Key gene for duck meat and egg character differentiation and method for excavating key gene based on whole genome sequencing

Abstract

The invention relates to a key gene for duck meat and egg character differentiation and a method for excavating the key gene based on whole genome sequencing, the key gene is a duck SIK1 gene, and the nucleotide sequence of the key gene is shown as SEQ ID NO: 1. The key gene SIK1 influencing the differentiation and domestication process of laying ducks and meat ducks is found by using a method for analyzing the key gene in the domestication process of duck meat traits and egg traits through whole genome re-sequencing. The gene SIK1 plays a role in regulating glycometabolism and cell proliferation. The genome of the Jinding duck is assembled by using a whole genome re-sequencing technology for the first time and is compared with the genome of other duck species such as Beijing duck, another key gene SIK1 for meat and egg trait differentiation of ducks is excavated, molecular breeding of ducks is facilitated, and a new thought is provided for searching key genes for meat / egg duck trait differentiation in future.

Description

Technical Field

[0001] The present invention belongs to the technical field of agriculture and animal husbandry, and more specifically relates to a whole genome resequencing method for analyzing the domestication process of duck meat traits and egg traits, and the method obtains the duck SIK1 gene, a key gene affecting the differentiation and domestication process of laying ducks and meat ducks. Background Art

[0002] Duck is a general term for waterfowl of the Anatidae family, subfamily Anatidae. It is one of the most widely distributed waterfowl species in the world and has important economic value. Scientific research on duck breeding and improvement helps to improve the quality of duck products and production efficiency. According to economic value, ducks are generally divided into three types: meat-producing type, egg-producing type and dual-purpose type. Among them, Beijing duck is the representative of meat-producing type, Jinding duck is the representative of egg-producing type, and Gaoyou duck is the representative of dual-purpose type. There are great differences in body shape, growth rate and egg production among the above three types of ducks. Although there have been many literature reports, the differentiation of duck egg-producing and meat-producing traits and their controlling genes are still unclear.

[0003] Since the breeding characteristics of ducks are closely related to the environment, it is difficult for traditional breeding methods to directly select the correct genotype from the phenotype, so duck breeding and trait improvement are greatly limited. The combination of molecular breeding and traditional breeding methods to efficiently and quickly cultivate new varieties with high yield and high quality is a new trend in poultry breeding, providing new ideas and approaches for poultry breeding. However, the premise of molecular breeding is to have a clear understanding of the molecular genetic basis and functional genes of the target traits.

[0004] In recent years, with the continuous development of sequencing technology, many poultry breeds, such as ducks, chickens and geese, have completed the sequencing of reference genomes, which enables the exploration of the relationship between genetic variation and phenotypic polymorphism at the whole genome level, greatly accelerating the breeding process of livestock and providing a solid theoretical basis for molecular breeding. The present invention sequences the whole genome of Jinding duck and compares it with the genomes of other duck species such as Beijing duck to explore the key genes for the differentiation of duck egg and meat traits, which is helpful for the molecular breeding of ducks. Summary of the invention

[0005] The purpose of the present invention is to analyze the key genes in the domestication process of duck meat traits and egg traits using the whole genome resequencing method. The SIK1 gene, a key gene affecting the differentiation and domestication process of laying ducks and meat ducks, was discovered through the whole genome resequencing method of the present invention.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] The invention provides a key gene for differentiation of duck meat and egg traits, which is duck SIK1 gene, and the nucleotide sequence of the gene is shown in SEQ ID NO:1.

[0008]

[0009] The application of the key gene, the duck SIK1 gene is used in the molecular breeding of duck meat traits and egg traits; during the domestication process of egg-laying ducks and meat ducks, egg-laying ability and fat deposition are differentiated, and the duck SIK1 gene plays a role in regulating sugar metabolism and cell proliferation in meat ducks.

[0010] The invention relates to an application of the key gene in the preparation of a duck growth promoter. The duck SIK1 gene plays a role in regulating sugar metabolism and cell proliferation, so the duck SIK1 gene can be used in the preparation of a duck growth promoter. It can also be used in the preparation of a reagent for detecting the growth rate of a duck.

[0011] A kit for detecting the growth rate of ducks comprises a reagent for detecting duck SIK1 gene.

[0012] The reagent includes primers for detecting duck SIK1 gene; the primers are Real-Time PCR primers, and the sequence thereof is:

[0013] Upstream primer: 5'-CTGTACGTCCTTGTCTGTGGTT-3' (SEQ ID NO: 2),

[0014] Downstream primer: 5'-CGGATTAGCGTTTCACAGTCTTC-3' (SEQ ID NO: 3).

[0015] A siRNA targeting the key gene, wherein the siRNA is 3-si-SIK1, the sense strand of which is: 5'-GCUUCAGCAUCACCAGCUATT-3' (SEQ ID NO: 4), and the antisense strand of which is: 5'-UAGCUGGUGAUGCUGAAGCTT-3' (SEQ ID NO: 5).

[0016] 3-si-SIK1 has a high interference efficiency and can be used in cell transfection experiments. It can also be used in molecular breeding for duck meat and egg traits.

