Molecular cloning,alternative splicing and mRNA expression analysis of MAGI1 and its correlation with laying performance in geese

1. In a previous study, a laying-related single nucleotide polymorphism (SNP) Record-106975 (A–G) was found in the intron of Membrane-associated Guanylate Kinase with Inverted Domain Structure-1 (MAGI1) gene by restriction site-associated DNA sequencing. This study was performed to further identify the function of MAGI1 on laying performance in Yangzhou geese.

2. The complete MAGI1 cDNA sequence was cloned from the ovary of the Yangzhou goose. A total of 4 alternative splice variants were identified, and the characterisation of their nucleic acid and amino acid (AA) sequences were analysed. The AA sequence of goose MAGI1 was highly conserved with other avian species.

3. MAGI1 was ubiquitously expressed in 11 tested tissues, with greater amounts of mRNA in the granulosa cell layer and ovary. GG (n = 14) and GA (n = 6) genotypes of SNP Record-106975 showed significantly lower MAGI1 mRNA levels than those of the AA genotype.

4. Over-expression and knockdown experiments revealed that MAGI1 mRNA expression was associated with caspase-3 mRNA levels in granulosa cells, and may play a role in regulating apoptosis of granulosa cells.     

北京鸭PRLR基因部分序列的克隆及分析

利用PCR方法克隆了北京鸭催乳素受体(PRLR)基因部分序列,该序列长1325bp。序列分析结果表明,该序列由第9外显子的一部分(57bp)、第9内含子(635bp)以及第10外显子的一部分(633bp)组成;在核苷酸水平上,北京鸭的该序列(除去内含子)与鹅(U07694)、鸡(D13154)、野鸽(DQ209271)、火鸡(L76587)的相似性分别为94.2%、88.7%、86.8%、86.1%;在氨基酸水平上,与鹅(ABA71353)、鸡(BAA02439)、野鸽(AAA20646)、火鸡(AAB01544)的相似性分别为93.5%、84.3%、80.8%、80.9%。     

松辽黑猪OAS1基因多态性及其与繁殖性状的关联分析

为探究寡腺苷酸合成酶1（oligoadenylate synthase 1,OAS1）基因多态性与松辽黑猪繁殖性状的关联性,试验选取130头松辽黑猪母猪为研究对象,利用Sanger直接测序法测序查找OAS1基因外显子1~8的SNP位点,使用SPSS 19.0软件分析OAS1基因SNP位点与松辽黑猪繁殖性状的关联性。结果显示,在松辽黑猪OAS1基因外显子2、3和6上共检测到33个突变位点;其中在外显子2的110 bp处存在1个SNP位点（G110C）,存在3种基因型：GG、GC和CC;在外显子3的176 bp处存在1个SNP位点（C176T）,存在3种基因型：CC、CT和TT;在外显子6的145 bp处存在1个SNP位点（C145T）,存在3种基因型：CC、CA和AA;在166 bp处存在1个SNP位点（G166A）,存在3种基因型：GG、GA和AA;在206 bp处存在1个SNP位点（A206G）,存在3种基因型：AA、AG和GG。卡方适合性检验结果显示,松辽黑猪OAS1基因G110C突变位点符合Hardy-Weinberg平衡状态,C176T、C145A、G166A和G206A位点均偏离Hardy-Weinberg平衡状态。群体遗传参数分析结果显示,各SNPs位点遗传杂合度均位于中等水平,为中度多态（0.25<PIC<0.5）。关联分析结果发现,G110C位点GC基因型个体总产仔数、产活仔数和断奶仔猪数均显著高于GG基因型个体（P<0.05）;C176T位点CT基因型个体断奶仔猪数显著高于CC基因型个体（P<0.05）;C145T位点CC基因型个体总产仔数和产活仔数均显著高于AA基因型个体（P<0.05）;G166A位点GA基因型个体断奶仔猪数显著高于GG基因型个体（P<0.05）;A206G位点GG基因型个体总产仔数和产活仔数显著高于AA基因型个体（P<0.05）。结果表明,OAS1基因外显子区存在突变位点,对松辽黑猪部分繁殖性状有显著性影响。     

