Molecular marker related to Hu sheep body type character and application of molecular marker

Abstract

The invention discloses a molecular marker related to Hu sheep body type characters and application of the molecular marker, and relates to agricultural genetic engineering.The molecular marker CHST3 related to the Hu sheep body type characters is screened out by conducting chip sequencing on Hu sheep and adopting a method of combining GWAS, QTL and functional region annotation to analyze candidate genes related to the body type characters, and the molecular marker CHST3 related to the Hu sheep body type characters is obtained. The molecular marker is a molecular marker, a reference genome is ARS-UIRamv2.0, an SNP site is SNP2527304876, the molecular marker can be applied to establishment of a Hu sheep core breeding group and can also be used for breeding of new strains, the genetic mechanism of Hu sheep body type characters can be known, and Hu sheep breeding is further guided.

Description

Technical Field

[0001] The invention relates to agricultural genetic engineering, in particular to a molecular marker related to the body shape traits of Hu sheep and application thereof. Background Art

[0002] Sheep body size is the most important growth indicator in production, which is directly related to meat production, fat deposition and reproductive performance. Global meat consumption continues to increase, and a larger body size will bring higher profits to sheep farmers. However, compared with other meat sheep, Hu sheep are smaller in size and urgently need to be improved. Traditional breeding methods are time-consuming and slow to take effect, which restricts the development of the animal husbandry industry. In view of this, in order to speed up the breeding process of Hu sheep, it is urgent to screen out the key genes that affect the body size of Hu sheep. The body size of sheep is affected by many factors, especially genetic factors. At present, most studies on sheep body size only focus on weight traits, while ignoring other body size traits. Cannula girth is an important body size trait of sheep. Increasing the cannula girth of sheep can increase the load-bearing capacity of the legs and obtain a larger body size. So far, few studies have conducted in-depth discussions on the cannula girth trait of sheep and its effect on body size. Through the study of cannula girth traits, it is helpful to propose new breeding strategies to improve sheep body size. In addition, most studies on sheep body size traits use a single method to screen related loci. There is no focus on the differences between genotypes at specific loci, nor on the expression of candidate genes in tissues and systems. Therefore, it is crucial to use more comprehensive analysis methods to identify relevant loci and candidate genes and explore the potential molecular mechanisms.

[0003] Hu sheep is a world-renowned multi-lamb sheep breed and a first-level local protected breed in my country. It has the advantages of early sexual maturity, high fertility, high slaughter rate, and delicious meat. Hu sheep has huge potential waiting for us to explore. Therefore, this study used the sheep 50K chip to sequence 558 Hu sheep, and estimated the heritability of each trait and the correlation between them. The relevant sites and candidate genes were screened out using genome-wide association analysis (GWAS), quantitative trait loci (QTL) and functional region annotation. The present invention discovered and verified the candidate genes related to the body shape traits of Hu sheep, which can provide reference information for molecular marker-assisted breeding of Hu sheep body shape, and can be used to guide future breeding decisions. Summary of the invention

[0004] To address the deficiencies in the prior art, the present invention aims to provide a molecular marker related to the body shape traits of Hu sheep and its application. The present invention screens out CHST3 which can be used as a molecular marker for the body shape traits of Hu sheep, and can be used to establish a core breeding group of Hu sheep and cultivate new varieties.

[0005] In order to achieve the above object, the present invention adopts the following technical solution:

[0006] A molecular marker related to the body shape traits of Hu sheep, the molecular marker is CHST3, the reference genome is ARS-UI_Ramb_v2.0, and the SNP site is SNP25_27304876.

[0007] The aforementioned molecular marker related to the body shape trait of Hu sheep, SNP25_27304876, has three genotypes, namely TT, TC, and CC. Among the tube girth traits, the TT genotype has the largest tube girth, and the CC genotype has the smallest tube girth.

[0008] The aforementioned molecular marker related to the body shape trait of Hu sheep, SNP25_27304876, has three genotypes, namely TT, TC, and CC. In the weight trait, there are significant differences between the genotypes TT and TC. The TT genotype has the largest weight, and the CC genotype has the smallest weight.

