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Molecular cloning, expression and association study with reproductive traits of the duck LRP8 gene ab
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C. Wang , S.J. Li , C. Li , G.H. Yu , Y.P. Feng , X.L. Peng & Y.Z. Gong a
Key Laboratory of Agricultural Animal Genetics, Breeding, and Reproduction of Ministry of Education, Huazhong Agricultural University, Wuhan, Hubei 430070, P.R. China b
National Poultry Engineering Research Center, Shanghai Academy of Agricultural Sciences, Shanghai 201106, P.R. China Accepted author version posted online: 08 Oct 2013.Published online: 29 Nov 2013.
To cite this article: C. Wang, S.J. Li, C. Li, G.H. Yu, Y.P. Feng, X.L. Peng & Y.Z. Gong (2013) Molecular cloning, expression and association study with reproductive traits of the duck LRP8 gene, British Poultry Science, 54:5, 567-574, DOI: 10.1080/00071668.2013.819488 To link to this article: http://dx.doi.org/10.1080/00071668.2013.819488
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British Poultry Science, 2013 Vol. 54, No. 5, 567–574, http://dx.doi.org/10.1080/00071668.2013.819488
Molecular cloning, expression and association study with reproductive traits of the duck LRP8 gene C. WANG1,2, S.J. LI1, C. LI1, G.H. YU1, Y.P. FENG1, X.L. PENG1
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Y.Z. GONG1
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Key Laboratory of Agricultural Animal Genetics, Breeding, and Reproduction of Ministry of Education, Huazhong Agricultural University, Wuhan, Hubei 430070, P.R. China, and 2National Poultry Engineering Research Center, Shanghai Academy of Agricultural Sciences, Shanghai 201106, P.R. China
Abstract 1. Two splice variants of duck LRP8 were identified, one containing 8 ligand-binding repeats (LRP8-1) and the other containing only 7 repeats (LRP8-2). The two transcripts share ~71–91% nucleic acid identity and ~65–94% amino acid identity with their counterparts in other species. A phylogenetic tree based on amino acid sequences shows that duck LRP8 proteins are closely related to those of chicken, turkey and zebra finch. 2. The semi-quantitative reverse transcription polymerase chain reaction (RT-PCR )analysis indicates that the two transcripts are expressed in all the examined tissues, and the LRP8-1 transcript is more highly expressed in hypothalamus, ovary and pituitary gland than in other detected tissues. 3. Six single nucleotide polymorphisms (SNPs) were identified in the coding region. Association analysis demonstrated that the c.528C > T genotypes were associated with egg production (EP) (EP210d, EP300d and EP360d), age at laying the first egg (AFE) and body weight at sexual maturity (BWSM). The c.1371A > G genotypes were associated with egg production (EP210d, EP300d and EP360d). 4. The haplotypes of c.528C > T and c.1371A > G were associated with EP (EP210d, EP300d and EP360d), yolk weight (YW), albumen weight (AW), egg weight (EW), BWSM and the first egg weight (FEW). 5. Duck LRP8 gene was associated with some reproductive traits and is an important candidate gene for the genetic selection of improved reproductive traits.
INTRODUCTION Low-density lipoprotein receptor-related protein 8 (LRP8), also known as apolipoprotein E receptor 2 (ApoER2, in mammals), is a member of the lowdensity lipoprotein receptor (LDLR) gene family, which includes the LDLR and the very-low-density lipoprotein receptor (VLDLR). Except for mediating the lipid-rich extracellular lipoprotein uptake, LRP8 also participates in endocytosis and multiple intracellular signalling cascades (Vassiliou et al., 2001; Argov et al., 2004; Fayad et al., 2007). Moreover, LRP8 plays a crucial role in mouse sperm maturation and bovine follicular growth (Andersen et al., 2003; Argov et al., 2004; Olson et al., 2007).
A complete cDNA sequence containing one open reading frame of 2754 bp encoding a 918residue protein was identified in chicken and a 6.5 kb receptor transcript was detected in the brain and ovary (Brandes et al., 2001). Mahon et al. (1999) reported that chicken clusterin was a major product of the somatic granulosa cells and correlated with the developmental phases of individual follicles. Furthermore, they documented that LRP8 may internalise cell-derived granulosa clusterin. Subsequently, the role of LRP8 gene on follicular growth has been examined. Recently, a study in dwarf chickens showed that the LRP8 gene was associated with egg yolk colour, shape index and some eggshell traits, with the authors suggesting that the gene may be a
Correspondence to: Yan-Zhang Gong, College of Animal Science and Technology of Huazhong Agricultural University, No. 1, Shizishan Street, Hongshan District, Wuhan City, Hubei Province, P.R. China. E-mail: [email protected] Accepted for publication 7 May 2013.
© 2013 British Poultry Science Ltd
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candidate gene associated with egg traits (Yao et al., 2010). As an important agricultural species, the reproductive traits (for example, age at laying the first egg, body weight at sexual maturity, egg production and egg weight) of some local duck breeds need to be improved genetically. Although the functions of chicken LRP8 gene on follicular growth and egg traits have been studied, the information on duck LRP8 gene is scarce. In this study, the cloning of the gene sequence, analysis of gene structure and expression pattern analysis will facilitate future research into gene function. Association analysis identifies potential markers to improve the reproductive traits of ducks in future breeding programmes.
MATERIALS AND METHODS
cross using a standard phenol-chloroform method (Joseph and David, 2002). DNA concentration and quality were measured with spectrophotometer ND-1000 (Nano-Drop, USA), and the concentrations were adjusted between 50 and 300 ng/μl. DNA samples were stored at 4°C for later use. Total RNA was isolated from different tissues with Trizol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. The quality of total RNA samples was evaluated by electrophoresis on 1.2 % agarose gels stained with ethidium bromide, and their concentrations were measured with spectrophotometer ND-1000 (Nano-Drop, USA). cDNA was obtained by reverse transcription polymerase chain reaction (RT-PCR) from 1 μg of DNase-treated (DNaseI, TOYOBO Co., Japan,) total RNA according to M-MLV reverse transcriptase kit (TOYOBO Co, Japan) at 42°C.
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Ducks, tissue and data collection Ducks from the second (n = 296) and third (n = 251) generation of white Liancheng × white Kaiya cross (Gong et al., 2010) were provided by the Hankou Jingwu Industry Garden Ltd. The ducks were reared in cages in a semi-open house and were subjected to conventional management conditions. Recorded traits included age at laying the first egg (AFE), first egg weight (FEW), body weight at sexual maturity (BWSM) and egg production (EP, during 210 d, 300 d and 360 d) of each individual duck. Eggs were obtained from the above two generations for 3 consecutive days when the ducks were 295–300 d old. Data were averaged after cracked, soft-shell and double-yolked eggs had been removed. The egg quality traits measured were egg weight (EW), Haugh units (HU), eggshell index (ESI), albumen weight (AW), yolk weight (YW), yolk colour (YC), albumen height (AH), albumen ratio (AR), yolk ratio (YR) and eggshell strength (ESS) (Zhang et al., 2005). Three healthy female ducks (aged 25 weeks) were selected from the second generation of white Liancheng × white Kaiya cross reared under normal management conditions. A total of 12 tissues, including heart, liver, spleen, lungs, kidneys, skin, muscle, hypothalamus, intestine, pituitary gland, ovary and oviduct, were collected from each duck. The dissected tissues were immediately frozen in liquid nitrogen and subsequently stored at –80°C for later total RNA extraction. All animal procedures were performed according to protocols approved by the Biological Studies Animal Care and Use Committee of Hubei Province, P.R. China. DNA isolation, RNA isolation and cDNA synthesis Genomic DNA was extracted from 547 blood samples of the second (n = 296) and third (n = 251) generations of the white Liancheng × white Kaiya
Molecular cloning and sequence analysis of duck LRP8 gene Based on the conserved region of LRP8 gene in Gallus gallus (GI: 45383975), Meleagris gallopavo (GI: 326925389), Homo sapiens (GI: 66082553), Mus musculus (GI: 124286869) and Taeniopygia guttata (GI: 224058238), 4 pairs of primers (LRP8-F1/R1~LRP8-F4/R4) were designed to obtain the coding region sequence of the duck LRP8 gene (primers shown in Supplementary Table S1, accessible via the online version of the journal at: http://dx.doi.org/10.1080/ 00071668.2013.819488). The PCR programme included denaturation for 5 min at 94°C, followed by 38 cycles of 35 s at 94°C, 35 s at annealing temperature, 35–60 s at 72°C and an extension step of 5 min at 72°C. The purified PCR products were cloned into the PEASY-T1 vector (TransGen Biotech Co., Ltd., Beijing, China) and sequenced commercially (Invitrogen, Shanghai, China). The sequences were matched using SeqMan software (DNAstar). Secondary structure prediction was performed using online tools on the ExPASy website (http://cn.expasy.org/tools/). The phylograms were created by MEGA 4.0 NeighbourJoining (NJ) software with 1000 bootstrap trials after multiple alignments of sequence data by ClustalW (http://www.ebi.ac.uk/Tools/msa/ clustalw2/) (Edgar, 2004; Tamura et al., 2007). Expression profiling To determine the tissue expression of the two types of splice variants, semi-quantitative RT-PCR was carried out using total RNA from twelve duck tissues of three female ducks and a pair of primers (LRP8-A/S) encompassing the region corresponding to the 8th ligand-binding repeats (primers shown in Supplementary Table S1, accessible
GENE RELATED WITH DUCK REPRODUCTIVE TRAITS
via the online version of the journal at: http://dx. doi.org/10.1080/00071668.2013.819488). The PCR programme included a denaturation step of 5 min at 94°C, followed by 36 cycles of 30 s at 94°C, 30 s at 60°C, 30 s at 72°C and a final step of 5 min at 72°C. As control, a pair of primers (β-actin-A/S) (Supplementary Table S1, accessible via the online version of the journal at: http://dx. doi.org/10.1080/00071668.2013.819488) were used under the same conditions. PCR products were visualised on 2.0 % agarose gels stained with ethidium bromide under ultraviolet light.
