Yellow feather broiler abdominal fat character related genome and application thereof

Abstract

The invention discloses a yellow feather broiler abdominal fat character related genome, which comprises one or a combination of more of FOS, DDAH1 and PTN genes, and the nucleotide sequences of the FOS, the DDAH1 and the PTN genes are respectively shown as SEQ ID NO.1-3. According to the genome related to the abdominal fat character of the yellow-feathered broiler chicken and the application of the genome, key genes FOS, DDAH1 and PTN closely related to the abdominal fat character of the chicken are screened out on the basis of a transcriptome sequencing platform for the first time, and the significant difference of the key genes in high and low abdominal fat tissues of the yellow-feathered broiler chicken is clear. The FOS and the DDAH1 are remarkably and highly expressed in abdominal fat tissues, and the FOS, the DDAH1 and the PTN genes can be used as molecular markers of abdominal fat characters of yellow feather broilers and are applied to chicken variety character prediction and molecular assisted breeding. The method is simple and convenient to operate, stable in result and high in repeatability, and has good popularization and application prospects.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of genetic breeding, in particular to a genome related to abdominal fat traits of yellow-feathered broilers and application thereof. BACKGROUND

[0002] Yellow-feathered broilers are local characteristic chicken germplasm resources in China, which are bred by local high-quality chicken breeds (such as Shiqi hybrid chicken and Lingnan yellow chicken, etc.). Compared with white-feathered broilers with rapid growth, yellow-feathered broilers have moderate growth cycle and rich meat flavor, which is more suitable for Chinese cooking and occupies a dominant position in the domestic live chicken consumption market. However, in the process of intensive high-yield breeding, the increase of growth rate of yellow-feathered broilers is accompanied by the problem of excessive abdominal fat deposition. As the core fat deposition area, abdominal fat accumulation directly affects the economic benefits of the industry.

[0003] Abdominal fat refers to the fat tissue deposited in the mesentery and around the gizzard of poultry in the abdominal cavity, which is often referred to as "board oil" in poultry production. This trait is one of the core economic indicators of broilers, which directly affects the dressing percentage, feed conversion efficiency and carcass quality. Yellow-feathered broilers, as a Chinese local characteristic chicken breed, have significantly higher abdominal fat deposition capacity than white-feathered broilers, with an abdominal fat rate of 3.5%-5.8% of the live body weight in adult chickens. Therefore, it is of great significance to find key candidate genes related to abdominal fat traits of yellow-feathered broilers and to mine markers that can be used for molecular breeding for the improvement of poultry breeds.

[0004] The information disclosed in this BACKGROUND section is only for the purpose of increasing the understanding of the background of the present application and should not be taken as an acknowledgment or any form of suggestion that this information forms prior art that is publicly known. SUMMARY

[0005] The present application aims to provide a genome related to abdominal fat traits of yellow-feathered broilers, which is specifically highly expressed in abdominal fat tissue and can be applied to chicken shape prediction and molecular assisted breeding.

[0006] Another object of the present application is to provide a genome related to abdominal fat traits of yellow-feathered broilers and application thereof.

[0007] To achieve the above-mentioned objects, the present application provides a genome related to abdominal fat traits of yellow-feathered broilers, which comprises FOS 、 DDAH1 、 PTN one or more combinations of the genes, and the nucleotide sequences of the genes are shown in SEQ ID NO. 1-3, respectively. FOS 、 DDAH1 、 PTN

[0008] ​A genome related to abdominal fat traits of yellow-feathered broilers, comprising FOS 、 DDAH1 one or more combinations of genes.

[0009] Application of the genome as described above in the prediction of abdominal fat traits of yellow-feathered broilers and in molecular-assisted breeding.

[0010] Application of the genome as described above in the identification of abdominal fat traits of yellow-feathered broilers.

[0011] A screening method of the genome as described above, comprising the following steps:

[0012] (1) population selection and phenotype collection;

[0013] (2) RNA extraction, reverse transcription and sequencing of the individual to be tested;

[0014] (3) Transcription data alignment: align and analyze the gene data obtained by sequencing through bioinformatics analysis software;

[0015] (4) Screening and obtaining key genes affecting abdominal fat traits of yellow-feathered broilers: using QRT-PCR technology, through quantitative detection of significantly different genes FOS 、 DDAH1 、 PTN in high and low abdominal fat groups, primer design and synthesis of candidate genes, design β-actin 、 FOS 、 DDAH1 、 PTN Real-time fluorescent quantitative PCR primers of the genes, after checking the specificity of the primers, synthesize the primers; verify FOS 、 DDAH1 、 PTN The results of the three genes in high and low abdominal fat groups are consistent with the results of transcriptome sequencing.

