Development of innovative marker detection methods for high-fertility ducks (Anas plastyrhynchos).

We previously analyzed the genome-wide gene expression at the transcription level in pre-hierarchical ovarian follicles (approximate 5 mm in diameter) between two groups of ducks representing high and low fertility. Orthodenticle homeobox 2 (otx2) was identified with significantly differential expression in the high-fertility group versus the low-fertility group. To identify the relationships between genotypes and phenotypes, we recorded the reproductive performance in advance, including fertility, hatchability, and fertile period of female ducks. To ensure coverage of the entire duration of the fertile period, we extended the egg collection period after artificial insemination. Naturally, sperm cannot survive after a certain period of time in the female reproductive tract (sperm is not immortal); therefore, lower average values for fertility were observed in this study than that observed after a normal egg collection period, i.e., the lower average values of fertility (18 days after artificial insemination), were not due to the effect of otx2. The otx2 genomic sequence of Tsaiya ducks was firstly amplified with a primer pair of i3F and i3R for polymerase chain reaction based on Pekin duck sequence and a resultant 444-base pair fragment was obtained for DNA sequencing. Using multiple sequence alignment, new single-nucleotide polymorphisms g.366T > C and g.182G > T were discovered in the otx2 gene. With respect to g.366T > C, ducks were classified into CC, CT, and TT genotypes. For g.182G > T, three genotypes (GG, GT, and TT) were identified. Ducks were genotyped using novel specific primers and probes to rapidly screen their single-nucleotide polymorphisms. The results indicated that ducks with the CC genotype of g.366T > C exhibited the highest fertility among the CC, CT, and TT genotypes (p < 0.05). No significant difference was found in the fertile period and hatchability among three genotypes of g.366T > C. Moreover, no association was found between g.182G > T genotypes and the three reproductive phenotypes examined in this study. Collectively, the otx2 g.366T > C genotype is associated with duck females, and can be used as a marker for farming a flock of ducks with high fertility, as well as for genetic selection of breeders.

A new DNA marker of the TMIGD1 gene used to identify high fertilization rates in Tsaiya ducks (Anas platyrhynchos).

In a prior study, comparisons of individuals of Anas platyrhynchos with higher/lower reproductive performances showed that the expression of the transmembrane and immunoglobulin domain containing 1 (TMIGD1) gene significantly differed between the two groups. Here, we demonstrate that ducks with the TMIGD1 GG genotype have a significantly higher fertilization rate than other TMIGD1 genotypes. Primers designed based on the TMIGD1 sequence of Pekin duck were able to successfully amplify a TMIGD1 fragment from Tsaiya ducks, and sequencing results indicated that a single nucleotide polymorphism (SNP) of the TMIGD1 gene existed. We also developed a cost-effective method of restriction fragment length polymorphism. Using the above methods, ducks were classified into three genotypes. To identify the relationships between genotypes and traits, we recorded the ducks' performance; to ensure the coverage of the entire duration of the fertile period, the egg collection period was extended to 18 days, and therefore, lower than usual fertilization rates were observed. Further assessment using a high-throughput system showed that the ducks with the GG genotype exhibited the highest fertilization rates among genotypes (P < 0.05). We suggest that TMIGD1 may affect the release of sperm protection factors from the female genital tract, and thus alter fertilization rate. In conclusion, the results of this study demonstrate that the TMIGD1 GG genotype can be used as a new DNA marker to identify animals with high fertilization rates at a young age, a process which could improve farming efficiency.

A new method for detection of single nucleotide polymorphism in a female reproduction-associated gene, tmigd1, of Anas platyrhynchos using a strip biosensor with gold nanoparticles.

In this study, we first reported a lateral flow assay combined with primer extension (PEXT) and gold nanoparticles for single-nucleotide polymorphism (SNP) genotyping of the tmigd1 gene of the Tsaiya ducks (Anas platyrhynchos), which has the advantages of simplicity of operation, cost-effectiveness, and time-saving. Gold nanoparticles were tailed with thiol-thymine oligodeoxyribonucleotides (thiol-(dT)30) using the salt-aging method at 25°C and used as a label in a lateral flow assay. The lateral flow device was composed of test and control zones on a nitrocellulose membrane containing streptavidin and adenosine oligodeoxyribonucleotides ((dA)30), respectively. When the specific SNP existed, the corresponding primers were extended, and the reaction product was captured by streptavidin at the test zone owing to the introduction of biotin-deoxyuridine triphosphate (biotin-dUTP) into the reaction product during PEXT. Gold nanoparticles hybridized with the reaction product to render it visible. Here, we developed a new system for detection of single nucleotide polymorphism in a female reproduction-associated gene, tmigd1, of Anas platyrhynchos using the strip biosensor, and identified the optimized parameters for the concentration of Mg2+ in the PEXT reaction and the amount of streptavidin used on membranes for signal specificity.