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SNP screening and genotyping According to the cDNA sequence of the duck LRP8 gene obtained from the above RT-PCR reactions, 10 pairs of primers (LRP8-AF/AR~LRP8-JF/JR, Supplementary Table S1, accessible via the online version of the journal at: http://dx.doi.org/ 10.1080/00071668.2013.819488), were designed to screen the potential single nucleotide polymorphisms (SNPs) in the coding region. Ninety-six DNA samples from the second generation were used for PCR-SSCP (Single-Strand Conformation Polymorphism). The PCR reaction was performed in a 10 μl final volume, containing 50–300 ng of genomic DNA, 1X PCR buffer, 0.5 μM of each primer, 25 μM of dNTPs, 2.0 mM MgCl2 and 0.2 units Taq DNA Polymerase (TransGen Biotech Co., Ltd., Beijing, China). The PCR parameters were 5 min at 94°C followed by 35 cycles of 30 s at 94°C, 30 s at annealing temperature, 30 s at 72°C and a common final extension at 72°C. Aliquots of 3 μl of the PCR products were mixed with 10 μl of the denaturing solution (95% formamide, 25 mM EDTA, 0.025% xylene-cyanole and 0.025 % bromophenol blue), heated for 10 min at 98°C and chilled on ice rapidly. Denatured DNA was subjected to 12% PAGE (polyacrylamide gel electrophoresis) analysis, which was run with 1XTBE buffer for 16–20 h at 4°C under a constant voltage (120–150 V). The gel was stained with 0.1% silver nitrate and visualised with 2% NaOH solution (Zhang et al., 2007). The PCR products of two samples representing different PCR-SSCP genotypes were cloned into PEASY-T1 vector and were sequenced M1 1
M2 2
commercially. Polymorphism sites were analysed by sequence comparisons using DNAStar software (DNAstar Inc., Madison, WI, USA). A total of 547 samples from the second and third generations of white Liancheng × white Kaiya were subjected to genotyping analysis using the PCR-RFLP (Restriction Fragment Length Polymorphism) technique. The PCR reaction was performed as described above. A volume of 3 μl of the PCR products was digested with 3 or 5 units of restriction enzyme (RE) BseGI (HhaI/BcII) (TaKaRa, Tokyo, Japan) for 12 or 16 h at 37°C or 55°C, respectively, and separated by electrophoresis on 1.5% or 2.5% agarose gels stained with ethidium bromide; they were visualised under ultraviolet light, and the genotype of each sample was recorded. Haplotype reconstruction Haplotypes were constructed based on the SNPs identified in all 547 experimental birds using PHASE 2.0 programmer (Stephens et al., 2001; Zhou et al., 2010). The genetic status of the subjects was expressed as the combination of two haplotypes (diplotype configuration). Genetic effects of the diplotypes were performed with the model mentioned in the following section. Statistical analysis The general linear model (GLM) procedures of SAS software package (SAS Inst. Inc., Cary NC, USA) was used to determine associations of the different genotypes with reproductive traits according to the following model: Y ¼μþS þF þG þe where Y is the observed values of different reproductive traits, µ is the population mean, S is the fixed effects of generation, F is the fixed effects of sire family, G is the fixed effects of genotype or haplotype and e is the random error. Multiple comparisons were performed among the least squares means. Values were considered significant at P < 0.05 and are presented as least square means ± standard error. M1 4
M2 3
M3
1410 bp 1287 bp 406 bp
569
5 3391 bp
902 bp 326 bp
Figure 1. The PCR amplification results of duck LRP8 gene. Lane M1, Marker I; M2, DL2000 Marker; M3, DL2000 Plus. Lanes 1, 2, 3 and 4: amplification fragment of primer LRP8-F1/R1, LRP8-F2/R2, LRP8-F3/R3 and LRP8-F4/R4, respectively. All these primers were used to obtain the whole sequence of duck LRP8 gene. Lane 5, amplification fragment of primer LRP8-F2/R2 using duck genomic DNA.
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RESULTS
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Sequence analysis of duck LRP8 gene Primers LRP8-F1/R1, LRP8-F2/R2, LRP8-F3/R3 and LRP8-F4/R4 amplified a total 2867 bp sequence of duck LRP8 gene from brain tissue, and clear amplified bands are shown in Figure 1. The cDNA (GenBank: JN108873) contains an open reading frame (ORF) of 2751 bp, a 2 bp 5′-terminal untranslated region (5′-UTR) and a 114 bp 3′-UTR including a TGA termination codon (nucleotides 2754–2756 bp). The duck LRP8 nucleotide sequence shows high identity to its counterparts in Gallus gallus (GI: 45383975, 91%), Homo sapiens (GI: 65301118, 80%), Mus musculus (GI: 124286869, 78%), Sus scrofa (GI: 315468530, 76%), Bos taurus (GI: 148231248, 76%), Danio rerio (GI: 374428822, 71%), Oryctolagus cuniculus (GI: 291398861, 78%) and Taeniopygia guttata (GI: 224058238, 91%). The bioinformatic analysis indicated that the ORF encoded a protein of 917 amino acid residues with a molecular weight of 101.526 kDa and an isoelectric point (pI) of 4.68. A 24-amino-acid sequence (residues 1 to 24) constitutes the putative signal peptide at the N-terminus, two potential N-linked glycosylation sites (N-X-S or N-X-T), a 23amino-acid sequence (residues 836–858) in the putative transmembrane domain and a FDNPVY sequence in the cytoplasmic domain at the C-terminus (Supplementary Figure S1, accessible via the online version of the journal at: http://dx. doi.org/10.1080/00071668.2013.819488). Using the LRP8-F2/R2 primers, another shorter product was amplified (Figure 1). The sequence alignment result of the two PCR products showed that the shorter product had lost a 123 bp fragment, which exactly encoded the 8th repeat; the rest of the sequence was identical to the long product (Supplementary Figure S2, accessible via the online version of the journal at: http://dx.doi. org/10.1080/00071668.2013.819488). We postulated that the short one was another LRP8 mRNA transcription splice isoform. To confirm our conclusion, a single, approximately 3391 bp fragment (reference chicken genome DNA sequence), was obtained following amplification by PCR using duck genomic DNA and the LRP8-F2/R2 primers (Figure 1). The fragment was subcloned into a T-vector and sequenced commercially. The alignment between the genomic DNA sequence and mRNA sequence showed that the genomic DNA sequence contained the deletion fragment (123 nucleotides). Therefore, we concluded that we had identified two forms of LRP8, one containing 8 (LRP8-1, GenBank: JN108873) and the other with 7 (LRP8-2, GenBank: JN108874) ligand-binding repeats.
To investigate the evolutionary origin of duck LRP8, a phylogenetic tree was constructed with the amino acid sequences of duck and 9 other animal species, including Gallus gallus (GI: 45383976), Meleagris gallopavo (GI: 326925390), Homo sapiens-1 (GI: 61744471), Homo sapiens-2 (GI: 66082554), Homo sapiens-3 (GI: 61744467), Homo sapiens-4 (GI: 65301119), Sus scrofa (GI: 315468531), Mus musculus-1 (GI: 123229814), Mus musculus-2 (GI: 123229810), Mus musculus-3 (GI: 123229812), Mus musculus-4 (GI: 13928141), Oryctolagus cuniculus (GI: 291398862), Bos taurus (GI: 148231249), Danio rerio (GI: 326668703) and Taeniopygia guttata (GI: 224058239). The phylogenetic tree shows three groups, with duck, chicken, turkey and zebra finch belonging to one group, indicating that duck LRP8 proteins are closer to the three bird species than those of other animal species (Supplementary Figure S3, accessible via the online version of the journal at: http://dx.doi. org/10.1080/00071668.2013.819488). Expression profile Semi-quantitative RT-PCR was applied to detect the tissue distribution of the two duck LRP8 splice variants. The PCR product indicates that if the LRP8-1 variant was expressed, the amplification segment size would be 373 bp, and if the LRP8-2 variant was expressed, the amplification segment size would be 250 bp. The expression profile indicated that both of the PCR products could be detected in the twelve tissues, which means that the two duck LRP8 transcripts were widely expressed (Figure 2). The LRP8-1 transcript was more highly expressed in hypothalamus, ovary and pituitary gland than in other tissues, whereas the LRP8-2 transcript was equally expressed in all the examined tissues. SNP screening and association analysis By aligning the PCR sequences from different individuals, 6 synonymous polymorphisms, c.528C > T, c591C > T, c621C > T, c.642C > T, c.1371A > G and c2664A > G, were identified in the coding region of the duck LRP8 gene. The variations on three sites, c.528C > T, c.642C > T
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9 10 11 12 LRP8-1 LRP8-2 β-action
Figure 2. Expression patterns of two LRP8 transcripts in different duck tissues. M: Marker I; Lanes1 to 12 represent heart, liver, spleen, lung, kidney, skin, muscle, hypothalamus, intestine, pituitary gland, ovary and oviduct, respectively. β-actin was used as the control.
