A miRNA related to heterosis of egg laying number and continuous laying traits in chickens and its application

Abstract

The present invention discloses a miRNA related to the heterosis of the number of eggs and continuous laying traits of chickens and its application. The present invention has the beneficial effect that the present invention detects the miRNA expression profile of the ovarian tissue of a heterosis population by high-throughput sequencing technology, and screens out the miRNA significantly related to the heterosis of the number of eggs and continuous laying traits of chickens, which can be used as a molecular marker of the heterosis of the number of eggs and continuous laying traits, and applied to the breeding of the heterosis of the number of eggs and continuous laying traits of chickens, so as to improve the production performance of laying hens.

Description

Technical Field

[0001] The invention relates to the technical field of molecular markers, and in particular to a miRNA related to the heterosis of egg laying number and continuous laying traits of chickens and an application thereof. Background Art

[0002] Hybrid vigor refers to the phenomenon that the offspring of two populations with different genetic structures are superior to the average of their parents in terms of vitality, fertility and adaptability. Hybrid vigor is widely used to increase the yield of agricultural animals and plants, enhance their stress resistance and adaptability, and plays an important role in improving the efficiency of agricultural production. However, the utilization of hybrid vigor does not mean that hybridization alone can achieve satisfactory results. The same trait shows great differences in hybrid vigor between different parent combinations. In the actual breeding of livestock and poultry, it is often necessary to carry out hybridization experiments between different pure line parent groups, and screen parent combinations with high hybrid vigor for target traits based on the results of production performance measurement of hybrid offspring. However, hybridization experiments are time-consuming, labor-intensive and costly, and can usually only be carried out between a limited number of pure lines, which limits the development and utilization of hybrid vigor. In recent years, with the development of high-throughput sequencing technology and molecular biology, it has provided a platform for analyzing the genetic mechanism of hybrid vigor and predicting hybrid vigor for complex traits. More advanced technical means have been adopted; the number of eggs and continuous production traits of chickens are complex traits regulated by multiple factors and are the most important production performance indicators of laying hens; in the breeding practice of laying hens, it is necessary to measure or predict the heterosis of the number of eggs and continuous production traits; but there is currently a lack of effective molecular markers; according to the classical hypothesis of the mechanism of heterosis formation, the non-additive expression of genes or related regulatory elements is an important reason for the formation of heterosis; non-additive expression patterns include high-parent expression (the expression level of hybrid offspring is higher than the average expression level of parents), low-parent expression (the expression level of hybrid offspring is lower than the average expression level of parents), super-high-parent expression (the expression level of hybrid offspring is higher than the expression level of the higher parent), and super-low-parent expression (the expression level of hybrid offspring is lower than the expression level of the lower parent); mining genes or regulatory elements with non-additive expression as molecular markers has become an important way to predict heterosis and utilize it in breeding;

[0003] miRNA is a non-coding RNA with a length of about 22nt. It plays an important regulatory role in processes such as cell differentiation and proliferation. Previous studies have shown that miRNA is expressed in large quantities in the reproductive system and is a regulator of the reproductive function of livestock and poultry. It is closely related to the regulation of complex traits and is therefore a potential molecular marker. Summary of the invention

[0004] The purpose of the present invention is to solve the above problems, and to design a miRNA related to the heterosis of egg production and continuous production traits of chickens and its application.

[0005] The technical solution of the present invention to achieve the above object is that the present invention first provides a miRNA related to the heterosis of egg production and continuous production in chickens, and the nucleotide sequence thereof is SEQ ID No. 1: 5'-UACCCUGUAGAACCGAAUUUGU-3'.

[0006] As a preferred specific manner, a miRNA related to the heterosis of egg production and continuous production traits in chickens is used as a molecular marker in predicting the heterosis of egg production and continuous production traits in chickens.

[0007] As a preferred specific manner, a method for predicting heterosis of egg production and continuous egg production in chickens comprises the following steps:

[0008] (1) extracting total RNA from chicken ovarian tissue samples to be tested;

[0009] (2) Use miScript II RT kit for miRNA reverse transcription;

[0010] (3) Amplify the miRNA using a specific upstream primer for amplification of the target miRNA and a fluorescent quantitative PCR kit suitable for the tailing method; after amplification, use 2 -ΔΔCt The quantitative results were analyzed by the method;

[0011] (4) Compare the expression level of miRNA in the detected hybrid population with the average expression level of miRNA in the parental ovarian tissue, analyze the expression pattern, and predict hybrid vigor.

