Molecular identification method for forage grass ingestion habit of pasturing livestock

Abstract

The invention discloses a molecular identification method for foraging habits of pasture livestock forage grass, and belongs to the technical field of animal husbandry. The molecular identification method comprises the following steps: by taking DNA extracted from livestock manure as a template, carrying out PCR amplification and then purifying to obtain a PCR product; performing electrophoresis detection on the PCR product, and constructing a library; carrying out high-throughput sequencing on the PCR product by using Illumina; and performing mass filtering, sequence removal, sequence splicing and species annotation on the result of the high-throughput sequencing to obtain the ingestion habits and ingestion nutrients of the livestock. According to the method, the nutritional status ingested by the livestock is obtained by using the composition of feeding habits of the natural grassland pasture livestock and the nutritional quality of the pasture, a direct means is provided for researching the nutritional requirements of the pasture livestock and the change of grassland vegetation community composition, and the method has important significance for researching pasture livestock nutriology and grassland management science.

Description

Technical Field

[0001] The invention belongs to the technical field of animal husbandry and relates to a molecular identification method for the forage feeding habits of grazing livestock. Background Art

[0002] In the field of grazing livestock nutrition, it is very important to measure the composition of livestock feed and understand the nutrient intake of livestock to understand the nutritional needs of grazing livestock and grassland management. However, under grazing conditions, the composition of grazing livestock feed is often very complex and difficult to monitor due to the seasonal changes in palatability and forage nutrients. In addition, the current determination of livestock nutrient intake under grazing conditions only stays at monitoring the nutritional status of grassland vegetation to estimate the nutrient content of livestock intake, and does not consider the actual status of livestock feeding on forage. In recent years, DNA macrobarcoding technology (molecular identification method) has been widely used to quantify animal diet composition, and this technology solves the problem of cumbersome and low accuracy of traditional methods under grazing conditions. Macrobarcoding can quantify the composition of livestock diet according to the relative abundance of each species, and by further combining the diet ratio with the nutritional status of grassland vegetation, the actual nutrient content of livestock intake can be obtained. However, the current technology for combining livestock diet composition with the nutrient status of vegetation communities to discuss livestock nutrient intake is not mature. Summary of the invention

[0003] In view of the defects in the prior art, the purpose of the present invention is to use molecular identification methods to quantitatively determine the diet composition and nutrient content of grazing livestock, calculate the actual nutrient content ingested by livestock, and further explore the difference between the nutrients ingested by livestock and the nutrients that can be provided by grassland.

[0004] In one aspect, the present invention provides a molecular identification method for the forage-eating habits of grazing livestock, comprising: extracting DNA from livestock feces as a template, amplifying by PCR and purifying to obtain a PCR product;

[0005] After the PCR product is subjected to electrophoresis detection, a library is constructed;

[0006] The PCR product was sequenced using Illumina.

[0007] The results of the high-throughput sequencing were subjected to quality filtering, sequence removal, sequence splicing and species annotation.

[0008] Furthermore, the present invention first samples the grassland where livestock graze, determines the biomass of the grazing grassland, the species that livestock can consume and the nutritional quality content of the grass, and obtains the sequence information of the species that livestock can consume from the NCBI database.

[0009] Furthermore, the present invention adopts the rectal feces collection method to collect fresh feces of livestock, collects equal amounts of livestock feces for five consecutive days and mixes them as a sample, and then extracts DNA from the sample.

[0010] Furthermore, in the molecular identification method of forage feeding habits of grazing livestock provided by the present invention, the primer sequence for PCR amplification is:

[0011] rbcL P1-F(CTTACCAGYCTTGATCGTTACAAAGG);

[0012] rbcL P1-R(GTAAAATCAAGTCCACCRCG).

[0013] Furthermore, the present invention constructs a library according to the sequence information obtained by electrophoresis detection of the PCR product, and on this basis, the constructed library is verified by the Qsep-400 method.

