CN117568492A - Sand pig SNP molecular marker, chip prepared from sand pig SNP molecular marker and application of sand pig SNP molecular marker - Google Patents
Sand pig SNP molecular marker, chip prepared from sand pig SNP molecular marker and application of sand pig SNP molecular marker Download PDFInfo
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- 238000009395 breeding Methods 0.000 claims abstract description 19
- 230000001488 breeding effect Effects 0.000 claims abstract description 17
- 238000004458 analytical method Methods 0.000 claims abstract description 5
- 238000002864 sequence alignment Methods 0.000 claims abstract description 3
- 239000000523 sample Substances 0.000 claims description 19
- 239000007791 liquid phase Substances 0.000 claims description 16
- 230000002068 genetic effect Effects 0.000 claims description 9
- 238000003205 genotyping method Methods 0.000 claims description 8
- 238000010276 construction Methods 0.000 claims description 5
- 108090000623 proteins and genes Proteins 0.000 claims description 4
- 238000012098 association analyses Methods 0.000 claims description 2
- 238000007621 cluster analysis Methods 0.000 claims description 2
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- 238000009394 selective breeding Methods 0.000 claims description 2
- 241000894007 species Species 0.000 claims description 2
- 238000011161 development Methods 0.000 abstract description 7
- 241000282898 Sus scrofa Species 0.000 description 65
- 238000001514 detection method Methods 0.000 description 11
- 238000012163 sequencing technique Methods 0.000 description 11
- 108091092584 GDNA Proteins 0.000 description 10
- 238000000034 method Methods 0.000 description 9
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- 108020004414 DNA Proteins 0.000 description 4
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- 210000004369 blood Anatomy 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 238000013461 design Methods 0.000 description 4
- 238000012165 high-throughput sequencing Methods 0.000 description 4
- 230000000694 effects Effects 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 2
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Abstract
The invention discloses a sand ridge pig SNP molecular marker, a chip prepared from the sand ridge pig SNP molecular marker and application of the sand ridge pig SNP molecular marker, and relates to the technical field of biology. The sand-ridge pig SNP molecular marker comprises 410 SNP molecular markers, and the genomic positions of the 410 SNP molecular markers are determined by sequence alignment based on a reference genome Sscofa 11.1 of a pig. The SNP molecular marker can be used for specifically identifying sand pigs, can realize variety identification, germplasm resource and kindred relation identification and analysis, protection and development of the germplasm resource and the like of the sand pigs, and has higher application value in a plurality of fields of sand pig breeding.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a sand pig SNP molecular marker, a chip prepared from the sand pig SNP molecular marker and application of the sand pig SNP molecular marker.
Background
Sand-kaolin pigs are the main groups of two black pigs in China and are listed in the directory of the national livestock and poultry genetic resource protection varieties, and have the excellent genetic characteristics of good hybridization effect, coarse feeding resistance, wide adaptability, strong disease resistance and the like. In addition, sand-ridge pigs have great medical development value, are most suitable for being used as pig xenograft donors in a plurality of local pig breeds, and are precious genetic resources. As local pig species, sand and pig are lack of a system and a perfect molecular detection means, and mainly conventional breeding and seed conservation are adopted. Therefore, the development of the liquid-phase chip product of the sand-green pig meets the development requirement of the sand-green pig industry at the present stage.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems in the prior art described above. Therefore, the invention provides a sand-ridge pig SNP molecular marker.
The invention also provides an SNP chip for detecting the sand ridge pig SNP molecular marker.
The invention also provides a kit for detecting the sand-kaolin pig SNP molecular marker.
The invention also provides application of the sand-ridge pig SNP molecular marker, SNP chip or kit.
The invention also provides a breeding method of the sand-ridge pigs.
According to one aspect of the invention, a sand-ridge pig SNP molecular marker is provided, comprising at least one of 410 SNP molecular markers, wherein the physical positions of the 410 SNP molecular markers are determined based on sequence alignment of a pig reference genome Srcrofa 11.1, and the site information is specifically as follows:
in some embodiments of the invention, the positional information of the SNP site is expressed in the form of chromosome number_physical position.
In a second aspect of the present invention, a sand pig SNP chip is provided, which comprises a primer set and/or a probe for detecting the sand pig SNP molecular marker.
In a third aspect of the present invention, a kit is provided, which comprises a primer set and/or a probe for detecting the sand-ridge pig SNP molecular marker.
