23
<212> DNA
<213> unknown
<400> 93
tcntcyccma cwgatgayga gcg 23
<210> 94
<211> 23
<212> DNA
<213> unknown
<400> 94
ggrctyttyt cmacrctygc yac 23
<210> 95
<211> 30
<212> DNA
<213> unknown
<400> 95
rgaracagat garcghmgdg chcarytkca 30
<210> 96
<211> 29
<212> DNA
<213> unknown
<400> 96
tgkgtbtgtg gytcctcytc ytcywcctc 29
<210> 97
<211> 23
<212> DNA
<213> unknown
<400> 97
gcmggrctbg aggtscacca gtt 23
<210> 98
<211> 23
<212> DNA
<213> unknown
<400> 98
gtgatbccsa ckatcatggt cat 23
<210> 99
<211> 40
<212> DNA
<213> unknown
<400> 99
ggvtgygarg csctsatgaa yaarttyggy ttccagtggc 40
<210> 100
<211> 40
<212> DNA
<213> unknown
<400> 100
tcatgaacac bgtgaagtcb ggvgwcatsg gggcraagtt 40
<210> 101
<211> 23
<212> DNA
<213> unknown
<400> 101
gytcwcgttc dcgcwsmyac agc 23
<210> 102
<211> 23
<212> DNA
<213> unknown
<400> 102
ggdgtgtgrt gragbgcaga rgc 23
<210> 103
<211> 23
<212> DNA
<213> unknown
<400> 103
agrcgbagca gctayagycg nag 23
<210> 104
<211> 29
<212> DNA
<213> unknown
<400> 104
agrtgrtgyt grggwatrtg rtgyacctg 29
<210> 105
<211> 23
<212> DNA
<213> unknown
<400> 105
aayatgwcya acctsrgygg ctc 23
<210> 106
<211> 23
<212> DNA
<213> unknown
<400> 106
tcrgargasc kvagattgtt cgc 23
<210> 107
<211> 30
<212> DNA
<213> unknown
<400> 107
acaactayat yttycarggn ggbcagcatc 30
<210> 108
<211> 30
<212> DNA
<213> unknown
<400> 108
ccargaagcg rtccttctcy ttbaggctga 30
<210> 109
<211> 23
<212> DNA
<213> unknown
<400> 109
gcdtgtgcma arccmagygt gct 23
<210> 110
<211> 23
<212> DNA
<213> unknown
<400> 110
gwvtgwccng gwgtdgtdgg cat 23
<210> 111
<211> 30
<212> DNA
<213> unknown
<400> 111
gctkctmaaa cacatmmgrt cycacacagg 30
<210> 112
<211> 28
<212> DNA
<213> unknown
<400> 112
grgrdggwac rgaragvagr aayactgg 28
<210> 113
<211> 23
<212> DNA
<213> unknown
<400> 113
aaraaccaga gtkgcyhtga acc 23
<210> 114
<211> 23
<212> DNA
<213> unknown
<400> 114
ccagctggtt gttctcyagc cag 23
<210> 115
<211> 40
<212> DNA
<213> unknown
<400> 115
gtbctgcagr atcatcaayg agaccsacrg cdgcmgccat 40
<210> 116
<211> 40
<212> DNA
<213> unknown
<400> 116
ttgctsagbc kgccyttrtc rttggtgatk gtgatyttgt 40
<210> 117
<211> 23
<212> DNA
<213> unknown
<400> 117
ccbttygtgg argartcwga cct 23
<210> 118
<211> 23
<212> DNA
<213> unknown
<400> 118
aaadgccatg tgggadygrt agc 23
<210> 119
<211> 29
<212> DNA
<213> unknown
<400> 119
agrccywtbg aaggyatgac yatcatgga 29
<210> 120
<211> 29
<212> DNA
<213> unknown
<400> 120
grtagctggg rcttgyycty ckgctgacc 29
<210> 121
<211> 23
<212> DNA
<213> unknown
<400> 121
gagcgyctbm gragytctat gtc 23
<210> 122
<211> 23
<212> DNA
<213> unknown
<400> 122
atgctgtcrt trtcvccrtc nga 23
<210> 123
<211> 30
<212> DNA
<213> unknown
<400> 123
gytttcbtty garggacctg araaagtcca 30
<210> 124
<211> 30
<212> DNA
<213> unknown
<400> 124
gaartcvgac tcngayttgc tytgbckgtt 30
<210> 125
<211> 23
<212> DNA
<213> unknown
<400> 125
garmgrgtgg tcatyaacat ctc 23
<210> 126
<211> 23
<212> DNA
<213> unknown
<400> 126
gcytcytgra tytccatrta gtc 23
<210> 127
<211> 38
<212> DNA
<213> unknown
<400> 127
agratgcgst ayttygaycc dctraggaay gartactt 38
<210> 128
<211> 40
<212> DNA
<213> unknown
<400> 128
tcdcgrtgrt agaagtagtt raarttkgas acdatgacag 40
<210> 129
<211> 23
<212> DNA
<213> unknown
<400> 129
gagmgvgact tyttyttcca cga 23
<210> 130
<211> 23
<212> DNA
<213> unknown
<400> 130
taraacatra csgtkgcraa gat 23
<210> 131
<211> 40
<212> DNA
<213> unknown
<400> 131
ttccacgagg agasvaryga rtwcttytty gaccgygacc 40
<210> 132
<211> 40
<212> DNA
<213> unknown
<400> 132
