CN106048038B - Chinese giant salamander population division method based on mitochondrial DNA sequence - Google Patents
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Abstract
The invention discloses a Chinese giant salamander population division method based on a mitochondrial DNA sequence, which comprises the following steps: (1) collecting a giant salamander sample, and extracting genome DNA; (2) synthesizing a giant salamander specific mitochondrial DNA primer; (3) carrying out PCR amplification on the giant salamander sample and sequencing a PCR amplification product; (4) comparing the obtained sequence with a reference sequence; (5) and judging the group to which the sample belongs according to the sequence characteristics of the corresponding sites. The invention takes the giant salamander mitochondrial DNA sequence as a sequence marker for distinguishing the giant salamander population in China, and can quickly and accurately identify the target population by comparing the sequence marker with the sequence characteristics of a reference sequence. The method can be used for identifying the giant salamander individuals in the giant salamander farm and determining the source of the individuals; the breeding selection can be assisted when the parents are propagated. The method can also be applied to the identification of the proliferation and releasing samples, the population to which the proliferation and releasing samples belong is determined, and then the population is released to a suitable area, so that the germplasm of the released population is prevented from being mixed with the germplasm of the wild population.
Description
Technical Field
The invention belongs to the technical field of biological identification, and particularly relates to a Chinese giant salamander colony division method based on a mitochondrial DNA sequence.
Background
The Chinese giant salamander (Andrias davidianus) is one of the existing amphibious classes, the largest body size is one, the largest individual body length can reach more than 1 meter, and the body weight can reach more than 8 kilograms. The giant salamander in China is only distributed in China, is unique in China, and the distribution areas comprise provinces, cities and regions such as Beijing, Hebei, Henan, Shanxi, Gansu, Qinghai, Sichuan, Guizhou, Hubei, Hunan, Anhui, Jiansu, Zhejiang, Jiangxi, Fujian, Guangdong and Guangxi. Due to important scientific research and economic values and large market requirements, the giant salamander is excessively fished in China, and the habitat is reduced, so that the wild giant salamander resources are extremely rare at present, and China is listed as a national secondary protection animal.
At present, the state continuously strengthens the protection force of wild giant salamanders, and is provided with more than ten Chinese giant salamander protection areas of national level, provincial level, city and county level and the like so as to protect wild Chinese giant salamander resources. Meanwhile, China also has a plurality of farms with different scales, and the bred and cultured giant salamanders are not only used for market consumption, but also partially used for breeding and releasing to supplement wild resources. The giant salamander is protected, so that the number of the giant salamanders is protected, and the genetic diversity of the giant salamanders is also protected. Giant salamanders belong to amphibians, and live in river water bodies and act slowly. Isolation of mountain water systems and disruption of habitats cause geographical differentiation and genetic structural variation of giant salamanders in long-term evolution. The genetic markers for distinguishing different geographical populations are searched, and the method has high application value. On one hand, in the practice of protection work, each population with genetic differentiation is used as a different germplasm resource management unit to be protected to avoid loss of genetic diversity. On the other hand, the method is also used for identifying parent sources of giant salamander farms, distinguishing germplasm resources and avoiding germplasm mixing in artificial propagation. Meanwhile, the giant salamanders used for releasing can be identified, so that the situation that the germplasms of wild groups are mixed due to the fact that the giant salamanders of different geographical groups are released mutually is avoided.
At present, some workers identify giant salamanders of different geographical groups through body surface patterns, but the body surface patterns are unstable, have no fixed marks and are difficult to be used as the standard for group division. Some reports exist at home and abroad, and mitochondrial DNA is adopted to analyze a small amount of samples to find that Chinese giant salamander groups have certain genetic difference, but clear group markers are not provided. Mitochondrial DNA sequence variation rate is fast, and different isolated populations can easily generate recognizable sequence markers. Therefore, the mitochondrial DNA sequence is convenient and reliable to be used as a marker for distinguishing the Chinese giant salamander population.
Disclosure of Invention
In view of this, the technical problem to be solved by the present invention is that there is no method for conveniently and reliably identifying giant salamander population in the prior art. Provides a Chinese giant salamander population division method based on mitochondrial DNA sequences.
