Metabolomics technology and its applications in agricultural animal and plant research.

Metabolomics is a discipline that uses Chromatography-Mass Spectrometry and Nuclear Magnetic Resonance techniques to identify and quantify all small molecule metabolites in living organisms and biological samples, which relies on sensitive, stable analytical procedures and improving databases. Metabolomics has been widely used in medicine, food science, crop and farm animal research, and other fields. Metabolomics can establish a more direct relationship between changes in the type and content of metabolites and phenotypes. Metabolomics has gradually become a new research method for the analysis of genetic mechanisms of complex traits following genomics, transcriptomics, and proteomics with the advances in omics technology. In this review, we firstly introduce common analytical strategies, metabolomics platforms, and metabolomics databases. Then, we review the application of metabolomics in metabolic molecular identification of important economic traits in agricultural animals, disease diagnosis, meat quality and safety detection of animal products. We also introduce the latest achievements in the development, formation and analysis of important traits of animals and plants by using metabolomics, transcriptome, and genomics. Overall, the integrated analysis of metabolomics and other omics can comprehensively explain the genetic mechanism of all kinds of complex traits and help to improve the complete biological process of "mutation-gene-expression-metabolism-phenotype". Metabolomics provides a new method for the mechanism analysis of complex characters and a novel idea for new agricultural breeding.

Application of genomic selection in farm animal breeding.

Genomic selection (GS) has become a widely accepted method in animal breeding to genetically improve economic traits. With the declining costs of high-density SNP chips and next-generation sequencing, GS has been applied in dairy cattle, swine, poultry and other animals and gained varying degrees of success. Currently, major challenges in GS studies include further reducing the cost of genome-wide SNP genotyping and improving the predictive accuracy of genomic estimated breeding value (GEBV). In this review, we summarize various methods for genome-wide SNP genotyping and GEBV prediction, and give a brief introduction of GS in livestock and poultry breeding. This review will provide a reference for further implementation of GS in farm animal breeding.

[Expression of Myogenin and MCK genes regulated by PI3K/AKT pathway].

Many intracellular signaling pathways regulate skeletal muscle differentiation. Among them, PI3K/AKT pathway plays an important role. But the mechanisms of chromatin regulation remain unclear. In this study, the murine C2C12 myoblast cell line was used to investigate the expression of Myogenin and MCK genes during the skeletal muscle differentiation. Western blotting analysis showed that the expression of Myogenin and MCK protein was increased significantly after PI3K/AKT activator treatment for 24 h during the C2C12 cell differentiation and the expression of H3K27me3 demethylase UTX was also increased. Chromatin immunoprecipitation (ChIP) and quantitative PCR (Q-PCR) analysis showed that the enrichment of H3K27me3 on the promoter regions of Myogenin and MCK genes and the enhancer region of MCK gene were decreased. It was opposite to the PI3K/AKT inhibitor treatment. We concluded that the PI3K/AKT pathway maybe regulate skeletal muscle differentiation by regulating the expression of UTX gene to change the enrichment of H3K27me3 on the target genes.

Chicken chromatin accessibility atlas accelerates epigenetic annotation of birds and gene fine-mapping associated with growth traits.

The development of epigenetic maps, such as the ENCODE project in humans, provides resources for gene regulation studies and a reference for research of disease-related regulatory elements. However, epigenetic information, such as a bird-specific chromatin accessibility atlas, is currently lacking for the thousands of bird species currently described. The major genomic difference between birds and mammals is their shorter introns and intergenic distances, which seriously hinders the use of humans and mice as a reference for studying the function of important regulatory regions in birds. In this study, using chicken as a model bird species, we systematically compiled a chicken chromatin accessibility atlas using 53 Assay of Transposase Accessible Chromatin sequencing (ATAC-seq) samples across 11 tissues. An average of 50 796 open chromatin regions were identified per sample, cumulatively accounting for 20.36% of the chicken genome. Tissue specificity was largely reflected by differences in intergenic and intronic peaks, with specific functional regulation achieved by two mechanisms: recruitment of several sequence-specific transcription factors and direct regulation of adjacent functional genes. By integrating data from genome-wide association studies, our results suggest that chicken body weight is driven by different regulatory variants active in growth-relevant tissues. We propose CAB39L (active in the duodenum), RCBTB1 (muscle and liver), and novel long non-coding RNA ENSGALG00000053256 (bone) as candidate genes regulating chicken body weight. Overall, this study demonstrates the value of epigenetic data in fine-mapping functional variants and provides a compendium of resources for further research on the epigenetics and evolution of birds and mammals.

A genome-wide scan of selective sweeps in two broiler chicken lines divergently selected for abdominal fat content.