[0016] (5) Tissue expression profile analysis: using QRT-PCR to detect FOS 、 DDAH1 and PTN the tissue expression profile of the genes and analyze, detecting the expression amount of the above genes in liver, breast muscle, back fat, leg muscle and abdominal fat five kinds of tissues.

[0017] Preferably, in the above technical solution, step (2) uses Trizol method to extract total RNA of the tissue, uses reverse transcription kit to synthesize cDNA as QRT-PCR template.

[0018] Preferably, in the above technical solution, in step (5), the QRT-PCR reaction system is: a 15.0 μL reaction system, including 7.5 μL 2×TB Green Premix Ex Taq II (Tli RNaseH Plus), 4.6 μL ddH2O, 1.5 μL cDNA template, and 0.6 μL each of upstream and downstream primers; the PCR reaction program is: pre-denaturation 95 ℃, 30 s; cyclic denaturation 95 ℃, 5 s; annealing temperature 60 ℃ for 30 s, for a total of 35 cycles.

[0019] Preferably, in the above technical solution, in steps (4)-(5), the QRT-PCR uses an internal reference gene for correction, the internal reference gene being β-actin, all reaction solutions are prepared on ice throughout the process, each gene is replicated three times, and data are presented in 2... -△△CT The method is used to calculate the relative expression level.

[0020] Compared with existing technologies, the present invention has the following beneficial effects: The present invention discloses a genome related to the abdominal fat trait in yellow-feathered broilers and its application, and for the first time screens key genes closely related to the abdominal fat trait in chickens based on a transcriptome sequencing platform. FOS , DDAH1 , PTN This study clarified the significant differences in abdominal fat tissue between high and low-fat regions in yellow-feathered broilers. FOS and DDAH1 It is significantly highly expressed in abdominal fat tissue. FOS , DDAH1 , PTN All genes can serve as molecular markers for abdominal fat traits in yellow-feathered broilers, applicable to breed trait prediction and molecular-assisted breeding. The method of this invention is simple to operate, yields stable results, and has strong reproducibility, showing promising prospects for widespread application. Attached Figure Description

[0021] Figure 1 This is the high and low abdominal fat weight and abdominal fat percentage grouping phenotype of Embodiment 1 of the present invention.

[0022] Figure 2 This shows the differential expression of the three candidate genes in Example 1 of the present invention in the high and low abdominal fat groups.

[0023] Figure 3 This is a comparison of the quantitative detection and transcriptome sequencing results of the three candidate genes in chicken abdominal fat according to Example 1 of the present invention.

[0024] Figure 4 This is a candidate gene from Example 1 of the present invention. FOS , DDAH1 , PTN Tissue expression profile. Detailed Implementation

[0025] The specific embodiments of the present application are described in detail below with reference to the accompanying drawings, but the scope of protection of the present application is not limited by the specific embodiments.

[0026] Unless otherwise clearly indicated, throughout the specification and claims, the terms "comprise", "comprising", "including" and "includes" are not intended to exclude other elements or steps. Throughout the specification and claims, the singular encompasses the plural unless otherwise indicated.

[0027] The animals, instruments, reagents, kits used in the following examples can be obtained commercially. Examples

[0028] Transcriptome sequencing and verification of yellow-feathered broiler high and low abdominal fat groups

[0029] I. Population selection and phenotype collection

[0030] Yellow-feathered broilers were fed at Guangxi University Animal Experiment Base until 120 days of age, and then slaughtered after 24 hours of stopping feeding. Abdominal fat was removed and weighed. According to the two-tailed method, the population was divided into high abdominal fat group (H) and low abdominal fat group (L), with 3 in each group. The abdominal fat weight and abdominal fat rate of the high and low groups are shown in Table 1. Figure 1

[0031] II. RNA extraction and sequencing

[0032] (1) Put the abdominal fat sample into a pre-cooled mortar, grind while adding liquid nitrogen to prevent RNA degradation, until the abdominal fat tissue becomes powder. Pour the powder into a 2 ml EP tube, and then add 1.5 ml Trizol.

[0033] (2) Add 200 μL of chloroform, shake vigorously for 30 s, mix thoroughly, and let stand on ice for 5 min.

[0034] (3) After standing, centrifuge at 4 ℃, 12000 rpm, 15 min, and transfer the supernatant to a new 1.5 ml EP tube (note to avoid contamination of RNA by other impurities).

[0035] (4) Add an equal volume of isopropanol, gently invert and let stand on ice for 10 min.

