GENE-40367; No. of pages: 5; 4C: Gene xxx (2015) xxx–xxx
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Effect of polymorphism within miRNA-1606 gene on growth and carcass traits in chicken
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Hong Li, Shanhe Wang, Fengbin Yan, Xiaojun Liu, Ruirui Jiang, Ruili Han, Zhuanjian Li, Guoxi Li, Yadong Tian, Xiangtao Kang ⁎, Guirong Sun ⁎
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College of Animal Science and Veterinary Medicine, Henan Agricultural University, Henan Innovative Engineering Research Center of Poultry Germplasm Resource, Zhengzhou 450002, PR China
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Article history: Received 18 November 2014 Received in revised form 28 February 2015 Accepted 16 March 2015 Available online xxxx
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Keyword: Chicken Polymorphisms MiRNA-1606 Phenotypic variation Association analysis
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Genetic variations in microRNAs (miRNAs) including primary miRNAs, precursor miRNAs and mature miRNAs can lead to phenotypic variation by altering the biogenesis of miRNAs and/or their binding to target mRNAs. Increasing functional studies suggest that polymorphisms occurring in miRNAs can lead to phenotypic variation in farm animal. Here, we identified a single nucleotide polymorphism (SNP) located in the precursor of chicken miRNA-1606 gene. The association study on body indexes, body weight at different growth stages, and carcass traits was performed in a Gushi–Anka F2 population resource. The SNP was not only significantly associated with body weight at 10 and 12 weeks, respectively, but also with chicken shank length, chest depth and body slanting length at 8 weeks; shank length, pectoral angle, body slanting length and pelvis breadth at 12 weeks, respectively. And the polymorphism was also significantly associated with carcass traits including semievisceration weight, evisceration weight, breast muscle weight, leg weight and carcass weight as well. The observed values of individuals with CA genotype were significantly higher than CC genotype both in body weight at different stages and carcass traits. This SNP altered the predicted second structure of pre-mir-1606, with the altering of the free energy values. And the relative expression level of mature miRNA between CA and AA was significantly changed in leg muscle. Our data suggested that miRNA-1606 may be a candidate gene associated with chicken growth traits. © 2015 Published by Elsevier B.V.
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1. Introduction
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MicroRNAs (miRNAs) are short, non-coding RNA molecules that post-transcriptionally modulate gene expression in a sequence specific manner. Emerging evidence suggests that miRNAs are involved in a broad range of biological processes, such as embryonic development (Wang et al., 2013), cellular differentiation and proliferation (Shi et al., 2014; Zhang et al., 2012), skeletal muscle growth (Luo et al., 2013) and fat deposition (Lin et al., 2012), and oocyte maturation and ovarian follicular development (Fiedler et al., 2008). Genetic variations in miRNAs including primary miRNA (pri-miRNA), precursor miRNA (pre-miRNA) and mature miRNAs can alter the biogenesis of miRNAs and/or their binding to target mRNAs (Duan et al., 2007; Georges
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Abbreviations: miRNA, microRNA; SNP, single nucleotide polymorphism; PCR-RFLP, polymerase chain reaction-restriction fragment length polymorphism; qRT-PCR, quantitative real-time polymerase chain reaction; Pri-miRNA, primary miRNA; Pre-miRNA, precursor miRNA; gga, Gallus gallus; BW, body weight; SL, shank length; SG, shank girth; CD, chest depth; CB, chest breadth; BBL, breast bone length; PA, pectoral angle; BSL, body slanting length;PB, pelvisbreadth;CW, carcass weight;SEW, semi-evisceration weight; EW, evisceration weight; AFW, abdominal fat weight; LW, leg weight; LMW, leg muscle weight; BMW, breast muscle weight. ⁎ Corresponding authors. E-mail addresses: [email protected] (X. Kang), [email protected] (G. Sun).
et al., 2007; Slaby et al., 2012). Single nucleotide polymorphisms (SNPs) that reside within the miRNA genes may modulate the transcription of pri-miRNA transcripts and the stability or processing of pri- and pre-miRNA, thereby leading to either an increase or decrease in mature miRNA levels. On the other hand, changes in the sequence of mature miRNAs can have an impact on their interaction with target mRNAs. Because miRNAs potentially regulate the expression of multiple targets, base changes in miRNA genes can have a functional effect and contribute to phenotypic variation via deregulation of target genes (Hong et al., 2012). Limited functional researches about miRNAs in farm animals indicate that the miRNAs play key roles in muscle development (Lin et al., 2012), oocyte maturation (Kang et al., 2013) and early embryonic development (Darnell et al., 2006), and adipose tissue growth (Li et al., 2011; Yao et al., 2011). Increasing evidence suggests that SNP in miRNA genes or miRNA target sites may contribute to change in production or health traits in farm animals. Mutation created in the 3′ UTR of GDF8 gene of Texel sheep resulted in a new target site that allowed miR-1 and miR-206 to bind simultaneously, which led to the expression of the target gene MSTN reduction and muscle hypertrophy (Clop et al., 2006). Our previous work suggested that a point mutation occurring in Gallus gallus (gga) miR-1657 was significantly associated with body indexes and meat quality traits (Li et al., 2012).
http://dx.doi.org/10.1016/j.gene.2015.03.037 0378-1119/© 2015 Published by Elsevier B.V.
Please cite this article as: Li, H., et al., Effect of polymorphism within miRNA-1606 gene on growth and carcass traits in chicken, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.03.037
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2.1. Animals and trait measurements
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A Gushi–Anka F2 resource population which was created by Henan Innovative Engineering Research Center of Poultry Germplasm Resource as described previously (Han et al., 2012; Li et al., 2012) was used as the study material. In detail, the population was a cross of a slow-growing Chinese native breed (Gushi chicken) and a fastgrowing broiler (Anka chicken). Through two hatches for an interval of two weeks, a total of 860 F2 chickens were produced. All the chickens were managed in cages under the same environment with free access to feed and water, and fed according to the Chinese National Research Council (1994) recommendations. Body weight (BW) was weighed individually at birth, 2, 4, 6, 8, 10, and 12 weeks, and body indexes such as shank length (SL), shank girth (SG), chest depth (CD), chest breadth (CB), breast bone length (BBL), pectoral angle (PA), body slanting length (BSL) and pelvis breadth (PB) were measured at 4, 8 and 12 weeks, respectively. Body slanting length (BSL) was measured as the distance between chicken anterior/superior joint of the clavicle to the ischial tuberosity with a tape. PA was measured as the angle of breastbone front end using Pectoral Angle caliper. CB, CD and PB were measured with vernier caliper. CB was measured as the distance between chicken shoulder joint. CD was measured as the distance between the first thoracic vertebrae and the front of breastbone. PB also named back width was measured as the distance between the outer edges of chicken lions in both sides. All the chickens were slaughtered at the age of 12 weeks. Blood samples were collected from the F2 individuals and stored at − 80 °C. The carcass traits including carcass weight (CW), semi-evisceration weight (SEW, the carcass weight excluding the trachea, esophagus, crop, intestine, spleen, pancreas, gallbladder and reproductive organs), evisceration weight (EW, the SEW excluding the heart, liver, proventriculus, gizzard, head, feet and abdominal fat), abdominal fat weight (AFW), leg weight (LW), leg muscle weight (LMW), and breast muscle weight (BMW) were measured. All the detailed measuring methods were described previously by Han et al. (2011).
