CN113943821B - Insertion deletion marker related to FGF7 gene and goat growth traits and application thereof - Google Patents
Insertion deletion marker related to FGF7 gene and goat growth traits and application thereof Download PDFInfo
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Abstract
The invention disclosesFGF7An indel marker related to goat growth character in the gene and application thereof. The indel marker is located in goatFGF7The nucleotide sequence of the second intron region of the gene is shown as SEQ ID NO.3, the 93 rd to 114 th insertions or deletions are arranged at the 5' end, and the nucleotide sequence of the insertion or deletion fragment is shown as SEQ ID NO. 4. The indel marker provided by the invention can be used in the field of goat growth trait molecular marker assisted breeding, is used for screening goat growth traits, provides a theoretical basis and a genetic basis for breeding or cultivating goat varieties with excellent growth traits, is beneficial to improving the breeding efficiency and accelerating the goat breeding process.
Description
Technical Field
The invention relates to the technical field of goat molecular marker screening, in particular to a goat molecular marker screening methodFGF7Indel markers associated with the growth traits of the gene and goat and application thereof.
Background
The goat meat is rich in protein and vitamin B 1 、B 2 、B 6 Along with the improvement of living standard and dietary structure of people, the proportion of the goat meat in the food structure of people is higher and higher. And due to the supply of pork in recent yearsNot to be required, quality goat meat will be a place in future market competition. Along with the rapid development of goat industry, all parties in the industry are creating hybridization matching among specialized strains meeting different requirements, and realizing specificity and high yield by using limited variety resources. When the male parent and the female parent matched with hybridization are selected, the growth characters including the body weight and the body size are the most important economic characters in goat production. Therefore, the research on how to improve the growth character of the goat through breeding has great significance for the research of goat hybridization breeding.
Molecular markers multi-finger DNA markers are DNA sequences on chromosomes that can identify differences in individuals or species, resulting from different types of mutations, such as insertions, deletions, substitutions, reconstructions, and the like. Molecular markers are of many kinds, such as amplified fragment length polymorphism (Amplified fragment length polymorphism, AFLP), restriction fragment length polymorphism (Restriction fragment length polymorphism, RFLP), and InDel polymorphism (Insertion/deletion polymorphism, inDel) markers, etc. Indel polymorphism refers to the insertion or deletion of nucleotide fragments of different sizes in the sequence at the same locus in the genome between closely related species or different individuals of the same species, i.e., one or more bases in one sequence at a locus that is homologous to another sequence. The InDel marker has the characteristics of large quantity, wide distribution, good genetic stability, suitability for high-throughput automatic detection and the like, and is a first choice tool for researching molecular breeding, gene positioning and the like. Molecular Marker assisted selection (Marker-assisted selection, MAS) utilizes molecular markers associated with specific characters as an auxiliary means for selective breeding, has the advantages of rapidness, accuracy and no environmental influence, can accelerate the breeding process, has greater advantages for growth characters with low genetic force, and can accelerate the selection process of goat economic characters more rapidly and effectively along with the continuous development of high-tip biotechnology means such as RNA-Seq, non-coding RNA sequencing and the like, thereby being beneficial to the good and rapid development of the goat industry in China and bringing great economic benefits.
Fibroblast growth factor 7 (fibroblastist growth)h factor 7, fgf 7) gene was originally found to be closely related to stem cell growth and survival, tissue regeneration and canceration during embryogenesis. Later, more and more researches find thatFGF7Genes also play an important role in animal growth. The study shows that the preparation method has the advantages that,FGF7the gene is taken as a QTL candidate gene of the body type character of the mouse, plays an important role in bone growth and differentiation of the mouse, and has research and discovery at the same time,FGF7the genes are closely related to carcass weight and meat quality traits of poultry. The above studies suggest that, in goats,FGF7whether the gene may affect the productivity of goats by affecting bone growth and development. To date, there is no relevance to goatFGF7The gene is used as a relevant report of research on molecular markers of growth traits.
Disclosure of Invention
The invention aims to provideFGF7An indel marker related to goat growth character in the gene and application thereof. The invention provides a theoretical basis for cultivating high-quality goats and breeding goat breeds by developing indels related to the goat growth traits.
In order to achieve the above purpose, the invention adopts the following technical scheme:
an indel marker associated with a goat growth trait, said indel marker being located atFGF7The nucleotide sequence of the second intron region of the gene is shown as SEQ ID NO.3, the 93 rd to 114 th insertions or deletions are arranged at the 5' end, and the nucleotide sequence of the insertion or deletion fragment is shown as SEQ ID NO. 4.