[0036] (5) After standing, centrifuge at 4 ℃, 12000 rpm, 15 min, discard the supernatant, and use a pipette to remove the remaining liquid from the centrifuge tube to avoid sucking the bottom white precipitate.

[0037] (6) Add 1 ml of 75 % ethanol (freshly prepared), gently tap the bottom of the centrifuge tube to remove the white precipitate, and let the liquid contact the precipitate on ice for 2 min.​

[0038] (7) Again centrifuge at 4 °C, 7500 rpm, 5 min setting, remove supernatant.

[0039] (8) Add 1 ml of anhydrous ethanol to the centrifuge tube, shake slightly, centrifuge at 4 °C, 7500 rpm, 5 min.

[0040] (9) Discard the supernatant, remove the excess anhydrous ethanol in the tube with a pipette, open the cap and dry on ice, then add 20 μL of RNase Free dH2O.

[0041] (10) Finally, after the RNA precipitate is completely dissolved, 1 μL of the RNA stock solution is diluted 5 times and used for agarose gel electrophoresis to detect the integrity of the RNA.

[0042] The purity of the RNA is detected by ultraviolet spectrophotometer, and after the detection is qualified, it is sent to Beijing Novogene Bioinformatics Technology Co., Ltd. for gene resequencing, the sequencing platform is Novaseq, the effective information is obtained after filtering the sequencing data, and the filtering standards are mainly:

[0043] 1) Filter out reads containing adapter sequences;

[0044] 2) When the content of N in single-end sequencing read is more than 10% of the length of the read, remove the read;

[0045] 3) When the number of low-quality (<=5) bases contained in the single-end sequencing read is more than 50% of the length of the read, remove the read. After obtaining the effective data, further sequencing quality assessment is carried out to check the error rate distribution.

[0046] III. Transcriptome data alignment and analysis

[0047] The data is again subjected to quality control and detection by bioinformatics analysis software fastp (v0.23.4), aligned with Gallus_gallus.bGalGal1.mat.broiler.GRCg7b.dna.toplevel.fa gene sequence file using Hisat2 (v2.2.1) software, and finally quantified using HTseq-count (v0.6.1p1), SAMtools (v1.21), DESeq2 R (v1.34.0) to arrange and analyze the gene data obtained by sequencing.

[0048] IV. RNA reverse transcription

[0049] Total RNA was reverse transcribed using a Thermo Fisher Scientific reverse transcription kit. First, genomic DNA was removed. The first step reaction solution was prepared on ice: 1.0 μg total RNA, 1.0 μL 10X Reaction Buffer with MgCl2, 1.0 μL LDNase I, RNase-free (1 U / μL), and RNase-free dH2O to a final volume of 10 μL. The mixture was incubated at 37°C for 30 min in a standard PCR instrument, followed by the addition of 1.0 μL 50 mM EDTA and incubation at 65°C for 10 min. Reverse transcription was then performed. The second step reaction solution was prepared on ice: 9.0 μL of the first step reaction solution, 10.0 μL 2X RT Buffer Mix, 1.0 μL 20X RT Enzyme Mix, and RNase-free dH2O to a final volume of 20 μL. The reaction tubes were briefly centrifuged, and then incubated at 42°C for 30 min in a standard PCR instrument; followed by incubation at 95°C for 5 min, and then maintained at 4°C.

[0050] V. Primer Design and Synthesis of Candidate Genes

[0051] Designed using Oligo7 software and the online website NCBI β-actin , FOS , PTN and DDAH1 The primers for real-time quantitative PCR of the gene were tested for specificity using the online tool Primer-BLAST and then sent to Sangon Biotech (Shanghai) Co., Ltd. for primer synthesis. The primer sequence information is shown in Table 1 (as shown in SEQ ID NO: 4-11).

[0052] Table 1 Gene Primer Information Table

[0053]

[0054] VI. Screening and obtaining key genes affecting abdominal fat traits in yellow-feathered broilers

[0055] QRT-PCR technology was used to analyze candidate genes. FOS , DDAH1 and PTN The expression differences between high-abdominal-fat and low-abdominal-fat chickens were quantitatively detected, and the results are as follows: Figure 2 As shown. Figure 3 To verify FOS , DDAH1 , PTN The results for the three genes in the high and low abdominal fat groups were consistent with the transcriptome sequencing results.

