Method for tracing mutton producing area by combining whole-genome recombination sequencing and meat quality characteristics

Abstract

The invention discloses a method for tracing a mutton production place by combining whole-genome recombination sequencing and meat quality characteristics, belongs to the technical field of tracing the mutton production place, and aims to solve the problem that how to distinguish or select different varieties of sheep meat with different sensory characteristics and nutritional values but similar shapes of the mutton entering the market become a great difficulty. Comprising the following steps: extracting sample DNA to obtain a DNA sample; according to the method, the relation graph of the difference sites and the meat quality is generated, the mutton producing area is traced according to the relation graph, according to the three difference sites on one chromosome and the specific meat quality markers of the three difference sites, the Wuzhu sheep and the Western-flag sheep can be completely distinguished, accurate producing area traceability is effectively conducted on the producing areas of the three kinds of mutton, and the mutton producing area traceability is improved. The method for tracing the origin of mutton by combining whole-genome recombination sequencing and meat quality characteristics has a positive promotion effect on inspection and detection technologies for preventing doping and adulteration of geographical indication mutton in Inner Mongolia, identifying authenticity and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of tracing the origin of mutton, and particularly relates to a method for tracing the origin of mutton by combining whole-genome recombinant sequencing with meat quality characteristics. Background Art

[0002] Compared with pork, mutton is more popular among consumers because of its lower fat and cholesterol content, higher protein content and more delicate texture. Inner Mongolia Autonomous Region in China has a vast territory and extensive grasslands. Thanks to the unique natural environment and the long-term breeding and cultivation by herdsmen, Inner Mongolia has a variety of excellent sheep breeds, such as Wuzhumuqin sheep, Chahar sheep, Sunite sheep and Hulunbuir sheep. Among them, the Wuzhumuqin sheep produced in the Wuzhumuqin Grassland in Xilingol League, Inner Mongolia, is adaptable and is a fat and wool dual-purpose coarse-wool sheep breed. Wuzhumuqin mutton is rich in minerals such as calcium, iron and phosphorus, has a low water content, is tender, has a strong ability to accumulate fat, a high meat production rate, and sufficient fat precipitation between myofibrils and muscle fibers. The Chahar sheep is mainly distributed in the southern part of Xilingol League, Inner Mongolia. This breed of sheep is a meat and wool dual-purpose type, with soft and non-greasy meat, a moderate fat content, a high meat yield, a high lean meat rate, and a large proportion of lean meat. The Xiqiyang sheep, a special product of Xin Barag Right Banner in Hulunbuir City, Inner Mongolia, has a low water content, a high protein and fat content, and has rich nutritional value. The sensory characteristics and nutritional values of the meat of different sheep breeds are different, but the mutton on the market has a similar appearance. How to distinguish or select it has become a major difficulty.

[0003] In population genetics, genetic evolution is a very important part. The primary research content of genetic evolution is the genetic structure of populations and their evolutionary relationships with each other. Using this relationship to reconstruct phylogenetic trees, calculate the degree of relatedness between individuals, etc. Such analyses are very necessary for us to further understand the genetic structure and evolutionary history of species and even to conduct larger-scale research on species in combination with sampling geography. Another important research content is selective sweep. By calculating genetic indicators such as Fst and yuan for different populations, we can understand which regions have experienced strong selection effects between different populations, and combine traits to judge the sites most likely to be affected by selection between two groups of samples.

[0004] Calculation, etc. Such analyses are very necessary for us to further understand the genetic structure, evolutionary history of species and even to conduct larger-scale research on species in combination with sampling geography. Another important research content is selective sweep. By calculating genetic indicators such as Fst and yuan for different populations, we can understand which regions have experienced strong selection effects between different populations, and combine traits to judge the sites most likely to be affected by selection between two groups of samples.

