A method for identifying and selecting seed source of high-altitude feeding forest musk deer based on mtDNA

Abstract

The application discloses a kind of based on mtDNA's high altitude feeding forest musk deer's provenance identification and selection method, it is related to genetic identification technical field.The identification and selection method includes the following steps: selecting low altitude forest musk deer sample and high altitude forest musk deer sample with same genetic background, respectively collecting low altitude forest musk deer sample and high altitude forest musk deer sample's feces;Mitochondrial DNA is extracted from feces, and the amplification and bidirectional sequencing of object fragment are carried out;The dominant haplotype typing of ATP8-6, Cytb, D-loop with clear genetic function of low altitude forest musk deer sample and high altitude forest musk deer sample is determined respectively, and the sample quantity of each dominant haplotype typing in ATP8-6, Cytb, D-loop of low altitude sample and high altitude forest musk deer sample is obtained;The dominant haplotype typing of low altitude forest musk deer sample and high altitude forest musk deer sample is valued respectively, and score F is obtained;The high altitude adaptability of dominant haplotype typing is evaluated according to the assignment of dominant haplotype typing;The genetic characteristics of forest musk deer adapted to high altitude environment are determined.The identification and selection method of provenance of high altitude feeding forest musk deer based on mtDNA in the application is based on three mtDNA haplotype parameters to analyze the genetic characteristics of high altitude survival and healthy forest musk deer, which is the first time to establish an identification and selection evaluation method for forest musk deer suitable for high altitude area feeding from the genetic characteristics approach, and provides a scientific basis for subsequent selection and transportation of forest musk deer.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of gene identification, and in particular to a method for identifying and selecting a high-altitude breeding forest musk deer source based on mtDNA. BACKGROUND

[0002] China's artificial breeding of forest musk deer began in the late 1950s, and after more than 60 years of continuous exploration, a technical system for forest musk deer breeding has been initially established, including the forest musk deer breeding cage model, the survival rate of seed source breeding, the prevention and control of basic diseases, and the artificial live body musk taking. However, due to the small population effect of the founders and the lack of subsequent genetic management, the germplasm quality of the existing forest musk deer breeding population still lacks a scientific evaluation system.

[0003] The eastern part of Tibet is an important ecological fragile zone in China, and forest musk deer breeding is an important way to develop local ecological economy. Since the establishment of the Xizang Bianba Forest Musk Deer Breeding Base in 2018, the migration population has successfully passed the adaptation period of high-altitude hypoxia and cold, and a stable population of 150 high-altitude breeding forest musk deer has been formed, with a good growth trend of the breeding population.

[0004] In the field of economic animal breeding, there are many technical methods for selecting individuals with excellent genetic characteristics to form new populations. The identification markers for high-altitude adaptability are often specific to the genetic characteristics of the species, and the reference value between different species is difficult to directly adopt.

[0005] Forest musk deer has the biological characteristics of timidity, alertness, and high physiological stress, and deviating from the suitable living environment can easily lead to illness and even death. High-altitude hypoxia and cold environment are undoubtedly unfavorable environmental conditions. So far, forest musk deer lacks species-specific genetic identification indicators for adapting to high altitudes. Current genetic indicators are all evaluation indicators of genetic diversity, including mitochondrial genes and nuclear gene microsatellite markers. The former mainly uses D-loop fragments and Cytb fragments for maternal genetic structure and population genetic diversity research, and the latter mainly uses microsatellite markers. Both of them are used in the research of genetic structure and genetic diversity of forest musk deer population, but cannot establish a genetic characteristic link between forest musk deer population and high-altitude environment adaptation. Therefore, there is no genetic marker technology for establishing a new population of forest musk deer that adapts to high altitudes.

[0006] Whether a wild animal can adapt to high-altitude environment can be expressed in terms of genetic, physiological, morphological, and behavioral characteristics. Morphology and behavior are the most commonly used biological trait indicators, but there is no inherent correlation between morphological and behavioral parameters and animal adaptation to high altitudes, and it is impossible to establish an identification technology for high-altitude adaptability based on morphological and behavioral indicators. At its root, genetic characteristics are the biological basis for determining whether an animal individual can adapt to high altitudes. Therefore, identifying whether an animal individual can adapt to high-altitude environment from the genetic characteristics can provide a basis for selecting a source suitable for high altitudes. Summary of the Invention

[0007] The purpose of this invention is to provide a method for the identification and selection of musk deer raised at high altitudes based on mtDNA. The method analyzes the genetic characteristics of musk deer that survive and are healthy at high altitudes based on three mtDNA haplotype parameters. For the first time, a method for identifying and selecting musk deer suitable for raising in high-altitude areas is established from the perspective of genetic characteristics, providing a scientific basis for the subsequent selection and transportation of musk deer.