In order to solve the technical problems, the invention discloses a Chinese giant salamander colony dividing method based on a mitochondrial DNA sequence, which is characterized by comprising the following steps of: (1) collecting a giant salamander sample, and extracting genome DNA; (2) synthesizing a giant salamander specific mitochondrial DNA primer; (3) carrying out PCR amplification on a giant salamander sample and sequencing a PCR amplification product; (4) comparing the obtained sequence with a reference sequence; (5) and judging the group to which the sample belongs according to the sequence characteristics of the corresponding sites.
Further, in the step (2), the giant salamander specific mitochondrial DNA primers are L14764 and H16062, wherein the L14764 is shown in SEQ ID NO.1, and the H16062 is shown in SEQ ID NO. 2.
Further, the PCR amplification reaction system in the step (3) is as follows: the total volume is 50 mu L, the PCR primer comprises 2 mu L of 10 XPCR buffer5 mu L, 0.5 mu L of 10mmol/L dNTPs, 2 mu L of 10 mu mol/L primers L14764 and H16062, 2 units of Taq DNA polymerase and 20-100 ng template DNA, and sterile double distilled water is supplemented to 50 mu L.
Further, the PCR amplification reaction conditions in the step (3) are as follows: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 30s, extension at 72 ℃ for 90s, and 35 cycles; further extension was carried out at 72 ℃ for 8 min.
Further, the sequencing method in the step (3) is as follows: and detecting the PCR amplification product by 1% agarose gel electrophoresis, purifying and recovering by adopting a PCR product gel recovery kit, and performing bidirectional sequencing on the recovered product by using primers L14764 and H16062.
Further, the step (4) is specifically as follows: and (4) comparing the sequence obtained in the step (3) with a reference sequence group, and determining the group to which the detected sample belongs by referring to the sequence characteristics of the corresponding sites based on the sites set by the initial sequences of the sequence group.
Further, the characteristic sequences of the population in the step (4) are as follows:
POP 1: sites 654-903, 910-920, 927-972 are absent, 904-909 is GCAGTG, GCAGAG or GCAGGG, and sites 921-926 are CGAATT;
P0P 2: sites 654-659, 664-903, 910-920 and 930-974 are deleted, sites 660-663 are T, and sites 904-909 are ACAGAG;
POP 3: sites 654-659, 664-903, 910-920 and 929-974 are deleted, and sites 660-663 are T; sites 904-909 are ACAGGG;
POP 4: sites 653-659 and 664-895 are deleted, sites 593-605 are TTTTCACTTTTA, and sites 660-663 are TTTT;
POP 5: sites 653-659 and 664-895 are deleted, sites 593-605 are TTTCACTTTTTCA, and sites 660-663 are TTTT;
POP 6: 656-659, 664-896, 610-619 TCTAAGGGT, 653-655 ATA, 897-901 GAGAGAT, 973-977 GAAA, 1005-1007 CAA;
POP 7: sites 664-895 are deleted, sites 653-663 are AAAATATTTTT, and sites 897-901 are ATGTT;
POP 8: the sites 364-368 are GGGGG, and the sites 664-802 are
GATGTACTTTCTTTTTGCCTATGCAATTCTCCGATCAACCCCAAACAAACTCGGAGGCGTCCTAGCCTTAGGGGCCTCCATTTTCATTTTAATCCTATACCCCTTCTTCATACATCAAAACAACGAGGTTTAATATTCC。
Further, the distribution range of the population POP1-POP8 in the step (4) is as follows:
POP1 population is mainly distributed in Hunan, Hubei, Henan, Shanxi, Anhui, Gansu, Chongqing, Guizhou, Yunnan, Sichuan, Guangxi melt water and other areas;
the POP2 population is mainly distributed in Guangxi resource county;
POP3 colony is mainly distributed in Henan Koelreuteria, Anhui Laoban and Shaanxi Ankang;
POP4 population is mainly distributed in Huangshan region of Anhui province;
POP5 colony is mainly distributed in Jiangxi, Zhejiang and Anhui leaderboard;
POP6 population is mainly distributed in Anhui Huangshan and Zhejiang Lishui;
POP7 colony is mainly distributed in Hunan Yongding and Longshan, Hunbei Enshi, Chongqing Fuling and Wulong, Guizhou, Yunnan, Guangxi melt water and other areas;
the POP8 population was mainly distributed in guangxi xingan county.