[0005] Whole-genome resequencing is to sequence the genomes of different individuals of a species with a known genome sequence, and on this basis, perform differential analysis on individuals or populations. For the individuals subjected to whole-genome resequencing, through sequence alignment, a large number of single nucleotide polymorphism sites (SNPs), insertion / deletion sites (InDels, Insertion / Deletion), and other variant information can be found. After obtaining SNP information by using resequencing methods in population genetics and performing subsequent analysis, it can more accurately reflect the differences between different individuals or populations at the whole-genome level, obtain more reliable analysis results, and provide convenience for subsequent sample typing and data mining, etc.

[0006] Therefore, the present invention uses whole-genome resequencing technology to determine and analyze the gene differential sites of different breeds of sheep, providing technical support for scientifically and efficiently distinguishing the origins of multiple sheep breeds. For this purpose, we propose a method for tracing the origin of mutton by combining whole-genome recombination sequencing with meat quality characteristics. Summary of the Invention

[0007] The purpose of the present invention is to provide a method for tracing the origin of mutton by combining whole-genome recombination sequencing with meat quality characteristics to solve the problems raised in the above background technology.

[0008] To achieve the above purpose, the present invention provides the following technical solution: A method for tracing the origin of mutton by combining whole-genome recombination sequencing with meat quality characteristics, including the following steps:

[0009] Extract the sample DNA to obtain a DNA sample and perform detection;

[0010] Perform library construction, sequencing, and quality control on the qualified DNA sample to obtain a sample;

[0011] Analyze the sample;

[0012] Detect the meat quality and flavor of multiple sheep;

[0013] Analyze and obtain the origin of the traced mutton according to the relationship between the meat quality and flavor detection results and the sample analysis results.

[0014] It should be noted in the solution that: The methods for detecting the DNA sample include agarose gel electrophoresis detection, Nanodrop detection, and Qubit detection.

[0015] Furthermore, it is worth noting that the library construction is to randomly break the qualified DNA sample into DNA fragments by a crusher, and complete the library preparation after the DNA fragments are processed.

[0016] Even further, it should be noted that the sequencing process is to perform high-throughput sequencing on the DNA sample that has passed the library quality inspection on the machine.

[0017] As a preferred embodiment, the quality control process is to obtain CleanData after removing low-quality sequences and adapter sequences, align the obtained CleanData to the reference genome, then detect single nucleotide polymorphism sites and insertion / deletion sites, and perform quality filtering on the detected variant sites.

[0018] As a preferred embodiment, the sample analysis is to perform population structure analysis and principal component analysis on the sample based on single nucleotide polymorphism site data, and perform selective sweep analysis to obtain differential sites on the chromosome, and obtain the principal component clustering of the sample.

[0019] As a preferred embodiment, the selective sweep analysis includes calculating population genetics indexes of all SNP sites within a sliding window of a step size.

[0020] As a preferred embodiment, the population genetics indexes include population differentiation, nucleotide polymorphism, and combined analysis of population differentiation and nucleotide polymorphism to detect selected regions.

[0021] As a preferred embodiment, the meat quality and flavor detection obtains volatile flavor substances in the longissimus dorsi muscle among multiple sheep populations.

[0022] As a preferred embodiment, the origin tracing of the mutton is to trace the origin according to the number of differential sites of multiple volatile flavor substances and the corresponding chromosomes of the sheep.

[0023] Compared with the prior art, the method for tracing the origin of mutton by combining whole-genome recombination sequencing and meat quality characteristics provided by the present invention has at least the following beneficial effects:

[0024] Based on 5 differential sites and unique meat quality markers on two chromosomes, the present invention can completely distinguish Chahar sheep, and then be used for tracing the origin of mutton. Based on 3 differential sites and unique meat quality markers on one chromosome, Ujimqin sheep and Xiqi sheep can be completely distinguished, effectively tracing the origin of the three kinds of mutton accurately. The method for tracing the origin of mutton by combining whole-genome recombination sequencing and meat quality characteristics will play a positive role in promoting the inspection and detection technologies for preventing adulteration and authenticity identification of Inner Mongolia geographical indication mutton, enhancing the brand influence of Inner Mongolia geographical indication mutton, promoting the development of the livestock product industry in Inner Mongolia Autonomous Region, and improving the recognition of Inner Mongolia mutton by consumers nationwide. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is a schematic diagram of the Fst result of CHR_vs_SNT of the present invention;