[0014] Furthermore, the molecular identification method of forage feeding habits of grazing livestock provided by the present invention, which performs quality filtering, sequence removal, sequence splicing and species annotation on the results of high-throughput sequencing, specifically: using the DADA2 method in QIIME2 (versoin 2020.6) to denoise the data after quality control, and using 0.005% of all sequence numbers as the threshold to filter ASVs by default; using the trained annotator to annotate all sequences for species.

[0015] Furthermore, the present invention provides a molecular identification method for the forage feeding habits of grazing livestock, wherein the annotator is obtained after training using QIIME2 to which sequence information of the edible species of the livestock is added, using a naive Bayes classifier algorithm.

[0016] Furthermore, the present invention provides a molecular identification method for the forage feeding habits of grazing livestock, wherein the trained annotator is used to perform species annotation on all sequences to obtain the composition of the livestock's forage feeding habits.

[0017] Furthermore, the present invention obtains the nutritional composition ingested by the livestock by multiplying the obtained composition of the livestock forage by the nutritional quality content of the forage.

[0018] On the other hand, the present invention provides the application of the molecular identification method of forage feeding habits of grazing livestock in studying the nutritional status of grazing livestock.

[0019] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages:

[0020] The present invention measures the nutrient composition of grazing livestock and multiplies the nutrient composition ratio of livestock with the nutrient quality of forage provided by grassland to obtain the nutrient status actually ingested by livestock. This not only realizes the application of molecular identification methods on the nutrient composition of livestock, but also further determines the nutrient status actually ingested by livestock and compares the difference between the ingested nutrients and the nutrients that can be provided by grassland. This method provides a direct means for studying the nutritional status of grazing livestock and the changes in grassland community structure, and has certain reference significance for the research on related grazing livestock nutrition and grassland ecology. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Flowchart of techniques for molecular characterization of forage feeding habits in grazing livestock.

[0022] Figure 2 The diet composition of grazing livestock in different months.

[0023] Figure 3 This is a comparison of the nutrients that grazing livestock can take in at different months and the nutrients that grassland can provide. A is neutral detergent fiber; B is acid detergent fiber; and C is crude protein. DETAILED DESCRIPTION

[0024] The technical scheme of the present invention is described below in conjunction with the embodiments, but the present invention is not limited to the following embodiments. The experimental methods and detection methods described in each embodiment are conventional methods unless otherwise specified; the reagents and materials described are all commercially available unless otherwise specified.

[0025] Example 1

[0026] This example provides a method for extracting DNA from fresh feces of grazing livestock.

[0027] Plants in grazing areas are sampled to determine the biomass of the various species in the grazing pasture, the species available to livestock in the area, and the nutritional quality of the forage.

[0028] The present invention utilizes a rectal sampling method to collect fresh feces of grazing livestock, collects the feces continuously for 5 days, mixes the feces in equal amounts as a sample, and stores the sample in a -80°C refrigerator for a long term.

[0029] Add 0.25-0.5g of sample to a 2mL centrifuge tube, add 500μL buffer SA, 100μL buffer SC and 0.25g grinding beads, (there may be residual RNA in the fecal sample. If RNA needs to be removed, it is recommended to add 10μL RNase A (TIANGEN, RT405-02, self-prepared)) vortex to mix or use a tissue homogenizer to mix, and heat to lyse at 70℃ for 15min. Then centrifuge at 12000rpm (centrifugal force is about 13400×g) for 1min, and transfer the supernatant (about 500μL) to a new 2mL centrifuge tube. Add 200μL buffer SH to mix, vortex for 5s, and place at 4℃ for 10min. Centrifuge at 12000rpm (centrifugal force is about 13400×g) for 3min, transfer the supernatant to a new 2mL centrifuge tube, add 500μL buffer GFA (check whether isopropanol has been added before use), and mix by inversion. Add 10 μL of magnetic bead suspension and vortex to mix for 5 min.