GENE RELATED WITH DUCK REPRODUCTIVE TRAITS (A)
M
(B)
A1A1 A2A2 A1A2
M
300 bp
300 bp
200 bp
163 bp 200 bp 128 bp
100 bp
B1B1 B2B2 B1B2
297 bp 153 bp 144 bp
100 bp (C)
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M C1C1 C1C1 C2C2 C2C2 C1C2 C1C2
300 bp 200 bp 131 bp
100 bp
83 bp 48 bp
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Figure 3. PCR-RFLP results of three SNPs in the duck LRP8 gene. For locus A (c.528C > T): A1A1 (163 bp), A2A2 (35/128 bp) and A1A2 (35/128/163 bp) genotypes were generated (A); for locus B (c.642C > T): B1B1 (297 bp), B2B2 (144/153 bp) and B1B2 (144/ 153/297 bp) genotypes were generated (B) and for locus C (c.1371A > G): C1C1 (131 bp), C2C2 (48/83 bp) and C1C2 (48/83/131 bp) genotypes were generated (C). The genotypes are shown at the top of each lane (M: Marker 1, a lane representing a fragment of 35 bp is not shown).
and c.1371A > G, led to changes in recognised restriction enzyme (RE) sites of BseGI, BcII and HhaI, respectively, and the variations on these sites could be genotyped by PCR-RFLPs. The results of RE digestion showed that the digested fragments were completely consistent with the predicted fragment size. For locus A (c.528C > T), A1A1 (163 bp), A2A2 (35 + 128 bp) and A1A2 (35 + 128 + 163 bp) were generated (Figure 3(A)). For locus B (c.642C > T), B1B1 (297 bp), B2B2 (144 + 153 bp) and B1B2 (144 + 153 + 297 bp) were generated (Figure 3 (B)). And for locus C (c.1371A>G), C1C1 (131 bp), C2C2 (48 + 83 bp) and C1C2 (48 + 83 +131 bp) were generated (Figure 3(C)). For these three polymorphic sites, all the three genotypes were detected in our resource population. The results of the association analyses between genotypes of locus A and locus C on the duck LRP8 gene and 14 reproductive traits (EP, BWSM, AFE, EW, FEW, YW, AW, AH, ESI, ESS, AR, YR, YC and HU) are summarised in Table 1: for locus B no association was found. In locus A, genotypes had
significant associations with EP (EP210d (P = 0.014), EP300d (P = 0.002) and EP360d (P < 0.001)), AFE (P = 0.027) and BWSM (P = 0.041), but no association was observed for the other 11 traits. The ducks harbouring genotypes A1A1 and A1A2 had higher EP (P < 0.01), earlier AFE and lower BWSM (P < 0.05) than those harbouring A2A2 but showed no difference between A1A1 and A1A2. In locus C, different genotypes had an effect on EP (EP210d (P = 0.015), EP300d (P = 0.019) and EP360d (P = 0.002)). The EP210d and EP300d of C2C2 and C1C2 were higher than those of genotype C1C1 (P < 0.05), the EP360d of C2C2 was higher than that of the other two genotypes. For locus B, no significant association was observed for the 14 reproductive traits (P > 0.05) (data not shown). Construction of haplotypes and association analysis Table 2 shows all haplotypes that were reconstructed from the two SNPs (locus A and C) in all 547 experimental ducks. Three haplotypes with
Table 1. Association analysis between the duck reproductive traits and LRP8 gene SNPs (values are means ± SE for the different genotypes) Locus A Traits1 EP210d (n) EP300d (n) EP360d (n) AFE(d) BWSM (kg)
A1A1(52)2 77.4 156.5 209.1 120.6 1.796
± ± ± ± ±
2.3a 3.1A 3.8 A 2.0a 0.032a
A2A2(241) 72.8 150.1 200.2 122.7 1.843
± ± ± ± ±
1.0b 1.4B 1.8B 0.9b 0.015b
A1A2(254) 77.3 ± 1.0a 157.2 ± 1.4A 210.0 ± 1.7A 119.1 ± 0.9a 1.797 ± 0.014a
Locus C
EP210d (n) EP300d (n) EP360d (n)
C1C1(23)
C2C2(401)
67.0 ± 3.3a 142.6 ± 4.7a 192.0 ± 5.7A
76.4 ± 0.8b 155.8 ± 1.1b 208.6 ± 1.3B
C1C2(123) 73.3 ± 1.4b 151.5 ± 2.0b 201.0 ± 2.4AB
EP210d = egg production within 210 d; EP300d = egg production within 300 d; EP360d = egg production within 360 d; AFE = age at laying the first egg; BWSM = body weight at sexual maturity. 2The numbers in the brackets are the chicken individuals of respective genotypes. Within a column, values not sharing a common superscript letter are significantly different (a,bP < 0.05; A,BP < 0.01). 1
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Table 2. Haplotypes
c.528 C > T
c.1371 A > G
Frequency (%)
A2 A2 A1 A1
C2 C1 C2 C1
32.19 0.90 52.80 14.11
H1 H2 H3 H4
Table 3.
Diplotypes of duck LRP8 gene
Diplotypes
Frequency (%)
H1H1 H1H3 H1H4 H3H3 H3H4 H4H4
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binding repeats (LRP8-1) and the other containing only 7 repeats (LRP8-2), and this represented the alternative splicing of an extra exon. It is supposed that the alternative splicing is evolutionally conserved. LRP8 (also named as ApoER2) was first cloned and characterised from a human placenta library, and subsequently, a novel receptor termed LR8B was discovered in the brain of chicken and mouse (Kim et al., 1996; Novak et al., 1996). Later studies showed that the receptors were not two distinct members of the LDLR family, but they originated from alterative splicing of the mRNA transcribed from a single gene (Brandes et al., 1997). Recently, several splicing variants of LRP8 mRNA have been detected and the corresponding proteins have been predicted in chicken, mouse, human and bovine, and their structures in these different species have been elucidated (Novak et al., 1996; Kim et al., 1997; Brandes et al., 2001; Argov and Sklan, 2004). For the products of these spliced variants, if and when they are translated, constitute receptors with (i) variant numbers of cysteine-rich repeats in the binding domain, (ii) with or without a potential furin-cleavage site downstream of the LA (type A binding repeats) repeats, (iii) with or without the putative O-linked sugar domain, and (iv) different by an insertion sequence in the cytoplasmic domain (Clatworthy et al., 1999; Schneider et al., 1999; Brandes et al., 2001; Koch et al., 2002). In this study, the products of two spliced variants of the duck LRP8 belong to the first spliced mode. The same mode has been reported in chicken and mouse (Brandes et al., 1997). Similar to LDLR and VLDLR, LRP8 also consists of 5 functional domains: (i) an amino-terminal ligand-binding domain composed of multiple cysteine-rich repeats, (ii) an epidermal growth factor (EGF) precursor homologous domain, (iii) an O-linked sugar domain, (iv) a 23-aminoacid sequence in the putative transmembrane domain and (v) a cytoplasmic domain with a FDNPVY sequence (Supplementary Figure S1,
Haplotypes inferred on the basis of two SNPs in the LRP8 gene
46 (8.49 ) 216 (39.85) 51 (9.41) 146 (26.94) 61 (11.25) 20 (3.69)
frequency higher than 2%, including H1 (A2C2, 32.19%), H3 (A1C2, 52.8%) and H4 (A1C1, 14.11%) were identified. The three main haplotypes accounted for 99.1% of the observations. Eight diplotypes were obtained based on the above three haplotypes. Among them, 6 diplotypes had a frequency higher than 1% and accounted for 99.63% of the genotypes (Table 3). The association analysis indicated that there was a significant association of diplotypes with reproductive traits (Table 4). Diplotypes had a significant effect on EP (EP210d (P = 0.008), EP300d (P = 0.003) and EP360d (P < 0.001)), YW (P < 0.001), AW (P = 0.005), BWSM (P < 0.001), EW (P < 0.001) and FEW (P < 0.001). The H4H4 diplotype presented higher BWSM and FEW but lower EP, EW, YW and AW, whereas the H1H4 diplotype presented lower BWSM and FEW but higher EP, EW, YW and AW.
