CN113186302B - Sex specific molecular marker of Megalobrama amblycephala hybrid Pioneer No. 2 and application - Google Patents
Sex specific molecular marker of Megalobrama amblycephala hybrid Pioneer No. 2 and application Download PDFInfo
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- CN113186302B CN113186302B CN202110624804.9A CN202110624804A CN113186302B CN 113186302 B CN113186302 B CN 113186302B CN 202110624804 A CN202110624804 A CN 202110624804A CN 113186302 B CN113186302 B CN 113186302B
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Abstract
The invention belongs to the field of fish sex identification in the field of aquaculture, and particularly relates to a sex specific molecular marker of culter bream hybrid Pioneer No. 2 and application thereof. According to the invention, a culter bream hybrid fish male sex specific DNA fragment is successfully screened by a high-throughput sequencing method, wherein the specific DNA fragment is shown in SEQ ID NO. 1. Aiming at the specific sequence, a primer is designed, the sex of the culter bream hybrid fish and the male parent ancherythroculter nigrocauda can be accurately and quickly identified by utilizing conventional PCR amplification and common agarose electrophoresis, and the primer sequence, the PCR reaction system and the conditions are disclosed. Compared with the prior dissection detection or the observation of the outburst, the technology has the advantages of accuracy, simplicity, rapidness, less damage to fish bodies and the like, solves the difficult problem of sex identification of the culter breams and the ancherythroculter nigrocauda, and is beneficial to large-scale cultivation of the culter bream hybrid fish hologynic system.
Description
Technical Field
The invention belongs to the field of fish sex identification in the field of aquaculture, and particularly relates to a sex specific molecular marker of culter bream hybrid Pioneer No. 2 and application thereof.
Background
In many fish species, males and females exhibit distinct biological traits with sex-allergenicity, such as differences in growth rate, maturation age, mode of reproduction, body color, body type, individual size, etc. (Kobayashi et al, 2013; Mei and Gui, 2015). Therefore, the development of the parthenocarpic breeding and culture technology of the fishes has great significance in the fish culture industry. The culter bream hybrid 'Pioneer No. 2' is a new species of Chinese characteristic economic fishes which is formed by multi-generation directional breeding after distant hybridization of megalobrama amblycephala (male parent) and Ancherythroculter nigrocauda (male parent), and has the excellent characteristics of high growth speed, strong disease resistance and easy catching and transportation resistance. In pond culture, the female of "pioneer No. 2" has a faster growth rate, i.e., higher market economic value, than the male. The continuous breeding of the all-female fish can accumulate the excellent characters of stable species in a short time and can also avoid gene pollution to natural resources. Before the gonads mature, the female fish and the male fish are difficult to distinguish from each other in external morphology, and the female fish and the male fish in the embryo stage are completely indistinguishable from each other in morphology; there is no heterochromosome, and the genetic sex cannot be identified from the cytological point of view. If a sex specificity marker capable of accurately identifying the culter bream hybrid can be developed, the accuracy and convenience of large-scale cultivation of the full-female population can be greatly improved.
With the rapid development of high-throughput sequencing technology, simplified genome sequencing (RAD-Seq) based on enzyme digestion is used as a method for genotyping through sequencing, so that possibility is provided for developing sex-specific molecular markers of fishes without complete genome Sequence information (Gamble et al, 2014; Liu et al, 2018; Zhou et al, 2019; Han et al, 2020). The invention discloses a simplified genome sequencing method by utilizing RAD (Radar induced fluorescence) and provides a male specific sex molecular marker for the culter bream hybrid fish, and the marker is successfully applied to the rapid identification of the sex of the culter bream hybrid fish and the male parent ancherythroculter nigrocauda.
Disclosure of Invention
The invention aims to provide a specific DNA fragment for sex identification of 'Pioneer No. 2' of a culter bream hybrid fish, wherein the DNA fragment is shown in SEQ ID NO. 1.
The invention provides a primer designed aiming at a sequence shown in SEQ ID NO. 1.
The invention provides an application of a sequence shown in SEQ ID NO.1 or a primer designed aiming at the sequence shown in SEQ ID NO. 1. The fragment or the primer can be used for sex identification of the culter bream hybrid fish and the ancherythroculter nigrocauda, the problem of early sex identification is solved, a method for quickly, simply, conveniently and accurately identifying genetic sex based on PCR is provided, and a foundation is established for smoothly carrying out sex control breeding work of the culter bream hybrid fish and large-scale culture of a unisexual culture population.
