Novel SNPs in the <Emphasis Type="Italic">PRDM16</Emphasis> gene and their associations with performance traits in chickens

The PR domain containing 16 (PRDM16) is a member of the Prdm family, and is known to regulate cell differentiation. In the present study, DNA pool sequencing methods were employed to screen genetic variations in the chicken PRDM16 gene. The results revealed four novel single nucleotide polymorphisms (SNPs): NC_006108.2: g.92188G>A, XM_417551: c.1161C>T (Ala/Ala, 387aa), c.1233C>T (Ser/Ser, 411aa) and c.1433G>A (Ser/Asn, 478aa). The BglI polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) was used to detect c.1161C>T, while HhaI Forced PCR-RFLP methods were used to detect 1233C>T and c.1433G>A in 964 chickens. The chickens comprised 38 grandparents, 66 F₁ parents and 860 F₂ birds derived from an F₂ resource population of Gushi chickens crossed with Anka broilers. The associations of the polymorphisms in the chicken PRDM16 gene with performance traits were analyzed in the 860 F₂ chickens. The results indicated that the three SNPs were significantly associated with growth, fatness and meat quality traits in the chickens. In particular, the polymorphisms of the missense SNP (c.1433G>A) had positive effects on chicken body weight and body size at different stages. It affected also fatness traits significantly. Comparison of the different genotypes of c.1433G>A showed that the GG genotype favored chicken growth and fatness traits.     

Polymorphisms of <Emphasis Type="Italic">STAT5A</Emphasis> gene and their association with milk production traits in Holstein cows

The STAT5A gene was studied as a candidate gene for five milk production traits (milk yield at 305 days, protein percentage, fat percentage, lactose percentage and dry matter percentage) in Holstein cows. According to the sequence of bovine STAT5A gene, two pairs of primers (P1 and P2) were designed to detect polymorphisms of STAT5A gene in 401 Holstein cows by PCR-RFLP and PCR-SSCP. The results showed that the products amplified by primers P1 and P2 displayed polymorphisms. For P1, three genotypes (AA, AG, and GG) were detected, and the frequency of AA/AG/GG was 0.252/0.486/0.262, respectively. Sequence analysis revealed a single nucleotide substitution A–G at 14217 bp (GenBank NC_007317) of bovine STAT5A gene while compared GG genotype with AA genotype. The differences of the least squares means for the four milk production traits (milk yield at 305 days, fat percentage, lactose percentage and dry matter percentage) between AA, AG and GG were not significant (P > 0.05). Least squares mean of protein percentage for AG or GG was significantly higher than that for AA (P < 0.05); the difference of the least squares mean for protein percentage was not significant between AG and GG (P > 0.05). For P2, three genotypes (CC, CT, and TT) were detected in Holstein cows, and the frequency of CC/CT/TT was 0.751/0.234/0.015, respectively. Sequencing revealed an insertion CCT at 17266 (NC_007317) of bovine STAT5A gene while compared CC genotype with TT genotype. The differences of the least squares means for the three milk production traits (protein percentage, lactose percentage and dry matter percentage) between CC, CT and TT were not significant (P > 0.05). Least squares mean of milk yield at 305 days for TT or CT was significantly higher than that for CC (P < 0.05); the difference of the least squares mean for milk yield at 305 days was not significant between TT and CT (P > 0.05). Least squares mean of fat percentage for CC or CT was significantly higher than that for TT (P < 0.05); the difference of the least squares mean for fat percentage was not significant between CC and CT (P > 0.05). The results preliminarily indicated that allele G of A14217G polymorphic site of STAT5A gene is a potential DNA marker for improving protein percentage in dairy cattle, 17266indelCCT polymorphic site of STAT5A gene is a potential DNA marker for improving milk yield at 305 days and fat percentage in dairy cattle.     

Two novel cSNPs of <Emphasis Type="Italic">weaver</Emphasis> gene in Chinese indigenous goat and their associations with milk yield