DISCUSSION In the current study, two transcripts of duck LRP8 gene were identified, one containing 8 ligandTable 4. Reproductive traits1 BWSM(kg) FEW(g) EW(g) YW(g) AW(g) EP210d EP300d EP360d
Associations between diplotypes and duck reproductive traits (values are means ± SE for the different diplotypes) Diplotype H1H1 1.77 47.6 68.8 20.6 36.4 78.4 158.1 210.2
± ± ± ± ± ± ± ±
H1H3 a
0.03 1.4ab 0.9A 0.6AB 0.8AB 2.3A 3.5A 3.9A
1.80 46.2 67.8 20.1 35.1 77.8 157.1 210.9
± ± ± ± ± ± ± ±
H1H4 ab
0.02 0.6ab 0.4A 0.3AB 0.5AB 1.1A 1.8A 1.8A
1.77 44.7 69.4 20.7 36.8 78.3 159.8 210.3
± ± ± ± ± ± ± ±
H3H3 a
0.03 1.3b 0.9A 0.6AB 0.8A 2.2 A 3.3A 3.7A
1.84 46.6 68.6 20.7 35.1 75.2 154.1 205.7
± ± ± ± ± ± ± ±
H3H4 ab
0.02 0.8ab 0.5A 0.4AB 0.6AB 1.3 AB 2.1AB 2.2AB
1.84 46.6 67.2 22.1 36.1 70.2 146.1 193.6
± ± ± ± ± ± ± ±
H4H4 ab
0.03 1.2ab 0.8A 0.6A 0.8AB 2.0BC 3.0BC 3.4B
1.88 50.7 63.5 18.9 33.8 66.7 143.2 191.8
± ± ± ± ± ± ± ±
0.05b 2.1a 1.5B 0.9B 1.2B 3.5C 5.4C 5.9B
EP210d = egg production within 210 d; EP300d = egg production within 300 d; EP360d = egg production within 360 d; AFE = age at laying the first egg; BWSM = body weight at sexual maturity; FEW = first egg weight; EW = egg weight; AW = albumen weight; YW = yolk weight. Within a row, values not sharing a common superscript letter are significantly different (a,bP < 0.05; A,B,CP < 0.01).
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accessible via the online version of the journal at: http://dx.doi.org/10.1080/ 00071668.2013.819488). In this study, we cloned the gene sequence containing these 5 functional domains and showed that they shared striking homology to LRP8 gene sequence in other species. These data on the gene sequence, especially the coding region of the duck LRP8 gene, will facilitate future research into the function of this gene. The close relationship through evolution and the highly conservative structure suggest that the gene probably performs similar functions in different avian species. In the coding region, 6 synonymous mutations revealed that the duck LRP8 gene was rich in polymorphisms but that the sequence of LRP8 amino acids is highly conserved in different ducks. In humans, ApoER2 mRNA is predominantly expressed in brain and placenta and is undetectable in other tissues. In rabbits, ApoER2 mRNA is detected most intensely in brain and testis and, to a much lesser extent, in ovary, but is undetectable in other tissues. In chickens, the 6.5-kb transcript is present at high levels in brain and at much lower levels in ovary. In mouse, the same transcript of 6.5 kb is detected only in brain (Kim et al., 1996; Novak et al., 1996). Argov et al. (2004) have detected the presence of bovine LRP8 mRNA in ovarian granulose cells, which play pivotal roles in many aspects of ovarian functions including folliculogenesis and steroidogenesis (Argov and Sklan, 2004; Argov et al., 2004). In the present study, the two duck LRP8 transcripts were detected in all the examined tissues, and the expression of the LRP8-1 transcript was higher in the hypothalamic-pituitary-gonadal (HPG) axis, which participates in the regulation of reproductive activities, including folliculogenesis, ovulation, oviposition and incubation behaviour (Chen et al., 2007; Liu et al., 2011). The differential tissue distribution of LRP8 in different species implies that the receptor plays an important role in the central nervous system and in reproductive activities. Wang et al. (2006) reported that a SNP in LRP8 was associated with birth weight of progeny in black women. Yao et al. (2010) demonstrated that the C1623T polymorphism of chicken LRP8 was associated with egg yolk colour, shape index and some eggshell traits in dwarf chicken. In this study, these polymorphisms of duck LRP8 gene were significantly associated with the reproductive traits EP, AFE, BWSM, EW, YW and AW. Although the SNPs were synonymous mutations and did not directly change the amino acid sequence of the protein, they may alter gene function by affecting translation with codon bias, changing the stability of the mRNA or controlling the transcription of the gene (Duan, 2003). The results indicate that the LRP8 gene
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may be an important candidate gene in duck reproduction. The heavier body weight at sexual maturity, the larger size of the first egg, and, generally, if the birds lay their first eggs earlier, they will lay more eggs. Our results suggest that the LRP8 gene may be a good candidate gene of EP and AFE traits and provide some marker information for duck breeding. In summary, two variants of duck LRP8 transcripts were identified and characterised, and their tissue expression patterns were analysed. Association analysis showed that the duck LRP8 gene was related with some reproductive traits, and it is suggested that LRP8 is an important candidate gene for these traits and can be used as a potential marker to improve them.
ACKNOWLEDGEMENTS The authors gratefully acknowledge Dr Xiangdong Liu and Dr Ming Xiong for help on the statistical analysis. This work was supported by the Project of Scientific Research and Development in Hubei Province (2011BBB096), NSFC (31172201) and Fundamental Research Funds for the Central Universities (2011QC006).
REFERENCES ANDERSEN, O.M., YEUNG, C.H., VORUM, H., WELLNER, M., ANDREASSEN, T.K., ERDMANN, B., MUELLER, E.C., HERZ, J., OTTO, A., COOPER, T.G. & WILLNOW, T.E. (2003) Essential role of the apolipoprotein E receptor-2 in sperm development. Journal of Biological Chemistry, 278: 23989–23995. ARGOV, N. & SKLAN, D. (2004) Expression of mRNA of lipoprotein receptor related protein 8, low density lipoprotein receptor, and very low density lipoprotein receptor in bovine ovarian cells during follicular development and corpus luteum formation and regression. Molecular Reproduction and Development, 68: 169–175. ARGOV, N., MOALLEM, U. & SKLAN, D. (2004) Lipid transport in the developing bovine follicle: messenger RNA expression increases for selective uptake receptors and decreases for endocytosis receptors. Biology of Reproduction, 71: 479–485. BRANDES, C., NOVAK, S., STOCKINGER, W., HERZ, J., SCHNEIDER, W.J. & NIMPF, J. (1997) Avian and murine LR8B and human apolipoprotein E receptor 2: differentially spliced products from corresponding genes. Genomics, 42: 185–191. BRANDES, C., KAHR, L., STOCKINGER, W., HIESBERGER, T., SCHNEIDER, W.J. & NIMPF, J. (2001) Alternative splicing in the ligand binding domain of mouse ApoE receptor-2 produces receptor variants binding reelin but not alpha 2-macroglobulin. Journal of Biological Chemistry, 276: 22160–22169. CHEN, C.F., SHIUE, Y.L., YEN, C.J., TANG, P.C., CHANG, H.C. & LEE, Y.P. (2007) Laying traits and underlying transcripts, expressed in the hypothalamus and pituitary gland, that were associated with egg production variability in chickens. Theriogenology, 68: 1305–1315. CLATWORTHY, A.E., STOCKINGER, W., CHRISTIE, R.H., SCHNEIDER, W.J., NIMPF, J., HYMAN, B.T. & REBECK, G.W. (1999) Expression and alternate splicing of apolipoprotein E receptor 2 in brain. Neuroscience, 90: 903–911. DUAN, J. (2003) Synonymous mutations in the human dopamine receptor D2 (DRD2) affect mRNA stability and synthesis of the receptor. Human Molecular Genetics, 12: 205–216.
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EDGAR, R.C. (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Research, 32: 1792–1797. FAYAD, T., LEFEBVRE, R., NIMPF, J., SILVERSIDES, D.W. & LUSSIER, J. G. (2007) Low-density lipoprotein receptor-related protein 8 (LRP8) is upregulated in granulosa cells of bovine dominant follicle: molecular characterisation and spatio-temporal expression studies. Biology of Reproduction, 76: 466–475. GONG, Y., YANG, Q., LI, S., FENG, Y., GAO, C., TU, G. & PENG, X. (2010) Grey plumage colouration in the duck is genetically determined by the alleles on two different, interacting loci. Animal Genetics, 41: 105–108. JOSEPH, S. & DAVID, W.R. (2002) Preparation and analysis of eukaryotic genomic DNA, in: HUANG, P.T. (Ed) Molecular Cloning a Laboratory Manual, 3rd edn (Beijing, Science Press). KIM, D.H., IIJIMA, H., GOTO, K., SAKAI, J., ISHII, H., KIM, H.J., SUZUKI, H., KONDO, H., SAEKI, S. & YAMAMOTO, T. (1996) Human apolipoprotein E receptor 2. Journal of Biological Chemistry, 271: 8373–8380. KIM, D.H., MAGOORI, K., INOUE, T.R., MAO, C.C., KIM, H.J., SUZUKI, H., FUJITA, T., ENDO, Y., SAEKI, S. & YAMAMOTO, T.T. (1997) Exon/intron organization, chromosome localization, alternative splicing, and transcription units of the human apolipoprotein E receptor 2 gene. Journal of Biological Chemistry, 272: 8498–8504. KOCH, S., STRASSER, V., HAUSER, C., FASCHING, D., BRANDES, C., BAJARI, T.M., SCHNEIDER, W.J. & NIMPF, J. (2002) A secreted soluble form of ApoE receptor 2 acts as a dominant-negative receptor and inhibits reelin signaling. EMBO Journal, 21: 5996–6004. LIU, L.Q., LI, F.E., DENG, C.Y. & XIONG, Y.Z. (2011) Molecular cloning, tissue expression and association of porcine NR4A1 gene with reproductive traits. Molecular Biology Reports, 38: 103–114. MAHON, M.G., LINDSTEDT, K.A., HERMANN, M., NIMPF, J. & SCHNEIDER, W.J. (1999) Multiple involvement of clusterin in chicken ovarian follicle development. Journal of Biological Chemistry, 274: 4036–4044. NOVAK, S., HIESBERGER, T., SCHNEIDER, W.J. & NIMPF, J. (1996) A new low density lipoprotein receptor homologue with 8 ligand binding repeats in brain of chicken and mouse. Journal of Biological Chemistry, 271: 11732–11736.
