CN111560421B - Detection method and application of marker of HMGA2 gene Indel of cattle in south of summer - Google Patents

Detection method and application of marker of HMGA2 gene Indel of cattle in south of summer Download PDF

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CN111560421B
CN111560421B CN202010550697.5A CN202010550697A CN111560421B CN 111560421 B CN111560421 B CN 111560421B CN 202010550697 A CN202010550697 A CN 202010550697A CN 111560421 B CN111560421 B CN 111560421B
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陈宏�
曹修凯
程杰
王晓刚
黄永震
蓝贤勇
雷初朝
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Abstract

The invention discloses a detection method and application of a summer cattle HMGA2 gene Indel mark; the genome DNA of the summer south cattle is used as a template, the partial fragment of the HMGA2 gene of the summer south cattle is amplified by PCR, and 7bp insertion deletion mutation sites of the HMGA2 gene chr5:48094736-48094742 of the individual summer south cattle are rapidly typed by agarose gel electrophoresis; the detection method can screen and detect molecular genetic markers closely related to the south-summer cattle body on the DNA level, and is applied to the auxiliary selection of the molecular markers of the south-summer cattle body, so that the breeding process of the south-summer cattle variety improvement is quickened.

Description

Detection method and application of marker of HMGA2 gene Indel of cattle in south of summer
Technical Field
The invention belongs to the field of livestock molecular breeding, and relates to a method for detecting a high-related HMGA2 gene Indel marker of a summer south cattle body and application thereof.
Background
The summer south cattle is a special beef cattle breeding variety formed by taking a summer solenoidal cattle as a male parent and carrying out open breeding in three stages of hybridization, cross fixation and self-group breeding. The cattle in summer has strong physique and temperament, the body of the adult cattle is rectangular, the structure is symmetrical, the chest is deep and round, the back and waist are flat, the length of the long and wide jirime part is long, the tail is slender, the limbs are thick, and the meat characteristics are obvious. Adult bull (142.5+ -8.5) cm, weight 850kg; adult cows have a height (135.5.+ -. 9.2) cm and a weight of 600kg. The summer and south cattle are resistant to coarse feeding, strong in adaptability, can be raised in barn feeding and grazing, can be raised in agricultural areas and semi-agricultural and pasture areas, and has the characteristics of fast growth and development and easiness in fattening.
Indel mutations (indels) refer to differences in the entire genome between two genotypes, with a certain number of nucleotide insertions or deletions in the genome of one genotype relative to the other genotype, a type of co-dominant marker. Indels are widely distributed in the genome, dense, numerous, far higher than SSR (simple sequence repeats) markers. SNP markers have great theoretical potential, but detection methods and costs are generally higher than indels. Indel is most to utilize electrophoresis platform to carry out the typing at present, electrophoresis typing platform does not need complicated experimental facilities, and typing is quick, accurate, economical. Indel, a molecular marker, has been widely used in molecular marker-assisted selection (molecular mark-assisted selection, MAS) of livestock and poultry. MAS is the genetic improvement of accelerating the comprehensive characters of livestock and poultry varieties by selecting genetic resources or breeding materials by means of DNA molecular markers.
The HMGA family includes four members, HMGA1a, HMGA1b, HMGA1c, and HMGA2, all of which are produced by variable cleavage of the HMGA1 gene. The HMGA family members all contain three AT hooks AT the N end, can be combined with DNA minor groove rich in AT sequences, induce chromatin conformation change, recruit transcription complex and promote gene transcription, so that the HMGA2 gene can be used as a candidate gene for bovine growth and development. At present, a Single Nucleotide Polymorphism (SNP) locus rs29016809 which is obviously related to Siemens cattle body height has been identified in the HMGA2 gene intron 3, but mutation of the locus has no phenotypic effect in summer south cattle.
Disclosure of Invention
The invention aims to provide a detection method and application of a summer south cattle HMGA2 gene Indel marker, so that the breeding process of the summer south cattle variety improvement is quickened.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a detection method of the HMGA2 gene indel mutation of the cattle in the south of summer comprises the following steps:
the genome DNA of the individual of the south-summer cattle to be detected is used as a template, a primer pair P is used as a primer, the partial fragment of the HMGA2 gene of the south-summer cattle is amplified by PCR, agarose gel electrophoresis is carried out on the PCR amplified product, and the genotype of the HMGA2 gene indel mutation site (bovine HMGA2 gene chr5:48094736-48094742 (UMD3.1.1)) of the individual of the south-summer cattle is identified according to the agarose gel electrophoresis result.
Preferably, the primer pair P is:
upstream primer F1:5'-CAGGTGGTGTTCAGATCTGCTCTG-3';
downstream primer R1:5'-CAGGTTGTCTGAGGTCCATCG-3'.
Preferably, the amplification system used for PCR is 25. Mu.L: 50 ng/. Mu.L of template DNA, 1.0. Mu.L of 10pmol/L of each of the upstream and downstream primers of primer pair P, 2X Taq PCR Master Mix. Mu.L, and 9. Mu.L of deionized water.
Preferably, the amplification reaction procedure used for the PCR is: pre-denaturation at 95 ℃ for 5min; denaturation at 94℃for 30s, annealing at 58℃for 30s, elongation at 72℃for 20s,35 cycles; extending at 72℃for 10min.
Preferably, the agarose gel electrophoresis adopts agarose gel with the mass concentration of 4 percent.
Preferably, the genotype of the obtained summer south cattle HMGA2 gene insert deletion mutation site (cattle HMGA2 gene chr5:48094736-48094742 (UMD3.1.1)) can be identified according to agarose gel electrophoresis result as follows: insertion/insertion (II) genotype appears as a band of 112 bp; insertion/deletion (ID) genotypes appear as two bands of 112bp and 105 bp; the deletion/deletion (DD) genotype appears as a 105bp band.
A kit for detecting the insertion deletion mutation of the HMGA2 gene of the cattle in the south of summer comprises the primer pair P.