[0030] Place the centrifuge tube on the magnetic rack and let it stand for 30 seconds. After the magnetic beads are completely adsorbed, carefully remove the liquid. Remove the centrifuge tube from the magnetic rack, add 700μL of deproteinization solution RD (check whether anhydrous ethanol has been added before use), and shake and mix for 5 minutes. Place the centrifuge tube on the magnetic rack and let it stand for 30 seconds. After the magnetic beads are completely adsorbed, carefully remove the liquid. Remove the centrifuge tube from the magnetic rack, add 700μL of rinse solution PWD (check whether anhydrous ethanol has been added before use), and shake and mix for 3 minutes. Place the centrifuge tube on the magnetic rack and let it stand for 30 seconds. After the magnetic beads are completely adsorbed, carefully remove the liquid. Place the centrifuge tube on the magnetic rack and dry it at room temperature for 5 to 10 minutes. Remove the centrifuge tube from the magnetic rack, add 50 to 100μL of elution buffer TB, shake and mix, place it at 56℃, incubate for 5 minutes, shake 5 times during the period, and repeat the shaking 3 times. Place the centrifuge tube on the magnetic stand and let it stand for 2 minutes. After the magnetic beads are completely adsorbed, the sample DNA is obtained. Transfer the sample DNA to a new centrifuge tube and store it under appropriate conditions.

[0031] The concentration of the extracted sample DNA was detected using an enzyme reader (GeneCompang Limited, model synergy HTX), and PCR detection amplification was performed according to the primer sequences shown in Table 1. After PCR amplification, the integrity of the sample DNA was detected by electrophoresis detection using 1.8% agarose (Beijing Bomei Fuxin Technology Co., Ltd.).

[0032] Table 1 PCR amplification primer sequence list

[0033]

[0034] Example 2

[0035] This example provides a method for constructing a molecular library of forage feeding habits of grazing livestock.

[0036] 1. PCR purification of target region

[0037] Table 2 Target region PCR system

[0038] Reagent name Dosage Sample DNA 5~50ng Forward primer (10 μM) 0.3 Reverse primer (10 μM) 0.3 KOD FX Neo Buffer 5 dNTP (2mM each) 2 KOD FX Neo 0.2 <![CDATA[ddH 2 The]]> Make up to 10 μL

[0039] Add the reagents shown in Table 2 to the amplified PCR to construct the target region PCR product system. Mix the target region PCR product system and magnetic beads at a volume ratio of 1:1 and perform magnetic bead screening of fragments. The specific operation is as follows: Add 10 μL of VAHTSTM DNA Clean Beads magnetic beads to the target region PCR product system, mix for 5 minutes at room temperature, place on a magnetic stand for 5 minutes, and remove the supernatant. Then add 200 μL of 80% ethanol to wash the magnetic beads, remove the supernatant after standing at room temperature for 30 seconds, and repeat once. Finally, place on a magnetic stand to dry for 3 minutes, and use 8-10 μL ddH 2 Resuspend the magnetic beads in 5% CO, incubate at room temperature for 2 min, place on a magnetic stand for 2 min, and pipette 8-10 μL of supernatant into a new PCR tube to obtain the PCR product.

[0040] 2. Electrophoresis detection

[0041] Prepare 1.8% agarose gel and spot 3.5μL of 100bp marker. Add 2μL of xylene cyanol to the PCR product and spot it on the agarose gel. Use marker to separate every 10 PCR products. Then add the gel to the electrophoresis tank, add the newly prepared buffer solution, and run the electrophoresis at 120V for 40-45min. After the electrophoresis is completed, place the gel block in the staining solution for 20min, and then observe the electrophoresis results under a gel imager.

[0042] 3. Library detection

[0043] The results of electrophoresis detection were constructed into a library and verified using the Qsep-400 method.

[0044] 4. Sequencing Methods

[0045] The constructed library was sequenced using Illumina Novasek6000.

[0046] Example 3

[0047] This example provides data analysis for molecular identification of forage feeding habits of grazing livestock.

[0048] 1. Sequencing data quality assessment

[0049] First, Trimmomatic was used to filter the quality of the raw data, and then Cutadapt was used to identify and remove primer sequences. After that, USEARCH was used to splice the double-end reads and remove chimeras, and finally high-quality sequences were obtained for subsequent analysis.