[0008] To achieve the above objectives, this invention provides a method for the identification and selection of musk deer raised at high altitudes based on mtDNA, comprising the following steps:

[0009] S1. Select low-altitude and high-altitude musk deer samples with the same genetic background, and collect feces from the low-altitude and high-altitude musk deer samples respectively.

[0010] S2. Extract mitochondrial DNA from feces, amplify the target fragment, and perform bidirectional sequencing.

[0011] S3. Identify low-altitude musk deer samples with clearly defined genetic functions. ATP8-6, Cytb, Dominant haplotype typing in D-loop was used to obtain low-altitude samples. ATP8-6, Cytb, The number of samples for each dominant haplotype in the D-loop was determined; samples from high-altitude forest musk deer with clear genetic functions were identified. ATP8-6, Cytb, Dominant haplotype typing in D-loop was used to obtain high-altitude samples. ATP8-6, Cytb, The number of samples for each dominant haplotype in the D-loop;

[0012] S4. Assign values ​​to the dominant haplotypes of low-altitude and high-altitude musk deer samples respectively, and obtain scores. F ;

[0013] S5. Evaluate the high-altitude adaptability of dominant haplotypes based on the assignment of dominant haplotype classification values;

[0014] S6. Determine the genetic characteristics of forest musk deer adapted to high-altitude environments.

[0015] Preferably, step S3 specifically includes the following steps:

[0016] S31. Identify low-altitude musk deer samples separately. ATP8-6, Cytb, D-loop haplotype typing was used to calculate the number of samples for each haplotype; the number of samples from high-altitude musk deer was determined. ATP8-6, Cytb, D-loop haplotype typing calculates the number of samples for each haplotype.

[0017] S32. Calculate the values ​​of each species in the low-altitude forest musk deer samples. ATP8-6, Cytb,The proportion of D-loop haplotype typing in low-altitude forest musk deer samples is gradually accumulated from high proportion to low proportion, and when the accumulated value exceeds the set value, the accumulated haplotype typing is the dominant haplotype typing of the low-altitude forest musk deer sample;

[0018] The proportion of D-loop haplotype typing in low-altitude forest musk deer samples is gradually accumulated from high proportion to low proportion, and when the accumulated value exceeds the set value, the accumulated haplotype typing is the dominant haplotype typing of the low-altitude forest musk deer sample; ATP8-6, Cytb, The proportion of D-loop haplotype typing in low-altitude forest musk deer samples is gradually accumulated from high proportion to low proportion, and when the accumulated value exceeds the set value, the accumulated haplotype typing is the dominant haplotype typing of the low-altitude forest musk deer sample.

[0019] Preferably, in the S32, the set value of the accumulated value is 80%.

[0020] Preferably, in the S4, the assignment method is:

[0021]

[0022]

[0023]

[0024] wherein, is the first dominant haplotype typing, ATP8-6 is the first dominant haplotype typing, is the first D-loop dominant haplotype typing, Cytb , , , , is the number of dominant haplotype typing.

[0025] Preferably, in the S5, the specific evaluation method of high-altitude adaptability is:

[0026] The adaptability of each haplotype typing in the high-altitude area is evaluated in three levels:

[0027] If high-altitude = low-altitude , then no difference in adaptability between high altitude and low altitude is shown;

[0028] If high-altitude > low-altitude , then the adaptability in high altitude is higher than that in low altitude;

[0029] If high-altitude < low-altitude , then the adaptability in high altitude is lower than that in low altitude;

[0030] wherein, is .

[0031] Preferably, in S6, when the musk deer individual has ATP8-6, Cytb, D-loop three haplotype scores with high altitude >= low altitude, i.e., the dominant haplotype of high altitude environment adaptation.

[0032] The advantages and positive effects of the mtDNA-based high-altitude feeding musk deer provenance identification and selection method of the present application are:

[0033] 1. The present application adopts a non-invasive sampling technique, i.e., collecting fresh feces of musk deer, extracting the genetic material of the intestinal shedding cells of the feces, i.e., mitochondrial DNA (mtDNA), which has a stable circular structure, is easy to extract from the intestinal shedding cells of the feces, does not affect the highly stressed musk deer, and has a success rate close to 100%. It is suitable for scale sampling of highly stressed species such as musk deer and does not affect or even disturb the normal life of musk deer.

