Method for identifying and selecting seed sources of high-altitude feeding forest musk deer based on mtDNATechnical Field
The invention relates to the technical field of gene identification, in particular to a method for identifying and selecting a seed source of high-altitude feeding forest musk deer based on mtDNA.
Background
The artificial domestication and propagation of the forest musk deer in China starts from the end of the 50 th century and goes through continuous exploration for over sixty years, and a technical system of forest musk deer breeding colony mode, seed source breeding survival rate, prevention and control of basic diseases, artificial living body fragrance taking and the like is initially established. However, the germplasm quality of existing forest musk breeding populations still lacks a scientific evaluation system due to small population effects of founder and the lack of subsequent genetic management.
The eastern part of the Tibetan is an important ecological fragile zone in China, and the forest musk feeding industry is an important way for developing local ecological economy. From 2018, a breeding base of the Tibetan Bianba forest musk deer is established, the transferred population successfully spends the adaptation period of high altitude hypoxia and coldness, and a stable population of 150 high altitude raising forest musk deer is formed and the raising population has good growing trend.
In the field of economic animal breeding, a plurality of technical methods are provided for constructing individuals with excellent genetic characteristics of new populations, and identification markers aiming at high altitude adaptability are often specific excavation, design and application of the genetic characteristics of species, and have reference values among different species, but are difficult to directly adopt.
Forest musk deer has the biological characteristics of timidity, police and high physiological stress, and deviation from a proper living environment is extremely easy to cause illness and even death, and high-altitude hypoxia and cold environments are certainly unfavorable environmental conditions. The prior forest musk deer lacks species-specific genetic identification indexes suitable for high altitude, and the prior genetic indexes are all evaluation indexes of genetic diversity, including mitochondrial gene microsatellite markers and nuclear gene microsatellite markers, wherein the former mainly adopts D-loop fragments and Cytb fragments to study maternal genetic structure and population genetic diversity, and the latter mainly adopts microsatellite markers, and the latter are both applied to study genetic structure and genetic diversity of the forest musk deer population, but cannot establish genetic characteristic connection with whether the forest musk deer population is suitable for high altitude environment or not. Thus, currently, there is no established genetic marker technology for woody musk deer that accommodates new populations at high altitudes.
Whether the wild animal is in a high altitude environment or not can be expressed from biological characteristics such as inheritance, physiology, morphology, behavior and the like, animal morphology and behavior characteristics are the most common biological property indexes, but the morphological and behavior parameters and the animal adaptation to the high altitude lack of inherent relevance, and an identification technology of the high altitude adaptation based on the morphological and behavior indexes cannot be established. At its root, the genetic trait is the biological basis that determines whether an animal individual is adapted to high altitude. Therefore, whether the animal individuals adapt to the high-altitude environment or not can be identified from the genetic characteristics, and a basis can be provided for selecting the seed sources suitable for the high altitude.
Disclosure of Invention
The invention aims to provide a seed source identification and selection method of high-altitude feeding forest musk deer based on mtDNA, which is based on three mtDNA haplotype parameters to analyze the genetic characteristics of the high-altitude survival and good-health forest musk deer, and a forest musk deer evaluation method suitable for feeding in a high-altitude area is established from a genetic characteristic path for the first time, so that scientific basis is provided for subsequent selection and transportation of the forest musk deer.
In order to achieve the above purpose, the invention provides a method for identifying and selecting the seed source of the high-altitude feeding forest musk deer based on mtDNA, which comprises the following steps:
S1, selecting a low-altitude forest musk deer sample and a high-altitude forest musk deer sample with the same genetic background, and respectively collecting excrement of the low-altitude forest musk deer sample and the high-altitude forest musk deer sample;
s2, extracting mitochondrial DNA from the feces, and performing target fragment amplification and bidirectional sequencing;
S3, respectively determining dominant haplotypes in ATP8-6, cytb and D-loop of the low-altitude forest musk deer sample with definite genetic functions, and respectively obtaining the sample number of each dominant haplotype in ATP8-6, cytb and D-loop of the low-altitude forest musk deer sample; respectively determining dominant haplotypes in ATP8-6, cytb and D-loop of the high-altitude forest musk deer samples with definite genetic functions, and respectively obtaining the sample number of each dominant haplotype in ATP8-6, cytb and D-loop of the high-altitude forest musk deer samples;
s4, assigning a value to the dominant haplotype type of the low-altitude forest musk deer sample and the dominant haplotype type of the high-altitude forest musk deer sample respectively to obtain a score F;
s5, evaluating the high altitude adaptability of the dominant haplotype according to the assignment of the dominant haplotype;
s6, determining the genetic characteristics of the forest musk deer adapting to the high-altitude environment.