Polymorphism of inhibin βB gene and its relationship with litter size in sheep

The inhibin β_B (INHBB) gene was studied as a candidate gene for the prolificacy of Small Tail Han and Hu sheep. According to the sequence of exon 1 and 2 of bovine INHBB gene, six pairs of primers were designed to detect single nucleotide polymorphisms of exon 1 and 2 of INHBB gene in both high (Small Tail Han and Hu sheep) and low prolificacy breeds (Dorset, Texel and German Mutton Merino sheep) by polymerase chain reaction‐single strand conformation polymorphism (PCR‐SSCP). Three pairs of primers (primers 1‐1, 1‐2 and 1‐3) were used to amplify the exon 1, and others (primers 2‐1, 2‐2 and 2‐3) to the exon 2. Only the products amplified by primer 2‐3 displayed polymorphism. For primer 2‐3, three genotypes (AA, AB and BB) were detected in Hu sheep and only AA genotype in other breeds. In Hu sheep, frequency of AA, AB and BB genotypes was 0.636, 0.046 and 0.318, respectively. Sequencing revealed 276A > G mutation (based on the amplification region of primer 2‐3) which did not cause any amino acid change because it lay in the 3′ untranslated region. The ewes with genotype BB had 0.58 (P < 0.01) lambs more than those with AA in Hu sheep.     

Association Analysis of Polymorphism in the CYP2C45 Gene with Fatty Liver Production in Geese

In order to study the influence of genetic variation in CYP2C45 gene on fatty liver production performance, PCR and sequencing methods were used to identify polymorphism of CYP2C45 gene exons in 113 Landes goose, and the association of polymorphism with production performance of goose fatty liver were investigated. The results indicated that there were 4 SNPs in exon 5 at 111 bp of C>T transition and at 147 bp of C>T transition, and in exon 7 at 71 bp of A>G transition and at 135 bp of G>A transition, respectively. These SNPs were in the stage of complete linkage by linkage disequilibrium analysis. Combining 4 SNPs as one genotype, there were AA (CCAG/CCAG) and AB (CCAG/TTGA) genotypes, but BB genotype (TTGA/TTGA) was not found， the genotype frequencies were 0.9027 and 0.0973, respectively, and the gene frequencies of alleles A and B were 0.9513 and 0.0487, respectively. Analysis of variance showed that the body weight of AB genotype was higher significantly than AA genotype (P<0.05), although the liver weight and ratio of liver weight versus body weight of AA genotype were higher than AB genotype, there were no significant difference （P>0.05）. It suggested that, in the case of the same yield of liver production, less body weight less feeding, AA genotype maybe contribute to the improvement of production efficiency.     

豁眼鹅GH基因外显子2多态性与体重和体尺性状的关联分析

实验旨在探究对豁眼鹅体重和体尺性状进行选育的有效分子标记,根据GenBank鹅GH基因的cDNA序列以及前人的研究设计引物,以饲养于辽宁省和江苏省同批出雏的豁眼鹅,共计552只为实验材料,利用PCR-SSCP方法对GH基因外显子2进行SNPs检测,并将纯合基因型进行克隆测序。结果表明:豁眼鹅GH基因外显子2共有2个SNPs突变位点,得到10种基因型AA、BB、CC、DD、AB、AC、AD、BC、BD和CD,等位基因D的频率较高;经χ2检验,公、母鹅群体以及整个群体均处于Hardy-Weinberg平衡状态;将氨基酸序列突变和未突变的基因型分别合并后得到的3种基因型与10周龄体重和体尺性状进行关联分析发现,基因型对体重和部分体尺性状均有极显著(P<0.01)或显著(P<0.05)影响;在公鹅和母鹅群体中,合并后的AB型与BB型分别为高生产性能基因型,可以作为对豁眼鹅体重和体尺性状进行选育的分子标记。     

边鸡MSTN基因C.234位点对生长性状的遗传效应分析

采用PCR-RFLP技术检测连续2个世代边鸡群体肌肉生长抑制素基因外显子1的多态性,分析了多态位点对边鸡生长性状的遗传效应。结果表明,边鸡MSTN基因的C.234位点存在多态性,在外显子1编码区的234bp处发生G→A的碱基突变,产生了GG、GA和AA 3种基因型。关联分析表明,无论是一世代还是二世代的边鸡群体,在6~16周龄时,AA和GA基因型边鸡的体质量均显著或极显著的高于GG基因型（P〈0.05或P〈0.01）。结果提示,MSTN基因外显子1的C.234位点突变对边鸡的生长性状具有显著的遗传效应,初步推断该位点可用于边鸡生长性状的标记辅助选择。     

Association of MYF5 and KLF15 gene polymorphisms with carcass traits in domestic pigeons (Columba livia)

1. Single nucleotide polymorphisms (SNPs) in the exons of the myogenic factor 5 (MYF5) and Kruppel-like factor 15 (KLF15) genes were identified and analysed by using DNA sequencing methods in 60 female domestic pigeons (Columba livia).