tgatggccat ggtgagvgar aakagbagga agccsagytc 40
<210> 133
<211> 23
<212> DNA
<213> unknown
<400> 133
atgatycgnc gcaayttcca caa 23
<210> 134
<211> 23
<212> DNA
<213> unknown
<400> 134
ttytgbggca tkgggtavcg ctg 23
<210> 135
<211> 40
<212> DNA
<213> unknown
<400> 135
gcaayttcca caargtyatc caggaygasg arttytacac 40
<210> 136
<211> 38
<212> DNA
<213> unknown
<400> 136
ttgcgrtact gcttrtchac rtcrtcrctr ttcttcca 38
<210> 137
<211> 23
<212> DNA
<213> unknown
<400> 137
gshccmccyg tbatgaacta cat 23
<210> 138
<211> 23
<212> DNA
<213> unknown
<400> 138
cvggbgabgt ytgratctgc ttc 23
<210> 139
<211> 26
<212> DNA
<213> unknown
<400> 139
atgtacmchc cdggtccvgc mcmgca 26
<210> 140
<211> 22
<212> DNA
<213> unknown
<400> 140
ggrtavggtg ghggnggrca gc 22
<210> 141
<211> 23
<212> DNA
<213> unknown
<400> 141
ttygarmgrg agcgvatggc nta 23
<210> 142
<211> 23
<212> DNA
<213> unknown
<220>
<221> misc_feature
<222> (7)..(7)
<223> n is a, c, g, or t
<400> 142
cagytcngcc ardgtcgatg cac 23
<210> 143
<211> 29
<212> DNA
<213> unknown
<400> 143
ggrgargcng gdgayttygc yvkyatggc 29
<210> 144
<211> 26
<212> DNA
<213> unknown
<400> 144
gatvacngag ttgcrbggyt cdatgt 26
<210> 145
<211> 23
<212> DNA
<213> unknown
<400> 145
tyccvtacct bgtbgtbatc cac 23
<210> 146
<211> 23
<212> DNA
<213> unknown
<400> 146
gttbtcyyta caggtctcar ctc 23
<210> 147
<211> 30
<212> DNA
<213> unknown
<400> 147
ctbgvsaaga tgyttygayt cnccbtggac 30
<210> 148
<211> 30
<212> DNA
<213> unknown
<400> 148
aggctraara tcrgcrctbg ggatncgytc 30
<210> 149
<211> 23
<212> DNA
<213> unknown
<400> 149
cccagyttyc crtggasyga gtt 23
<210> 150
<211> 23
<212> DNA
<213> unknown
<400> 150
tcnccraart ckgacagsga ctc 23
<210> 151
<211> 29
<212> DNA
<213> unknown
<400> 151
atycagacgc gyaaygaygg vagcaagtt 29
<210> 152
<211> 28
<212> DNA
<213> unknown
<400> 152
gcvgaragcg ghggctgncc rttgtcct 28
<210> 153
<211> 23
<212> DNA
<213> unknown
<400> 153
gchaayatht cngargacat gcc 23
<210> 154
<211> 22
<212> DNA
<213> unknown
<400> 154
ccbgtyccha craaggtgtt gg 22
<210> 155
<211> 29
<212> DNA
<213> unknown
<400> 155
gaytcyggna ayccdgcnct vtchagcac 29
<210> 156
<211> 30
<212> DNA
<213> unknown
<400> 156
gtcctgvacn gcytggtgyt tctkdgaggc 30
<210> 157
<211> 23
<212> DNA
<213> unknown
<400> 157
gtsgcbgara aygagmmrga gca 23
<210> 158
<211> 23
<212> DNA
<213> unknown
<400> 158
crchamrtct gacscctcvt cca 23
<210> 159
<211> 30
<212> DNA
<213> unknown
<400> 159
ctctgaracn ccbtgygtka acytncagct 30
<210> 160
<211> 30
<212> DNA
<213> unknown
<400> 160
aavacvtcmc krttccavcc catrgccatg 30
<210> 161
<211> 23
<212> DNA
<213> unknown
<400> 161
atggagggnr tcashgagtt yac 23
<210> 162
<211> 23
<212> DNA
<213> unknown
<400> 162
ttgbgtkgch achggrtabc kca 23
<210> 163
<211> 30
<212> DNA
<213> unknown
<400> 163
ttygacctbc tkgagccncc macntcygga 30
<210> 164
<211> 30
<212> DNA
<213> unknown
<400> 164
gttytgyggy gcrttyccrg graacarmac 30
<210> 165
<211> 23
<212> DNA
<213> unknown
<400> 165
cghccbaayc arctbgtsgg ytc 23
<210> 166
<211> 23
<212> DNA
<213> unknown
<400> 166
gtrcyrtcry tgtactgytc cca 23
<210> 167
<211> 30
<212> DNA
<213> unknown
<400> 167
gcdgcdytma gcaacatgyt dggbggmatg 30
<210> 168
<211> 27
<212> DNA
<213> unknown
<400> 168
agngcyctva cwgccarrta gttgatg 27
<210> 169
<211> 23
<212> DNA
<213> unknown
<400> 169
cacdgscaar ctgmggwctg act 23
<210> 170
<211> 23
<212> DNA
<213> unknown
<400> 170
agrttyccyc tkggrcghac ctc 23
<210> 171
<211> 29
<212> DNA
<213> unknown
<400> 171
caytcvccya aycacaacac hytdcargc 29