[0034] 2. The present application evaluates the effects of high-altitude musk deer feeding population from the composite angle of oxidation cycle, energy metabolism and genetic diversity through three known genetic markers, selects individuals based on the comprehensive scores of the three haplotypes, more fully evaluates the germplasm quality, and uses it as the standard for identification and selection.

[0035] 3. The 80% is used as the judgment standard of the dominant haplotype, which avoids the evaluation deviation caused by the haplotype with low proportion, and at the same time, greatly simplifies the evaluation process. Through the comparison of the three genetic markers of low-altitude and high-altitude feeding musk deer, three types of haplotypes are distinguished, i.e., high-altitude adaptability higher than low-altitude, high-altitude adaptability lower than low-altitude, and no difference in high-altitude and low-altitude adaptability.

[0036] 4. The low-altitude and high-altitude musk deer feeding populations with the same genetic background are used to construct the high-altitude adapted ATP8-6, Cytb and D-loop haplotype databases of the three indicators, respectively, and 57, 51 and 39 haplotype sequences of the three indicators are collected.

[0037] 5. This invention establishes a method for identifying the high-altitude adaptability of individual musk deer from low-altitude populations, allowing them to undergo adaptive domestication at higher altitudes and expand the population. Genetic data serves as the foundation for this selection method. Candidate high-altitude musk deer individuals are evaluated based on three indicators outlined in this method, with those showing excellent evaluation results being recommended as breeding stock. This invention enables the identification and selection of high-altitude musk deer individuals. Furthermore, for existing high-altitude musk deer populations, individuals with one or two high-altitude haplotypes or none can be identified and their breeding characteristics can be carefully monitored. These individuals can be avoided as much as possible during the selective breeding of high-altitude captive populations, thus improving the genetic management and scientific breeding guidance for high-altitude musk deer populations. Attached Figure Description

[0038] Figure 1 This is a flowchart of an embodiment of the present invention. Detailed Implementation

[0039] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0040] like Figure 1 As shown. A method for progeny identification and selection of high-altitude captive musk deer based on mtDNA includes the following steps:

[0041] S1. Select low-altitude and high-altitude musk deer samples with the same genetic background, and collect feces from the low-altitude and high-altitude musk deer samples respectively.

[0042] S2. Extract mitochondrial DNA from feces, amplify the target fragment, and perform bidirectional sequencing.

[0043] Mitochondrial DNA was extracted using the TIANGEN Blood / Cell / Tissue Genomic DNA Extraction Kit (DP304), and the target fragment was amplified and bidirectionally sequenced.

[0044] S3. Identify low-altitude musk deer samples with clearly defined genetic functions. ATP8-6, Cytb, Dominant haplotype typing in D-loop was used to obtain low-altitude samples. ATP8-6, Cytb, The number of samples for each dominant haplotype in the D-loop was determined; samples from high-altitude forest musk deer with clear genetic functions were identified. ATP8-6, Cytb, Dominant haplotype typing in D-loop was used to obtain high-altitude samples. ATP8-6, Cytb, The number of samples for each dominant haplotype in the D-loop;

[0045] S3 specifically includes the following steps:

[0046] S31. Identify low-altitude musk deer samples separately. ATP8-6, Cytb,D-loop haplotype typing, calculate the number of samples owned by each haplotype typing; determine the proportion of D-loop haplotype typing in low-altitude forest musk deer samples, and gradually accumulate from high to low, and when the accumulated value exceeds the set value, the accumulated haplotype typing is the dominant haplotype typing of low-altitude forest musk deer samples. ATP8-6, Cytb, D-loop haplotype typing, calculate the number of samples owned by each haplotype typing.

[0047] S32, respectively calculate the proportion of each D-loop haplotype typing in low-altitude forest musk deer samples, and gradually accumulate from high to low, and when the accumulated value exceeds the set value, the accumulated haplotype typing is the dominant haplotype typing of low-altitude forest musk deer samples. ATP8-6, Cytb, D-loop haplotype typing in low-altitude forest musk deer samples, and gradually accumulate from high to low, and when the accumulated value exceeds the set value, the accumulated haplotype typing is the dominant haplotype typing of low-altitude forest musk deer samples.

[0048] respectively calculate the proportion of each D-loop haplotype typing in high-altitude forest musk deer samples, and gradually accumulate from high to low, and when the accumulated value exceeds the set value, the accumulated haplotype typing is the dominant haplotype typing of high-altitude forest musk deer samples. ATP8-6, Cytb, D-loop haplotype typing in high-altitude forest musk deer samples, and gradually accumulate from high to low, and when the accumulated value exceeds the set value, the accumulated haplotype typing is the dominant haplotype typing of high-altitude forest musk deer samples.