In a fourth aspect of the invention, the application of at least one of the sand-ridge pig SNP molecular marker, the sand-ridge pig SNP chip and the kit is provided.
In some embodiments of the invention, the application may be specifically realized by the following method:
s1, genotyping a sample to be tested by adopting at least one of the sand-ridge pig SNP molecular marker, the sand-ridge pig liquid phase chip and the kit to obtain a genotyping result;
s2, analyzing the genotyping result obtained in the step S1.
In some embodiments of the invention, the use is in genotyping of sand-bearing pigs.
In some embodiments of the invention, the use is in whole genome association analysis of sand-kaolin pigs.
In some embodiments of the invention, the application is in cluster analysis and genetic relationship identification of sand-bearing pigs.
In some embodiments of the invention, the use is in the identification of genetic diversity of sand-lined pigs.
In some embodiments of the invention, the use is in the breeding or assisted breeding of sand-lined pigs.
In some embodiments of the invention, the breeding or assisted breeding comprises at least one of assisted major gene selection, molecular assisted breeding, whole genome selective breeding, sand-ridge pig breed identification, genetic map construction, gene localization, species evolution analysis, and germplasm resource identification.
In a fifth aspect of the present invention, a method for breeding a sand-lined pig is provided, comprising the steps of: detecting DNA of the pig to be detected by using at least one of the sand-ridge pig SNP molecular markers, the sand-ridge pig liquid phase chip and the kit, and selecting the sand-ridge pig for subsequent breeding.
In some embodiments of the invention, the detection is performed based on liquid phase probe capture sequencing typing techniques.
The invention has at least the following beneficial effects:
the SNP molecular marker can be used for specifically identifying sand-lined pigs, can realize variety identification, germplasm resource and kindred relation identification and analysis, protection and development of germplasm resources and the like of the sand-lined pigs, and has higher application value in a plurality of fields of sand-lined pig breeding.
The liquid-phase chip of the sand-green pig can be used for genotyping the sand-green pig in a low-cost and large-scale manner, is suitable for variety research of the sand-green pig, and is beneficial to development and research of sand-green pig germplasm resources. Based on the liquid phase chip principle, the site is flexible, and new functional marking sites can be added at any time in the later stage.
Drawings
The invention is further described with reference to the accompanying drawings and examples, in which:
FIG. 1 is a schematic diagram of the flow detection of a cGPS liquid phase chip in embodiment 3 of the present invention;
FIG. 2 is a statistical result of the site detection rate of 16 local pig samples according to example 4 of the present invention;
FIG. 3 is a graph showing the average uniformity of genotype of the repeated samples according to example 4 of the present invention;
FIG. 4 is a cluster typing result of example 5 of the present invention.
Detailed Description
The conception and the technical effects produced by the present invention will be clearly and completely described in conjunction with the embodiments below to fully understand the objects, features and effects of the present invention. It is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments, and that other embodiments obtained by those skilled in the art without inventive effort are within the scope of the present invention based on the embodiments of the present invention.
Example 1
The embodiment provides a sand-ridge pig SNP molecular marker, which comprises 410 SNP loci, wherein the physical positions of the 410 SNP loci are determined based on the alignment of the reference genome (Sscofa 11.1) sequences of pigs. The 410 SNP molecular markers are as follows:
the screening process is specifically as follows:
(1) Obtaining a set of SNP candidate positions from a whole genome resequencing source:
total genome re-sequencing was performed on 52 sand-producing pigs (from the Hunan Xiangtan market, sand-producing pig breeding farm) to obtain a rich and diverse whole genome locus. The library was sequenced on-machine using DNBSEQ-T7 high throughput sequencing platform of MGI. SNP gating was performed sequentially using fastp (v0.20.0) filtration according to the procedure, burows-Wheeler Aligner (0.7.12-r 1039) aligned to the pig reference group (Sscofa 11.1), picard 1.107 software (http:// www.psc.edu/index. Php/user-resources/software/picard) sequenced and converted to bam files, and PCR duplex was removed, GATK software was used for SNP detection and filtration, and the following filtration criteria were used based on genome data: QD <2.0 < FS >60.0 < MQ <35.0 < MQRankSum < -12.5 < Read PosRankSum < -8.0 < DP >6950. The vcf file containing all sample SNP variation information was obtained by filtering for a total of 7593680 SNP sites.