2. Five SNPs (T5067A, C5084T, C5101T, T5127A and C5154G) were detected in exon 3 of MYF5 and 6 SNPs (C1398T, C1464T, G1542A, C1929T, G1965A and A2355G) were found in exon 2 of KLF15, respectively. The analysis revealed three genotypes, in which the AA genotype was dominant and the A allele showed a dominant advantage.

3. For the MYF5 gene, the C5084T and T5127A SNP genotypes were significantly associated with carcass traits of pigeons. Within those two SNPs, the BB genotype showed relatively higher trait association values than those of AA or AB genotypes. No significant association was observed between the KLF15 SNP genotypes and carcass traits.

4. These results indicated that the MYF5 gene is a potential major gene affecting carcass traits in domestic pigeons. The BB genotype of the C5084T and T5127A SNPs could be a potential candidate genetic marker for marker-assisted selection in pigeon.

     

石歧杂鸡催乳素基因外显子2的SNP检测与产蛋就巢性状的关联分析

利用PCR-SSCP技术对石歧杂鸡的催乳素(prolactin,PRL)基因外显子2进行SNP检测和测序分析,并对该基因与石歧杂鸡产蛋、就巢性状的相关性进行了研究。结果表明,PRL基因外显子2存在多态性位点,该位点由于在3838位点发生C→T的碱基突变,产生了AA、AB和BB3种基因型,并导致氨基酸的改变,由亮氨酸变为苯丙氨酸。基因型AA和等位基因A的频率最高;BB基因型个体首次就巢时间显著晚于AA和AB基因型(P0.01);BB基因型的就巢次数显著多于AA和AB基因型(P0.05);3种基因型与产蛋性状相关性不显著。     

Paired box 7 (Pax7) gene: molecular characterisation,polymorphism and its association with growth performance in goose (Anser cygnoides)

1. Paired box (Pax7) gene is a member of the paired box family and plays a critical role in animal growth and muscle development. However, the molecular characterisation of the goose Pax7 gene is unknown.

2. The open-reading frame of goose Pax7 is composed of 1509 bp, which encodes a protein of 503 amino acids and shares high homology with Pax7 of other birds.

3. Ten single-nucleotide polymorphisms were identified in the genomic DNA sequence, 8 located in the intron region and two located in the exon region.

4. Association analysis showed the C122T locus was significantly associated with the body weight of Zhedong-White geese in week 4, 6, 8, 10 and 12.

5. It was concluded that the goose Pax7 gene may be an important candidate gene for goose growth traits and marker-assisted selection.     

吉林白鹅类胰岛素生长因子Ⅱ的SNP及其与屠体性状的相关性分析

试验设计了5对引物,采用PCR-SSCP方法对吉林白鹅肉用品系85只个体类胰岛素生长因子Ⅱ（IGFⅡ）进行了SNP检测,分析其群体遗传结构,并对SNP与屠体性状的相关性进行了研究。结果表明,在IGFⅡ外显子3中存在3种基因型AA、AB、BB基因型,基因型频率分别为0.7647、0.1412、0.0941,AA为优势基因型;存在2种等位基因A、B,基因频率分别为0.8353、0.1647,A为优势等位基因。IGFⅡ外显子3中发现1处SNP位点（A→G）,该突变为沉默突变,未引起氨基酸序列改变。对3种基因型进行方差分析,结果发现,BB基因型个体半净膛重、肝脏重显著高于AA、AB基因型（P＜0.05）;腿肉重BB基因型极显著高于AA基因型（P＜0.01）;基因型对全净膛重、翅重、胸肉重无显著影响（P＞0.05）。     

Association of allelic polymorphisms of IGFALS gene with growth traits in Makouei and Ghezel sheep breeds