[0026] Figure 2 Schematic diagram of the Fst results of CHR_vs_WZMQ of the present invention;

[0027] Figure 3 Schematic diagram of the Fst results of CHR_vs_XQ of the present invention;

[0028] Figure 4 Schematic diagram of the Fst results of SNT_vs_WZMQ of the present invention;

[0029] Figure 5 Schematic diagram of the Fs results of SNT_vs_XQ of the present invention;

[0030] Figure 6 Schematic diagram of the Fst results of WZMQ_vs_XQ of the present invention;

[0031] Figure 7 Schematic diagram of the principal component clustering of the samples of the present invention;

[0032] Figure 8 Schematic diagram of the heat map analysis of muscle volatile flavor substances of different sheep breeds of the present invention;

[0033] Figure 9 Schematic diagram of the comparison of muscle characteristic flavor substances of different sheep breeds of the present invention;

[0034] Figure 10 Schematic diagram of the network relationship diagram between differential sites and meat quality among four sheep populations of the present invention. Detailed implementation manners

[0035] The method for tracing the origin of mutton by combining whole-genome recombination sequencing and meat quality characteristics provided by the present invention will be described in detail below in conjunction with the accompanying drawings and specific embodiments. At the same time, it should be noted here that in order to make the embodiments more detailed, the following embodiments are the best and preferred embodiments. For some well-known technologies, those skilled in the art can also adopt other alternative methods for implementation; moreover, the accompanying drawings are only for more specific description of the embodiments and are not intended to specifically limit the present invention.

[0036] It should be pointed out that in the specification, the mention of "an embodiment", "embodiment", "exemplary embodiment", "some embodiments", etc. indicates that the described embodiment may include specific features, structures or characteristics, but not necessarily every embodiment includes the specific feature, structure or characteristic. In addition, when combining an embodiment to describe a specific feature, structure or characteristic, implementing such a feature, structure or characteristic in combination with other embodiments (whether explicitly described or not) should be within the knowledge of those skilled in the relevant art.

[0037] Generally, terms can be understood at least in part from their use in context. For example, depending at least in part on the context, the term "one or more" as used herein can be used to describe any feature, structure, or property in a singular sense, or can be used to describe a combination of features, structures, or properties in a plural sense. Additionally, the term "based on" can be understood to not necessarily be intended to convey a set of exclusive factors, but rather can alternatively, depending at least in part on the context, allow for the existence of other factors that are not necessarily explicitly described.

[0038] It is to be understood that the meanings of "on", "above", and "over" in the present invention should be construed in the broadest manner such that "on" not only means "directly on" something, but also includes the meaning of being "on" something with intervening features or layers therebetween, and "above" or "over" not only means "above" or "over" something, but can also include the meaning of being "above" or "over" something with no intervening features or layers therebetween.

[0039] In addition, spatial relative terms such as "under", "below", "lower", "above", "upper", etc. may be used herein for convenience of description to describe the relationship of one element or feature to another or other elements or features, as shown in the drawings. Spatial relative terms are intended to cover different orientations in the use or operation of the device in addition to the orientation depicted in the drawings. The device may be oriented in other ways, and the spatial relative descriptors used herein may be similarly interpreted accordingly.

[0040] The following examples are used to illustrate the present invention, but cannot be used to limit the protection scope of the present invention. The conditions in the examples can be further adjusted according to specific conditions, and simple improvements to the method of the present invention under the premise of the concept of the present invention all fall within the scope of protection required by the present invention.