[0017] A method for analyzing key genes in the domestication process of duck meat and egg traits using whole genome resequencing, comprising the following steps:

[0018] S1. Nanopore sequencing and Hi-C technology were used to construct egg-laying duck chromosomes. After constructing the Hi-C library, the Hi-C assembly data was analyzed to annotate the genome and repetitive sequences, predict the gene structure, non-coding RNA and pseudogenes of the genome, and annotate the gene function;

[0019] S2. Resequence some local duck breeds; by comparing the genomes of laying ducks and meat ducks, detect candidate differential regions (CDRs) of autosomes, search for extreme differences in allele frequency (FST) and regions with the largest differences in genetic diversity (πlog ratio), perform principal component analysis on genetic data to analyze population structure, detect homozygous SNPs and indels, and obtain differential sites between sequenced samples and reference genomes;

[0020] S3. When predicting gene mutation regions with potential high / moderate functional effects, it was found that the high mutation regions may contain SIK1, a key gene that affects the differentiation and domestication process of egg-laying ducks and meat ducks.

[0021] Before step S1, the present invention also performs sample collection, library preparation and sequencing steps, and the specific method is: downloading the genomes of wild ducks and meat ducks, identifying potential target genome regions; taking a certain number of wild ducks for resequencing, taking a certain number of laying ducks for third-generation and second-generation genome sequencing analysis, and reassembling the genome; rapidly freezing the blood and tissue samples of all the wild ducks and laying ducks to -20°C, and then extracting total DNA; in addition, slaughtering under the same processing conditions, and measuring the weight and appearance of the ducks. The laying ducks selected in this method are Jinding ducks, and the meat ducks are Beijing ducks.

[0022] (1) Compared with the prior art, the advantages of the present invention are as follows: the present invention is the first to use whole genome resequencing technology to assemble the genome of Jinding duck, and compare it with the genomes of other duck species such as Beijing duck, thereby discovering another key gene SIK1 gene for the differentiation of duck meat and egg traits, which is helpful for the molecular breeding of ducks and provides a new idea for exploring the key genes for the differentiation of meat / eating duck traits in the future.

[0023] (2) The present invention uses Nanopore and Hi-C sequencing technologies to obtain high-quality genome assembly, and found more than 25 million domestication-related SNPs, insertions and deletions, and structural variations in wild ducks / meat ducks / laying ducks. SIK1 and IGF2 genes were screened out as possible key genes for the differentiation of laying ducks and meat ducks. The experiment found that high-sugar culture led to increased SIK1 mRNA and protein expression levels in duck embryo fibroblasts. High-sugar culture and silencing of SIK1 can both induce cell proliferation, while overexpression of SIK1 can inhibit cell proliferation. Duck SIK1 may have the function of regulating sugar metabolism and cell proliferation.

[0024] (3) The technical solution of the invention is complete and comprehensive, the data is detailed, and the experimental results are reliable. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the qPCR result of SIK1 gene interference and overexpression in duck embryo fibroblasts, among which, Figure 1a is the SIK1 mRNA expression level after SIK1 gene interference, showing that 3-si-SIK1 expression level is low and has a high interference efficiency; Figure 1 b shows the expression level of SIK1 mRNA after overexpression of SIK1 gene.

[0026] Figure 2 is the Jinding duck genome information, including Figure 2 a is the genome assembly and annotation map of Jinding duck, Figure 2 b is the heat map of Hi-C assembled chromosomes, Figure 2 c is the collinearity between Beijing duck and Jinding duck.

[0027] Figure 3 is the resequencing data of different duck breeds; among them, Figure 3 a is the duck phylogeny and population structure analysis, Figure 3 b is the population structure of wild ducks and domestic ducks, Figure 3 c is some pictures of Chinese duck breeds.

[0028] Figure 4 It is a selective sweep gene map.

[0029] Figure 5 This is the KEGG pathway map of IGF2.

[0030] Figure 6 This is the KEGG pathway map of SIK1.

[0031] Figure 7 It is the expression of SIK1 gene in different parts of Jinding duck.

[0032] Figure 8 These are the CCK8 detection results of the effects of high glucose culture and SiRNA-SIK1 and pcDNA3.1-SIK1 on the proliferation of duck embryo fibroblasts. Note: **P<001NC vs NC(25mM Glucose); Δp<0.05SiRNA-NC vs SiRNA-NC(25mM Glucose) / SiRNA-SIK1; ΔΔp<0.01SiRNA-NC vs SiRNA-SIK1(25mM Glucose); #p<0.05pcDNA3.1 vs pcDNA3.1(25mM Glucose) / pcDNA3.1-SIK1(25mM Glucose); ##p<0.01pcDNA3.1 vs pcDNA3.1-SIK1.

[0033] Fig. 9The graph shows the mRNA expression levels of SIK1 and IGF2 after SIK1 intervention and high glucose culture. Note: **P<001NC vs NC(25mM Glucose); ΔΔp<0.01SiRNA-NC vs SiRNA-NC(25mM Glucose); NSp>0.05SiRNA-NC vs SiRNA-SIK1 / SiRNA-SIK1(25mM Glucose); ##p<0.01pcDNA3.1 vs pcDNA3.1(25mMGlucose)

[0034] / pcDNA3.1-SIK1 / pcDNA3.1-SIK1 (25mM Glucose).