Weaver gene plays an essential physiological role in the function of many organs, including brain, heart, kidney and endocrine cells, and also in the regulation of insulin secretion by glucose and/or neurotransmitters. Thus, weaver gene is an important potential candidate gene effecting on performance traits. The objective of this study was to detect the genetic variation of five loci within weaver gene by PCR-SSCP, DNA sequencing and forced PCR-RFLP methods in 1,019 Chinese indigenous goats. Two novel coding SNPs (XM_598993:m.864G>A; XM_598993:m.1224T>A) locating on P3 and P4 loci were identified and detected by MluI and AsuII forced PCR-RFLP, respectively. In the MluI analysis, the frequencies of goat MluI-A allele in the analyzed populations were 0.226, 0.248, 0.096 and 0.088 for XNSN, GZ, SBWC and XJWC, respectively. Genotypic frequencies were found to be significantly different in four breeds (χ² = 75.842, df = 6, P < 0.001); In the AsuII analysis, the frequencies of goat AsuII-A allele in the analyzed populations were 0.584, 0.441, 0.073 and 0.063 for XNSN, GZ, SBWC and XJWC, respectively. Genotypic frequencies were found to be significantly different among four breeds (χ² = 399.464, df = 6, P < 0.001). The frequencies of allele MluI-A and AsuII-A in XNSN and GZ populations were significantly higher than those of SBWC and XJWC goats. Association analysis with adjusted milk yield in the XNSN breed indicated that the animals with AsuII-AA genotype owned significantly higher adjusted milk yield than the ones with AsuII-TT genotype in the second lactation (P < 0.05). The observation suggested that the allele “AsuII-A” had the positive effects on adjusted milk yield in the second lactation.     

An <Emphasis Type="Italic">Msp</Emphasis>I polymorphism in the inhibin alpha gene and its associations with superovulation traits in Chinese Holstein cows

To identify a predictor to forecast superovulation response on the basis of associations between superovulation performance and gene polymorphism, the PCR–RFLP method was applied to detect an A>G transition determining an MspI polymorphism at position 192 in the exon I of the bovine inhibin alpha (INHA) gene and evaluate its associations with superovulatory response in 118 Chinese Holstein cows treated for superovulation. Association analysis showed that cows with the GG genotype resulted in a significant increase in the number of ova (TNO) than AG and AA genotypes in the first (P = 0.023), second (P = 0.004) and third (P = 0.002) superovulation treatments and produced more transferable embryos (NTE) than that of AG and AA genotypes in the third (P = 0.045) superovulation treatment. Moreover, individuals with GG genotype produced more transferable embryos than AA (P < 0.05) genotype in the second superovulation treatment and all cows without superovulation response were mutations with genotypes of AA and AG. These results indicate that INHA gene can be used as a predictor for superovulation in Chinese Holstein cows, and imply that cows with AA genotype should be excluded for superovulation practices.     

Identification of novel SNPs of ovine <Emphasis Type="Italic">PRL</Emphasis> gene and their association with milk production traits

Prolactin (PRL) is a lactogenic hormone that plays a significant role in milk production; its depletion in sheep provokes a severe reduction of milk secretion. Thus, PRL also could be used as a positional marker gene associated with milk production and composition traits. Therefore, the purpose of the study was to identify genotype frequencies of single nucleotide polymorphisms the intron 2 in ovine PRL gene and its possible association genotypes with milk traits in dairy sheep breeds. The genetic structures of ovine PRL gene were examined by PCR-RFLP and DNA sequencing methods in three sheep populations. Four hundred and fifty blood and milk samples were used in the study, which included 150 samples from each of Sakiz, Akkaraman and Awassi ewes respectively. As a result, PRL genotype AA showed a strong association with milk yields content, whereas the animals carrying BB genotype had a higher fat percentage value in the three sheep breeds. Haplotype analysis of the obtained sequences showed the presence of 12 haplotypes in the PRL intron 2 region. In the present study, we have reported for the first time 48 SNPs of the PRL gene for intron 2 in dairy sheep breeds. These preliminary results indicate that the identified SNPs lend themselves readily for further research regarding physiological impacts such as milk production and reproductive traits in other dairy sheep populations.     

Single nucleotide polymorphisms in bovine <Emphasis Type="Italic">PRL</Emphasis> gene and their associations with milk production traits in Chinese Holsteins

Prolactin (PRL) plays a crucial role in the initiation and maintenance of lactation in mammals. In this study, seven PCR fragments representing most important functional domains of PRL gene were screened for single nucleotide polymorphisms (SNPs) in Chinese Holsteins by single-strand conformation polymorphisms and amplicons sequencing, and their genetic effects on milk production traits were evaluated. A total of four SNPs, including two in the promoter (−1043A>G and −402A>G), one in intron 1 (+2723C>T) and one in exon 4 (+8398G>A) were identified in PRL gene. Statistical results showed significant associations between the promoter genotypes and the milk performance traits in Chinese Holsteins. Cows with genotype P1-GG showed higher milk yields (P < 0.01), while cows with genotype P1-AA showed higher fat contents (P < 0.01). Haplotype analysis of two SNPs in promoter region revealed that the Hap(AG) was significantly associated with increased milk yields and Hap(AA) was associated with increased fat contents (P < 0.01). This is the second study reporting SNPs in the 5′-regulatory region of PRL gene, which interfere with milk production traits.     