[0009] The aforementioned molecular marker related to the body shape trait of Hu sheep, SNP25_27304876, has three genotypes, namely TT, TC, and CC. In the body height trait, there are significant differences between the genotypes TT and TC. The body height of the TT genotype is the largest, and the body height of the CC genotype is the smallest.

[0010] The aforementioned molecular marker related to the body shape trait of Hu sheep, SNP25_27304876, has three genotypes, namely TT, TC, and CC. In the chest circumference trait, there are significant differences between the genotypes TT and TC. The body height of the TT genotype is the largest, and the body height of the CC genotype is the smallest.

[0011] The aforementioned molecular marker related to the body shape trait of Hu sheep, SNP25_27304876, has three genotypes, namely TT, TC, and CC. In the body length trait, there is no significant difference between the genotypes TT and TC, and there is a significant difference between the genotypes TT and CC. The body length of the TT genotype is the largest, and the body length of the CC genotype is the smallest.

[0012] The aforementioned application of the molecular marker related to the body shape traits of Hu sheep is used to establish the core breeding group of Hu sheep.

[0013] The aforementioned molecular marker related to the body shape traits of Hu sheep is used in the breeding of new breeds.

[0014] The present invention is beneficial in that:

[0015] The molecular marker CHST3 screened out by the present invention can be used as a candidate gene for the body shape traits of Hu sheep;

[0016] The present invention verifies that the candidate genes are closely related to the body shape traits of Hu sheep. These molecular markers have a wide range of applications. For example, they can be used to establish a core breeding group of Hu sheep, or to cultivate new strains and guide Hu sheep breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 is a correlation diagram of the genome-wide association analysis of body shape traits of the present invention; (AE: Manhattan diagram of genome-wide association analysis of body shape traits, including weight, height, body length, chest circumference and tube circumference; FJ: Quantile-Quantile (QQ) diagram of genome-wide association results of body shape traits, including weight, height, body length, chest circumference and tube circumference);

[0018] Figure 2 It is the quantitative trait locus enrichment correlation diagram of the body shape trait of the present invention; (AE: QTL diagram of significant SNP loci enrichment, including body weight, body height, body length, chest circumference and tube circumference);

[0019] Figure 3It is a correlation diagram of the key site function annotation and key gene expression level of the present invention; (A: The site after SNP25_27304876 (ARS-UI_Ramb_v2.0) conversion is SNP25_27834432 (Oar_v4.0), the sheep enhancer region annotation map of SNP25_27834432, the red line is the site of SNP25_27834432; in Figure A: Cerebellum: cerebellum, Macrophage: macrophage, Ovary: ovary, intest: prostate Gland, Stomach: stomach, Cortex: cortex, Testis: testis, Muscle: muscle, Heart: heart, Adipose: fat, Spleen: spleen, Lung: lung, Liver: liver, Colon: colon; B: APA event annotation map of SNP25_27304876, the percentage of distal poly (A) site usage index (PDUI) is used to quantify APA events; Figure B: Caecum: cecum, Lung: lung, Rumen: rumen, Thymus Gland: thymus, Cutin: stratum corneum, Lymph Nodes: lymph nodes, Esophagus: esophagus, Side Skin: side skin, Spleen: spleen, Subcutaneous Adipose: subcutaneous fat, Tail Adipose: tail fat, Tonsil: tonsil, Ventral Skin: abdominal skin; C: expression level of CHST3 gene in multiple tissues of Hu sheep, the darker the color, the higher the expression level; Figure C: abomasum: abomasum, latissimus dorsi: latissimus dorsi, omasum: omasum ovary: ovary, radial extensor of wrist: radial extensor of wrist, rhomboid muscle: rhomboid muscle, rumen dorsal sac: rumen dorsal sac, rumen predorsum blind sac: rumen pretumor blind sac, rumen venter posterior blind sac: rumen ventral sac, sternocephalicus muscle: sternocephalicus muscle, tail fat: tail fat, tail muscle: tail muscle, uterus: uterus; D: expression level of CHST3 gene in multiple systems of Hu sheep;: integumentary system: integumentary system, exercise system: exercise system, digestive system: digestive system, respiratory system: respiratory system, urinary system: urinary system, reproductive system: reproductive system, circulatory system: circulatory system, immune system: immune system, nervous system: nervous system, endocrine system: endocrine system);