Global transcriptional expression in ovarian follicles from Tsaiya ducks (Anas platyrhynchos) with a high-fertilization rate.

Novel candidates for biomarkers of a high-fertilization rate were identified here through global transcriptional profiling of ovarian follicles. Some other differentially expressed candidate genes were first noted to influence animal reproduction in our previous cDNA microarray analysis and are now recognized as markers for marker-assisted selection. In the present study, we compared gene expression in ovarian follicles from animals with high- and low-fertilization rates using an oligonucleotide array. On the basis of a fold change of greater than 1.2 and less than -1.2, a difference of >100 Affymetrix arbitrary units between the two groups, and a P value of less than 0.05, 47 genes were found to be associated with fertilization rate. GOEAST and MetaCore software were further used to identify the functional categories of genes that were differentially expressed. Then, we focused on three interesting genes associated with a high-fertilization rate: one of these genes was discovered to participate in signaling pathways of fertilization, and two genes take roles in lipid metabolism. An oligonucleotide array showed that the levels of orthodenticle homeobox 2 (OTX2) and lecithin:cholesterol acyltransferase (LCAT) gene expression were 1.62-fold and 1.95-fold higher in the high-fertilization rate group than in the low-fertilization rate group, respectively (P < 0.05). The level of apolipoprotein A-I (APOA1) gene expression was also higher in the high-fertilization rate group, with a difference of 2.31-fold (P < 0.05). The data were validated through quantitative polymerase chain reaction analysis. These results confirm the usefulness of the array technique and data mining methods in the discovery of new biomarkers and add knowledge to our understanding of the factors affecting fertilization rates in ovarian follicles.

A novel minisequencing single-nucleotide polymorphism marker of the lysozyme gene detects high hatchability of Tsaiya ducks (Anas platyrhynchos).

Lysozyme, one of the major albumen antimicrobials, can break down the polysaccharide walls of a broad spectrum of bacteria. This study presents a novel lysozyme marker of high hatchability in the form of minisequencing single-nucleotide polymorphisms (SNPs). Recently, lysozyme was identified by complementary DNA microarray analysis as one of several differentially expressed genes noted to influence hatchability and recognized as a marker candidate for animal marker-assisted selection. Higher levels (P < 0.05) of lysozyme mRNA (via real-time polymerase chain reaction analysis) and protein (in Western blotting results) were found to be associated with a high-hatchability phenotype. In the preliminary sequence analysis of this study, TsLy1-1 and TsLy1-2 primer pairs, designed according to the lysozyme sequence, were used to amplify small-scale genomic DNA samples from animals in two extreme groups of hatchability. Sequence analysis of the amplified 763-bp DNA products clearly showed that AA and GG genotypes of SNP g.390A > G were from the ducks of the low- and high-hatchability groups, respectively. The SNP g.390A > G also created a new specificity protein 1 transcription factor binding site in the lysozyme gene. Primer pairs of TsLy2-1 and TsLy2-2 then probed the amplified 763-bp DNA products to produce a shorter fragment for easier minisequencing analysis to divide 114 ducks into GG, GA, and AA genotypes. The GG ducks had the highest hatchability, representing that a new lysozyme SNP marker of good hatchability performance can be used for the purpose of marker-assisted selection in Tsaiya ducks.

A Molecular Biological and Biochemical Investigation on Mycobacterium tuberculosis MutT Protein.

BACKGROUND: Mycobacterium tuberculosis is a vicious microbe co-existing with the infected host. This pathogen exploited opportunities to spread during periods of urbanization and social upheaval, and got retreated with improved hygiene. OBJECTIVES: This investigation was designed to clone and characterize M. tuberculosis mutT gene, a homologue of a DNA repair protein in Escherichia coli. The aim was to depict the possible role of this homologue in the virulent microbe. MATERIALS AND METHODS: A DNA fragment of the mutT gene was amplified with PCR from the genomic DNA of strain H37Rv M. tuberculosis. The expression vector was transformed into E. coli strains BL21 (DE3) and MK602 (DE3) (mutT-). The protein activity assay was performed by biochemical methods. RESULTS: M. tuberculosis MutT shares 23% identity with the E. coli MutT protein. The mutT gene DNA fragment was subcloned into the expression vector pET28a(+) and the recombinant plasmid was overexpressed in E. coli. Purified and refolded M. tuberculosis MutT possesses a dGTPase activity, which is one of the most well-known preference nucleotidase activities of MutT in E. coli. This study also showed that the dGTPase activity of M. tuberculosis MutT was enhanced by magnesium and inhibited by Ni(2+) or EDTA. Endogenous MutT protein in M. tuberculosis lysate displayed a smear pattern in the Western blot, suggesting instability of this protein in the bacteria similar to the important proteins, such as P53 protein, tightly regulated by protein degradation. CONCLUSIONS: The cloned M. tuberculosis mutT gene and MutT protein were characterized. M. tuberculosis MutT has a dGTPase activity, which is one of the most well-known preference nucleotidase activities of MutT in E. coli. These findings provide further understanding about the vicious bacterium.