[0050] Trimmomatic: Trimmomatic is a read quality control filtering tool for Illumina high-throughput sequencing. It can filter FASTQ format double-end sequencing or single-end sequencing data with base quality format of phred 33 or phred 64. Single-end sequencing files require an input file and an output file name, plus parameters; double-end sequencing data requires two input files (FASTQ data of forward primer sequence and reverse primer sequence). The parameter setting is: 50bp window, if the average quality value in the window is less than 20, the back end base is truncated from the window.

[0051] Primer sequence identification and removal: Cutadapt software was used to identify primer sequences according to the parameters of a maximum mismatch rate of 20% and a minimum coverage of 80%.

[0052] PEreads splicing: Usearch v10 software was used to splice the reads of each sample according to the minimum overlap length of 10 bp, the minimum similarity allowed in the overlap region of 90%, and the maximum number of mismatched bases of 5 bp.

[0053] UCHIME splits the sequence to be detected into modules (chunks) without overlap, and then compares them to the database; selects the best match (match) of each module in the database, and finally selects the two best parent sequences; compares the sequence to be detected with the two parent sequences, and if the two parents have a sequence that is more than 80% similar to the sequence to be detected, the sequence to be detected is judged to be a chimera.

[0054] 2. Species annotation and taxonomic analysis

[0055] The NCBI database (ftp: / / ftp.ncbi.nlm.nih.gov / refseq / TargetedLoci / ) was used to obtain sequence information of edible species of livestock grazing grasslands, and the sequence information was imported into QIIME2. QIIME 2 was used to annotate the DNA in livestock feces for species.

[0056] The QIIME2 species annotation training method is as follows:

[0057] Alignment-based method: classify-consensus-blast in QIIME2 is an alignment-based method that can find the most consistent one among the N (default 3) best alignment results for classification, with a minimum sequence similarity of 90%, a minimum coverage of 90%, and a minimum consistency of 51%.

[0058] Based on the Naive Bass classifier: implemented by classify-sklearn in QIIME2. The classifier must be trained before use so that the software can "learn" which features can best distinguish each classification group. The classifier confidence is 0.7.

[0059] Alignment combined with classifier method: Use classify-consensus-blast to align the characteristic sequence with the sequence information of edible species of livestock grazing grasslands in the NCBI database. Use classify-sklearn classifier to classify the sequence information of edible species of livestock grazing grasslands that cannot be accurately aligned with the NCBI database.

[0060] In the present invention, the collected fresh feces of grazing livestock are crushed and preserved, and then sent to Biomaker Biotechnology Co., Ltd. for DNA extraction, PCR amplification, high-throughput sequencing, and preliminary filtering of the data. The ASVs sequence information table initially returned by Biomaker Biotechnology Co., Ltd. is processed. The specific method is to use the DADA2 method in QIIME2 (versoin2020.6) to denoise the data after quality control, and the default threshold is 0.005% of all sequence numbers sequenced to filter ASVs. In QI IME2, DADA2 is a tool for microbiome data analysis, which can help remove noise from sequencing data and improve the accuracy of the analysis. The denoising process includes quality filtering, removal of primer and label sequences, and identification and removal of chimeras.

[0061] Example 4

[0062] This example provides a survey on the feeding components of sheep on the summer grazing and winter feeding production grazing platform of China Agricultural University in Xilinhot, Inner Mongolia.

[0063] like Figure 1As shown, rectal sampling method or picking up fresh sheep dung samples and crushing them for preservation. The obtained sheep dung samples are marked and sent to Biomark Biotechnology Co., Ltd. for DNA extraction, PCR amplification, high-throughput sequencing and preliminary filtering of the data. The ASVs sequence information table initially returned by Biomark Biotechnology Co., Ltd. is processed and converted to obtain a relative proportion table of the barcodes of the main species eaten by sheep. According to the vegetation survey, the dominant species of grassland are determined, and the nutrient content of the dominant species is determined, including crude protein (CP), neutral detergent fiber (NDF), and acid detergent fiber (ADF). According to the feeding components of sheep, the actual nutrient intake ratio of sheep is calculated, that is, the relative proportion of the main species eaten by sheep is multiplied by the nutrient content.