[0020] Figure 4 It is a box plot showing different phenotypic differences according to genotypes of the present invention (A: body weight; B: body height; C: body length; D: chest circumference; E: tube circumference). DETAILED DESCRIPTION

[0021] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0022] The following experiment demonstrates the process of screening out the molecular marker CHST3 as the key gene for the body conformation traits of Hu sheep, and screening out the three molecular markers ADGRL4, FAT4 and ARK2N as candidate genes for the body conformation traits of Hu sheep.

[0023] 1. Materials and Methods

[0024] 1.1 Phenotypic measurements and sample preparation

[0025] The Hu sheep (n=558) used in this study were all from Huzhou Yihui Ecological Agriculture Co., Ltd. All Hu sheep were randomly selected and raised in the same environment. The weight of Hu sheep was measured using a special livestock scale, the height and length of Hu sheep were measured using a special measuring stick, and the chest circumference and canal circumference of Hu sheep were measured using a special tape measure. Blood samples were collected from the posterior vein of Hu sheep and stored in a -80 degree refrigerator in preparation for DNA extraction. We used CWE9600 MagbeadBlood DNAKit and magnetic bead method to extract DNA. The extracted DNA samples were spotted on 1.5% agarose gel for DNA sample integrity detection; then the purity of DNA was detected using NanoDrop 2000 nucleic acid protein analyzer; finally, the samples were accurately quantified using dsDNAHS Assay Kit for Qubit.

[0026] 1.2 Library construction and genotyping

[0027] During library construction, a universal library construction kit (for MGI) was used. DNA. Genomic DNA with a total amount greater than 1 μg was crushed by Covaris TM The fragments were randomly cut into about 300-350 bp fragments, and then the ends were repaired, A-tailed, and connected to sequencing adapters. SPBeads screened DNA of around 300-350 bp, amplified it by PCR, and used it again SP Beads were used to purify the PCR products and finally obtain the sequencing library. After the library was constructed, Qubit2.0 was used for preliminary quantification and then (Agilent) was used to detect the insert fragments of the library. Then, sheep 50K chip was used for genotyping. The chip was produced and designed by Compson. Plink software was used to filter SNPs with a detection rate of less than 90% and a minor allele frequency (MAF) of less than 5%.

[0028] 1.3 Heritability and correlation calculations

[0029] The following model was used to calculate the heritability and correlation between each trait:

[0030] y=Xb+Za+e#(1)

[0031] Among them, y is the trait vector; b is the fixed effect, including sex, measurement year and measurement age; a is the random effect, and its mean is 0 and its variance is The normal distribution of is the genetic variance; X and Z are the correlation matrices of b and a respectively; e is the residual effect, which follows a normal distribution is the residual variance; G is the kinship matrix between individuals. The heritability calculation formula is:

[0032] The genetic correlation and phenotypic correlation between traits were calculated using HIBLUP software. The genetic correlation (r g ) and phenotypic correlation (r p ), the model used is still as shown in (1). Among them, a and e follow and Normal distribution; H is the additive genetic variance matrix between each team of traits; R is the residual variance matrix between traits; is the tensor product of matrices. Calculate genetic correlation r g and phenotypic correlation p The equation is as follows:

[0033]

[0034] in, and are the additive genetic covariance and residual covariance between each pair of traits; and are the additive genetic variance and residual variance of each trait.

[0035] 1.4 Genome-wide association analysis

[0036] GWAS analysis was performed using rMVP software. The model used in GWAS analysis is a mixed linear model, which can well correct for population structure and complex kinship within the population. The formula of the model is as follows:

[0037] y=Xβ+Z k γ k +ξ+e

[0038] Among them, y is the phenotypic vector, Xβ is the fixed effect, including population structure, sex, measurement year and measurement month, Z k γ k To test the marker effect, is a polygenic effect, e~N(0,Iσ 2 ) is the residual effect. K in the polygenic effect is the kinship matrix inferred by the markers. Then, the Manhattan plot and QQ plot were drawn using the qqman package of R software.