OLSON, G.E., WINFREY, V.P., NAGDAS, S.K., HILL, K.E. & BURK, R. F. (2007) Apolipoprotein E receptor-2 (ApoER2) mediates selenium uptake from selenoprotein P by the mouse testis. Journal of Biological Chemistry, 282: 12290–12297. SCHNEIDER, W.J., NIMPF, J., BRANDES, C. & DREXLER, M. (1999) The low-density lipoprotein receptor family: genetics, function, and evolution. Current Atherosclerosis Reports, 1: 115–122. STEPHENS, M., SMITH, N.J. & DONNELLY, P. (2001) A new statistical method for haplotype reconstruction from population data. American Journal of Human Genetics, 68: 978–989. TAMURA, K., DUDLEY, J., NEI, M. & KUMAR, S. (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Molecular Biology and Evolution, 24: 1596–1599. VASSILIOU, G., BENOIST, F., LAU, P., KAVASLAR, G.N. & MCPHERSON, R. (2001) The low density lipoprotein receptor-related protein contributes to selective uptake of high density lipoprotein cholesteryl esters by SW872 liposarcoma cells and primary human adipocytes. Journal of Biological Chemistry, 276: 48823–48830. WANG, L., WANG, X., LAIRD, N., ZUCKERMAN, B., STUBBLEFIELD, P. & XU, X. (2006) Polymorphism in maternal LRP8 gene is associated with fetal growth. American Journal of Human Genetics, 78: 770–777. YAO, J.F., CHEN, Z.X., XU, G.Y., WANG, X.L., NING, Z.H., ZHENG, J.X., QU, L.J. & YANG, N. (2010) Low-density lipoprotein receptor-related protein 8 gene association with egg traits in dwarf chickens. Poultry Science, 89: 883–886. ZHANG, L.C., NING, Z.H., XU, G.Y., HOU, Z.C. & YANG, N. (2005) Heritabilities and genetic and phenotypic correlation of egg quality traits in brown-egg dwarf layers. Poultry Science, 84: 1209–1213. ZHANG, C.L., WANG, Y., CHEN, H., LAN, X.Y. & LEI, C.Z. (2007) Enhance the efficiency of single-strand conformation polymorphism analysis by short polyacrylamide gel and modified silver staining. Analytical Biochemistry, 365: 286–287. ZHOU, Y., LIU, Y., XIAOSONG, J., DU, H., XIAOCHENG, L. & ZHU, Q. (2010) Polymorphism of chicken myocyte-specific enhancer-binding factor 2A gene and its association with chicken carcass traits. Molecular Biology Reports, 37: 587–594.
British Poultry Science Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/cbps20
Molecular cloning, expression and association study with reproductive traits of the duck LRP8 gene ab
a
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C. Wang , S.J. Li , C. Li , G.H. Yu , Y.P. Feng , X.L. Peng & Y.Z. Gong a
Key Laboratory of Agricultural Animal Genetics, Breeding, and Reproduction of Ministry of Education, Huazhong Agricultural University, Wuhan, Hubei 430070, P.R. China b
National Poultry Engineering Research Center, Shanghai Academy of Agricultural Sciences, Shanghai 201106, P.R. China Accepted author version posted online: 08 Oct 2013.Published online: 29 Nov 2013.
To cite this article: C. Wang, S.J. Li, C. Li, G.H. Yu, Y.P. Feng, X.L. Peng & Y.Z. Gong (2013) Molecular cloning, expression and association study with reproductive traits of the duck LRP8 gene, British Poultry Science, 54:5, 567-574, DOI: 10.1080/00071668.2013.819488 To link to this article: http://dx.doi.org/10.1080/00071668.2013.819488
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British Poultry Science, 2013 Vol. 54, No. 5, 567–574, http://dx.doi.org/10.1080/00071668.2013.819488
Molecular cloning, expression and association study with reproductive traits of the duck LRP8 gene C. WANG1,2, S.J. LI1, C. LI1, G.H. YU1, Y.P. FENG1, X.L. PENG1
AND
Y.Z. GONG1
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Key Laboratory of Agricultural Animal Genetics, Breeding, and Reproduction of Ministry of Education, Huazhong Agricultural University, Wuhan, Hubei 430070, P.R. China, and 2National Poultry Engineering Research Center, Shanghai Academy of Agricultural Sciences, Shanghai 201106, P.R. China
Abstract 1. Two splice variants of duck LRP8 were identified, one containing 8 ligand-binding repeats (LRP8-1) and the other containing only 7 repeats (LRP8-2). The two transcripts share ~71–91% nucleic acid identity and ~65–94% amino acid identity with their counterparts in other species. A phylogenetic tree based on amino acid sequences shows that duck LRP8 proteins are closely related to those of chicken, turkey and zebra finch. 2. The semi-quantitative reverse transcription polymerase chain reaction (RT-PCR )analysis indicates that the two transcripts are expressed in all the examined tissues, and the LRP8-1 transcript is more highly expressed in hypothalamus, ovary and pituitary gland than in other detected tissues. 3. Six single nucleotide polymorphisms (SNPs) were identified in the coding region. Association analysis demonstrated that the c.528C > T genotypes were associated with egg production (EP) (EP210d, EP300d and EP360d), age at laying the first egg (AFE) and body weight at sexual maturity (BWSM). The c.1371A > G genotypes were associated with egg production (EP210d, EP300d and EP360d). 4. The haplotypes of c.528C > T and c.1371A > G were associated with EP (EP210d, EP300d and EP360d), yolk weight (YW), albumen weight (AW), egg weight (EW), BWSM and the first egg weight (FEW). 5. Duck LRP8 gene was associated with some reproductive traits and is an important candidate gene for the genetic selection of improved reproductive traits.
INTRODUCTION Low-density lipoprotein receptor-related protein 8 (LRP8), also known as apolipoprotein E receptor 2 (ApoER2, in mammals), is a member of the lowdensity lipoprotein receptor (LDLR) gene family, which includes the LDLR and the very-low-density lipoprotein receptor (VLDLR). Except for mediating the lipid-rich extracellular lipoprotein uptake, LRP8 also participates in endocytosis and multiple intracellular signalling cascades (Vassiliou et al., 2001; Argov et al., 2004; Fayad et al., 2007). Moreover, LRP8 plays a crucial role in mouse sperm maturation and bovine follicular growth (Andersen et al., 2003; Argov et al., 2004; Olson et al., 2007).
A complete cDNA sequence containing one open reading frame of 2754 bp encoding a 918residue protein was identified in chicken and a 6.5 kb receptor transcript was detected in the brain and ovary (Brandes et al., 2001). Mahon et al. (1999) reported that chicken clusterin was a major product of the somatic granulosa cells and correlated with the developmental phases of individual follicles. Furthermore, they documented that LRP8 may internalise cell-derived granulosa clusterin. Subsequently, the role of LRP8 gene on follicular growth has been examined. Recently, a study in dwarf chickens showed that the LRP8 gene was associated with egg yolk colour, shape index and some eggshell traits, with the authors suggesting that the gene may be a
Correspondence to: Yan-Zhang Gong, College of Animal Science and Technology of Huazhong Agricultural University, No. 1, Shizishan Street, Hongshan District, Wuhan City, Hubei Province, P.R. China. E-mail: [email protected] Accepted for publication 7 May 2013.
© 2013 British Poultry Science Ltd
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candidate gene associated with egg traits (Yao et al., 2010). As an important agricultural species, the reproductive traits (for example, age at laying the first egg, body weight at sexual maturity, egg production and egg weight) of some local duck breeds need to be improved genetically. Although the functions of chicken LRP8 gene on follicular growth and egg traits have been studied, the information on duck LRP8 gene is scarce. In this study, the cloning of the gene sequence, analysis of gene structure and expression pattern analysis will facilitate future research into gene function. Association analysis identifies potential markers to improve the reproductive traits of ducks in future breeding programmes.