[0041] Please refer to Figure 1-10 , the present invention provides a method for tracing the origin of mutton by combining whole-genome recombination sequencing with meat quality characteristics, including the following steps:

[0042] Extract sample DNA to obtain a DNA sample and perform detection;

[0043] Perform library construction, sequencing, and quality control on the qualified DNA sample to obtain a sample;

[0044] Analyze the sample;

[0045] Detect various mutton meat quality flavors;

[0046] Analyze the relationship between the meat quality flavor detection results and the sample analysis results to obtain the traced origin of the mutton.

[0047] Extract the sample DNA to obtain a DNA sample. During the detection, a DNA extraction kit is used to extract DNA according to the standard procedure. The detection of the DNA sample mainly includes three methods:

[0048] 1. Analyze the purity and integrity of DNA by agarose gel electrophoresis;

[0049] 2. Detect the purity of DNA (OD260 / 280 ratio) using Nanodrop;

[0050] 3. Accurately quantify the DNA concentration using Qubit;

[0051] For the qualified DNA samples, construct libraries, perform sequencing and quality control. In the obtained samples, the qualified DNA samples are randomly fragmented into DNA fragments with a length of 330 - 370 bp by a crusher. The DNA fragment processing includes end repair, adding a polyA tail, adding sequencing adapters, purification, and PCR amplification. Then, use Qubit 2.0 for preliminary quantification, dilute the library to 1 - 5 ng / μl, and then use Agilent 2100 to detect the insert size of the library. After the insert size detection meets the requirements, use the Q-PCR method to accurately quantify the effective concentration of the library, where the effective concentration of the library is greater than 1 nM.

[0052] For the qualified DNA samples, construct libraries, perform sequencing and quality control. In the obtained samples, the sequencing platform is Illumina Nova6000, and the sequencing mode is PE150.

[0053] For the qualified DNA samples, construct libraries, perform sequencing and quality control. In the obtained samples, after removing low-quality sequences and adapter sequences, CleanData is obtained. Align the obtained CleanData to the reference genome v4.0, and use the GATK software to detect SNPs and InDels, and perform quality filtering on the detected variant sites.

[0054] Sample analysis is based on single nucleotide polymorphism (SNP) site data to perform population structure analysis and principal component analysis on samples, and perform selective sweep analysis to obtain differential sites on chromosomes, obtaining the principal component clustering of samples. The content of sample analysis includes phylogenetic trees, population structure, principal component analysis, and the genetic relationship between samples. Subsequent selective sweep analysis is also based on SNP data. Selective sweep analysis will calculate indicators such as the population genetic distance Fst and population nucleotide diversity π, and view the differences in different genomic segments among groups. Based on these differences, it is judged which positions have experienced strong selection effects, and finally differential sites are selected. Among them, SNP is the single nucleotide polymorphism site, and InDel is the insertion-deletion site.

[0055] In the sample analysis, the selective sweep analysis includes calculating the population genetics indicators of all SNP sites within a sliding window (such as 100 kb) with a specific step size (such as 10 kb). In this project, the population differentiation degree (Fst), nucleotide polymorphism (π), and the combined analysis of Fst and π were performed to detect the selected regions. The fixation coefficient Fst of the population reflects the level of allelic heterozygosity of the population.

[0056] Fst is a traditional basic indicator for measuring genetic differentiation between populations, and its calculation formula is as follows:

[0057]

[0058] Performing pairwise Fst analysis on the four groups of samples, we obtained Figure 1 、 Figure 2 、 Figure 3 、 Figure 4 、 Figure 5 、 Figure 6 , from Figure 1 it can be seen that the highest Fst value between the CHR and SNT populations is located on the chromosome NC_019470.2, and its value is 0.73, indicating a relatively large genetic differentiation between these two populations. There are more differential sites with strong selection in this region. Through Figure 1 、 Figure 2 、 Figure 3 comprehensive analysis, it can be seen that the CHR population has a relatively large genetic differentiation from the other three populations, and the regions experiencing strong selection are similar. Compared with the other three populations, it can be found that the Fst value is relatively small, close to about 0.3, but the frequency of the Fst value appearing in the top 1% of the selected regions increases.

