Massively parallel reporter assay: a novel technique for analyzing the regulation of gene expression.

Massively parallel reporter assay (MPRA) is a high-throughput analysis method that can simultaneously investigate the activity of thousands of regulatory elements in the genome. MPRA introduces a uniquely identified barcode on a conventional luciferase reporter gene vector, sequences the DNA barcode before transfection and the mRNA barcode after transfection by next-generation sequencing technology, and uses the ratio of mRNA and DNA barcode reads to analyze the activity of cis-regulatory elements. Since MPRA was proposed, it has been widely used in the identification of genomic cis-regulatory elements and functional variants, the effect of post-transcriptional regulation on phenotypes and so on. In this review, we summarize the development history, basic principles, experimental procedures and statistical analysis methods of MPRA, and its applications in post-transcriptional regulation and cis-regulatory elements. It also provides prospects for its development and useful references for researchers in related fields to understand and apply MPRA.

A Study of Combined Genotype Effects of SHCBP1 on Wool Quality Traits in Chinese Merino.

SHCBP1 (Shc SH2-domain binding protein 1) is a member of the Src and collagen homolog (Shc) protein family and is closely associated with multiple signaling pathways that play important roles during hair follicle induction, morphogenesis, and cycling. The purpose of this study was to investigate SHCBP1 gene expression, polymorphisms, and the association between SHCBP1 and wool quality traits in Chinese Merino sheep. The SHCBP1 gene was shown, by qPCR, to be ubiquitously expressed in sheep tissues and differentially expressed in the adult skin of Chinese Merino and Suffolk sheep. Four SNPs (termed SHCBP1SNPs 1-4) were identified by Sanger sequencing and were located in exon 2, intron 9, intron 12, and exon 13 of the sheep SHCBP1 gene, respectively. SHCBP1SNPs 3 and 4 (rs411176240 and rs160910635) were significantly associated with wool crimp (P < 0.05). The combined polymorphism (SHCBP1SNP3-SHCBP1SNP4) was significantly associated with wool crimp (P < 0.05). Bioinformatics analysis showed that the SNPs associated with wool crimp (SHCBP1SNPs 3 and 4) might affect the pre-mRNA splicing by creating binding sites for serine-arginine-rich proteins and that SHCBP1SNP4 might alter the SHCBP1 mRNA and protein secondary structure. Our results suggest that SHCBP1 influences wool crimp and SHCBP1SNPs 3 and 4 might be useful markers for marker-assisted selection and sheep breeding.

A phenome database (NEAUHLFPD) designed and constructed for broiler lines divergently selected for abdominal fat content.

Effective management and analysis of precisely recorded phenotypic traits are important components of the selection and breeding of superior livestocks. Over two decades, we divergently selected chicken lines for abdominal fat content at Northeast Agricultural University (Northeast Agricultural University High and Low Fat, NEAUHLF), and collected large volume of phenotypic data related to the investigation on molecular genetic basis of adipose tissue deposition in broilers. To effectively and systematically store, manage and analyze phenotypic data, we built the NEAUHLF Phenome Database (NEAUHLFPD). NEAUHLFPD included the following phenotypic records: pedigree (generations 1-19) and 29 phenotypes, such as body sizes and weights, carcass traits and their corresponding rates. The design and construction strategy of NEAUHLFPD were executed as follows: (1) Framework design. We used Apache as our web server, MySQL and Navicat as database management tools, and PHP as the HTML-embedded language to create dynamic interactive website. (2) Structural components. On the main interface, detailed introduction on the composition, function, and the index buttons of the basic structure of the database could be found. The functional modules of NEAUHLFPD had two main components: the first module referred to the physical storage space for phenotypic data, in which functional manipulation on data can be realized, such as data indexing, filtering, range-setting, searching, etc.; the second module related to the calculation of basic descriptive statistics, where data filtered from the database can be used for the computation of basic statistical parameters and the simultaneous conditional sorting. NEAUHLFPD could be used to effectively store and manage not only phenotypic, but also genotypic and genomics data, which can facilitate further investigation on the molecular genetic basis of chicken adipose tissue growth and development, and expedite the selection and breeding of broilers with low fat content.

Polymorphisms of FST gene and their association with wool quality traits in Chinese Merino sheep.