Further, the giant salamander sample collected in the step (1) comprises fin rays, blood, muscle, fresh molt, mucus or oral epithelial cells.
Compared with the prior art, the invention can obtain the following technical effects:
1) the invention takes the giant salamander mitochondrial DNA sequence as a sequence marker for distinguishing the giant salamander population in China, and can quickly and accurately identify the target population by comparing the sequence marker with the sequence characteristics of a reference sequence.
2) The technical scheme of the invention has the advantages of simple steps, easy operation, good repeatability and easy learning and mastering of technicians.
3) The experimental reagents selected in the invention are conventional reagents, no expensive and rare reagents are used, and the experimental operation cost is low.
4) The method can be used for identifying the giant salamander individuals in the giant salamander farm and determining the source of the individuals; the breeding selection can be assisted when the parents are propagated.
5) The invention can also be applied to the identification of the proliferation and releasing samples, the population to which the proliferation and releasing samples belong is determined, and then the proliferation and releasing samples are released to a proper area, so that the germplasm of the releasing population is prevented from being mixed with the germplasm of the wild population.
Of course, it is not necessary for any one product in which the invention is practiced to achieve all of the above-described technical effects simultaneously.
Detailed Description
The following embodiments are described in detail with reference to the accompanying drawings, so that how to implement the technical features of the present invention to solve the technical problems and achieve the technical effects can be fully understood and implemented.
Examples
(1) Sample collection and DNA extraction
Chinese giant salamander individuals were collected in Guangxi Xingan (GXXA), Hunan Longshan (HNLS), Jiangxi Jingan (JXJA), Anhui Huangshan (AHHS), Hubei Laifeng (HBLF), Zhejiang Lishui (ZJLS) and the like in China. The collected part or tissue may be small amount of fin ray, blood, muscle, fresh molt, or oral epithelial cells scraped with sterilized cotton swab, etc. And extracting the genome DNA by adopting a DNA extraction kit or a phenol chloroform method. The purity and content of DNA were determined by a spectrophotometer and stored at-20 ℃ for further use. The DNA concentration was adjusted to 10-100 ng/. mu.L at the time of use.
(2) Primer synthesis
The following primers were synthesized by Shanghai Biotechnology Ltd:
L14764：5′-ATGAAACAGGGTCAAGCAATCCAAC-3′
H16062：5′-AGATGAGGGCAGACTCAGTTATG-3′。
the primers were dissolved and diluted to a concentration of 10. mu. mo/L with sterilized double distilled water.
(3) PCR amplification and sequencing
The total volume of PCR reaction was 50. mu.L, containing 10 XBuffer (Mg)2+)5 mu L, Taq enzyme (5U/L)2 units, dNTPs (10mmol/L)0.5 mu L, 10 mu mol/L primers each 2 mu L, template DNA 20-100 ng, sterilized double distilled water to 50 mu L.
Reaction procedure: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 30s, extension at 72 ℃ for 90s, and 35 cycles; further extension was carried out at 72 ℃ for 8 min.
After the PCR was completed, the objective DNA fragment (800-1200bp) was excised by 1% agarose gel electrophoresis, and recovered and purified by a gel recovery kit.
The purified DNA fragments were sent to Shanghai Biotechnology Ltd for sequencing, and the same primers as those used for PCR amplification were used for bidirectional sequencing.
(4) Sequence splicing and alignment
And splicing the sequencing result by using Seqman in a Lasergene software package, and manually correcting. The measured sequences were aligned with 8 sequences provided by the present invention (POP1-POP8) using CLUSTALX.
(5) Analysis of results
Comparing the measured sequence with POP1-POP8 one by sites, the result is shown in Table 1, and the number on the sequence represents the position of the sequence; the sequence features of the samples are underlined.
Table 1 comparison of the determined sequences with POP1-POP8 by one-by-one and by one-by-one sites
As can be seen from Table 1, the giant salamander sequences from the provinces of Phoenix, Hubei (number: HBLF517) and Longshan, Hunan (number: HNLS70) are deleted at sites 654 to 903, 910 to 920, 927 to 972; the sequence of sites 921-926 is CGAATT; the sequences of the sites 904-909 are GCAGTG. These sites were characterized by the same sequence as POP1, so these samples belong to the POP1 population.
