Ovine alpha-amylase genes: isolation, linkage mapping and association analysis with milk traits

On the basis of comparisons between cattle and sheep genome mapping information the ovine alpha-amylase gene was examined as a possible genetic marker for milk traits in sheep. The objective of the present study was to isolate, map and determine whether this gene is a candidate gene for milk traits. DNA fragments (832 and 2360 bp) corresponding to two different AMY genes were isolated, and one SNP in intron 3 and one GTG deletion in exon 3 of the 2360 bp DNA fragment were found. The 2360 bp ovine AMY DNA fragment was located on chromosome 1 by linkage mapping using the International Mapping Flock. No association was found between estimated breeding values for milk yield, protein and fat contents and AMY genotypes in a daughter design comprising 13 Manchega families with an average of 29 daughters (12-62) per sire.     

Porcine skeletal muscle differentially expressed gene ATP5B: molecular characterization, expression patterns, and association analysis with meat quality traits

The 2-DE/MS-based proteomics approach was used to investigate the differences of porcine skeletal muscle, and ATP5B was identified as one differential expression protein. In the present study, ATP5B gene was further cloned by RT-PCR, the sequence was analyzed using the bioinformatics method, and the mRNA expression was detected by qRT-PCR. Sequence analysis showed that the porcine ATP5B gene contains an ORF encoding 528-amino-acid residues with 49 and 166 nucleotides in the 5′ and 3′ UTRs, respectively. The mRNA of ATP5B was widely expressed in all 14 tissues tested, but especially highly expressed in parorchis and fat. The expression pattern of ATP5B was similar in Large White and Meishan breeds, showing that the expression was upregulated by 3 days after birth and downregulated during postnatal development of skeletal muscle. Comparing the two breeds, the mRNA abundance of ATP5B in Large White was more highly expressed than in Meishan at all developmental stages (P < 0.05). Moreover, a synonymous mutation, G75A in exon 8, was identified and association analysis with the traits of meat quality showed that it was significantly associated with the RLF, FMP, IFR, IMF, and IMW (P < 0.05). These results suggested that ATP5B probably plays a key role in porcine skeletal muscle development and may provide further insight into the molecular mechanisms responsible for breed-specific differences in meat quality.     

Cloning, mapping and association study with carcass traits of the porcine SDHD gene

A 1320-bp cDNA containing the full coding region of the porcine succinate dehydrogenase complex, subunit D (SDHD) gene was obtained by random sequencing of clones from a Chinese Tongcheng pig 55-day fetal longissimus dorsi muscle cDNA library. Analysis of the SDHD gene across the INRA-University of Minnesota porcine radiation hybrid panel indicated close linkage with microsatellite marker SW2401, located on SSC9p21. The open reading frame of this cDNA covers 480 bp and encodes 159 amino acids. The deduced porcine amino acid sequence showed greater similarity with human and bovine protein sequences than with those from mouse and rat. The BLAST analysis of the porcine SDHD to NCBI identified Unigene Cluster Ssc.2586. Possible single nucleotide polymorphisms (SNP) were identified by alignment of expressed sequence tags in the cluster. The polymerase chain reaction (PCR) single strand conformation polymorphism, sequencing, and PCR restriction fragment length polymorphism were used to confirm and detect a synonymous polymorphic MboI site within the open-reading frame. Allele frequencies of this SNP were investigated in two commercial and five Chinese local pig breeds. These five Chinese breeds had very high frequencies for one allele, whereas frequencies of both alleles were intermediate in Large White and Duroc. An association analysis suggested that different SDHD genotypes have significant differences in loin-muscle area (P < 0.01).     