[0035] Fig.10 The cells are duck embryo fibroblasts after high glucose culture and SiRNA-SIK1 and pcDNA3.1-SIK1. Note: **P<0.01NC vs NC(25mM Glucose); Δp<0.05; ΔΔp<0.01SiRNA-NC vs SiRNA-NC(25mM Glucose) / SiRNA-SIK1 / SiRNA-SIK1(25mM Glucose); #p<0.05; ##p<0.01pcDNA3.1 vspcDNA3.1(25mM Glucose)

[0036] / pcDNA3.1-SIK1 / pcDNA3.1-SIK1 (25mM Glucose). DETAILED DESCRIPTION

[0037] The present invention is described in detail below with reference to the accompanying drawings and embodiments:

[0038] The present invention uses a whole genome resequencing method to analyze the key genes in the domestication process of duck meat traits and egg traits. To achieve the above purpose, the present invention adopts the following technical solutions:

[0039] 1.1 Sample collection, library preparation, and sequencing

[0040] The genomes of wild ducks and meat ducks (Peking ducks) were downloaded from the NCBI website to identify potential target genomic regions. In addition, the inventors collected wild ducks (Anas platyrhynchos) from Zhejiang Province and took 20 individuals for resequencing. Jinding ducks, as the main representative of laying ducks, come from Fujian Province. A total of 20 individuals (10 males and 10 females) were taken for third-generation and second-generation genome sequencing analysis, and genome reassembly. All blood and tissue samples were quickly frozen to -20°C, and total DNA was extracted using the traditional phenol-chloroform method. The ducks were slaughtered under the same treatment conditions, and the weight and appearance of the ducks were measured.

[0041] 1.2 Jinding duck genome assembly

[0042] 1.2.1 Nanopore sequencing

[0043] Nanopore sequencing technology was used to perform third-generation single-molecule sequencing on Jinding duck: extract high-quality DNA, randomly cut genomic DNA after passing the test; use magnetic beads to enrich and purify large DNA fragments, and then cut and recover large DNA fragments; fragment repair: damage repair, end repair and 3' end A addition to the selected large DNA fragments, and purify the reaction products; ligation reaction: ligation and purification of the fragment repair products to obtain the final library; quantitative detection: use Qubit to accurately quantify the established DNA library; library loading: take a certain amount of DNA library, mix it with the relevant reagents on the library, and add it to the Flowcell. Real-time single-molecule sequencing was performed on the Nanopore PromethION sequencer to obtain the original sequencing data.

[0044] 1.2.2 Hi-C library construction and data collection

[0045] The type of Hi-C was in situ Hi-C, which included steps such as cell cross-linking, endonuclease digestion, end repair, circularization, DNA purification capture, and sequencing. High-throughput sequencing was performed using the Illumina HiSeq 2000 platform with a read length of PE150.

[0046] Fine grapHI-C assembly. Clean Data was corrected using Canu software and assembled based on the corrected data. The third-generation sequencing data was corrected three times using Racon software, and the second-generation data was corrected three times using Pilon software.

[0047] Evaluation of assembly results. The assembly results were evaluated from the following three aspects: alignment rate of second-generation sequencing reads; core gene integrity; and BUSCO evaluation.

[0048] Clean Reads data was counted, with one unit for every four lines, and the two ends were counted separately, that is, read1 and read2 were recorded as two Reads. The Samtools flagstat command was used to calculate the percentage of clean reads that matched the reference genome. The samtools flagstat command located the two end sequencing sequences on the reference genome, and the distance was consistent with the length distribution of the sequencing fragments. BWAMEM was used for alignment (%) without parameters; BWA software compared the short sequences obtained by second-generation high-throughput sequencing (Illumina HiSeq sequencing platform) with the reference genome, and then the alignment rate was statistically evaluated to evaluate the completeness of the assembled genome.

[0049] The CEGMA v2.5 (default parameters) database contains 458 conserved core genes of eukaryotic organisms. CEGMA v2.5 assessed the completeness of the final genome assembly.

[0050] The BUSCO V2 database contains 2586 conserved core genes. The completeness of the genome assembly was assessed by BUSCO v2.0 software. BUSCO parameters: -evalue 1e-05 (BLAST search E-value cutoff), -sp generic (generic model).

[0051] 1.2.3HI-C assembly and analysis

[0052] The raw data of HI-C sequencing is filtered to remove the adapter sequences and low-quality reads to obtain high-quality Clean Data. The raw data is compared with the preliminarily assembled genome to obtain the mapped data. Based on the statistics of the mapped data, the comparison efficiency, insertion length, effective Hi-C data volume and coverage are calculated as the basis for evaluating the quality of the sequencing library. Finally, the Draft Genome sequence is further assembled using the effective Hi-C data, including the classification, sequencing and post-sequencing direction of the Draft Genome sequence, and the genome sequence is finally obtained, and the assembly results are evaluated. At the same time, the genome is finely annotated, including repetitive sequence annotation and transposon classification, coding gene prediction, non-coding RNA annotation and pseudogene annotation; gene function annotation, including NR library annotation, KEGG metabolic pathway annotation, COG annotation (geneontology), TrEMBL annotation and regulatory motif prediction.