[0059] From Figure 1 、 Figure 4 、 Figure 5 、 Figure 6By comparison, there are significant differences in the Fst results between the SNT population and the three sheep breeds. The maximum Fst value between SNT and WZMQ is located on chromosome NC_019484.2, and the frequency of screening regions above the threshold of 1% on this chromosome is significantly higher than that on other chromosomes. However, the maximum Fst value is close to 0.3, which is significantly lower than the value between SNT and CHR, indicating that the selection experienced between them is relatively weak. The maximum Fst value between SNT and X0 appears on chromosome NC_019462.2 and does not exceed 0.4. From the above, it can be shown that there has been a strong selection between SNT and CHR, with a large genetic differentiation, while the genetic differentiation between SNT and the other two sheep breeds is smaller, and there may be fewer differential sites.

[0060] The Fst value between the WZMQ and XQ populations is less than 0.25, which is different from the other two sheep breeds; the genetic differentiation between WZMQ and XQ is smaller. However, there are more screening regions above the threshold of 1%. Finally, the differentiation of WZMQ from the other three sheep meat products can be achieved by analyzing the multi-locus differences.

[0061] Analyzing the PCA results as Figure 7 shown, after clustering with PC1 and PC2, the WZMQ and SNT samples are relatively close, indicating that the genetic relationship between these two populations is relatively close. Clustering with PC2 and PC3 still shows that the genetic relationship between the WZMQ and SNT populations is relatively close; while clustering with PC1 and PC3, the distances between the three populations of CHR, WZMQ, and SNT are relatively close, and XQ is completely separated from the samples of other populations. Part of the reason for the relatively close genetic relationship among the three major populations of SNT, CHR, and WZMQ may be that their geographical locations are close and their living environments are similar. Therefore, the environment has less selectivity for the three populations, and their positions on the evolutionary tree are close.

[0062] Among them, it is further worth noting that:

[0063] XQ refers to the Western Flag sheep flock

[0064] WZMQ refers to the Ujimqin sheep flock

[0065] SNT refers to the Sunite sheep flock

[0066] CHR refers to the Chahar sheep flock

[0067] During the detection of the meat quality and flavor of various sheep, a FlavourSpec gas chromatography-ion mobility spectrometer and an MXT-Wax chromatographic column were used for the detection of meat quality and flavor, and the composition of volatile flavor substances in the longissimus dorsi muscle among the four sheep populations was obtained. See Figure 8 , as can be seen from the figure, there are certain differences in the volatile flavor substances among the four sheep breeds. Through cluster analysis, it was found that SNT and WZMQ are clustered together, and CHR and XQ are clustered together. Further, the characteristic flavor substances among the four sheep breeds were selected. SeeFigure 9 , the characteristic flavors of Sunite sheep include 1-pentanol, nonanal, benzaldehyde, and 2,3-octanedione. These flavor substances endow Sunite mutton with fruity, grassy, milky, almond-like flavors, etc. The characteristic flavors of Wuzhumuqin sheep include 1-hexanol, ethyl acetate, benzaldehyde, and 2-pentylfuran. These flavors endow Wuzhumuqin mutton with floral, fruity, grilled meat flavors, etc. The characteristic flavors of Chahar sheep include leaf alcohol, 1-octen-3-ol, and 2-butanone. These flavors endow Chahar mutton with leafy, mushroomy, spicy flavors, etc. The characteristic flavors of Xiqi sheep include 1-octanol, 2-ethylhexanol, 2-decen-1-ol, and 4-allylanisole. These flavors endow Xiqi mutton with fatty, floral, grassy flavors, etc.