[0064] Table 3 Nutritional quality of dominant species in different months

[0065]

[0066]

[0067] Figure 2 The dietary composition of sheep in July, August and September. Figure 2 It can be seen that the diet of sheep mainly consists of grass species, Rosaceae species, Cyperus species and other non-grass plants. Table 3 is an analysis of the nutritional components of grass species, Cyperus species and other non-grass plants in July, August and September. Figure 3 It is the comparison between the nutrients taken by sheep and the nutrients that can be provided by grassland. It can be seen from the above results that the method provided by the present invention can not only detect the dietary composition of grazing livestock, but also combine the dietary composition with the nutritional quality of forage, calculate the actual nutrient intake of livestock, and compare it with the nutrients that can be provided by grassland, so as to obtain the preference of livestock for forage in different grazing seasons and grasp the nutritional needs of livestock at different stages. Compared with traditional methods, the present invention not only quantitatively analyzes the dietary composition of grazing livestock, but also obtains the actual nutrient content of livestock intake. The method of the present invention is fast, accurate and in-depth.

[0068] The embodiments described above are part of the embodiments of the present invention, but not all of them. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but only represents selected embodiments of the present invention. All other embodiments obtained without creative work and related deductions and substitutions made by ordinary technicians in the field under the conditions of the concept of the present invention belong to the scope of protection of the present invention.

Claims

1. A molecular identification method for the forage feeding habits of grazing livestock, characterized in that: include: The DNA extracted from livestock feces was used as a template, and the PCR product was obtained by PCR amplification and purification; After the PCR product is subjected to electrophoresis detection, a library is constructed; The PCR product was sequenced using Illumina. The results of the high-throughput sequencing were subjected to quality filtering, sequence removal, sequence splicing and species annotation.

2. The molecular identification method of forage feeding habits of grazing livestock according to claim 1, characterized in that: Prior to extracting DNA from livestock feces, the livestock grazing grassland is sampled to determine the biomass of the grassland, the species of food available to the livestock, and the nutritional quality content of the grass.

3. The molecular identification method of forage feeding habits of grazing livestock according to claim 1, characterized in that: The livestock feces were collected by rectal feces collection, and equal amounts of livestock feces were collected for five consecutive days and mixed to form a sample.

4. The molecular identification method of forage feeding habits of grazing livestock according to claim 1, characterized in that: The primer sequences for the PCR amplification are: rbcL P1-F(CTTACCAGYCTTGATCGTTACAAAGG); rbcL P1-R(GTAAAATCAAGTCCACCRCG).

5. The molecular identification method of forage feeding habits of grazing livestock according to claim 1, characterized in that: After the library was constructed, it was validated using the Qsep-400 method.

6. The molecular identification method of forage feeding habits of grazing livestock according to claim 1, characterized in that: The high-throughput sequencing results are subjected to quality filtering, sequence removal, sequence splicing and species annotation, including: using the DADA2 method in QIIME2 to perform denoising on the quality-controlled data, using 0.005% of all sequence numbers as a threshold to filter ASVs by default; and using the trained annotator to perform species annotation on all sequences.

7. The molecular identification method of forage feeding habits of grazing livestock according to claim 6, characterized in that: The annotator is obtained after training using the QIIME2 with the sequence information of the livestock edible species added as the training set and using the Naive Bayes classifier algorithm.

8. The molecular identification method of forage feeding habits of grazing livestock according to claim 6, characterized in that: The trained annotator is used to annotate species of all sequences to obtain the composition of livestock forage intake.

9. The molecular identification method of forage feeding habits of grazing livestock according to claim 7, characterized in that: The composition of the forage consumed by the livestock is multiplied by the nutritional quality content of the forage to obtain the nutritional composition ingested by the livestock.

10. Use of the molecular identification method of forage feeding habits of grazing livestock according to any one of claims 1 to 9 in studying the nutritional status of grazing livestock.

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