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2.2. Polymorphism detection
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Genomic DNA was extracted from the 860 chickens EDTAanticoagulated peripheral blood samples using the standard phenol–chloroform methods. Considering the DNA work concentration requirement for MALDI-TOF MS genotyping, all the DNA samples were modulated to a adjust concentration and stored at − 80 °C until use. One hundred DNA samples were selected randomly, and taken the equal amount from each DNA sample to construct a DNA pool. A pair of amplification primers was designed according to the public chicken gene sequence (GenBank accession no: NC_006091. 3). The primers (F: 5′-ATGGCAGGTAAACATTTGAG-3′, R: 5′-GATT TGCTCAGCCCCAGCTTC-3′) were used to amplify a fragment containing the precursor region of miR-1606 gene sequence (GenBank accession no: MI0007333). The PCR product was sequenced by Sangon Bio Co., Shanghai to detect polymorphisms. All the DNA samples were sent to the company to test the SNP by using MALDI-TOF MS assay technology.
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2.4. The prediction of pre-miR-1606 secondary structure
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Due to the rs313467992 SNP occurring in the precursor region of miRNA-1606, the most stable secondary structure of pre-gga-mir-1606 with the lowest free energy was calculated by using M-fold software (Zuker, 2003). The absolute difference in the free energy for pre-mir1606 with different alleles was used as the parameter for the assessment of the impact on the secondary structure of pre-miR-1606.
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2.5. Expression detection of miR-1606
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To evaluate the effect of different alleles on the expression of mature miR-1606, real time PCR was conducted. A quantitative reverse transcription-PCR approach was used to determine the levels of mature miR-1606. The frozen leg muscle tissue and breast muscle of F2 chickens of the indicated genotypes were selected randomly each for six. Total RNA was isolated by Trizol (Invitrogen) according to the manufacturer's instruction. RNA quality was detected by electrophoresis (Formaldehyde RNA gel) and the NanoDropTM1000 spectrophotometer (Thermo Scientific). cDNA was synthesized using a cDNA Synthesis kit (Invitrogen) according to the manufacturer's instruction. Real-time PCR was performed using SYBR Green method in Roche instrument LightCycler®96. The level of mature miR-1606 was normalized to U6 snRNA. The loop primers used for reverse-transcription and the real time of miR-1606 and U6 snRNA genes were designed (Chen et al., 2005) and sent to the company to synthesize (Supplementary Table 1). The real-time PCR amplification process was as follows: 95 °C for 3 min, 40 cycles of 95 °C for 12 s, 62 °C for 40 s, 72 °C for 30 s, and followed by a further 10 min extension at 72 °C. The expression values of different genotypes were analyzed by using 2−△△ct method (Schmittgen and Livak, 2008), and significant effects of different genotypes were statistically tested by one-way ANOVA.
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All statistical analyses were performed by Statistical Package Service Solution software (SPSS for Windows, Standard version 19.0; SPSS, USA). The correlations between polymorphism and chicken related economic characteristics were analyzed by linear mixed models. Model I was used to evaluate the body weight and body index traits. Considering the effect of body weight difference on carcass traits, model II was applied to analyze the carcass traits, and carcass weight was used as the concomitant variable in model II. In case the effect of genotype was significant, the Bonferroni test was carried out to control for multiple comparisons of the genotypes. The P-value b 0.05 was considered statistically significant. The mixed linear models were as follows:
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Model I : Y ijklm ¼ μ þ Gi þ S j þ H k þ f l þ eijkl m
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The rs313467992 SNP occurring in miR-1606 (+ 35 CNA) in the Gushi–Anka F2 resource population was genotyped by Sequenom MassArray_ iPLEX GOLD System. For the SNP, a pair of amplification primers and an extension primer was designed using Assay Design 3.1 software (Sequenom Inc., USA). The 1st PCR primer sequence of premir-1606 polymorphism was 5′-ACGTTGGATGAACAAGCAGCGTGAAT GGTC-3′; the 2nd PCR primer sequence was 5′-ACGTTGGATGTTCAGG ACTGTTCTGTTGCC-3′, and the extension primer was 5′-GTTGCCCCCT CTTTCAGT-3′. The SNP was genotyped by MassArray matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (Sequenom) following the manufacturer's instructions (Oeth et al., 2005). Related data as the call rate and peak area were acquired through using the software provided by the manufacturer. Alleles were automatically assigned by the software.
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In this study, we identified a SNP located in the precursor region of the gga-miR-1606 and investigated its association with body weight, body indexes at different developmental stages, and carcass traits in chickens. The effect of the miR-1606 SNP on the secondary structure of pre-miRNA and the expression level of mature miR-1606 were also examined. Collectively, our data suggested a potential role for miR-1606 in regulating the growth development.
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Model II : Y ijklm ¼ μ þ Gi þ S j þ H k þ f l þ b W ijklm −W þ eijklm
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where Yijklm represents the observe value; μ the overall mean, Gi the fixed effect of genotype (i = CC, CA, AA); fl the random effect of family (l = 7); Sj fixed effect of sex (j = f, m), Hk the fixed effect of hatch (k = 2), and eijklm the random error. b is the regression coefficient for carcass weight; Wijklm the individual carcass weight; W the average carcass weight.
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3. Results
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3.1. Identification and genotyping of miR-1606 SNP
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The PCR product sequencing results were compared with the published chicken genome sequence, and the result suggested that a CNA mutation located in +35 of pre-miR-1606 (Supplementary Fig. 1) existed in the population. The genotyping calls for the variant (Supplementary Fig. 2) were the same as our sequencing alignment result, and the mass spectrogram of variant genotypes CC, CA, and AA is summarized in Supplementary Fig. 3, respectively.
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3.2. Association of the miR-1606 SNP with growth and carcass traits
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To investigate the functional effect of the +35 CN A polymorphism in miR-1606 precursor on chicken phenotypic traits, the association of the SNP with body weight at different developmental stages (Table 1), body indexes at 4 weeks, 8 weeks and 12 weeks (Table 2), and carcass traits at 12 weeks (Table 3) was performed. To the Gushi–Anka F2 resource population, the + 35 C N A had a significant effect on BW at the age of 10 weeks and 12 weeks. The SNP was significantly associated with chicken SL, CD and BSL at 8 weeks (P b 0.05). Significance was also found among traits such as SL, BSL, PA and PD at 12 weeks (P b 0.05). The effect of the SNP on carcass traits, including SEW, EW, BMW, LW and CW was significant (P b 0.05) as listed in Table 3. These suggest that the mutation indirectly contributes to body index at different times and carcass traits in chicken. As for the effect of the genotypes on growth and carcass traits, the results showed that the values of CA genotype individuals were the highest, and significance was observed between CA and CC genotypes (P b 0.05), but the difference between CA and AA genotypes was not significant (P N 0.05).