<210> 172
<211> 29
<212> DNA
<213> unknown
<400> 172
gcrctggabc cyggrctbgg raaratcat 29
<210> 173
<211> 23
<212> DNA
<213> unknown
<400> 173
aygcmtgyga rytgagrtgy cag 23
<210> 174
<211> 23
<212> DNA
<213> unknown
<400> 174
cdbccctgtc crtavgaytt gct 23
<210> 175
<211> 30
<212> DNA
<213> unknown
<400> 175
garrccrgtb gargtbcgyr tbgtgcarcg 30
<210> 176
<211> 29
<212> DNA
<213> unknown
<400> 176
tccakrccva rhccytcyct ggwraactg 29
<210> 177
<211> 23
<212> DNA
<213> unknown
<400> 177
gacacvgtkc tdgyrtcygg rga 23
<210> 178
<211> 23
<212> DNA
<213> unknown
<400> 178
cyacdggdcc ratrttgaca gta 23
<210> 179
<211> 30
<212> DNA
<213> unknown
<400> 179
ggvaaragca cmykrctyca gmgrctrcac 30
<210> 180
<211> 29
<212> DNA
<213> unknown
<400> 180
tgcatgctyt tcttytchcc yksyacagg 29
<210> 181
<211> 23
<212> DNA
<213> unknown
<400> 181
gghccrctba tygacmgrca gat 23
<210> 182
<211> 23
<212> DNA
<213> unknown
<400> 182
tcyckbyknc ccatgtgsac cag 23
<210> 183
<211> 30
<212> DNA
<213> unknown
<400> 183
gatyttymgn ttcagygarg arggcatggt 30
<210> 184
<211> 29
<212> DNA
<213> unknown
<400> 184
casgcmacrt araactcrtc sccrctgct 29
<210> 185
<211> 23
<212> DNA
<213> unknown
<400> 185
tcyagcachg ccatyctsca rgt 23
<210> 186
<211> 23
<212> DNA
<213> unknown
<400> 186
ttgatytgra abgtdggctg agg 23
<210> 187
<211> 23
<212> DNA
<213> unknown
<400> 187
gacaaygtyc chtcyataga cac 23
<210> 188
<211> 23
<212> DNA
<213> unknown
<400> 188
gtrgggtcdt caggcttgga ttc 23
<210> 189
<211> 23
<212> DNA
<213> unknown
<400> 189
casacgacca svgcytcmam cac 23
<210> 190
<211> 23
<212> DNA
<213> unknown
<400> 190
tgcttcatra actcvckgck ytg 23
<210> 191
<211> 30
<212> DNA
<213> unknown
<400> 191
gactayrcng acrchgactt yytgggtgac 30
<210> 192
<211> 30
<212> DNA
<213> unknown
<400> 192
tcytcyttgc tccagtascg dcccatcttc 30
<210> 193
<211> 23
<212> DNA
<213> unknown
<400> 193
cactayaaca cmaagctggg mta 23
<210> 194
<211> 23
<212> DNA
<213> unknown
<400> 194
tgctcctgag gttghggttg caa 23
<210> 195
<211> 30
<212> DNA
<213> unknown
<400> 195
cgvaargact tyctrtgcca gtactgygcc 30
<210> 196
<211> 30
<212> DNA
<213> unknown
<400> 196
cccagcagrt grgaraartc catgttagca 30
<210> 197
<211> 23
<212> DNA
<213> unknown
<400> 197
atggtstgya cyggsaagat gca 23
<210> 198
<211> 23
<212> DNA
<213> unknown
<400> 198
atgttytcwg araavgtrta gtt 23
<210> 199
<211> 40
<212> DNA
<213> unknown
<400> 199
cyggsaagat gcacachgay cgcatytgcc gcttygacta 40
<210> 200
<211> 40
<212> DNA
<213> unknown
<400> 200
trtaaggcat rtabgtrtty tctccytgct cytgkatcca 40
<210> 201
<211> 23
<212> DNA
<213> unknown
<400> 201
gactacycrc tgcagagcaa cag 23
<210> 202
<211> 23
<212> DNA
<213> unknown
<400> 202
ttytcyttct gtcttcctgt tac 23
<210> 203
<211> 40
<212> DNA
<213> unknown
<400> 203
ctgcagagca acagycacmc gctcagccac gcrcaccagt 40
<210> 204
<211> 40
<212> DNA
<213> unknown
<400> 204
ttcctgttac aaaaccaaac ycksacyacy tctttytcca 40
<210> 205
<211> 23
<212> DNA
<213> unknown
<400> 205
gtkccwgayc chcarytdga tga 23
<210> 206
<211> 23
<212> DNA
<213> unknown
<400> 206
tcccartcrc aytcytgrct ctg 23
<210> 207
<211> 29
<212> DNA
<213> unknown
<400> 207
garacvgarv tsaaagargg rgcwcagca 29
<210> 208
<211> 30
<212> DNA
<213> unknown
<400> 208
gartgytcyt tytcytgytg rggrtctctg 30
<210> 209
<211> 23
<212> DNA
<213> unknown
<400> 209
acvctbaayg aratgtggtg cca 23
<210> 210
<211> 23
<212> DNA
<213> unknown
<400> 210
agcagrtcrt tsatctgbtc cag 23
<210> 211
<211> 29
<212> DNA
<213> unknown
<400> 211