BACKGROUND: Genomic regions controlling abdominal fatness (AF) were studied in the Northeast Agricultural University broiler line divergently selected for AF. In this study, the chicken 60KSNP chip and extended haplotype homozygosity (EHH) test were used to detect genome-wide signatures of AF. RESULTS: A total of 5357 and 5593 core regions were detected in the lean and fat lines, and 51 and 57 reached a significant level (P<0.01), respectively. A number of genes in the significant core regions, including RB1, BBS7, MAOA, MAOB, EHBP1, LRP2BP, LRP1B, MYO7A, MYO9A and PRPSAP1, were detected. These genes may be important for AF deposition in chickens. CONCLUSIONS: We provide a genome-wide map of selection signatures in the chicken genome, and make a contribution to the better understanding the mechanisms of selection for AF content in chickens. The selection for low AF in commercial breeding using this information will accelerate the breeding progress.

Targeting lentiviral vectors to primordial germ cells (PGCs): An efficient strategy for generating transgenic chickens.

Recent advances in avian transgenic studies highlight the possibility of utilizing lentiviral vectors as tools to generate transgenic chickens. However, low rates of gonadal chimerism and germ line transmission efficiency still limit the broad usage of this method in creating transgenic chickens. In this study, we implemented a simple strategy using modified lentiviral vectors targeted to chicken primordial germ cells (PGCs) to generate transgenic chickens. The lentiviral vectors were pseudotyped with a modified Sindbis virus envelope protein (termed M168) and conjugated with an antibody specific to PGC membrane proteins. We demonstrated that these optimized M168-pseudotyped lentiviral vectors conjugated with SSEA4 antibodies successfully targeted transduction of PGCs in vitro and in vivo. Compared with the control, 50.0%-66.7% of chicken embryos expressed green fluorescent protein (GFP) in gonads transduced by the M168-pseudotyped lentivirus. This improved the targeted transduction efficiency by 30.0%-46.7%. Efficient chimerism of exogenous genes was also observed. This targeting technology could improve the efficiency of germ line transmission and provide greater opportunities for transgenic poultry studies.

[Effects of microsatellite in the regulatory region of IGF1 on growth traits in Jinhua swine].

Insulin-like growth factor 1 (IGF1) and its associated binding proteins and transmembrane receptors (IGFR) play an important role in the physiologic process of mammalian growth. The objectives of present work were to estimate the effects of microsatellite markers located in the 5'-regulatory region of the IGF1 on birth weight (BW), weaning weight (WW), weight at the 120th day, the 180th day and litter weight at birth (LWB) by the least square method in Jinhua pig. Significant effect was found for IGF1 genotype on BW (P<0.05), with positive effects associated with the 286/286 genotype, and 280/286 genotype on LWB in second parity (P>0.05). Furthermore, according to analysis of allele average substitution effect, alleles 274 bp and 286 bp was favourable for BW increase, allele 280 bp was favourable for LWB increase in the second parity. By correlation analysis, total number of birth, number of birth alive and LWB of the second parity in Jinhua pig had highly significant correlation (P<0.01), therefore increasing LWB of the second parity could improve litter performance of Jinhua pig.

[A genetic linkage map of chicken chromosome 1 in an F2 resource population].

Three hundred and sixty-nine F2 individuals produced from Northeast Agricultural University Resource Population (NEAURP) were genotyped by 23 fluorescent microsatellite markers on chromosome 1. The characterization of these microsatellites was moderate or high polymorphic except marker MCW0058 which was low polymorphic. The length of the sex averaged linkage map was 637.9 cM. The order of MCW0115 and ROS0025 disagreed with that of EL map, but consisted with that of W map. The intervals of markers were larger than those of three reference families.

[Polymorphism analysis of the exon 2 of SLA-DQB gene in Xiao Meishan, Zhong Meishan and Yorkshire pigs with PCR-RFLP].

