CN108359736B - SNP genetic marker for sow limb hoof bone density character - Google Patents
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Abstract
The invention belongs to the technical field of animal molecular marker screening, and particularly relates to an SNP genetic marker for sow limb hoof bone density traits. The molecular marker is cloned from a gene segment with the accession number of ALGA0082045, and the gene is subjected to typing screening by a gene chip technology to obtain the molecular marker related to the density of the bones of the sow hooves, wherein the nucleotide sequence of the marker is shown as SEQ ID NO:1, there is an allelic mutation of C/T at the 51 st base of the sequence, resulting in SEQ ID NO:1 and when the nucleotide at position 51 of said sequence is C, the mare's hoof has a higher bone density. The invention also discloses a screening method of the genetic marker related to the sow limb ungula bone density character and application of the genetic marker in association analysis.
Description
Technical Field
The invention relates to the technical field of pig genetic marker preparation, in particular to an SNP genetic marker for sow limb hoof bone density traits. The genetic marker can be used for predicting the pig limb hoof bone density character.
Background
With the development of science and technology, the pig breeding industry makes significant breakthrough in the aspects of genetic breeding, nutrition regulation, disease prevention and control and the like, and the lean meat percentage, the feed-meat ratio and the growth speed of commercial pigs are greatly improved. However, the intensive breeding is accompanied by the increasingly prominent limb and hoof problem, the range of motion of the pig herds is limited for intensive production, and the limb and hoof disease of the pigs is further aggravated by the design of the stall (slotted floor). It was reported that 20-50% of all replacement boars passing the performance measurement were rejected due to weak limb hooves, and that sows rejected due to weak limb hooves among the production sows were as high as 6.1-15% (Zhang Heng, association of site function candidate genes HMGA1, C6orf106 and NSSSCG00000023160 with pig limb hoof firmness [ J ] Chinese agricultural science, 2016,49(20): 4030-. In addition, due to the limitation of production cost, once the pig limb hoofs have problems, only a few of the pig limb hoofs can be cured (Guo Chun, shallow talk about the causes of the pig limb hoof diseases and prevention measures [ J ] farmer leading to rich consultants, 2017(16):20-20), and most of the pig limb hoofs adopt elimination measures, thereby causing huge economic loss to the pig breeding industry.
The crux of the limbs and hoofs is the comprehensive reaction of the health degree of the limbs and hoofs and the coordination degree of the movement of the limbs, but for the convenience of the study, the crux of the opposite face is generally measured by the weakness (leg weakness) of the limbs and hoofs. Generally speaking, the crura solidness can be measured in terms of a plurality of aspects such as the hoof score, the gait score, the leg type score, the bone mineral content and bone density, the osteomalacia score, the articular cartilage score and the length of the biceps brachii (beautiful silk, etc., the progress of genetic analysis of the crura solidness of pigs, pig industry science 2013: 4-7). Bone density is an important reference standard for the diagnosis of osteoporosis in humans (dawn, bone density measurement and osteoporosis diagnosis [ J ]. International J. endocrine metabolism, 2005,25(5):308- & 310). In addition, it has been reported that Bone Mineral Density (BMD) can reflect 58-70% of Bone strength (Caochai Wei et al, application and evaluation of quantitative ultrasound and dual-energy Bone Density measurement in osteoporosis diagnosis [ J ]. China journal of osteoporosis, 2001,7(2): 110-. Therefore, the bone density is an important index in the research of animal bone strength, but due to the limitation of various production conditions, the research of livestock bones is very rare.
Bone Density, i.e. the content of bone mineral per unit volume, in g/cm3Common methods for measuring bone density include radioabsorption, dual-energy X-ray, quantitative computed tomography, and axial ultrasound propagation (Caojiao, bone Density (BMD) ("J")]The dual energy X-ray measurement and quantitative computed tomography techniques are most widely used, but the dual energy X-ray measurement technique measures the bone density in area, influenced by the geometric volume of the object, while the quantitative computed tomography technique can measure the bone density of animals more accurately, but the equipment cost is expensive (liu jade, dual energy X-ray bone densitometer and quantitative CT comparative studies on bone density measurement [ D]Zhong nan university, 2005) and is cumbersome to manipulate (after anesthesia is required for large livestock), greatly limiting its use in animal bone density studies. The ultrasonic bone-axis propagation technique is a bone-density measuring technique which has been developed in recent years, can measure not only the bone density of animals but also the strength and the bone structure of the bones of the animals to a certain extent, and has the advantages of simplicity, no radiation damage, high repetition accuracy, low price, and portability (SUZUO et al, introduction of bone-density nondestructive measuring method and recent progress thereof [ J J.]Chinese medical equipment, 2009,24(1): 49-51). Therefore, the pig limb hoof bone density is measured by using an ultrasonic bone axis propagation technology, and the SNP genetic marker related to the middle limb hoof bone density is screened by using GWAS, so that a feasible way is provided for genetic selection of the pig limb hoof traits, and the pig limb hoof genetic marker has important significance for the pig breeding industry.