Molecular characterization of differentially expressed TXNIP gene and its association with porcine carcass traits

Thioredoxin interacting protein (TXNIP), which plays a regulatory role in lipid metabolism and immune regulation, is down-regulated expressed in F₁ hybrids Landrace?×?Yorkshire skeletal muscle. Here we described the molecular characterization of porcine TXNIP gene. The full-length cDNA contains a coding sequence of 1,176?bp nucleotides with untranslated regions of 263?bp at 5′-end and 441?bp at 3′-end, respectively. The predicted molecular mass and isoelectric point of porcine TXNIP is 43.81?kDa and 7.385, respectively. The deduced 391 amino acids exhibit high identity with other mammalian TXNIP. The TXNIP gene contains eight coding exons and seven non coding introns, spans approximately 3,348?bp. The expression of porcine TXNIP mRNA is almost absent in Landrace?×?Yorkshire and lower level in 6-month-old pigs during skeletal muscle development. Other stages and breeds were high level expressed. Statistical analysis showed the TXNIP gene polymorphism (c.575-4T>C) was different between F₁ hybrids and their parents, was highly associated with dressing percentage (DP) and thorax–waist fat thickness (TFT) in the Yorkshire?×?Meishan F₂ population. The possible role of TXNIP was discussed.     

Polymorphism identification, RH mapping and association of placental lactogen gene with milk production traits of dairy cows

Bovine placental lactogen (bPL) is structurally related to prolactin (PRL) and growth hormone (GH). In synergism with steroid and thyroid hormones, bPL is crucial in stimulating the development of the mammary gland, mammary cell differentiation and function. To further explore whether bPL gene is associated with milk production traits, we herein analyzed single-nucleotide polymorphisms (SNPs) within eight regions of bPL gene, which are potentially associated with five milk production traits on 1028 Chinese Holstein cows. Among these, two SNPs, NT7409(T-C) and Nt11246(G-A), were identified. The former is within exon 2; it induces an alteration of amino acid from Val to Ala. The later is within exon 4. It is a synonymous mutation. We found that there were significant associations between NT7409(T-C) and milk and protein yield. Cows of the AA genotype yielded less milk (P = 0.001) and less protein (P = 0.003) than those of genotypes AB and BB. However, on the NT11246(G-A) locus, no significant association was observed in the five milk production traits studied. In addition, bPL has been localized near markers RM185 and CC549051 with a distance of 23.2 cR on BTA 23. It is at the same position as the region including quantitative trait loci (QTLs) affecting milk and protein yields by previous linkage analysis. In summary, our findings demonstrated that the SNP within exon 2 of bPL (NT7409(T-C)) is associated with two milk production traits, and this provided further evidence that bPL could be a major gene-controlling milk production trait in Holstein dairy cattle.     

Porcine NAMPT gene: search for polymorphism,mapping and association studies

NAMPT encodes an enzyme catalysing the rate‐limiting step in NAD biosynthesis. The extracellular form of the enzyme is known as adipokine visfatin. We detected SNP AM999341:g.669T>C (referred to as 669T>C) in intron 9 and SNP FN392209:g.358A>G (referred to as 358A>G) in the promoter of the gene. RH mapping linked the gene to microsatellite SW944. Linkage analysis placed the gene on the current USDA – USMARC linkage map at position 92 cM on SSC9. Association analyses were performed in a wild boar × Meishan F₂ family (W × M), with 45 traits recorded (growth and fattening, fat deposition, muscling, meat quality, stress resistance and other traits), and in a commercial Landrace × Chinese‐European (LCE) synthetic population with records for 15 traits (growth, fat deposition, muscling, intramuscular fat, meat colour and backfat fatty acid content). In the W × M, SNP 669T>C was associated with muscling, fat deposition, growth and fattening, meat quality and other traits and in the LCE with muscling, meat quality and backfat fatty acid composition. In the W × M, SNP 358A>G was associated with muscling, fat deposition, growth and other traits. After correction for multiple testing, the NAMPT haplotypes were associated in the W × M with, in descending order, muscling (q = 0.0056), growth (q = 0.0056), fat deposition (q = 0.0109), fat‐to‐meat ratio (q = 0.0135), cooling losses (q = 0.0568) and longissimus pH_U (q = 0.0695). The SNPs are hypothesized to be in linkage disequilibrium with a causative mutation affecting energy metabolism as a whole rather than fat metabolism alone.     