Follistatin (FST) is involved in hair follicle morphogenesis. However, its effects on hair traits are not clear. This study was designed to investigate the effects of FST gene single nucleotide polymorphisms (SNP) on wool quality traits in Chinese Merino sheep (Junken Type). We performed gene expression analysis, SNP detection, and association analysis of FST gene with sheep wool quality traits. The real-time RT-PCR analysis showed that FST gene was differentially expressed in adult skin between Chinese Merino sheep (Junken Type) and Suffolk sheep. Immunostaining showed that FST was localized in inner root sheath (IRS) and matrix of hair follicle (HF) in both SF and Suffolk sheep. Sequencing analysis identified a total of seven SNPs (termed SNPs 1-7) in the FST gene in Chinese Merino sheep (Junken Type). Association analysis showed that SNP2 (Chr 16. 25,633,662 G>A) was significantly associated with average wool fiber diameter, wool fineness SD, and wool crimp (P < 0.05). SNP4 (Chr 16. 25,633,569 C>T) was significantly associated with wool fineness SD and CV of fiber diameter (P < 0.05). Similarly, the haplotypes derived from these seven identified SNPs were also significantly associated with average wool fiber diameter, wool fineness SD, CV of fiber diameter, and wool crimp (P < 0.05). Our results suggest that FST influences wool quality traits and its SNPs 2 and 4 might be useful markers for marker-assisted selection and sheep breeding.

TCF21 is related to testis growth and development in broiler chickens.

BACKGROUND: Large amounts of fat deposition often lead to loss of reproductive efficiency in humans and animals. We used broiler chickens as a model species to conduct a two-directional selection for and against abdominal fat over 19 generations, which resulted in a lean and a fat line. Direct selection for abdominal fat content also indirectly resulted in significant differences (P < 0.05) in testis weight (TeW) and in TeW as a percentage of total body weight (TeP) between the lean and fat lines. RESULTS: A total of 475 individuals from the generation 11 (G11) were genotyped. Genome-wide association studies revealed two regions on chicken chromosomes 3 and 10 that were associated with TeW and TeP. Forty G16 individuals (20 from each line), were further profiled by focusing on these two chromosomal regions, to identify candidate genes with functions that may be potentially related to testis growth and development. Of the nine candidate genes identified with database mining, a significant association was confirmed for one gene, TCF21, based on mRNA expression analysis. Gene expression analysis of the TCF21 gene was conducted again across 30 G19 individuals (15 individuals from each line) and the results confirmed the findings on the G16 animals. CONCLUSIONS: This study revealed that the TCF21 gene is related to testis growth and development in male broilers. This finding will be useful to guide future studies to understand the genetic mechanisms that underlie reproductive efficiency.

Detection of genome-wide copy number variations in two chicken lines divergently selected for abdominal fat content.

BACKGROUND: The chicken (Gallus gallus) is an important model organism that bridges the evolutionary gap between mammals and other vertebrates. Copy number variations (CNVs) are a form of genomic structural variation widely distributed in the genome. CNV analysis has recently gained greater attention and momentum, as the identification of CNVs can contribute to a better understanding of traits important to both humans and other animals. To detect chicken CNVs, we genotyped 475 animals derived from two broiler chicken lines divergently selected for abdominal fat content using chicken 60 K SNP array, which is a high-throughput method widely used in chicken genomics studies. RESULTS: Using PennCNV algorithm, we detected 438 and 291 CNVs in the lean and fat lines, respectively, corresponding to 271 and 188 CNV regions (CNVRs), which were obtained by merging overlapping CNVs. Out of these CNVRs, 99% were confirmed also by the CNVPartition program. These CNVRs covered 40.26 and 30.60 Mb of the chicken genome in the lean and fat lines, respectively. Moreover, CNVRs included 176 loss, 68 gain and 27 both (i.e. loss and gain within the same region) events in the lean line, and 143 loss, 25 gain and 20 both events in the fat line. Ten CNVRs were chosen for the validation experiment using qPCR method, and all of them were confirmed in at least one qPCR assay. We found a total of 886 genes located within these CNVRs, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses showed they could play various roles in a number of biological processes. Integrating the results of CNVRs, known quantitative trait loci (QTL) and selective sweeps for abdominal fat content suggested that some genes (including SLC9A3, GNAL, SPOCK3, ANXA10, HELIOS, MYLK, CCDC14, SPAG9, SOX5, VSNL1, SMC6, GEN1, MSGN1 and ZPAX) may be important for abdominal fat deposition in the chicken. CONCLUSIONS: Our study provided a genome-wide CNVR map of the chicken genome, thereby contributing to our understanding of genomic structural variations and their potential roles in abdominal fat content in the chicken.

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.

[Association of single nucleotide polymorphism of RB1 gene with body weight traits in chicken].

The Northeast Agricultural University broiler lines divergently selected for abdominal fat content (NEAUHLF) were used in the current study to investigate the effects of Retinoblastoma1 (RB1) gene on chicken body weight (BW). The single nucleotide polymorphisms (SNPs) of the individuals were detected by MALDI-TOF-MS and PCR-RFLP methods and the genotypes of 27 SNPs were obtained. Haplotypes were constructed by liding window approach. Association analysis between single SNPs and haplotypes and body weight were conducted, respectively. Based on the results of single SNPs and haplotype association analyses. Four SNPs with significant effects on body weight at 1 week of age (BW1) and 2 SNPs with significant effects on BW1 and BW3 were determined. These results suggested that RB1 is an important candidate gene that affects chicken early growth and development.