A novel SNP marker of ovalbumin gene in association with duck hatchability.

Our previous transcriptome analysis using a cDNA microarray identified differentially-expressed transcripts in Tsaiya ducks (Anas platyrhynchos); we concluded that the ovalbumin gene might be involved in duck hatchability. In the present study, associations of single nucleotide polymorphism (SNP) genotypes of the duck ovalbumin gene with hatchability were investigated. To confirm the cDNA microarray analysis, real-time polymerase chain reaction (PCR) and Western blot analysis were used to validate ovalbumin gene expression. The messenger RNA and protein expression of the ovalbumin gene were higher (P < 0.05) in the low-hatchability group (1.00 ± 0.19; 30.36 ± 3.51 arbitrary units) than in high-hatchability counterparts (0.56 ± 0.07; 8.53 ± 2.97 arbitrary units), consistent with the previous cDNA microarray analysis. The PCR products (506 base pairs) of ovalbumin gene amplified by the primer pair of TovaF and TovaR from the genomic DNA templates of 10 ducks were sequenced and a g.385 C>T SNP site in the 506-base pair sequence of the ovalbumin gene identified. Genotyping of SNP of 187 ducks was then carried out by PCR restriction fragment length polymorphism and minisequencing methods. Based on SNP genotypes of the duck ovalbumin gene, there were three types: CC, TT, and CT. Birds with the CC and TT genotypes had higher hatchability (79.59 ± 3.40, 76.35 ± 1.77) (P < 0.05) than those with a CT genotype (65.77 ± 2.07). In conclusion, the ovalbumin gene was an important candidate gene that can be used for marker-assisted selection to increase hatchability in Tsaiya ducks.

Genome-wide transcript expression analysis in the uterovaginal junction in association with fertile period in Tsaiya ducks.

We performed the first genome-wide expression analysis to compare the differences in gene expression in the female sperm reservoir of the duck reproductive tract between two groups with long and short fertile periods to identify factors that may be associated with the fertile period using an oligonucleotide microarray. RNA was extracted from the uterovaginal junction (UV junction) of the two groups. Affymetrix chips containing comprehensive coverage of 32773 transcripts were hybridized with biotin-labeled cRNA, and three biological repeats were performed. We identified 27 transcripts as being differentially regulated. Interestingly, by mapping the differentially expressed transcripts to annotated pathways, we found that Neuropeptide Y (NPY), the RNA expression of which was increased by 2.96-fold in the short-fertile-period group as compared with the long-fertile-period group in our experiment, has been shown to reduce blood flow and substance supply to local tissues. Enah/Vasp-like (EVL), the RNA expression of which was significantly increased by 1.77-fold in the short-fertile-period group as compared with the long-period group, has been demonstrated to be important in activated T-cells. In contrast, trafficking kinesin-binding protein 1 (TRAK1), the expression of which was increased by 2.33-fold in the long-period group as compared with its counterparts, has been suggested to inhibit precocious activation of sperm and prolong sperm life in the female sperm reservoir. The results of real-time PCR confirmed the data obtained by microarray analysis. Our study demonstrated that combining global gene expression investigation with annotated pathway resources contributes to the understanding of sperm life when sustained in the UV junction.

Duck (Anas platyrhynchos) linkage mapping by AFLP fingerprinting.

