Xingtang Fang

Comparative Transcriptome Profiling of Dairy Goat MicroRNAs from Dry Period and Peak Lactation Mammary Gland Tissues

Background MicroRNAs (miRNAs) are small noncoding RNA molecules that serve as important post-transcriptional gene expression regulators by targeting messenger RNAs for post-transcriptional endonucleolytic cleavage or translational inhibition. miRNAs play important roles in many biological processes. Extensive high-throughput sequencing studies of miRNAs have been performed in several animal models. However, little is known about the diversity of these regulatory RNAs in goat (Capra hircus), which is one of the most important agricultural animals and the oldest domesticated species raised worldwide. Goats have long been used for their milk, meat, hair (including cashmere), and skins throughout much of the world. Results In this study, two small RNA libraries were constructed based on dry period and peak lactation dairy goat mammary gland tissues and sequenced using the Illumina-Solexa high-throughput sequencing technology. A total of 346 conserved and 95 novel miRNAs were identified in the dairy goat. miRNAs expression was confirmed by qRT-PCR in nine tissues and in the mammary gland during different stages of lactation. In addition, several candidate miRNAs that may be involved in mammary gland development and lactation were found by comparing the miRNA expression profiles in different tissues and developmental stages of the mammary gland. Conclusions This study reveals the first miRNAs profile related to the biology of the mammary gland in the dairy goat. The characterization of these miRNAs could contribute to a better understanding of the molecular mechanisms of lactation physiology and mammary gland development in the dairy goat.

Novel 12-bp deletion in the coding region of the bovine NPM1 gene affects growth traits.

The nucleophosmin 1 gene (NPM1) encodes a multifunctional nucleolar phosphoprotein that plays a crucial role in the control of various aspects of cell growth and homeostasis. In this study, the coding region of theNPM1 gene was screened in 1035 individuals of 4 Chinese cattle breeds by DNA sequencing and polyacrylamide gel electrophoresis. A novel 12-bp deletion mutation was identified in the coding region of theNPM1 gene. The PCR products of primerNPM1-P2 exhibited 3 genotypes and 2 alleles: 178 bp (denoted asW) and 166 bp (denoted asD). GenotypeDD and alleleD were predominant in the studied populations. Association analysis with growth traits in the Nanyang breed (N = 265) showed that the animals with genotypeDD had significantly greater birth weight, body weight, body length, and heart girth than those with genotypeWD (P < 0.01 orP < 0.05) at birth and after 6 months and 12 months, but not at 18 and 24 months of age. Results of this study suggest that theNPM1 gene is a candidate gene for growth traits in cattle.

A new insight into cattle's maternal origin in six Asian countries.

The domestication of cattle fuelled the development of agricultural society in the history of human being. The evolution and genetic relationship of cattle can be elucidated by investigating the variation of mitochondrial DNA (mtDNA) D-loop sequence. In this study, we built a cattle phylogeny with a pool of 856 individual D-loop sequences, of which 264 Chinese cattle D-loop sequences were obtained in this study (141 ones were first analyzed, and 123 were first submitted) and the rest sequences of cattle from six Asian countries (Japan, Korea, Mongolia, Nepal, India and China) were retrieved from GenBank. Our results indicated that cattle from six Asian countries fell into three clades, Bos taurus (taurine), Bos indicus (zebu) and yak. Four main haplogroups T1A, T2, T3 (including T3A and T3B) and T5 were found in taurine, and two haplogroups I1 and I2 in zebu. Furthermore, we found that I1 and I2 haplogroups were separated by four variable sites rather than five ones and four haplogroups or sub-haplogroups of T1A, T3A, T3B and T5 were found for the first time in these Asian cattle. These data brought us a new insight into cattles genetic structure in these six Asian countries. The geographical distribution of haplogroups was also outlined to provide systematic information on cattle genetic resources.