[0053] 1.2.4 Genome annotation analysis

[0054] With reference to relevant literature, the software was used to analyze the information such as repetitive sequence annotation, coding gene prediction, non-coding RNA prediction, pseudogene annotation, gene function annotation in the Jinding duck genome, and the prediction results were evaluated.

[0055] 1.2.5 Repeat sequence annotation

[0056] Based on the principles of structure prediction and de novo prediction, the repetitive sequence database of the genome was established using LTR_FINDER and RepeatScout. The database was classified using PASTEClassifier and then compared with Repbase 21 The databases were merged to form the final repeat sequence database. Using RepeatMasker 22 The software predicts the repetitive sequences of the genome based on the constructed repetitive sequence database.

[0057] Repeat sequence software parameters: LTR_FINDER, RepeatScout and PASTEClassifier used default parameters. RepeatMasker parameters were -norna-engine-wublast.

[0058] 1.2.6 Prediction of coding genes

[0059] Three different strategies were used to predict the gene structure of the genome, including de novo prediction, homologous species prediction, and single gene prediction. EVM v1.1.1 software was used to integrate the prediction results; Genscan, Augustus V2.4, GlimmerHMM v3.0.4, GeneID v1.4, and SNAP (version 2006-07-28) were used for de novo prediction; Gemoma V1.3.1 was used to predict homologous species; HisatV2.0.4 and Stringtie V1.2.3 were used for reference transcript assembly, TransDecoderV2.0 and Genemarks-TV5.1 were used for gene prediction; PASAV2.0.2 was used for Unigene sequence prediction based on unreferenced transcriptome data. Finally, EVM V1.1.1 was used to integrate the prediction results of the above three methods, and PASAV2.0.2 was used to modify the results. The results are shown in the progress table.

[0060] 1.2.7 Non-coding RNA Prediction

[0061] Non-coding RNAs (ncRNAs) are non-protein coding RNAs, including microRNAs, rRNAs, and tRNAs with various known functions. Different strategies are used for prediction according to the structural characteristics of different non-coding RNAs.36 Database, perform Blastn alignment on the whole genome, and identify microRNA and rRNA.

[0062] 1.2.8 Pseudogene Annotation

[0063] Pseudogenes have sequences similar to functional genes, but lose their original functions due to mutations such as insertions or deletions. Use the predicted protein sequence and BLAST alignment to search for homologous gene sequences (possible genes) on the genome. Then, use GeneWise to search for premature stop codons and frameshift mutations in gene sequences to obtain pseudogenes. GenBlastA: -e1E-5 (Blast parameter: e value), other parameters are default. GeneWise software parameters are default.

[0064] 1.2.9 Gene function annotation

[0065] The predicted gene sequences were compared with the functional databases NR, KOG, GO, KEGG, and TrEMB by BLAST V2.2.31 (-EVALue 1E-5). KEGG pathway analysis, KOG functional analysis, and GO functional analysis were used for gene function annotation.

[0066] 1.3 Whole-genome resequencing

[0067] Agarose gel electrophoresis was used to determine the quality and quantity of the DNA samples for resequencing analysis of Fujian local varieties. Paired-end libraries of each qualified sample were generated using standard procedures. The average insert length was 500 bp and the average read length was 150 bp. All libraries were The average raw read sequence coverage of each sample was 10×. The sequencing depth ensured the accuracy of mutation screening and genotyping, meeting the requirements of population genetic analysis. The uploaded resequencing data were downloaded from NCBI, and a total of 119 DNA samples from 13 varieties were screened.

[0068] 1.3.1SNP / indel

[0069] In order to find out the key genes for the differentiation of laying ducks, meat ducks and wild ducks, we aligned the paired end reads of different samples with the reference genome, detected homozygous SNPs and indels, and obtained the differential sites between the sequenced samples and the reference genome. The raw reads of the double-end 150bp sequencing were aligned with the reference genome using Burrows-Wheeler alignment (BWAAlN) (default parameters). According to the gene annotation of the reference genome, the identified SNP / indel was further classified using SnpEff software.

[0070] 1.3.2 Selective Scan Detection

[0071] A genome-wide search for highly differentiated regions between wild ducks, laying ducks, and broiler ducks was performed. The fitness index (FST) and p-values ​​of the likelihood ratio test were calculated using vcfflibs (https: / / github.com / vcflflib / vcflflib#vcflflib).

[0072] 1.3.3 Principal Component Analysis

[0073] All SNPs were analyzed by principal component analysis using EIGENSOFT software (version 4.2). This software package was used to perform principal component analysis on genetic data and analyze population structure.

[0074] 1.3.4 SIK1 gene sequence analysis

[0075] Based on the resequencing data, the differential SNP sites of different samples were found. Primers were designed using Primer Premier software, and the annealing temperature is shown in Appendix 3. The PCR products were sequenced using Sanger sequencing technology to further correct the GAP sequence.

[0076] 1.4 SIK1 gene function verification

[0077] 1.4.1 Cultivation and transfection of duck embryo fibroblasts

[0078] Duck embryo cells (Shanghai Cell Bank, Chinese Academy of Sciences) were subcultured in duck embryo-specific culture medium (IMMOCELL, Xiamen, China) at 37°C and 5% CO2 for subsequent RNA interference efficiency, overexpression efficiency analysis and high glucose induction experiments.