Preferably, the step S3 specifically includes the following steps:
S31, determining ATP8-6, cytb and D-loop haplotypes of the low-altitude forest musk deer samples respectively, and calculating the number of samples owned by each haplotype; respectively determining ATP8-6, cytb and D-loop haplotype types of the high-altitude forest musk deer samples, and calculating the number of samples owned by each haplotype type;
S32, respectively calculating the duty ratio of each ATP8-6, cytb and D-loop haplotype in the low-altitude forest musk deer sample, gradually accumulating from the high duty ratio to the low duty ratio, and when the accumulated value exceeds a set value, performing the haplotype typing to be the dominant haplotype typing of the low-altitude forest musk deer sample;
And (3) respectively calculating the duty ratio of each ATP8-6, cytb and D-loop haplotype in the high-altitude forest musk deer sample, gradually accumulating from the high duty ratio to the low duty ratio, and when the accumulated value exceeds a set value, performing the haplotype typing to be the dominant haplotype typing of the high-altitude forest musk deer sample.
Preferably, in S32, the set value of the accumulated value is 80%.
Preferably, in the step S4, the assigning method is as follows:
Wherein Hai is i ATP8-6 dominant haplotype, hci is i Cytb dominant haplotype, hdi is i D-loop dominant haplotype, i=1, 2,3,..n, n is the number of dominant haploids.
Preferably, in the step S5, the specific evaluation method of the high altitude adaptability is as follows:
the adaptability of each haplotype type in a high altitude area is evaluated in three stages:
if high altitude FHxi is approximately equal to low altitude FHxi, hxi does not exhibit the adaptability difference between high altitude and low altitude;
if high altitude FHxi > low altitude FHxi, hxi is higher in high altitude adaptation than low altitude;
if high altitude FHxi < low altitude FHxi, hxi is less adaptive at high altitude than at low altitude;
wherein x is a, c, d.
Preferably, in the step S6, when the forest musk deer individual has the high altitude ≡low altitude on the three haplotype scores of ATP8-6, cytb and D-loop, namely, the dominant haplotype suitable for the high altitude environment.
The mtDNA-based high-altitude breeding forest musk deer source identification and selection method has the advantages and positive effects that:
1. The invention adopts a non-invasive sampling technology, namely, fresh excrement of the forest musk deer is collected, genetic materials of animal intestinal exfoliated cells of the excrement, namely mitochondrial DNA (mtDNA), have a stable annular structure, are convenient to extract from the intestinal exfoliated cells of the excrement, cannot influence the highly stressed forest musk deer, successfully extract the highly stressed forest musk deer to be close to 100%, are suitable for large-scale sampling of highly stressed species such as the forest musk deer, and do not influence or even interfere with normal life of the forest musk deer.
2. According to the invention, through known three genetic markers, the effect of high altitude adaptation is initially established from the composite angles of oxidation circulation, energy metabolism and genetic diversity of the high altitude forest musk deer breeding population, individuals are comprehensively selected by comprehensive scoring of three haplotypes, and the germplasm quality is more fully evaluated and used as a standard for identification and selection.
3. 80% Is adopted as a judgment standard of the dominant haplotype, so that evaluation deviation caused by the haplotype with low duty ratio is avoided, and meanwhile, the evaluation flow is greatly simplified. Three types of haplotypes with high altitude adaptability higher than low altitude, with high altitude adaptability lower than low altitude, and without showing adaptability difference between high altitude and low altitude are screened by comparing three genetic markers of the low altitude and high altitude feeding forest musk deer.