[0056] VII. Tissue Expression Profile Analysis

[0057] Detection using QRT-PCRFOS , DDAH1 and PTN Gene expression profiles were analyzed in five tissues: liver, pectoral muscles, back fat, leg muscles, and abdominal fat. Figure 4 As shown FOS , DDAH1 The gene was most highly expressed in the long-range abdominal fat tissue of chickens; while PTN The gene is most highly expressed in leg muscles, but its expression level differs significantly between individuals with high and low abdominal fat. Therefore... FOS , DDAH1 and PTN It was selected as a key candidate gene closely related to the abdominal fat trait in chickens and can be used as a molecular marker for trait prediction and molecular-assisted breeding in chicken breeding.

[0058] QRT-PCR system and reaction program: The internal control gene was β-actin. All reaction solutions were prepared on ice throughout the process. Three technical replicates were used for each gene, and real-time quantitative PCR was performed using the SYBR Green method. Relative expression levels were calculated using the 2-ΔΔCT method. All obtained 2-ΔΔCT values ​​were plotted and differential expression significance analyzed using Prism software. QRT-PCR reaction system: A 15.0 μL reaction system consisted of 7.5 μL 2×TB Green Premix Ex Taq II (Tli RNase HPlus), 4.6 μL ddH2O, 1.5 μL cDNA template, and 0.6 μL each of forward and reverse primers. The PCR reaction program was: pre-denaturation at 95 ℃ for 30 s; cyclic denaturation at 95 ℃ for 5 s; annealing at 60 ℃ for 30 s, for a total of 35 cycles.

[0059] As can be seen from the above, the molecular markers related to chicken abdominal fat traits screened by this invention... FOS , DDAH1 and PTN The gene is closely related to the abdominal fat trait in chickens and has a specific expression pattern. It can be used as a molecular marker for trait prediction and molecular-assisted breeding in chicken breeding.

[0060] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A genome related to abdominal fat traits in yellow-feathered broiler chickens, characterized in that, include FOS , DDAH1 , PTN One or more combinations of genes, the FOS , DDAH1 , PTN The nucleotide sequences of the genes are shown in SEQ ID NO.1-3, respectively.

2. The genome related to abdominal fat traits in yellow-feathered broiler chickens according to claim 1, characterized in that, include FOS , DDAH1 One or more combinations of genes.

3. The application of the genome as described in claim 1 or 2 in the prediction of abdominal fat traits and molecular-assisted breeding of yellow-feathered broilers.

4. The application of the genome as described in claim 1 or 2 in identifying abdominal fat traits in yellow-feathered broilers.

5. A method for screening genomes as described in claim 1 or 2, characterized in that, Includes the following steps: (1) Group selection and phenotypic collection; (2) RNA extraction, reverse transcription, and sequencing of the individuals to be tested; (3) Transcription data alignment: The gene data obtained from sequencing were aligned and analyzed using bioinformatics analysis software; (4) Screening and obtaining key genes affecting abdominal fat traits in yellow-feathered broilers: QRT-PCR technology was used to screen genes with significant differences. FOS , DDAH1 , PTN Quantitative detection was performed in chickens with high and low abdominal fat groups. Primers for candidate genes were designed and synthesized. β-actin , FOS , DDAH1 , PTN Primers for real-time quantitative PCR of genes were synthesized after checking their specificity. verify FOS , DDAH1 , PTN The results for the three genes in the high and low abdominal fat groups were consistent with the transcriptome sequencing results. (5) Tissue expression profile analysis: QRT-PCR was used for detection. FOS , DDAH1 and PTN The tissue expression profiles of the genes were analyzed, and the expression levels of the above genes in five tissues, namely liver, pectoral muscle, back fat, leg muscle and abdominal fat, were detected.

6. The screening method according to claim 5, characterized in that, Step (2) Total RNA was extracted from tissues using the Trizol method, and cDNA was synthesized using a reverse transcription kit as a template for QRT-PCR.

7. The screening method according to claim 5, characterized in that, In step (5), the QRT-PCR reaction system is: 15.0 μL reaction system, including 7.5 μL 2×TB Green Premix Ex Taq II (Tli RNaseH Plus), 4.6 μL ddH2O, 1.5 μL cDNA template, and 0.6 μL each of forward and reverse primers; the PCR reaction program is: pre-denaturation 95 ℃, 30 s; cyclic denaturation 95 ℃, 5 s; annealing temperature 60 ℃ for 30 s, for a total of 35 cycles.

8. The screening method according to claim 5, characterized in that, In steps (4)-(5), the QRT-PCR is corrected using an internal reference gene, which is... β-actin All reaction solutions were prepared on ice throughout the process, with three technical replicates for each gene. Data were presented in 2... -△△CT The method is used to calculate the relative expression level.

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