ggytccaagc arttyaarat ccacaccat 29
<210> 212
<211> 30
<212> DNA
<213> unknown
<400> 212
agvggraarc gytgcytgtr yctgvagatg 30
<210> 213
<211> 23
<212> DNA
<213> unknown
<400> 213
tctayatyat caacsttygg cat 23
<210> 214
<211> 23
<212> DNA
<213> unknown
<400> 214
gtgagvaggg agcayttgaa cag 23
<210> 215
<211> 39
<212> DNA
<213> unknown
<400> 215
atcaacstty ggcatgggwg cyagcccytt yacyaacat 39
<210> 216
<211> 30
<212> DNA
<213> unknown
<400> 216
agtgtavagr tgkggkgcac agtgatccat 30
<210> 217
<211> 23
<212> DNA
<213> unknown
<400> 217
tgggtscgsc acasctacar cca 23
<210> 218
<211> 23
<212> DNA
<213> unknown
<400> 218
catccctttc ttyttscggt tga 23
<210> 219
<211> 28
<212> DNA
<213> unknown
<400> 219
aayagygayg argacagcaa agayggca 28
<210> 220
<211> 29
<212> DNA
<213> unknown
<400> 220
cgngcnggcc accargggaa kccrtgrat 29
<210> 221
<211> 23
<212> DNA
<213> unknown
<400> 221
ttycaysmvt ttgartggca gcc 23
<210> 222
<211> 23
<212> DNA
<213> unknown
<400> 222
ctgaasagyt tgttbschga ctc 23
<210> 223
<211> 35
<212> DNA
<213> unknown
<400> 223
aargayctbg agagaggaca thatggargg dctga 35
<210> 224
<211> 38
<212> DNA
<213> unknown
<400> 224
gtcatyaryy kbcgrgcrta gttbccattc atcctcat 38
<210> 225
<211> 23
<212> DNA
<213> unknown
<400> 225
ttwggncara arggntggcc naa 23
<210> 226
<211> 24
<212> DNA
<213> unknown
<400> 226
catrcaytgn gcrtgnaccc artg 24
<210> 227
<211> 43
<212> DNA
<213> unknown
<400> 227
agggttttcc cagtcacgac ggwggkaara cnccnaayaa yga 43
<210> 228
<211> 40
<212> DNA
<213> unknown
<400> 228
agataacaat ttcacacagg carcayttda tccartancc 40
<210> 229
<211> 23
<212> DNA
<213> unknown
<400> 229
atgctstcvg gvtcyccdca gtc 23
<210> 230
<211> 23
<212> DNA
<213> unknown
<400> 230
agmccyctrg cytkctccac cca 23
<210> 231
<211> 29
<212> DNA
<213> unknown
<400> 231
cagtcytact gcrtbgtrga raartggag 29
<210> 232
<211> 30
<212> DNA
<213> unknown
<400> 232
chagytgsgt tytkatcctc tcytcytcca 30
<210> 233
<211> 23
<212> DNA
<213> unknown
<400> 233
tccagaycch acntaygarc tca 23
<210> 234
<211> 23
<212> DNA
<213> unknown
<400> 234
acrgtdcgnc cdgtctccat gca 23
<210> 235
<211> 30
<212> DNA
<213> unknown
<400> 235
ggyaaagtyc cyhtrmgrca gctkgtstac 30
<210> 236
<211> 29
<212> DNA
<213> unknown
<400> 236
gttcatyttk gcyggrtcva grgcccagt 29
<210> 237
<211> 23
<212> DNA
<213> unknown
<400> 237
garcagacyc argtggtggc yat 23
<210> 238
<211> 23
<212> DNA
<213> unknown
<400> 238
tytgwkgbkg gtggtgsakc tgc 23
<210> 239
<211> 30
<212> DNA
<213> unknown
<400> 239
gtrrtyacya argavcagcc matgagcatg 30
<210> 240
<211> 30
<212> DNA
<213> unknown
<400> 240
atsggkatyt gcatgggrta raagttctgc 30
<210> 241
<211> 23
<212> DNA
<213> unknown
<400> 241
aayathacna aygcntgyta yaa 23
<210> 242
<211> 23
<212> DNA
<213> unknown
<400> 242
gcraartgnc crttnacrtg raa 23
<210> 243
<211> 43
<212> DNA
<213> unknown
<400> 243
agggttttcc cagtcacgac tgytayaayg aytgyccntg gat 43
<210> 244
<211> 43
<212> DNA
<213> unknown
<400> 244
agataacaat ttcacacagg ckgtgrggyt tyttrtartt rtg 43
<210> 245
<211> 23
<212> DNA
<213> unknown
<400> 245
ggarcagytr gtswgyctyc agc 23
<210> 246
<211> 23
<212> DNA
<213> unknown
<400> 246
caratkggrc angagtgytg cat 23
<210> 247
<211> 30
<212> DNA
<213> unknown
<400> 247
carcarctbt ctgcwgcagc wgchctvatw 30
<210> 248
<211> 29
<212> DNA
<213> unknown
<400> 248
gtkgwgaang cvckgccrca gatyttgca 29
<210> 249
<211> 23
<212> DNA
<213> unknown
<400> 249
tcatyttytc yatmttyggc atg 23
<210> 250
<211> 23
<212> DNA
<213> unknown
<400> 250