[0049] The set value of the accumulated value is 80%.

[0050] S4, respectively, the dominant haplotype typing of low-altitude forest musk deer samples, and the dominant haplotype typing of high-altitude forest musk deer samples are valued, and the score is obtained. F .

[0051] The valuation method is:

[0052]

[0053]

[0054]

[0055] Among them, is the first dominant haplotype typing, ATP8-6 is the first dominant haplotype typing, is the first D-loop dominant haplotype typing, Cytb , is the number of dominant haplotype typing. The specific evaluation method of high-altitude adaptability is:

[0056] The specific evaluation method of high-altitude adaptability is:

[0057] The specific evaluation method of high-altitude adaptability is:

[0058] The specific evaluation method of high-altitude adaptability is:

[0059] If the high-altitude​​ = Low altitude ,but No difference in adaptability between high and low altitudes was observed;

[0060] If high altitude > Low altitude ,but It adapts better at high altitudes than at low altitudes;

[0061] If high altitude < Low altitude ,but Adaptability at high altitudes is lower than at low altitudes;

[0062] in, for .

[0063] S6. Determine the genetic characteristics of forest musk deer adapted to high-altitude environments.

[0064] When individual musk deer are ATP8-6, Cytb, The three haplotypes in the D-loop simultaneously exhibit dominant haplotypes at altitudes ≥ low altitudes, indicating high-altitude adaptation. Based on this, a species of musk deer adapted to high-altitude environments can be established. ATP8-6, Cytb, The D-loop database is used to identify and select individuals suitable for high altitudes from low-altitude populations.

[0065] Example

[0066] 584 individual samples of low-altitude musk deer were selected from the low-mountain musk deer population in Feng County, Shaanxi Province, and 97 individual samples of high-altitude musk deer were selected from the high-altitude area of ​​Bianba, Tibet, which has the same genetic background. Fresh feces were collected from the samples, with 10 fecal pellets per sample, and stored at -80℃.

[0067] Mitochondrial DNA was extracted using the TIANGEN Blood / Cell / Tissue Genomic DNA Extraction Kit (DP304), and the target fragment was amplified and bidirectionally sequenced.

[0068] By adopting ATP8-6, Cytb Three mitochondrial gene molecular markers, including D-loop, were detected in low-altitude musk deer samples, totaling 57. ATP8-6 ( Ha ) Haplotype typing, 51 Cytb ( Hc Haplotype typing was performed on 39 D-loop (Hd) haplotypes. A total of 12 haplotypes were determined from high-altitude musk deer samples. ATP8-6 (Ha) haplotype typing, 8 Cytb (Hc) haplotype typing, 16 D-loop (Hd) haplotype typing. (Regarding...) ATP8-6, CytbThe haplotypes of the D-loop were numbered. The haplotype comparison results between low-altitude and high-altitude musk deer samples are shown in Table 1.

[0069] Table 1. Comparison of haplotypes between low-altitude and high-altitude musk deer samples.

[0070]

[0071] Among them, the haplotypes with filled background color represent the dominant haplotypes of low-altitude samples, while the haplotypes with bold text represent the dominant haplotypes of high-altitude samples. Haplotypes with a higher frequency in high-altitude samples are labeled "...". The label "High-altitude samples have a lower frequency than low-altitude samples" indicates this. ".

[0072] The proportion of high-altitude dominant haplotypes in low-altitude and high-altitude samples is shown in Table 2.

[0073] Table 2. Proportion of high-altitude dominant haplotypes in low-altitude and high-altitude samples.

[0074]

[0075] As shown in Table 1, the results of the high-altitude adaptability assessment of the high-altitude musk deer samples were: 62 individuals. ATP8-6 Haplotypes accounted for 63.9%; 33 individuals Cytb Haplotype accounted for 60%; 38 individuals had the D-loop haplotype, accounting for 67.9%. The high-altitude adaptability assessment results for low-altitude musk deer samples were: 102 individuals. ATP8-6 Haplotype (Ha) accounted for 17.5% and consisted of 48 individuals. Cytb ATP8-6 Haplotype Hc accounted for 13.8% of individuals, and 116 individuals had haplotype D-loop (Hd), accounting for 23.25%. High-altitude adaptability refers to the fact that the proportion of the dominant haplotype in high-altitude musk deer samples is greater than that in low-altitude musk deer samples. During the initial establishment of high-altitude adaptability in captive high-altitude musk deer populations, the proportions of different haplotypes changed due to individual mortality and reproductive development. Certain haplotypes were more adapted to high-altitude environments than low-altitude environments, resulting in a higher proportion of individuals possessing that haplotype.