4. And respectively constructing haplotype databases of three indexes of ATP8-6, cytb and D-loop suitable for high altitude by adopting low altitude and high altitude forest musk feeding groups with the same genetic background, and respectively recording 57, 51 and 39 haplotype sequences of the three indexes.
5. The invention establishes an individual high-altitude adaptability identification method for adaptively domesticating and expanding a population from a low-altitude population to a high-altitude population, genetic data is used as a basic support of the selection method, three indexes in the selection method are used for evaluating forest musk candidate high-altitude seed source individuals, and evaluation results are good and are used as recommended seed sources. The invention can implement identification and selection of individuals of the high-altitude forest musk deer, and simultaneously, the existing high-altitude forest musk deer group can pay attention to the individuals of the forest musk deer with 1-2 or no high-altitude haplotypes, and avoid the individuals as much as possible in the process of selecting, breeding and breeding the high-altitude breeding population, thereby improving the guiding basis of genetic management and scientific breeding of the high-altitude forest musk deer group.
Drawings
FIG. 1 is a flow chart of an embodiment of the present invention.
Detailed Description
The technical scheme of the invention is further described below through the attached drawings and the embodiments.
As shown in fig. 1. A method for identifying and selecting a seed source of high-altitude feeding forest musk deer based on mtDNA comprises the following steps:
S1, selecting a low-altitude forest musk deer sample and a high-altitude forest musk deer sample with the same genetic background, and respectively collecting excrement of the low-altitude forest musk deer sample and the high-altitude forest musk deer sample.
S2, extracting mitochondrial DNA from the feces, and performing target fragment amplification and bidirectional sequencing.
Mitochondrial DNA is extracted by TIANGEN blood/cell/tissue genome DNA extraction kit (DP 304), and target fragment amplification and bidirectional sequencing are carried out.
S3, respectively determining dominant haplotypes in ATP8-6, cytb and D-loop of the low-altitude forest musk deer sample with definite genetic functions, and respectively obtaining the sample number of each dominant haplotype in ATP8-6, cytb and D-loop of the low-altitude forest musk deer sample; respectively determining dominant haplotypes in ATP8-6, cytb and D-loop of the high-altitude forest musk deer samples with definite genetic functions, and respectively obtaining the sample number of each dominant haplotype in ATP8-6, cytb and D-loop of the high-altitude forest musk deer samples;
The step S3 specifically comprises the following steps:
s31, determining ATP8-6, cytb and D-loop haplotypes of the low-altitude forest musk deer samples respectively, and calculating the number of samples owned by each haplotype; and respectively determining ATP8-6, cytb and D-loop haplotypes of the high-altitude forest musk deer samples, and calculating the number of samples owned by each haplotype.
S32, respectively calculating the duty ratio of each ATP8-6, cytb and D-loop haplotype in the low-altitude forest musk deer sample, gradually accumulating from the high duty ratio to the low duty ratio, and when the accumulated value exceeds a set value, performing the haplotype typing to be the dominant haplotype typing of the low-altitude forest musk deer sample.
And (3) respectively calculating the duty ratio of each ATP8-6, cytb and D-loop haplotype in the high-altitude forest musk deer sample, gradually accumulating from the high duty ratio to the low duty ratio, and when the accumulated value exceeds a set value, performing the haplotype typing to be the dominant haplotype typing of the high-altitude forest musk deer sample.
The accumulated value was set at 80%.
And S4, assigning the dominant haplotype of the low-altitude forest musk deer sample and the dominant haplotype of the high-altitude forest musk deer sample to obtain a score F.
The assignment method comprises the following steps:
Wherein Hai is i ATP8-6 dominant haplotype, hci is i Cytb dominant haplotype, hdi is i D-loop dominant haplotype, i=1, 2,3,..n, n is the number of dominant haploids.
S5, evaluating the high altitude adaptability of the dominant haplotype according to the assignment of the dominant haplotype.