A specific primer pair for amplifying the indel marker associated with goat growth trait, said primer pair having the nucleotide sequence:
the upstream primer InDel1-F:5'-GTTGCCATTTCCATCATAGTTG-3' the number of the individual pieces of the plastic,
downstream primer InDel1-R: 5'-CATGCCCTTATTATACTTGCATTAC-3'.
The indel mark or the specific primer pair is applied to the auxiliary breeding of goat growth character molecular markers.
Further, the growth trait is one or more of Body Weight (BW), height (BH), body Length (BL), circumference (ChC), chest depth (ChD), chest width (ChW), jirimwidth (HhW), circumference (ChC), body Length Index (BLI), circumference index (ChCI), circumference index (CaCI), hip index (HuWI), body Trunk Index (TI).
Further, the above application includes the following steps:
extracting genome DNA of the goat to be detected, and carrying out PCR amplification by using the specific primer pair to obtain the indel mark.
Further, the application further comprises the following steps:
judging the growth advantage of the goat according to the length difference of the amplified fragments and/or the sequencing result of the PCR products; if the amplified fragment contains a fragment with the size of 216bp or contains a fragment with the sequence shown as SEQ ID NO.1, the goat to be detected is judged to be an individual with growth advantages, and the goat to be detected is determined to be used as a backup parent for breeding.
Further, the PCR amplification system was 10. Mu.L: genomic DNA (50 ng/. Mu.L) 0.8. Mu.L, upstream and downstream primers (10 pmol) 0.2. Mu.L each, 2 XPAmix Taq Mix 5. Mu.L, deionized water 3.8. Mu.L.
Further, the PCR reaction conditions are as follows: pre-denaturation at 95℃for 3min; denaturation at 95℃for 30s, annealing for 30s, extension at 72℃for 15s,40 cycles, extension at 72℃for 5min.
Further, by subjecting the above PCR products to 3.5% agarose gel electrophoresis, inDel1 exists in 3 genotypes: insertion type (II), deletion type (DD) and heterozygous type (DI); electrophoresis results of different genotypes were: one band of type II 216bp, one band of type DD 194bp, and two bands of DI 216bp and 194 bp.
The invention has the remarkable advantages that:
(1) The goat provided by the inventionFGF7The gene insertion deletion mutation detection method is not limited by age, can be used for early breeding of goats, and can be used for selection even just after birth.
(2) Detection goatFGF7The method for gene insertion deletion variation is simple and convenient to operate, rapid and accurateThe cost is low and is not influenced by the factors of the culture environment conditions.
(3) By preference of goatsFGF7Dominant alleles of the InDel1 locus can be used for rapidly screening dominant individuals, increasing genetic progress of goat growth traits, shortening breeding time and improving economic benefits of effective goat individuals.
Drawings
FIG. 1 is a goatFGF7Gene InDel1 (rs 671485500) site sequencing (forward) and sequence comparison; the triangles in the figure represent insertion positions and the grey parts represent insertion sequences.
FIG. 2 is a goatFGF7Gene InDel1 locus typing electrophoretogram.
Detailed Description
In order to make the contents of the present invention more easily understood, the technical scheme of the present invention will be further described with reference to the specific embodiments, but the present invention is not limited thereto.
EXAMPLE 1 development of indel markers associated with growth traits
1. Sample collection and DNA extraction
In the invention, 437 adult goats are specifically taken as detection objects, the number of Fuqing goats F1 generation (FQF 1) is 87, the number of Fuqing goats F2 generation (FQF 2) is 85, the number of Nubian goats (NB) is 144, and the number of simple male large-ear goats (JY) is 121. Blood samples were collected from the Fuqing sheep raising platform of the institute of livestock and veterinary at the institute of fool agriculture (sampling time: 12 months 1 day in 2016). Extracting genomic DNA from blood by conventional phenol-chloroform method, dissolving in ultrapure water, measuring its concentration and purity with NanoDrop-2000 micro nucleic acid tester, diluting with ultrapure water to 50 ng/. Mu.L according to its concentration, and storing at-20deg.C. Meanwhile, body size data of the above 437 adult goat individuals, including Body Weight (BW), height (BH), body Length (BL), chest circumference (ChC), chest depth (ChD), chest width (ChW), jirimwidth (HhW) and tube circumference (ChC), were measured according to the method of erbet et al (specific method reference: linear body measurements of cattle before and after twenty years of selection for post weaning gain when fed two different diets), and Body Length Index (BLI), chest circumference index (ChCI), tube circumference index (CaCI), hip bone index (HuWI) and body Trunk Index (TI) were calculated.