MATERIALS AND METHODS
cross using a standard phenol-chloroform method (Joseph and David, 2002). DNA concentration and quality were measured with spectrophotometer ND-1000 (Nano-Drop, USA), and the concentrations were adjusted between 50 and 300 ng/μl. DNA samples were stored at 4°C for later use. Total RNA was isolated from different tissues with Trizol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. The quality of total RNA samples was evaluated by electrophoresis on 1.2 % agarose gels stained with ethidium bromide, and their concentrations were measured with spectrophotometer ND-1000 (Nano-Drop, USA). cDNA was obtained by reverse transcription polymerase chain reaction (RT-PCR) from 1 μg of DNase-treated (DNaseI, TOYOBO Co., Japan,) total RNA according to M-MLV reverse transcriptase kit (TOYOBO Co, Japan) at 42°C.
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Ducks, tissue and data collection Ducks from the second (n = 296) and third (n = 251) generation of white Liancheng × white Kaiya cross (Gong et al., 2010) were provided by the Hankou Jingwu Industry Garden Ltd. The ducks were reared in cages in a semi-open house and were subjected to conventional management conditions. Recorded traits included age at laying the first egg (AFE), first egg weight (FEW), body weight at sexual maturity (BWSM) and egg production (EP, during 210 d, 300 d and 360 d) of each individual duck. Eggs were obtained from the above two generations for 3 consecutive days when the ducks were 295–300 d old. Data were averaged after cracked, soft-shell and double-yolked eggs had been removed. The egg quality traits measured were egg weight (EW), Haugh units (HU), eggshell index (ESI), albumen weight (AW), yolk weight (YW), yolk colour (YC), albumen height (AH), albumen ratio (AR), yolk ratio (YR) and eggshell strength (ESS) (Zhang et al., 2005). Three healthy female ducks (aged 25 weeks) were selected from the second generation of white Liancheng × white Kaiya cross reared under normal management conditions. A total of 12 tissues, including heart, liver, spleen, lungs, kidneys, skin, muscle, hypothalamus, intestine, pituitary gland, ovary and oviduct, were collected from each duck. The dissected tissues were immediately frozen in liquid nitrogen and subsequently stored at –80°C for later total RNA extraction. All animal procedures were performed according to protocols approved by the Biological Studies Animal Care and Use Committee of Hubei Province, P.R. China. DNA isolation, RNA isolation and cDNA synthesis Genomic DNA was extracted from 547 blood samples of the second (n = 296) and third (n = 251) generations of the white Liancheng × white Kaiya
Molecular cloning and sequence analysis of duck LRP8 gene Based on the conserved region of LRP8 gene in Gallus gallus (GI: 45383975), Meleagris gallopavo (GI: 326925389), Homo sapiens (GI: 66082553), Mus musculus (GI: 124286869) and Taeniopygia guttata (GI: 224058238), 4 pairs of primers (LRP8-F1/R1~LRP8-F4/R4) were designed to obtain the coding region sequence of the duck LRP8 gene (primers shown in Supplementary Table S1, accessible via the online version of the journal at: http://dx.doi.org/10.1080/ 00071668.2013.819488). The PCR programme included denaturation for 5 min at 94°C, followed by 38 cycles of 35 s at 94°C, 35 s at annealing temperature, 35–60 s at 72°C and an extension step of 5 min at 72°C. The purified PCR products were cloned into the PEASY-T1 vector (TransGen Biotech Co., Ltd., Beijing, China) and sequenced commercially (Invitrogen, Shanghai, China). The sequences were matched using SeqMan software (DNAstar). Secondary structure prediction was performed using online tools on the ExPASy website (http://cn.expasy.org/tools/). The phylograms were created by MEGA 4.0 NeighbourJoining (NJ) software with 1000 bootstrap trials after multiple alignments of sequence data by ClustalW (http://www.ebi.ac.uk/Tools/msa/ clustalw2/) (Edgar, 2004; Tamura et al., 2007). Expression profiling To determine the tissue expression of the two types of splice variants, semi-quantitative RT-PCR was carried out using total RNA from twelve duck tissues of three female ducks and a pair of primers (LRP8-A/S) encompassing the region corresponding to the 8th ligand-binding repeats (primers shown in Supplementary Table S1, accessible
GENE RELATED WITH DUCK REPRODUCTIVE TRAITS
via the online version of the journal at: http://dx. doi.org/10.1080/00071668.2013.819488). The PCR programme included a denaturation step of 5 min at 94°C, followed by 36 cycles of 30 s at 94°C, 30 s at 60°C, 30 s at 72°C and a final step of 5 min at 72°C. As control, a pair of primers (β-actin-A/S) (Supplementary Table S1, accessible via the online version of the journal at: http://dx. doi.org/10.1080/00071668.2013.819488) were used under the same conditions. PCR products were visualised on 2.0 % agarose gels stained with ethidium bromide under ultraviolet light.
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SNP screening and genotyping According to the cDNA sequence of the duck LRP8 gene obtained from the above RT-PCR reactions, 10 pairs of primers (LRP8-AF/AR~LRP8-JF/JR, Supplementary Table S1, accessible via the online version of the journal at: http://dx.doi.org/ 10.1080/00071668.2013.819488), were designed to screen the potential single nucleotide polymorphisms (SNPs) in the coding region. Ninety-six DNA samples from the second generation were used for PCR-SSCP (Single-Strand Conformation Polymorphism). The PCR reaction was performed in a 10 μl final volume, containing 50–300 ng of genomic DNA, 1X PCR buffer, 0.5 μM of each primer, 25 μM of dNTPs, 2.0 mM MgCl2 and 0.2 units Taq DNA Polymerase (TransGen Biotech Co., Ltd., Beijing, China). The PCR parameters were 5 min at 94°C followed by 35 cycles of 30 s at 94°C, 30 s at annealing temperature, 30 s at 72°C and a common final extension at 72°C. Aliquots of 3 μl of the PCR products were mixed with 10 μl of the denaturing solution (95% formamide, 25 mM EDTA, 0.025% xylene-cyanole and 0.025 % bromophenol blue), heated for 10 min at 98°C and chilled on ice rapidly. Denatured DNA was subjected to 12% PAGE (polyacrylamide gel electrophoresis) analysis, which was run with 1XTBE buffer for 16–20 h at 4°C under a constant voltage (120–150 V). The gel was stained with 0.1% silver nitrate and visualised with 2% NaOH solution (Zhang et al., 2007). The PCR products of two samples representing different PCR-SSCP genotypes were cloned into PEASY-T1 vector and were sequenced M1 1
M2 2
commercially. Polymorphism sites were analysed by sequence comparisons using DNAStar software (DNAstar Inc., Madison, WI, USA). A total of 547 samples from the second and third generations of white Liancheng × white Kaiya were subjected to genotyping analysis using the PCR-RFLP (Restriction Fragment Length Polymorphism) technique. The PCR reaction was performed as described above. A volume of 3 μl of the PCR products was digested with 3 or 5 units of restriction enzyme (RE) BseGI (HhaI/BcII) (TaKaRa, Tokyo, Japan) for 12 or 16 h at 37°C or 55°C, respectively, and separated by electrophoresis on 1.5% or 2.5% agarose gels stained with ethidium bromide; they were visualised under ultraviolet light, and the genotype of each sample was recorded. Haplotype reconstruction Haplotypes were constructed based on the SNPs identified in all 547 experimental birds using PHASE 2.0 programmer (Stephens et al., 2001; Zhou et al., 2010). The genetic status of the subjects was expressed as the combination of two haplotypes (diplotype configuration). Genetic effects of the diplotypes were performed with the model mentioned in the following section. Statistical analysis The general linear model (GLM) procedures of SAS software package (SAS Inst. Inc., Cary NC, USA) was used to determine associations of the different genotypes with reproductive traits according to the following model: Y ¼μþS þF þG þe where Y is the observed values of different reproductive traits, µ is the population mean, S is the fixed effects of generation, F is the fixed effects of sire family, G is the fixed effects of genotype or haplotype and e is the random error. Multiple comparisons were performed among the least squares means. Values were considered significant at P < 0.05 and are presented as least square means ± standard error. M1 4
M2 3
M3
1410 bp 1287 bp 406 bp
569
5 3391 bp
902 bp 326 bp
Figure 1. The PCR amplification results of duck LRP8 gene. Lane M1, Marker I; M2, DL2000 Marker; M3, DL2000 Plus. Lanes 1, 2, 3 and 4: amplification fragment of primer LRP8-F1/R1, LRP8-F2/R2, LRP8-F3/R3 and LRP8-F4/R4, respectively. All these primers were used to obtain the whole sequence of duck LRP8 gene. Lane 5, amplification fragment of primer LRP8-F2/R2 using duck genomic DNA.