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P-value
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SL (cm) SG (cm) CD (cm) CB (cm) BBL (cm) PA (°) BSL (cm) PB (cm) SL (cm) SG (cm) CD (cm) CB (cm) BBL (cm) PA (°) BSL (cm) PB (cm) SL (cm) SG (cm) CD (cm) CB (cm) BBL (cm) PA (°) BSL (cm) PB (cm)
5.50 ± 0.08 2.69 ± 0.03 4.79 ± 0.049 4.05 ± 0.05 6.17 ± 0.07 73.83 ± 0.45 11.40 ± 0.24 5.14 ± 0.06 7.84 ± 0.07 3.41 ± 0.03 6.45 ± 0.08ab 5.65 ± 0.06 8.91 ± 0.08 75.90 ± 0.52 16.28 ± 0.12ab 6.85 ± 0.08 9.43 ± 0.08 3.86 ± 0.03 7.88 ± 0.07 6.31 ± 0.08 11.05 ± 0.10 78.25 ± 0.48B 19.85 ± 0.13a 8.67 ± 0.07AB
5.46 ± 0.07 2.71 ± 0.03 4.86 ± 0.04 4.09 ± 0.04 6.23 ± 0.06 74.13 ± 0.36 11.61 ± 0.18 5.17 ± 0.04 7.96 ± 0.06 3.42 ± 0.03 6.62 ± 0.07a 5.71 ± 0.05 8.88 ± 0.07 76.80 ± 0.44 16.30 ± 0.10a 6.95 ± 0.06 9.40 ± 0.08 3.84 ± 0.03 7.91 ± 0.06 6.35 ± 0.07 10.98 ± 0.09 79.46 ± 0.42A 19.80 ± 0.12a 8.77 ± 0.05A
5.50 ± 0.08 2.66 ± 0.03 4.84 ± 0.05 4.13 ± 0.05 6.18 ± 0.07 74.28 ± 0.47 11.34 ± 0.26 5.17 ± 0.06 7.80 ± 0.07 3.40 ± 0.03 6.40 ± 0.09b 5.58 ± 0.06 8.91 ± 0.08 76.09 ± 0.54 16.00 ± 0.12b 6.81 ± 0.08 9.26 ± 0.09 3.79 ± 0.03 7.81 ± 0.08 6.27 ± 0.08 10.86 ± 0.10 79.01 ± 0.50AB 19.50 ± 0.14b 8.50 ± 0.07B
0.796 0.069 0.419 0.445 0.329 0.703 0.521 0.836 0.032 0.666 0.012 0.051 0.768 0.096 0.017 0.158 0.046 0.052 0.434 0.369 0.062 0.008 0.013 0.004
t2:5 t2:6 t2:7 t2:8 t2:9 t2:10 t2:11 t2:12 t2:13 t2:14 t2:15 t2:16 t2:17 t2:18 t2:19 t2:20 t2:21 t2:22 t2:23 t2:24 t2:25 t2:26 t2:27 t2:28
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Note: SL = shank length; SG = shank girth, CD = chest depth; CB = chest breadth; BBL = t2:29 breast bone length, PA = pectoral angle; PB = pelvis breadth, BSL = body slanting length. t2:30 Means with different superscripts in the line mean significant difference: small letter t2:31 means P b 0.05, capital letter means P b 0.01. t2:32
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Table 2 Effects of the miR-1606 SNP on the growth traits investigated in chicken.
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To estimate the functional relevance of the SNP with mature miR1606 expression, we first determined the levels of mature miR-1606 in F2 chickens with different homozygous and heterozygote genotypes using qRT-PCR. The result showed that the alteration of the relative expression value of mature miR-1606 between AA and CA genotypes was significant in leg muscle tissue (P b 0.05), but the alteration of the expression level of miR-1606 between the CC and AA genotypes was not significant in either breast or leg muscle (P N 0.05) (Fig. 2).
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3.3. Second structure alterations of the miR-1606 gene precursor
4. Discussion
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miRNA is a class of important regulators in the biology process. Many miRNAs have been identified in all kinds of farm animal species. Related researches have revealed that the miRNA plays a crucial role in many aspects of animal development (Kloosterman and Plasterk, 2006; Plasterk, 2006) and physiology (Fatima et al., 2014). Understanding the functions of miRNAs in farm animals is essential to know how
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M-fold software was used to check the alteration of RNA secondary structure led by the alleles of miR-1606 SNP. The result showed that the C NA substitution located in the precursor region of miR-1606 could introduce a base-pairing mismatch, and led to generation of new RNA bulges in the predicted second structure, with the altering of the free energy values (Fig. 1).
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Table 1 Effects of the miR-1606 SNP on body weight at different stages in chicken.
Table 3 Effects of the miR-1606 SNP on carcass traits in chicken.
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BW0 (g) BW2 (g) BW4 (g) BW6 (g) BW8 (g) BW10 (g) BW12 (g)
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30.47 ± 0.94 121.81 ± 3.25 317.82 ± 8.12 554.40 ± 14.46 816.14 ± 22.03 1103.23 ± 27.58AB 1341.42 ± 30.40
31.58 ± 0.75 124.14 ± 2.96 323.55 ± 7.64 565.64 ± 13.59 818.00 ± 20.77 1126.39 ± 26.01A 1365.10 ± 28.46
30.46 ± 0.99 119.75 ± 3.38 315.14 ± 8.31 553.86 ± 14.79 790.23 ± 22.43 1078.29 ± 28.02B 1319.61 ± 31.14
0.350 0.172 0.153 0.218 0.087 0.007 0.041
Note: BW0, 2, 4, 6, 8, 10, 12 = body weight at the age of 0 day, 2, 4, 6, 8, 10 and 12 weeks, respectively. Means with different superscripts in the line mean significant difference: small letter means P b 0.05, capital letter means P b 0.01.
Carcass traits SEW (g) EW (g) BMW (g) LW (g) LMW (g) CW (g)
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1094.10 ± 27.74ab 915.21 ± 25.17 69.00 ± 2.78ab 149.21 ± 4.52ab 98.81 ± 3.26 1204.91 ± 29.48ab
1114.19 ± 26.35a 930.43 ± 23.99 71.71 ± 2.66a 150.37 ± 4.34a 100.03 ± 3.12 1226.46 ± 27.93a
1070.48 ± 28.35b 893.35 ± 25.65 67.87 ± 2.84b 144.34 ± 4.60b 96.60 ± 3.32 1184.17 ± 30.15b
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P-value
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0.016 0.020 0.012 0.031 0.119 0.032
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Note: SEW = semi-evisceration weight; EW = evisceration weight; BMW = breast mus- t3:12 cle weight; LW = leg weight; LMW = leg muscle weight; CW = carcass weight. Means t3:13 with different superscripts in the line mean significant difference: small letter means P b t3:14 0.05.
Please cite this article as: Li, H., et al., Effect of polymorphism within miRNA-1606 gene on growth and carcass traits in chicken, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.03.037
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the various biological processes are regulated and to explore novel strategies to improve production efficiency in farm animals. Mutations in miRNAs resulting in aberrant miRNA-mediated gene regulation may modulate the transcription of pri-miRNA transcripts and the stability
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Fig. 1. The predicted secondary structure of the pre-mir-1606 with different alleles.