2. Primer design and Synthesis
With goatsFGF7The full-length sequence of the gene (NC_ 030817.1) was used as a reference sequence, the indel screening primer was designed using NCBI on-line primer design software and Premier 5.0 software, and the designed primer was synthesized by the assigned Boshang Biotechnology Co., ltd. The specific primer sequence designed by the invention is as follows:
the upstream primer InDel1-F (SEQ ID NO. 1): 5'-GTTGCCATTTCCATCATAGTTG-3' the number of the individual pieces of the plastic,
the downstream primer InDel1-R (SEQ ID NO. 2): 5'-CATGCCCTTATTATACTTGCATTAC-3'.
3 PCR amplification
PCR amplification was performed using the above-mentioned extracted genomic DNA as a template and the above-mentioned synthesized specific primer pair.
The PCR amplification system was 10. Mu.L: genomic DNA (50 ng/. Mu.L) 0.8. Mu.L, upstream and downstream primers (10 pmol) 0.2. Mu.L each, 2 XPAmix Taq Mix 5. Mu.L, deionized water 3.8. Mu.L.
The PCR reaction conditions were: pre-denaturation at 95℃for 3min; denaturation at 95℃for 30s, annealing for 30s, extension at 72℃for 15s,40 cycles, extension at 72℃for 5min.
Agarose gel electrophoresis analysis of 4 PCR products
The PCR amplified products were subjected to 3.5% agarose gel electrophoresis, and the amplified products were identified by typing by Ethidium Bromide (EB) staining. The PCR products were submitted to the biological engineering (Shanghai) Co., ltd for sequencing, and the sequencing results were aligned by DNAStar to select potential InDel sites.
After sequencing (FIG. 1), it was found in goatsFGF7An InDel mark (InDel 1) exists in the second intron region of the gene, the nucleotide sequence of the InDel mark is shown as SEQ ID NO.3, the 93 rd to 114 th insertions or deletions are arranged at the 5' end, and the nucleotide sequence of the insertion or deletion fragment is shown as SEQ ID NO. 4.
The result of PCR product electrophoresis is shown in FIG. 2. In agarose gel electrophoresis results, there are 3 genotypes at the InDel1 site: insertion (II), deletion (DD) and heterozygous (DI), the insertion (II) shows a band of 216bp, the deletion (DD) shows a band of 194bp, and the heterozygous (DI) shows two bands of 216bp and 194 bp.
5. Genotype frequency and allele frequency distribution and genetic parameter analysis
Goat was calculated using excel softwareFGF7Genotype frequency and allele frequency of gene insertion deletion sites were detected and Polymorphism Information Content (PIC) using a GDIcall on-line calculator (http:// www.msrcall.com/Gdicall. Aspx). Usingχ 2 Inspection to determineFGF7Whether the gene locus polymorphism is in HardyWeinberg equilibrium (HWE).
As shown in Table 1, F1 generation of Fuqing goats and Nubian goats had three genotypes of insertion type (II), deletion type (DD) and heterozygous type (DI), while concha large-ear goats had only two genotypes of insertion type (II) and heterozygous type (DI). The genotype frequencies of dominant genotype II were 11.5%, 35% and 95.4% in the F1 generation, nubian and concha large ear sheep populations, respectively.
TABLE 1 InDel1 locus genotype frequencies and allele frequencies for FGF7 Gene
6. Correlation analysis of Indel marker (Indel 1) and growth trait
Correlation between indel markers and goat population growth traits was calculated using analysis of variance (ANOVA). The statistical software used was SPSS (version 18.0) (IBM corporation, armonk, N.Y., USA). Analyzing the relationship between the genotype and the growth traits of the goat by adopting a basic linear model:
Y i =u+G i +e
wherein the method comprises the steps ofY i Actual measurement data of each animal trait;uis the over-average value for each feature;G i in order for the genotype to be affected,eis a random error.
The results are shown in table 2, in the F1 generation group of the ford goats, inDel1 was very significantly correlated with body weight, body diagonal length, circumference, chest depth, chest width, body length index and circumference index; in the nubian goat population, inDel1 is significantly correlated with body length and extremely significantly correlated with body length index and body trunk index; in the conclusive large ear sheep population, inDel1 was significantly correlated with the chest circumference index and tube circumference index.
TABLE 2 InDel site and growth trait association analysis Table of goat FGF7 Gene
Note that: the different lowercase letters (a, B) are marked to indicate that the difference is significant, and the different uppercase letters (a, B) are marked to indicate that the difference is extremely significant.