ttbtcrggyt tggtyacrct gtc 23
<210> 251
<211> 39
<212> DNA
<213> unknown
<400> 251
gachtgtaya acttygarac nttyggmaac agyatgatc 39
<210> 252
<211> 30
<212> DNA
<213> unknown
<400> 252
gtgcyctcyt tyttytcttg ytcytgrttg 30
<210> 253
<211> 23
<212> DNA
<213> unknown
<400> 253
ccdcaygcyc trtchctscg sag 23
<210> 254
<211> 23
<212> DNA
<213> unknown
<400> 254
acctcrcavs crsaactgct gcc 23
<210> 255
<211> 30
<212> DNA
<213> unknown
<400> 255
aghtggaggt rtchtcwccm aarmtctaca 30
<210> 256
<211> 30
<212> DNA
<213> unknown
<400> 256
tcctcctmct mctscrccts cryyggscat 30
<210> 257
<211> 23
<212> DNA
<213> unknown
<400> 257
gayagytcwg ghggdgchgt bca 23
<210> 258
<211> 23
<212> DNA
<213> unknown
<400> 258
gcmacyytyt cnggytcrct gtc 23
<210> 259
<211> 29
<212> DNA
<213> unknown
<400> 259
gaymgmtgya cyacbgtvag yccngtggt 29
<210> 260
<211> 30
<212> DNA
<213> unknown
<400> 260
aggmaywtgg carggyttya gdakytgctc 30
<210> 261
<211> 23
<212> DNA
<213> unknown
<400> 261
gtvatagayg gycargagag act 23
<210> 262
<211> 23
<212> DNA
<213> unknown
<400> 262
ggyctgaang cvgabaygta gct 23
<210> 263
<211> 30
<212> DNA
<213> unknown
<400> 263
gtbgcbctkg gvatcacytg ygtscagatg 30
<210> 264
<211> 30
<212> DNA
<213> unknown
<400> 264
gyrggccara acatsggraa rgarkgrtag 30
<210> 265
<211> 23
<212> DNA
<213> unknown
<400> 265
atgctbtaca tggtvatygt gcc 23
<210> 266
<211> 23
<212> DNA
<213> unknown
<400> 266
agcagmagca rvgcyggygc gta 23
<210> 267
<211> 29
<212> DNA
<213> unknown
<400> 267
ctbttygcnt chaargccat ybtscagct 29
<210> 268
<211> 30
<212> DNA
<213> unknown
<400> 268
tchgcdatdg crtamatcrc trccrtacac 30
<210> 269
<211> 23
<212> DNA
<213> unknown
<400> 269
tacatgttcy tgggyatgtc cat 23
<210> 270
<211> 21
<212> DNA
<213> unknown
<400> 270
trgcctcrtg rcagctcacc a 21
<210> 271
<211> 40
<212> DNA
<213> unknown
<400> 271
tgttcytggg yatgtccatc athgcvgayc gbttcatgtc 40
<210> 272
<211> 38
<212> DNA
<213> unknown
<400> 272
gcrtgcttyt tcarratgtt yccngcyccg atcatcat 38
<210> 273
<211> 23
<212> DNA
<213> unknown
<400> 273
gargaratmg aygagccytg ctt 23
<210> 274
<211> 23
<212> DNA
<213> unknown
<400> 274
atvagvachg asaghggvac agc 23
<210> 275
<211> 30
<212> DNA
<213> unknown
<400> 275
cargayathc aygybggngc dttyaatcgg 30
<210> 276
<211> 29
<212> DNA
<213> unknown
<400> 276
atrtcrcang agcavtccca dggrttctg 29
<210> 277
<211> 23
<212> DNA
<213> unknown
<400> 277
acygtstaym grccbacrca gct 23
<210> 278
<211> 23
<212> DNA
<213> unknown
<400> 278
tcvtcvagyt crtcratggt gct 23
<210> 279
<211> 30
<212> DNA
<213> unknown
<400> 279
garaacytgg aatacctyca ggcygactac 30
<210> 280
<211> 30
<212> DNA
<213> unknown
<400> 280
aggcaraarg cacacraava cwgtnaggat 30
<210> 281
<211> 23
<212> DNA
<213> unknown
<400> 281
gayttygtca tgctycagca gcc 23
<210> 282
<211> 23
<212> DNA
<213> unknown
<400> 282
taytgyagvg arcgcacttg cat 23
<210> 283
<211> 28
<212> DNA
<213> unknown
<400> 283
gargtvaaag cycagmgrcc cytnagat 28
<210> 284
<211> 29
<212> DNA
<213> unknown
<400> 284
ccytcbacrt cdggctgctc rtaraagct 29
<210> 285
<211> 23
<212> DNA
<213> unknown
<400> 285
atycchcgng aggtggagat gga 23
<210> 286
<211> 23
<212> DNA
<213> unknown
<400> 286
cgytchagna rccactgcat ggt 23
<210> 287
<211> 30
<212> DNA
<213> unknown
<400> 287
acvccngagt gggaygtktc nggggagact 30
<210> 288
<211> 29
<212> DNA
<213> unknown
<400> 288
tthcgcagaw gtgccdgact tkgwgtagc 29
<210> 289
<211> 23
<212> DNA
<213> unknown
<400> 289
gcycgtytga rrgargcbcg dga 23
<210> 290
<211> 23
<212> DNA
<213> unknown
