CN117568486A - Xinglong buffalo liquid-phase chip and application thereof - Google Patents
Xinglong buffalo liquid-phase chip and application thereof Download PDFInfo
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Abstract
The invention discloses a liquid-phase chip for a Khinghing buffalo, wherein a genotyping object of the chip comprises 1208 SNP loci, and 1208 SNP loci are positioned on a buffalo reference genome CUSA_SWP. The Xinglong buffalo liquid-phase chip can be applied to Xinglong buffalo blood system analysis, buffalo genetic diversity evaluation, germplasm resource and kindred relation identification, genetic map construction and gene positioning, whole genome association analysis and buffalo molecular marker assisted breeding.
Description
Technical Field
The invention belongs to the technical field of molecular detection, and particularly relates to a liquid-phase chip for a water buffalo and application thereof.
Background
The Xinglong buffalo is an excellent buffalo variety in tropical areas, has the variety characteristics of high humidity resistance, high heat resistance, strong disease resistance and the like, is mainly distributed in the south and north-south China of Wanning county of Hainan province, and the germplasm characteristic of the Xinglong buffalo is gradually formed under the interaction of a long-term tropical climate environment and gene mutation and the long-term breeding of local masses. In recent years, due to blind hybridization improvement and lack of resource protection consciousness, the production performance and the product quality of buffalo in various places are greatly influenced, and the number of buffalo is gradually reduced or even is about to disappear. The molecular marker assisted breeding technology is a technology for assisting a traditional breeding method by utilizing molecular markers and genetic information, and can accelerate the breeding process, improve the selection efficiency, expand the genetic diversity, reduce the cost and improve the breeding precision and accuracy.
Single nucleotide polymorphism (Single Nucleotide Polymorphism, SNP) refers to variation of a single nucleotide at the genomic level. With the development of high-throughput sequencing and array technology, the cost of large-scale genotyping has been greatly reduced, and the selection of SNP as a genetic marker has become a trend, so that the SNP has been widely applied to animal molecular genetic studies such as trait association analysis, genetic identification and the like. Among them, the targeted sequencing genotyping (Genotyping By Target Sequencing, GBTS) technique is a technique for deep resequencing of target sites. Compared with the endonuclease-mediated sequencing genotyping-based technology, the GBTS technology has stronger targeting, higher site coverage and sample uniformity. GBTS technology is unique in its "targeting" properties, and its system consists of the GenoBaits technology and the GenoPlexs technology. The liquid phase chip developed in recent years is a SNP-based targeted sequencing genotype detection technology, and the existence of a target molecule and the genotype or specific variation thereof can be determined by detecting the combination condition of the target molecule and a probe.
The existing buffalo SNP breeding chips in the market at present, such as axiom buffalo genotyping chip, buffalo 50KSNP chip and the like, are not suitable for researching and evaluating the genetic diversity of the prosperous buffalo variety. In order to better excavate and protect the valuable germplasm resource of the prosperous buffalo, the design of SNP liquid phase chips belonging to the prosperous buffalo is urgently needed.
Disclosure of Invention
The invention aims to provide a liquid-phase chip for the Xinglong buffalo, so as to realize variety identification and genetic relationship analysis of the Xinglong buffalo.
The invention also aims to provide the application of the liquid-phase chip for the water buffalo in breeding of the water buffalo.
The technical scheme adopted by the invention is that the genotyping object of the Dairy water buffalo liquid chip comprises 1208 SNP loci, and the position information of 1208 SNP loci positioned on the water buffalo reference genome CUSA_SWP (GWHAAJZ 00000000) is as follows:
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the invention adopts another technical scheme that the liquid-phase chip of the prosperous buffalo is applied to the blood system analysis, the genetic diversity evaluation, the germplasm resource and kindred relation identification, the genetic map construction, the gene location, the whole genome association analysis and the buffalo molecular marker assisted breeding of the prosperous buffalo.
The beneficial effects of the invention are as follows: the liquid-phase chip for the prosperous buffalo has the advantage of low-cost genotyping; variety identification and genetic relationship analysis of the Xinglong buffalo are facilitated; can provide scientific guidance for the hybridization improvement work of the prosperous buffalo; is helpful for protecting and developing germplasm resources of the prosperous buffalo. In addition, by using the chip, a more accurate genotyping result can be realized, and more accurate information is provided for buffalo breeding work.
Drawings
FIG. 1 is a graph showing the results of a phylogenetic tree of all re-sequenced buffalo samples in the examples;
FIG. 2 is a statistical map of the chromosome distribution of SNP markers (200 Kb in plot units) in the examples;
FIG. 3 is a GenoBaits operating principle and flow chart;
fig. 4 is a schematic and flow chart of the GenoPlexs operation;
FIG. 5 is a statistical diagram of SNP annotations;
Detailed Description
The invention will be described in detail below with reference to the drawings and the detailed description.