[Association between chicken A-FABP gene polymorphisms and growth and body composition traits].

This experiment was designed to study the effects of polymorphism of A-FABP gene on growth and body composition traits in chicken. The 10th generation broiler population, derived from the Northeast Agricultural University broiler lines divergently selected for abdominal fat content (NEAUHLF) was used. Polymorphism among individuals was detected by DNA sequencing, PCR-RFLP, PCR-LP, and DHPLC. Linkage disequilibrium analysis for eight SNPs was performed, and five htSNPs were selected to construct haplotypes. The association analysis between the individual SNPs and haplotypes and growth and body composition traits were investigated, respectively. The results showed that there were consistently significant effects on muscle stomach weight (MSW) and percentage of muscle stomach (MSW/BW) (Pamp;0.05) in the seven SNPs (except for SNP 5) and haplotypes, but no significant effect on any other trait (P>0.05). Due to no evidence on effects of A-FABP for digestion system in other species, future experiments need to be developed to confirm whether A-FABP could be a major gene of MSW and MSW/BW traits in broiler chicken.

[Genetic analysis of epistatic effects between ApoB and UCP on abdominal fat trait in chicken].

It has been found that epistasis for selective response plays an indispensible role in animal genetics and breeding. In this study, the polymorphisms of T123G in apoliprotein B (ApoB) and C1197A in uncoupling protein (UCP) among individuals from the 8th to the 10th generation populations of the Northeast Agricultural University broiler lines divergently selected for abdominal fat content (NEAUHFL) were detected, and genetic analysis of the epistatic effects between the two SNPs on abdominal fat percentage (AFP) was performed using Natural and Orthogonal InterActions (NOIA) model. According to these assays, we concluded that at least one out of four epistatic components between these two SNPs was significantly associated with AFP (Plt;0.05) in fat lines from the 8th to the 10th generations of NEAUHFL; on the contrary, none was significantly associated with AFP (P>0.05) in lean lines. Our results suggested that epistatic interactions among QTLs and functional SNPs in candidate genes affecting fat traits might lead to differences in growth patterns of fat traits between lean and fat chicken lines.

[Research advances on RB1 gene].

The retinoblastoma1 (RB1) gene is the first cloned tumor suppressor gene. As a negative regulator of the cell cycle, RB1 gene could maintain a balance between cell growth and development through binding to transcription factors and regulating the expression of genes involved in cell proliferation and differentiation. Thus, it is involved in cell cycle, cell senescence, growth arrest, apoptosis and differentiation. This review summarizes recent advances on the structure, expression, and function of RB1 gene.

[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.

Profiling of chicken adipose tissue gene expression by genome array.

BACKGROUND: Excessive accumulation of lipids in the adipose tissue is a major problem in the present-day broiler industry. However, few studies have analyzed the expression of adipose tissue genes that are involved in pathways and mechanisms leading to adiposity in chickens. Gene expression profiling of chicken adipose tissue could provide key information about the ontogenesis of fatness and clarify the molecular mechanisms underlying obesity. In this study, Chicken Genome Arrays were used to construct an adipose tissue gene expression profile of 7-week-old broilers, and to screen adipose tissue genes that are differentially expressed in lean and fat lines divergently selected over eight generations for high and low abdominal fat weight. RESULTS: The gene expression profiles detected 13,234-16,858 probe sets in chicken adipose tissue at 7 weeks, and genes involved in lipid metabolism and immunity such as fatty acid binding protein (FABP), thyroid hormone-responsive protein (Spot14), lipoprotein lipase(LPL), insulin-like growth factor binding protein 7(IGFBP7) and major histocompatibility complex (MHC), were highly expressed. In contrast, some genes related to lipogenesis, such as leptin receptor, sterol regulatory element binding proteins1 (SREBP1), apolipoprotein B(ApoB) and insulin-like growth factor 2(IGF2), were not detected. Moreover, 230 genes that were differentially expressed between the two lines were screened out; these were mainly involved in lipid metabolism, signal transduction, energy metabolism, tumorigenesis and immunity. Subsequently, real-time RT-PCR was performed to validate fifteen differentially expressed genes screened out by the microarray approach and high consistency was observed between the two methods. CONCLUSION: Our results establish the groundwork for further studies of the basic genetic control of growth and development of chicken adipose tissue, and will be beneficial in clarifying the molecular mechanism of obesity in chickens.