[0079] In order to confirm the role of SIK1 under high glucose induction, siRNA-SIK1 and pcDNA3.1-SIK1 (duck SIK1 gene interference and overexpression vectors, see below) were constructed, and duck embryo fibroblasts were transiently transfected and then cultured in high glucose induction. The steps are as follows:

[0080] Group setting: control group, siRNA-NC group, siRNA-SIK1 group, pcDNA3.1 group, pcDNA3.1-SIK1 group, three replicates in each group;

[0081] Transfection reagent preparation:

[0082] siRNA-NC and siRNA-SIK1 groups: 125 μl serum-free medium + 5 μl RNA reagent + 4 μl LipoRNAi reagent mixed;

[0083] pcDNA3.1 and pcDNA3.1-SIK1 groups: 125 μl serum-free culture medium (Thermo Fisher Scientific, Waltham, USA, the same below) + 2.5 μl plasmid (1 ug / μl) + 4 μl Lipo8000 (Beyotime, Shanghai, China) reagent were mixed.

[0084] When the confluence of duck embryo fibroblasts reached about 60%, fresh culture medium was replaced; 125 μl of transfection reagent was added to each well of the experimental group, and 125 μl of serum-free culture medium was added to each well of the control group. After 8 hours of transfection, the supernatant was removed, and the cells were washed with PBS, and 2 mL of fresh culture medium was added to continue culturing; 48 hours after transfection, normal (RPMI 1640 sugar-free culture medium (Procell, Wuhan, China) + 10% FBS + 5mM D-glucose solution (Procell, Wuhan, China)) and high-glucose culture medium (RPMI 1640 sugar-free culture medium + 10% FBS + 25mM D-glucose solution) were added for culture, and the cells were collected after 24 hours. qPCR was used to detect the mRNA levels of SIK1 and IGF2 in duck embryo fibroblasts; Western Blot was used to detect the protein level, and CCK8 colorimetric method was used to detect the proliferation activity of cells.

[0085] 1.4.2 siRNA synthesis and interference efficiency analysis

[0086] According to the duck SIK1 gene sequence (sequence number: XM_005026639.5), 1-si-SIK1, 2-si-SIK1 and 3-si-SIK1 siRNAs and negative control siRNA (si-NC) were designed and synthesized by Huzhou Hippo Biotechnology Co., Ltd. The siRNA sequences are shown in Table 1.

[0087] Duck embryo fibroblasts in the logarithmic growth phase were taken and when the cells grew close to 50% to 60% confluence, siRNA transfection was performed according to the instructions of the Lipofectmine 3000 kit (Thermo Fisher Scientific, Waltham, USA, the same below). siRNA (si-NC, 1-si-SIK1, 2-si-SIK1 and 3-si-SIK1) and liposome Lipofectamine3000 were diluted with 2 ml of serum-free Opi-MEM medium, so that the final concentration of the four siRNAs was 100 nmol / L. Cells were collected 48 hours after transfection, and the SIK1 mRNA level was detected by qPCR to analyze the interference efficiency. 3-si-SIK1 has a higher interference efficiency ( Figure 1), used in cell transfection experiments, or as siRNA of SIK1 gene, involved in molecular breeding for duck meat and egg traits.

[0088] Table 1 Specific siRNA sequences

[0089]

[0090] 1.4.3 Analysis of the efficiency of pcDNA3.1-SIK1 overexpression vector

[0091] Duck embryo fibroblasts were cultured at 5×10 ^5 The cells were inoculated into 6-well plates at a density of 100 cells / ml and incubated overnight at 37°C in a 5% CO2 incubator. When the confluence of duck embryo fibroblasts reached 70-80%, the cells were inoculated with Lipofectamine TM 3000 instructions were used to transfect pcDNA3.1-SIK1 overexpression plasmid and pcDNA3.1 control plasmid, respectively. Fresh complete culture medium was replaced after 8 hours. Cells were collected after 48 hours, and SIK1 mRNA levels were detected by qPCR to analyze the overexpression efficiency.

[0092] 1.4.4 Cell proliferation assay

[0093] In order to explore the regulatory effect of SIK1 on the proliferation of duck embryo fibroblasts, after overexpression / interference of SIK1 under normal and high-glucose culture conditions (specific transfection methods and grouping are the same as above), 6 duplicate wells were set up in each group, and the proliferation of duck embryo fibroblasts was detected by CCK8 colorimetry. After the cell culture was completed, 10 μl of CCK8 reagent (Dojin Chemical Research Institute, Japan) was added to each well, and the cells were placed in the incubator for 2-4 hours; the absorbance of each well was measured at 450 nm using an ELISA reader (Molecular Devices, USA), and the values ​​were statistically analyzed and plotted using GraphPadPrism 8.0.

[0094] 1.4.5 Western Blot Analysis

[0095] Total protein was extracted using a total protein extraction kit (containing Protease Inhibitor Cocktail) (Thermo Pierce, USA), and then total protein was quantified using a BCA quantification kit (Biyuntian). Equal amounts of protein were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Bio-Rad) and electrotransferred to a PVDF membrane. After transfer, T-TBS (containing 5% BSA) was blocked at room temperature for 1 hour. SIK1 (Affinity, 1:500), IGF2 (Affinity, 1:500) and β-actin (Affinity, 1:10000) primary antibodies were incubated at 4°C overnight. Then, it was incubated with goat anti-mouse or goat anti-rabbit IgG (1:5000) at room temperature for 1 hour. Finally, an enhanced chemiluminescence (ECL) kit ( WestDura Extended Duration Substrate, Thermo Pierce) was used for band visualization. Immunoreactive bands were quantified by optical density using Image J software and normalized to β-actin signal.