In order to achieve the purpose, the invention adopts the following technical measures:
the invention provides a screening method of specific molecular markers of sex of a culter bream hybrid, which comprises the steps of utilizing a culter bream hybrid sample with known sex, taking a tail fin to extract genome DNA, sequencing by a 2b-RAD method, constructing a library, and performing sequencing on a computer after the test is qualified; and filtering sequencing data, and identifying a sex specific sequence by using UStags to obtain a candidate sex specific molecular marker. And further screening candidate molecular markers to obtain a universally adapted specific sex molecular marker which can be used for identifying the culter bream hybrid fish and the ancherythroculter nigrocauda, wherein the molecular marker is a segment of DNA fragment, and the nucleotide sequence of the DNA fragment is shown as SEQ ID NO. 1.
The protection content of the invention also comprises:
the reagent for detecting the gene shown in SEQ ID NO.1 is applied to sex identification of culter bream hybrid fish or ancherythroculter nigrocauda.
In the above application, preferably, the reagent is a primer;
in the above application, preferably, the primer is: ATTCGCCAAAGGGCCGAATC and R TTT CTGCTGCAGTCGGTTCA.
In the above application, the result is determined as: the primer can be used for amplifying a 412bp fragment which is a male culter bream hybrid fish or an ancherythroculter nigrocauda male fish.
Compared with the prior art, the invention has the following advantages:
(1) the genetic sex identification method provided by the invention can identify the sex by using genome DNA as a template and only using simple P CR and an electrophoresis result. The method is not influenced by the development period and environment of individuals, and can stably identify the genetic sex of different individuals in each group of the hybrid culter, including embryos, larvae and adults, thereby overcoming the defects of more complicated operation, longer time consumption and the like of other methods, and being suitable for quickly identifying large samples.
(2) The DNA molecular marker is low in cost, simple, rapid and accurate in operation and suitable for popularization and application, and can effectively identify and identify genetic sex in various development stages by applying the DNA molecular marker to production practice, and is beneficial to distinguishing normal female fish and pseudo-female fish (physiological male fish), so that culter bream hybrid fish hologynic aquaculture populations are cultured in a large scale, the aquaculture benefit is further improved, and the economic benefit of fishpond aquaculture is increased.
(3) The detection accuracy of the molecular marker of the invention reaches 100%.
Drawings
FIG. 1 is a schematic diagram showing the result of genetic sex identification of male specific DNA fragment "Pioneer No. 2" of a culter bream hybrid fish in RAD test sample population;
in the figure, the lanes of female 1-10 individuals can not amplify a band, the lanes of male 1-10 individuals can amplify a specific band of 412bp, and M represents DL2000DNA marker.
FIG. 2 is a schematic diagram showing the result of genetic sex identification of male specific DNA fragment "Pioneer No. 2" of culter bream hybrid fish in natural culter bream hybrid fish;
in the figure, lanes of female 1-15 individuals can not amplify a band, lanes of male 1-15 individuals can amplify a specific band of 412bp, and M represents DL2000DNA marker.
FIG. 3 is a schematic diagram showing the result of genetic sex identification of male specific DNA fragment "Pioneer No. 2" of a culter bream hybrid fish in an ancherythroculter nigrocauda population;
in the figure, lanes of female 1-15 individuals can not amplify a band, lanes of male 1-15 individuals can amplify a specific band of 412bp, and M represents DL2000DNA marker.
Detailed Description
The features and advantages of the present invention will be further understood from the following detailed description taken in conjunction with the accompanying drawings. The examples provided are merely illustrative of the method of the present invention and do not limit the remainder of the disclosure in any way. The examples do not specify particular techniques or conditions, and are performed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Example 1:
obtaining specific DNA fragments of the hybrid fish sex of the culter breams:
(1) and (4) screening male specific sequences. 10-female 10-male culter bream hybrid fish samples determined by dissecting and observing gonads are taken, genomic DNA is extracted from the tail fins, a sequencing library is constructed by a 2b-RAD method, and the machine is used for sequencing after the sequencing library is qualified; after filtering the sequencing data, a total of 5.49G of raw data and 312164 high quality tags were obtained after filtering, with the average depth of the tags being 38.73X. Using UStags to carry out sex sequence identification, and finally obtaining 9 tags which appear in 10 males and do not appear in 10 females, wherein the tags are male specific short sequences, namely candidate specific sex molecular markers;
(2) and constructing a male fish reference genome. The short sequence screened in the step (1) is only 27bp, common PCR primer design can not be carried out, and a longer genome sequence needs to be obtained. Selecting 1 male fish to perform personalized analysis, and performing de novo assembly by using Soap Denov o v1.2.0 software to obtain survey of the male fish as a reference genome sequence;
(3) BL AST comparison search is carried out on the male specific short sequence obtained in the step (1) in the male reference genome sequence obtained in the step (2), a corresponding longer nucleotide sequence is obtained, a corresponding primer is designed to carry out population verification on the validity, a male specific DNA fragment (shown in SEQ ID NO. 1) is finally obtained, and no homologous sequence is found through comparison of a GenBank database on NCBI.