Polymorphism of <Emphasis Type="Italic">DLK1</Emphasis> and <Emphasis Type="Italic">CLPG</Emphasis> gene and their association with phenotypic traits in Chinese cattle

DLK1 and CLPG were located in DLK1-GTL2 imprinted cluster. They all affected muscle growth and meat tenderness. The functional importance of DLK1 and CLPG imply that the variation of the genes could affect the growth traits of animal. PCR-SSCP and sequencing were used to analyze the four loci of DLK1 gene and CLPG gene in 1109 individuals, which belong to eight breeds/species of bovidae, including cattle, buffalo and yak. A synonymous mutation (C451T) was detected in exon 5 of DLK1 in Qinchuan cattle, but didn’t change significantly with phenotypic traits. Three genotypes AA, AB and AC of CLPG were identified in Jiaxian cattle. The associations analyst of different genotypes showed that the individuals with genotypes AA and AC had a greater body weight and longer body length than those with genotype AB (P < 0.05 and P < 0.01, respectively); the AA individuals were different from those AB (P < 0.05) in the circumference of cannon bone. No polymorphism was observed in the other populations at other loci. These results were in agreement with the homology analysis: DLK1 and CLPG genes were in a highly conserved.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.     

The polymorphisms of bovine <Emphasis Type="Italic">VEGF</Emphasis> gene and their associations with growth traits in Chinese cattle

PCR-SSCP and DNA sequencing methods were employed to screen the genetic variation of VEGF gene in 671 individuals belonging to three Chinese indigenous cattle breeds including Nanyang, Jiaxian Red and Qinchuan. Three haplotypes (A, B and C), four observed genotypes (AA, AB, BB and AC) and three new SNPs (6765T>C ss130456744, 6860A>G ss130456745, 6893T>C ss130456746) were detected. The analysis suggested that one SNP (ss130456744) in the bovine VEGF gene had significant effects on birth weight, body weight and heart girth at 6 months old in the Nanyang breed (P < 0.05). The results showed that the SNP (ss130456744) in intron 2 of the VEGF gene is associated with early development and growth of Chinese cattle. These findings raise hope that this polymorphism can be a molecular breeding marker in breeding strategies through marker assisted selection (MAS) in Chinese domestic cattle.     

<Emphasis Type="Italic">Galleria</Emphasis><Emphasis Type="Italic">mellonella</Emphasis> are Resistant to <Emphasis Type="Italic">Pneumocystis</Emphasis><Emphasis Type="Italic">murina</Emphasis> Infection

Studying Pneumocystis has proven to be a challenge from the perspective of propagating a significant amount of the pathogen in a facile manner. The study of several fungal pathogens has been aided by the use of invertebrate model hosts. Our efforts to infect the invertebrate larvae Galleria mellonella with Pneumocystis proved futile since P. murina neither caused disease nor was able to proliferate within G. mellonella. It did, however, show that the pathogen could be rapidly cleared from the host.     

Revision of the <Emphasis Type="Italic">Serrulati</Emphasis> species of <Emphasis Type="Italic">Penstemon</Emphasis> section <Emphasis Type="Italic">Saccanthera</Emphasis> subsection <Emphasis Type="Italic">Serrulati</Emphasis>

A revision of Penstemon sect. Saccanthera subsect. Serrulati includes a new species (P. salmonensis), a new variety (P. triphyllus var. infernalis), and the elevation of a subspecies to species (P. curtiflorus), bringing the total number of species to eight, which are keyed and described, complete with nomenclature and type citations.     

Novel SNPs of the bovine <Emphasis Type="Italic">NUCB2</Emphasis> gene and their association with growth traits in three native Chinese cattle breeds

In this study, polymorphism in the exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 11 of bovine NUCB2 gene was detected by PCR-SSCP and DNA sequencing methods in 686 individuals from three Chinese cattle breeds. Two haplotypes (M and N), three observed genotypes (MM, MN and NN) and two SNPs (NC_007313: g. 27451G>A, NC_007313: g. 27472T>C) were detected. The frequencies of haplotypes M and N in inland Chinese three breeds were 0.531–0.721 and 0.279–0.469 respectively. The studied showed that Nanyang, Jiaxian Red and Qinchuan cattle populations were in Hardy–Weinberg equilibrium at SNPs locus of NUCB2 gene (P > 0.05). Polymorphism of the NUCB2 gene was shown to be associated with growth traits in Qingchuan and Nanyang cattle breed. The linkage of two mutant sites in the bovine NUCB2 gene had significant effects on body length, body weight, heart girth, and average daily gain at 24 months (P < 0.05). Results of this study suggested that the NUCB2-gene-specific SNP may be a useful marker for growth traits in future marker-assisted selection programmes in inland Chinese cattle.     