[0012] Among them, the chickens to be tested are Rock Island Red Chicken and White Leghorn Chicken.

[0013] The present invention screens out a miRNA significantly correlated with the heterosis of egg laying number and continuous laying traits of chickens by establishing pure breeding offspring of Rock Island Red chickens and White Leghorn chickens and reciprocal hybrid offspring, and by using high-throughput sequencing technology and identification of miRNA expression profiles of ovarian tissues in different combinations and differential expression pattern analysis. The nucleotide sequence of the miRNA is SEQ ID No. 1: 5'-UACCCUGUAGAACCGAAUUUGU-3'.

[0014] The present invention further verified through qRT-PCR experiments that the expression level of miRNA in the ovarian tissue of the hybrid population of Rock Island Red Chicken and White Leghorn Chicken was higher than the average expression level of the purebred offspring of the two strains, which showed hybrid vigor.

[0015] Therefore, the miRNAs related to the number of eggs laid and the heterosis of continuous laying traits in chickens can be used as molecular markers, including:

[0016] Predicting heterosis: detecting the expression level of the miRNA in the ovaries of hybrid chickens, comparing it with the average expression level of the miRNA in the ovarian tissues of the parents, and predicting the heterosis of the number of eggs and continuous production traits of the chickens based on the heterosis of the miRNA expression levels;

[0017] As a preferred specific method, a fluorescent quantitative PCR kit for predicting the hybrid vigor of egg production and continuous production traits in chickens includes: RNA reverse transcription reagent, specific amplification forward primer of target miRNA, specific amplification forward primer of internal reference gene U6, miRNA fluorescent quantitative PCR universal reverse primer, and fluorescent quantitative PCR reaction solution.

[0018] As a preferred specific manner, the specific amplification forward primer sequence of the target miRNA is SEQ ID No. 2, and its sequence is: 5'-CGCGTACCCTGTAGAACCGA-3';

[0019] The specific amplification forward primer sequence of the internal reference gene U6 is SEQ ID No. 3, and its sequence is: 5'-GTGACCCACGATGTGTATTCGC-3';

[0020] The universal reverse primer sequence of miRNA fluorescent quantitative PCR is SEQ ID No. 4, and its sequence is: 5'-CAGTGCGTGTCGTGGAGT-3'.

[0021] As a preferred specific manner, a fluorescent quantitative PCR kit for predicting the hybrid vigor of egg production and continuous production traits in chickens is used to predict the hybrid vigor of egg production and continuous production traits in Rock Island Red chickens and White Leghorn chickens.

[0022] The beneficial effect is that the present invention detects the miRNA expression profile of ovarian tissue of a population with heterotic vigor through high-throughput sequencing technology, and screens out miRNA that is significantly correlated with the heterotic vigor of egg laying number and continuous laying traits in chickens. The miRNA can be used as a molecular marker for the heterotic vigor of egg laying number and continuous laying traits, and can be applied to the breeding of heterotic vigor of egg laying number and continuous laying traits in chickens to improve the production performance of laying hens. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is the number of differentially expressed miRNAs in different comparison groups of hybrid and pure line populations in Example 2 of the present invention. DETAILED DESCRIPTION

[0024] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0025] Example 1 Determination of heterosis of egg production and continuous laying traits in chickens and collection of related samples

[0026] 1. Group building

[0027] Rhode Island Red (RR) and White Leghorn (WW) chickens were selected and purebred and hybridized in a male-female ratio of 1:8, that is, RR roosters and RR hens mated to produce RR purebred offspring, WW roosters and WW hens mated to produce WW purebred offspring, RR roosters and WW hens hybridized to produce RW orthogonal hybrid groups, WW roosters and RR hens hybridized to produce reciprocal hybrid groups, all groups were hatched in the same batch and raised in the same chicken house with the same management method.

[0028] 2. Phenotypic Analysis

[0029] When the chickens were 52 weeks old, the cumulative number of eggs laid and the longest consecutive laying days of each individual were counted.

[0030] 3. Test results

[0031] The results of the significance test showed that the cumulative number of eggs laid in 52 weeks and the longest consecutive laying days showed significant heterosis, among which the heterosis of egg laying number of RW and WR were 20.28 and 25.73, respectively, and the heterosis of the longest consecutive laying days were 1.57 days and 2.28 days, respectively (Table 1).

