Correction: Deciphering Transcriptome and Complex Alternative Splicing Transcripts in Mammary Gland Tissues from Cows Naturally Infected with Staphylococcus aureus Mastitis.

[This corrects the article DOI: 10.1371/journal.pone.0159719.].

Deciphering Transcriptome and Complex Alternative Splicing Transcripts in Mammary Gland Tissues from Cows Naturally Infected with Staphylococcus aureus Mastitis.

Alternative splicing (AS) contributes to the complexity of the mammalian proteome and plays an important role in diseases, including infectious diseases. The differential AS patterns of these transcript sequences between the healthy (HS3A) and mastitic (HS8A) cows naturally infected by Staphylococcus aureus were compared to understand the molecular mechanisms underlying mastitis resistance and susceptibility. In this study, using the Illumina paired-end RNA sequencing method, 1352 differentially expressed genes (DEGs) with higher than twofold changes were found in the HS3A and HS8A mammary gland tissues. Gene ontology and KEGG pathway analyses revealed that the cytokine-cytokine receptor interaction pathway is the most significantly enriched pathway. Approximately 16k annotated unigenes were respectively identified in two libraries, based on the bovine Bos taurus UMD3.1 sequence assembly and search. A total of 52.62% and 51.24% annotated unigenes were alternatively spliced in term of exon skipping, intron retention, alternative 5' splicing and alternative 3' splicing. Additionally, 1,317 AS unigenes were HS3A-specific, whereas 1,093 AS unigenes were HS8A-specific. Some immune-related genes, such as ITGB6, MYD88, ADA, ACKR1, and TNFRSF1B, and their potential relationships with mastitis were highlighted. From Chromosome 2, 4, 6, 7, 10, 13, 14, 17, and 20, 3.66% (HS3A) and 5.4% (HS8A) novel transcripts, which harbor known quantitative trait locus associated with clinical mastitis, were identified. Many DEGs in the healthy and mastitic mammary glands are involved in immune, defense, and inflammation responses. These DEGs, which exhibit diverse and specific splicing patterns and events, can endow dairy cattle with the potential complex genetic resistance against mastitis.

[Functional haplotypes of INCENP affect promoter activity and bovine semen quality].

To explore the association between single nucleotide polymorphisms (SNPs) in the promoter region of the inner centromere protein (INCENP) gene and bovine semen quality, the haplotypes in 250 Chinese Holstein bulls were detected using PCR-RFLP method in this study. Two SNPs (g.-556 G>T, rs 136823901 and g.-692 C>T, rs 211010999) and three haplotypes (CG, TT, TG) were identified in the promoter region of INCENP. The genotype frequency and allele frequency of these two SNPs as well as the correlation between different SNP haplotype combinations and bovine semen quality were then analyzed. Our results showed that fresh sperm motility of the GT genotype was significantly higher than that of the GG genotype (P<0.05) at the SNP site g.-556 G>T, while fresh and frozen-thawed sperm motilities of the haplotype combinations H1H1(CCGG), H1H3(CTGT), H2H3(TTGT) and H3H3(TTTT) were significantly higher than that of H1H2 (P<0.05). To further study the possible mechanisms by which g.-556 G>T and g.-692 C>T affect semen quality, three haplotype plasmids were respectively transfected into MLTC-1 cells. The TG haplotype demonstrated the highest luciferase activity, suggesting that g.-556 G>T and g.-692 C>T are functional mutations which could regulate INCENP gene expression by affecting promoter activity and thus affect semen quality.

Two novel SNPs in HSF1 gene are associated with thermal tolerance traits in Chinese Holstein cattle.

Heat-shock transcription factors (HSFs) play an important role in regulating heat stress response. The activation of heat-shock protein (HSP) genes is mediated by HSFs, which bind to promoters of HSP genes. In this research, two novel single nucleotide polymorphisms, T909C and G4693T, and their association with thermal tolerance were investigated in 951 Chinese Holstein cattle. Linkage disequilibrium and haplotype construction were analyzed using SHEsis software. Four haplotypes were constructed, and nine haplotype combinations were found. Potassium content in erythrocytes (PCE), decreased rate of milk production (R), rectal temperature (RT), and heat-tolerance coefficient (HTC) were selected for the thermotolerance index. Association analysis showed that thermal tolerance in Chinese Holstein cattle was significantly affected by T909C and G4693T. The PCE of cows with CC or TC genotype was lower than that of TT at the 909 position (p < 0.05). Cows with TT genotype had lower PCE (p < 0.01) and higher HTC (p < 0.05) at the 4693 position. Cows with H2H4 haplotype combination had lower PCE (p < 0.01), R (p < 0.05) and RT (p < 0.05) and higher HTC (p < 0.05) than those with H1H3 haplotype combination. Bioinformatic analysis predicted that the 4693 position was located in the microRNA-binding (bta-miR-484) region. Quantitative reverse transcription-polymerase chain reaction demonstrated that 4693-T mutation caused the disruption of microRNA target binding, resulting in the relief of the transcriptional repression, which, in turn, resulted in increased expression. Thus, the HSF1 gene is useful in dairy cattle thermal tolerant breeding.

[Genetic polymorphism in 5'-flanking region of the lactoferrin gene and its associations with mastitis in Chinese Holstein cows].

Bovine lactoferrin (LF) is a multifunctional glycoprotein found in milk, which acts mainly as a defense factor in the mammary gland. Polymorphism has been found in the bovine LF gene. However, there is no report on genetic polymorphism of LF gene and its associations with mastitis in dairy cattle. In this study, the promoter fragment of LF gene containing -926(G/A), -915(T/G), -478(/G), and +72(T/C) mutations were genotyped by the PCR-RFLP and CRS-PCR method. Two hundred and sixty-eight Chinese Holstein cows were screened. Least square linear model (LSM) analysis was applied to evaluate the associations of LF gene with somatic cell score (SCS). The results indicated that the SCS was significantly affected by -478(/G) and +72(T/C), but not by the other two loci (P >0.05). The SCS of cow with genotype AB in +72(T/C) position was significantly lower than that of genotype AA (P<0.01) or AB (P<0.05). In position -478(/G), the cow with genotype CC showed significantly lower SCS in contrast to cow with genotype CD and DD (P < 0.01). In conclusion, genotype AB in position +72(T/C) and genotype CC in position -478(/G) of LF gene were advantageous genotype, which can be used as candidate markers for mastitis resistance selection in dairy cattle.

[The development and application of method for detecting bovine complex vertebral malformation].

Complex vertebral malformation (CVM), a lethal autosomal recessive inherited defect in Holstein calves, was newly reported worldwide. The molecular cause of CVM was a substitution of guanine by thymine (G-->T) in a solute carrier family 35 member 3 gene (SLC35A3), encoding UDP-N-acetylglucosamine transporter. It was characterized by stillborn, abortion, and premature birth. The objective of this study was to study the actual carrier frequency of the CVM mutation in a population of Chinese Holstein (=Chinese Black-and-White) normal cattle. The normal 436 Holstein cows and 93 Holstein bulls were genotyping by using the Created Restriction Site PCR (CRS-PCR) and Allele-specific PCR (AS-PCR) methods. There were two bulls and one cow in three observed CVM-carriers. In the Holstein dairy cattle and Holstein bull population, the percentages of CVM carriers were estimated as 0.60% and 2.20% respectively. This study provided a more reliable and useful method for extensive screening of CVM and also offers a theoretical basis for molecular diagnosis in Holstein calves.