Example 2:
the method for using sex-specific DNA fragments of the culter bream hybrid fish comprises the following steps:
(1) the primer designed aiming at the sequence shown in SEQ ID NO.1 is as follows:
ATTCGCCAAAGGGCCGAATC and R TTTCTGCTGCAGTCGGTTCA.
(2) And (3) PCR amplification:
the PCR reaction system was a mixture of 0.5. mu.l (100 ng/. mu.l) of template DNA template, 5. mu.l of 2x Hieff PCR Master Mix (Yeasen), and 0.25. mu.l each of the upstream and downstream primers (10. mu. mol/. mu.l), supplemented with ddH2O to 10. mu.l of PCR conditions: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 52.2 ℃ for 30s, and extension at 72 ℃ for 30s for 35 cycles; finally, extension was carried out at 72 ℃ for 7min and stored at 4 ℃.
(3) After PCR amplification is finished, a male specific DNA fragment is screened out through 1% agarose gel electrophoresis detection, and amplified specific bands exist in male individuals and do not exist in female individuals.
Example 3:
the application of the male specificity DNA fragment in sex identification of the culter bream hybrid fish and the ancherythroculter nigrocauda:
(1) a culter bream hybrid fish (25 female and male) and a culter nigrocauda (15 female and male) group with known sex are stored in absolute ethyl alcohol by using fin ray tissue samples, extracted with an ammonium acetate method to obtain genome DNA, diluted to 100 ng/mu L and stored at-20 ℃ for later use.
(2) Performing PCR amplification on the sample population by using the method of example 2;
(3) the amplification results were as follows:
FIG. 1 shows the amplification results of male-specific DNA fragments of a culter bream hybrid in RAD test sample populations; in the figure, the lanes of female 1-10 individuals can not amplify a band, the lanes of male 1-10 individuals can amplify a specific band of 412bp, and M represents DL2000DNA marker.
FIG. 2 shows the amplification result of male specific DNA fragments of a culter bream hybrid fish in a natural culter bream hybrid fish population; in the figure, lanes of female 1-15 individuals can not amplify a band, lanes of male 1-15 individuals can amplify a specific band of 412bp, and M represents DL2000DNA marker.
FIG. 3 shows the amplification result of male specific DNA fragment of culter bream hybrid in Ancherythroculter nigrocauda population; in the figure, lanes of female 1-15 individuals can not amplify a band, lanes of male 1-15 individuals can amplify a specific band of 412bp, and M represents DL2000DNA marker.
Sequence listing
<110> university of agriculture in Huazhong
WUHAN ACADEMY OF AGRICULTURAL SCIENCES
WUHAN XIANFENG AQUATIC PRODUCT SCIENCE & TECHNOLOGY Co.,Ltd.
Sex specific molecular marker of <120> culter bream hybrid Pioneer No. 2 and application
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 412
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
attcgccaaa gggccgaatc ttatatacag cgacagcgtc atttaaaaaa ataaaaaatt 60
atgtctgtaa ccccaaccct ttgaaaaaaa aagaaaaagt attttgaata aactgtgatt 120
tgataaagta tcatatatat gctaacttaa tgaatcaaag tttattcaat attcagtaca 180
gtacagtaca gtatatatgc agatttaatg ttttctggat tgaaaacact gcattcttcc 240
agagatgcaa cagattctcc attgaaacgt cacttttctt aaaaaaatga aagatttcct 300
tggacgtgaa ttcatgtgta actacatata cttcattata caatcattat cattcatttg 360
attttgtaaa ggagaaagac gtggaaacat catgaaccga ctgcagcaga aa 412
<210> 2
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
attcgccaaa gggccgaatc 20
<210> 3
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
tttctgctgc agtcggttca 20
Claims (4)
1. Identifying specific sex molecular markers of the culter bream hybrid fish and the ancherythroculter nigrocauda, wherein the molecular markers are DNA fragments, and the nucleotide sequences of the DNA fragments are shown in SEQ ID NO. 1.
2. The reagent for detecting the gene shown in SEQ ID NO.1 is applied to sex identification of culter bream hybrid fish or ancherythroculter nigrocauda.
3. The use of claim 2, wherein the agent is a primer.
4. The use of claim 3, wherein the primers are: ATTCGCCAAAGGGCCGAATC and R TTTCTGCTGCAGTCGGTTCA.
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