The giant salamander sequence from Jiangxi Jingan (number: JXJA322) and Zhejiang Lishui (number: ZJLS535) is deleted at sites 653-659 and 664-895; sites 593-605 are TTTCACTTTTTCA; sites 660-663 are TTTT. These sites were characterized by the same sequence as POP5, so these samples belong to the POP5 population.
A giant salamander sequence from Anhui Huangshan (AHHS 474) is deleted at sites 664-895, sites 653-663 are AAAATATTTTT, and sites 897-901 are ATGTT. These sites were characterized by the same sequence as POP7, so this sample belongs to the POP7 population.
The sequence of giant salamander from Guangxi Xingan (No. GXXA616) is GGGGG at 364-368, and is shown at 664-802
GATGTACTTTCTTTTTGCCTATGCAATTCTCCGATCAACCCCAAACAAACTCGGAGGCGTCCTAGCCTTAGGGGCCTCCATTTTCATTTTAATCCTATACCCCTTCTTCATACATCAAAACAACGAGGTTTAATATTCC are provided. These sites were characterized by the same sequence as POP8, so this sample belongs to the POP8 population.
The invention takes the giant salamander mitochondrial DNA sequence as a sequence marker for distinguishing the giant salamander population in China, and can quickly and accurately identify the target population by comparing the sequence marker with the sequence characteristics of a reference sequence. The method can be used for identifying the giant salamander individuals in the giant salamander farm and determining the source of the individuals; the breeding selection can be assisted when the parents are propagated. The method can also be applied to the identification of the proliferation and releasing samples, the population to which the proliferation and releasing samples belong is determined, and then the population is released to a suitable area, so that the germplasm of the released population is prevented from being mixed with the germplasm of the wild population.
The technical scheme of the invention has the advantages of simple steps, easy operation, good repeatability and easy learning and mastering of technicians. The selected experimental reagents are conventional reagents, no expensive and rare reagents are used, and the experimental operation cost is low.
As used in the specification and claims, certain terms are used to refer to particular components or methods. As one skilled in the art will appreciate, different regions may refer to a component by different names. The present specification and claims do not intend to distinguish between components that differ in name but not in name. In the following description and in the claims, the terms "include" and "comprise" are used in an open-ended fashion, and thus should be interpreted to mean "include, but not limited to. "substantially" means within an acceptable error range, and a person skilled in the art can solve the technical problem within a certain error range to substantially achieve the technical effect. The following description is of the preferred embodiment for carrying out the invention, and is made for the purpose of illustrating the general principles of the invention and not for the purpose of limiting the scope of the invention. The scope of the present invention is defined by the appended claims.
It is also noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a good or system that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such good or system. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other like elements in a commodity or system that includes the element.