[0076] Therefore, the method for identifying and selecting musk deer raised at high altitudes based on mtDNA described in this invention analyzes the genetic characteristics of musk deer that survive and are in good health at high altitudes based on three mtDNA haplotype parameters. For the first time, a method for identifying and selecting musk deer suitable for raising in high-altitude areas is established from the perspective of genetic characteristics, providing a scientific basis for the subsequent selection and transportation of musk deer.

[0077] It should be pointed out finally that the above examples are only used to illustrate the technical solutions of the present application but not to limit it, and although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present application can still be modified or replaced equivalently, and these modifications or equivalent replacements should not make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present application.

Claims

1. A method for identifying and selecting seed source of forest musk deer (Moschus berezovskii) based on mtDNA at high altitude, characterized in that, The method comprises the following steps: S1, selecting low-altitude forest musk deer samples and high-altitude forest musk deer samples with the same genetic background, and collecting feces of the low-altitude forest musk deer samples and the high-altitude forest musk deer samples respectively; S2, extracting mitochondrial DNA from the feces, and performing target fragment amplification and bidirectional sequencing; S3, respectively, the low-altitude forest musk deer sample with clear genetic function ATP8-6, Cytb, Haplotype typing in the D-loop, respectively, the low-altitude sample ATP8-6, Cytb, The number of samples typed for each haplotype in the D-loop; respectively, the high-altitude forest musk deer sample with clear genetic function ATP8-6, Cytb, Haplotype typing in the D-loop, respectively, the high-altitude sample ATP8-6, Cytb, The number of samples typed for each haplotype in the D-loop; S4, respectively, to the low altitude forest musk deer sample of the dominant haplotype typing, high altitude forest musk deer sample of the dominant haplotype typing is assigned, get the score F ; S5, evaluating the high-altitude adaptability of the advantageous haplotype according to the assignment value of the advantageous haplotype; S6, determining the genetic characteristics of the forest musk deer adapted to the high-altitude environment; In the S4, the assignment method is: ； ； ； wherein, is the th ATP8-6 advantageous haplotype, is the th Cytb advantageous haplotype, is the th D-loop advantageous haplotype, , is the number of advantageous haplotypes. In the S5, the specific evaluation method of the high-altitude adaptability is: The adaptability of each haplotype in the high-altitude area is evaluated in three levels: If high altitude = low altitude , then No difference in adaptation between high altitude and low altitude was observed; If high altitude Low altitude Then High altitude adaptation is higher than low altitude; If high altitude < low altitude then high altitude adaptation is lower than low altitude; wherein is ; In S6, when the individual of forest musk deer is in ATP8-6, Cytb, The D-loop three haplotype scores simultaneously have high altitude >= low altitude, that is, the dominant haplotype of high altitude adaptation.

2. The method for mtDNA-based provenance identification and selection of forest musk deer raised at high altitude according to claim 1, characterized in that, The S3 specifically comprises the following steps: S31, respectively determine the number of samples of each haplotype typing of the low-altitude forest musk deer sample ATP8-6, Cytb, D-loop haplotype typing, calculate the number of samples owned by each haplotype typing; respectively determine the number of samples of each haplotype typing of the high-altitude forest musk deer sample ATP8-6, Cytb, D-loop haplotype typing, calculate the number of samples owned by each haplotype typing; respectively determine the number of samples of each haplotype typing of the high-altitude forest musk deer sample S32, calculate the proportion of each D-loop haplotype in the low-elevation forest musk deer samples, respectively ATP8-6, Cytb, The proportion of D-loop haplotype typing in low-elevation forest musk deer samples is gradually accumulated from high to low, and when the cumulative value exceeds the set value, the accumulated haplotype typing is the dominant haplotype typing of low-elevation forest musk deer samples; The proportion of each D-loop haplotype in the high-altitude forest musk deer samples was calculated respectively ATP8-6, Cytb, The proportions of D-loop haplotype typing in high-altitude forest musk deer samples were gradually accumulated from high to low, and when the cumulative value exceeded the set value, the accumulated haplotype typing was the dominant haplotype typing of high-altitude forest musk deer samples.

3. The method for mtDNA-based provenance identification and selection of forest musk deer raised at high altitude according to claim 2, characterized in that: In the S32, the set value of the cumulative value is 80%.

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