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Fig. 2. The relative expression level of mature miR-1606 in three different genotypes. The different superscripts mean significant difference P b 0.05.
or processing of pri- and pre-miRNAs, thereby leading to either an increase or decrease in mature miRNA levels, and may contribute to phenotypic variation (Georges et al., 2007). For example, Lei et al. reported that a T NC variation in porcine miR-27a gene was associated with litter size (Lei et al., 2011). SNPs in porcine miR-1 significantly altered the expression in the level of both precursor and mature miR-1, and associated with type I and type IIa muscle fibers in number and area compositions, respectively (Hong et al., 2012). SNPs that reside within the miRNA genes could regulate miRNA biogenesis and alter target selection, thereby potentially having profound biological effects (Duan et al., 2007). A single mutation in pre-miR-155 creating a mismatch near the 3′end of miR-155 leads to a shift in strand selection, thereby fine-tunes their targets and results in a butterfly effect on global gene expression (Lee et al., 2011). In this study, we identified a SNP in the precursor of chicken miR-1606 locus. The effects of the miR-1606 SNP on growth development and carcass traits were evaluated. Genotype analysis revealed significant associations with the body weight, body indexes at different developmental stages and carcass traits in chickens. The above statistical results showed that chickens with CA genotype related with greater body weight at 10 and 12 weeks (Table 1), and some body indexes at 8 and 12 weeks (Table 2). Many miRNAs are found to be expressed in a spatiotemporal-specific manner, which indicates that miRNAs play roles in specific tissue types or at specific developmental stages (Wang
Please cite this article as: Li, H., et al., Effect of polymorphism within miRNA-1606 gene on growth and carcass traits in chicken, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.03.037
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Conflict of interest
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This study was supported by the National Natural Science Foundation of China (No. 31201795), the Earmarked Fund for Modern Agro-industry Technology Research System (No. CARS-41-K04), the Ministry of Education Innovation Team Development Plan (No. IRT1236) and the Postdoctoral Science Foundation of China (2013M531675).
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In conclusion, we detected and identified the rs313467992 SNP within miR-1606 gene in the Gushi–Anka F2 population. The association analysis results indicated a promising impact of the SNP in miR-1606 gene on body weight and body indexes at different growth stages, and carcass traits. These results facilitate gene mining and provide a preliminary evidence for the possible functional role of miR-1606 in a large population, which could provide new insights into the potential role to be used as a genetic marker or molecular assist selection in animal breeding. However, we conclude that further research and validation of the allelic effects and functional mechanisms are needed. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.gene.2015.03.037.
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Common SNP in pre-miR-146a decreases mature miR expression and predisposes to papillary thyroid carcinoma. Proc. Natl. Acad. Sci. U. S. A. 105, 7269–7274. http://dx.doi.org/10.1073/pnas.0802682105. Kang, L., Cui, X.X., Zhang, Y.J., Yang, C.H., Jiang, Y.L., 2013. Identification of miRNAs associated with sexual maturity in chicken ovary by Illumina small RNA deep sequencing. BMC Genomics 14, 352. http://dx.doi.org/10.1186/1471-2164-14-352. Kloosterman, W.P., Plasterk, R.H.A., 2006. The diverse functions of microRNAs in animal development and disease. Dev. Cell 11, 441–450. http://dx.doi.org/10.1016/j.devcel.2006.09.009. Kunej, T., Skok, D.J., Simon, H., Peter, D., Jiang, Z.H., 2010. The glypican 3-hosted murine mir717 gene: sequence conservation, seed region polymorphisms and putative targets. Int. J. Biol. Sci. 6, 769–772. Lee, H.C., Yang, C.W., Chen, C.Y., Au, L.C., 2011. Single point mutation of microRNA may cause butterfly effect on alteration of global gene expression. Biochem. 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Let-7b regulates the expression of the growth hormone receptor gene in deletion-type dwarf chickens. BMC Genomics 13, 306. http://dx.doi.org/10.1186/14712164-13-306. Luo, W., Nie, Q., Zhang, X., 2013. MicroRNAs involved in skeletal muscle differentiation. J. Genet. Genomics 40, 107–116. http://dx.doi.org/10.1016/j.jgg.2013.02.002. Oeth, P., Martin, B., Park, C., K, Dominik, del M, Guy, van den B, Dirk, Jurinke, C., 2005. iPLEX™ assay: increased plexing efficiency and flexibility for MassARRAY® system through single base primer extension with mass-modified terminators. Sequenom Appl. Note 8876-006. Plasterk, R.H.A., 2006. Micro RNAs in animal development. Cell 124, 877–881. http://dx.doi.org/ 10.1016/j.cell.2006.02.030. Schmittgen, T.D., Livak, K.J., 2008. Analyzing real-time PCR data by the comparative CT method. Nat. Protoc. 3, 1101–1108. http://dx.doi.org/10.1038/nprot.2008.73. Shi, X.E., et al., 2014. MicroRNA-199a-5p affects porcine preadipocyte proliferation and differentiation. Int. J. Mol. Sci. 15, 8526–8538. http://dx.doi.org/10.3390/ijms15058526. Slaby, O., Bienertova-Vasku, Julie, Svoboda, M., Vyzula, R., 2012. Genetic polymorphisms and microRNAs: new direction in molecular epidemiology of solid cancer. J. Cell. Mol. Med. 16, 8–21. http://dx.doi.org/10.1111/j.1582-4934.2011.01359.x. Slezak-Prochazka, I., Durmus, S., Kroesen, B.J., van den Berg, A., 2010. MicroRNAs, macrocontrol: regulation of miRNA processing. RNA 16, 1087–1095. http://dx.doi.org/10.1261/rna. 1804410. Sun, G.H., Yan, J., Noltner, K., Feng, J.N., Li, H.T., Sarkis, D.A., Sommer, S.S., Rossi, J.J., 2009. SNPs in human miRNA genes affect biogenesis and function. RNA 15, 1640–1651. http://dx.doi.org/ 10.1261/rna.1560209. Wang, X., Gu, Z., Jiang, H., 2013. MicroRNAs in farm animals. Animal 7, 1567–1575. http://dx.doi. org/10.1017/S1751731113001183. Yao, J., Wang, Y.X., Wang, W.S., Wang, N., Li, H., 2011. Solexa sequencing analysis of chicken preadipocyte microRNAs. Biosci. Biotechnol. Biochem. 75, 54–61. http://dx.doi.org/10.1271/ bbb.100530. Zhang, J., Ying, Z.Z., Tang, Z.L., Long, L.Q., Li, K., 2012. MicroRNA-148a promotes myogenic differentiation by targeting the ROCK1 gene. J. Biol. Chem. 287, 21093–21101. http://dx.doi.org/ 10.1074/jbc.M111.330381. Zhu, Z., Gao, W., Qian, Z., Miao, Y., 2009. Genetic variation of miRNA sequence in pancreatic cancer. Acta Biochim. Biophys. Sin. 41, 407–413. http://dx.doi.org/10.1093/abbs/gmp023. Zuker, M., 2003. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 31, 3406–3415. http://dx.doi.org/10.1093/nar/gkg595.