Isolation and imprinting analysis of the porcine DLX5 gene and its association with carcass traits

Imprinted genes play important roles in mammalian growth and development. However, reports on imprinted genes are limited in livestock. In this study, the complete ORF containing 289 amino acids of the porcine DLX5 gene was obtained. A C-to-T SNP mutation in exon 1 of the DLX5 gene was used to detect imprinting status with an RT-PCR/RFLP test (using HhaI) in eight heterozygous pigs from a population of Large White × Meishan F₁ hybrids. Imprinting analysis showed that the porcine DLX5 gene was maternally expressed in skeletal muscle, fat, lung, spleen, stomach and small intestine, but not imprinted in heart, liver, kidney, uterus, ovary, testicle or pituitary. A PCR–RFLP test was also used to detect the polymorphism in 310 pigs of a Large White × Meishan F₂ resource population. The statistical results showed significant association ( P  < 0.01) of the genotypes and fat meat percentage, carcass length, bone percentage, 6–7 rib fat thickness, average backfat thickness, thorax-waist fat thickness and buttock fat thickness.     

猪ATF4基因多态性与生产性状的关联及基因表达分析

为进一步了解和认识ATF4基因的功能,揭示ATF4对猪脂肪代谢的影响,寻找与肉质性状相关联的分子标记,文章采用PCR方法扩增了ATF4基因部分序列,通过序列比对发现在翻译起始密码子ATG下游159 bp处存在A159G转换,通过PCR-AluⅠ-RFLP对大白猪、长白猪、梅山猪和通城猪进行酶切分型,发现在大白猪和长白猪中均为AA基因型,在梅山猪和通城猪中均为GG基因型。进一步对大白猪×梅山F2群体资源家系进行了酶切分型,并分析该位点的多态性与生产性状的关系。结果表明,ATF4的多态性与臀部平均膘厚存在极显著相关(P<0.01),与胸腰椎间膘厚、平均膘厚、眼肌高、眼肌面积存在显著相关(P<0.05)。采用Real-time PCR分析了ATF4基因在大白猪与梅山猪背最长肌不同发育阶段的表达模式。结果表明,ATF4基因在大白猪和梅山猪胚胎期65 d和出生后3 d中的表达水平相对都比较低,且在两品种间无明显差异;而在出生后60 d和120 d,ATF4基因在大白猪中与梅山猪均出现了上调表达,并且在梅山猪中的相对表达水平要显著高于大白猪。研究结果为进一步深入研究猪ATF4基因在脂肪代谢中的分子机理奠定了基础。     

Gene mapping by linkage and association analysis

Genetic analysis is used to map genes, including disease loci, to positions within the human genome. Linkage analysis depends on the co-segregation of a gene (locus) and a phenotype through a pedigree, while association analysis, or linkage disequilibrium mapping, depends on measuring deviation from the random occurrence of alleles in a haplotype in unrelated individuals or nuclear families. Complex computer programs may be used in both forms of analysis. In recent years most interest has focused on identifying genes involved in common, multifactorial diseases. Here I review some current and developing techniques of genetic analysis and give references to where further information can be obtained.     

Molecular characterization,transcriptional regulation and association analysis with carcass traits of porcine TCAP gene

TCAP (also known as titin-cap or telethonin) is one of the titin interacting Z-disk proteins involved in the regulation and development of normal sarcomeric structure. In this study, we cloned the cDNA and promoter sequences of porcine TCAP gene, which contained a 504 bp full-length coding region. Quantitative real-time PCR (qRT-PCR) analyses showed that porcine TCAP was highly expressed in the skeletal muscle, heart, and kidney. During postnatal muscle development, TCAP expression was down-regulated from 30 days to 120 days in Large White and Meishan pigs. One single nucleotide polymorphism c.334G>A in exon 2 of the TCAP gene was identified and detected by allele-specific primer-polymerase chain reaction (ASP-PCR). Association analysis revealed that the polymorphism had significant associations (P < 0.05 and P < 0.01) with some carcass traits. Analysis of the porcine TCAP promoter in different cell lines demonstrated that it is a muscle-specific promoter. In addition, we found that the porcine TCAP promoter can be activated by MyoD, MyoG and MEF2 in myotubes, which indicated that TCAP may play a role in the regulation of porcine skeletal muscle development. These findings provide useful information for the further investigation of the function of TCAP in porcine skeletal muscle.     