The correlation between the SNP and the pig leg hoof bone density character reaches a remarkable level, and a new genetic resource is provided for the research of the pig leg hoof compactness character.
Disclosure of Invention
The invention aims to perfect the disease-resistant breeding genetic marker of the domestic pig, utilize a 50K gene chip to carry out typing on SNP, and use GWAS to screen SNP related to the sow limb hoof, thereby providing new genetic marker resources and application for disease-resistant breeding of the pig.
The technical scheme of the invention is as follows:
the applicant obtains the upstream and downstream 50bp nucleotide sequences with the login number of ALGA0082045 single nucleotide mutation by a genotyping technology and referring to an Ensembl database, and the sequences are shown as SEQ ID NO. 1. The details are as follows:
GCTTAGGCCAAAAAGATAATTTATAGCTATATGTTATTGGAGAGTTCAAGR(T/C)AAGGCTGGATCCAGGGGTTCGGATAATGTCATCAGGAAAGTGTCTGTTTC
r at the 51bp base of the sequence is an allelic mutation of T51-C51, which makes the nucleotide sequence shown in SEQ ID NO:1 sequence generates nucleotide polymorphisms. The genetic marker can be used as a genetic marker for detecting the pig limb hoof bone density, and when the nucleotide sequence shown in SEQ ID NO: when the 51 st nucleotide in 1 is C, the pig limb hoof has higher bone density.
The sequence can be used as a genetic marker for detecting the character related to the pig bone density.
The applicant provides a method for screening a pig bone density trait related SNP genetic marker, which comprises the following steps:
extracting genome DNA of pig ear tissue by a conventional method, and carrying out quality detection on the DNA.
Secondly, the gene chip technology is utilized to carry out typing (the concrete steps are described in the specification at the end).
Thirdly, adopting a method based on a single-mark correlation regression model, taking individual gender as a fixed effect, and carrying out GWAS analysis by utilizing an enaBEL software package under the R statistical environment. The specific regression model is as follows: y ═ Xb + Sa + Zu + e, where Y represents the "vector of phenotypes" (the vector of phenotypes); b represents "mean value of estimated and phenotypic values μ" of fixation effects (including gender); α represents "substitution effect of SNP"; u represents a "random additive genetic effect"; subject to a multidimensional normal distribution, u-N (0, G σ α)2) G denotes the genome similarity matrix (based on SNP markers), G denotes the polygenic additive variance (from which the heritability is estimated); x, S, Z are incidence matrices (affinity matrix) of b, α, u, respectively; e represents a "residual vector"(a vector of residual errors) following a normal distribution, e-N (0, I σ e)2) And represents the residual variance.
The genetic marker screened by the invention can be used for non-diagnosis purpose correlation analysis between the genotype of the pig limb hoof bone density related gene or the bone density in the pig limb hoof character, and provides a new genetic marker resource for the genetic marker-assisted selection of the pig bone density character.
Compared with the prior art, the invention has the following beneficial effects:
the invention can detect the genotype of the pig by adopting a gene chip technology in vitro, is used for evaluating the acra and hoof firmness of the pig for non-diagnosis purpose, and has the outstanding advantages of simplicity, rapidness, high sensitivity, good specificity and the like compared with the prior PCR-RFLP and other methods.
For a more detailed technical solution, refer to the embodiments in the description of the drawings and the detailed description of the invention.
Drawings
Sequence listing SEQ ID NO:1 is the upstream and downstream 50bp nucleotide sequence of the genetic marker (i.e. the gene fragment with the login number of ALGA 0082045) screened by the invention. This fragment is the genetic marker screened by the present invention. The sequence length is 101bp, and R at 51 base of the sequence has a T/C allele mutation.
FIG. 1: the general technical process schematic diagram of the invention.
FIG. 2: is the upstream and downstream 50bp nucleotide sequence of the cloned ALGA0082045 gene and the nucleotide sequence of the genetic marker of the invention. Description of reference numerals: a T/C allele mutation is present at the 51 st base of the nucleotide sequence shown in FIG. 2 (i.e., the sequence shown in SEQ ID NO:1 of the sequence Listing) (the English letter "R" at the 51 st base of the sequence is a mutation site).
FIG. 3: is a manhattan diagram of the present invention. Description of reference numerals: the black circles and arrows indicate the genetic markers selected by the present invention, which are located on pig chromosome 14.