Insulin-like growth factor-binding protein acid-labile subunit (IGFALS) encodes a protein which binds to IGF1 and IGFBP-3 to regulate the growth, differentiation, and other physiological processes. The aim of this study was the identification of allelic polymorphisms of the IGFALS gene using the PCR-RFLP technique and evaluation of their association with growth traits. For this end, 120 blood samples were randomly collected from each breed. Following amplification of an 1113-bp fragment of exon 1 and a part of intron 1 of the IGFLAS gene, genotyping was conducted by three restriction enzymes including HinfI, MscI, and PvuII. The results showed that only one allele was observed in IGFALS-PvuII site, while in IGFLAS-MscI site, three AA, AB, and BB genotypes with the frequencies of 17.5%, 32%, and 50.5% and 11%, 37.5%, and 51.5% were observed in Makouei and Ghezel sheep breeds, respectively. Additionally, in the IGFLAS-HinfI site, two AB and BB genotypes with the frequency of 34.2% and 65.8% were observed in Makouei sheep and AA, AB, and BB genotypes with the frequency of 9%, 21%, and 70% were observed in Ghezel sheep. So that, Makouei sheep with AB genotype had more chest girth (CG) compared with other genotypes. Furthermore, a significant association was observed between the genotypes of IGFLAS-HinfI with birth weight (BW) in Ghezel and BW, weaning weight (BW3), and CG in Makouei sheep. Haplotype analysis revealed an association between paternal haplotypes and BW in both Ghezel and Makouei breeds. So that, AAB and ABB haplotypes showed more BW than others in Makouei and Ghezel sheep, respectively.

     

DNA polymorphism of insulin-like growth factor-binding protein-3 gene and its association with birth weight and body weight in cattle

Insulin‐like growth factor‐binding protein‐3 (IGFBP‐3) is a protein that binds the majority of insulin‐like growth factors in circulation for regulation of its action on growth and metabolism of the animals. Animals belonging to Hariana, Holstein‐Friesian (HF) and their crossbreds (HF × Hariana) were studied using polymerase chain reaction‐restriction fragment length polymorphism and nucleotide sequencing of the IGFBP‐3 gene. A 651‐bp fragment of the IGFBP‐3 gene spanning over a part of exon 2, complete intron 2, exon 3 and a part of intron 3 was amplified and digested with HaeIII restriction enzyme. Three patterns of restriction fragments were observed in HF and crossbred cattle revealing polymorphism in both the populations. The frequency of AA, AB and BB genotypes was 0.65, 0.32 and 0.03 in crossbreds and 0.29, 0.65 and 0.06 in HF respectively. The allelic frequency of the A and B allele was 0.81 and 0.19 in crossbreds and 0.62 and 0.38 in HF cattle respectively. Only one restriction pattern (AA genotype) was observed in all the animals of Hariana breed of Bos indicus showing the absence of polymorphism. Nucleotide sequencing revealed a C → A mutation in the intron 2 region of the IGFBP‐3 gene as the cause of the polymorphism. Least squares analysis revealed a significant effect (p < 0.05) of genotypes on birth weight and body weight (weight at 12, 18 and 24 months of age) of the animals. Animals of AB genotype showed higher birth weight and body weight than the animals possessing AA genotype.     

Study on the Polymorphisms of IGF-ⅠR Gene Alu Ⅰ Locus and Genetic Effect on Growth and Reproduction Traits in Bian Chicken

Single nucleotide polymorphisms (SNPs) in exon 2 of insulin-like growth factor Ⅰ receptor (IGF-ⅠR) gene were detected by PCR-RFLP in Bian chicken,which was to find the correlation markers of growth and reproduction traits in Bian chicken.The results showed that AluⅠsite displayed polymorphic, and a G→A mutation at the position of the 376 bp in exon 2 was occurred. There were three genotypes (GG, GA and AA), the genotypes frequencies were 0.687, 0.296 and 0.017, respectively, GG genotype was the dominant genotype, the frequency of G allele was 0.835, which was the dominant allele. This site was in Hardy-Weinberg equilibrium (P>0.05).Correlation analysis showed that GG genotype had significantly higher body weight than those of GA genotype at the age of 8, 14, 16 and 18 weeks (P<0.05);The GG geno type had extremely significantly higher body weight at first egg than those of GA genotype (P<0.01),but it didn't mean that this locus could be used as a genetic marker for reproduction traits in Bian chicken. This results suggested that the AluⅠsite (G→A) in exon 2 of IGF-ⅠR gene could be used as a genetic marker for growth traits in Bian chicken.     