[0039] 1.5 Functional region annotation and gene expression level analysis

[0040] The reference genome information of sheep Ovis aries (assembly ARS-UI_Ramb_v2.0) was downloaded from the ENSEMBL website, and the significant SNPs were annotated to their corresponding genes using ANNOVAR software. The Animal QTL Database (http: / / www.animalgenome.org / QTLdb) was used to annotate the potential functions of the selected regions, and the GALLO package of R software was used for QTL enrichment analysis. The significant SNP sites were annotated to the reference genome Oar_v4.0 using LiftOver software. Subsequently, the significant SNPs were functionally annotated in the Sheep Enhancer Database (http: / / genome.ucsc.edu / s / zhypan / oviAri4_chr_state). Alternative polyadenylation (APA) annotations were performed on the significant SNPs in the Animal-APA database (http: / / gong_lab.hzau.edu.cn / Animal-APAdb). The expression levels of candidate genes in tissues and systems were queried in the Herbivore Transcriptome Information Resource Database (https: / / yanglab.hzau.edu.cn / HTIR), and KEGG pathway enrichment analysis was performed on the candidate genes.

[0041] 2 Results

[0042] 2.1 Phenotypic statistics and heritability calculation

[0043] In this study, 558 Hu sheep were randomly selected and their body shape traits, including weight, height, body length, chest circumference, and tube circumference, were counted. Data statistics for these traits provided a preliminary overall characteristic analysis. In addition, the heritability of all traits was estimated by the GBLUP model, ranging from 0.18 to 0.76. Except for the heritability of weight and body length traits, which was less than 0.2, the heritability of the remaining traits was greater than 0.2. This indicates that these body shape traits can be effectively improved in future breeding.

[0044] Table 1. Descriptive statistics of body conformation traits of Hu sheep and estimates of their heritability.

[0045]

[0046] 2.2 Genetic and phenotypic correlations

[0047] As shown in Table 2, the tube circumference trait shows good genetic correlation with the weight (0.98), height (0.79) and chest circumference (0.73) traits. At the same time, the tube circumference trait also has a good phenotypic correlation with the weight (0.48), height (0.40) and chest circumference (0.52) traits. This shows that by improving the tube circumference trait, the weight, height and chest circumference traits will also be improved. The genetic correlation (-0.05) and phenotypic correlation (0.18) between the tube circumference trait and the body length trait are both low. The genetic correlation (0.99) and phenotypic correlation (0.40) between the weight trait and the height trait are higher than the correlation between the weight trait and the body length trait (-0.38, 0.29). This shows that the body height trait has a greater effect on weight than the body length trait. The genetic correlation (-0.53) and phenotypic correlation (-0.38) between the height trait and the body length trait are both negatively correlated.

[0048] Table 2. Genetic and phenotypic correlations of body shape traits in Hu sheep

[0049]

[0050] The values ​​in the upper right corner are genetic correlations, and the values ​​in the lower left corner are phenotypic correlations.

[0051] 2.3SNP25_27304876 is a key locus affecting tube circumference traits

[0052] In the tube circumference trait, 11 significant SNPs were identified. The significant SNPs of the tube circumference trait were annotated with enhancers. SNP25_27304876 was found to be annotated in enhancer regions of multiple tissues, including ovary, stomach, testis, muscle and lung ( Figure 3 A). In addition, through the Animal-APA database, it was found that SNP25_27304876 was annotated in multiple tissue APA events, including rumen, spleen, and esophagus ( Figure 3 B). SNP25_27304876 is annotated to the CHST3 gene. The CHST3 gene is highly expressed in the tail fat (3.26), sternocephalic muscle (3.23) and tail muscle (3.09) of Hu sheep, and moderately expressed in the dorsal sac of the rumen (2.99), uterus (2.92), ovary (2.87), rhomboid muscle (2.79) and wrist radial extensor muscle (2.09). Figure 3 C). The CHST3 gene is expressed at a high level in the circulatory system and epidermal system of Hu sheep, and at a moderate level in the respiratory system, immune system, and reproductive system ( Figure 3 D). In addition, CHST3 genes were enriched in glycosyltransferases and glycosaminoglycanbiosynthesis-chondroitin sulfate / dermatan sulfate pathways.