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RESULTS
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Sequence analysis of duck LRP8 gene Primers LRP8-F1/R1, LRP8-F2/R2, LRP8-F3/R3 and LRP8-F4/R4 amplified a total 2867 bp sequence of duck LRP8 gene from brain tissue, and clear amplified bands are shown in Figure 1. The cDNA (GenBank: JN108873) contains an open reading frame (ORF) of 2751 bp, a 2 bp 5′-terminal untranslated region (5′-UTR) and a 114 bp 3′-UTR including a TGA termination codon (nucleotides 2754–2756 bp). The duck LRP8 nucleotide sequence shows high identity to its counterparts in Gallus gallus (GI: 45383975, 91%), Homo sapiens (GI: 65301118, 80%), Mus musculus (GI: 124286869, 78%), Sus scrofa (GI: 315468530, 76%), Bos taurus (GI: 148231248, 76%), Danio rerio (GI: 374428822, 71%), Oryctolagus cuniculus (GI: 291398861, 78%) and Taeniopygia guttata (GI: 224058238, 91%). The bioinformatic analysis indicated that the ORF encoded a protein of 917 amino acid residues with a molecular weight of 101.526 kDa and an isoelectric point (pI) of 4.68. A 24-amino-acid sequence (residues 1 to 24) constitutes the putative signal peptide at the N-terminus, two potential N-linked glycosylation sites (N-X-S or N-X-T), a 23amino-acid sequence (residues 836–858) in the putative transmembrane domain and a FDNPVY sequence in the cytoplasmic domain at the C-terminus (Supplementary Figure S1, accessible via the online version of the journal at: http://dx. doi.org/10.1080/00071668.2013.819488). Using the LRP8-F2/R2 primers, another shorter product was amplified (Figure 1). The sequence alignment result of the two PCR products showed that the shorter product had lost a 123 bp fragment, which exactly encoded the 8th repeat; the rest of the sequence was identical to the long product (Supplementary Figure S2, accessible via the online version of the journal at: http://dx.doi. org/10.1080/00071668.2013.819488). We postulated that the short one was another LRP8 mRNA transcription splice isoform. To confirm our conclusion, a single, approximately 3391 bp fragment (reference chicken genome DNA sequence), was obtained following amplification by PCR using duck genomic DNA and the LRP8-F2/R2 primers (Figure 1). The fragment was subcloned into a T-vector and sequenced commercially. The alignment between the genomic DNA sequence and mRNA sequence showed that the genomic DNA sequence contained the deletion fragment (123 nucleotides). Therefore, we concluded that we had identified two forms of LRP8, one containing 8 (LRP8-1, GenBank: JN108873) and the other with 7 (LRP8-2, GenBank: JN108874) ligand-binding repeats.
To investigate the evolutionary origin of duck LRP8, a phylogenetic tree was constructed with the amino acid sequences of duck and 9 other animal species, including Gallus gallus (GI: 45383976), Meleagris gallopavo (GI: 326925390), Homo sapiens-1 (GI: 61744471), Homo sapiens-2 (GI: 66082554), Homo sapiens-3 (GI: 61744467), Homo sapiens-4 (GI: 65301119), Sus scrofa (GI: 315468531), Mus musculus-1 (GI: 123229814), Mus musculus-2 (GI: 123229810), Mus musculus-3 (GI: 123229812), Mus musculus-4 (GI: 13928141), Oryctolagus cuniculus (GI: 291398862), Bos taurus (GI: 148231249), Danio rerio (GI: 326668703) and Taeniopygia guttata (GI: 224058239). The phylogenetic tree shows three groups, with duck, chicken, turkey and zebra finch belonging to one group, indicating that duck LRP8 proteins are closer to the three bird species than those of other animal species (Supplementary Figure S3, accessible via the online version of the journal at: http://dx.doi. org/10.1080/00071668.2013.819488). Expression profile Semi-quantitative RT-PCR was applied to detect the tissue distribution of the two duck LRP8 splice variants. The PCR product indicates that if the LRP8-1 variant was expressed, the amplification segment size would be 373 bp, and if the LRP8-2 variant was expressed, the amplification segment size would be 250 bp. The expression profile indicated that both of the PCR products could be detected in the twelve tissues, which means that the two duck LRP8 transcripts were widely expressed (Figure 2). The LRP8-1 transcript was more highly expressed in hypothalamus, ovary and pituitary gland than in other tissues, whereas the LRP8-2 transcript was equally expressed in all the examined tissues. SNP screening and association analysis By aligning the PCR sequences from different individuals, 6 synonymous polymorphisms, c.528C > T, c591C > T, c621C > T, c.642C > T, c.1371A > G and c2664A > G, were identified in the coding region of the duck LRP8 gene. The variations on three sites, c.528C > T, c.642C > T
M
1
2
3
4
5
6
7
8
9 10 11 12 LRP8-1 LRP8-2 β-action
Figure 2. Expression patterns of two LRP8 transcripts in different duck tissues. M: Marker I; Lanes1 to 12 represent heart, liver, spleen, lung, kidney, skin, muscle, hypothalamus, intestine, pituitary gland, ovary and oviduct, respectively. β-actin was used as the control.
GENE RELATED WITH DUCK REPRODUCTIVE TRAITS (A)
M
(B)
A1A1 A2A2 A1A2
M
300 bp
300 bp
200 bp
163 bp 200 bp 128 bp
100 bp
B1B1 B2B2 B1B2
297 bp 153 bp 144 bp
100 bp (C)
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M C1C1 C1C1 C2C2 C2C2 C1C2 C1C2
300 bp 200 bp 131 bp
100 bp
83 bp 48 bp
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Figure 3. PCR-RFLP results of three SNPs in the duck LRP8 gene. For locus A (c.528C > T): A1A1 (163 bp), A2A2 (35/128 bp) and A1A2 (35/128/163 bp) genotypes were generated (A); for locus B (c.642C > T): B1B1 (297 bp), B2B2 (144/153 bp) and B1B2 (144/ 153/297 bp) genotypes were generated (B) and for locus C (c.1371A > G): C1C1 (131 bp), C2C2 (48/83 bp) and C1C2 (48/83/131 bp) genotypes were generated (C). The genotypes are shown at the top of each lane (M: Marker 1, a lane representing a fragment of 35 bp is not shown).
and c.1371A > G, led to changes in recognised restriction enzyme (RE) sites of BseGI, BcII and HhaI, respectively, and the variations on these sites could be genotyped by PCR-RFLPs. The results of RE digestion showed that the digested fragments were completely consistent with the predicted fragment size. For locus A (c.528C > T), A1A1 (163 bp), A2A2 (35 + 128 bp) and A1A2 (35 + 128 + 163 bp) were generated (Figure 3(A)). For locus B (c.642C > T), B1B1 (297 bp), B2B2 (144 + 153 bp) and B1B2 (144 + 153 + 297 bp) were generated (Figure 3 (B)). And for locus C (c.1371A>G), C1C1 (131 bp), C2C2 (48 + 83 bp) and C1C2 (48 + 83 +131 bp) were generated (Figure 3(C)). For these three polymorphic sites, all the three genotypes were detected in our resource population. The results of the association analyses between genotypes of locus A and locus C on the duck LRP8 gene and 14 reproductive traits (EP, BWSM, AFE, EW, FEW, YW, AW, AH, ESI, ESS, AR, YR, YC and HU) are summarised in Table 1: for locus B no association was found. In locus A, genotypes had
significant associations with EP (EP210d (P = 0.014), EP300d (P = 0.002) and EP360d (P < 0.001)), AFE (P = 0.027) and BWSM (P = 0.041), but no association was observed for the other 11 traits. The ducks harbouring genotypes A1A1 and A1A2 had higher EP (P < 0.01), earlier AFE and lower BWSM (P < 0.05) than those harbouring A2A2 but showed no difference between A1A1 and A1A2. In locus C, different genotypes had an effect on EP (EP210d (P = 0.015), EP300d (P = 0.019) and EP360d (P = 0.002)). The EP210d and EP300d of C2C2 and C1C2 were higher than those of genotype C1C1 (P < 0.05), the EP360d of C2C2 was higher than that of the other two genotypes. For locus B, no significant association was observed for the 14 reproductive traits (P > 0.05) (data not shown). Construction of haplotypes and association analysis Table 2 shows all haplotypes that were reconstructed from the two SNPs (locus A and C) in all 547 experimental ducks. Three haplotypes with
Table 1. Association analysis between the duck reproductive traits and LRP8 gene SNPs (values are means ± SE for the different genotypes) Locus A Traits1 EP210d (n) EP300d (n) EP360d (n) AFE(d) BWSM (kg)
A1A1(52)2 77.4 156.5 209.1 120.6 1.796
± ± ± ± ±
2.3a 3.1A 3.8 A 2.0a 0.032a
A2A2(241) 72.8 150.1 200.2 122.7 1.843
± ± ± ± ±
1.0b 1.4B 1.8B 0.9b 0.015b
A1A2(254) 77.3 ± 1.0a 157.2 ± 1.4A 210.0 ± 1.7A 119.1 ± 0.9a 1.797 ± 0.014a
Locus C
EP210d (n) EP300d (n) EP360d (n)
C1C1(23)
C2C2(401)
67.0 ± 3.3a 142.6 ± 4.7a 192.0 ± 5.7A
76.4 ± 0.8b 155.8 ± 1.1b 208.6 ± 1.3B
C1C2(123) 73.3 ± 1.4b 151.5 ± 2.0b 201.0 ± 2.4AB
EP210d = egg production within 210 d; EP300d = egg production within 300 d; EP360d = egg production within 360 d; AFE = age at laying the first egg; BWSM = body weight at sexual maturity. 2The numbers in the brackets are the chicken individuals of respective genotypes. Within a column, values not sharing a common superscript letter are significantly different (a,bP < 0.05; A,BP < 0.01). 1
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Table 2. Haplotypes
c.528 C > T
c.1371 A > G
Frequency (%)
A2 A2 A1 A1
C2 C1 C2 C1
32.19 0.90 52.80 14.11
H1 H2 H3 H4
Table 3.