[0068] Based on the analysis of the relationship between the meat quality flavor detection results and the sample analysis results, in the traceability of the mutton origin, according to the different quality indicators and different loci of the four geographical indication sheep, the OmicStudio tool is used to draw a network diagram on https: / / www.omicstudio.cn / tool / 56 to obtain a relationship diagram. According to Figure 10 , thus, based on the 5 different loci and specific meat quality markers on two chromosomes, Chahar sheep can be completely distinguished, and then used for the traceability of the mutton origin. Based on the 3 different loci and specific meat quality markers on one chromosome, Wuzhumuqin sheep and Xiqi sheep can be completely distinguished.

[0069] Unless otherwise defined, the technical terms or scientific terms used in the present invention shall have the ordinary meaning understood by those of ordinary skill in the art to which the present invention pertains. The words such as "including" or "comprising" used in the present invention mean that the elements or objects appearing before the word cover the elements or objects listed after the word and their equivalents, without excluding other elements or objects. The words such as "connected" or "linked" do not limit to physical or mechanical connections, and may also include electrical connections, whether direct or indirect. The words such as "up", "down", "left", "right" are only used to represent relative positional relationships. When the absolute position of the object being described changes, the relative positional relationship may also change accordingly.

[0070] Although the embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for tracing the origin of mutton by combining whole-genome recombination sequencing with meat quality characteristics, characterized in that, It includes the following steps: Extract sample DNA to obtain a DNA sample and conduct detection; Perform library construction, sequencing, and quality control on the qualified DNA sample to obtain a sample; Analyze the sample; Detect the meat quality and flavor of multiple sheep; Analyze the relationship between the meat quality and flavor detection results and the sample analysis results to trace the origin of the mutton.

2. The method for tracing the origin of mutton by combining whole-gene recombinant sequencing with meat quality characteristics according to claim 1, wherein: The methods for detecting the DNA sample include agarose gel electrophoresis detection, Nanodrop detection, and Qubit detection.

3. The method for tracing the origin of mutton by combining whole-gene recombinant sequencing with meat quality characteristics according to claim 1, wherein: The library construction is to randomly break the qualified DNA sample into DNA fragments by a crusher, and complete the library preparation after the DNA fragments are processed.

4. The method for tracing the origin of mutton by combining whole-gene recombinant sequencing with meat quality characteristics according to claim 1, wherein: The sequencing process is to perform high-throughput sequencing on the DNA sample that has passed the library quality inspection on a machine.

5. The method for tracing the origin of mutton by combining whole-gene recombinant sequencing with meat quality characteristics according to claim 1, wherein: The quality control process is to obtain CleanData data after removing low-quality sequences and adapter sequences, align the obtained CleanData data to the reference genome, then detect single nucleotide polymorphism sites and insertion / deletion sites, and perform quality filtering on the detected variant sites.

6. The method for tracing the origin of mutton by combining whole-genome recombination sequencing with meat quality characteristics according to claim 1, wherein: The sample analysis is to perform population structure analysis and principal component analysis on the sample based on the single nucleotide polymorphism site data, and perform selective sweep analysis to obtain the differential sites of the chromosome, and obtain the principal component clustering situation of the sample.

7. The method for tracing the origin of mutton by combining whole-genome recombination sequencing with meat quality characteristics according to claim 6, wherein: The selective sweep analysis includes calculating the population genetics indicators of all SNP sites within a sliding window with a step size.

8. The method for tracing the origin of mutton by combining whole-gene recombination sequencing with meat quality characteristics according to claim 7, characterized in that: The population genetics indicators include population divergence, nucleotide polymorphism, and the combined analysis of population divergence and nucleotide polymorphism to detect the selected regions.

9. The method for tracing the origin of mutton by combining whole-gene recombination sequencing with meat quality characteristics according to claim 1, wherein: The meat quality and flavor detection obtains the volatile flavor substances in the longissimus dorsi muscle among multiple sheep populations.

10. The method for tracing the origin of mutton by combining whole-gene recombinant sequencing with meat quality characteristics according to claim 1, wherein: The origin tracing of the mutton is to trace the origin according to the number of differential sites on the chromosome corresponding to multiple volatile flavor substances and the corresponding sheep.

Images