[0096] 1.5 Tissue-specific expression of SIK1

[0097] The heart, liver, spleen, lung, kidney, stomach, ovary, chest, skin and other tissues of Jinding duck were taken for qPCR detection of SIK1 gene. Duck embryo fibroblasts from each group were collected for qPCR detection of SIK1 gene and IGF2. Primers were designed by PrimerPremier software, and the annealing temperature is shown in Table 2. Total RNA of cells was extracted using a total RNA kit (Thermo Fisher, USA). The RNA concentration was determined using a Nanodrop spectrophotometer (Thermo Fisher, USA). All samples were subjected to 3 physical replicates, using 2 -△△Ct Methods Data were processed. The reference gene was glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

[0098] Table 2 Real-Time PCR primers and conditions

[0099]

[0100] 1.6 Statistical analysis

[0101] SPSS19.0 software was used for statistical analysis, and the results were expressed as mean±SEM. The means between the two groups were compared with the independent sample t-test, and the means between multiple groups were compared with one-way ANOVA. The difference was considered significant when P<0.05(*), and extremely significant when P<0.01(**).

[0102] This invention uses whole genome resequencing technology for the first time to assemble the genome of Jinding duck, and compares it with the genomes of other duck species such as Beijing duck, to explore the key genes for the differentiation of duck meat and egg-laying traits, which will help the molecular breeding of ducks and provide new ideas for exploring the key genes for the differentiation of meat / laying duck traits in the future.

[0103] 2. Results

[0104] 2.1 Genome assembly and annotation

[0105] In section 1.2 of the present invention, Jinding duck chromosomes were constructed based on nanopore and HI-C technology, in which the third-generation nanopore sequencing obtained a total of 170.26Gb of data. After data quality control, the data volume was 155.14Gb, and the average read length reached 21.35Kb. The genome sequence was 1.15Gb, and the contig N50 was 13.17Mb.

[0106] Hi-C obtained 31 chromosomes (29 autosomes and chromosomes Z and W) with a length of 1.13Gb, accounting for 98.06%. The length of the sequence and direction on the chromosome that can be determined is 1.11Gb, accounting for 97.71% of the total length of the chromosome sequence. A total of 141.72Mb of repetitive sequences, 18672 genes, 393 tRNAs, 97 rRNAs, 216 miRNAs and 776 pseudogenes were predicted. In the NR, GO, KOG and KEGG databases, 94.15% of the genes can be annotated.

[0107] Compared with previous studies (Peking duck genome: https: / / www.ncbi.nlm.nih.gov / genome / ?term= txi d8839 , Table 3), the present invention significantly improves the assembly quality by combining multiple sequencing technologies, further improving the integrity of the Jinding duck genome and the accurate assembly of highly complex regions. According to the BUSCO score (95.28%), the conservative genomic elements in the genome are relatively complete (Table 4), while the BUSCO score of the previous Beijing duck genome assembly was less than 91%. The Jinding duck genome has significantly improved continuity and integrity compared to the Beijing duck.

[0108] Table 3 Genome information: Peking and Jinding ducks

[0109]

[0110] Table 4 BUSCO evaluation results

[0111]

[0112] After Hi-C assembly and manual adjustment, the 1.13Gb genome sequence was mapped to 31 chromosomes, accounting for 98.06%, and the number of sequences was 400, accounting for 77.67% ( Figure 2 a). The total length of the sequences on the chromosome that can determine the sequence and direction is 1.11Gb, accounting for 97.71% of the total length of the chromosome sequence. After error correction, Contig N50 and Scaffold are 10.90Mb and 76.08Mb respectively. The genome at the chromosome level obtained by Hi-C assembly is cut into 100Kb / bin, and then Hi-C ReadPairs are inserted between any two bins as the intensity signal of the interaction between the two bins. The heat map is drawn as follows Figure 1 As shown in b. It can be seen that the chromosomes are clearly divided into 31 groups; there is a strong interaction between adjacent sequences near the diagonal of the chromosome, while the interaction signal intensity between adjacent sequences in the non-diagonal position is weak, which is consistent with the principle of Hi-C assisted genome assembly and proves that the genome assembly is effective ( Figure 2 b). The perfect match between marker sequences in the radiation hybridization map indicates the accuracy and completeness of the assembly. The colinearity of the three genomes indicates that the assembly structure is consistent ( Figure 2 c).

[0113] In part 1.3 of this invention, 13 Chinese native duck breeds, 4 wild ducks and 36 ducks (including Peking ducks), were resequenced with an average depth of ×10. Principal component analysis (PCA) divided the samples into four groups: wild ducks, Muscovy ducks, meat ducks, and laying ducks. Adjacent native duck breeds (JD / SM / PT / LC(R)) clustered more closely, suggesting that they have a closer genetic relationship and may have a similar domestication history ( Figure 3 a / b). Figure 3 b is the phylogenetic tree of 119 duck species based on Bayesian clustering model, with K ranging from 2 to 5. The branch colors of the developmental tree represent different species (consistent with the colors shown in a). In besk K=4, there are four subgroups. FF: Muscovy duck, MDZ: wild duck, MEAT: meat duck, EGG: laying duck.