Deep RNA Sequencing Reveals that MicroRNAs Play a Key Role in Lactation in Rats

Understanding the regulatory contribution of maternal physiology to difficulties with lactation is beneficial to both mother and infant. MicroRNAs (miRNAs), a type of noncoding RNA, may be involved in the regulation of mammary gland development and function. In the present study, a deep RNA sequencing (RNA-seq) technique was used to compare the expression profile of miRNAs and mRNAs of 2 pooled RNA samples from day 1 and day 7 postpartum (n = 1/d) rat (Rattus norvegicus) mammary glands to identify key miRNAs and their target genes that may control the rate-limiting steps of lactation. A total of 395 and 400 known miRNAs were identified in days 1 and 7 postpartum rat mammary samples, respectively. Compared with day 1 postpartum, 27 miRNAs were differentially expressed at day 7 postpartum. The expression differences between lactation periods were further analyzed by real-time quantitative polymerase chain reaction (qPCR) (n = 5). The ΔΔCt values of rno-miR-30, rno-miR-1, rno-miR-145-3p, rno-miR-142, rno-miR-7a-5p, rno-miR-3571, rno-miR-224-5p, rno-miR-362-5p, rno-miR-342-3p, rno-miR-322-3p, rno-miR-18a-5p, and rno-miR-202-5p between the 2 libraries varied from 0.64 to 9.44; the ΔΔCt values of rno-miR-133, rno-miR-190a-5p, rno-miR-27a-5p, rno-miR-451-5p, rno-miR-3120, rno-miR-23a-5p, rno-miR-20a-3p, rno-miR-92a-1-5p, and rno-miR-134-5p between the 2 libraries varied from -1.02 to -4.37 (P < 0.05). The intersection of the expressed mRNA genes from RNA-seq and putative target genes of differentially expressed miRNAs, termed mammary gland target genes (MTGs), was analyzed. The results indicated that 1259 MTGs overlapped between the 2 gene sets. The expression of 14 randomly selected genes of the MTGs was further confirmed by real-time qPCR (R(2) = 0.86, P < 0.01). The downregulated MTGs were enriched for the pathways involved in lipid biosynthesis. This gene cluster included 24 lipid metabolic process-related genes, which were putative targets of 10 differentially expressed miRNAs. These results will be helpful in discovering the biologic underpinnings of poor lactation performance in women attempting to breastfeed.

Circular RNA of cattle casein genes are highly expressed in bovine mammary gland

Recent studies have revealed that, in addition to hormones and other protein factors, noncoding RNA molecules play an important regulatory role in milk protein synthesis. Circular RNAs (circRNAs) are universally expressed noncoding RNA species that have been proposed recently to regulate the expression of their parental genes. In the present study, the deep RNA-sequencing technique known as RNA-seq was used to compare expression profiles of circRNAs from 2 pooled RNA samples from cow mammary gland on d 90 and 250 postpartum and to identify the key circRNAs involved in lactation. A total of 4,804 and 4,048 circRNAs were identified in the cow mammary gland on d 90 and 250 postpartum, respectively, of which only 2,231 circRNAs were co-expressed at both lactation stages, suggesting high stage specificity in the circRNAs. The enrichment of some Gene Ontology terms for the circRNA parental genes differed between lactation stages. Among the top 10 enriched Gene Ontology terms, vesicle, endoplasmic reticulum, and mitochondrial lumen were more common on lactation d 90. All 4 casein-coding genes (CSN1S1, CSN1S2, CSN2, and CSN3) produced circRNAs in the cattle mammary gland. In total, 80 circRNAs were identified from these 4 genes; circRNAs from CSN1S1 had very high abundance, and 3 of them accounted for 36% of all the circRNAs expressed in the mammary gland on lactation d 90. Three circRNAs from CSN1S1, 1 circRNA from CSN1S2, and 1 circRNA from CSN2 were all more highly expressed on lactation d 90 than on lactation d 250, as confirmed by quantitative PCR. These circRNAs had several target sites for the microRNA miR-2284 family and were predicted to target CSN1S1 and CSN2 mRNA, suggesting their potential involvement in regulating expression of the casein genes.

Identification and profiling of microRNAs and their target genes from developing caprine skeletal Muscle.

Goat is an important agricultural animal for meat production. Functional studies have demonstrated that microRNAs (miRNAs) regulate gene expression at the post-transcriptional level and play an important role in various biological processes. Although studies on miRNAs expression profiles have been performed in various animals, relatively limited information about goat muscle miRNAs has been reported. To investigate the miRNAs involved in regulating different periods of skeletal muscle development, we herein performed a comprehensive research for expression profiles of caprine miRNAs during two developmental stages of skeletal muscles: fetal stage and six month-old stage. As a result, 15,627,457 and 15,593,721 clean reads were obtained from the fetal goat library (FC) and the six month old goat library (SMC), respectively. 464 known miRNAs and 83 novel miRNA candidates were identified. Furthermore, by comparing the miRNA profile, 336 differentially expressed miRNAs were identified and then the potential targets of the differentially expressed miRNAs were predicted. To understand the regulatory network of miRNAs during muscle development, the mRNA expression profiles for the two development stages were characterized and 7322 differentially expressed genes (DEGs) were identified. Then the potential targets of miRNAs were compared to the DEGs, the intersection of the two gene sets were screened out and called differentially expressed targets (DE-targets), which were involved in 231 pathways. Ten of the 231 pathways that have smallest P-value were shown as network figures. Based on the analysis of pathways and networks, we found that miR-424-5p and miR-29a might have important regulatory effect on muscle development, which needed to be further studied. This study provided the first global view of the miRNAs in caprine muscle tissues. Our results help elucidation of complex regulatory networks between miRNAs and mRNAs and for the study of muscle development.