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et al., 2013). Compared with CC and AA genotypes, the observed values of the CA genotype in CW, SEW, EW, BMW and LW are significantly larger (Table 3). The results implied that the CA genotype may be superior to the other genotypes in improving production performance. A point mutation within murine miR-717 was proven to affect fat deposition (Kunej et al., 2010). SNPs in the primary RNA miR-206/miR-133b cluster were found to be associated with muscle fiber characteristics, meat quality and lean meat production in Berkshire, Landrace and Yorkshire pig breeds (Lee et al., 2013). SNP in the primary region of miRNA can lead to a pronounced effect on the efficiency of the miRNA processing machinery (Slezak-Prochazka et al., 2010). Previous research reported that a mutation in pre-miR146a led to decrease in mature miRNA expression and predisposes to papillary thyroid carcinoma (Jazdzewski et al., 2008). The CNA SNP located in the precursor of miRNA-1606 led to a change in the secondary structure of pre-miRNA, and notably decreased the expression of mature miRNA in leg muscle tissue. Sun et al. had detected several SNPs occurring in human miRNA genes that could affect the biogenesis and function of miRNAs (Sun et al., 2009). Previous report showed that an AN G mutation at 29 nt downstream of pre-miR-21 led to a conformational change of the secondary structure and a relative reduction of the mature miR-21 expression in vivo (Zhu et al., 2009). Our result suggested that the SNP may alter the level of processing efficiency of premiRNA to mature miRNA or pri-miRNA to pre-miRNA, thereby resulting in the function alteration. Several functional studies of miRNAs in farm animal have indicated that miRNAs involve in a range of biology processes and play important roles in growth development in farm animals. Geng et al. suggested that the rs16681031 SNP in miR-1658* gene may play an important role in the formation of some phenotype of chicken (Geng et al., 2011). Given the important effect of rs313467992 SNP residing in miR-1606 on chicken body weight, body indexes and carcass traits, and the functional role of miR-1606 has not been revealed yet in any other species, it is necessary to further study the function of miR-1606 in different aspects.
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Please cite this article as: Li, H., et al., Effect of polymorphism within miRNA-1606 gene on growth and carcass traits in chicken, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.03.037
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Effect of polymorphism within miRNA-1606 gene on growth and carcass traits in chicken
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Hong Li, Shanhe Wang, Fengbin Yan, Xiaojun Liu, Ruirui Jiang, Ruili Han, Zhuanjian Li, Guoxi Li, Yadong Tian, Xiangtao Kang ⁎, Guirong Sun ⁎
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College of Animal Science and Veterinary Medicine, Henan Agricultural University, Henan Innovative Engineering Research Center of Poultry Germplasm Resource, Zhengzhou 450002, PR China
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Article history: Received 18 November 2014 Received in revised form 28 February 2015 Accepted 16 March 2015 Available online xxxx
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Keyword: Chicken Polymorphisms MiRNA-1606 Phenotypic variation Association analysis
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Genetic variations in microRNAs (miRNAs) including primary miRNAs, precursor miRNAs and mature miRNAs can lead to phenotypic variation by altering the biogenesis of miRNAs and/or their binding to target mRNAs. Increasing functional studies suggest that polymorphisms occurring in miRNAs can lead to phenotypic variation in farm animal. Here, we identified a single nucleotide polymorphism (SNP) located in the precursor of chicken miRNA-1606 gene. The association study on body indexes, body weight at different growth stages, and carcass traits was performed in a Gushi–Anka F2 population resource. The SNP was not only significantly associated with body weight at 10 and 12 weeks, respectively, but also with chicken shank length, chest depth and body slanting length at 8 weeks; shank length, pectoral angle, body slanting length and pelvis breadth at 12 weeks, respectively. And the polymorphism was also significantly associated with carcass traits including semievisceration weight, evisceration weight, breast muscle weight, leg weight and carcass weight as well. The observed values of individuals with CA genotype were significantly higher than CC genotype both in body weight at different stages and carcass traits. This SNP altered the predicted second structure of pre-mir-1606, with the altering of the free energy values. And the relative expression level of mature miRNA between CA and AA was significantly changed in leg muscle. Our data suggested that miRNA-1606 may be a candidate gene associated with chicken growth traits. © 2015 Published by Elsevier B.V.
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1. Introduction
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MicroRNAs (miRNAs) are short, non-coding RNA molecules that post-transcriptionally modulate gene expression in a sequence specific manner. Emerging evidence suggests that miRNAs are involved in a broad range of biological processes, such as embryonic development (Wang et al., 2013), cellular differentiation and proliferation (Shi et al., 2014; Zhang et al., 2012), skeletal muscle growth (Luo et al., 2013) and fat deposition (Lin et al., 2012), and oocyte maturation and ovarian follicular development (Fiedler et al., 2008). Genetic variations in miRNAs including primary miRNA (pri-miRNA), precursor miRNA (pre-miRNA) and mature miRNAs can alter the biogenesis of miRNAs and/or their binding to target mRNAs (Duan et al., 2007; Georges
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Abbreviations: miRNA, microRNA; SNP, single nucleotide polymorphism; PCR-RFLP, polymerase chain reaction-restriction fragment length polymorphism; qRT-PCR, quantitative real-time polymerase chain reaction; Pri-miRNA, primary miRNA; Pre-miRNA, precursor miRNA; gga, Gallus gallus; BW, body weight; SL, shank length; SG, shank girth; CD, chest depth; CB, chest breadth; BBL, breast bone length; PA, pectoral angle; BSL, body slanting length;PB, pelvisbreadth;CW, carcass weight;SEW, semi-evisceration weight; EW, evisceration weight; AFW, abdominal fat weight; LW, leg weight; LMW, leg muscle weight; BMW, breast muscle weight. ⁎ Corresponding authors. E-mail addresses: [email protected] (X. Kang), [email protected] (G. Sun).
et al., 2007; Slaby et al., 2012). Single nucleotide polymorphisms (SNPs) that reside within the miRNA genes may modulate the transcription of pri-miRNA transcripts and the stability or processing of pri- and pre-miRNA, thereby leading to either an increase or decrease in mature miRNA levels. On the other hand, changes in the sequence of mature miRNAs can have an impact on their interaction with target mRNAs. Because miRNAs potentially regulate the expression of multiple targets, base changes in miRNA genes can have a functional effect and contribute to phenotypic variation via deregulation of target genes (Hong et al., 2012). Limited functional researches about miRNAs in farm animals indicate that the miRNAs play key roles in muscle development (Lin et al., 2012), oocyte maturation (Kang et al., 2013) and early embryonic development (Darnell et al., 2006), and adipose tissue growth (Li et al., 2011; Yao et al., 2011). Increasing evidence suggests that SNP in miRNA genes or miRNA target sites may contribute to change in production or health traits in farm animals. Mutation created in the 3′ UTR of GDF8 gene of Texel sheep resulted in a new target site that allowed miR-1 and miR-206 to bind simultaneously, which led to the expression of the target gene MSTN reduction and muscle hypertrophy (Clop et al., 2006). Our previous work suggested that a point mutation occurring in Gallus gallus (gga) miR-1657 was significantly associated with body indexes and meat quality traits (Li et al., 2012).
http://dx.doi.org/10.1016/j.gene.2015.03.037 0378-1119/© 2015 Published by Elsevier B.V.