Genetic basis of maize stalk strength decoded via linkage and association mapping

Stalk lodging is a severe problem that limits maize production worldwide, although little attention has been given to its genetic basis. Here we measured rind penetrometer resistance (RPR), an effective index for stalk lodging, in a multi-parent population of 1948 recombinant inbred lines (RILs) and an association population of 508 inbred lines (AMP508). Linkage and association mapping identified 53 and 29 single quantitative trait loci (QTLs) and 50 and 19 pairs of epistatic interactions for RPR in the multi-parent population and AMP508 population, respectively. Phenotypic variation explained by all identified epistatic QTLs (up to ~5%) was much less than that explained by all single additive QTLs (up to ~33% in the multi-parent population and ~ 60% in the AMP508 population). Among all detected QTLs, only eight single QTLs explained >10% of phenotypic variation in single RIL populations. Alleles that increased RPR were enriched in tropical/subtropical (TST) groups from the AMP508 population. Based on genome-wide association studies in both populations, we identified 137 candidate genes affecting RPR, which were assigned to multiple biological processes, such as the biosynthesis of cell wall components. Sixty-six candidate genes were cross-validated by multiple methods or populations. Most importantly, 23 candidate genes were upregulated or downregulated in high-RPR lines relative to low-RPR lines, supporting the associations between candidate genes and RPR. These findings reveal the complex nature of the genetic basis underlying RPR and provide loci or candidate genes for developing elite varieties that are resistant to stalk lodging via molecular breeding.     

转座子引起的猪ktn1基因结构变异及其与生产性能的关联分析

为探明转座子对猪的ktn1基因及其侧翼区结构变异的贡献,从全基因组测序(WGS)数据库中获取14个猪基因组中的ktn1基因序列和侧翼序列,通过ClustalX多序列比对和RepeatMasker转座子注释,全面解析转座子对ktn1的影响。通过PCR检测到一个SINEA1转座子插入多态,在苏姜猪群体中与相关性状进行关联分析。结果显示,ktn1基因及其侧翼区中含有至少77个转座子片段,其中绝大部分(98.70%)为SINE类转座子,并鉴定到9个小结构变异和4个由转座子引起的大结构变异,表明转座子是基因变异的重要来源。其中一个SINEA1插入多态引起的结构变异,在不同品种中呈现丰富的多态性,且无插入个体(SINE-/-)苏姜猪的断奶窝重((64.20±10.6) kg)比纯合有插入个体(SINE+/+)((74.14±9.0) kg)和杂合有插入个体(SINE+/-)((69.71±7.7) kg)轻(P<0.05),表明基于转座子插入多态研发分子标记具有可行性,提示转座子插入多态分子标记在分子辅助育种中具有较强的应用潜力。     

Characterization of the porcine KIT ligand gene: expression analysis, genomic structure, polymorphism detection and association with coat colour traits

Kit ligand (KITLG) is the ligand for the type III receptor tyrosine kinase KIT. Studies of the KIT/KITLG pathway in a number of mammalian species have shown that it is important for the development of stem cell populations in haematopoietic tissues, germ cells in reproductive organs and the embryonic migrating melanoblasts that give rise to melanocytes. Consequently, mutations in the pathway may result in a range of defects including anaemia, sterility and de-pigmentation. The cDNA sequence of the porcine KITLG gene has been reported previously, and is an attractive candidate locus for moderating coat colour in pigs. In this paper we report the gene structure and physical mapping of the porcine gene. We also report the identification of polymorphisms in the gene, one of which was used to confirm linkage to chromosome 5. Preliminary RNA expression studies using a panel of tissues have shown that in addition to the known variant lacking exon 6, there is alternative splicing of exon 4. However, little evidence was found for the KITLG gene being linked to variation in colour in a Meishan x Large White cross.     

Analysis of genome-wide structure, diversity and fine mapping of Mendelian traits in traditional and village chickens