[0114] 2.2 Screening of candidate differentially expressed regions (CDRs)

[0115] By comparing the egg-laying and meat-duck genomes, we detected candidate differential regions (CDRs) on the autosomes and searched for regions with the greatest differences in allele frequency (FST) and genetic diversity (πlog ratio). We identified two CDRs (SIK1 and IGF2) that reached a significant level of P<0.005 ( Figure 4 ).

[0116] IGF2 is related to individual growth rate, lean meat rate and back fat thickness. IGF2 (insulin-like growth factor 2) is located on chromosome 5, and there are significant differences between egg-laying ducks and meat ducks. IGF is the most complex and diverse growth factor known to date. IGF2 can promote cell mitosis and differentiation, participate in postpartum genome reconstruction, and play an important role in X chromosome inactivation. These functions all show that IGF2 plays an important role in growth and development. IGF2 is closely related to production performance such as individual growth rate, lean meat rate, back fat thickness, and has been used as a candidate gene in some species ( Figure 5 ).

[0117] During the analysis, we found a CDR gene located on chromosome 1, the SIK1 gene (salt-induced kinase 1), which encodes a serine / threonine protein kinase, with significant differences between laying ducks and meat ducks. It contains a ubiquitin-associated (UBA) domain-encoded protein and is a member of the adenosine phosphate-activated protein kinase (AMPK) subfamily of kinases in the conserved signal transduction pathway. The SIK gene has not been reported in poultry, but it plays an important role in human cancer research, mouse insulin regulation, and the regulation of HepG2 cell lipid deposition. In particular, SIK1 can phosphorylate CRTC2 (cAMP-regulated transcriptional coactivator), inactivating CRTC2 and inhibiting the expression of the gluconeogenesis rate-limiting enzyme gene downstream of CRTC2-CREB ( Figure 6 Abnormal gluconeogenesis leads to increased hepatic glucose output and fasting blood glucose, which is closely related to metabolic diseases. Therefore, we speculate that SIK1 mutation may be one of the key factors in the differentiation of egg-laying ducks and meat ducks.

[0118] The present invention conducted a tissue-specific expression experiment of SIK1 in section 1.5. The expression results of SIK1 gene in different tissues of Jinding duck showed that SIK1 gene was expressed in all parts of the body, with the highest expression level in the lung and the lowest in the liver. There was no significant difference in other tissues ( Figure 7 ). We speculate that this phenomenon is related to the function of SIK1. SIK1 is mainly involved in gluconeogenesis and affects the regulation of intracellular lipid deposition. Under normal circumstances, the lung is very active and the glycogen consumption rate is accelerated. It is necessary to increase the supply of glycogen by increasing gluconeogenesis and other reactions. The liver is mainly involved in detoxification reactions. Under normal circumstances, it does not need to consume a large amount of sugar, so the expression level of the SIK1 gene is slightly low.

[0119] 2.3 Identification of SIK1 gene function

[0120] Part 1.4 of the present invention verifies the function of SIK1 gene, and CCK8 is detected in duck embryo fibroblasts after transfection with si-SIK1, pcDNA3.1 SIK1 and high glucose culture. The results show that:

[0121] Compared with the pure blank group (NC group), the OD450 value of the high sugar group increased, and the difference was statistically significant (P<0.01).

[0122] After silencing SIK1, the OD450 values ​​of the SiRNA-NC high glucose group, SiRNA-SIK1 OD450, and SiRNA-SIK1 high glucose group were significantly increased compared with those of the SiRNA-NC group (P<0.01).

[0123] After overexpression of SIK1, compared with the pcDNA3.1 group, the OD450 value of the pcDNA3.1 high-glucose group was upregulated, while the OD450 of the pcDNA3.1-SIK1 and pcDNA3.1-SIK1 high-glucose groups was significantly downregulated (P<0.01).

[0124] The results showed that both high-glucose culture and Si-SIK1 could induce cell proliferation, while pcDNA3.1-SIK1 could inhibit cell proliferation, and this inhibition could not be relieved by high-glucose culture. Figure 8 What I see.

[0125] 2.4 High glucose and SIK1 interference affect SIK1 and IGF2 expression

[0126] After transfection of si-SIK1 and pcDNA3.1 SIK1, duck embryo fibroblasts were cultured under high glucose conditions, and the expression trends of SIK1 and IGF2 mRNA were similar, such as Fig. 9 As shown. Compared with the pure blank group (NC group), the expression of SIK1 and IGF2 in the NC (25mM Glucose) group was significantly increased (P<0.01); compared with the SiRNA-NC empty group, the expression levels of SIK1 and IGF2 in the SiRNA-NC (25mM Glucose) group were significantly increased (P<0.01); there was no significant change in SIK1 expression between the SiRNA-SIK1 group and the SiRNA-SIK1 (25mM Glucose) group (P>0.05); compared with the pcDNA3.1 group, the expression of SIK1 and IGF2 in the pcDNA3.1 (25mM Glucose) group, pcDNA3.1-SIK1 group, and pcDNA3.1-SIK1 (25mM Glucose) group were significantly increased (P<0.01). These results indicate that high glucose and overexpression of SIK1 can change the expression levels of SIK1 and IGF2 mRNA, but silencing SIK1 has no significant effect on the expression levels of SIK1 and IGF2 mRNA.