Please cite this article as: Li, H., et al., Effect of polymorphism within miRNA-1606 gene on growth and carcass traits in chicken, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.03.037
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2.1. Animals and trait measurements
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A Gushi–Anka F2 resource population which was created by Henan Innovative Engineering Research Center of Poultry Germplasm Resource as described previously (Han et al., 2012; Li et al., 2012) was used as the study material. In detail, the population was a cross of a slow-growing Chinese native breed (Gushi chicken) and a fastgrowing broiler (Anka chicken). Through two hatches for an interval of two weeks, a total of 860 F2 chickens were produced. All the chickens were managed in cages under the same environment with free access to feed and water, and fed according to the Chinese National Research Council (1994) recommendations. Body weight (BW) was weighed individually at birth, 2, 4, 6, 8, 10, and 12 weeks, and body indexes such as shank length (SL), shank girth (SG), chest depth (CD), chest breadth (CB), breast bone length (BBL), pectoral angle (PA), body slanting length (BSL) and pelvis breadth (PB) were measured at 4, 8 and 12 weeks, respectively. Body slanting length (BSL) was measured as the distance between chicken anterior/superior joint of the clavicle to the ischial tuberosity with a tape. PA was measured as the angle of breastbone front end using Pectoral Angle caliper. CB, CD and PB were measured with vernier caliper. CB was measured as the distance between chicken shoulder joint. CD was measured as the distance between the first thoracic vertebrae and the front of breastbone. PB also named back width was measured as the distance between the outer edges of chicken lions in both sides. All the chickens were slaughtered at the age of 12 weeks. Blood samples were collected from the F2 individuals and stored at − 80 °C. The carcass traits including carcass weight (CW), semi-evisceration weight (SEW, the carcass weight excluding the trachea, esophagus, crop, intestine, spleen, pancreas, gallbladder and reproductive organs), evisceration weight (EW, the SEW excluding the heart, liver, proventriculus, gizzard, head, feet and abdominal fat), abdominal fat weight (AFW), leg weight (LW), leg muscle weight (LMW), and breast muscle weight (BMW) were measured. All the detailed measuring methods were described previously by Han et al. (2011).
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Genomic DNA was extracted from the 860 chickens EDTAanticoagulated peripheral blood samples using the standard phenol–chloroform methods. Considering the DNA work concentration requirement for MALDI-TOF MS genotyping, all the DNA samples were modulated to a adjust concentration and stored at − 80 °C until use. One hundred DNA samples were selected randomly, and taken the equal amount from each DNA sample to construct a DNA pool. A pair of amplification primers was designed according to the public chicken gene sequence (GenBank accession no: NC_006091. 3). The primers (F: 5′-ATGGCAGGTAAACATTTGAG-3′, R: 5′-GATT TGCTCAGCCCCAGCTTC-3′) were used to amplify a fragment containing the precursor region of miR-1606 gene sequence (GenBank accession no: MI0007333). The PCR product was sequenced by Sangon Bio Co., Shanghai to detect polymorphisms. All the DNA samples were sent to the company to test the SNP by using MALDI-TOF MS assay technology.
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2.4. The prediction of pre-miR-1606 secondary structure
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Due to the rs313467992 SNP occurring in the precursor region of miRNA-1606, the most stable secondary structure of pre-gga-mir-1606 with the lowest free energy was calculated by using M-fold software (Zuker, 2003). The absolute difference in the free energy for pre-mir1606 with different alleles was used as the parameter for the assessment of the impact on the secondary structure of pre-miR-1606.
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2.5. Expression detection of miR-1606
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To evaluate the effect of different alleles on the expression of mature miR-1606, real time PCR was conducted. A quantitative reverse transcription-PCR approach was used to determine the levels of mature miR-1606. The frozen leg muscle tissue and breast muscle of F2 chickens of the indicated genotypes were selected randomly each for six. Total RNA was isolated by Trizol (Invitrogen) according to the manufacturer's instruction. RNA quality was detected by electrophoresis (Formaldehyde RNA gel) and the NanoDropTM1000 spectrophotometer (Thermo Scientific). cDNA was synthesized using a cDNA Synthesis kit (Invitrogen) according to the manufacturer's instruction. Real-time PCR was performed using SYBR Green method in Roche instrument LightCycler®96. The level of mature miR-1606 was normalized to U6 snRNA. The loop primers used for reverse-transcription and the real time of miR-1606 and U6 snRNA genes were designed (Chen et al., 2005) and sent to the company to synthesize (Supplementary Table 1). The real-time PCR amplification process was as follows: 95 °C for 3 min, 40 cycles of 95 °C for 12 s, 62 °C for 40 s, 72 °C for 30 s, and followed by a further 10 min extension at 72 °C. The expression values of different genotypes were analyzed by using 2−△△ct method (Schmittgen and Livak, 2008), and significant effects of different genotypes were statistically tested by one-way ANOVA.
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All statistical analyses were performed by Statistical Package Service Solution software (SPSS for Windows, Standard version 19.0; SPSS, USA). The correlations between polymorphism and chicken related economic characteristics were analyzed by linear mixed models. Model I was used to evaluate the body weight and body index traits. Considering the effect of body weight difference on carcass traits, model II was applied to analyze the carcass traits, and carcass weight was used as the concomitant variable in model II. In case the effect of genotype was significant, the Bonferroni test was carried out to control for multiple comparisons of the genotypes. The P-value b 0.05 was considered statistically significant. The mixed linear models were as follows:
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Model I : Y ijklm ¼ μ þ Gi þ S j þ H k þ f l þ eijkl m
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The rs313467992 SNP occurring in miR-1606 (+ 35 CNA) in the Gushi–Anka F2 resource population was genotyped by Sequenom MassArray_ iPLEX GOLD System. For the SNP, a pair of amplification primers and an extension primer was designed using Assay Design 3.1 software (Sequenom Inc., USA). The 1st PCR primer sequence of premir-1606 polymorphism was 5′-ACGTTGGATGAACAAGCAGCGTGAAT GGTC-3′; the 2nd PCR primer sequence was 5′-ACGTTGGATGTTCAGG ACTGTTCTGTTGCC-3′, and the extension primer was 5′-GTTGCCCCCT CTTTCAGT-3′. The SNP was genotyped by MassArray matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (Sequenom) following the manufacturer's instructions (Oeth et al., 2005). Related data as the call rate and peak area were acquired through using the software provided by the manufacturer. Alleles were automatically assigned by the software.
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In this study, we identified a SNP located in the precursor region of the gga-miR-1606 and investigated its association with body weight, body indexes at different developmental stages, and carcass traits in chickens. The effect of the miR-1606 SNP on the secondary structure of pre-miRNA and the expression level of mature miR-1606 were also examined. Collectively, our data suggested a potential role for miR-1606 in regulating the growth development.