<400> 290
ctvagagcng ccacdatyct vag 23
<210> 291
<211> 30
<212> DNA
<213> unknown
<400> 291
agcarcarcc ygcyctrtcy gargccatga 30
<210> 292
<211> 30
<212> DNA
<213> unknown
<400> 292
cmacytgcat rgccatbgcy tkrtggtcct 30
<210> 293
<211> 23
<212> DNA
<213> unknown
<400> 293
gacagcacvm grgtyagctt cac 23
<210> 294
<211> 23
<212> DNA
<213> unknown
<400> 294
tartctcykc tcbccraagt gca 23
<210> 295
<211> 39
<212> DNA
<213> unknown
<400> 295
gacmggcaga thttycagst tcagygarga yggcatggt 39
<210> 296
<211> 30
<212> DNA
<213> unknown
<400> 296
taraactcyt cnccrctrct dattctccat 30
<210> 297
<211> 23
<212> DNA
<213> unknown
<400> 297
agyccstgyt ccaayggyta cat 23
<210> 298
<211> 23
<212> DNA
<213> unknown
<400> 298
cctgrtagkc yggmggrgaa ggt 23
<210> 299
<211> 30
<212> DNA
<213> unknown
<400> 299
cctsgtcatg ctctaygtgg tytacctkgt 30
<210> 300
<211> 30
<212> DNA
<213> unknown
<400> 300
tgghvcgrtg gcagcgytcr carttctgcc 30
<210> 301
<211> 23
<212> DNA
<213> unknown
<400> 301
gagcarbtgg crkcngayca cag 23
<210> 302
<211> 23
<212> DNA
<213> unknown
<400> 302
cccaryctyy tytccttctg ctc 23
<210> 303
<211> 31
<212> DNA
<213> unknown
<400> 303
ggnatgaarg argaraacag ycayytgaaa g 31
<210> 304
<211> 31
<212> DNA
<213> unknown
<400> 304
ttvacygtbg tgatsacctg cwkcttccac t 31
<210> 305
<211> 23
<212> DNA
<213> unknown
<400> 305
aactcyccac tyagcaatgg cac 23
<210> 306
<211> 23
<212> DNA
<213> unknown
<400> 306
ggagytgrta rgcrgcrtga atg 23
<210> 307
<211> 30
<212> DNA
<213> unknown
<400> 307
tgcaaayytg atytcdgayg cytcytggtc 30
<210> 308
<211> 29
<212> DNA
<213> unknown
<400> 308
tgrgcyttrc tgatwgcact kgaracwgt 29
<210> 309
<211> 23
<212> DNA
<213> unknown
<400> 309
gagctggtga argtratggg dct 23
<210> 310
<211> 23
<212> DNA
<213> unknown
<400> 310
tactggaacc artamacngt ctc 23
<210> 311
<211> 36
<212> DNA
<213> unknown
<400> 311
ggvagcctba tmtacctsca ygacacyytg gaggag 36
<210> 312
<211> 35
<212> DNA
<213> unknown
<400> 312
cchtcccagw artgyttgaa sccrcabccr cactt 35
<210> 313
<211> 23
<212> DNA
<213> unknown
<400> 313
catatcdcca cgdgayagta cca 23
<210> 314
<211> 23
<212> DNA
<213> unknown
<400> 314
atggcttcry thacaatgag gtc 23
<210> 315
<211> 30
<212> DNA
<213> unknown
<400> 315
tgagrcaygt rcgaagggct caycccactg 30
<210> 316
<211> 30
<212> DNA
<213> unknown
<400> 316
tggctgaaga tccagtgcra gcatttctgc 30
<210> 317
<211> 23
<212> DNA
<213> unknown
<400> 317
acbaacacyg ccatywccca gct 23
<210> 318
<211> 23
<212> DNA
<213> unknown
<400> 318
gctggcytgc aysgggggca tga 23
<210> 319
<211> 29
<212> DNA
<213> unknown
<400> 319
gacttcaayg actayaagct satgatggc 29
<210> 320
<211> 29
<212> DNA
<213> unknown
<400> 320
ttggacagrt argccargtt sarmggctc 29
<210> 321
<211> 23
<212> DNA
<213> unknown
<400> 321
ggbgagsysa adttyggnga cac 23
<210> 322
<211> 23
<212> DNA
<213> unknown
<400> 322
tctkrygctc kvtcngagaa gtc 23
<210> 323
<211> 29
<212> DNA
<213> unknown
<400> 323
ggrctcttyt ggggnccnyt stgctgctc 29
<210> 324
<211> 29
<212> DNA
<213> unknown
<400> 324
ggraggaarg cvggrtcrkt rgwcttctg 29
<210> 325
<211> 23
<212> DNA
<213> unknown
<400> 325
gycarccwat yagcagycag atg 23
<210> 326
<211> 23
<212> DNA
<213> unknown
<400> 326
gtsgtggadc ggctgatgga ctg 23
<210> 327
<211> 30
<212> DNA
<213> unknown
<400> 327
tgyggcaarc gyttccgytt yaacagcatc 30
<210> 328
<211> 30
<212> DNA
<213> unknown
<400> 328
gckgvtgctg stgctgtgmt cyttnggcat 30

Claims (10)

1. A universal nuclear gene molecular marker primer for fish is characterized by comprising one or more of primers shown as SEQ NO.1-SEQ NO. 328.