SLA is an important genetic region which influences disease resistance in pigs, and the gene family is one of important candidates for disease resistance and susceptibility. In present research the exon 2 of SLA-DQB gene was amplified and a uniform fragment of 273 bp was obtained in Xiao Meishan, Zhong Meishan and Yorkshire Pigs. The 273 bp PCR product was digested with restriction endonuclease Rsa I and genetic Polymorphism was investigated with PCR-RFLP (restriction fragment length polymorphism). Polymorphic sites were detected at base position 11, 94 and 124 of the exon 2 of SLA-DQB gene. There were two kinds of genotypes in Xiao Meishan pigs, 246 bp/27 bp(AA) and 132 bp/84 bp/30 bp/27 bp(BB), the frequencies of genotype AA and BB are 0.571, and 0.439 respectively. Four kinds of genotypes were found in Zhong Meishan pigs, 246 bp/27 bp (AA), 132 bp/84 bp/30 bp/27 bp(BB), 132 bp/114 bp/27 bp(CC) and 246 bp/132 bp/84 bp/30 bp/27 bp(AB), their frequencies are 0.037, 0.593, 0.037 and 0.333 respectively. Five kinds of genotypes were found in Yorkshire pigs, 246 bp/27 bp(AA), 246 bp/132 bp/114 bp/27 bp(AC), 132 bp/114 bp/27 bp(CC), 246 bp/132 bp/84 bp/30 bp/27 bp(AB) and 273 bp/246 bp/27 bp(AD), the frequencies of five genotypes are 0.467, 0.300, 0.100, 0.100 and 0.033 respectively. Statistical results indicated that there is no favorable allele in Xiao Meishan pig breed; B is the favorable allele in Zhong Meishan and its gene frequency is 0.759; while in Yorkshire pigs A is the favorable allele and its gene frequency is 0.683. Four alleles (A, B, C and D) were found in SLA-DQB locus among three pig breeds; The results of chi 2 showed that four alleles of this locus fit with Hardy-Weinberg equilibrium. The research showed that conservation of Chinese local pig breeds is necessary in order to protect our rich genetic resources and make use of them in the future.

[A new method for phylogenic analysis].

With ESTs from porcine fatty tissue and cDNA sequences from human, bovine and mouse in non-reduncdant database and dbEST in GenBank,we sampled cDNA sequences of 70 function-known genes in four species on the base of randomly sampling method, analyzed the mutation pattern of 70 x 150 bp linking sequences between them, and established an integrated phylogenic analysis method. The results showed that 391 single bases mutations were found in 70 x 150 bp linking sequences alignment among four species. The number of mutation bases between them were greatly exceeded the 1/1000 predicted in the human genome analysis. C/T(T/C) and A/G (G/A) transitions were the major types of single base mutation. The genetic relationship between pig and bovine who are both Artiodactylous is the nearest, the next is human, and the farthest is mouse. The differentiation sequence taken place in four species from the same ancestor is that mouse is the earliest one, and the latter human, and pig and bovine are the latest.

[Single nucleotide polymorphism analysis in chicken insulin-like growth factor-II gene and its associations with growth and carcass traits].

In this experiment, F2 chicken derived from Broilers crossing with Silky was used to study the effect of insulin-like growth factor-II gene on growth and carcass traits. The partial gene was amplified by two pairs of primers, and single nucleotide polymorphism (SNPs) was detected by the technique of restriction fragment length polymorphism (RFLP), and then confirmed by DNA sequencing. The mutation was found in the exon-2 of the gene, and can be clarified by cutting of restriction enzyme Aci-I. The result of least square analysis showed the gene was significantly related with growth and carcass traits. It implied that the insulin-like growth factor-II gene could be a genetic locus or linked to a major gene affecting greatly the growth and carcass traits in chicken.

[Identification and analysis of a novel microsatellite marker flanking porcine myostatin gene (MSTN)].

In animal breeding, microsatellite marker plays an important role in constructing genetic maps, QTL mapping and function analysis of structural genes. Myostatin, also known as GDF8, is a negative regulator of skeletal muscle mass and, in swine, it is evidenced to be related to birth weight and average daily gain from 60 kg to 100 kg of body weight. In present study, by subcloning and sequencing,we identified a novel microsatellite marker which is useful for fine QTL mapping for meat traits. A BAC clone containing porcine MSTN was extracted and digested with EcoR I to recover the fragment of > 4 kb for subcloning in pGEM-3zf (+). Sequencing and alignment results showed that this subcloned fragment was not from porcine MSTN, but included a tandem repeat of (TG) 13, which is a novel microsatellite marker (GenBank accession number: AF454400) flanking MSTN. To exclude its vector origin we designed specific primers flanking this marker and successfully amplified this fragment from porcine genome. Through a pedigree analysis of a double-muscled Yorshire strain, we found that it is inherited in a co-dominant manner. We also checked the gene frequencies of this locus in 381 unrelated individuals of 7 pig breeds, namely Laiwu,Landrace, Yorkshire,Duroc, Peterian, Min and Erhualian. Only two alleles were detected, the repeating number of which are 13 (allele A) and 19 (allele B) respectively, which indicated that it is a low poly morphic microsatellite marker. In addition, the frequencies of the two alleles are different between the two types of pig breeds, while allele A is dominant in Chinese local breeds, allele B is dominant in imported breeds. Alignment with AY208121 indicate that this locus is located 42 kb downstream of porcine MSTN. We speculate that this microsatellite DNA is an important marker both in fine QTL mapping for meat traits and in the expression study of porcine MSTN.

[Preliminary analysis on China Agricultural University chicken resource population based on genomic scanning].

Combining the technique of multiplex-PCR and the fluorescent semi-automated detection, a large-scale genome scanning was performed for 440 chickens, which was derived from China Agricultural University chicken resource families, within three generations. Fifty-five microsatellite markers were analyzed for this study. Those 55 microsatellite loci accorded with the characters of Mendelian co-inheritance. The heterozygosities ranged from zero to 0.89, with 72% of loci having a heterozygosity of more than 0.60. The polymorphism information content (PIC) ranged from 0 to 0.85, in which 70% of those loci had a PIC of more than 0.50 but their distribution varied in line A and line C. The allele frequency was significantly different between line A and line C at most loci (P < 0.01). At the same time, gene accordance inclination was found in line C. The Nei population resemble coefficient and standard genetic distance were 0.1002 and 0.8928.

[The factors affecting the efficiency of mutiplex PCR].

In the research,the outputs of different cycle parameters, PCR buffer and reaction volumes are compared. The results indicated that the annealing temperature, annealing time, elongated time and ingredient of PCR buffer affected mutiplex PCR, but the reaction volume and cycle number had few effect on it.

[Genetic diversity in Tibetan chicken].

Morphological traits and living habit of Tibetan chicken, which is an aboriginal chicken breed on plateau with its own characteristic populational genetic features, are in great common with the Red Jungle Fowl, the assumed ancestry of domestic chicken. To fully exploit this chicken resource, Multiplex PCR with semi-automated polyacrilamide gel electrophoresis (PAGE) using fluorescently labeled microsatellite primers was used to detect the polymorphism at 20 microsatellite loci. At the same time, we randomly test the individual morphology and performance. It showed that numbers of polymorphic alleles were 4 approximately 10, with mean value 7.25 per locus. Polymorphism Information Content (C(PI)) and Heterozygosity (H) had mean values 0.67 and 0.74, respectively. Macrochromosomes had relatively higher polymorphism than microchromosomes(P > 0.05). In all, high polymorphisms at microsatellite loci related to the uneven production performance and morphological discrepancy of population genetic characteristics in Tibetan chicken.

Repression of Slc24a5 can reduce pigmentation in chicken.

Slc24a5 is a putative cation transporter, which is a member of the potassium-dependent sodium-calcium ion exchanger family. Association of the Slc24a5 gene with pigmentation has been established in Zebrafish, mice and humans. Despite these findings, its function in chicken remains unknown. The intent of this study was to describe the association of Slc24a5 with respect to melanin deposition in the chicken using RNAi. The objective was to detect the variety of melanin deposition caused by the down-regulation of Slc24a5 in chicken retinal pigment epithelium (RPE) cells. Nine siRNAs that targeted against Slc24a5 mRNA were found to be effective in suppressing Slc24a5 gene expression in 293FT cells. The most effective target tested effectively inhibited Slc24a5 expression in mRNA and subsequently reduced protein levels in RPE cells. These results show that down-regulation of Slc24a5 results in a reduction of melanin content, as well as a decrease of melaeneous type ß and type χ melanosomes simultaneously. Taken together, this work suggests that Slc24a5 function is conserved in the chicken, and necessary for melanin synthesis.

Isolation and characterization of 70 novel microsatellite markers from ostrich (Struthio camelus) genome.

Microsatellite markers are widely used in linkage mapping, parentage testing, population genetic studies, and molecular evolution studies in many agricultural species, while only a limited number of ostrich (Struthio camelus) microsatellites have been isolated. Thus, we constructed a random small-insert genomic library and a microsatellite-enriched library containing CA repeats. Fourteen clones containing CA repeats were isolated from 3462 clones in the non-enriched library by radioactive screening and 248 positive clones were isolated from 300 sequenced clones from the enriched library by PCR screening. After the enrichment procedures, the proportion of clones containing CA repeats was raised to 78.8%, compared with 0.4% in the non-enriched libraries, indicating that the enrichment value approaches 200 fold, which decreased the time and cost of cloning. The number of complete simple CA repeats in these positive clones ranged from 5 to 29. The primers for 94 of these microsatellites were developed and used to detect polymorphisms, of which 61 loci exhibited length polymorphisms in 17 unrelated ostrich individuals. The new polymorphic microsatellite markers we have identified and characterized will contribute to the ostrich genetic map, parentage testing, and comparative genomics between avian species.