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Model II : Y ijklm ¼ μ þ Gi þ S j þ H k þ f l þ b W ijklm −W þ eijklm
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where Yijklm represents the observe value; μ the overall mean, Gi the fixed effect of genotype (i = CC, CA, AA); fl the random effect of family (l = 7); Sj fixed effect of sex (j = f, m), Hk the fixed effect of hatch (k = 2), and eijklm the random error. b is the regression coefficient for carcass weight; Wijklm the individual carcass weight; W the average carcass weight.
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3.1. Identification and genotyping of miR-1606 SNP
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The PCR product sequencing results were compared with the published chicken genome sequence, and the result suggested that a CNA mutation located in +35 of pre-miR-1606 (Supplementary Fig. 1) existed in the population. The genotyping calls for the variant (Supplementary Fig. 2) were the same as our sequencing alignment result, and the mass spectrogram of variant genotypes CC, CA, and AA is summarized in Supplementary Fig. 3, respectively.
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3.2. Association of the miR-1606 SNP with growth and carcass traits
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To investigate the functional effect of the +35 CN A polymorphism in miR-1606 precursor on chicken phenotypic traits, the association of the SNP with body weight at different developmental stages (Table 1), body indexes at 4 weeks, 8 weeks and 12 weeks (Table 2), and carcass traits at 12 weeks (Table 3) was performed. To the Gushi–Anka F2 resource population, the + 35 C N A had a significant effect on BW at the age of 10 weeks and 12 weeks. The SNP was significantly associated with chicken SL, CD and BSL at 8 weeks (P b 0.05). Significance was also found among traits such as SL, BSL, PA and PD at 12 weeks (P b 0.05). The effect of the SNP on carcass traits, including SEW, EW, BMW, LW and CW was significant (P b 0.05) as listed in Table 3. These suggest that the mutation indirectly contributes to body index at different times and carcass traits in chicken. As for the effect of the genotypes on growth and carcass traits, the results showed that the values of CA genotype individuals were the highest, and significance was observed between CA and CC genotypes (P b 0.05), but the difference between CA and AA genotypes was not significant (P N 0.05).
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SL (cm) SG (cm) CD (cm) CB (cm) BBL (cm) PA (°) BSL (cm) PB (cm) SL (cm) SG (cm) CD (cm) CB (cm) BBL (cm) PA (°) BSL (cm) PB (cm) SL (cm) SG (cm) CD (cm) CB (cm) BBL (cm) PA (°) BSL (cm) PB (cm)
5.50 ± 0.08 2.69 ± 0.03 4.79 ± 0.049 4.05 ± 0.05 6.17 ± 0.07 73.83 ± 0.45 11.40 ± 0.24 5.14 ± 0.06 7.84 ± 0.07 3.41 ± 0.03 6.45 ± 0.08ab 5.65 ± 0.06 8.91 ± 0.08 75.90 ± 0.52 16.28 ± 0.12ab 6.85 ± 0.08 9.43 ± 0.08 3.86 ± 0.03 7.88 ± 0.07 6.31 ± 0.08 11.05 ± 0.10 78.25 ± 0.48B 19.85 ± 0.13a 8.67 ± 0.07AB
5.46 ± 0.07 2.71 ± 0.03 4.86 ± 0.04 4.09 ± 0.04 6.23 ± 0.06 74.13 ± 0.36 11.61 ± 0.18 5.17 ± 0.04 7.96 ± 0.06 3.42 ± 0.03 6.62 ± 0.07a 5.71 ± 0.05 8.88 ± 0.07 76.80 ± 0.44 16.30 ± 0.10a 6.95 ± 0.06 9.40 ± 0.08 3.84 ± 0.03 7.91 ± 0.06 6.35 ± 0.07 10.98 ± 0.09 79.46 ± 0.42A 19.80 ± 0.12a 8.77 ± 0.05A
5.50 ± 0.08 2.66 ± 0.03 4.84 ± 0.05 4.13 ± 0.05 6.18 ± 0.07 74.28 ± 0.47 11.34 ± 0.26 5.17 ± 0.06 7.80 ± 0.07 3.40 ± 0.03 6.40 ± 0.09b 5.58 ± 0.06 8.91 ± 0.08 76.09 ± 0.54 16.00 ± 0.12b 6.81 ± 0.08 9.26 ± 0.09 3.79 ± 0.03 7.81 ± 0.08 6.27 ± 0.08 10.86 ± 0.10 79.01 ± 0.50AB 19.50 ± 0.14b 8.50 ± 0.07B
0.796 0.069 0.419 0.445 0.329 0.703 0.521 0.836 0.032 0.666 0.012 0.051 0.768 0.096 0.017 0.158 0.046 0.052 0.434 0.369 0.062 0.008 0.013 0.004
t2:5 t2:6 t2:7 t2:8 t2:9 t2:10 t2:11 t2:12 t2:13 t2:14 t2:15 t2:16 t2:17 t2:18 t2:19 t2:20 t2:21 t2:22 t2:23 t2:24 t2:25 t2:26 t2:27 t2:28
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Note: SL = shank length; SG = shank girth, CD = chest depth; CB = chest breadth; BBL = t2:29 breast bone length, PA = pectoral angle; PB = pelvis breadth, BSL = body slanting length. t2:30 Means with different superscripts in the line mean significant difference: small letter t2:31 means P b 0.05, capital letter means P b 0.01. t2:32
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Table 2 Effects of the miR-1606 SNP on the growth traits investigated in chicken.
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To estimate the functional relevance of the SNP with mature miR1606 expression, we first determined the levels of mature miR-1606 in F2 chickens with different homozygous and heterozygote genotypes using qRT-PCR. The result showed that the alteration of the relative expression value of mature miR-1606 between AA and CA genotypes was significant in leg muscle tissue (P b 0.05), but the alteration of the expression level of miR-1606 between the CC and AA genotypes was not significant in either breast or leg muscle (P N 0.05) (Fig. 2).
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miRNA is a class of important regulators in the biology process. Many miRNAs have been identified in all kinds of farm animal species. Related researches have revealed that the miRNA plays a crucial role in many aspects of animal development (Kloosterman and Plasterk, 2006; Plasterk, 2006) and physiology (Fatima et al., 2014). Understanding the functions of miRNAs in farm animals is essential to know how
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M-fold software was used to check the alteration of RNA secondary structure led by the alleles of miR-1606 SNP. The result showed that the C NA substitution located in the precursor region of miR-1606 could introduce a base-pairing mismatch, and led to generation of new RNA bulges in the predicted second structure, with the altering of the free energy values (Fig. 1).
t1:1 t1:2
Table 1 Effects of the miR-1606 SNP on body weight at different stages in chicken.
Table 3 Effects of the miR-1606 SNP on carcass traits in chicken.
t3:1 t3:2
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3.4. Expression of miR-1606
t1:3 t1:4 t1:5
Body weight
t1:6 t1:7 t1:8 t1:9 t1:10 t1:11 t1:12
BW0 (g) BW2 (g) BW4 (g) BW6 (g) BW8 (g) BW10 (g) BW12 (g)
t1:13 t1:14 t1:15
Mean ± SD
P-value
AA
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CC
30.47 ± 0.94 121.81 ± 3.25 317.82 ± 8.12 554.40 ± 14.46 816.14 ± 22.03 1103.23 ± 27.58AB 1341.42 ± 30.40
31.58 ± 0.75 124.14 ± 2.96 323.55 ± 7.64 565.64 ± 13.59 818.00 ± 20.77 1126.39 ± 26.01A 1365.10 ± 28.46
30.46 ± 0.99 119.75 ± 3.38 315.14 ± 8.31 553.86 ± 14.79 790.23 ± 22.43 1078.29 ± 28.02B 1319.61 ± 31.14
0.350 0.172 0.153 0.218 0.087 0.007 0.041
Note: BW0, 2, 4, 6, 8, 10, 12 = body weight at the age of 0 day, 2, 4, 6, 8, 10 and 12 weeks, respectively. Means with different superscripts in the line mean significant difference: small letter means P b 0.05, capital letter means P b 0.01.