The specific evaluation method of the high altitude adaptability comprises the following steps:
the adaptability of each haplotype type in a high altitude area is evaluated in three stages:
if high altitude FHxi is approximately equal to low altitude FHxi, hxi does not exhibit the adaptability difference between high altitude and low altitude;
if high altitude FHxi > low altitude FHxi, hxi is higher in high altitude adaptation than low altitude;
if high altitude FHxi < low altitude FHxi, hxi is less adaptive at high altitude than at low altitude;
wherein x is a, c, d.
S6, determining the genetic characteristics of the forest musk deer adapting to the high-altitude environment.
When the forest musk deer individuals have high altitude +.low altitude on the three haplotype scores of ATP8-6, cytb and D-loop, namely the dominant haplotype adapting to the high altitude. Accordingly, a forest musk ATP8-6, cytb, D-loop database is established for the high altitude environment for identifying and selecting individuals from the low altitude population for the high altitude.
Examples
584 Low-altitude forest musk deer individual samples are selected from the forest musk deer population in the low mountain area of Fengshan county of Shaanxi, 97 high-altitude forest musk deer individual samples are selected from the high-altitude area of the Tibetan side dam with the same genetic background, and the fresh feces of the samples are collected, and 10 feces grains are stored at the temperature of minus 80 ℃.
Mitochondrial DNA is extracted by TIANGEN blood/cell/tissue genome DNA extraction kit (DP 304), and target fragment amplification and bidirectional sequencing are carried out.
By adopting three mitochondrial gene molecular markers of ATP8-6, cytb and D-loop, 57 ATP8-6 (Ha) haplotypes, 51 Cytb (Hc) haplotypes and 39D-loop (Hd) haplotypes are measured in a low-altitude forest musk deer sample. 12 ATP8-6 (Ha) haplotypes, 8 Cytb (Hc) haplotypes and 16D-loop (Hd) haplotypes were determined in total in the high altitude woody musk samples. Haplotypes for ATP8-6, cytb and D-loop were numbered. The haplotype comparison of the low elevation forest musk samples with the high elevation forest musk samples is shown in Table 1.
The method comprises the steps of filling ground color, wherein the typing of the ground color is a dominant haplotype of a low-altitude sample, and the thickening typing is a dominant haplotype of a high-altitude sample. The frequency of the high-altitude samples is greater than the label "+.h" of the low-altitude samples, and the frequency of the high-altitude samples is less than the label "+.h" of the low-altitude samples.
The ratio of high altitude advantage haplotypes in the low altitude sample and the high altitude sample is shown in Table 2.
TABLE 2 duty cycle of high altitude dominant haplotypes in low altitude samples and high altitude samples
As can be seen from table 1, the high altitude fitness evaluation results of the high altitude woody musk samples are: 62 ATP8-6 haplotypes with a ratio of 63.9%;33 Cytb haplotypes, accounting for 60%; 38D-loop haplotypes were found to be 67.9%. The high altitude adaptability evaluation result of the low altitude woody musk samples is as follows: 102 ATP8-6 haplotypes (Ha), 17.5% by weight, 48 Cytb haplotypes (Hc), 13.8% by weight, 116D-loop haplotypes (Hd) and 23.25% by weight. High altitude adaptation means that the duty cycle of the dominant haplotype in the high altitude forest musk sample is greater than the duty cycle of the dominant haplotype in the low altitude forest musk sample. In the process of initially establishing high-altitude adaptability of a high-altitude forest musk deer breeding population, the duty ratio of different haplotypes changes under the influence of death and breeding development of individuals, and specific haplotypes are more suitable for the high-altitude environment relative to the low-altitude environment and are higher in duty ratio of the number of individuals possessed by the haplotypes.
Therefore, by adopting the seed source identification and selection method of the high-altitude feeding forest musk deer based on the mtDNA, the genetic characteristics of the high-altitude survival and good-health forest musk deer are analyzed based on three mtDNA haplotype parameters, and the forest musk deer evaluation method suitable for feeding in the high-altitude area is established from the genetic characteristic path for the first time, so that scientific basis is provided for subsequent selection and transportation of the forest musk deer.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention and not for limiting it, and although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that: the technical scheme of the invention can be modified or replaced by the same, and the modified technical scheme cannot deviate from the spirit and scope of the technical scheme of the invention.