Diplotypes of duck LRP8 gene
Diplotypes
Frequency (%)
H1H1 H1H3 H1H4 H3H3 H3H4 H4H4
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binding repeats (LRP8-1) and the other containing only 7 repeats (LRP8-2), and this represented the alternative splicing of an extra exon. It is supposed that the alternative splicing is evolutionally conserved. LRP8 (also named as ApoER2) was first cloned and characterised from a human placenta library, and subsequently, a novel receptor termed LR8B was discovered in the brain of chicken and mouse (Kim et al., 1996; Novak et al., 1996). Later studies showed that the receptors were not two distinct members of the LDLR family, but they originated from alterative splicing of the mRNA transcribed from a single gene (Brandes et al., 1997). Recently, several splicing variants of LRP8 mRNA have been detected and the corresponding proteins have been predicted in chicken, mouse, human and bovine, and their structures in these different species have been elucidated (Novak et al., 1996; Kim et al., 1997; Brandes et al., 2001; Argov and Sklan, 2004). For the products of these spliced variants, if and when they are translated, constitute receptors with (i) variant numbers of cysteine-rich repeats in the binding domain, (ii) with or without a potential furin-cleavage site downstream of the LA (type A binding repeats) repeats, (iii) with or without the putative O-linked sugar domain, and (iv) different by an insertion sequence in the cytoplasmic domain (Clatworthy et al., 1999; Schneider et al., 1999; Brandes et al., 2001; Koch et al., 2002). In this study, the products of two spliced variants of the duck LRP8 belong to the first spliced mode. The same mode has been reported in chicken and mouse (Brandes et al., 1997). Similar to LDLR and VLDLR, LRP8 also consists of 5 functional domains: (i) an amino-terminal ligand-binding domain composed of multiple cysteine-rich repeats, (ii) an epidermal growth factor (EGF) precursor homologous domain, (iii) an O-linked sugar domain, (iv) a 23-aminoacid sequence in the putative transmembrane domain and (v) a cytoplasmic domain with a FDNPVY sequence (Supplementary Figure S1,
Haplotypes inferred on the basis of two SNPs in the LRP8 gene
46 (8.49 ) 216 (39.85) 51 (9.41) 146 (26.94) 61 (11.25) 20 (3.69)
frequency higher than 2%, including H1 (A2C2, 32.19%), H3 (A1C2, 52.8%) and H4 (A1C1, 14.11%) were identified. The three main haplotypes accounted for 99.1% of the observations. Eight diplotypes were obtained based on the above three haplotypes. Among them, 6 diplotypes had a frequency higher than 1% and accounted for 99.63% of the genotypes (Table 3). The association analysis indicated that there was a significant association of diplotypes with reproductive traits (Table 4). Diplotypes had a significant effect on EP (EP210d (P = 0.008), EP300d (P = 0.003) and EP360d (P < 0.001)), YW (P < 0.001), AW (P = 0.005), BWSM (P < 0.001), EW (P < 0.001) and FEW (P < 0.001). The H4H4 diplotype presented higher BWSM and FEW but lower EP, EW, YW and AW, whereas the H1H4 diplotype presented lower BWSM and FEW but higher EP, EW, YW and AW.
DISCUSSION In the current study, two transcripts of duck LRP8 gene were identified, one containing 8 ligandTable 4. Reproductive traits1 BWSM(kg) FEW(g) EW(g) YW(g) AW(g) EP210d EP300d EP360d
Associations between diplotypes and duck reproductive traits (values are means ± SE for the different diplotypes) Diplotype H1H1 1.77 47.6 68.8 20.6 36.4 78.4 158.1 210.2
± ± ± ± ± ± ± ±
H1H3 a
0.03 1.4ab 0.9A 0.6AB 0.8AB 2.3A 3.5A 3.9A
1.80 46.2 67.8 20.1 35.1 77.8 157.1 210.9
± ± ± ± ± ± ± ±
H1H4 ab
0.02 0.6ab 0.4A 0.3AB 0.5AB 1.1A 1.8A 1.8A
1.77 44.7 69.4 20.7 36.8 78.3 159.8 210.3
± ± ± ± ± ± ± ±
H3H3 a
0.03 1.3b 0.9A 0.6AB 0.8A 2.2 A 3.3A 3.7A
1.84 46.6 68.6 20.7 35.1 75.2 154.1 205.7
± ± ± ± ± ± ± ±
H3H4 ab
0.02 0.8ab 0.5A 0.4AB 0.6AB 1.3 AB 2.1AB 2.2AB
1.84 46.6 67.2 22.1 36.1 70.2 146.1 193.6
± ± ± ± ± ± ± ±
H4H4 ab
0.03 1.2ab 0.8A 0.6A 0.8AB 2.0BC 3.0BC 3.4B
1.88 50.7 63.5 18.9 33.8 66.7 143.2 191.8
± ± ± ± ± ± ± ±
0.05b 2.1a 1.5B 0.9B 1.2B 3.5C 5.4C 5.9B
EP210d = egg production within 210 d; EP300d = egg production within 300 d; EP360d = egg production within 360 d; AFE = age at laying the first egg; BWSM = body weight at sexual maturity; FEW = first egg weight; EW = egg weight; AW = albumen weight; YW = yolk weight. Within a row, values not sharing a common superscript letter are significantly different (a,bP < 0.05; A,B,CP < 0.01).
1
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GENE RELATED WITH DUCK REPRODUCTIVE TRAITS
accessible via the online version of the journal at: http://dx.doi.org/10.1080/ 00071668.2013.819488). In this study, we cloned the gene sequence containing these 5 functional domains and showed that they shared striking homology to LRP8 gene sequence in other species. These data on the gene sequence, especially the coding region of the duck LRP8 gene, will facilitate future research into the function of this gene. The close relationship through evolution and the highly conservative structure suggest that the gene probably performs similar functions in different avian species. In the coding region, 6 synonymous mutations revealed that the duck LRP8 gene was rich in polymorphisms but that the sequence of LRP8 amino acids is highly conserved in different ducks. In humans, ApoER2 mRNA is predominantly expressed in brain and placenta and is undetectable in other tissues. In rabbits, ApoER2 mRNA is detected most intensely in brain and testis and, to a much lesser extent, in ovary, but is undetectable in other tissues. In chickens, the 6.5-kb transcript is present at high levels in brain and at much lower levels in ovary. In mouse, the same transcript of 6.5 kb is detected only in brain (Kim et al., 1996; Novak et al., 1996). Argov et al. (2004) have detected the presence of bovine LRP8 mRNA in ovarian granulose cells, which play pivotal roles in many aspects of ovarian functions including folliculogenesis and steroidogenesis (Argov and Sklan, 2004; Argov et al., 2004). In the present study, the two duck LRP8 transcripts were detected in all the examined tissues, and the expression of the LRP8-1 transcript was higher in the hypothalamic-pituitary-gonadal (HPG) axis, which participates in the regulation of reproductive activities, including folliculogenesis, ovulation, oviposition and incubation behaviour (Chen et al., 2007; Liu et al., 2011). The differential tissue distribution of LRP8 in different species implies that the receptor plays an important role in the central nervous system and in reproductive activities. Wang et al. (2006) reported that a SNP in LRP8 was associated with birth weight of progeny in black women. Yao et al. (2010) demonstrated that the C1623T polymorphism of chicken LRP8 was associated with egg yolk colour, shape index and some eggshell traits in dwarf chicken. In this study, these polymorphisms of duck LRP8 gene were significantly associated with the reproductive traits EP, AFE, BWSM, EW, YW and AW. Although the SNPs were synonymous mutations and did not directly change the amino acid sequence of the protein, they may alter gene function by affecting translation with codon bias, changing the stability of the mRNA or controlling the transcription of the gene (Duan, 2003). The results indicate that the LRP8 gene
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may be an important candidate gene in duck reproduction. The heavier body weight at sexual maturity, the larger size of the first egg, and, generally, if the birds lay their first eggs earlier, they will lay more eggs. Our results suggest that the LRP8 gene may be a good candidate gene of EP and AFE traits and provide some marker information for duck breeding. In summary, two variants of duck LRP8 transcripts were identified and characterised, and their tissue expression patterns were analysed. Association analysis showed that the duck LRP8 gene was related with some reproductive traits, and it is suggested that LRP8 is an important candidate gene for these traits and can be used as a potential marker to improve them.
ACKNOWLEDGEMENTS The authors gratefully acknowledge Dr Xiangdong Liu and Dr Ming Xiong for help on the statistical analysis. This work was supported by the Project of Scientific Research and Development in Hubei Province (2011BBB096), NSFC (31172201) and Fundamental Research Funds for the Central Universities (2011QC006).
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