鸭脂肪酸结合蛋白基因的克隆和序列多态性分析

根据鸡脂肪型脂肪酸结合蛋白（A-FABP）基因序列设计1对引物对鸭基因组进行PCR扩增，将扩增出的475bp片段进行克隆和测序，序列分析表明该基因片段包含部分外显子2、3和完整的内含子2序列，与鸡、鹅、猪的A-FABP基因分别有84％、94％和77％的同源性。在此基础上又设计3对引物利用PCR—SSCP方法对获得的序列进行多态性研究。结果引物P3在樱桃谷鸭、山麻鸭等品种中发现4处单碱基突变，分别为：241处“A～T”突变；243处的“T—C”突变；258处的“T—C”突变；299处的“C—T”突变。这些点突变共产生了3种基因型，且基因型分布在各鸭品种间有显著差异。     

Agouti signalling protein (ASIP) gene: molecular cloning,sequence characterisation and tissue distribution in domestic goose

1. Agouti signalling protein (ASIP) is an endogenous antagonist of melanocortin-1 receptor (MC1R) and is involved in the regulation of pigmentation in mammals. The objective of this study was to identify and characterise the ASIP gene in domestic goose.

2. The goose ASIP cDNA consisted of a 44-nucleotide 5?-terminal untranslated region (UTR), a 390-nucleotide open-reading frame (ORF) and a 45-nucleotide 3?-UTR. The length of goose ASIP genomic DNA was 6176 bp, including three coding exons and two introns.

3. Bioinformatic analysis indicated that the ORF encodes a protein of 130 amino-acid residues with a molecular weight of 14.88 kDa and an isoelectric point of 9.73.

4. Multiple sequence alignments and phylogenetic analysis showed that the amino-acid sequence of ASIP was conserved in vertebrates, especially in the avian species.

5. RT-qPCR showed that the goose ASIP mRNA was differentially expressed in the pigment deposition tissues, including eye, foot, feather follicle, skin of the back, as well as in skin of the abdomen. The expression level of the ASIP gene in skin of the abdomen was higher than that in skin of the back.

6. Those findings will contribute to further understanding the functions of the ASIP gene in geese plumage colouring.

     

DGAT1, GH, GHR, PRL and PRLR polymorphism in water buffalo (Bubalus bubalis)

The polymorphism of several genes has been shown to affect the milk composition traits in dairy cattle, including DGAT1‐exon8 K232A, GH‐intron3 MspI, GH‐exon5 AluI, GHR‐exon8 F279Y, PRL‐exon3 RsaI and PRLR‐exon3 S18N. However, the polymorphism and effects of these genes on the milk traits of water buffalo are still unclear. In this study, four DNA pooling samples from Murrah, Nili‐ravi, Murrah‐Nili‐Swamp crossbreed and Chinese swamp buffalo were constructed, respectively, and polymorphism of these sites was investigated using PCR–Single‐strand conformation polymorphism and sequencing. Twenty‐eight inter‐specific single‐nucleotide polymorphism (SNPs) were found in these six assayed gene fragments between buffalo and dairy cattle, including nine intra‐specific SNPs among buffalo groups. All buffalo fixed a K allele genotype in DGAT1‐exon8, MspI⁺ restriction site(c nucleotide) and AluI⁺ site(c nucleotide) at intron3 and exon5 of GH gene, F allele genotype of F279Y mutation in GHR gene, RsaI^? restriction site at PRL‐exon3/exon4 and N allele genotype of S18N mutation at PRLR‐exon3. It provides an indirect evidence that water buffalo have fixed alleles with genotypes reported in dairy cattle, which is thought to be responsible for high milk fat, high protein content and low milk yield. Moreover, three new intra‐specific SNPs were found including 275th bp (c/t) in DGAT1 of Murrah buffalo, 109th bp (t/a) in PRL‐exon3/exon4 and 43rd bp (c/t) in PRLR‐exon3 of Chinese swamp buffalo. Information provided in this study will be useful in further studies to improve buffalo breeding for better lactation performances.