(2) Site selection:
the Fst value of each SNP variation site between the sand-and non-sand-ridge pig reference groups is calculated by using VCFtools-0.1.13, and 6104 sites are screened based on parameters that the Maf is more than or equal to 0.1 and the detection rate is more than or equal to 0.9, and are used for probe design. The design principle of the probe is as follows: probes are designed for all target sites within the range of about 100bp, the length of the probes is generally 100bp, and the GC content is between 20% and 80%. According to the design result of the probe, the probe which cannot be uniquely aligned on the genome and contains the repetitive sequence in the flanking sequence is removed. Finally, 410 candidate SNP loci with Fst >0.6 are obtained.
Example 2
The embodiment provides a sand-ridge pig liquid-phase chip, which is prepared by synthesizing a specific liquid-phase capture probe based on the SNP molecular marker of the embodiment 1 by Huazhi biotechnology limited company.
The accurate positioning sequencing typing technology (cGPS) is based on an optimized thermodynamic stability algorithm model, the genome sequences of different target areas are subjected to probe design, the synthesized specific probes are utilized to perform liquid-phase hybridization capture enrichment on a plurality of different target sequences positioned at different genome positions, then the target genome sequences subjected to capture enrichment are subjected to sequencing library construction and high-throughput sequencing, and thus genotypes of all SNP/InDel loci in the target areas are obtained.
Example 3
The embodiment provides a method for using the sand-ridge pig liquid-phase chip in the embodiment 2 in identifying sand-ridge pigs, which specifically comprises the following steps:
(1) Extraction and detection of gDNA:
collecting fresh blood of a pig to be tested, and extracting gDNA from the pig blood by a magnetic bead method. The integrity and purity of gDNA was analyzed by 1% agarose gel electrophoresis, and the concentration was accurately quantified using Qubit.
(2) cGPS experimental procedure
(2.1) taking 200ng of gDNA qualified in quantitative quality inspection, enzyme-cutting the gDNA into fragments with the size of 100-500 bp by using an enzyme-cutting reagent, and adding Taq enzyme for terminal repair;
(2.2) using T4 ligase to ligate the adaptor fragments to both ends of the DNA, purifying the ligation products using fragment sorting magnetic beads and amplifying the library to complete the library construction;
(2.3) placing the qualified library, the blocking reagent, the RNase inhibitor Rnase Block and 410 SNP liquid phase chip probes on a PCR instrument for hybridization reaction, and performing hybridization incubation at 55 ℃ for overnight (16-24 h);
(2.4) capturing the hybridization product by using streptavidin, amplifying and enriching the captured library, and sequencing PE150 by using a Huada sequencing platform;
(2.5) the raw data after high throughput sequencing is subjected to quality control filtration and the like, and the fastp software is used for removing the Reads containing the joint pollution and the Reads with low quality. And (3) comparing the BWA software with a reference genome (Sscofa 11.1), and then analyzing mutation sites of the sequencing result by using GATK software to obtain the genotyping result of the target site.
The flow chart is shown in fig. 1.
Example 4
The present example was based on the sand-kaolin pig SNP molecular markers provided in example 1 to detect 16 Hunan local pigs (including 4 parts of repeated samples, band-re) as 410 SNP liquid phase chip development system verification samples, including Guangyi black pig 2 heads (GYHZ-1, GYHZ-36), hunan black pig 1 head (XXHZ-48), temple front pig 1 head (SMQZ-5), east mountain pig 1 head (DSZ-6), sand-kaolin pig 2 heads (SZLZ-19, SZLZ-20), large girl pig 2 heads (DWZZ-19, DWZZ-3), ningsu pig 2 heads (NXZ-10, NXZ-23), qian Shaoxiang flower pig 1 head (QSHZ-1) to verify their effectiveness in detecting local pig DNA samples. The method comprises the following steps:
(1) Extraction and detection of gDNA:
12 parts of fresh blood of a local pig in Hunan province is collected, and gDNA is extracted from the pig blood by a magnetic bead method. The integrity and purity of gDNA was analyzed by 1% agarose gel electrophoresis, and the concentration was accurately quantified using Qubit.