The beneficial effects of the invention are as follows:
the invention can carry out rapid typing detection on the deletion mutation site (chr 5:48094736-48094742 (UMD3.1.1)) inserted into the HMGA2 gene of the summer south cattle through PCR amplification and agarose gel electrophoresis, and the detection result can obtain a molecular genetic marker (Indel marker) which is closely related to the high body of the summer south cattle and is screened and detected on the DNA level. The invention can be applied to molecular marker assisted selection of the south China cattle, thereby accelerating the breeding process of the south China cattle variety improvement.
Compared with the prior art, the invention has the following advantages:
(1) The method for detecting the HMGA2 gene indels of the summer south cattle is not limited by age, can be used for early breeding of male cows, and can be selected even when the male cows are born;
(2) The method for detecting the cattle HMGA2 gene indel mutation is accurate and reliable and is simple and convenient to operate;
(3) Detection of the cattle HMGA2 gene insertion deletion site (chr 5:48094736-48094742 (UMD3.1.1)) provides scientific basis for molecular marker assisted selection of cattle growth and development.
Drawings
FIG. 1 shows the result of electrophoresis of amplification products of insertion deletion sites (chr 5:48094736-48094742 (UMD3.1.1) 7bp insertion deletion mutation sites) of samples detected by cattle in the south of summer in the examples of the present invention.
Detailed Description
The invention will be described in further detail with reference to the drawings and examples. The examples are given solely for the purpose of illustration and are not intended to limit the scope of the invention.
(one) HMGA2 gene Indel marker related to Calf
The invention screens and discovers the Indel site of the HMGA2 gene (intron 3) of cattle in south of summer, namely chr5:48094736-48094742 (UMD3.1.1), and sorts the Indel site; performing association analysis on Indel types and higher traits; finally, the molecular genetic markers at Indel sites are used for early breeding of the high traits of the cattle in the south of summer.
(II) detection of marker of HMGA2 gene Indel of summer south cattle and application thereof
1. Summer south cow sample collection
The invention specifically takes the south-summer cattle as a detection object, and a blood sample of 224 south-summer cattle is collected from the south-summer cattle scientific and technological development Co., ltd in the Xueyang county of the standing-marry of Henan province in the month of 2016.
2. Isolation, extraction and purification of genomic DNA
Reference sambroock et al (2002).
3. Amplification of target sequences
PCR primers (see Table 1) were designed using the published bovine HMGA2 gene sequence (GenBank AC 000162.1) of NCBI database (http:// www.ncbi.nlm.nih.gov /) as reference sequences using Primer 5.0 for amplifying the HMGA2 gene Indel site (chr 5:48094736-48094742 (UMD3.1.1)), and identification of Indel types could be performed using PCR and agarose gel electrophoresis.
TABLE 1 PCR primer information
Figure BDA0002542406850000031
Wherein, the amplification system used for PCR is calculated as 25. Mu.L: 50 ng/. Mu.L of template DNA, 1.0. Mu.L of 10pmol/L of each of the upstream and downstream primers of primer pair P, 2X Taq PCR Master Mix. Mu.L, and 9. Mu.L of deionized water.
The amplification reaction procedure used for PCR was: pre-denaturation at 95 ℃ for 5min; denaturation at 94℃for 30s, annealing at 58℃for 30s, elongation at 72℃for 20s,35 cycles; extending at 72℃for 10min.
4. Electrophoresis detection and genotype determination
The PCR amplification products of each individual were subjected to 4% agarose gel electrophoresis, and bands corresponding to the insertion/insertion (II), insertion/deletion (ID) and deletion/deletion (DD) types were observed (see FIG. 1), and the results of Indel type statistical analysis are shown in Table 2-1.
TABLE 2-1 distribution of HMGA2 Gene Indel locus Gene frequency in summer south cattle
Figure BDA0002542406850000041
5. Correlation analysis of summer south cattle HMGA2 gene Indel locus and body size character
Genotype data: genotypes identified (II, ID and DD).
Body size trait data: high, chest and abdomen circumference.
Correlation analysis model: firstly, carrying out description analysis on data, determining whether outliers exist or not, and correcting the data by utilizing least square analysis; based on the data characteristics, SPSS 19 software was used to analyze phenotypic effects between genotypes. A fixed model was used in the analysis of genotype effects:
Y ij ＝μ+G i +e ij
wherein: y is Y ij As observed values of the characters, mu is the overall mean value, G i For the fixed effect of the ith genotype, e ij Is a random error. The variability between the data sets was tested using LSD multiplex comparison and the test results are expressed in mean+ -SD.
Additive effect:
Figure BDA0002542406850000042
dominant effect:
Figure BDA0002542406850000043
TABLE 2-2 correlation analysis of HMGA2 Gene Indel loci with California cattle height
Figure BDA0002542406850000051
The correlation analysis results showed (see Table 2-2): the HMGA2 gene Indel locus obviously affects the height of the cattle in summer, the dominant genotype is DD type, and the height of DD type individuals is obviously higher than that of II and ID type individuals. The DD type of HMGA2 gene Indel locus (chr 5:48094736-48094742 (UMD3.1.1)) can be used as a molecular marker (Indel marker) for breeding of cattle in summer south.
6. Application of Indel marker in breeding of summer south cattle
According to the analysis results of tables 2-1 and 2-2, in the auxiliary selection of early molecular markers, individuals of II and ID types are eliminated, and the DD type individuals are reserved for propagation. According to the propagation result, DD type individual can form a large excellent summer south cattle population, thereby accelerating the breeding process of the improvement of the summer south cattle variety.
<110> university of agriculture and forestry science and technology in northwest
<120> detection method and application of marker of HMGA2 gene Indel of cattle in south of summer
<160>2
<210>1
<211>24
<212>DNA
<213> Synthesis
<400>1
caggtggtgt tcagatctgc tctg 24
<210>2
<211>21
<212>DNA
<213> Synthesis
<400>2
caggttgtct gaggtccatc g 21