2. The fish universal nuclear gene molecular marker primer as claimed in claim 1, characterized by comprising one or more primer pairs of NP 1-NP 82, wherein the primer pair NP1 is shown as SEQ NO.1-SEQ NO.4, the primer pair NP2 is shown as SEQ NO.5-SEQ NO.8, the primer pair NP3 is shown as SEQ NO.9-SEQ NO.12, the primer pair NP4 is shown as SEQ NO.13-SEQ NO.16, the primer pair NP5 is shown as SEQ NO.17-SEQ NO.20, the primer pair NP6 is shown as SEQ NO.21-SEQ NO.24, the primer pair NP7 is shown as SEQ NO.25-SEQ NO.28, the primer pair NP8 is shown as SEQ NO.29-SEQ NO.32, the primer pair NP9 is shown as SEQ NO.33-SEQ NO.36, the primer pair NP10 is shown as SEQ NO.37-SEQ NO.40, the primer pair NP11 is shown as SEQ NO.41-SEQ NO.44, the primer pair NP12 is shown as SEQ NO. 48-SEQ NO.48, the primer pair NP49 is shown as SEQ NO.52, the primer pair NP14 is shown as SEQ NO.53-SEQ NO.56, the primer pair NP15 is shown as SEQ NO.57-SEQ NO.60, the primer pair NP16 is shown as SEQ NO.61-SEQ NO.64, the primer pair NP17 is shown as SEQ NO.65-SEQ NO.68, the primer pair NP18 is shown as SEQ NO.69-SEQ NO.72, the primer pair NP19 is shown as SEQ NO.73-SEQ NO.76, the primer pair NP20 is shown as SEQ NO.77-SEQ NO.80, the primer pair NP21 is shown as SEQ NO.81-SEQ NO.84, the primer pair NP22 is shown as SEQ NO.85-SEQ NO.88, the primer pair NP23 is shown as SEQ NO.89-SEQ NO.92, the primer pair NP24 is shown as SEQ NO.93-SEQ NO.96, the primer pair NP25 is shown as SEQ NO.97-SEQ NO.100, the primer pair NP26 is shown as SEQ NO.101-SEQ NO.104, the primer pair NP27 is shown as SEQ NO.105-SEQ NO.108, and SEQ NO. 112-SEQ NO.109, the primer pair NP29 is shown as SEQ NO.113-SEQ NO.116, the primer pair NP30 is shown as SEQ NO.117-SEQ NO.120, the primer pair NP31 is shown as SEQ NO.121-SEQ NO.124, the primer pair NP32 is shown as SEQ NO.125-SEQ NO.128, the primer pair NP33 is shown as SEQ NO.129-SEQ NO.132, the primer pair NP34 is shown as SEQ NO.133-SEQ NO.136, the primer pair NP35 is shown as SEQ NO.137-SEQ NO.140, the primer pair NP36 is shown as SEQ NO.141-SEQ NO.144, the primer pair NP37 is shown as SEQ NO.145-SEQ NO.148, the primer pair NP38 is shown as SEQ NO.149-SEQ NO.152, the primer pair NP39 is shown as SEQ NO.153-SEQ NO.156, the primer pair NP40 is shown as SEQ NO.157-SEQ NO.160, the primer pair NP41 is shown as SEQ NO.161-SEQ NO.164, the primer pair NP42 is shown as SEQ NO.165-SEQ NO.168, the primer pair NP42, the primer pair NP 44-SEQ NO.169 is shown as SEQ NO.169-SEQ NO.176, and SEQ NO.169, the primer pair NP45 is shown as SEQ NO.177-SEQ NO.180, the primer pair NP46 is shown as SEQ NO.179-SEQ NO.184, the primer pair NP47 is shown as SEQ NO.185-SEQ NO.188, the primer pair NP48 is shown as SEQ NO.189-SEQ NO.192, the primer pair NP49 is shown as SEQ NO.193-SEQ NO.196, the primer pair NP50 is shown as SEQ NO.197-SEQ NO.200, the primer pair NP51 is shown as SEQ NO.201-SEQ NO.204, the primer pair NP52 is shown as SEQ NO.233-SEQ NO.208, the primer pair NP53 is shown as SEQ NO.209-SEQ NO.212, the primer pair NP54 is shown as SEQ NO.213-SEQ NO.216, the primer pair NP55 is shown as SEQ NO.217-SEQ NO.220, the primer pair NP56 is shown as SEQ NO.233-SEQ NO.224, the primer pair NP57 is shown as SEQ NO.225-SEQ NO.228, the primer pair NP58 is shown as SEQ NO.229-SEQ NO.232, and the primer pair NP59, the primer pair NP60 is shown as SEQ NO.237-SEQ NO.240, the primer pair NP61 is shown as SEQ NO.241-SEQ NO.244, the primer pair NP62 is shown as SEQ NO.245-SEQ NO.248, the primer pair NP63 is shown as SEQ NO.249-SEQ NO.252, the primer pair NP64 is shown as SEQ NO.253-SEQ NO.256, the primer pair NP65 is shown as SEQ NO.257-SEQ NO.260, the primer pair NP66 is shown as SEQ NO.261-SEQ NO.264, the primer pair NP67 is shown as SEQ NO.265-SEQ NO.268, the primer pair NP68 is shown as SEQ NO.269-SEQ NO.272, the primer pair NP69 is shown as SEQ NO.273-SEQ NO.276, the primer pair NP70 is shown as SEQ NO.277-SEQ NO.280, the primer pair NP71 is shown as SEQ NO.281-SEQ NO.284, the primer pair NP72 is shown as SEQ NO.285-SEQ NO.288, the primer pair NP73 is shown as SEQ NO.289-SEQ NO.292, the primer pair NP74 is shown as SEQ NO.293-SEQ NO.296, the primer pair NP75 is shown as SEQ NO.297-SEQ NO.300, the primer pair NP76 is shown as SEQ NO.301-SEQ NO.304, the primer pair NP77 is shown as SEQ NO.305-SEQ NO.308, the primer pair NP78 is shown as SEQ NO.309-SEQ NO.312, the primer pair NP79 is shown as SEQ NO.313-SEQ NO.316, the primer pair NP80 is shown as SEQ NO.317-SEQ NO.320, the primer pair NP81 is shown as SEQ NO.321-SEQ NO.324, and the primer pair NP82 is shown as SEQ NO.325-SEQ NO. 328.
3. A universal nuclear gene molecular marker for fish, which is formed by amplifying the molecular marker primer according to any one of claims 1 to 2.
4. A fish universal nuclear gene molecular marker database, comprising the fish universal nuclear gene molecular marker of claim 3.
5. Use of the fish universal nuclear gene molecular marker primer according to claim 1 or 2, the fish universal nuclear gene molecular marker according to claim 3, and the fish universal nuclear gene molecular marker database according to claim 4 in preparation of reagents for identifying fish germplasm resources and categories.
6. A method for identifying a species of a fish, comprising the steps of:
a1, obtaining DNA of a fish to be identified;
a2, carrying out PCR amplification through the primer of claim 1;
and A3, performing high-throughput sequencing on the PCR amplification product obtained in the step A2, comparing the PCR amplification product with the fish universal type nuclear gene molecular marker database of claim 4, and acquiring species information of the fish to be identified according to molecular markers matched with the fish to be identified in the molecular marker database and a phylogenetic classification tree established on the basis of the molecular marker database.
7. A kit comprising the fish universal nuclear gene molecular marker of claim 3 and/or the fish universal nuclear gene molecular marker primer of claim 1 or 2.
8. The kit according to claim 7, further comprising reagents required for the PCR amplification system of the fish universal nuclear gene molecular marker primer according to claim 1 or 2.
9. A phylogenetic model constructed from the fish universal nuclear gene molecular marker primers of claim 1 or 2, the fish universal nuclear gene molecular markers of claim 3, and/or the fish universal nuclear gene molecular marker database of claim 4.
10. The phylogenetic model of claim 9, comprising a phylogenetic tree.
CN202210345227.4A 2021-06-11 2022-03-31 Fish nuclear gene molecular marker primer, molecular marker and molecular marker database Active CN115323060B (en)

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