[0032] Table 1 Phenotype and heterosis of egg production and longest continuous laying days in purebred and crossbred groups

[0033]

[0034]

[0035] Note: ψ1: pure line vs hybrid, ψ2: RR vs WW, ψ3: RW vs WR

[0036] 4. Sample Collection

[0037] When the chickens were 53 weeks old, 4 individuals were randomly selected from each of the four groups, WW, RR, WR, and RW, for ovarian tissue sample collection. The samples were temporarily stored in liquid nitrogen and then transferred to a -80°C refrigerator for later use.

[0038] Example 2 Identification of miRNAs associated with heterosis in egg production and continuous production

[0039] 1. RNA Sample Preparation

[0040] In a sterile environment, TRIzol kit (Invitrogen, USA) was used to extract total RNA from ovarian tissues of four individuals in each group of RR, RW, WR and RR, and the purity and concentration of RNA were detected by Nano Drop ND-1000, and the RIN value of RNA was detected by Bioanalyzer 2100. Qualified samples required that the ratio of OD260nm and OD280nm was within the range of 1.8 to 2.2, RIN ≥ 7 and 28S / 18S ≥ 1.0, and the concentration was ≥ 65 ng / μL.

[0041] 2. Small RNA library construction and sequencing

[0042] Small RNA includes miRNA, siRNA, piRNA and others. Due to the large number of miRNA genes and their rich expression, miRNA occupies a major position in the data obtained by Small RNA sequencing. The construction of the Small RNA library is carried out according to the steps required by the MirVanakit library construction kit. After the total RNA is subjected to PAGE gel electrophoresis, the gel is cut to recover 10-40nt fragments, and adapter sequences are added to both ends; then the obtained fragments are reverse transcribed and amplified by PCR, and the purified and quality-checked small RNA library is single-end sequenced on the Illumina TruSeq sequencer to obtain 50bp of original sequencing sequence data.

[0043] 3. Identification of miRNA

[0044] The original sequencing data was quality controlled to obtain clean reads. The Gallus-gallus 6.0 version genome was used as the reference genome by Bowtie 1 (http: / / bowtie-bio.sourceforge.net / index.shtml) to obtain the evaluation value of sequencing quality. The clean reads were compared with the database and small RNA was annotated. The small RNA was compared with the known chicken miRNA precursor sequences included in the miRBase database, and the sequencing sequences that could be compared with the chicken miRNA precursor sequences were identified as known chicken miRNAs. Unannotated small RNAs were used for new miRNA prediction. MirDeep2 software was used to evaluate whether each sequence had miRNA potential based on its secondary structure, Dicer restriction site, energy and other information.

[0045] 4. Analysis of miRNA expression patterns and screening of miRNA molecular markers

[0046] The expression levels of miRNAs in each sample were statistically analyzed and normalized using the TPM method. DEGseq (v1.18.0) was used to analyze the differentially expressed miRNAs, and Log2|Fold Change|>1 and FDR<0.05 were used as the differential gene screening criteria to identify the differentially expressed miRNAs in each comparison group. According to the classical hypothesis of the mechanism of hybrid vigor formation, non-additive expression of genes or related regulatory elements is an important reason for the formation of hybrid vigor. Non-additive expression patterns include high-parent expression (the expression level of the hybrid offspring is significantly higher than the average of the parental expression levels), low-parent expression (the expression level of the hybrid offspring is significantly lower than the average of the parental expression levels), ultra-high-parent expression (the expression level of the hybrid offspring is significantly higher than the expression level of the higher parent), and ultra-low-parent expression (the expression level of the hybrid offspring is significantly lower than the expression level of the lower parent). The average expression level of the pure line parents WW and RR is defined as A. The miRNAs differentially expressed between WR and A and the miRNAs differentially expressed between RW and A are non-additively expressed miRNAs, which are related to hybrid vigor. For example Figure 1 As shown in the figure, 21 differentially expressed miRNAs were identified in RW and A, of which 8 were highly expressed (up-regulated miRNAs) and 13 were low-expressed (down-regulated miRNAs). In WR and A, 40 differentially expressed miRNAs were identified, of which 14 were highly expressed (up-regulated miRNAs) and 26 were low-expressed (down-regulated miRNAs). The expression patterns of the 20 identified miRNAs are shown in Table 2. Moreover, gga-miR-10b-5p, gga-miR-19a-3p and gga-miR-7b had high basal expression levels in ovarian tissue (Raw read counts), and showed a low-expression pattern in both orthogonal and reciprocal populations, which was related to hybrid vigor and was a potential factor in the formation of hybrid vigor.