[0053] 2.4CHST3 gene is a key gene affecting the body shape of Hu sheep

[0054] In the tube circumference trait, there are three genotypes at the SNP25_27304876 locus, namely TT, TC, and CC. There is a significant difference in tube circumference between the genotypes TT and TC, and there is also a significant difference in tube circumference between the genotypes TT and CC. In addition, the tube circumference of the genotype TT is the largest, and the tube circumference of the genotype CC is the smallest ( Figure 4 E). In addition, at the SNP25_27304876 site, the body weight trait ( Figure 4 A), body height traits ( Figure 4 B) and chest circumference traits ( Figure 4 D), the same situation exists. In the body length trait, there is no significant difference between the genotypes TT and TC, but there is a significant difference between the genotypes TT and CC. In addition, the genotype TT still has the largest body length, and the genotype CC has the smallest body length ( Figure 4 C). Therefore, SNP25_27304876 can be used as a key locus for body shape traits, and CHST3 gene can be used as a key gene for body shape traits.

[0055] In summary: CHST3 screened out by the present invention can be used as a key gene for the body shape traits of Hu sheep. This candidate gene is closely related to the body shape traits of Hu sheep, can be used as a molecular marker, can be used to establish a core breeding group of Hu sheep, and can also be used for the cultivation of new strains. It will help to understand the genetic mechanism of the body shape traits of Hu sheep and further guide Hu sheep breeding.

[0056] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any form, and any technical solution obtained by equivalent replacement or equivalent transformation falls within the protection scope of the present invention.

Claims

1. A molecular marker related to the body shape traits of Hu sheep, characterized in that: The molecular marker is CHST3, the reference genome is ARS-UI_Ramb_v2.0, and the SNP site is SNP25_27304876.

2. The molecular marker associated with the body shape traits of Hu sheep according to claim 1, characterized in that: There are three genotypes at the SNP25_27304876 site, namely TT, TC, and CC. Among the tube circumference traits, the TT genotype has the largest tube circumference, and the CC genotype has the smallest tube circumference.

3. The molecular marker associated with the body shape traits of Hu sheep according to claim 1, characterized in that: There are three genotypes at the SNP25_27304876 site, namely TT, TC, and CC. In the body weight trait, there is a significant difference between the genotypes TT and TC, the TT genotype has the largest body weight, and the CC genotype has the smallest body weight.

4. The molecular marker associated with the body shape traits of Hu sheep according to claim 1, characterized in that: There are three genotypes at the SNP25_27304876 site, namely TT, TC, and CC. In the body height trait, there is a significant difference between the genotypes TT and TC, the TT genotype has the largest body height, and the CC genotype has the smallest body height.

5. The molecular marker associated with the body shape traits of Hu sheep according to claim 1, characterized in that: There are three genotypes at the SNP25_27304876 site, namely TT, TC, and CC. In the chest circumference trait, there is a significant difference between the genotypes TT and TC, the TT genotype has the largest body height, and the CC genotype has the smallest body height.

6. The molecular marker associated with the body shape traits of Hu sheep according to claim 1, characterized in that: There are three genotypes at the SNP25_27304876 site, namely TT, TC, and CC. In the body length trait, there is no significant difference between the genotypes TT and TC, and there is a significant difference between the genotypes TT and CC. The body length of the TT genotype is the largest, and the body length of the CC genotype is the smallest.

7. The use of a molecular marker related to the body shape traits of Hu sheep according to claim 1, characterized in that: Used to form the core breeding group of Hu sheep.

8. The use of a molecular marker related to the body shape traits of Hu sheep according to claim 1, characterized in that: Used in the cultivation of new varieties.

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