Detailed Description
Example 1: genotyping assay
(1) Method for extracting ear tissue DNA of population related to bone density by using kit
1) Cutting ear-like tissue of bone density related group (the group comprises rhizoma Bletillae of Danish (Denmark) and Changbai, which is a conventional variety and provided by Guangxi Yangxiang GmbH) into paste with ophthalmic scissors, adding 200ul GA (provided by kit), and shaking for mixing; then put into a 56 ℃ water bath kettle to be digested overnight.
2) The digested tissue sample was added to 200ul of buffer GB (kit-in-one), mixed well by inversion, and left at 70 ℃ for 10 minutes, the solution cleared and centrifuged briefly to remove water droplets on the tube wall.
3) 200ul of absolute ethanol was added, mixed well for 15 seconds with sufficient shaking, at which time a flocculent precipitate may appear, and centrifuged briefly to remove water droplets on the tube wall.
4) Adding the solution and flocculent precipitate into adsorption column CB3, placing the adsorption column into a collecting tube, centrifuging at 12000rpm for 30s, pouring off waste liquid, and placing the adsorption column back into the collecting tube.
5) 500ul of buffer GD (kit-in-kit) was added to adsorption column CB3, centrifuged at 12000rpm for 30s, the waste liquid was decanted, and the adsorption column was placed in a collection tube.
6) 600ul of the rinsing solution PW (kit tape by itself) was added to the adsorption column CB3, centrifuged at 12000rpm for 30s, the waste liquid was decanted, and the adsorption column was placed in a collection tube, and the procedure was repeated.
7) The adsorption column CB3 was left at room temperature for several minutes to completely dry the rinse remaining in the adsorption column material.
8) Transferring the adsorption column CB3 into a clean centrifuge tube, suspending and dripping 50-200ul of eluent TE into the middle part of the adsorption column membrane, standing for 2-5 minutes at room temperature, centrifuging for 2 minutes at 12000rpm, and collecting the solution into the centrifuge tube.
(2) SNP genotype determination and quality control
Typing was performed using a 50K gene chip (GeneSeek Porcine 50K SNP chip) and quality testing was performed on the genotype data, with 895 individuals and 48909 SNPs finally being used for genome-wide analysis (GWAS) studies.
Example 2: application of genetic marker screened by the invention and a typing method thereof in pig limb hoof bone density character correlation analysis (1) correlation analysis of genetic marker typing result and limb hoof bone density character
The experimental pig group used for detecting and analyzing the correlation between the genotype and the bone density character of the limb hoof is from a pure sow group cultivated by Yang Xiang GmbH in Guangxi, including a large white pig and a long white pig (which are conventional varieties). The DNA used for genotyping is extracted from ear samples of pure white pigs and long white pigs (called as "pure white and long white" in the text and table of the specification and simply called as "pigs"). The GWAS analysis is carried out by adopting a method based on a single-marker association regression model, adopting the sex of an individual as a fixed effect and using a GenABEL software package under an R statistical environment. The specific regression analysis model is as follows: y ═ Xb + Sa + Zu + e
Wherein: y represents a "vector of phenotyps" (the vector of phenotyps); b represents "mean value of estimated and phenotypic values μ" of fixation effects (including gender); α represents "substitution effect of SNP"; u represents a "random additive genetic effect"; subject to a multidimensional normal distribution, u-N (0, G σ α)2) G represents a genome similarity matrix (based on SNP markers), G represents a polygenic additive variance (estimation of heritability by this variance); x, S, Z are incidence matrices (affinity matrix) of b, sigma, u, respectively; e represents a "residual vector" (a vector of residual errors) obeying a normal distribution, e-N (0, I σ e)2) And represents the residual variance.
The correlation analysis results are shown in table 1.
TABLE 1 results of bone density detection of sows of different genotypes by using ALGA0082045 gene polymorphism
Table 1 illustrates: p <0.05 is significantly different; p <0.01 is very significantly different.
As can be seen from Table 1, the bone density of the hoof and limb of the individual with the genotype CC is significantly higher than that of the individual with CT, so C is an allele which is favorable for the increase of the bone density.
Appendix: description of the Gene chip technology:
the invention uses the Illumina company to develop GeneSeek Porcine 50K SNP chip scanning, the chip contains more than 50000 SNP sites, and covers the genome of pigs. The chip integrates gene differences of a plurality of pigs, including Duroc pigs, Changbai pigs, Pietrain pigs and big white pigs, can provide enough SNP density, and can be applied to genome-wide association analysis (GWAS).