Example 1 design and preparation of a Carnis Corvus liquid phase chip
The invention utilizes the whole genome re-sequencing data of 143 samples of nineteen different varieties of buffalo, including 12 Chinese varieties (5 head De Hongzhu buffalo, 9 head Yunnan buffalo, 5 head Dongyou buffalo, 12 head Fu Zhong Shuiniu, 15 head Guizhou buffalo, 10 head Hai buffalo, 5 head Poyang lake buffalo, 6 head Wenzhou buffalo, 5 head Xinyang buffalo, 10 head Yanjin buffalo, 10 head Yibin buffalo and 15 head Hainan Dairy buffalo) and 7 foreign varieties (5 head Laos buffalo, 5 head Vietnam buffalo, 5 head Philippine buffalo, 5 head Burmesn buffalo, 5 head Thailand 10 head Indonesia buffalo), and obtains highly reliable SNP sites for subsequent screening through data analysis.
The GenoBaits DNA-seq Library Prep kit is adopted to construct a resequencing Library for the qualified DNA, and the steps are as follows: (1) 200ng of DNA with quantitative quality control is placed in a 0.2uL PCR tube, 4 mu L GenoBaits End Repair Buffer and 2.7 mu L GenoBaits End Repair Enzyme are added into the tube, water is added to 20 mu L, the tube is placed in an ABI9700 PCR instrument for incubation at 37 ℃ for 20 minutes and denaturation at 72 ℃ for 20 minutes, and the processes of DNA fragmentation, end repair and A tail addition are completed. (2) The PCR tube was removed from the PCR apparatus, added with 2. Mu. L GenoBaits Ultra DNA ligase, 8. Mu. L GenoBaits Ultra DNA Ligase Buffer and 2. Mu. L GenoBaits Adapter, made up to 40. Mu.L, and placed on an ABI9700 PCR apparatus for reaction at 22℃for 30 minutes to complete the ligation of the sequencing adapter. 48uL of Beackman AMPure XP Beads is added to the ligation product, the ligation product is purified, and after purification, fragment screening is performed by using 0.65+0.2 times of magnetic beads, so that the ligation product with the inserted fragment of 200-300bp is reserved. (3) mu.L of sequencing adapter with Barcode sequence, 1 mu L P adapter, 10 mu L GenoBaits PCR Master Mix are added to the PCR tube of the previous step, and the mixture is supplemented to 20 mu L with water; amplification was performed using an ABI9700 PCR instrument, the amplification procedure being: pre-denaturation at 95℃for 5 min, denaturation at 95℃for 30 sec, annealing at 60℃for 30 sec, extension at 72℃for 30 sec; repeating the steps 2-4 for 6 cycles; extension was carried out at 72℃for 5 minutes. Different Barcode was used to distinguish between different samples. (4) After adding 24 mu L Beckmen AMPure XP Beads to the second round of PCR products and pipetting up and down evenly, the 0.2PCR tube was placed on a magnetic rack until the solution was clear, the supernatant was discarded and the beads were washed twice with 75% ethanol and the library DNA eluted with pH=8.0 Tris-HCl.
After the library construction is completed, quality detection is performed to ensure library quality. And after the quality of the library is detected to be qualified, sequencing by utilizing a Huada MGI-2000/MGI-T7 sequencing platform, wherein the sequencing mode is a PE150 mode. Sequencing to obtain an original sequencing sequence (sequential Reads) or raw Reads, and then filtering the sequence Reads to obtain clean Reads, and performing subsequent analysis by using the clean Reads. Comparing the clear Reads after quality control with a reference genome sequence by using software BWA (mem comparison), detecting variation by using a HaplotyCaller module of software GATK (version 4.0) according to the comparison result of the clear Reads in the reference genome, and filtering by using a variant filtration module. After having annotated the file with the reference genome, position annotation can be performed on the basis of the dataset of SNPs, for a total of 34,757,694 SNPs detected.
In order to construct a reference population of the prosperous buffalo and a reference population of the non-prosperous buffalo, phylogenetic tree analysis is carried out on all samples, and the results are shown in fig. 1, so that the non-prosperous buffalo population of the prosperous buffalo is successfully constructed. After the reference population of the prosperous buffalo and the non-prosperous buffalo is determined, all samples are divided into two groups of the prosperous buffalo and the non-prosperous buffalo, and then the value of each SNP mutation site Fst is calculated.