Genotypes,haplotypes and diplotypes of <Emphasis Type="Italic">IGF</Emphasis>-<Emphasis Type="Italic">II</Emphasis> SNPs and their association with growth traits in largemouth bass (<Emphasis Type="Italic">Micropterus salmoides</Emphasis>)

Insulin-like growth factor II (IGF-II) is involved in the regulation of somatic growth and metabolism in many fishes. IGF-II is an important candidate gene for growth traits in fishes and its polymorphisms were associated with the growth traits. The aim of this study is to screen single nucleotide polymorphisms (SNPs) of the largemouth bass (Micropterus salmoides) IGF-II gene and to analyze potential association between IGF-II gene polymorphisms and growth traits in largemouth bass. Four SNPs (C127T, T1012G, C1836T and C1861T) were detected and verified by DNA sequencing in the largemouth bass IGF-II gene. These SNPs were found to organize into seven haplotypes, which formed 13 diplotypes (haplotype pairs). Association analysis showed that four individual SNPs were not significantly associated with growth traits. Significant associations were, however, noted between diplotypes and growth traits (P < 0.05). The fish with H1H3 (CTCC/CGCC) and H1H5 (CTCC/TTTT) had greater body weight than those with H1H1 (CTCC/CTCC), H1H2 (CTCC/TGTT) and H4H4 (TGCT/TGCT/) did. Our data suggest a significant association between genetic variations in the largemouth bass IGF-II gene and growth traits. IGF-II SNPs could be used as potential genetic markers in future breeding programs of largemouth bass.     

Novel SNPs of butyrophilin (<Emphasis Type="Italic">BTN1A1</Emphasis>) and milk fat globule epidermal growth factor (EGF) 8 (<Emphasis Type="Italic">MFG</Emphasis>-<Emphasis Type="Italic">E8</Emphasis>) are associated with milk traits in dairy goat

Butyrophilin (BTN1A1) and milk fat globule epidermal growth factor (EGF) 8 (MFG-E8) genes are both milk fat globule membrane proteins. BTN1A1 plays a key role in the secretion of milk lipid and production which has effects on performance traits, while the MFG-E8 is vital for the development of the mammary gland and phagocytic clearance of apoptotic cells. Therefore, BTN1A1 and MFG-E8 gene are candidate genes for quantitative traits in mammalian animals with respect to milk performance traits. The objective of this study is to investigate variations in goat BTN1A1 and MFG-E8 gene and analyze their associations with growth trait and milk performance. In this study, the goat BTN1A1 gene showed a novel single-nucleotide polymorphism (SNP): XM_001494179:g.8659C>T, resulting in a missense mutation: CTT (Leu)>TTT (Phe) at position 377 aa of the BTN1A1 (526 aa); the goat MFG-E8 gene showed four novel SNPs: NC_007319: g.843delA, 6417delC, 14892T>C and 14996A>C, only the 14892T>C result in a synonymous mutation. The associations between genotypes and production traits were analyzed. Significant statistical results implied that HinfI locus of BTN1A1 gene is associated with milk fat yield (P = 0.004), total solid (P = 0.002), solid-non fat (P = 0.018) and first milk yield (P = 0.030). The DA and EcoRV loci of MFG-E8 gene are associated with milk fat yield (DA locus: P = 0.000; EcoRV locus: P = 0.033) and total solid (DA locus: P = 0.002; EcoRV locus: P = 0.015) in the Xinong Saanen dairy goat.     

<Emphasis Type="Italic">Drosophila melanogaster</Emphasis> gene <Emphasis Type="Italic">Merlin</Emphasis> interacts with the clathrin adaptor protein gene <Emphasis Type="Italic">lap</Emphasis>

The protein Merlin is involved in the regulation of cell proliferation and differentiation in the eyes and wings of Drosophila and is a homolog of the human protein encoded by the Neurofibromatosis 2 (NF2) gene whose mutations cause auricular nerve tumors. Recent studies show that Merlin and Expanded cooperatively regulate the recycling of membrane receptors, such as the epidermal growth factor receptor (EGFR). By performing a search for potential genetic interactions between Merlin (Mer) and the genes important for vesicular trafficking, we found that ectopic expression in the wing pouch of the clathrin adapter protein Lap involved in clathrin-mediated receptor endocytosis resulted in the formation of extra vein materials. On the one hand, coexpression of wild-type Merlin and lap in the wing pouch restored normal venation, while overexpression of a dominant-negative mutant Mer ^DBB together with lap enhanced ectopic vein formation. Using various constructs with Merlin truncated copies, we showed the C-terminal portion of the Merlin protein to be responsible for the Merlin-lap genetic interaction. Furthermore, we showed that the Merlin and Lap proteins colocalized at the cortex of the wing imaginal disc cells.     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.