Amplified fragment length polymorphism (AFLP) with multicolored fluorescent molecular markers was used to analyze duck (Anas platyrhynchos) genomic DNA and to construct the first AFLP genetic linkage map. These markers were developed and genotyped in 766 F2 individuals from six families from a cross between two different selected duck lines, brown Tsaiya and Pekin. Two hundred and ninety-six polymorphic bands (64% of all bands) were detected using 18 pairs of fluorescent TaqI/EcoRI primer combinations. Each primer set produced a range of 7 to 29 fragments in the reactions, and generated on average 16.4 polymorphic bands. The AFLP linkage map included 260 co-dominant markers distributed in 32 linkage groups. Twenty-one co-dominant markers were not linked with any other marker. Each linkage group contained three to 63 molecular markers and their size ranged between 19.0 cM and 171.9 cM. This AFLP linkage map provides important information for establishing a duck chromosome map, for mapping quantitative trait loci (QTL mapping) and for breeding applications.

Differential regulation of telomerase in endothelial cells by fibroblast growth factor-2 and vascular endothelial growth factor-a: association with replicative life span.

In cultured human umbilical vein endothelial cells (HUVECs), fibroblast growth factor-2 (FGF-2), but not vascular endothelial growth factor-A (VEGF-A), upregulates telomerase activity. Here, we examined the functional significance of this differential regulation on the replicative life span of HUVECs. HUVECs were serially passaged until senescence under four different conditions: (1) EGM-2, a medium containing both VEGF-A and FGF-2; (2) basal medium (BM), consisting of EGM-2 devoid of FGF-2 and VEGF-A; (3) BM supplemented with FGF-2; and (4) BM supplemented with VEGF-A. Cells cultured in BM demonstrated decreased growth rate and ceased to proliferate at approximately 15 population doublings (PDs), whereas those cultured with VEGF-A alone initially proliferated vigorously but arrested growth abruptly at a PD level comparable with cultures grown in BM. In contrast, cells maintained in EGM-2 or in BM/FGF-2 attained a normal replicative life span (approximately 40 PDs). These differences in replicative behavior were reflected by the early appearance of a senescent phenotype in cultures grown in BM or BM/VEGF-A. HUVECs grown in the presence of VEGF-A alone have a decreased life span compared with cultures maintained with FGF-2. This suggests that the upregulation of telomerase activity by FGF-2, an effect not achieved with VEGF-A, plays a functional role in preventing the early onset of senescence.

Early atherogenesis in senescence-accelerated mice.

We studied atheromatous lesion formation in an animal model of accelerated ageing. The senescence-accelerated prone mouse (SAM-P) has a reduced life-span and exhibits clinical features characteristic of human ageing. Our aim was to establish whether these mice are more susceptible to atherosclerosis than a related strain, senescence-accelerated resistant mice (SAM-R), which age normally. We fed a Western-type diet to 14 SAM-P/8 and 14 SAM-R/1 mice for 17 weeks, starting at 28 weeks of age, measuring their serum lipid profiles before and after this diet. We stained aortic root cryostat cross-sections with Oil red O, and assessed lipid deposition morphometrically. We used immunohistochemistry to detect macrophages in the aortic roots. We found that despite showing similar alterations in lipid profile, SAM-P/8 mice developed more prevalent and extensive fatty lesions than SAM-R/1 mice. Furthermore, the lipid lesions in SAM-P/8 mice showed a greater frequency of invasion by macrophages. We conclude that mice, which age at an accelerated rate, are more prone to early atherogenesis than mice which age normally. We suggest that this increased susceptibility may result from abnormalities in the oxidative status and cellular replicative capacity of these mice.

Fibroblast growth factor-2, but not vascular endothelial growth factor, upregulates telomerase activity in human endothelial cells.

OBJECTIVE: Telomerase plays a major role in the control of replicative capacity, a critical property for successful angiogenesis and maintenance of endothelial integrity. In this study, we examined the relationship between telomerase activity and endothelial cell proliferation as well as the regulation of this enzyme by fibroblast growth factor-2 (FGF-2) and vascular endothelial growth factor-A (VEGF). METHODS AND RESULTS: Telomerase was repressed in endothelial cells freshly derived from intact endothelium, whereas activity was present during logarithmic growth in culture. In cultured human umbilical vein endothelial cells (HUVECs), mRNA levels of hTERT-the catalytic subunit of telomerase-and enzyme activity decreased reversibly on induction of quiescence. Treatment of quiescent HUVECs with FGF-2 restored telomerase activity in a time- and dose-dependent manner, whereas VEGF had no such effect, although both factors induced comparable mitogenic responses. FGF-2, but not VEGF, upregulated the mRNA levels for hTERT and for the hTERT gene transactivation factor Sp1. Serial passage in the presence of individual growth factors accelerated the accumulation of senescent cells in VEGF-treated cultures compared with cultures treated with FGF-2. CONCLUSIONS: FGF-2, but not VEGF, restores telomerase activity and maintains the replicative capacity of endothelial cells.