The main references:
[1] zhang Xuanwei, relevance of position function candidate genes HMGA1, C6orf106 and ENSSSCG00000023160 and the compactness of pig limb and hoof [ J ]. Chinese agricultural science, 2016,49(20): 4030-4039;
[2] guo Chunyang, who is a short talk about the causes of the diseases of the limbs and hooves of sows and preventive measures [ J ], farmer's rich consultant, 2017(16) 20-20;
[3] the genetic analysis progress of the pig limb and hoof firmness degree through the beautiful silk waiting and the like, pig industry science 2013: 94-7;
[4] dawn light, bone density measurement and osteoporosis diagnosis [ J ]. International journal of endocrine metabolism, 2005,25(5): 308-;
[5] caochilawei et al, application and evaluation of quantitative ultrasound and dual-energy bone density measurement in osteoporosis diagnosis [ J ], China journal of osteoporosis, 2001,7(2): 110-;
[6] application evaluation of Caojia, bone Density (BMD) assay [ J ] pharmaceutical frontier, 2013 (2);
[7] liuyu, comparative study of bone density measurement by a dual-energy X-ray bone densitometer and quantitative CT [ D ], Zhongnan university, 2005;
[8] SUGAO et al, introduction to the non-destructive determination of bone density and its latest developments [ J ], Chinese medical devices, 2009.
Sequence listing
<110> university of agriculture in Huazhong
<120> SNP genetic marker for sow limb and hoof bone density character
<141> 2018-05-02
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 101
<212> DNA
<213> pig (sus scrofa)
<220>
<221> gene
<222> (1)..(101)
<220>
<221> mutation
<222> (51)..(51)
<400> 1
gcttaggcca aaaagataat ttatagctat atgttattgg agagttcaag caaggctgga 60
tccaggggtt cggataatgt catcaggaaa gtgtctgttt c 101
Claims (1)
1. The application of the SNP genetic marker in the marker-assisted selection of the pig leg and hoof bone density traits is characterized in that the sow breeds are Dan line white pigs and long white pigs, the leg and hoof bone density is the leg and hoof firmness, and the nucleotide sequence of the genetic marker is as follows:
GCTTAGGCCAAAAAGATAATTTATAGCTATATGTTATTGGAGAGTTCAAGRAAGGCTGGATCCAGGGGTTCGGATAATGTCATCAGGAAAGTGTCTGTTTC，
r at the base 51 of the sequence is C or T, the density of the foot and limb bone of an individual with the genotype CC is obviously higher than that of the individual with the genotype CT, and C is an allele which is favorable for increasing the bone density.
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Citations (4)
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|---|---|---|---|---|
| WO2001042511A2 (en) * | 1999-12-10 | 2001-06-14 | Whitehead Institute For Biomedical Research | Ibd-related polymorphisms |
| WO2002101022A2 (en) * | 2001-06-13 | 2002-12-19 | Invitrogen Corporation | Method for rapid amplification of dna |
| CN107988388A (en) * | 2017-12-11 | 2018-05-04 | 华南农业大学 | A kind of and relevant SNP marker of pig dewclaw undue growth character and application thereof |
| CN108004332A (en) * | 2017-12-20 | 2018-05-08 | 华南农业大学 | A kind of molecular labeling for influencing the main hoof growth of pig and its application |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001042511A2 (en) * | 1999-12-10 | 2001-06-14 | Whitehead Institute For Biomedical Research | Ibd-related polymorphisms |
| WO2002101022A2 (en) * | 2001-06-13 | 2002-12-19 | Invitrogen Corporation | Method for rapid amplification of dna |
| CN107988388A (en) * | 2017-12-11 | 2018-05-04 | 华南农业大学 | A kind of and relevant SNP marker of pig dewclaw undue growth character and application thereof |
| CN108004332A (en) * | 2017-12-20 | 2018-05-08 | 华南农业大学 | A kind of molecular labeling for influencing the main hoof growth of pig and its application |
Non-Patent Citations (5)
| Title |
|---|
| Field study of hind limb claw lesions and claw measures in sows;Yosuke Sasaki等;《Animal Science Journal》;20141204;第86卷(第3期);第351-357页 * |
| rs80886555;Ensembl release 92;《EMBL-EBI》;20180430;第1-2页"Flanking sequence" * |
| 位置功能候选基因 HMGA1、C6orf106和 ENSSSCG00000023160与猪肢蹄结实度的关联性;张徐非等;《中国农业科学》;20161021;第49卷(第20期);第4030-4039页 * |
| 在四个实验猪群中利用全基因组关联分析及其荟萃分析定位影响四肢骨骼长度的基因位点;侯利娟;《中国优秀硕士学位论文全文数据库农业科技辑》;20170315(第03期);D050-223 * |
| 猪肢蹄结实度评估方法的研究进展;马官军等;《猪业科学》;20171231;第34卷(第12期);第117-118页 * |
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