The foregoing description shows and describes several preferred embodiments of the invention, but as aforementioned, it is to be understood that the invention is not limited to the forms disclosed herein, but is not to be construed as excluding other embodiments and is capable of use in various other combinations, modifications, and environments and is capable of changes within the scope of the inventive concept as expressed herein, commensurate with the above teachings, or the skill or knowledge of the relevant art. And that modifications and variations may be effected by those skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (7)
1. A Chinese giant salamander population division method based on mitochondrial DNA sequences is characterized by comprising the following steps: (1) collecting a giant salamander sample, and extracting genome DNA; (2) synthesizing a giant salamander specific mitochondrial DNA primer; (3) carrying out PCR amplification on a giant salamander sample and sequencing a PCR amplification product; (4) comparing the obtained sequence with a reference sequence; (5) judging the population to which the sample belongs according to the sequence characteristics of the corresponding sites, wherein the reference sequence in the step (4) is as follows:
POP 1: sites 654-903, 910-920, 927-972 are absent, 904-909 is GCAGTG, GCAGAG or GCAGGG, and sites 921-926 are CGAATT;
P0P 2: sites 654-659, 664-903, 910-920 and 930-974 are deleted, sites 660-663 are T, and sites 904-909 are ACAGAG;
POP 3: sites 654-659, 664-903, 910-920 and 929-974 are deleted, and sites 660-663 are T; sites 904-909 are ACAGGG;
POP 4: sites 653-659 and 664-895 are deleted, sites 593-605 are TTTTCACTTTTA, and sites 660-663 are TTTT;
POP 5: sites 653-659 and 664-895 are deleted, sites 593-605 are TTTCACTTTTTCA, and sites 660-663 are TTTT;
POP 6: 656-659, 664-896, 610-619 TCTAAGGGT, 653-655 ATA, 897-901 GAGAGAT, 973-977 GAAA, 1005-1007 CAA;
POP 7: missing sites 664-895, AAAATATTTTT at sites 653-663, and ATGTT at sites 897-901;
p O P8: sites 364-368 are G G G G G, sites 664-802 are GATGTACTTTCTTTTTGCCTATGCAATTCTCCGATCAACCCCAAACAAACTCGGAGGCGTCCTAGCCTTAGGGGCCTCCATTTTCATTTTAATCCTATACCCCTTCTTCATACATCAAAACAACGAGGTTTAATATTCC;
the giant salamander specific mitochondrial DNA primers in the step (2) are L14764 and H16062, wherein the L14764 is shown as SEQ ID NO.1, and the H16062 is shown as SEQ ID NO. 2.
2. The method for dividing Chinese giant salamander population based on mitochondrial DNA sequences according to claim 1, wherein the PCR amplification reaction system in step (3) is as follows: the total volume is 50 mu L, the PCR primer comprises 5 mu L of 10 XPCR buffer, 0.5 mu L of 10mmol/L dNTPs, 2 mu L of each of 10 mu mol/L primers L14764 and H16062, 2 units of Taq DNA polymerase and 20-100 ng template DNA, and sterile double distilled water is supplemented to 50 mu L.
3. The method for dividing Chinese giant salamander population based on mitochondrial DNA sequences according to claim 2, wherein the PCR amplification reaction conditions in step (3) are as follows: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 30s, extension at 72 ℃ for 90s, and 35 cycles; further extension was carried out at 72 ℃ for 8 min.
4. The method for dividing Chinese giant salamander population based on mitochondrial DNA sequence according to claim 3, wherein the sequencing method in step (3) is as follows: and detecting the PCR amplification product by 1% agarose gel electrophoresis, purifying and recovering by adopting a PCR product gel recovery kit, and performing bidirectional sequencing on the recovered product by using the primers L14764 and H16062.
5. The method for dividing Chinese giant salamander population based on mitochondrial DNA sequence according to claim 4, wherein the step (4) is specifically as follows: and (4) comparing the sequence obtained in the step (3) with a reference sequence group, and determining the group to which the detected sample belongs by referring to the sequence characteristics of the corresponding sites based on the sites set by the initial sequences of the sequence group.
6. The method for dividing Chinese giant salamander population based on mitochondrial DNA sequences according to claim 1, wherein the distribution range of POP1-POP8 population in the step (4) is as follows:
POP1 population is mainly distributed in Hunan, Hubei, Henan, Shanxi, Anhui, Gansu, Chongqing, Guizhou, Yunnan, Sichuan, Guangxi melt water and other areas;
the POP2 population is mainly distributed in Guangxi resource county;
POP3 colony is mainly distributed in Henan Koelreuteria, Anhui Laoban and Shaanxi Ankang;
POP4 population is mainly distributed in Huangshan region of Anhui province;
POP5 colony is mainly distributed in Jiangxi, Zhejiang and Anhui leaderboard;
POP6 population is mainly distributed in Anhui Huangshan and Zhejiang Lishui;
POP7 colony is mainly distributed in Hunan Yongding and Longshan, Hunbei Enshi, Chongqing Fuling and Wulong, Guizhou, Yunnan, Guangxi melt water and other areas;
the POP8 population was mainly distributed in guangxi xingan county.
7. The method for Chinese giant salamander population division based on mitochondrial DNA sequences of claim 1, wherein the Chinese giant salamander sample collected in step (1) comprises fin streak, blood, muscle, fresh molt, mucus or oral epithelial cells.
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