Carcass traits SEW (g) EW (g) BMW (g) LW (g) LMW (g) CW (g)
Mean ± SD AA
CA
CC
1094.10 ± 27.74ab 915.21 ± 25.17 69.00 ± 2.78ab 149.21 ± 4.52ab 98.81 ± 3.26 1204.91 ± 29.48ab
1114.19 ± 26.35a 930.43 ± 23.99 71.71 ± 2.66a 150.37 ± 4.34a 100.03 ± 3.12 1226.46 ± 27.93a
1070.48 ± 28.35b 893.35 ± 25.65 67.87 ± 2.84b 144.34 ± 4.60b 96.60 ± 3.32 1184.17 ± 30.15b
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P-value
t3:3 t3:4 t3:5
0.016 0.020 0.012 0.031 0.119 0.032
t3:6 t3:7 t3:8 t3:9 t3:10 t3:11
Note: SEW = semi-evisceration weight; EW = evisceration weight; BMW = breast mus- t3:12 cle weight; LW = leg weight; LMW = leg muscle weight; CW = carcass weight. Means t3:13 with different superscripts in the line mean significant difference: small letter means P b t3:14 0.05.
Please cite this article as: Li, H., et al., Effect of polymorphism within miRNA-1606 gene on growth and carcass traits in chicken, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.03.037
H. Li et al. / Gene xxx (2015) xxx–xxx
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the various biological processes are regulated and to explore novel strategies to improve production efficiency in farm animals. Mutations in miRNAs resulting in aberrant miRNA-mediated gene regulation may modulate the transcription of pri-miRNA transcripts and the stability
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The relative expression level
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Fig. 1. The predicted secondary structure of the pre-mir-1606 with different alleles.
a ab b
CC CA AA
Fig. 2. The relative expression level of mature miR-1606 in three different genotypes. The different superscripts mean significant difference P b 0.05.
or processing of pri- and pre-miRNAs, thereby leading to either an increase or decrease in mature miRNA levels, and may contribute to phenotypic variation (Georges et al., 2007). For example, Lei et al. reported that a T NC variation in porcine miR-27a gene was associated with litter size (Lei et al., 2011). SNPs in porcine miR-1 significantly altered the expression in the level of both precursor and mature miR-1, and associated with type I and type IIa muscle fibers in number and area compositions, respectively (Hong et al., 2012). SNPs that reside within the miRNA genes could regulate miRNA biogenesis and alter target selection, thereby potentially having profound biological effects (Duan et al., 2007). A single mutation in pre-miR-155 creating a mismatch near the 3′end of miR-155 leads to a shift in strand selection, thereby fine-tunes their targets and results in a butterfly effect on global gene expression (Lee et al., 2011). In this study, we identified a SNP in the precursor of chicken miR-1606 locus. The effects of the miR-1606 SNP on growth development and carcass traits were evaluated. Genotype analysis revealed significant associations with the body weight, body indexes at different developmental stages and carcass traits in chickens. The above statistical results showed that chickens with CA genotype related with greater body weight at 10 and 12 weeks (Table 1), and some body indexes at 8 and 12 weeks (Table 2). Many miRNAs are found to be expressed in a spatiotemporal-specific manner, which indicates that miRNAs play roles in specific tissue types or at specific developmental stages (Wang
Please cite this article as: Li, H., et al., Effect of polymorphism within miRNA-1606 gene on growth and carcass traits in chicken, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.03.037
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Conflict of interest
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The authors have declared no conflict of interest.
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Acknowledgments
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This study was supported by the National Natural Science Foundation of China (No. 31201795), the Earmarked Fund for Modern Agro-industry Technology Research System (No. CARS-41-K04), the Ministry of Education Innovation Team Development Plan (No. IRT1236) and the Postdoctoral Science Foundation of China (2013M531675).
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In conclusion, we detected and identified the rs313467992 SNP within miR-1606 gene in the Gushi–Anka F2 population. The association analysis results indicated a promising impact of the SNP in miR-1606 gene on body weight and body indexes at different growth stages, and carcass traits. These results facilitate gene mining and provide a preliminary evidence for the possible functional role of miR-1606 in a large population, which could provide new insights into the potential role to be used as a genetic marker or molecular assist selection in animal breeding. However, we conclude that further research and validation of the allelic effects and functional mechanisms are needed. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.gene.2015.03.037.
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et al., 2013). Compared with CC and AA genotypes, the observed values of the CA genotype in CW, SEW, EW, BMW and LW are significantly larger (Table 3). The results implied that the CA genotype may be superior to the other genotypes in improving production performance. A point mutation within murine miR-717 was proven to affect fat deposition (Kunej et al., 2010). SNPs in the primary RNA miR-206/miR-133b cluster were found to be associated with muscle fiber characteristics, meat quality and lean meat production in Berkshire, Landrace and Yorkshire pig breeds (Lee et al., 2013). SNP in the primary region of miRNA can lead to a pronounced effect on the efficiency of the miRNA processing machinery (Slezak-Prochazka et al., 2010). Previous research reported that a mutation in pre-miR146a led to decrease in mature miRNA expression and predisposes to papillary thyroid carcinoma (Jazdzewski et al., 2008). The CNA SNP located in the precursor of miRNA-1606 led to a change in the secondary structure of pre-miRNA, and notably decreased the expression of mature miRNA in leg muscle tissue. Sun et al. had detected several SNPs occurring in human miRNA genes that could affect the biogenesis and function of miRNAs (Sun et al., 2009). Previous report showed that an AN G mutation at 29 nt downstream of pre-miR-21 led to a conformational change of the secondary structure and a relative reduction of the mature miR-21 expression in vivo (Zhu et al., 2009). Our result suggested that the SNP may alter the level of processing efficiency of premiRNA to mature miRNA or pri-miRNA to pre-miRNA, thereby resulting in the function alteration. Several functional studies of miRNAs in farm animal have indicated that miRNAs involve in a range of biology processes and play important roles in growth development in farm animals. Geng et al. suggested that the rs16681031 SNP in miR-1658* gene may play an important role in the formation of some phenotype of chicken (Geng et al., 2011). Given the important effect of rs313467992 SNP residing in miR-1606 on chicken body weight, body indexes and carcass traits, and the functional role of miR-1606 has not been revealed yet in any other species, it is necessary to further study the function of miR-1606 in different aspects.
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Please cite this article as: Li, H., et al., Effect of polymorphism within miRNA-1606 gene on growth and carcass traits in chicken, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.03.037
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