Polymorphisms in BMP-2 gene and their associations with growth traits in goats

BMPs plays an important role in skeletal development. In this study, PCR-SSCP and DNA sequencing methods were employed to screen the genetic variation of caprine BMP-2 gene in 299 goats from three breeds (Boer goat, Chinese Xuhuai white goat and Chinese Haimen goat). Three fragments of BMP-2 gene were investigated, only 3′ flanking region of BMP-2 gene showed polymorphism. The alignment between nucleotide sequences of AY714781.1 in GenBank and the sequencing results of three PCR products with different patterns revealed that there was one mutation (AY714781.1: g.A172G) in 3′ flanking region of BMP-2 gene, which constructed three genotypes (AA, AB, BB). The frequencies of allele A and genotype AA were dominant in all three breeds. Frequency of allele B in Haimen goat breed was significantly lower than that of the other two breeds. The genotype distributions were in good agreement with Hardy-Weinberg equilibrium (P>0.05) in each breed. The PIC (Polymorphism Information Content) values in three populations were 0.3261, 0.2558, 0.1663 for Boer goat, Xuhuai White goat and Haimen goat respectively. It was indicated that individuals of Boer goats with genotype BB were significantly higher than those of individuals with genotype AA in body trunk index (P<0.01). No polymorphism was detected in the exon3.

Expression Patterns of Circular RNAs from Primary Kinase Transcripts in the Mammary Glands of Lactating Rats

Purpose Circular RNAs (circRNAs), a novel class of RNAs, perform important functions in biological processes. However, the role of circRNAs in the mammary gland remains unknown. The present study is aimed at identifying and characterizing the circRNAs expressed in the mammary gland of lactating rats. Methods Deep sequencing of RNase R-enriched rat lactating mammary gland samples was performed and circRNAs were predicted using a previously reported computational pipeline. Gene ontology terms of circRNA-producing genes were also analyzed. Results A total of 6,824 and 4,523 circRNAs were identified from rat mammary glands at two different lactation stages. Numerous circRNAs were specifically expressed at different lactation stages, and only 1,314 circRNAs were detected at both lactation stages. The majority of the candidate circRNAs map to noncoding intronic and intergenic regions. The results demonstrate a circular preference or specificity of some genes. DAVID analysis revealed an enrichment of protein kinases and related proteins among the set of genes encoding circRNAs. Interestingly, four protein-coding genes (Rev3l, IGSF11, MAML2, and LPP) that also transcribe high levels of circRNAs have been reported to be involved in cancer. Conclusion Our findings provide the basis for comparison between breast cancer profiles and for selecting representative circRNA candidates for future functional characterization in breast development and breast cancer.

The polymorphisms of bovine PCSK1 gene and their associations with growth traits

Proprotein convertase 1 (PCSK1) is an endopeptidase involved in proteolytic processing of peptide hormone precursors in granules of the regulated secretory pathway of endocrine cells and mutations in PCSK1 gene are thought to cause obesity. In the present study, based on PCR-SSCP and DNA sequencing methods, polymorphisms of the PCSK1 gene were detected in 858 individuals from five breeds (Nanyang cattle, Qinchuan cattle, Jiaxian cattle, Luxi cattle and Chinese Holstein). The results showed that only P8 locus showed polymorphisms and 3 synonymous SNPs of PCSK1 gene were identified. Additionally, significant statistical difference was found in bovine birth weight and diplotype MM were 7.35% higher than diplotype XY.

Polymorphism in a Microsatellite of the Acrp30 Gene and Its Association with Growth Traits in Goats

Acrp30 plays a critical role in the regulation of glucose and lipid homeostasis. In this study, polymorphism of the Acrp30 gene was detected by PCR-SSCP and DNA sequencing methods in 321 individuals from three goat breeds, and the association of Acrp30 gene polymorphism with growth traits in the three goat breeds was analyzed. A novel insert/deletion (GT)5 microsatellite sequence was detected in the 5′ flanking region of the gene. Three genotypes (AA, AB, and BB) were found in three breeds. There was moderate genetic diversity in the locus in the analyzed populations. Significant associations were observed between the genotypes of the locus and growth traits in the Boer goat population. The chest circumference of individuals with genotype BB was significantly greater than that of individuals with genotype AA.