(2) cGPS experimental procedure
(2.1) taking 200ng of gDNA qualified in quantitative quality inspection, enzyme-cutting the gDNA into fragments with the size of 100-500 bp by using an enzyme-cutting reagent, and adding Taq enzyme for terminal repair;
(2.2) using T4 ligase to ligate the adaptor fragments to both ends of the DNA, purifying the ligation products using fragment sorting magnetic beads and amplifying the library to complete the library construction;
(2.3) placing the qualified library, the blocking reagent, the RNase inhibitor Rnase Block and 410 SNP liquid phase chip probes on a PCR instrument for hybridization reaction, and performing hybridization incubation at 55 ℃ for overnight (16-24 h);
(2.4) capturing the hybridization product by using streptavidin, amplifying and enriching the captured library, and sequencing PE150 by using a Huada sequencing platform;
(2.5) the raw data after high throughput sequencing is subjected to quality control filtration and the like, and the fastp software is used for removing the Reads containing the joint pollution and the Reads with low quality. And comparing the BWA software with a target genome, and analyzing the mutation site of the sequencing result by using GATK software to obtain the genotyping result of the target site.
Through sequencing and data analysis, the detection rate of 16 sample sites is between 99.02% and 99.76%, and the average detection rate is 99.27%. The results are shown in FIG. 2.
In the consistency comparison, if one of the two results at a site is missing, the site does not account for consistency statistics. Genotype results of 4 duplicate samples (i.e., labeled "-re" in fig. 2) were aligned with an average concordance rate of 100.00%. The results are shown in FIG. 3.
The result shows that the sand ridge pig SNP molecular marker provided in the embodiment 1 has good stability, high detection rate of target sites and accurate and reliable typing result.
Example 5
The test example is based on 403 parts of sand-kaolin pig SNP molecular markers provided in the example 1 for carrying out group structure analysis on different local pigs in Hunan, including Guangyi black pig 51, hunan black pig 50, temple front pig 51, east mountain pig 46, sand-kaolin pig 52, large girl pig 50, ningxiang pig 54 and Qian Shao Hua pig 49.
The sand-green pig SNP molecular markers provided in example 1 are effective in distinguishing sand-green pigs of different sources from other local pig breeds. As shown in fig. 4. Consistent with the expected results.
The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of one of ordinary skill in the art without departing from the spirit of the present invention. Furthermore, embodiments of the invention and features of the embodiments may be combined with each other without conflict.
Claims (10)
1. The sand-ridge pig SNP molecular marker is characterized by comprising at least one of 410 SNP molecular markers, wherein the physical positions of the 410 SNP molecular markers are determined based on sequence alignment of a pig reference genome Sscofa 11.1, and the site information is specifically as follows:
。
2. a sand-pig SNP chip comprising a primer set and/or a probe for detecting the sand-pig SNP molecular marker of claim 1.
3. A kit comprising a primer set and/or a probe for detecting the sand-ridge pig SNP molecular markers described in claim 1.
4. Use of at least one of the sand-ridge pig SNP molecular markers as defined in claim 1, the sand-ridge pig SNP chip as defined in claim 2 and the kit as defined in claim 3 for genotyping sand-ridge pigs.
5. Use of at least one of the sand-pig SNP molecular markers as defined in claim 1, the sand-pig SNP chip as defined in claim 2 and the kit as defined in claim 3 for whole genome association analysis of sand-pigs.
6. Use of at least one of the sand-ridge pig SNP molecular markers as defined in claim 1, the sand-ridge pig SNP chip as defined in claim 2 and the kit as defined in claim 3 in cluster analysis and genetic relationship identification of sand-ridge pigs.
7. Use of at least one of the sand-pig SNP molecular markers as defined in claim 1, the sand-pig SNP chip as defined in claim 2 and the kit as defined in claim 3 for the identification of genetic diversity of sand-pigs.
8. Use of at least one of the sand-lined pig SNP molecular markers as defined in claim 1, the sand-lined pig liquid phase chip as defined in claim 2 and the kit as defined in claim 3 for breeding or assisted breeding of sand-lined pigs.
9. The use of claim 8, wherein the breeding or assisted breeding comprises at least one of assisted major gene selection, molecular assisted breeding, whole genome selective breeding, sand-ridge pig breed identification, genetic map construction, gene localization, species evolution analysis, and germplasm resource identification.
10. The breeding method of the sand-ridge pig is characterized by comprising the following steps of: detecting DNA of a pig to be detected by using at least one of the sand-ridge pig SNP molecular markers of claim 1, the sand-ridge pig liquid-phase chip of claim 2 and the kit of claim 3, and selecting the sand-ridge pig for subsequent breeding.
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Application publication date: 20240220 |