[0047] Table 2 Specific expression patterns of non-additively expressed miRNAs associated with heterosis

[0048]

[0049] Example 4 Prediction of heterosis of egg laying number and continuous laying traits by detecting miRNA expression by fluorescence quantitative PCR

[0050] Three individuals were selected from each of the four groups, RR, WW, WR and RW, to measure the number of eggs and continuous laying traits, including the number of eggs laid at 32 weeks, the number of eggs laid at 53 weeks, the longest continuous laying days at 32 weeks, and the longest continuous laying days at 53 weeks. Ovarian tissue was extracted and the expression level of gga-miR-10b-5p was quantified. The sequence of gga-miR-10b-5p was 5'-UACCCUGUAGAACCGAAUUUGU-3' (SEQ ID No. 1).

[0051] 1. Reverse Transcription and qRT-PCR

[0052] According to the RNA concentration, the total RNA of all samples was diluted and balanced to a concentration of 12.5 ng / μL using RNase-free ddH2O. The RNA was reverse transcribed into cDNA using the miScript II reverse transcription kit from Qiagen. The miScript SYBR Green PCR kit from Qiagen was used for quantification. The fluorescence quantitative PCR reaction was performed on an ABI7500 fluorescence quantitative PCR instrument, and U6 was selected as the internal reference gene to adjust the relative quantification of the detected miRNA. The universal reverse primer sequence of miRNA fluorescence quantitative PCR was 5'-CAGTGCGTGTCGTGGAGT-3' (SEQ ID No. 4), the specific amplification forward primer sequence of gga-miR-10b-5p was 5'-CGCGTACCCTGTAGAACCGA-3' (SEQ ID No. 2), and the specific amplification forward primer sequence of the internal reference gene U6 was 5'-GTGACCCACGATGTGTATTCGC-3' (SEQ ID No. 3). The fluorescence quantitative PCR reaction system was 20 μL, including 2×QuantiTect SYBR Green PCR Master Mix 10 μL, 10×miScript Universal Primer 2 μL, 10×miScript Primer Assay 2 μL, cDNA 1 μL, RNase-free water 5 μL. The reaction program was: 95°C for 15 min, 40 cycles (94°C for 15 s, 55°C for 30 s, 70°C for 30 s).

[0053] 2. Data Analysis and Results

[0054] Exploit 2 -△△CtThe relative expression of miRNAs between samples in each group was calculated by the method, and the phenotype and gene expression results of the above individuals are shown in Table 3. The expression pattern of gga-miR-10b-5p was a low-parent expression pattern in both WR and RW individuals. Both WR and RW showed significant heterosis in the number of eggs laid at 32 weeks (EN32) and the longest consecutive days of laying at 32 weeks (CS32), which once again proved that the identified miRNA gga-miR-10b-5p can be used to predict the heterosis of egg laying and continuous laying traits in chickens. In addition, since gga-miR-10b-5p was highly expressed in ovarian tissue and showed a consistent low-parent expression pattern in both orthogonal and reciprocal populations, the correlation coefficient between the heterosis of individual EN32 and the heterosis of gga-miR-10b-5 expression was 0.66, and the correlation coefficient between the heterosis of individual CS32 and the heterosis of gga-miR-10b-5 expression was 0.76. Therefore, it can be used as a preferred molecular marker for predicting egg production and continuous production traits and their heterosis.

[0055]

[0056]

[0057] The above technical solutions only reflect the preferred technical solutions of the technical solutions of the present invention. Some changes that may be made to certain parts thereof by technicians in this technical field all reflect the principles of the present invention and fall within the protection scope of the present invention.

Claims

1. An application of miRNA related to heterosis of egg production and continuous production in chickens as a molecular marker in predicting heterosis of egg production and continuous production in chickens, characterized in that: The nucleotide sequence of the miRNA is SEQ ID No. 1: 5'-UACCCUGUAGAACCGAAUUUGU-3'; The chicken is a Rock Island Red chicken or a White Leghorn chicken.

2. The use according to claim 1, characterized in that The steps include: (1) extracting total RNA from chicken ovarian tissue samples to be tested; (2) Use miScript II RT kit for miRNA reverse transcription; (3) Amplifying the mi RNA using a specific upstream primer for amplification of the target mi RNA and a fluorescent quantitative PCR kit suitable for the tailing method; after amplification, using 2 -ΔΔCt The quantitative results were analyzed by the method; (4) Compare the expression level of miRNA in the detected hybrid population with the average expression level of miRNA in the parental ovarian tissue, analyze the expression pattern, and predict hybrid vigor.

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