The sites and sequences were all converted to information consistent with the buffalo reference genome. And (3) using all the loci for probe design synthesis, screening qualified SNP loci according to locus screening requirements and probe design results, and uniformly distributing the loci in each chromosome of a genome, as shown in figure 2. And designing probes according to the position and the sequence information on two sides of the SNP locus, and carrying out probe synthesis and test by using Shijia Boruidi biotechnology limited company, and finally picking out the locus meeting the requirements to finish the development of the SNP marker detection kit of the prosperous buffalo, thereby obtaining the liquid-phase chip of the prosperous buffalo.
Example 2 detection and analysis Process of Carnis Corvus Nippon liquid phase chip
Sample DNA was extracted using a high throughput DNA extraction kit. The extracted DNA sample was then subjected to two assays: (1) DNA purity and integrity: the DNA samples were analyzed using a 1% agarose gel electrophoresis method. (2) accurate quantification of DNA concentration: a Qubit gauge was used. Taking a certain amount of DNA from a sample qualified in quality inspection, carrying out random physical crushing by using an ultrasonic crusher, controlling the peak value of the crushed fragment to be in the range of 200-300bp, repairing the tail end of the crushed DNA, and connecting the tail A. And (3) connecting the DNA fragments after the addition of A with a sequencing joint by using a ligase, purifying the library by using carboxyl modified magnetic beads, selecting fragments, and reserving a connecting product of the inserted fragments with about 200-300 bp. And adding a sequencing primer with Barcode into the connection product and performing PCR amplification by a high-fidelity PCR reaction system. After purification by carboxyl magnetic beads, the amplified product can be used in probe hybridization experiments. 500ng of the constructed sequencing library is taken, the sequencing library is freeze-dried, then the probe and the hybridization reagent are added, and the hybridization reaction is completed after denaturation and 2 hours incubation at 65 ℃, as shown in figure 3.
And adding a multiplex PCR Panel mix and a multiplex PCR amplification enzyme system into DNA with qualified quantitative quality inspection, and placing the DNA on a PCR instrument to complete PCR reaction. After the PCR product is purified by using carboxyl magnetic beads, sequencing primers with Barcode and a high-fidelity PCR reaction system are counted again for PCR amplification, and different Barcode are used for distinguishing different samples. The amplified products after purification by carboxyl magnetic beads are subjected to multiplex PCR capture and library construction, as shown in FIG. 4. And constructing a target sequencing library by using the sample qualified in the DNA quality inspection by using a corresponding product, and finally completing library preparation of all the samples. And then, carrying out preliminary quantification by using the Qubit2.0, accurately quantifying the effective concentration of the library by using a qPCR method, and entering an on-machine sequencing stage after the library is detected to be qualified.
The content of the information analysis includes: data quality control (removal of adaptors and low quality data), alignment with reference genome, mutation detection and annotation, and the like. ANNOVAR is a highly efficient software tool that can make functional annotations of detected genetic variations using up-to-date information. The location of the mutation site on the reference genome and the gene location information on the reference genome can be used to obtain the region (intron region, intergenic region, coding region, 5-terminal UTR region, 3-terminal UTR region, etc.) where the mutation site occurs in the genome and the influence of the mutation (synonymous, nonsensical mutation, etc.), and the results are shown in FIG. 5.
Example 3 application of Xinglong buffalo liquid chip
According to the invention, 93 cattle DNA samples qualified in quality inspection are detected by utilizing the developed Dairy buffalo liquid phase chip according to analysis such as data quality control (removing joints and low-quality data), comparison with a reference genome, mutation detection and annotation and the like. The detection rate of the qualified test sample at the target site is 99.565% -99.960%, and the average detection rate is 99.823%, which shows that the requirements of the liquid-phase chip of the water buffalo are met.
The chip has the advantage of low-cost genotyping, and is particularly suitable for variety identification and genetic relationship analysis of the Xinglong buffalo. Can provide scientific guidance for the hybridization improvement work of the prosperous buffalo, and is helpful for protecting and developing germplasm resources of the prosperous buffalo. In addition, by using the chip, a more accurate genotyping result can be realized, and more accurate information is provided for buffalo breeding work.
Claims (2)
1. The liquid-phase chip for the buffalo in the prospection is characterized in that the genotyping object of the chip comprises 1208 SNP loci, and the positional information of 1208 SNP loci positioned on the buffalo reference genome CUSA_SWP is as follows:
2. the use of the liquid-phase chip of prosperous buffalo according to claim 1 in prosperous buffalo pedigree analysis, buffalo genetic diversity evaluation, germplasm resource and kindred relationship identification, genetic map construction, gene positioning, whole genome association analysis and buffalo molecular marker assisted breeding.
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