Extensive phenotypic variation is a common feature among village chickens found throughout much of the developing world, and in traditional chicken breeds that have been artificially selected for traits such as plumage variety. We present here an assessment of traditional and village chicken populations, for fine mapping of Mendelian traits using genome-wide single-nucleotide polymorphism (SNP) genotyping while providing information on their genetic structure and diversity. Bayesian clustering analysis reveals two main genetic backgrounds in traditional breeds, Kenyan, Ethiopian and Chilean village chickens. Analysis of linkage disequilibrium (LD) reveals useful LD (r(2) ≥ 0.3) in both traditional and village chickens at pairwise marker distances of ~10 Kb; while haplotype block analysis indicates a median block size of 11-12 Kb. Association mapping yielded refined mapping intervals for duplex comb (Gga 2:38.55-38.89 Mb) and rose comb (Gga 7:18.41-22.09 Mb) phenotypes in traditional breeds. Combined mapping information from traditional breeds and Chilean village chicken allows the oocyan phenotype to be fine mapped to two small regions (Gga 1:67.25-67.28 Mb, Gga 1:67.28-67.32 Mb) totalling ~75 Kb. Mapping the unmapped earlobe pigmentation phenotype supports previous findings that the trait is sex-linked and polygenic. A critical assessment of the number of SNPs required to map simple traits indicate that between 90 and 110K SNPs are required for full genome-wide analysis of haplotype block structure/ancestry, and for association mapping in both traditional and village chickens. Our results demonstrate the importance and uniqueness of phenotypic diversity and genetic structure of traditional chicken breeds for fine-scale mapping of Mendelian traits in the species, with village chicken populations providing further opportunities to enhance mapping resolutions.     

Linkage disequilibrium pattern and genome‐wide association mapping for meat traits in multiple porcine F2 crosses

In the present study, data from four F2 crosses were analysed and used to study the linkage disequilibrium (LD) structure within and across the crosses. Genome‐wide association analyses (GWASes) for conductivity and dressing out meat traits were conducted using single‐marker and Bayesian multi‐marker models using the pooled data from all F2 crosses. Porcine F2 crosses generated from the distantly related founder breeds Wild Boar, Piétrain and Meishan, as well as from a porcine F2 cross from the closely related founder breed Piétrain and an F1 Large White × Landrace cross were pooled. A total of 2572 F2 animals were genotyped using a 62K SNP chip. The positions of the SNPs were based on genome assembly Sscrofa11.1. After post‐alignment and genotype filtering, approximately 50K SNPs were usable for LD studies and GWASes. The main findings of the present study are that the breakdown of LD was faster in crosses from closely related founder breeds compared to crosses from distantly related founders. The fastest breakdown of LD was observed by pooling the data. Based on the single‐marker results and LD structure, clusters and windows were built for 1‐Mb intervals. For conductivity and dressing out, 183 and 191 nominal significant associations respectively and six and five clusters respectively were found. Dominance was important for conductivity, and considering dominance in GWASes improved the mapping signals. Most clear signals were found for conductivity on SSC6, 8 and 15 and for dressing out on SSC2 and 7. Considering dominance might contribute to the accuracy of genomic selection and serve as a guide for choosing mating pairs with good combining abilities. However, further research is needed to investigate if dominance is also important in crossbreed pig breeding schemes.     

Association analysis of adiponectin and somatostatin polymorphisms on BTA1 with growth and carcass traits in Angus cattle

This study tested positional candidate genes adiponectin (ADIPOQ) and somatostatin (SST) for effects on carcass traits in a commercially relevant cattle population. Both genes are located within a region of BTA1 previously reported to harbour quantitative trait loci (QTL) that affect marbling, quality grade, yield grade, ribeye area and weaning weight in Bos taurus x Bos indicus crosses. Except for the first intron of ADIPOQ, both genes, including over 2 kb upstream of the promoters, were sequenced in five registered Angus sires to identify polymorphisms. A variable copy duplication and three single nucleotide polymorphisms (SNPs) in ADIPOQ and one SNP in SST were genotyped and tested for association with 19 traits in a 14-generation pedigree of 1697 registered Angus artificial insemination sires representing all the major USA lineages of the breed. Linear models that parameterized predicted genetic merits in terms of allele substitution effects were fit by weighted least squares, and goodness-of-fit tests were employed to differentiate causal mutations or polymorphisms in strong linkage disequilibrium (LD) with causal mutations from markers in weak LD with QTL. We confirmed the presence of QTL affecting marbling, ribeye muscle area and fat thickness in the vicinity of SST and ADIPOQ on BTA1 in Angus; excluded SST as underlying the ribeye muscle area QTL; and excluded ADIPOQ as underlying the marbling score QTL. However, association analysis provides very limited information about QTL location and has little intrinsic value when performed in the absence of linkage or LD analysis using flanking marker data to localize the QTL effect relative to positional candidate genes.