Analysis of genetic polymorphism of Indel marker (Indel 1) in F2-generation group of Fuqing goats
The Fuqing goat F2 generation (FQF 2) group is divided into two groups, 1 group is InDel1 locus selective breeding group (only II genotype, 41 in the group), and 2 group is self-propagating group (44). Culturing in a free feeding mode, collecting DNA and body size data of all individuals after two groups of individuals grow up by adopting the method, and analyzing the relation between the goat Indel (Indel 1) and the growth traits by adopting a signed basic linear model.
The results are shown in table 3, and the results of the analyses by comparing the FQF2 population selective breeding group and the self-propagation group show that: the body weight, height, body oblique length, chest circumference, chest depth, chest width and hip index of the breeding group are all significantly higher than those of the self-propagation group. Through the association analysis of F1 generation and F2 generation of Fuqing goats, the insertion genotype (II) is a dominant genotype, and the growth vigor of goats can be improved by screening individuals with the genotype, so that the breeding process of goats is accelerated.
TABLE 3 comparison analysis Table of growth characteristics between two groups of FQF2 population
Note that: the different lowercase letters (a, B) are marked to indicate that the difference is significant, and the different uppercase letters (a, B) are marked to indicate that the difference is extremely significant.
The foregoing description is only of the preferred embodiments of the invention, and all changes and modifications that come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
SEQUENCE LISTING
<110> institute of livestock and veterinary at the national academy of agricultural sciences of Fujian province
<120> indel marker associated with FGF7 gene and goat growth trait and application thereof
<130>
<160> 4
<170> PatentIn version 3.3
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tatgtctata ttttttcttt tgttaaatat ctgtttttct tttgttacat atgggttctt 120
tgatgaacac catatgtaaa aaacatgcat caaaattcag tatttttcaa aacaattttg 180
tattctctga agtaatgcaa gtataataag ggcatg 216
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Claims (8)
1. An indel marker associated with a goat growth trait, comprising: the indel marker is located in goatFGF7The nucleotide sequence of the second intron region of the gene is shown as SEQ ID NO.3, the 93 rd to 114 th insertions or deletions are arranged at the 5' end, and the nucleotide sequence of the insertion or deletion fragment is shown as SEQ ID NO. 4.
2. A specific primer pair for amplifying an indel marker associated with a goat growth trait according to claim 1, wherein: the nucleotide sequences of the primer pairs are as follows:
the upstream primer InDel1-F:5'-GTTGCCATTTCCATCATAGTTG-3' the number of the individual pieces of the plastic,
downstream primer InDel1-R: 5'-CATGCCCTTATTATACTTGCATTAC-3'.
3. Use of an indel marker associated with a goat growth trait according to claim 1 or a specific primer pair according to claim 2 in the molecular marker assisted selection of a goat growth trait.
4. A use according to claim 3, characterized in that: the growth character is one or more of weight, height, body length, chest circumference, chest depth, chest width, and jirimwidth, tube circumference, body length index, chest circumference index, tube circumference index, hip bone index, and body trunk index.
5. A use according to claim 3, characterized in that: the application comprises the following steps: extracting genome DNA of the goat to be detected, and carrying out PCR amplification by using the specific primer pair of claim 2 to obtain the indel marker.
6. The use according to claim 5, characterized in that: the application further comprises the steps of: judging the growth advantage of the goat according to the length difference of the amplified fragments and/or the sequencing result of the PCR products; if the amplified fragment contains a fragment with the size of 216bp or contains a fragment with the sequence shown as SEQ ID NO.3, the goat to be detected is judged to be an individual with growth advantages, and the goat to be detected is determined to be used as a backup parent for breeding.
7. The use according to claim 5, characterized in that: the PCR amplification system was 10. Mu.L: 50 ng/. Mu.L of genomic DNA, 10pmol of upstream primer InDel 1-F0.2. Mu.L, 10pmol of downstream primer InDel 1-R0.2. Mu.L, 2 XPromix Taq Mix 5. Mu.L, deionized water 3.8. Mu.L.
8. The use according to claim 5, characterized in that: the PCR reaction conditions are as follows: pre-denaturation at 95℃for 3min; denaturation at 95℃for 30s, annealing for 30s, extension at 72℃for 15s,40 cycles, extension at 72℃for 5min.
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| CN111394479A (en) * | 2020-04-28 | 2020-07-10 | 中国计量大学 | Molecular marker for assisting in fine wool sheep breeding and application method |
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