[0127] Western Blot results showed that ( Fig.10): Compared with the NC group, the expressions of SIK1 and IGF2 were upregulated in the NC(25mM Glucose) group (P<0.01). Compared with the Si-NC group, the expressions of SIK1 and IGF2 were upregulated in the siRNA-NC(25mM Glucose) group (P<0.01). The expressions of SIK1 and IGF2 were downregulated in the siRNA-SIK1 group (P<0.01). In the siRNA-SIK1(25mM Glucose) group, the expression of SIK1 was downregulated (P<0.01) while the expression of IGF2 was upregulated (P<0.05). Compared with the pcDNA3.1 group, the expressions of SIK1 and IGF2 were upregulated in pcDNA3.1(25mM Glucose), pcDNA3.1-SIK1 and pcDNA3.1-SIK1(25mM Glucose) (P<0.01 or P<0.05).

[0128] The present invention constructs a high-quality genome of Jinding duck. The 1.13GB length sequence was assembled onto 31 chromosomes (29 autologous, Z and W chromosomes), and SNPs, insertions and deletions and structural variations were screened out. These results are helpful for designing specific genome selection programs. More than 25 million SNPs, insertions and deletions and structural variations associated with domestication were found in 13 wild ducks / laying ducks / meat ducks. These sites will provide a reference for studying the genetic differences of domestic ducks / wild ducks and the differentiation of laying ducks / meat ducks. When predicting gene mutation regions with potential high / moderate functional effects, we found that some mutation regions may contain key genes that affect the differentiation of laying ducks and meat ducks, such as SIK1 and IGF2 genes, especially SIK1 gene. During the domestication process of laying ducks and meat ducks, egg-laying ability and fat deposition have differentiated. The role of duck SIK1 in regulating sugar metabolism and cell proliferation may play an important role in this, and its mechanism of action on ducks needs further study.

[0129] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. A key gene for differentiation of duck meat and egg traits, characterized by: It is a duck SIK1 gene, and its nucleotide sequence is shown in SEQ ID NO:

1.

2. An application of the key gene as claimed in claim 1, characterized in that: The duck SIK1 gene is used in molecular breeding for domestication of duck meat traits and egg traits.

3. Use of the key gene as claimed in claim 1 in the preparation of a duck growth promoter.

4. A kit for detecting the growth rate of ducks, characterized in that: It includes a reagent for detecting the duck SIK1 gene.

5. The kit according to claim 4, characterized in that: The reagent includes primers for detecting duck SIK1 gene; the primers are Real-Time PCR primers, and the sequence thereof is: Upstream primer: 5'-CTGTACGTCCTTGTCTGTGGTT-3', Downstream primer: 5′-CGGATTAGCGTTTCACAGTCTTC-3′.

6. A siRNA targeting the key gene as claimed in claim 1, characterized in that: The siRNA is 3-si-SIK1, the sense strand of which is: 5'-GCUUCAGCAUCACCAGCUATT-3', and the antisense strand of which is: 5'-UAGCUGGUGAUGCUGAAGCTT-3'.

7. The use of siRNA as claimed in claim 6, characterized in that: The siRNA is applied to the molecular breeding for domestication of duck meat traits and egg traits.

8. A method for analyzing key genes in the domestication process of duck meat and egg traits using whole genome resequencing, characterized in that: It includes the following steps: S1. Nanopore sequencing and Hi-C technology were used to construct egg-laying duck chromosomes. After constructing the Hi-C library, the Hi-C assembly data was analyzed to annotate the genome and repetitive sequences, predict the gene structure, non-coding RNA and pseudogenes of the genome, and annotate the gene function; S2. Resequence some local duck breeds; by comparing the genomes of laying ducks and meat ducks, detect candidate differential regions (CDRs) of autosomes, search for extreme differences in allele frequency (FST) and regions with the largest differences in genetic diversity (πlog ratio), perform principal component analysis on genetic data to analyze population structure, detect homozygous SNPs and indels, and obtain differential sites between sequenced samples and reference genomes; S3. When predicting gene mutation regions with potential high / moderate functional effects, it was found that the high mutation regions may contain SIK1, a key gene that affects the differentiation and domestication process of egg-laying ducks and meat ducks.

9. The method according to claim 7, characterized in that: Before step S1, sample collection, library preparation and sequencing steps were also performed. The specific method was as follows: download the genomes of wild ducks and meat ducks to identify potential target genomic regions; take a certain number of wild ducks for resequencing, and take a certain number of laying ducks for third-generation and second-generation genome sequencing analysis, and perform genome reassembly; quickly freeze the blood and tissue samples of all the above wild ducks and laying ducks to -20°C, and then extract total DNA; in addition, slaughter under the same processing conditions, and measure the ducks' weight, appearance and other traits.

10. The method according to claim 8 or 9, characterized in that: The egg-laying duck selected in the method is Jinding duck, and the meat duck is Beijing duck.

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