Claims (7)

1. The application of the detection method of the HMGA2 gene indel mutation of the south-summer cattle in molecular breeding of the south-summer cattle is characterized in that: the method for detecting the insertion deletion mutation of the HMGA2 gene of the summer south cattle comprises the following steps:
taking genomic DNA of the summer south cattle to be detected as a template, amplifying a portion of the HMGA2 gene fragment of the summer south cattle by PCR, performing agarose gel electrophoresis on the amplified product, and identifying the genotype of the HMGA2 gene of the summer south cattle inserted into the deletion mutation site according to the agarose gel electrophoresis result; the indel mutation site is positioned in the bovine HMGA2 gene chr5:48094736-48094742.
2. The use according to claim 1, characterized in that: the amplification primers of the PCR are as follows:
an upstream primer: 5'-CAGGTGGTGTTCAGATCTGCTCTG-3';
a downstream primer: 5'-CAGGTTGTCTGAGGTCCATCG-3'.
3. The use according to claim 1, characterized in that: the amplification system of the PCR included 50 ng/. Mu.L of template DNA 1.0. Mu.L and 10 pmol/. Mu.L of each of the upstream and downstream primers.
4. The use according to claim 1, characterized in that: the PCR amplification reaction program is as follows: pre-denaturation at 95 ℃ for 5min; denaturation at 94℃for 30s, annealing at 58℃for 30s, elongation at 72℃for 20s,35 cycles; extending at 72℃for 10min.
5. The use according to claim 1, characterized in that: the agarose gel electrophoresis adopts agarose gel with the mass concentration of 4 percent.
6. The use according to claim 1, characterized in that: the genotype of the indel mutation site is: the insertion/insertion type appears as a band of 112 bp; the insertion/deletion forms are represented by two bands of 112bp and 105 bp; the deletion/deletion pattern appears as a 105bp band.
7. The use according to claim 1, characterized in that: in the early molecular marker assisted selection, individuals with the genotype of the indel mutation site as a deletion/deletion form a large summer south cattle population.
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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1643162A (en) * 2002-03-15 2005-07-20 衣阿华州立大学研究基金公司 Novel HMGA alleles and use of the same as genetic markers for growth, fatness, meat quality and feed efficiency traits

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1643162A (en) * 2002-03-15 2005-07-20 衣阿华州立大学研究基金公司 Novel HMGA alleles and use of the same as genetic markers for growth, fatness, meat quality and feed efficiency traits

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Association of Copy Number Variation at Intron 3 of HMGA2 With Navel Length in Bos indicus;Aguiar TS等;《Front. Genet.》;20181207;第9卷;全文 *
rs524674056;Ensembl release 100;《Ensembl数据库》;20200430;序列 *

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